Original Articles - cu

Volume 11 • Number 12 • December 2012

Chinese-Germ

an Journal of Clinical Oncology Volum

e 11 • Num

ber 12 • Decem

ber 2012 pp 683–74410330

Volume 11 • Number 12 • December 2012

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Clinical Oncology

Chinese-GermanJournal of

ISSN 1610-1979 (Paper) 1613-9089 (Online)CN 42-1654/R

Chinese-German Journal of

Clinical OncologyReview Articles

DNA repair genes BRCA1 and DNA-PKcs have great potential in radiation therapy Jiao Yang, Ximing Xu, Yanrong Hao 683

Hypotheses explaining cancer metastasisHaijuan Wang, Chen Lin, Haili Qian 689

The progression of microRNA in human colorectal cancer Yeping Du, Jinhua Miao, Chunmei Wu, Liping Xu 691

Original Articles

Value of 99mTc-MDP SPECT/CT fusion imaging and CT in evaluating the extent of mandibular invasion by malignant tumor of oral cavityQingyun Duan, Muyun Jia, Rongtao Yuan, Lingxue Bu, Wei Shang, Xiaoming Jin, Ningyi Li, Jie Zhao, Guoming Wang 694

Prognostic factors in 408 elderly lung cancer patients over 70 years oldHua Zheng, Li Tong, Ying Hu, Weihua Wu, Hongmei Zhang, Baolan Li 699

Correlation of STAT3, CEA in lung adenocarcinoma cell A549 Debin Sun, Xiu Lan (Co-first author), Hongcheng Wang 705

Chinese-German Journal of Clinical OncologyDecember 2012 Volume 11 Number 12

Contents

Review Articles

DNA repair genes BRCA1 and DNA-PKcs have great potential in radiation therapy Jiao Yang, Ximing Xu, Yanrong Hao 683

Hypotheses explaining cancer metastasisHaijuan Wang, Chen Lin, Haili Qian 689

The progression of microRNA in human colorectal cancer Yeping Du, Jinhua Miao, Chunmei Wu, Liping Xu 691

Original Articles

Value of 99mTc-MDP SPECT/CT fusion imaging and CT in evaluating the extent of mandibular invasion by malignant tumor of oral cavityQingyun Duan, Muyun Jia, Rongtao Yuan, Lingxue Bu, Wei Shang, Xiaoming Jin, Ningyi Li, Jie Zhao, Guoming Wang 694

Prognostic factors in 408 elderly lung cancer patients over 70 years oldHua Zheng, Li Tong, Ying Hu, Weihua Wu, Hongmei Zhang, Baolan Li 699

Correlation of STAT3, CEA in lung adenocarcinoma cell A549 Debin Sun, Xiu Lan (Co-first author), Hongcheng Wang 705

Dosimetric study comparing photon and electron beams for boosting the tumor bed in early-stage breast cancerMohamed Mahmoud, Soha Ahmed, Ehab M. Attalla, Hassan S. Abouelenein, Shaimaa Shoier, Mohsen Barsoum 710

Individual detection significances of small breast epithelial mucin (SBEM) and human mammaglobin (hMAM) expressions in peripheral blood of breast cancer patientsZhaozhe Liu, Fang Guo, Xiaodong Xie 716

Reversing effects of traditional Chinese antitumor medicines on colorectal tumor immunosuppression of natural killer cell and T lymphocyte in vitroCheng Cui, Aixia Zhang, Jianjun Hu, Wenguang Zheng, Zhanjiang Fu, Lirong Qi, Meixiang Li, Wei Lv 721

Down-regulation of Bmi-1 by RNA interference inJurkat cellsShangen Zheng, Qibin Jing, Yaqiong Zheng, Yinjuan Ding, Qianchuan Huang, Guoqiang Zhao 732

Cell-cycle-dependent variation in UV absorption spectrum of Hela cells treated with Trichostatin AFengqiu Zhang, Xiaoxia Wang, Zhanguo Yang 737

Activties Report

Treat advanced cancer patient with care, strengthen multi-disciplinary cooperation – The 8th Conference of Chinese Cancer Rehabilitation and Palliative Care was held in Qingdao 741

A1

Chinese-German Journal of Clinical Oncology

Honorary Editors-in-ChiefW.-W. Höpker (Hamburg)Mengchao Wu (Shanghai) Yan Sun (Beijing) Editors-in-Chief Anmin Chen (Wuhan) Shiying Yu (Wuhan)Anthony D. Ho (Heidelberg) Administrative Associate Editor-in-ChiefShukui Qin (Nanjing)Associate Editors-in-Chief Yilong Wu (Guangzhou) Hanxiang An (Marburg) Ping Zou (Wuhan) Steering Committees of SpecialistsDatong Chu (Beijing) Feili Gong (Wuhan) Zhongzhen Guan (Guangzhou) Junyuan Guo (Wuhan)Xishan Hao (Tianjin)Guoliang Jiang (Shanghai) Meilin Liao (Shanghai) Yunyi Liu (Hongkong) Shuyou Peng (Hangzhou)Wenjiang Shen (Beijing)Zhenzhou Shen (Shanghai)Santai Song (Beijing)Zhaoyou Tang (Shanghai)Hongyang Wang (Shanghai)Pengzhi Wang (Tianjin)Zaide Wu (Wuhan)Jiamei Yang (Shanghai)Zihao Yu (Beijing)Yixin Zeng (Guangzhou) Shu Zheng (Hangzhou)Editorial Board Norbert Arnold (Kiel)Gunther Bastert (Heidelberg)Matthias W. Beckmann (Erlangen)H. J. Biersack (Bonn)Hubert E. Blum (Freiburg)

Markus W. Büchler (Heidelberg) Joe Y. Chang (Houston)Charles S. Cleeland (Houston) Jörg F. Debatin (Heidelberg) Joachim W. Dudenhausen (Berlin) Gerhard Ehninger (Dresden) Reg Gorczynski (Toronto) Adolf Grünert (Ulm) A. R. Hanauske (Hamburg)Ch. Herfarth (Heidelberg) Dieter Hoelzer (Frankfurt) W. Hohenberger (Erlangen) Arnulf H. Hölscher (Cologne) Ulrich T. Hopt (Freiburg) Karl-Walter Jauch (Munich) Gary A. Levy (Toronto) Guojun Li (Houston) Lei Li (Houston)Zhongxing Liao (Houston) Dolores J. Schendel (Munich) Helmut K. Seitz (Heidelberg) J. R. Siewert (Heidelberg) Katherine A. Siminovitch (Toronto) Christof von Kalle (Heidelberg) Xin Shelley Wang (Houston)Qingyi Wei (Houston) Jingyi Zhang (Toronto) Sanjun Cai (Shanghai) Xiaoping Chen (Wuhan) Yuan Chen (Wuhan) Ying Cheng (Changchun) Zhonghua Chen (Wuhan) Ruifang Fan (Lanzhou) Weijian Feng (Beijing) Xiaolong Fu (Shanghai) Jianping Gong (Wuhan) Benli Han (Chongqing)Jian Hou (Shanghai)Daoyu Hu (Wuhan) Xichun Hu (Shanghai) Huiqiang Huang (Guangzhou) Tao Huang (Wuhan)Zhiqiang Huang (Beijing) Jiafu Ji (Beijing)

Wenqi Jiang (Guangzhou) Zefei Jiang (Beijing) Changshu Ke (Wuhan) Jinyi Lang (Chengdu) Kaijian Lei (Yibin)Ting Lei (Wuhan) Jin Li (Shanghai) Shenjiang Li (Tai’an) Shuling Li (Tianjin) Yexiong Li (Beijing)Ziting Li (Shanghai) Zhiyong Li (Dalian) Liu Wei (Shijiazhuang) Shun Lu (Shanghai) Lu You (Chengdu) Rongcheng Luo (Guangzhou)Ding Ma (Wuhan)Hongbing Ma (Xi’an)Jun Ma (Harbin)Shenglin Ma (Hangzhou)Huaqing Min (Guangzhou)Xiaohui Niu (Beijing)Jun Ren (Beijing)Guanxin Shen (Wuhan)Lin Shen (Beijing)Zhixiang Shen (Shanghai)Qijia Shi (Wuhan)Yuankai Shi (Beijing)Yingqiang Shi (Shanghai)Lili Tang (Beijing)Dongmin Wang (Beijing)Guobin Wang (Wuhan)Huaqing Wang (Tianjin)Lvhua Wang (Beijing)Shixuan Wang (Wuhan)Wenwu Wang (Fuzhou)Yuquan Wei (Chengdu)Gang Wu (Wuhan)Jianlin Wu (Dalian)Lingying Wu (Beijing)Liangping Xia (Guangzhou)Xiaodong Xie (Shenyang)Jianping Xiong (Nanchang)Binghe Xu (Beijing)

A2

Ximing Xu (Wuhan)Yongjian Xu (Wuhan)Lvnan Yan (Chengdu)Baoming Yu (Shanghai)Ding Yu (Wuhan)Hongsheng Yu (Qingdao)Xiuchun Yu (Jinan)Guangjin Yuan (Huai’an)Li Zhang (Guangzhou)Lin Zhang (Wuhan)Qunhua Zhang (Shanghai)

Qingyuan Zhang (Harbin)Yicheng Zhang (Wuhan)Yongxue Zhang (Wuhan)Xiuyi Zhi (Beijing)Caicun Zhou (Shanghai)Jianfeng Zhou (Wuhan)Liqiang Zhou (Beijing)Qinghua Zhou (Chengdu)Yunfeng Zhou (Wuhan)Guangying Zhu (Beijing)Jun Zhu (Beijing)

Managing DirectorsWeiguo DongJun Xia

Executive Editors Yening WangJun XiaJing Chen Qiang WuTypesetting Editor Wenge Wang


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Chinese-German Journal of Clinical Oncology December 2012, Vol. 11, No. 12, P683–P688 DOI 10.1007/s10330-012-1088-4

DNA damage is responsible for a number of effects in cancer including being the cause of the radiation, being the target for many anticancer therapies. And DNA re-pair is a ubiquitous process throughout the living world. Wood et al reported that 130 genes directly involved in DNA repair in humans, and their cDNA sequence estab-lished in 2001 at the first time [1]. About 25 genes were added to DNA repair genes, and a few deleted in 2005 [2]. The DNA repair systems protect our genome from carci-nogenesis [3]. Double-strand breaks (DSBs) repair is one of the key factor in this system. An increasing body of evi-dence associates a reduction in mammalian DNA-repair capacity with increased rates of birth defects, cancer, and reduced lifespan. Breast cancer 1 (BRCA1) and DNA-de-pendent protein kinase catalytic subunit (DNA-PKcs) are two key players in the homologous recombination (HR) and non-homologous end joining (NHEJ) pathway of DNA double strand break repair, respectively. Although using the same treatment for the same clinical stages and the same pathology classification of patients the out-come are obvious difference. The goal of this article in an improved understanding of whether the expressions of BRCA1 and DNA-PKcs genes have great potential in radiation therapy (RT).

BRCA1 plays a pivotal role in radiotherapy through the HR

Homologous recombinationHR is the major pathway of DNA double-strand breaks

(DSBs) repair in all eukaryotes and thus, is essential for cell viability and genome stability and, as a consequence, plays an important role in prevention of mutations, chro-mosomal instability and cancer [4, 5]. Furthermore, HR uses an homologous DNA template and is highly accurate and conserved through eukaryotes. It dominates during S and G2 phases of cell cycle. HR has a distinct advantage over other repair mechanisms. It is an error-free pathway of damage tolerance, allowing replication bypass of lesions through a template switch [4, 5]. However, if single-strand breaks (SSBs) are left unrepaired, the resulting fork col-lapse and DSB formation [6], cannot be repaired in cells, which are HR incompetent [7]. BRCA1 and BRCA2 genes have crucial functions in the HR pathway and so muta-tions of these genes causes HR pathway failure [8]. The response of cancers, known to be deficient in HR due to BRCA1 or 2 mutations.

The biology of BRCA1 The BRCA1 was the first breast cancer susceptibility

gene identified in 1990 [9]. The BRCA1 gene is charac-terized by a unique protein–protein interaction surface,

DNA repair genes BRCA1 and DNA-PKcs have great potential in radiation therapy Jiao Yang1, 2, Ximing Xu1, Yanrong Hao2

1 Cancer Center, Renmin Hospital of Wuhan University, Wuhan 430060, China2 Department of Radiotherapy, Clinical Cancer Center, the People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China

Received: 20 September 2012 / Revised: 10 October 2012 / Accepted: 25 October 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Radiotherapy is a part of the front-line treatment regime for many cancers. The mechanisms of radiation-induced effects in cancers mainly involves double-strand breaks (DBS) which plays very important role in maintaining the stability of gene. As DNA repair gene breast cancer 1 (BRCA1) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) can act to maintain genetic stability though two distinct and complementary mechanisms for DNA DSB repair-homologous recombina-tion (HR) and non-homologous end joining (NHEJ). Therefor, BRCA1 and DNA-PKcs are closely associated with radiation sensitivity, which means that they may be used as a useful tool to predict radio sensitivity in human tumour cells.

Key words breast cancer 1 (BRCA1); DNA-dependent protein kinase catalytic subunit (DNA-PKcs); double-strand breaks (DBS) repair; radiation sensitivity

Correspondence to: Ximing Xu. Email: [email protected] Yanrong Hao. Email: [email protected]

684 www.springerlink.com/content/1613-9089

an N-terminal RING finger domain that dimerizes with BRCA1-associated ring domain protein 1 (BARD1) for ubiquitin ligase activity [10] and a C-terminal domain that possesses 2 copies of the BRCT motif, which is able to interact with several proteins [11]. Classically, BRCA1 has been implicated as a modulator of response to DNA dam-age induced by chemotherapy and RT [12, 13], the absence of functional BRCA1 expression leads to hypersensitivity of cells to various types of molecular damage, suggesting its participation in multiple DNA repair pathways.

BRCA1 contributes to the mechanism in HR BRCA1 is involved in a multitude of cellular functions,

including HR and perhaps some forms of NHEJ [14, 15]. The N-terminal RING and C-terminal BRCA1 COOH-ter-minal (BRCT) domains confer ubiquitin-ligase activity and specific phosphoprotein binding to BRCA1, respec-tively. Until recently, it was believed that mutations in the BRCA1 BRCT domain lead to reduced HR, resulting in genomic instability and ultimately, the development of cancer [16]. There is a new and growing literature sug-gesting that the disruption of specific BRCA1 protein complexes instead increases DNA resection, resulting in elevated HR [17], which is also likely to be aberrant and lead to increased genomic instability [18].

However, the mechanism by which BRCA1 contrib-utes to HR have been little studied. A single report claims that BRCA1 promotes single-stranded DNA (ssDNA) formation in response to ionizing radiation and BRCA1 then accumulates at the resulting ssDNA sites, possibly contributing to a later step in homology-dependent repair [19]. Later, other stduy found that ~300 residues at both termini of the BRCA1 protein were essential for homol-ogy-directed recombination. Whereas some mutations analyzed were neutral, mutations that altered any zinc-coordinating residue or generated M18T and T37R, which are variant proteins of BRCA1, alterations were defective for recombination [20]. Emerging evidence also suggests that E3 is of the BRCA1 complexes critical for HR. One remarkable feature for BRCA1 is its E3 ligase activity that is acquired by constitution of a RING heterodimer with BARD1 (BRCA1-associated RING domain protein), a core complex residing in most of BRCA1 complexes [21, 22]. The potential importance of the E3 ligase activity of BRCA1 in cellular pathways was supported by the fact that many missense mutations within RING finger domain of BRCA1, which causes familial breast cancer, abolished the E3 activity [21, 23, 24]. However, several hardships have been keeping researchers away from the significance of the E3 ligase activity of BRCA1 in HR, the identification of its bona fide substrate (s) in the process since the ac-tivity was discovered 10 years ago [21]. Nonetheless, two potential substrates, CtIP and NPM1 that are involved in HR have been demonstrated. CtIP can be ubiquitinated

by BRCA1–BARD1 in vitro and in vivo [25]. BRCA1-CtIP, in conjunction with the MRN complex, mediates exten-sive DSB end resection that generates ssDNA overhangs to support subsequent HR-mediated repair of DSB [26, 27]. The ubiquitination of CtIP in the chromatin extraction was clearly observed after IR in BRCA1-deficient HCC1937 cells only when enzymatically active BRCA1 was exog-enously expressed. Another candidate substrate, NPM1, was initially identified as a substrate for BRCA1 by two different screen methods using mass spectrometry [28, 29].

BRCA1 influence the radiosensitivity through the HR

As is mented above the BRCA1 contributes to the re-pair of DNA double-strand breaks through HR. Germ line mutations of BRCA1 have been detected in 50% of famil-ial breast cancers alone. And it is also found to be down-regulated in sporadic breast cancers [30, 31]. Due to BRCA1 is associated with both increased breast cancer risk and potentially DNA repair and radiation response, there are potential concerns regarding radiation treatment. Ruff-ner and colleagues reported that BRCA1 deficiency was able enhance sensitivity to RT, and retroviral restoration of BRCA1 in mutant HCC1937 cells led to an increase in DNA repair and reduced cytotoxicity caused by ionizing radiation [32]. The hypothesis that a possible enhanced ra-diosensitivity due to BRCA gene mutations is mainly a result of HR deficiency [33] and/or deficient G2 checkpoint activation after irradiation during the G2- to M-phase transition [34]. Ernestos and colleagues recently showed that BRCA1 and BRCA2 mutation carriers could enhance radiosensitivity, presumably because of the involvement of the BRCA genes in deoxyribonucleic acid repair and cell cycle control mechanisms [35]. The hypothesis also was supported by experimental and clinical data [36–38]. In contrast to these data, other investigators have not found an association between BRCA1/2 heterozygosity and ra-diosensitivity [39, 40]. Lori and colleagues concluted that there was no evidence of increased radiation sensitivity or sequelae in breast tissue heterozygous for a BRCA1/2 germline mutation compared with controls, and rates of tumor control in the breast and survival were comparable between BRCA1/2 carriers and controls at 5 years [41].

There are a number of possible explanations for the inconsistency in data in the literature. First, many stud-ies have used different methods to assess radiosensitivity. Another possible reason is that BRCA1 protein is impli-cated in many different complicated cellular procedures that is related to chromosome sensitivity in mutagens. Thus it is possible that different mutations in BRCA1 will have different effects on the recognition and processing of DNA damage. For example, Zhong et al [42] suggested a physical interaction between BRCA1 and RAD50 in the NHEJ repair pathway. Despite many experiments come

685Chinese-German J Clin Oncol, December 2012, Vol. 11, No. 12

to a different conclusion, but it is unquestionable BRCA1 has a close relationship with radiosensitivity. BRCA gene status also impact chemotherapy, this topic is beyond the scope of this article but has been reviewed in detail else-where [43–45].

DNA-PKcs plays a central role in radiotherapy through the NHEJ

Non-homologous end joiningNHEJ is the simplest mechanism to repair a DSB [8, 46].

This repair pathway rejoins juxtaposed ends in a man-ner that does not need to be error free, and it is usually precise for simple or two-ended breaks, characterized by blunt ends. It can, however, lead to sequence alterations at the break point when the ends are not compatible. Al-though the term “non-homologous” is used to describe this repair pathway, a short 1- to 6-bp region of sequence homology (microhomology) near the DNA end often fa-cilitates rejoining. A “clean” two-ended DSB, with either blunt ends or small 5’ or 3’ complementary overhangs, is a substrate for an NHEJ reaction that requires just its “core” components: Ku70, Ku80, DNA-PKcs, X-ray repair cross complementing gene4 (XRCC4) and DNA ligase IV [47].

DNA-PKcs catalytic actionIn the NHEJ pathway, one of the first enzymes to

be attracted to DSBs is the Ku70/80 heterodimer, sub-sequently, the DNA-Ku70/80 scaffold recruits a large 460-kDa serine/threonine kinase called the DNA-depen-dent protein kinase catalytic subunit (DNA-PKcs) [48–50]. DNA-PKcs can join two broken DNA ends together in a complex containing two DNA-PKcs molecules acting as a scaffold facilitating their re-joining [51, 52]. The Ku het-erodimer then binds to the free DNA ends ensuring the spatial arrangement is preserved. This is then followed by the recruitment of DNA-PKcs and the associated end-joining proteins, including Artemis, XRCC4 and DNA li-gase IV[53, 54], stimulating DNA-PK activity via phosphory-lation and enabling the NHEJ reaction to proceed [55]. And the protein complex formed after the association of both Ku70/80 and DNA-PKcs at the DNA ends is generally re-ferred to as the DNA dependent protein kinase (DNA-PK). It is based on amino acid sequence, is a member of the phosphatidylinositol-3-kinase (PI3K) family (along with ATM, ATR, mTOR, SMG-1 and TRRAP) [56, 57].

The DNA-PKcs is the key functional element in the DNA-PK complex that drives NHEJ. Several studies di-rectly examine the impact of such single nucleotide poly-morphism (SNP) on the function of the NHEJ protein DNA-PKcs. Kristin and colleagues recently reported that Prkdc polymorphisms can significantly influence DNA-PKcs function involving DNA repair capacity, telomere end-capping, and potentially cancer susceptibility [58].

With the similar concept, an association between poly-morphisms in DNA-PKcs (also known as Prkdc) and risk of breast cancer was recently reported in the U.S. Radio-logic Technologist cohort [59]. Decreased DNA-PKcs ki-nase activity has also been linked to individuals with lung cancer [60]. Most recently, a Prkdc mutation was found in a human patient that exhibited a severe combined im-mune-deficient (SCID) phenotype and a disorder of DNA repair [61, 62].

DNA-PKcs is a potential targeted therapy DNA-PKcs also play a critical role in RT. Targeted in-

hibition of DSB repair proteins is an attractive approach in the development of potent RT strategies [63, 64]. Azad et al validate the conclusion that inhibition of DNA-PK induces accelerated senescence in irradiated human can-cer cells. They additionally show that DNA-PKcs knock-down using siRNA promotes a striking accelerated senes-cence phenotype in irradiated cells [65]. Low DNA-PKcs expression has been associated with a better response af-ter RT for head-and-neck cancer [66] and with better over-all survival in patients treated with postoperative RT for glioblastoma [67]. On the contrary, an increased DNA-PK expression may be part of a radioresistance mechanism within cervical carcinoma [68]. Until now several data sup-port a central role for DNA-PKcs in radiation sensitivity in prostate cancer [69], lung cancer [70], cervical cancer [71], glioblastoma [72], nasopharyngeal carcinoma [73], esopha-geal cancer [74]. Moreover, targeting DNA-PKcs using a single-chain variable fragment antibody has been shown to induce radiosensitizing effects, together with growth inhibition of cancer cells [75]. However, some stduies have shown no correlation between DNA-PK activity and ra-diosensitivity [76–78]. Interestingly, some of the study's au-thors claimed that if the follow-up period was to be pro-longed or the number of patients increased, significant differences might be demonstrable. Taken together, these results suggest DNA-PKcs as a key therapeutic target in irradiated human cancer cells.

The relationship between BRCA1 and DNA-PKcs

To our knowledge, there are few studies about the re-lationship between BRCA1 and DNA-PKcs. A Japanese study reports radiation-induced centrosome over-dupli-cation is regulated by at least two mechanisms: a check-point-dependent pathway involved in wild-type cells, Ku70-deficient and DNA-PKcs-deficient cells, and a checkpoint-independent pathway as observed in BRCA1-deficient and NBS1-deficient cells. More researches are needed to analyze the internal relationship between them [79]. Nevertheless, the co-inhibition of both NHEJ and HR proteins would be a suitable strategy to enhance


the radiosensibility of cancer cells.

Conclusion

Since radiation sensitivity differs from patient to pa-tient and tumor to tumor, either genotyping of DNA re-pair genes in tumors or some type of pathway. Specific tumor biomarker will be needed to assess the DNA re-pair competence of individual tumors to identify which therapeutic targeting strategy to employ. The BRCA1 and DNA-PKcs expression profile of human tumors is strong-ly responsive to radiotherapy. Since new discoveries will elucidate the complex role of BRCA1 and DNA-PKcs in DNA repair systems, BRCA1 and DNA-PKcs as a poten-tial prognostic factors in cancer will play a key role in stratifying patients for therapies to identify the subsets that could benefit from a DNA repair targeted approach. Moreover the great potential of BRCA1 and DNA-PKcs will contribute to radiotherapy and clinical research.

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Metastasis is one of the landmark features and the ma-jor cause of cancer-related death. Malignancies without metastasis predict optimistic prognosis. However, once metastasis occurs, there is very slim chance for a com-plete cure. Therefore, it is an urgent task to explore the underlying mechanisms of cancer metastasis to develop new strategies benefiting clinical practice.

Cancer metastasis is a complex biological process in-volving multiple genes, multiple steps and phases. Be-sides the genetic and epigenetic alterations from the side of cancer cells, many factors from extracellular microen-vironment and even the entire orgasm system also play active roles in the self-promoting and positively-selecting process of cancer development and advancement. Though many efforts have been put into the investigation of can-cer metastasis mechanism, it is hard to say we had made a “comprehensive” or “systematic” understanding on that area. The following hypotheses on cancer metastasis were established based on the current understanding of cancer metastasis biology and emphasized on the interactive reg-ulation between cancer cells and environmental factors.

“Seed and soil” theory

In 1889, Steven Paget proposed the “seed and soil” hy-pothesis to explain the cancer metastasis mechanism [1]. In this theory, Paget, for the first time, indicated the impor-tance of tumor microenvironment in the development and metastasis of cancer. It proposed that the organ-pref-

erence of cancer metastasis was the favorable outcome of the interactive communication between the metastatic cancer cells as the seeds and the organ environment as the soil. For example, the hydrogen peroxide produced by the tumor cells and tumor-associated fibroblasts is able to “fertilize” the metastatic tumor cells by accelerating aging, DNA damage, inflammation and metabolism processes [1]. A specific type of metastasis, self-seeding, also exists in the tumor-environment interaction, which indicates the phenomenon that circulating tumor cells resettle to the primary tumors lesions [2]. By the self-seeding, the tumor cells gain more aggressive phenotype, accelerating the tu-mor advancement.

The significance of “seed and soil” hypothesis to cancer therapy lies in the concept to develop cancer treatment strategies considering both sides of tumor cells (e.g. che-motherapy) and microenvironment (e.g. antiangiogenesis therapy). This is the real macroscopic standpoint in the clinical cancer treatment practice [3].

Cancer stem cells (CSC) and metastasis

Metastasis is a biological process with low proficiency. In a patient with a tumor, though thousands and thou-sands of tumor cells are dispersed into blood circulation every day, only few of the cells have the ability to settle down and grow into metastatic lesions. Traditional metas-tasis hypothesis fails to explain why not all of the dissemi-nated tumor cells forms metastatic lesions [4]. New theory proposes that only the disseminated tumor cells with stem cell-like properties, termed CSC, grow into new metastat-ic lesions [5]. Based on this theory, the very small part of cancer cell population with stemness properties, such as

Hypotheses explaining cancer metastasis*Haijuan Wang, Chen Lin, Haili Qian

State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Beijing 100021, China

Received: 7 August 2012 / Revised: 5 September 2012 / Accepted: 25 September 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract　Cancer metastasis is the most important factor causing patients death. Cancer diseases will be controllable if metastasis does not happen. Concerning the mechanism of cancer metastasis, there are still many points to be clarified. Currently, the mechanism of cancer metastasis has been explained from several aspects of its biological behaviors. Here we briefly summarized some newly-developed metastasis models to provide a global glance at this topic.

Key words　metastasis; cancer stem cells; dormancy; epithelial-mesenchymal transition

Correspondence to: Haili Qian. Email: [email protected]* Supported by a grant from the National Natural Sciences Foundation of China (No. 81071773, 30973447, 30973471).


self-renewal, proliferation and differentiation potential is the root of cancer development, progression, relapse and metastasis. Hence, only therapies targeting the CSC popu-lation hold promise to completely cure cancer metastasis. However, cancer stem cell represents a cell population in a relatively undifferentiated state, lack of specific molec-ular markers usually expressed in differentiated terminal cells. This makes targeting CSCs even more difficult than targeting the common cancer cells.

Epithelial-mesenchymal transition (EMT) and tumor metastasis

EMT is a special biological process by which epithelial cells lose the epithelial characteristics such as adhesion and expression of E-cadherin and gain mesenchymal-like phenotypes. EMT is essential for numerous biological processes including embryo development, tissue recon-struction and tumor metastasis [6]. During EMT, epithe-lial cells lose the expression of intercellular adhesive molecules through which epithelial cells are attached to the base membrane, and reprogrammed to express mes-enchymal-like phenotype with enhanced invasion and migration potentials [7]. EMT in nature is a process during which cells undergo dedifferentiation, so the cancer cells going through EMT are with stem cell-like properties [7,

8]. This hypothesis supports the notion that some of the cancer stem cell may be derived from differentiated cells. Cancer therapy strategies blocking the EMT may reduce or eliminate the chance of metastasis.

Tumor dormancy and long-term metastasis Some of the tumor cells which are potentially capable

of booming into metastatic lesions settle down at resting state after they arrive at the destination organs, namely dormancy, instead of entering into proliferation cycles immediately. The dormant tumor cells will stay in a rest-ing condition for a considerable time period but not grow into new tumors [9]. About 20% of the breast cancer pa-tients will get a relapse even 5 to 20 years after the pri-mary tumors were removed, providing direct evidence supporting tumor dormancy. Moreover, even in the breast cancer patients without relapse 20 years after the removal of primary tumors, dormant tumor cells still can be found in the circulation. This line of evidence reminds the oncological clinicians that the status of tumor cells in dormancy or in activated-from-dormancy may make big difference for the prognosis of cancer patients [10]. Keep-ing the potentially booming tumor cells in dormancy may

result in a long-term survival of cancer patients in a tu-mor-bearing condition, while tumor cells activated from dormancy may grow into new spreading lesions, hence poor prognosis. Therefore, research in the regulation of tumor dormancy may create new areas of cancer therapy strategies.

The above hypotheses illustrate the facets of cancer cell characteristics and metastasis process from various viewpoints. Notably, these theories are not exclusive and isolated from each other, but interrelated, mutually transformed, sequentially linked and even collaboratedly resulted in the metastasis events. Exploration on the in-teractive crosstalk between these hypotheses may help to clarify some fundamental scientific questions, such as whether the “seeds” in the “seed and soil” hypothesis are cancer stem cells, what the difference between dormant cancer cells and CSCs is, and whether CSCs are induced and maintained through EMT. Further investigation to answer these questions will push forward the under-standing of cancer metastasis as well as bred new theo-retic framework.

References

Lisanti MP, Martinez-Outschoorn UE, Lin Z, et al. Hydrogen perox-ide fuels aging, inflammation, cancer metabolism and metastasis: the seed and soil also needs “fertilizer”. Cell Cycle, 2011, 10: 2440–2449.Aguirre-Ghiso JA. On the theory of tumor self-seeding: implications for metastasis progression in humans. Breast Cancer Res, 2010, 12: 304.Langley RR, Fidler IJ. The seed and soil hypothesis revisited–the role of tumor-stroma interactions in metastasis to different organs. Int J Cancer, 2011, 128: 2527–2535.Pece S, Tosoni D, Confalonieri S, et al. Biological and molecular het-erogeneity of breast cancers correlates with their cancer stem cell content. Cell, 2010, 140: 62–73.Setoguchi T, Taga T, Kondo T. Cancer stem cells persist in many can-cer cell lines. Cell Cycle, 2004, 3: 414–415.Kerosuo L, Bronner-Fraser M. What is bad in cancer is good in the embryo: Importance of EMT in neural crest development. Semin Cell Dev Biol, 2012, 23: 320–332.Biddle A, Mackenzie IC. Cancer stem cells and EMT in carcinoma. Cancer Metastasis Rev, 2012 Feb 3 [Epub ahead of print].Deng JJ, Xu XM. Epithelial-mesenchymal transition and cancerme-tastasis. Chinese-German J Clin Oncol, 2011, 10: 125–133.Reddy BY, Lim PK, Silverio K, et al. The microenvironmental effect in the progression, metastasis, and dormancy of breast cancer: a model system within bone marrow. Int J Breast Cancer, 2012. 2012: 721659.Karrison TG, Ferguson DJ, Meier P. Dormancy of mammary carci-noma after mastectomy. J Natl Cancer Inst, 1999, 91: 80–85.

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Colorectal cancer (CRC) is one of the most frequently occurring cancers worldwide [1], and its incidence is in-creasing in many countries including China. To data, there is no validated sensitivity and/or resistance predic-tive factors available in clinical settings and the mecha-nisms involved in cancer cell chemoresistance are still largely unknown. There is high focus on discovery and validation of early detection markers as well as on predic-tive and prognostic factors as reviewed by Asghar et al [2]. Since they have been discovered in 1993, microRNAs (miRNAs) have caused worldwide interest due to their characteristic function and modes of action, providing a new understanding of the central dogma of molecular bi-ology. Moreover, the importance of miRNAs in oncogen-esis has been recognized.

The structural feature of miRNAs

MiRNAs are the large family of highly conserved RNAs molecules 18–25 nucleotides in length which have important regulatory roles in animals and plants. MiRNAs are transcribed by RNA polymerase II as long primary transcripts (pri-miRNAs) and undergo sequen-tial processing to produce mature miRNAs [3, 4]. Aberrant expression of miRNAs is known to be involved in various human diseases, including cancer. MiRNAs participate in

diverse biological processes including tumorigenesis by sequence-specific targeting of particular messenger RNAs (mRNAs), primarily in the 3’-untranslated region (3’-UTR). They trigger translational repression and/or mRNA degradation mostly through complementary binding to the 3’-untranslated (3’-UTR) regions of target mRNAs [5].

The function of miRNAs

Many miRNAs regulate genes involved in the patho-genesis of cancer and are extensively deregulated in dif-ferent tumors. More than 500 miRNAs have been identi-fied in humans and more than half of human miRNAs are located at specific chromosomal regions, including fragile sites, as well as in regions that are frequently amplified, deleted, or rearranged in cancers [6, 7]. Each miRNA can target several different mRNAs and each mRNA can be targeted by multiple miRNAs, generating an intricate network of gene expression regulation. As master regula-tors of gene expression, miRNAs are involved in modu-lating multiple cellular pathways, including cell prolif-eration, differentiation, and apoptosis [8, 9]. MiRNAs have been suggested to play a vital role in tumor initiation and progression by negatively regulating oncogenes and tu-mor suppressors. The mir-21 has been shown to regulate invasiveness in cancer through translational repression of the metaloproteinase (MMP) inhibitor RECK [10]. More-over, Xiang et al [11] have reported that over-expression of miR-17-92 may inhibit cell proliferation via post-tran-

The progression of microRNA in human colorectal cancer Yeping Du, Jinhua Miao, Chunmei Wu, Liping Xu

Department of Laboratory, The 264 Hospital, Taiyuan 030001, China

Received: 17 September 2012 / Revised: 30 September 2012 / Accepted: 22 October 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that function as posttranscriptional gene regu-lators. The dysregulation of miRNAs has been linked to a series of diseases, including various types of cancer. Since their discovery in the miRNAs of cancer patients, there has been a steady increase in the study of miRNAs as stable, noninvasive biomarkers. Although several challenges remain to be concerned, miRNAs could be useful, non-invasive biomarkers for colorectal cancer diagnostics and prognosis. In this review, we summarized the discovery of miRNAs and their potential as biomarkers. We discussed their possible structural, function and further emphasized the significance of miRNAs in colorectal cancer (CRC). Key words microRNA (miRNA); colorectal cancer (CRC); function; progression

Correspondence to: Yeping Du. Email: [email protected]


scriptional repression of FOG-2 in embryonic cardiomyo-cytes. Recent evidence has shown that altered expression of miRNAs is associated with the pathogenesis of various human cancers and has indicated that some miRNAs may function as oncogenes or tumor suppressors [12, 13].

Quite recently, studies have identified some miRNAs operating to promote or suppress tumor invasion or me-tastasis via regulating cancer-related genes, providing potential therapeutic targets strategy. It is estimated that vertebrate genomes encode up to 1000 unique miRNAs, each of which is thought to regulate the expression level of a target gene. Up to 30% of human genes are thought to be regulated by miRNAs; however, most of the targets remain unknown [14].

The significance of miRNAs in colorectal cancer

As previously mentioned, miRNAs have proven to be expressed or under-expressed miRNAs in colorectal cancer. Recently, some studies have identified a number of miRNAs that activate the expression of certain target genes in a sequence-specific manner or silencing them in colorectal cancer (Table 1) [15–26].

There is an increasing amount of evidence for under-and overexpression of several miRNAs in colorectal can-cer, as compared to the normal tissue, and for the im-pact of miRNAs in epithelial-to-mesenchymal transition (EMT) and metastatic progression [27]. The study identi-fied 21 up-regulated miRNAs in human colorectal can-cers. MiR-31, miR-183, miR-18a, miR-17-5p, miR-20a and miR-92 were significantly overexpressed in cancer compared to normal tissues [28].

The expression of miR-143 and miR-145 are downreg-ulated in colorectal tumor and their in vitro transfection into human colon cancer cell lines (DLD-1, SW480) led to growth inhibition. Meanwhile, expression of miR-143 and miR-145 by real-time RT-PCR analysis were sig-

nificantly lower in 43 and 44 clinical colorectal tumors, respectively, than in normal tissues (P < 0.05) [29]. Schet-ter et al reported that 27 miRNAs were overexpressed in miRNA array expression profiling of 84 colorectal tumor and paired non-tumorous tissues, which included miR-17-5p, miR-20a and miR-92 [30].

Recently, it shown that eleven miRNAs (miR-183, -31, -20, -25, -92, -93, -17, -135a, -203, -133b, and -223) were over-expressed in colorectal tumors relative to mucosa, and nine (miR-192, -215, -26b, -143, -145, -191, -196a, -16, and let-7a) were under-expressed in colorectal tu-mors [31].

Quite recently, studies have identified some miRNAs operating to promote or suppress colorectal tumors inva-sion or metastasis via regulating metastasis-related genes, providing potential therapeutic targets on anti-metastasis strategy. Recently, miR-200c and miR-21 expression were reported to be associated with poor survival in colorectal tumors patients [32]. Zhang et al investigated that miRNA-143 is involved in the regulation of MACC1 and thus plays a functional role in colorectal tumors [26].

More study is required to clarify the precise contribu-tions of miRNAs to colorectal tumor progression. MiR-NAs are attractive candidates as multifunctional regula-tors of proliferation and metastatic progression because one miRNA can regulate an entire set of genes.

Conclusion

The discovery of miRNAs, are small non-coding RNAs, as potential diagnostic, prognostic markers and therapeu-tic targets. It will brought forward a new proof of the basic mechanisms of oncogenesis and opened up excit-ing prospects for diagnostics and prognostics. Although still a new field, with much to be explored, the advent of miRNA research may lead to possible applications to mo-lecular diagnostics and prognostics in colorectal cancer. More study is required to clarify the precise contributions

Table 1 The expression levels of up-regulated and down-regulated miRNAs and the targets mRNAs in colorectal cancermiRNAs Expression level Targets RNAs ContributorsMir-31 Up-regulation 5-FU Wang et al, BMC Cancer, 2010, 10: 616 [15]

Mir-20a Up-regulation BNIP2 Chai et al, Acta Biochim Biophys Sin, 2011, 43: 217 [16]

Mir-499-5p Up-regulation FOXO4/PDCD4 Liu et al, Carcinogenesis, 2011, 32: 1798 [17]

Mir-106a Up-regulation TGFBR2 Feng et al, PLoS One, 2012, 7: e43452 [18]

Mir-200c Up-regulation EMT Hur et al, Gut, 2012, July 10 [19]

Mir-17 Up-regulation RND3 Luo et al, Biochem J, 2012, 442: 311 [20]

Mir-135a Up-regulation APC Nagel et al, Cancer Res, 2008, 68: 5795 [21]

Mir-133b Up-regulation C-MET Hu et al, Cancer Biol Ther, 2010, 10: 190 [22]

Mir-221 Up-regulation CDKN1C/p57 Sun et al, Acta Pharmacol Sin, 2011, 32: 375 [23]

Mir-192/Mir-215 Down-regulation TYMS Boni et al, Mol Cancer Ther, 2010, 9: 2265 [24]

Mir-320a Down-regulation NRP-1 Zhang et al, Oncol Rep, 2012, 27: 685 [25]

Mir-143 Down-regulation MACC1 Zhang et al, Mol Cancer, 2012, 11: 23 [26]


of miRNAs to colorectal cancer progression.

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It well is recognized and documented that oral ma-lignant tumor invading mandibular bone require partial or total mandibulectomy in order to provide adequate margins. To predict the range of malignant tumor inva-sion into the mandible pre-operatively is very important for clinician. Exactly preoperative assessment the extent of mandibular invasion by malignant tumor is very im-portant to ensure getting a safe tissue margin in tumours abutting but not invading the mandible, which can re-serve most well-being bone tissue [1], and it is significance to patient’s prognosis. SPECT (single photon emission computed tomography) is functional imaging modality,

high sensitivity, but lack the structural delineation. So it is difficult to accurate locate focus [2]. CT (computered tomography) is anatomical imaging modality, provides accurate morphological information necessary for tumor localization and detection of structural abnormalities, but can not reflect the functional or metabolic activity of the tumor [3]. The coregistration of structural (CT) and functional (SPECT) imaging information can improve the identification of bone invasion. But the value of SPECT/CT to determine the extent of malignant tumour invading mandibule had not be reported [4].

The aim of this study was to compare the accuracy of SPECT/CT and helical CT to determine the extent of mandibule invasion by malignant tumour, and to evalu-

Value of 99mTc-MDP SPECT/CT fusion imaging and CT in evaluating the extent of mandibular invasion by malignant tumor of oral cavityQingyun Duan1, Muyun Jia2, Rongtao Yuan2, Lingxue Bu2, Wei Shang2, Xiaoming Jin2, Ningyi Li2, Jie Zhao3, Guoming Wang4

1 Department of Oral & Maxillofacial Sugery, Hangzhou First People’s Hospital, Hangzhou 310006, China2 Department of Oral & Maxillofacial Sugery, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China3 Pathology Deparment, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China4 Nuclear Medicine, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China

Received: 28 May 2012 / Revised: 22 October 2012 / Accepted: 25 November 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of our study was to compare the value of computed tomography (CT) and 99mTc-methylene-diphosphonate (MDP) SPECT (single photon emission computed tomography)/CT fusion imaging in determining the extent of mandibular invasion by malignant tumor of the oral cavity. Methods: This study had local ethical committee approval, and all patients gave written informed consent. Fifty-three patients were revealed mandibular invasion by malignant tumor of the oral cavity underwent CT and SPECT/CT. The patients were divided into two groups: group A (invasion-periphery-type) and group B (invasion-center- type). Two radiologists assessed the CT images and two nuclear medicine physicians separately assessed the SPECT/CT images in consensus and without knowledge of the results of other imaging tests. The extent of bone involvement suggested with an imaging modality was compared with pathological findings in the surgical specimen. Results: With pathological findings as the standard of reference, Group A: The extent of mandibular invasion by malignant tumor under-went SPECT/CT was 1.02 ± 0.20 cm larger than that underwent pathological findings. And the extent of mandibular invasion underwent CT was 1.42 ± 0.35 cm smaller than that underwent pathological examination. There were significant difference among the three methods (P < 0.01). Group B: The extent of mandibular invasion by malignant tumor underwent SPECT/CT was 1.3 ± 0.39 cm larger than that underwent pathological examination. The extent of mandibular invasion underwent CT was 2.55 ± 1.44 cm smaller than that underwent pathological findings. There were significant difference among the three methods (P < 0.01). The extent of mandibular invasion underwent SPECT/CT was the extent which surgeon must excise to get clear margins. Conclusion: SPECT/CT fusion imaging has significant clinical value in determining the extent of mandibular inva-sion by malignant tumor of oral cavity.

Key words SPECT/CT; fusion imaging; mandibular invasion; malignant tumor

Correspondence to: Qingyun Duan. Email: [email protected]


ate SPECT/CT’s diagnostic value in determining the ex-tent of mandibule invasion by malignant tumour of oral cavity and hoped to provide guidance for surgeon.

Materials and methods

Main instrumentsLightspeed 16 spiral CT (GE Co, USA); MillenniumVG

(HawKeye) SPECT/CT fusion imaging system (GE Co, USA); 99mTc generator (Beijing Atom High Technology Co, China).

Main experiment agentia MDP (methylene diphosphonate) kit (Beijing Shihong

Medicine Development Centre, China); 99mTc-MDP pho-tographic developer.

Patients Fifty-three patients with malignant tumour of oral

were investigated, they were determined that malignant tumour having invasion mandibule underwent SPECT/CT fusion imaging and CT in the Department of Oral & Max-illofacial Surgery of the Affiliated Hospital of Qingdao University Medical College from October, 2003 to March, 2009. The samples consisted of 34 men and 19 women (mean age, 49.24 ± 10.1 years; range, 28–71 years). All patients were treated with mandibulectomy mandibular as part of complete resection of the tumor.

Clinical procedureAll the patients underwent SPECT/CT and CT with-

in 1 week before surgery. Then they were treated with complete or near complete surgical resections of the tu-mor, which were determined by the surgeon based on the clinical, imaging, intraoperative, and fast-frozen histolog-ic findings. The results of SPECT/CT and CT scan were compared to the results of histopathology findings which was considered as the reference standard.

Imaging Technetium-99m-methylene diphosphonate (MDP,

740–1100 Mbq) were injected intravenously, then acqui-sitions were obtained 2.5 h later (± 30 min) using a low energy all purpose collimator through SPECT/CT fusion imaging system. This hybrid imaging system combines a dual-detector, variable angle gamma camera with a low-dose X-ray tube that is attached to the same rotating gan-try as the gamma camera. This device provides, together with SPECT data, cross-sectional X-ray transmission im-ages that accurately locate the anatomical sites of radio-tracer accumulations. Moreover, the gamma camera is capable of MDP imaging using coincidence detection.

For acquiring the transmission data, the X-ray tube and linear detector array rotate together around the patient in

a fixed geometry, with a single slice imaged in 14 seconds. Multiple transmission slices are obtained by moving the table, the full field of view consists of 40 slices with the slice thickness fixed at 1 cm. At the completion of the first type of acquisition (transmission or emission), the patient is automatically repositioned so that the 40-cm axial field that was just scanned matches the 40-cm axial field of view of the second imaging modality. Images are recon-structed in the workstation and transmission data are in-tegrated in the nuclear medicine database, the alignment of slices is automatically performed, then matching pairs of X-ray and scintigraphic images are fused generating new images overlying SPECT and CT data. The CT scan were performed in the axial plane with 3-mm-thick con-tiguous sections from the skull base to the thoracic inlet. All studies were reconstructed with soft-tissue and bone algorithms. The bone algorithm settings were a width of 3500 H and level of 700 H.

Image interpretationThe CT scans were displayed in gray scale and printed

on film. Two radiologists viewed the CT scans obtained in all patients with knowledge of the clinical data but without knowledge of the results of the other imaging examinations. The bone and soft-tissue windows were as-sessed in consensus. Bone invasion was suggested when tumor tissue was visible outside the cortical bone and the cortical bone was seen to be partially eroded or totally destroyed. Two radiologists were asked to mark extent of bone erosion.

The SPECT images were displayed in gray scale, in which dark areas corresponded to regions with increased tracer uptake and bright areas to regions with low or ab-sent tracer uptake. The CT images were assessed by us-ing bone and soft-tissue windows. The SPECT/CT images were displayed with tracer uptake overlaid in color on the CT images. The quality of image co-registration was visually controlled. The window settings were those used to evaluate bone and soft tissues. Two nuclear medicine physicians visually assessed all images on a computer screen. Images were read in consensus and with knowl-edge of clinical information but without knowledge of the results of other imaging examinations. Bone invasion was suggested when areas with locally increased tracer uptake corresponding to increased bone turnover were detected at the mandible bone.

Histopathology examinationThe fresh Mandibulectomy specimens after the soft

tissue was sectioned were fixed in 10% formalin solu-tion and decalcified in 5% nitric acid for a period of 1–4 days. Once the bone was soft, it was cut into 3 mm thick sequential slices in transverse, every slice was cut into several 1.5 mm long sequential smaller slice again, subse-


quently processed in paraffin wax, one 5 micron section stained with haematoxylin and eosin was examined from each block, with step serial sections examined as neces-sary. One pathologist who was unaware of the imaging findings examined the histologic material for evidence of bone invasion. The histological definition of bone in-vasion was in accordance with UICC TNM pathological classification of malignant tumours.

Statistical analysisDifferences among SPECT/CT, CT images and histopa-

thology examination were analyzed using the Statistical Package for the Social Sciences (SPSS) version 12.0. P < 0.05 was considered to indicate a significant difference.

Results

The diagnosis of mandibular invasion by malignant tumour was proved pathologically in all patients. Forty-three patients were with gingival carcinoma, 8 patients with central carcinoma of jaw. Two patients with adeno-carcinoma. A case of central carcinoma of jaw, CT scan showed no mandibular invasion before surgery (Fig. 1), but SPECT/CT scan showed increased uptake (“hot area”) in mandible (Fig. 2).

The way mandibule invasion of adenocarcinoma and central carcinoma (ento-ectad invaded mandibule) are difference with gingival carcinoma which invaded man-dibule from cortical bone to bone marrow. So we divided 53 patients into two groups, group A (periphery-invasion-type) included 43 patients with gingival carcinoma, group B (center-inasion-type) included 8 patients with central carcinoma of jaw and 2 patients with adenocarcinoma.

In the A group, the extent of mandibule invasion SPECT/CT finding was larger 1.02 ± 0.20 cm than that determined underwent histopathology examination, and that of CT scan was smaller 1.42 ± 0.35 cm than that underwent histopathology examination, while that of SPECT/CT scan was larger than that of CT scan 2.44 ±

0.33 cm. The difference between SPECT/CT and CT scan was high statistically significant (t = 14.44; P < 0.01). The difference among histopathology examination, SPECT/CT and CT were high statistically significant too (t = 8.0, 10.25; P < 0.01) (Table 1).

In the B group, the extent of mandibule tumor invasion on the SPECT/CT image is larger 1.3 ± 0.39 cm than that histopathology examination (t = 6.5, 10.067; P < 0.01), and that of SPECT/CT is larger 3.85 ± 1.3cm than that of CT (t = 5.632; P < 0.05), while that of CT is smaller 2.55 ± 1.44 cm than that histopathology examination, that are presented in Table 2.

The relation among SPECT/CT, CT and histopathology examination showing the extent of mandibular invasion were (S, P and C denote SPECT/CT, histopathology ex-amination and CT respectively, the unit is centimeter):

Group A (1) S = P + 10.2 ± 0.20 C = P – 1.42 ± 0.35 S = C + 2.44 ± 0.33 (2) S = P + 1.02 ± 0.20 = C + 2.44 ± 0.33Group B (1) S = P + 1.3 ± 0.39 C = P – 2.55 ± 1.44 S = C + 3.85 ± 1.34 (2) S = P + 1.3 ± 0.39 = C + 3.85 ± 1.34Between the two groups, the difference among the

three type examination methods are presented in Table 3. The difference between SPECT/CT with histological ex-amination was not statistical significance (t = 1.656, 0.446; P > 0.05). The difference between SPECT/CT with CT was statistically significance (t = 2.801, 2.566; P < 0.05).

Discussion

The mandibule which invaded by malignant tumor usually was resected to get complete excised primary tu-mor and decrease tumor recurring. Assessment of man-dibular invasion by oral carcinoma is very important for the success of ablative surgery. Such surgery in the mouth is often associated with significant loss of func-tion and deterioration of cosmetic appearance. How to obtain adequate resection margins? Somebody thought

Fig. 1 A case of central carcinoma of jaw, CT scan showed no mandibular invasion before surgery


that clear margins meant at least 5 mm off the tumour front [5], Lewis-Jones and Brown [5, 6] suggested the depth of tumor invasion mandibule within 5 mm tumor can be removed using the method of rim or marginal resection, but a segmental mandibulectomy is required when the depth of invasion is greater than this or in some large soft tissue cancers of the tongue and floor of mouth that were deeply invading the soft tissues and where tumor abut-

ment involved the whole depth of the mandible.Some investigation presented that a high percentage

of resected mandibles in oral cancer show no evidence of tumor invasion [7]. So, the information concerning the extent and depth of bone invasion in pre-operative and as far as possible to conserve well-being bone tissue would be significance to patients’ prognosis. But so far, it is only parvus article which discussed the extent of tumor inva-sion mandibule [8], and the article about using SPECT/CT image fuse technique to determine the extent of mandib-ule invasion was not yet published.

CT can provide morphological detail. The definition of structural abnormalities is accurate, but physiologic in-formation is not readily generated. It is at least 30%–50% mineral loss in the bone before any change is visible on the CT image [9], so CT’s sensitivity is low [10], and false-

Table 3 Differential analysis of examinations between group A and group BComparison t P

C–S 2.801 < 0.05C–P 2.566 < 0.05S–P 1.656 > 0.05C = CT; S = SPECT/CT; P = histopathology examination

Table 2 Comparison of bone invasion extent with three examinations in group BComparison Mean of (X1–X2) 95% CI t P

C : S –3.85 –3.85 ± 1.34 5.632 < 0.05C : P –2.55 –2.55 ± 1.44 4.368 < 0.05S : P 1.3 1.30 ± 0.39 6.5 < 0.01C = CT; S = SPECT/CT; P = histopathology examination

Fig. 2 As the same case, SPECT/CT scan showed increased tracer up-take (“hot area”) in mandible. which was carcinoma invasion demonstrated underwent pathological examination

Table 1 Comparison of bone invasion extent with three examinations in group A Comparison Mean of (X1–X2) 95% CI t P

C : S –2.44 –2.44 ± 0.33 14.44 < 0.01C : P –1.42 –1.42 ± 0.35 8.00 < 0.01S : P 1.02 1.02 ± 0.20 10.25 < 0.01C = CT; S = SPECT/CT; P = histopathology examination


negative rate is high to detect mandibular invasion [11]. SPECT is functional images which can provide physio-

logical information, his sensitivity is supernal, but it can’t present more anatomy information. So, it can’t accurately locate. SPECT/CT is a kind of registration and fusion im-age system with a structural image can be valuable as an adjunct to interpretation of functional images, as well as offering the possibility to overcome intrinsic limitations in nuclear medicine image, Image fusion is useful to ac-curately localize tracer accumulations, to detect occult pathology, to characterize metabolically active lesions, and to draw precisely regions of interest for quantitative studies. This hybrid imaging system combines a dual-de-tector, variable angle gamma camera with a low-dose X-ray tube that is attached to the same rotating gantry as the gamma camera. This device provides, together with SPECT data, cross-sectional X-ray transmission images that accurately locate the anatomical sites of radiotracer accumulations [12].

SPECT/CT had high sensitivity, it could discover fo-cus before the invaded mandibule had not morphological change. So on SPECT/CT images the extent of mandibular invasion were larger than that on the CT images. In A and B groups, the extent on the SPECT/CT images were larger 1.02 ± 0.20 cm and 1.3 ± 0.39 cm than that on histology examination, respectively.

Comparing A group and B group, the difference be-tween SPECT/CT and histopathology examination was not statistically significance, but the difference between CT and histopathology examination was statistically signifi-cance. It showed that the type of tumor do not obviously influenced on the extent of tumor invasion underwent SPECT/CT, but the influence is obvious underwent CT. So, SPECT/CT is more superior on showing the extent of mandibular invasion than CT. Some scholars considered that clear margins meant at least 5 mm of normal tissue between the tumour front and the edge of the resection [6]. On this view, the extent of tumor invasion mandibule on SPECT/CT image just is reasonable extent that must be resected for completely resecting tumor. Because the type of tumor influence on the extent of tumor invasion man-dibule on CT image, the extent of being resected must be determined on the type of tumor. Like periphery-inva-sion-type (A group), if we want get clear margin, it is at

least 1.21 (0.75 + 0.5) cm of normal tissue between the tumour front and the edge of the resection, While center-inasion-type (B group), it is 1.77 (1.27 + 0.5) cm.

In conclusion, the extent of mandibule invasion by malignant tumor determined on SPECT/CT image was more precise than CT, The result of SPECT/CT can be as guidance for oral surgeon.

References

Gu DH, Yoon DY, Park CH, et al. CT, MR, (18) F-FDG PET/CT, and their combined use for the assessment of mandibular invasion by squamous cell carcinomas of the oral cavity. Acta Radiol, 2010, 10: 1111–1119.Ljungberg M, Sjögreen-Gleisner K. The accuracy of absorbed dose estimates in tumours determined by quantitative SPECT: a Monte Carlo study. Acta Oncol, 2011, 50: 981–989.Perren E, Potter M, Golding S, et al. The accuracy of MRI and CT scans in assessing mandibular invasion in oral SCC. Brit J Oral Max Surg, 2011, S49–S91.Goerres GW, Schmid DT, Schuknecht B, et al. Bone invasion in pa-tients with oral cavity cancer: comparison of conventional CT with PET/CT and SPECT/CT. Radiology, 2005, 237: 281–287.Lewis-Jones HG, Rogers SN, Beime JC, et al. Radionuclide bone imaging for detection of mandibular invasion by squamous cell carci-noma. Br J Radiol, 2000, 73: 488–493.Brown JS, Kalavrezos N, D’Souza J, et al. Factors that influence the method of mandibular resection in the management of oral squamous cell carcinoma. Br J Oral Maxillofac Surg, 2002, 40: 275–284.Van Cann EM, Oyen WJ, Koole R, et al. Bone SPECT reduces the number of unnecessary mandibular resections in patients with squa-mous cell carcinoma. Oral Oncol, 2006, 42: 409–414.Brown JS, Lewis-Jones H. Evidence for imaging the mandible in the management of oral squamous cell carcinoma: a review. Br J Oral Maxillofac Surg, 2001, 39: 411–418.Huntley TA, Busmanis I, Desmond P, et al. Mandibular invasion by squamous cell carcinoma: a computed tomographic and histological study. Br J Oral Maxillofac Surg, 1996, 34: 69–74.Rybak LD, Rosenthal DI. Radiological imaging for the diagnosis of bone metastases. Q J Nucl Med, 2001, 45: 53–64.Mukherji SK, Isaacs DL,Creager A, et al. CT detection of mandibular Invasion by squamous cell carcinoma of the oral cavity. AJR Am J Roentgenol, 2001, 177: 237–243.Schillaci O, Simonetti G. Fusion imaging in nuclear medicine – ap-plications of dual-modality systems in oncology. Cancer Biother Ra-diopharm, 2004, 19: 1–10.

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Chinese-German Journal of Clinical Oncology December 2012, Vol. 11, No. 12, P699–P704DOI 10.1007/s10330-012-1016-7

As the population ages, the incidence of lung cancer in elderly is rising. It shows that, in the past 10 years, incidence and mortality of lung cancer in the 50 years of age and younger population has decreased, while in the population over the age of 70 increased [1]. A retrospec-tive analysis in elderly lung cancer revealed that the risk of chemotherapy-related toxicity was significantly higher in patients over 70 years old than in patients lower than 70 years old [2]. Thus, we defined 70 as the dividing line of the elderly in lung cancer and identify the prognostic factors of lung cancer patients in elderly over 70 years in order to provide a theoretical basis for clinical work.


Patients A retrospective review was conducted on 408 patients

aged 70 years or more with lung cancer who diagnosed from January 1998 to December 2002 at Beijing Chest Hospital of Capital Medical University. There were 330 men and 78 women with a mean age of 73.59 ± 3.53 years (70–93 years). One hundred and fifty-nine cases had no

complications and 249 cases were with at least one com-plication. Of the complications, cardiovascular diseases, tuberculosis, digestive diseases, chronic obstructive pul-monary disease and diabetes were most common. Thirty-two patients were discovered pulmonary shadow during physical examination, 337 patients visited hospital because of respiratory symptoms (such as cough, chest tightness, chest pain, hemoptysis, etc.), other 39 patients visited be-cause of non-respiratory symptoms (such as fever, weight loss, anorexia, neck and facial swelling, hoarseness, etc.). There were 167 cases of squamous cell carcinoma, 140 adenocarcinoma, 35 adenosquamous carcinoma, 52 small cell carcinoma and 14 others including large cell carci-noma, alveolar cell carcinoma, papillary adenocarcinoma and unclassified types of lung cancer. 173 patients were at stage Ia–IIIa and 235 patients were at stage IIIb–IV ac-cording to the 5th TNM staging of American Joint Com-mittee on Cancer (AJCC) in 1997.

Treatment Two hundred and forty-four patients received treat-

ment and 164 patients did not receive any treatment. The ratio of treated and untreated patients were 2.6:1 (125/48) in stage Ia–IIIa, 1:1 (119/116) in stage IIIb–IV. Among 93 patients undergone surgery, 19 cases with pneumonec-

Prognostic factors in 408 elderly lung cancer patients over 70 years oldHua Zheng1, Li Tong2, Ying Hu1, Weihua Wu2, Hongmei Zhang1, Baolan Li1

1 General Department, Beijing Chest Hospital of Capital Medical University, Beijing 101149, China2 Department of Oncology, Beijing Chest Hospital of Capital Medical University, Beijing 101149, China

Received: 10 May 2012 / Revised: 16 May 2012 / Accepted: 25 July 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The incidence and mortality of lung cancer in people over 70 years were increased in the past 10 years. We defined age 70 years as boundary line of the elderly patients in lung cancer and analyzed and identified the fac-tors affecting prognosis. Methods: A retrospective study had enrolled 408 cases of lung cancer aged over 70 years old and SPSS13.0 software was used in univariate analysis and COX regression analysis to analyze factors affecting prognosis, such as gender, age, complications, symptoms, pathological type, clinical stage, effusion, surgery, radiotherapy, chemotherapy and so on. Results: In univariate analysis, symptoms, stage, effusion, surgery, chemotherapy and chemotherapy cycles showed affecting prognosis significantly. In COX regression analysis, it showed that clinical stage (P = 0.000), surgery (P = 0.013), chemotherapy cycles (P = 0.001) were independent prognostic factors. Conclusion: Elderly lung cancer patients could be benefit from surgery and adjuvant chemotherapy while early stage. At late stage, their survival time may be prolonged when receive chemotherapy at least 4 cycles. Single-agent chemotherapy would be a good choice for elderly lung cancer. Effusion, particularly, pericardial effusion significantly influenced the prognosis, so that it should be effectively controlled.

Key words lung neoplasm; geriatrics; prognosis

Correspondence to: Baolan Li. Email: [email protected] Hua Zheng. Email: [email protected]


tomy, 2 cases with lobectomy and wedge resection of tumor, 5 cases with thoracic exploration. 146 patients received chemotherapy, 262 did not received chemo-therapy. There were 111 patients undergone chemo-therapy cycles < 4 cycles and 35 patients with ≥ 4 cycles of chemotherapy. The regimens for non-small cell lung cancer (NSCLC) are mainly vinorelbine + cisplatin or car-boplatin, paclitaxel + cisplatin or carboplatin, gemcitabi-ne + cisplatin or carboplatin and single agent paclitaxel or vinorelbine. The regimens for small cell lung cancer (SCLC) were mainly etoposide and cisplatin or carbopla-tin, topotecan + cisplatin or carboplatin and single agent etoposide or topotecan.

Survival Overall survival was measured from the day of diagno-

sis to the day of death or the day last seen alive in month. Date cut-off of this study was on 15th December, 2007.

Statistical analysis The analysis was performed using SPSS 13.0 (SPSS Inc.,

USA). Life table was used to calculate the survival rate and median survival time. Survival curves were estimated using the Kaplan-Meier method. The log-rank test was used in the univariate analysis of potential prognostic fac-tors. The Cox regression model was used for multivariate analysis to identify the factors with independent prog-nostic significance. A P < 0.05 was considered statistically significant.

Results

Follow-up results Until 15th December, 2007, 400 patients had died. 1-,

2-, 3- and 5-year survival rates were 41.2%, 14.2%, 8.3%, 1.5%, median survival time was 10.3 months.

Analysis of prognostic factorsUnivariate analysis was conducted on factors includ-

ing sex, age, complications, symptoms, pathology, clini-cal stage, serous effusion (including pleural effusion and pericardial effusion), surgery, chemotherapy, radiother-apy etc. Result showed that symptoms, clinical stage, se-rous effusion, surgery, chemotherapy and chemotherapy cycle affected prognosis and statistically significant (P < 0.05). Sex, age, pathology, and radiotherapy didn’t affect prognosis and combination chemotherapy was not supe-rior than single-agent chemotherapy. There were no dif-ference in survival between the two groups (P = 0.545; Table 1). Complications do not affect survival (P = 0.405; Fig. 1). Survival in patients undergone treatment or not in stage IIIb were 15.93 and 7.51 months (P = 0.000), 10.11 and 5.08 months in stage IV (P = 0.000; Fig. 2). In patients with stage IIIb, effusion significantly affected the survival

(P = 0.01), while in stage IV effusion had no significant effect on survival (P = 0.819; Fig. 3). The survival of pa-tients who had pericardial effusion was significantly less than the survival of the patients who had effusion but no pericardial effusion (P = 0.011) and no effusion (P = 0.000; Fig. 3).

COX regression multivariate analysis Statistically significant variables in univariate analysis

were get into the multivariate COX regression model. Re-sults showed that clinical stage, surgery and number of chemotherapy cycles were independent prognostic fac-tors (Table 2).

Table 1 Univariate analysis of lung cancer patients in the elderlyCharacteristics n % MST (months) P valueSex

Male 330 80.9 14.02 0.772Female 78 19.1 13.12

Age (years)70–75 311 76.2 14.20 0.284≥76 97 23.8 12.61

SymptomsNo 32 7.8 18.50 0.047Yes 376 92.2 13.42

Histological typeNSCLC 356 87.3 14.37 0.061SCLC 52 12.7 10.08

StageStage I 70 17.2 26.35 0.000Stage II 30 7.4 17.58Stage IIIa 73 17.9 14.18Stage IIIb 111 27.2 11.61Stage IV 124 30.4 7.72

Effusion fluidYes 107 26.2 8.47 0.000No 301 73.8 15.71

Surgical resectionYes 93 22.8 25.81 0.000No 315 77.2 10.41

ChemotherapyYes 146 35.8 16.02 0.007No 262 64.2 12.61

RadiotherapyYes 100 24.5 14.31 0.380No 308 75.5 13.64

Chemotherapy cycles< 4 373 91.4 13.15 0.003≥ 4 35 8.6 21.08

Chemotherapy regimenCombination 128 31.4 15.63 0.545Single agent 16 3.9 17.82

MST: middle survival time


Discussion

The incidence of lung cancer in China is gradually in-creased. Until 2015, China will become the country of world’s largest lung cancer incidence [3] and the incidence of lung cancer in elderly will increase accordingly. Al-though studies have shown that age is not an independent prognostic factor in lung cancer [4], the elders’ organ dys-function, complications and side effects of treatment lead to withdrawal from treatment, subsequently, affect the survival of elderly patients with lung cancer.

In this study, there are 408 cases of elderly lung cancer patients over 70 enrolled and only 224 patients received at least one kind of treatment, such as surgery, chemo-therapy and/or radiotherapy, while 164 cases of patients received only supportive care. The proportion of elder pa-tients’ withdrawal from treatment is higher in advanced stage than in early stage. However, our data shows that treatment can significantly prolong the survival of elderly lung cancer, especially in advanced stage (Fig. 2). More-over, more or less of complications didn’t affect the sur-vival (Fig. 1). Some studies have proved that there were

no difference in survival between elderly patients and non-elderly patients with lung cancer [5–7]. Therefore, el-derly patients should give treatment when their physical status permitted regardless of stage in order to improve survival.

In univariate analysis, the survival of patients among different consultation reasons are significantly different (P = 0.001), of which the longest survival were asymp-tomatic discoverers. It is because most of the symptoms

Fig. 1 The survival curve of complications in elderly lung cancer pa-tients

Fig. 2 Influence of treatment to survival in advanced elderly lung can-cer patients. (a) Influence of treatment to survival in stage IIIb; (b) Influ-ence of treatment to survival in stage IV

Fig. 3 Influence of effusion fluid to survival in advanced elderly lung cancer. (a) Influence of effusion fluid to survival in stage IIIb; (b) Influence of effusion fluid to survival in stage IV; (c) Influence of pericardial effusion fluid to survival in elderly lung cancer


are caused by airway obstruction, pulmonary atelectasis by pleural effusion or superior vena caval syndrome by lymph nodes and so on, and all these situations belong to advanced stage, in which survival time is limited. It is thus evident that the reason why different complaints have differences in survival is due to different stage. There-fore, in multivariate analysis, the reasons for the different consultation reason did not show significant difference (P = 0.676), but stage showed significant difference in both univariate analysis and multivariate analysis (P = 0.000) as reported in the literatures [8–10].

With or without malignant effusion impacts on sur-vival and showed a statistically significant (P = 0.000), which has relationship with the stage since only patients with advanced stage have effusion fluid. Stratified analy-sis showed that there are significant differences between survival in patients with or without effusion in stage IIIb (P = 0.01), but no differences in stage IV (0.819; Fig. 3). Stage IV patients present distant metastasis, including head, liver, bone, adrenal gland and other organs, which may lead to coma, liver failure, fracture, endocrine and electrolyte disorders, thus shortening survival time as same as effusion fluid. However, the presence of effusion would significantly affect the quality of life, lead to respi-ratory and circulatory failure and shorten survival time in patients with stage IIIb who exist only local lesions. The new TNM classification in 2009 [11] classified effusion fluid to M1a stage, concordant with our result.

Further, we conducted a stratified analysis on pericar-dial effusion. The result shows the survival of patients with pericardial effusion was significantly less than pa-tients with non-pericardial effusion fluid (P = 0.011) and with no effusion (P = 0.000; Fig. 3), indicating that pericardial effusion significant impact the prognosis than pleural effusion. It was reported that pericardial effusion is a sign of poor prognosis and malignant pericardial ef-fusion is a worse prognosis factor [12, 13], consistent with our results. In practice, small amount of pericardial effu-sion may significantly affect the quality of life of patients and large scale of pericardial effusion may lead to life-threatening cardiac tamponade. Pleural effusion is less harmful than pericardial effusion. For this reason, when patients have effusion fluid, they required drainage to re-lieve symptoms, and if necessary, inject drugs to reduce

exudation.There is no difference in survival between SCLC and

NSCLC (P = 0.061). In stratified analysis, there is no dif-ference in survival among different pathological types of lung cancer in which who did not receive any treatment (P = 0.955), while survival are different in whom under-gone treatment (P = 0.000). Thus, small-cell lung cancer has poor prognosis than NSCLC. Although SCLC patients gained benefit from treatment, but not as good as NSCLC. SCLC progresses rapidly, but is sensitive to chemotherapy and radiotherapy. Hence, mass can be rapidly reduced, but relapse quickly. It is difficult to control for long time for SCLC rather than NSCLC. Whatever the pathology, the patients’ survival can be prolonged from treatment.

Different treatments significantly affect the prognosis. In univariate analysis, surgery and chemotherapy has sig-nificant impacts on prognosis (P = 0.000 and 0.007, re-spectively), while radiotherapy has no significant effect on prognosis (P = 0.38). Probably, because radiotherapy can only control local lesion, but can not control the progression of systemic disease. In the comprehensive treatment, patients undergone surgery plus chemother-apy have the longest survival (30.36 months), compared with surgery alone (26.91 months), extend for the time of 3.45 months. Therefore, we suggest elderly patients in early stage after surgery conduct adjuvant chemotherapy. In multivariate analysis, chemotherapy or not does not appear as an independent prognostic factor, because the number of chemotherapy cycle has more influence on the prognosis (P = 0.001). In stratified analysis, patients con-ducted more than 4 cycles of chemotherapy have longer survival than patients with less than 4 cycles (P = 0.003). Thus, under the circumstances of postoperative adjuvant chemotherapy in early stage or first-line chemotherapy in late stage, at least 4 cycles of chemotherapy are recom-mended. This is also consistent with the NCCN guidelines in 2010.

Currently, chemotherapy in elderly lung cancer is controversial. To alleviate the toxicity of chemotherapy in elderly, single-agent of third-generation cytotoxic che-motherapy has become a hot research area. A phase II clinical trial has shown that vinorelbine combined with cisplatin chemotherapy in greater than 70-year-old elder-ly patients with advanced lung cancer had better survival

Table 2 Cox regression of variables in the equation of lung cancer in elderly

Variable B SE Wald df P Exp (B) 95.0% CI for Exp (B) Lower Upper

Symptom 0.060 0.143 0.175 1 0.676 1.061 0.803 1.404Stage 0.256 0.044 34.407 1 0.000 1.292 1.186 1.407Effusion fluid 0.009 0.129 0.005 1 0.945 1.009 0.784 1.299Surgery –0.208 0.084 6.199 1 0.013 0.812 0.689 0.957Chemotherapy 0.134 0.122 1.212 1 0.271 1.144 0.900 1.453Chemotherapy cycles –0.226 0.070 10.417 1 0.001 0.798 0.696 0.915


than single-agent vinorelbine (P = 0.030), on the other hand, the toxicity increased significantly (P < 0.01) [14]. Another study showed that single-agent gemcitabine had no difference in survival compared with platinum-based combined chemotherapy (P > 0.05), and single-agent reg-imen is better tolerated [15]. There are still many studies on single-agent chemotherapy in third generation cytotoxic [16–20], including vinorelbine, gemcitabine, taxol and peme-trexed, etc., and effects among them are similar [21, 22], well tolerated, even in patients with poor PS [16]. A study in 2010 ASCO meeting has come into notice [23]. The study enrolled 451 cases of aged from 70 to 89 years old (median 77.2 years), performance status (PS) score of 0 to 2, III / IV NSCLC patients. Compared with gemcitabine or vinorel-bine monotherapy, carboplatin (monthly treatment ) + paclitaxel (weekly treatment) in combination with che-motherapy significantly improved patients' overall sur-vival (10.4 months vs 6.2 months, P = 0.0001) and pro-gression-free survival (PFS, 6.3 months vs 3.2 months, P < 0.0001). However, 3/4 stage hematologic toxicity is more common in patients with combined therapy. Neutropenia rates were 54.3% and 14.3%, respectively. In our data, the survival of patients between platinum-based combined therapy and single drug therapy were no difference (P = 0.545). Even the survival of single-drug therapy had a slight extension of the survival than combined therapy. Therefore, an elder patient may select a single agent or in combined therapy according to his performance status, expected survival time and so on.

In short, elderly lung cancer patients may benefit from surgery and adjuvant chemotherapy when at early stage. At advanced stage, the patients may get prolonged sur-vival when proceed at least four cycles of chemotherapy. Single-agent chemotherapy for elderly lung cancer would be a good choice. Effusion, particularly for pericardial ef-fusion, will significantly affect the prognosis, so that it should be actively managed.

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《中德临床肿瘤学杂志》2013年征稿启事

《中德临床肿瘤学杂志》是由中华人民共和国教育部主管，华中科技大学主办的医学肿瘤学学术期刊（全英

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Lung cancer is the most common malignant tumor, where non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancer cases [1]. In NSCLC, inci-dence of adenocarcinoma is occult, non-specific symp-toms, more difficult to be done by biopsy and easy to transfer, so the early diagnosis is difficulty in clinical work. CEA as one of tumor markers which is more com-monly used on early diagnosis of NSCLC and evaluation of treatment. STAT3 is a recognized oncogene, the study have shown that blocking of JAK1-STAT3 signal trans-duction pathway can inhibit the growth of lung cancer [2], but if STAT3 can become a new tumor marker for clinical to improve the early diagnosis of lung adenocarcinoma, there is no aspects of clinical and experimental reports on home and abroad. This experiment analyze the rela-tionship between of STAT3 and CEA, so as to explore the value of STAT3 on the early diagnosis of lung adenocarci-noma, find a new and ideal tumor marker for the diagno-sis of lung adenocarcinoma for the clinic use. It provides a theoretical basis and experimental evidence


Materials Human lung adenocarcinoma A549 cell line, normal

human lung cells MRC-5 line from the Center Laboratory of Luzhou Medical College (China). Rabbit anti-STAT3 polyclonal antibody was purchased from the Bioworld technology Co. (USA), rabbit anti-human CEA poly-clonal antibody was purchased from Beijing Biosynthesis Biotechnology Co. Ltd. (China), the two-step immuno-histochemical detection kit PV-6001 was purchased from Beijing Zhongshan Jinqiao Biotechnology Co. Ltd. (Chi-na), total RNA extraction kit box purchase since Tiangen Company (China), 2008 BioBRK RT Kit was purchased from Chengdu Break Biotechnology Co. Ltd. (China), Ta-KaRa RNA PCR Kit (AMV) Ver.3.0 was purchased from Takara Co. Ltd. (Japan), STAT3, CEA, GAPDH primers were synthesized by Sanggon Biotech Co. Ltd. (China).

MethodsImmunohistochemistry Sterile coverslips was placed in 6-wells plate, human

lung adenocarcinoma cell A549 and normal human lung cell MRC-5 with cell density of 2 × 104/mL were seeded on glass respectively and cells climbed pieces, 5 days after-wards the specimens were washed with PBS, 4% parafor-maldehyde for 20 min, 0.5% of Triton X-100 were incu-

Correlation of STAT3, CEA in lung adenocarcinoma cell A549 Debin Sun1, Xiu Lan1 (Co-first author), Hongcheng Wang2

1 Department of Respiratory Medicine, Lishui Central Hospital, Lishui 323000, China2 Department of Respiratory Medicine, the Affliated Hospital of Luzhou Medical College, Luzhou 646000, China

Received: 27 June 2012 / Revised: 17 July 2012 / Accepted: 25 August 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The purpose of this study was to analyze the relationship between signal transducer and activator of transcription 3 (STAT3) and carcinoembryonic antigen (CEA) in lung adenocarcinoma cell A549, and to explore the value of STAT3 on early diagnosis of lung adenocarcinoma. Methods: The expression of CEA, STAT3 mRNA and it’s protein in human lung adenocarcinoma cell A549 and normal human lung cells MRC-5 were tested by immunohistochemistry staining (PV) and quantitative real time fluorescent PCR. The correlation between STAT3 and CEA in human lung adenocarcinoma cell A549 was analyzed. Results: The protein and mRNA levels of STAT3, CEA in lung adenocarcinoma cell A549 were apparently high-er than those in normal human lung cells MRC-5. The levels of STAT3 mRNA and it’s protein were positively correlated with CEA in lung adenocarcinoma cell A549. Conclusion: STAT3 have the same value in diagnosis of lung adenocarcinoma.

Key words lung adenocarcinoma; signal transducer and activator of transcription 3 (STAT3); carcinoembryonic antigen (CEA)

Correspondence to: Hongcheng Wang. Email: [email protected]


bated for 20 min, and the remaining operation referred to the kit instructions. Image acquisition, mean optical den-sity (MOD) was tested by IPP6.0 image analysis software. The average of the MOD of five fields of each slice were analyzed comparatively. MOD represented the staining intensity. It could reflect the relative concentration of the positive product of expression more accurately.

Extraction of total cellular RNAHuman lung adenocarcinoma cell A549 and normal

human lung cell MRC-5 which integrated up to about 80% respectively replaced with fresh medium 24 h before the experiment. PBS washed cells, added 1 mL of lysis buffer Trizol to cells per 10 cm2, beaten several times with a sampler, the rest of the operation referred to the kit in-structions. Added 1 μL of total RNA in micro-nucleic acid quantitative instrument to read the RNA samples con-centration and ratio of OD260/OD280, the ratio between 1.8–2.1 could be used for subsequent experiments. Ratio less than 1.8 indicated that the residual amount of pro-tein was too large and the sample should be re-extracted once.

Quantitative real time fluorescent PCR Accessed to mRNA sequences in Genbank and designed

primers by ABI primer express 3.0 software. Primers were synthesized by Sanggon Biotech Co., Ltd. (China). The STAT3 primer sequences, upstream prime: 5’-CCT GAAGCTGACCCAGGTAG-3’, down strem prime: 5’-TTC CAAACTGCATCAATGAATC-3’, CEA primers sequenc-es, upstream prime: 5’-GCACCTCA GACCAATCATC AACT-3’, down strem prime: 5’-CCACTTCTCAAGGAC CAAATACAC-3’, GAPDH primer sequences, upstream prime: 5’-ATGCTGGCGCTGAGTACGTC-3’, down strem prime: 5’-GGTCATGAGTCC TTCCACGATA-3’. Re-verse-transcription referred to the kit instructions, cDNA was synthesized after the establishment of SYBR Green I quantitative PCR reaction system, 95 ℃ for change 2 min, 95° ℃ denaturation 5 s, annealing at 59.5 ℃ 20 s, extension at 72 ℃ for 15 s, amplified 40 cycles. After the reaction, computer analyzed the results automatically.

Results calculation△Ct = Ct target gene – Ct reference gene

△△Ct = △Ct – (Ct random negative control sample – Ct the samples of

internal reference) The relative total amount of the target gene for the 2–

△△Ct. In this experiment, normal human embryonic lung fibroblasts MRC-5 was random negative controls group.

Statistical analysisAll the statistical analyses were performed using the

SPSS 16.0. Data were expressed as χ ± s. T-test for inde-pendent samples was used to compare the means between two samples. Pearson correlation test was used to analyze the correlation of the bivariate. A P value < 0.05 was con-sidered statistically significant.

Results

The expression of STAT3, CEA protein in human lung adenocarcinoma cell A549 and normal human lung cells MRC-5

Positive expression signal of STAT3 in human lung adenocarcinoma cell A549 were mainly located in cyto-plasm, a few in the nucleus. Positive expression signal of CEA in A549 cells were mainly located in cytoplasm and (or) cell membrane. Weak levels of expression of STAT3, CEA protein could also be found in the cytoplasm in the normal human lung cells MRC-5 (Fig. 1). The protein lev-els of STAT3 and CEA in A549 were significantly higher than those in MRC-5 (P < 0.01; Table 1).

Correlation of expression of STAT3 protein and CEA protein in human lung adenocarcinoma cell A549

After pearson correlation analysis, we found STAT3 protein were positively correlated with CEA protein in human lung adenocarcinoma cell A549 (r = 0.583, P = 0.007, P < 0.05).

Quantitative real time fluorescent PCR and its product identification

The melting curve showed each gene had a single product peak (Fig. 2), no non-specific products and prim-er-dimers, it indicated that the specificity of the primers, the experimental results could accurately reflect initial concentration of the samples.

Quantitative analysis of the target gene mRNAA certain amount of template of cDNA PCR products

were prepared, diluted from 103 to 109 respectively, a concentration gradient of 7, and then take 2 μL of dH2O as a zero tube. Quantitative real time fluorescent PCR amplified and established a standard curve. Amplification curve was a typical S-shaped, 10-fold serial dilutions of the standard PCR products of the Ct value as vertical axis (Y), natural logarithm of the standard PCR product con-centration as the abscissa (X), it got a strict linear standard curve. Bio-Rad IQ5 analysis software analyzed and calcu-lated standard curve (Fig. 3, Table 2 ). Amplification effi-ciency of the target gene and reference gene were similar, it ensured the accuracy and efficiency of the method.

The expression of STAT3 mRNA and CEA mRNA in human lung adenocarcinoma cell A549 and normal human lung cells MRC-5

The levels of STAT3 mRNA and CEA mRNA expres-sion in human lung adenocarcinoma cell A549 were sig-nificantly higher than those in normal human lung cells MRC-5 (P < 0.01; Table 3). After pearson correlation analysis, we found STAT3 mRNA were positively corre-


lated with CEA mRNA in human lung adenocarcinoma cell A549 (r = 0.521, P = 0.047 ).

Discussion

The sensitivity of non-invasive inspection methods is

not high in the early stages of lung cancer: CT can di-agnose 60% of peripheral lung cancer, sputum cytology diagnose only 60% of central type lung cancer. Bronchos-copy with high specificity, but its trauma is not easy to be accepted by patients [3]. Early diagnosis of lung can-cer is low, 70% of patients with lung cancer who were diagnosed had locally advanced or distant metastases (with stage IIIb / IV ), lost the chance of operation, lacked of effective treatment [4], so the early diagnosis of lung cancer become the important task of our clinicians and research workers. Tumor markers have merits of conve-nient, simple operation, lower prices, less invasive, exten-sively carried out, etc., appropriate serum levels of tumor marker have important clinical value in cancer screening, diagnosis, monitoring treatment, prognosis, etc.. CEA is

Table 1 Expression of STAT3, CEA protein in A549 and MRC-5 cell(χ ± s)Factor Group n MOD P

STAT3 protein A549 cella 20 0.133 ± 0.067 0.006MRC-5 cell 20 0.049 ± 0.010CEA protein A549 cella 20 0.106 ± 0.014 0.000MRC-5 cell 20 0.047 ± 0.011a P < 0.01 vs the MRC -5

Table 2 Results of standard curvesGene STAT3 CEA GAPDHEfficient 96.97 98.71 96.76Slop –3.746 –3.738 –3.709R2 0.995 0.996 0.996

Table 3 Expression of STAT3 mRNA in A549 and MRC-5 cell (χ ± s)Factor Group n 2-△△Ct P

STAT3 mRNA A549 cella 15 6.96 ± 3.37 0.001MRC-5 cell 5 1.01 ± 0.19CEA mRNA A549 cella 15 5.68 ± 2.70 0.001MRC-5 cell 5 1.02 ± 0.21a P < 0.01 vs MRC-5

Fig. 1 Expression of STAT3 and CEA. (a) Expression of STAT3 in A549 cell; (b) Expression of STAT3 in MRC-5 cell; (c) Expression of CEA in A549 cell; (d) Expression of CEA in MRC-5 cell

Fig. 2 The melting curves. (a) STAT3 gene; (b) CEA gene; (c) GAPDH gene


one of tumor marker which is currently the most com-mon in lung cancer diagnosis and treatment. The extent of the elevation is directly related to number of cancer cells [5]. Park [6] found that CEA had the highest positive rate in lung adenocarcinoma. STAT3 which can inhibit the generation of vascular NSCLC can be use as a impor-tant molecular target for NSCLC [7, 8] and effective way to targeted therapy [9]. Yan [10] who used Western blot analy-sis found that the expression of protein in human lung adenocarcinoma cell A549 were apparently higher than those in normal human lung cell HELF. In this study, we used immunohistochemistry and quantitative real time fluorescent PCR at the same time to detect two levels from the genes to proteins. At the level of proteins, im-munohistochemistry reflect intuitively the differences of expression of STAT3 protein in human lung adenocarci-

noma cell A549 and normal human lung cells MRC-5. At the level of genes, quantitative real time fluorescent PCR can be specific, efficient, sensitive and accurate to reflect of the difference of expression of STAT3 mRNA in hu-man lung adenocarcinoma cell A549 and normal human lung cells MRC–5 and improve detection the accuracy and reliability of the results. Our study observed that: ei-ther protein or gene levels of STAT3 which expressed in human lung adenocarcinoma cell A549 were apparently higher than those in normal human lung cells MRC-5. They were consistent with previously reported values. Moreover, either protein or gene levels of STAT3 were positively correlated with CEA in human lung adenocar-cinoma cell A549. Theses strongly suggest that they have the same biological effects. In summary, the results of this study show that STAT3 had good diagnostic value in lung

Fig. 3 The amplification curves and standard curves of the STAT3, CEA and GAPDH gene. (a) STAT3 gene; (b) CEA gene; (c) GAPDH gene


adenocarcinoma, it was worthy of being used in clinical practice.

References

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Breast-conserving surgery followed by whole breast radiation therapy (WBRT) and a boost to the tumor bed is the treatment of choice for most patients with stages I–II breast cancer. Not only are disease-free and overall sur-vival rates after such treatment comparable with those of patients treated by mastectomy [1, 2] but in addition breast-conserving therapy offers an obvious cosmetic advantage that may enhance quality of life and lead to less psycho-logical and emotional treatment-related distress [3].

The rationale for boosting the tumor bed is based on the hypothesis that higher local control rates may be achieved if a higher dose of radiation is administered to the region of the breast bearing the greatest tumor bur-

den [4]. Although the use of a tumor bed boost (10–20 Gy, depending on tumor size and surgical margins) is routine practice, there is no standard treatment delivery technique. Some authors recommend the use of intersti-tial implants but most studies report the use of electron beams (EBs) to boost the tumor bed [5, 6]. Most frequently, single 9–12 MeV EB with 2–3 cm margin around the es-timated tumor bed is used. Such energy range helps to adequately treat shallow targets inside the breast. Deep-seated tumors, however, may not adequately be treated with EB, though contemporary highly conformal photon beam techniques may be able to reduce the dose inhomo-geneity within the target while optimally decreasing the dose to the surrounding non-target tissues.

The present study aimed to assess the potential dosi-metric advantages and drawbacks of the following treat-

Dosimetric study comparing photon and electron beams for boosting the tumor bed in early-stage breast cancerMohamed Mahmoud1, Soha Ahmed2, Ehab M. Attalla1, 2, Hassan S. Abouelenein2, Shaimaa Shoier2, Mohsen Barsoum1

1 Radiation Oncology Department, National Cancer Institute, Cairo University, Cairo, Egypt2 Radiation Oncology Department, Children’s Cancer Hospital, Cairo, Egypt

Received: 11 September 2012 / Revised: 2 October 2012 / Accepted: 5 November 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of our study was to assess and compare the potential dosimetric advantages and drawbacks of photon beams and electron beams as a boost for the tumor bed in superficial and deep seated early-stage breast cancer. Methods: We planned CTs of 10 women with early breast cancer underwent breast conservative surgery were selected. Tumor bed was defined as superficial and deep with a cut of point 4 cm, those with less than 4 cm were defined as superficial tumors representing 4 patients and those with depth of 4 cm or more were classified as deep tumors representing 6 patients. The clinical target volume (CTV) was defined as the area of architectural distortion surrounded by surgical clips. The plan-ning target volume (PTV) was the CTV plus margin 1 cm. A dose of 10 Gy in 2 Gy fractions was given concurrently at the last week of treatment. Organs at risk (OARs) were heart, lungs, contra-lateral breast and a 5 mm thick skin segment of the breast surface. Dose volume histograms were defined to quantify the quality of concurrent treatment plans assessing target coverage and sparing OARs. The following treatment techniques were assessed: photon beam with 3D-conformal technique and a single electron beam. Results: For superficial tumors better coverage for CTV and PTV with good homogeneity with better CI was found for the 3D conformal radiotherapy (3DCRT) but with no significant planning objectives over electron beam. For deep tumors, the 3DCRT met the planning objectives for CTV, PTV with better coverage and fewer hot spots with better homogeneity and CI. For superficial tumors, OARs were spared by both techniques with better sparing for the electron beam where as for deep tumors also OARs were well spared by both techniques. Conclusion: Boosting the tumor bed in early-stage breast cancer with optimized photon may be preferred to electron beam for both superficial and deep tumors. The OARs dose sparing effect may allow for a potential long-term toxicity risk reduction and better cosmesis. Key words 3D conformal radiotherapy; electron beam; organs at risk

Correspondence to: Mohamed Mahmoud. Email: [email protected]


ment techniques: Conventional approaches with con-formal fields with photons (3DC) or also single field EB techniques for superficial and deeply seated tumors with a cutoff point 4 cm between superficially and deeply seat-ed tumors.


This study included 10 patients (age ranged from 30 to 60 years, median 45 years) who had received conservative surgery for early-stage unilateral breast cancer (six right-sided and 4 left-sided tumors). Distal tumor margins were located at different depths between 2.4 to 6.4 cm below the breast surface (i.e., deep-seated tumors) in all patients with cutoff point was taken to be 4 cm to differentiate be-tween superficially and deeply seated tumors. Tumor and target characteristics were summarized in Table 1.

The planning CT of the breast region in a free-breath-ing setting was performed postoperatively with the pa-tient in treatment position (i.e., patient on a breast board, lying supine, and with the ipsi-lateral arm above the head), radiopaque contrast material was placed at the su-perior, inferior, medial and lateral limits of the clinically palpable breast tissue, cuts were taken using a Semins To-moscan AV Helical CT scanner. CT images were acquired in 5 mm slice intervals from the mandible through the lung bases. The anatomic information from the CT scan was used to define the target volume and normal struc-tures at risk.

The following organs at risk (OARs) were outlined: ipsi-lateral and contra-lateral breasts and lungs, heart, and the skin covering the ipsi-lateral breast (a 5 mm thick segment on the breast surface). In all cases surgical clips were placed by the surgeon (IR) surrounding the tumor cavity at the time of lumpectomy.

All patients were first treated with 6 MV photon beams to the entire breast with two tangential fields. A total dose of 50 Gy in 25 daily fractions during 5 weeks was deliv-ered. The boost clinical target volume (CTV) was defined as the area of architectural distortion inside the breast (i.e., tumor bed) surrounded by surgical clips.

Seeds implanted around the resection cavity by the surgeon or defined by the tumor cavity. To account for treatment set-up uncertainties and breathing motion the boost planning treatment volume (PTV) was defined as a 1.0 cm expansion of the CTV as shown in Fig. 1. The prescribed dose was 10 Gy in 5 daily fractions given con-comitant at the last week of treatment.

All patients were panned by 2 techniques: 3D-confor-mal fields and electron beam with calculating the dose to the CTV, PTV and organ at risk.

3D-conformal static fieldsMultiple static fields (2 to 4 fields) were used with a

similar beam arrangement as in the conformal plans with simple field conformation to the PTV as shown in Fig. 2, 3. All static fields included an enhanced dynamic wedge (EDW). Plans were calculated with the pencil beam algo-rithm based on the work by Storchi et al [7, 8].

Electron beamsThe boost was planned with a single conformal por-

tal (Fig. 4, 5). The beam energy was selected in order to comply with the dosimetric goal mentioned above. The entry angle was selected so that the entrance surface was approximately perpendicular to the beam central axis Seven patients were planned with 12 MeV electron beam and three with 16 MeV, respectively. The dose distribu-tion was computed with the Generalized Gaussian Pencil Beam model [9, 10].

Table 1 Tumor characteristics of the 10 patients included in this studyCharacteristics No.Tumor site

Left breast 4Right breast 6

For tumors located at a distance < 4 cm Proximal depth tumor (cm)

Mean 0.5 Std dev +/–0.5

Distal depth tumor (cm) Mean 3.6 Std dev +/–0.4

For tumors located at a distance > 4 cm Proximal depth tumor (cm)


Distal depth tumor (cm) Mean 6.4 Std dev +/–1.5

For tumors located at a distance < 4 cm CTV (cc)


For tumors located at a distance > 4 cm CTV (cc)

Mean 64.96 Std dev +/–41.8

For tumors located at a distance < 4 cm PTV (cc) Mean 45.9 Std dev +/–5.3For tumors located at a distance > 4 cm PTV (cc) Mean 142.1 Std dev +/–87Std dev, standard deviation; CTV, clinical target volume; PTV, planning target volume


Tools for analysisQuantitative evaluation of plans was performed by

means of standard Dose-Volume Histogram (DVH). For PTV and CTV, the values of D 99% and D 1% (dose re-ceived by 99%, and 1% of the volume) were defined as metrics for minimum and maximum doses and conse-quently reported. To complement the appraisal of mini-mum and maximum dose, V95%, V107% (the volume receiving at least 95% or at most 107% of the prescribed dose) were reported. The inhomogeneity of the treatment was expressed in terms of D5%–D95%. The conformity of the plans was measured with a conformity index, (CI: ratio between the volume receiving at least 95% of the prescribed dose and the volume of the PTV).

For OARs, the analysis included the mean dose, the maximum dose expressed as D1% and a set of appropriate Vx and Dy values.

Average cumulative DVH for PTV, OARs and healthy tissue was built from the individual DVHs.

Results

Dose distribution are displayed for one patient for su-perficial tumor with axial views, average DVH plot for the CTV, PTV, OAR and healthy tissue as shown in Fig. 6, 7.

Data are represented as average over 4 patients with superficial tumors and for 6 patients with deep tumors, errors indicated inter-patient variability at one standard deviation level.

Table 2 shows DVH analysis for CTV and PTV for su-perficial tumors.

No specific planning objectives were imposed in CTV for superficial tumors except coverage and homogeneity in 3DCRT technique was better than that for electron beam.

Also no specific planning objectives were imposed in PTV of superficial tumors, except better coverage with V95% for the 3DCRT in range of 95.1%–96.7%, in com-parison with electron beam that showed V95 between

Fig. 1 After image registration, two volumes are contoured: the CTV around the area of architecture distortion (red) and the PTV by extending the CTV by 1 cm (green)Fig. 2 Dose distribution of 3DCRT for superficial tumorsFig. 3 Dose distribution of 3DCRT for deep tumorsFig. 4 Dose distribution of electron beam for superficial tumorsFig. 5 Dose distribution of electron beam for deep tumorsFig. 6 DVH including CTV, PTV, OAR for superficial tumorsFig. 7 DVH including CTV, PTV, OAR for deep tumors


84.3–85.2.Regarding the conformity index (CI), it was much bet-

ter for the 3DCRT than for electron beam.Table 3 shows DVH analysis for CTV and PTV for deep

tumors.The 3DCRT met the planning objectives for CTV, PTV

with better coverage and fewer hot spots with better ho-

mogeneity and CI.Table 4 shows DVH analysis for organs at risk includ-

ing healthy tissues for superficial tumors.There is significant differences were observed for the

dose to ipsilateral breast according to the treatment tech-nique whether 3DCRT or electron beam, where the V10 Gy and V3 Gy were in favor of the electron beam.

Better sparing of both contralateral lung and contralat-eral breast was achieved with electron beam technique.

The mean dose of ipsilateral lung ranged from 2% of the prescribed dose for 3DCRT technique and 6% for the electron beam.

The mean dose to the heart and maximum dose (D1) is worse with electron beam technique than 3DCRT.

No specific planning objectives regarding skin sparing between both 3DCRT and electron techniques as both showed decrease in sparing of the skin with minimal ad-vantage to 3DCRT over electron beam.

Regarding the integral dose to the healthy tissue, both

Table 4 Summary of DVH analysis for organs at risk ( including healthy tissue ) for superficial tumors < 4 cm

Superficial Mean ± SD (3DC) Mean ± SD (Electron)Healthy tissue 12949.3 ± 2466.9 12949.3 ± 2181.3 Mean (Gy) 0.05 ± 0 0.05 ± 0 V5% (Gy) 0.2 ± 0.1 0.2 ± 0.2 Doselnt 0.6 ± 0.3 0.7 ± 0.6Contra-lateral lung 1160.5 ± 28.2 1160.5 ± 228.4 Mean (Gy) 0.1 ± 0 0 ± 0 D1% (Gy) 0.9 ± 0 0.1 ± 0.1 V3 Gy (%) 0.5 ± 0 0 ± 0Contra-lateral breast 571.5 ± 143.3 571.5 ± 283.5 Mean (Gy) 0.2 ± 0 0 ± 0 D1% (Gy) 0.7 ± 0.1 0 ± 0 V3 Gy (%) 0 ± 0 0 ± 0Ipsi-lateral lung 1241.2 ± 61 1241.2 ± 164.2 Mean (Gy) 0.2 ± 0 0.6 ± 0.4 D1% (Gy) 1.9 ± 0.1 6 ± 2.1 V3 Gy (%) 1.5 ± 0 5.3 ± 4.6 V10 Gy (%) 0 ± 0 0 ± 0.1Ipsi-lateral breast 853.6 ± 173.4 853.6 ± 383.4 Mean (Gy) 2.5 ± 0.2 1.7 ± 0.5 D1% (Gy) 10.5 ± 0 10.3 ± 0.1 V3 Gy (%) 29.7 ± 2.8 19.9 ± 5.7 V10 Gy (%) 6.6 ± 0.3 3.4 ± 0.9Heart 490.8 ± 24.6 490.8 ± 107.3 Mean (Gy) 0.05 ± 0 0.1 ± 0.1 D1% (Gy) 0.7 ± 0 0.9 ± 0.9 V5 Gy (%) 0 ± 0 0 ± 0Skin (Upr) Mean 6.8 ± 0.8 8.9 ± 0.5 (Med) Mean 7.1 ± 0.5 8.7 ± 0.3 (Lwr) Mean 7 ± 0.8 8.8 ± 0.3DoseInt, integral dose; Dx%, dose received by the x% of the volume; Vx%, volume receiving at least x Gy of the prescribed dose

Table 2 Summary of DVH analysis for CTV, PTV for superficial tumors < 4 cm Mean ± SD (3DC) Mean ± SD (Electron)CTV 19.8 ± 9.1 19.8 ± 9.1 Mean (Gy) 10.3 ± 0.1 10 ± 0.2 D1% (Gy) 10.7 ± 0.1 10.6 ± 0.1 D5–95% (Gy) 0.5 ± 0.2 0.9 ± 0.1 D99% (Gy) 9.4 ± 0.5 9.5 ± 0.2 V95% (%) 99.5 ± 1.4 93.1 ± 6.9 V107% (%) 0.6 ± 0.7 1.1 ± 1.6PTV 45.9 ± 5.3 45.9 ± 13.3 Mean (Gy) 10.2 ± 0.1 9.9 ± 0.2 D1% (Gy) 10.7 ± 0 10.8 ± 0.1 D5–95% (Gy) 1.2 ± 0.2 1.1 ± 0.1 D99% (Gy) 9.1 ± 0.1 9.0 ± 0.2 V95% (%) 95.9 ± 0.8 84.3 ± 13.1 V107% (%) 1.0 ± 0.3 0.8 ± 0.7 CI 95% 1.4 ± 0.4 2.1 ± 0.9CTV, clinical target volume; PTV, planning target volume; 3D-conformal treatment. Dx%, dose received by the x% o f the volume; Vx%, volume receiving at least x% of the prescribed dose; CI, ratio between the patient volume receiving at least 95% of the prescribed dose and the volume of the total PTV

Table 3 Summary of DVH analysis for CTV, PTV for deep tumors > 4 cm Mean ± SD (3DC) Mean ± SD (Electron)CTV 64.96 ± 41.8 65 ± 41.8 Mean (Gy) 10.04 ± 0.2 9.8 ± 0.6 D1% (Gy) 10.40 ± 0.2 10.9 ± 0.4 D5–95% (Gy) 0.50 ± 0.1 2.3 ± 1 D99% (Gy) 9.60 ± 0.3 7.8 ± 1.6 V95% (%) 98.90 ± 1.5 76.3 ± 24.8 V107% (%) 0.60 ± 1.4 3.1 ± 3.7PTV 142.1 ± 87 129.8 ± 68.2 Mean (Gy) 9.90 ± 0.2 9.6 ± 0.7 D1% (Gy) 10.4 ± 0.2 10.8 ± 0.3 D95/D5% 0.9 ± 0 0.7 ± 0.2 D99% (Gy) 8.9 ± 0.4 7.2 ± 1.9 V95% (%) 89.6 ± 7.3 70.5 ± 23 V107% (%) 0.4 ± 0.9 2.8 ± 3.2 CI 95% 1.4 ± 0.4 1.8 ± 0.2CTV, clinical target volume; PTV, planning target volume; 3D-conformal treatment. Dx%, dose received by the x% o f the volume; Vx%, volume receiving at least x% o f the prescribed dose; CI, ratio between the patient volume receiving at least 95% of the prescribed dose and the volume of the total PTV


3CTR and electron beam showed comparable results par-ticularly in the lowest volume irradiated to mean low dose V5 Gy.

Table 5 shows DVH analysis for organ at risk including healthy tissues for deeper tumors. There is significant dif-ferences were observed for the dose to ipsilateral breast according to the treatment technique whether 3DCRT or electron beam, where the V10 Gy and V3 Gy were in fa-vor of the electron beam.

There is no difference in sparing the contra-lateral lung in both techniques with minimal difference regard-ing contra-lateral breast in favor of electron beam.

For the ipsilateral lung the dose received by electron beam was higher for the electron beam than for 3DCRT.

The mean dose to the heart is the same in comparing 3DCRT and electron beam for deep tumors; however the maximum dose (D1%) was higher for the electron beam.

Significant difference was observed for the skin dose that showed better skin sparing for 3DCRT than for elec-

tron beam.Regarding the integral dose to the healthy tissue, both

3CTR and electron beam showed comparable results par-ticularly in the lowest volume irradiated to mean low dose V5 Gy.

Discussion

The present study addressed a comparative analysis of two techniques with photons and electrons to irradiate the tumor bed after surgery for superficial and deep-seat-ed early-stage breast cancer patients with a cutoff point of 4 cm between superficial and deeply seated tumors. The rationale for this investigation was to search for proper coverage of the tumor bed for superficial and deeply seat-ed tumors as well as studying the side effects for normal tissue for both photon and electron beam.

Concerning electrons, the study design required the same dose prescription definition to that applied to the photon technique. This is different from the usual pre-scription definition for electrons defined by the 100% or 90% as a minimum dose within the PTV. The strategy of applying the same prescription to all techniques is neces-sary to perform an appropriate quantitative comparison between competing treatments and is standard in plan-ning investigations.

Single portal 9–12 MeV EB, with a 2–3 cm safety mar-gin around the tumor bed has several limitations. Indeed, high EB energies are required to optimally cover deep-seated PTVs while overdosing the skin, the heart, the breast, and the underlying lung. However, in the EORTC Trial 22881–10882 the 10-year risk of severe fibrosis in the tumor bed region increased significantly with higher EB energies [11, 12].

Therefore, only superficial tumors may be optimally treated with EB. Furthermore, it has been suggested that clinical delineation of the target volume based only on the surgical scar may frequently miss the target, there-by impairing local control [13]. Fiducial markers placed around the lumpectomy cavity can be easily identified with imaging techniques such as CT, thus helping to opti-mize treatment planning and dosimetry with potentially better local control and cosmetic results [14].

In our study the CTV was defined as the area of archi-tectural distortion inside the breast surrounded by surgi-cal clips around the resection cavity, and thereafter a 1.0 cm expansion was used for PTV definition, similar to the method described by Kirova et al [15].

For superficial tumors, PTV coverage was much better also for the 3DCRT but the V107% was higher than that for the electron beam, with better homogeneity and CI for the 3DCRT. This is not going by what is traditionally done in many centers where electron beam is considered to be standard for boosting superficial tumors less than 4

Table 5 Summary of DVH analysis for organs at risk ( including healthy tissue ) for deep tumors > 4 cmDeep Mean ± SD (3DC) Mean ± SD (Electron)Healthy tissue 23069.7 ± 4053.5 20822.1 ± 6592.1 Mean (Gy) 0.1 ± 0.1 0.1 ± 0.1 V5% (Gy) 0.3 ± 0.2 0.3 ± 0.3 Doselnt 2 ± 2.3 1.7 ± 0.9Contra-lateral lung 1182.4 ± 118.8 1181.1 ± 118.9 Mean (Gy) 0.1 ± 0.4 0.1 ± 0.1 D1% (Gy) 0.4 ± 1 0.5 ± 0.2 V3Gy(%) 0 ± 0 0 ± 0Contra-lateral breast 1344.2 ± 328.5 1344.2 ± 328.5 Mean (Gy) 0.2 ± 0.4 0 ± 0 D1% (Gy) 0.4 ± 0.8 0 ± 0 V3 Gy (%) 0 ± 0 0 ± 0Ipsi-lateral lung 1242.1 ± 225 1242.1 ± 225 Mean (Gy) 0.4 ± 0.6 1 ± 0.8 D1% (Gy) 2.2 ± 2.2 4.5 ± 3 V3 Gy (%) 5.3 ± 12.2 10.3 ± 10.7 V10 Gy (%) 0 ± 0 0 ± 0Ipsi-lateral breast 1479.5 ± 811.4 1479.4 ± 811.2 Mean (Gy) 3.1 ± 1.1 2.4 ± 0.9 D1% (Gy) 10.3 ± 0.3 10.5 ± 0.1 V3 Gy (%) 35.4 ± 11.6 26.8 ± 9.6 V10 Gy (%) 8.2 ± 7.3 7.5 ± 4.1Heart 613 ± 152.5 613 ± 152.5 Mean (Gy) 0.3 ± 0.7 0.3 ± 0.3 D1% (Gy) 0.8 ± 1.4 1.3 ± 1.1 V5 Gy (%) 0 ± 0 0 ± 0Skin (Upr) Mean 6.3 ± 1.5 9.6 ± 0.3 (Med) Mean 6.3 ± 1.3 9.6 ± 0.2 (Lwr) Mean 5.9 ± 1.4 9.6 ± 0.3DoseInt, integral dose; Dx%, dose received by the x% of the volume; Vx%, volume receiving at least x Gy of the prescribed dose


cm, as in the present study showed that PTV coverage is better for 3DCRT by photon.

In our study also, for deep tumors > 4 cm, coverage of both the CTV and PTV was much better by the 3DCRT than the electron beam with V95% > 95% for the CTV and about 90% for the PTV, where as the dose homoge-neity was better for 3DCRT than for the electron beam, the V107% was much lower for the 3DCRT than for elec-tron beam. This is coinciding with what was reported by Toscas et al [16], where the PTV coverage was better for 3DCRT than that of electron beam.

In addition to radiation-induced pneumonitis, worsen-ing of preexisting cardiovascular lesions leading to death has been reported after radiation therapy for early-stage breast cancer, especially in women with left-sided and in-ner quadrant breast tumors [17–19]. The present study none of the patients developed lung or cardiac complications as the dose for both of them was very low by both tech-niques.

ConclusionFrom the present study, we can conclude that tumor

bed breast cancer at a distance less than 4 cm can be ir-radiated either by photon or electron, where as deeply seated tumors which are found at a distance more than 4 cm are better to be irradiated by photon as it provides better coverage with sparing of ORAs.

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Breast cancer is a common malignant tumor of women. Micrometastases can occur in early stage of breast cancer lesions. It is confirmed that micrometastasis has occurred in about 30% breast cancer patients at initial diagnosis, and 30%–40% of the patients will occur distant metasta-ses in the following years. The detection of circulating tu-mour cells is an promising prognostic tool for risk stratifi-cation in early breast cancer [1]. Specific gene markers are of important significance to guide early diagnosis, prog-nosis prediction, recurrence prevention, and individual treatment for breast cancer patients [2]. Here, we mainly discussed the clinical significances of small breast epithe-lial mucin (SBEM) and human mammaglobin (hMAM) for the detection of hematogenous micrometastasis in breast cancers.

Patients and methods

Patients and specimensA cohort of 109 peripheral blood (PB) specimens was

obtained from breast cancer patients in Oncology De-partment, Cancer Treatment Center, General Hospital of Shenyang Military Region, China. The median age of pa-tients was 54 (27–76) years old. All cases were histopath-ologically diagnosed as invasive ductal cancer without exception. TNM staging was according to the Handbook of Cancer Stages, Edition 6, AJCC: 94 cases at stages I–III, and 15 cases at stage IV. The 5 mL blood samples from 5 healthy donors, 5 stomach cancers, 5 colorectal cancers, 5 lung cancers and 5 kidney cancer cases respectively were taken as controls.

ReagentsThe lymphocyte parting liquid was purchased from

Tianjin TBD Sciences Company, China. SBEM antibody of mouse monoclonal was purchased from Abcam plc. Cambridge, UK. hMAM antibody of mouse monoclonal

Individual detection significances of small breast epithelial mucin (SBEM) and human mammaglobin (hMAM) expressions in peripheral blood of breast cancer patientsZhaozhe Liu, Fang Guo, Xiaodong Xie

Oncology Department, Cancer Treatment Center, General Hospital of Shenyang Military Region, Shenyang 110840, China

Received: 2 September 2012 / Revised: 5 October 2012 / Accepted: 5 November 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of the study was to explore the individual detection significances of small breast epithelial mucin (SBEM) and human mammaglobin (hMAM) in peripheral blood (PB) of breast cancer patients. Methods: SBEM and hMAM expressions in PB samples of 109 primary breast cancer patients were detected by flow cytometry (FCM) and RT-PCR. Relationship between the biomarkers’ expression and prognostic parameters were analyzed. Results: SBEM and hMAM expressions in PB of breast cancer patients were much higher than those of healthy donors and other cancer patients. SBEM and hMAM expressed in 53.2% (50/94) and 39.4% (37/94) cases at stages I–III and expressed in 73.3% (11/15) and 46.7% (7/15) cases at stage IV respectively. SBEM and hMAM mRNA were only detected in PB samples of breast cancer patients, while no expression of them was found in that of healthy donors and other cancer patients. Conclusion: hMAM mRNA detection maybe helpful to predict hematogenous micrometastasis in ER-positive, well-differentiated breast cancers and SBEM mRNA detection maybe helpful to predict hematogenous micrometastasis in ER-negative, poorly-differentiated breast cancers.

Key words breast cancer; micrometastasis; small breast epithelial mucin (SBEM); human mammaglobin (hMAM)

Correspondence to: Xiaodong Xie. Email: [email protected]


was purchased from Beijing Zhongshan Goldenbridge Biotechnology Company, China. FITC labeled goat anti-mouse IgG was purchased from Sigma Company, USA. Blood RNA extraction kit was purchased from TIANGEN Company, Germany. Easy RT-PCR kit was purchased from Beijing TransGen Biotechnology Company, China.

Equipments and primersBench centrifuge (Micromax ThermoIEC) was pur-

chased from Thermo Fisher Science and Technology Company, USA. Ultraviolet spectrophotometer (ND-1000) was purchased from American NanoDrop Com-pany. PCR amplification instrument (9700) was pur-chased from Gene Company, USA. Gel imaging system (FireReader Series) was purchased from British UVItec Company. Flow cytometry was purchased from Ameri-can Becton Dickinson Company. Primers were synthe-sized by Beijing AuGCT Biotech Company, China.

MethodsDetection of SBEM and hMAM mRNA expressions in

PB by RT-PCRThe 5 mL venous blood was taken from patients and

total RNA was extracted using RNA extraction kit. A260/A280 was calculated by UV spectrophotometer. Reverse transcription was processed by the instruction of Easy RT-PCR. A total of 50 μL PCR reaction system included 2 μL cDNA, 1 μL each upstream and downstream primer (10 μM), 25 μL 2 × Trans Taq HiFi PCR mixture, 21 μL RNase-free double distilled water. The reaction conditions were as follows, initial denaturation at 94 ℃ for 5 min, denatur-ation 94 ℃ 30 s, annealing t ℃ 30 s (t: 56 ℃ for SBEM and hMAM, 59 ℃ for internal reference gene GAPDH), exten-sion at 72 ℃ 40 s, continuous n cycles (n: 35 for SBEM and hMAM, 29 for GAPDH), the total extension at 72 ℃ for 10 min. Primer sequences were listed as follows: SBEM [3] upstream 5’-GATCTTCAGGTCACCACCATG-3’, down-stream 5’-GGGACACACTCTACCATTCG-3’; hMAM up-stream 5’-CTCCCAGCACTGCTACGCAGGTC-3’, down-stream 5’-CACCTCAACATTGCTCAGTTTCATCCG-3’; GAPDH upstream 5’-ACCCACTCCTCCACCTTTG-3’,

downstream 5’-CTCTTGTGCTCTTGCTGGG-3’. PCR products were tested by 2% agarose gel electrophoresis. The gel imaging system was applied to record the results and semi-quantitatively analyze the gene expression.

Detection of SBEM and hMAM expressions in PB by FCM

The 5 mL venous blood was taken from patients in the morning. Diluted blood sample was slowly added to the surface of the lymphocyte parting liquid along the cen-trifuge tube wall to maintain a clearly hierarchical status. After centrifuged at 2000 rpm for 10 min, lymphocytes and mononuclear cells were sucked between the upper and middle layer. Cells was washed twice and the super-natant was discarded. Cells was counted to ensure that the approximate concentration of 1 × 106 /100 μL. The 200 μL diluted SBEM (1/800) and hMAM primary antibody were gently added, and incubated at 4 ℃ for 30 min. Cells without primary antibody was taken as negative control. After the cells was washed to remove excess unbound an-tibody, 200 μL FITC-labeled IgG (1/100) was added and the mixture was incubated at 4 ℃ in the dark for 30 min. The supernatant was discarded and cells was resuspended by 400 μL PBS before FCM testing.

Statistical methods SPSS 11.0 statistical software was used for data process.

χ2 test was used to analyze the relationship between mo-lecular markers and clinicopathological parameters. P < 0.05 was considered to be statistically different.

Results

Electrophoresis results showed that GAPDH gene expressed in all PB samples (Fig. 1a), while SBEM and hMAM bands expressed only in the PB samples of breast cancer patients. No band was detected in samples of healthy donors and other tumors (Fig. 1b and 1c).

FCM results showed that SBEM and hMAM expres-sions in PB of breast cancer patients were significantly higher than those of healthy donors and other tumors.

Fig. 1 GAPDH mRNA (a), SBEM mRNA (b) and hMAM mRNA (c) expressions in PB by RT-PCR. M: DNA marker; H: healthy donor; S: stomach cancer; C: colorectal cancer; L: lung cancer; K: kidney cancer; B: breast cancer


Positive staining rate of SBEM in breast cancer patients at stage IV was as high as 68% and that of hMAM was about 38%, while the rates of SBEM and hMAM in healthy do-nors were only about 6% (Fig. 2). Correlation analysis found that SBEM expression was related to TNM stage and lymph node metastasis (P = 0.005 and P = 0.009), in-dependent of age, tumor size, hormone receptor and C-erbB-2 (Table 1). hMAM expression also correlated with TNM stage and lymph node metastasis (P = 0.024 and P = 0.004), independent of age, tumor size, hormone receptor and C-erbB-2 (Table 2).

Discussion

In developed countries, breast cancer morbidity ranks the first in female malignancy and its mortality was sec-

ondary to lung cancer [4]. Although distant metastases is the leading cause of death, it can not be detected in majority of breast cancer patients at diagnosis. In recent years, researchers have focused on the study of breast cancer micrometastasis. Most studies show that microme-tastases is an independent poor prognosis factor in breast cancer patients and the early detection of micrometastasis is beneficial to early diagnosis, determine prognosis, and individual therapy in breast cancer patients.

From technical point of view, PB is an ideal specimen for the detection of micrometastasis. The collection of PB is simple, convenient, non-invasive and easily accept-able. It is helpful to acknowledge the change of circulat-ing tumor cells dynamically [5]. Detection of disseminated tumor cells in PB may indicate the possibility of early systemic metastasis, which will directly affect patient out-

Table 1 Relationship between SBEM mRNA expression and prognostic parameters

Prognostic parameters n SBEM positive SBEM negative χ2 Pn % n %Age (years)

≤ 45 31 19 61.3 12 38.7 0.499 0.480> 45 78 42 53.8 36 46.2

Tumor size (cm) ≤ 2 37 22 59.5 15 40.5 0.278 0.598> 2 72 39 54.2 33 45.8

TNMI and II 54 23 42.6 31 57.4 7.763 0.005III and IV 55 38 69.1 17 30.9

LNM Negative 44 18 40.9 26 59.1 6.785 0.009Positive 65 43 66.2 22 33.8

ER Negative 50 32 64.0 18 36.0 2.421 0.120Positive 59 29 49.2 30 50.8

PR Negative 63 37 58.7 26 41.3 0.464 0.496Positive 46 24 52.2 22 47.8

C-erbB-2Negative 47 25 53.2 22 46.8 0.258 0.612Positive 62 36 58.1 26 41.9

Fig. 2 The positive stainings of hMAM and SBEM were displayed by %Gated and representative FCM assay results were shown in the figure. (a) Control (without antibody); (b) Healthy donor; (c) hMAM expression in PB of breast cancer patients; (d) SBEM expression in PB of breast cancer pa-tients


comes and prognosis [6]. Methods based on specific genetic markers of metastatic tumor cells can provide important information for early screen and treatment of microme-tastasis [7]. Detection of breast tissue-specific protein and adhesion protein in PB maybe helpful to discover hema-togenous micrometastasis for breast cancer. Researchers have used a variety of molecular markers to detect dis-seminated cells in breast cancer, such as CK19 and MUC-1 [8, 9]. However, the expression of these markers is often associated with tumor differentiation, and the specificity is not much higher.

SBEM was first reported by the use of isotope label-ing technology and gene expression sequence analysis in 2002 [10]. SBEM gene encoded a kind of glycoprotein with low molecular weight, which was only expressed in the breast and salivary gland. SBEM was considered to be an attractive candidate for breast tumor marker. Skliris et al [3] analyzed the relationship between SBEM expression and other prognostic markers by tissue microarray and concluded that SBEM was able to identify a subgroup of breast cancer patients with poor prognosis. hMAM was a breast tissue-specific gene that separated by differential screening and amplification technology of cDNA ends. hMAM mRNA could not be detected in normal leuko-cytes and bone marrow, while it was found to be up-regu-lated in breast cancer tissues [11]. Roncella et al [12] applied RT-PCR to detect hMAM mRNA expression in PB and found that its specificity and sensitivity were higher than a set of markers (such as CK19, EGFR, CEA, etc.). It was expected to become a molecular marker for hematoge-

nous micrometastases detection in breast cancer. In addi-tion, researchers detected SBEM and hMAM mRNA ex-pression by RT-PCR in 54 cases of invasive ductal breast cancer. They found that SBEM mRNA expression corre-lated to hMAM mRNA expression (r = 0.34 and P = 0.011) with the rate above 90%. So far, rare research has been conducted to analyze individual detection significance of the two markers in PB of breast cancer patients.

RT-PCR and immunohistochemistry (IHC) offer the reliability and improved sensitivity for detection of dis-seminated tumor cells that are missed by conventional examination, whereas they are time-consuming and complicated for screening the out-patients in clinic. More importantly, IHC is unable to make an accurate measure-ment of the lower load of micrometastatic tumor cells in PB [13]. FCM technology aims at identifying and count-ing of positive tumor cells, which is objective fast, high specific and statistically reliable [14]. In this study, FCM analysis showed that SBEM and hMAM expressions in PB of breast cancer patients were significantly higher than those in healthy donors and other cancer patients, and were higher in PB of patients at stage IV than patients at stages I–III. In addition, RT-PCR results showed that SBEM and hMAM bands were not found in PB samples of healthy donors and other cancer patients. These results indicated that SBEM and hMAM expressed in breast can-cer patients with a higher specificity. Detecting SBEM and hMAM expressions for outpatients by FCM was help-ful to screen the sub-clinical stage of micrometastasis in breast cancer.

Table 2 Relationship between hMAM mRNA expression and prognostic parameters

Prognostic parameters n hMAM positive hMAM negative χ2 Pn % n %

Age (years)≤ 45 31 16 51.6 15 48.4 2.276 0.131> 45 78 28 35.9 50 64.1

Tumor size (cm) ≤ 2 37 19 51.4 18 48.6 2.808 0.094> 2 72 25 34.7 47 65.3

TNMI and II 54 16 29.6 38 70.4 5.126 0.024III and IV 55 28 50.9 27 49.1

LNM Negative 44 25 56.8 19 43.2 8.296 0.004Positive 65 19 29.2 46 70.8

ER Negative 50 17 34.0 33 66.0 1.556 0.212Positive 59 27 45.8 32 54.2

PR Negative 63 29 46.0 34 54.0 1.990 0.158Positive 46 15 32.6 31 67.4

C-erbB-2Negative 47 23 48.9 24 51.1 2.521 0.112Positive 62 21 33.9 41 66.1


In this study, we found that SBEM mRNA expression rate in PB of breast cancer patients was 55.9% (61/109). SBEM expression correlated with TNM staging and lymph node metastasis, independent of age, ER/PR and C-erbB-2. SBEM expression was found more frequently expressed in ER/PR-negative and C-erbB-2-positive specimens (64.0% vs. 49.2%, and 58.7% vs. 52.2% respectively). It was suggested that SBEM was highly associated with some markers of poor prognosis and might be useful as an independent marker to screen out subsets of breast cancer patients with higher recurrence and metastasis risk. The findings coincided with the domestic report [15]. hMAM expression also correlated with TNM staging and lymph node metastasis, independent of age, tumor size, ER/PR and C-erbB-2, while it was found more frequently ex-pressed in ER-positive and C-erbB-2-negative specimens. Circulating tumor cells usually maintain the molecular characteristic of the primary tumor. hMAM mRNA was highly expressed in ER-positive and well-differentiated breast cancer cells, and more easily to be detected.

In summary, this study verify that SBEM and hMAM mRNA have the potential to become specific markers for hematogenous micrometastasis in breast cancer. Our re-sults indicate that hMAM mRNA detection maybe help-ful to predict hematogenous micrometastasis in ER-posi-tive, well-differentiated breast cancers and SBEM mRNA detection maybe helpful to predict hematogenous micro-metastasis in ER-negative, poorly-differentiated breast cancers.

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Colorectal cancer is one of the malignant tumors with high incidence and mortality rates, and its occurrence and development are closely related with the antitumor immunity of patients. Therefore, when antitumor drugs were discovered and developed, it was undoubtedly to test their medical effects by killing tumor cells and en-hancing the immunity of patients. However, the dosage and approach needed for direct killing are very difficult to carry out in vivo, and decreased immune function usually cannot be found or detected until an advanced stage of cancer. Meanwhile, satisfactory curative effects

have not been observed with the standard radiotherapy and chemotherapy treatments practiced in clinics, and many antitumor bioremedial medicines enhancing the immunity of patients have not achieved expected results in vivo. This could be attributed to the ignorance of the relationship between development of tumors, curative ef-fects in clinics and tumor immunosuppression caused by tumor cells in previous studies [1, 2].

Along with the routine treatment of operation, radio-therapy and chemotherapy can limit the malignant prolif-eration of tumor cells. As an adjuvant treatment, although antitumor bioremedies can activate immunocytes and ele-vate immunity of patients to a certain degree, the curative effects in vivo are greatly decreased compared with that in vitro unexpectedly. This is due to the “black hole” zone of strong tumor immunosuppression in situ and through-

Reversing effects of traditional Chinese antitumor medicines on colorectal tumor immunosuppression of natural killer cell and T lymphocyte in vitro*Cheng Cui1, Aixia Zhang1, Jianjun Hu1, Wenguang Zheng1, Zhanjiang Fu1, Lirong Qi1, Meixiang Li2, Wei Lv2

1 Department of Training, Bethune Military Medical College, Shijiazhuang 050081, China2 Department of Immunology, Basic Medical College, Hebei Medical University, Shijiazhuang 050017, China

Received: 26 August 2012 / Revised: 28 September 2012 / Accepted: 5 November 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: Reversing effects of traditional Chinese medicines on colorectal tumor immunosuppressions of natural killer (NK) cell and T lymphocyte were analyzed to provide evidence on selecting medicines for patients according to the differ-ent types of tumor immunosuppression. Methods: Six traditional Chinese medicines, including Arsenious acid (AS), Ligustra-zine hydrochloride (LHC), Astragalus mongholicus bge (AMB), Matrine N-oxide (MOX), Polyporus umbellatus polysaccharide (PUPS) and Artesunate (ART), were enrolled. The reversing effects on suppression of murine splenocyte transformation and NK killing activity were measured by 3-{4,5-dimethyl-2-thiazolyl}-2,5-diphenyl tetrazolium (MTT), and the effects on the suppressed expression of interleukin 2 receptor α (IL-2Rα), CD3ε+ζ+ and CD3ε–ζ+ were detected by flow cytometry (FCM). The effects on immunosuppressive molecules were measured by enzyme linked immunosorbent assay (ELISA), including transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), interleukin 4 (IL-4), IL-6, IL-10 and pros-taglandin (PG) E2. Results: (1) The reversing effects of AMB on the inhibition of NK killing and CD3ε+ζ+ expression were the most significant; the effect of LHC on inhibition of CD3ε–ζ+ expression was the strongest; the effects of AMB, PUPS and ART on inhibition of transformation were the greatest; and the effect of ART on inhibition of IL-2Rα expression was the strongest. (2) The correlated molecules of these medicines that exerted reversing effects on colorectal tumor immunosuppression were TGF-β1 and IL-10. AMB had the highest down-regulating effect on the secretion of TGF-β1. AS and ART had the highest effects on IL-10. Conclusion: Reversing tumor immunosuppression through the down-regulation of immunosuppressive molecules is one of the novel antitumor mechanisms of traditional Chinese medicines. The clinical use of compounded prescriptions of ART combined with AMB and LHC should be considered to avoid the reduced treatment efficiency caused by tumor im-munosuppression.

Key words colorectal cancer; immunosuppression; immunosuppressive molecules; traditional Chinese medicines; mouse

Correspondence to: Cheng Cui. Email: [email protected]* Supported by grants from Hebei Province Nature Science Foundation (No. H2012509001) and Hebei Province Science & Technology Support-ing Foundation (No. 092061111D).


out the whole body of patients with colorectal cancer [3, 4], owing to the immunosuppressive molecules secreted by tumor cells that can suppress immunological surveillance of T lymphocytes and natural killer (NK) cells, such as transforming growth factor (TGF)-β1, vascular endothe-lial growth factor (VEGF), interleukin (IL) -4, IL-6, IL-10 and prostaglandin (PG) E2 [5–7]. Eventually, the effective or activated immunocytes, either in the tumor tissue or transferred passively into this “black hole” of tumor, will be doomed to become “silence cells” with immunosup-pression. Therefore, reversing tumor immunosuppression should be the essential goal for discovering and develop-ing antitumor medicines by ascertaining their antitumor mechanisms and guiding appropriate application in clin-ics.

Currently, synthesized medicines that can decrease tumor immunosuppression are still in the period of ex-perimental study, and their target sites are single [8–10]. Some non-steroidal anti-inflammatory drugs (NSAIDs) (such as asprin [11], indomethacin [12], celecoxib [13], rofe-coxib [14]) can inhibit the occurrence and development of tumors through down-regulating the productions of im-munosuppressive molecules (IL-10 and PGE2), but their toxicity and side effects are well-known, e.g., hypersen-sitivity, nephrotoxicity, and damages on gastrointestinal tract, cardiovascular organs, neural system and hemopoi-etic system [12, 15]. Traditional Chinese medicines have had more inspiring prospects due to their multiple pathways and targets, low toxicity, high efficiency, low cost and less harm to immunity. They can impede the proliferation of tumor cells and the growth of tumors [16–20], and elevate the immunofunction [21, 22]. Meanwhile, they also can en-hance the sensitivity of radiotherapy and chemotherapy, and reduce the toxicity and side effects of these two ther-apies [23, 24]. Moreover, as our preliminary study shown, it was possible that traditional Chinese medicines could ef-ficiently eliminate the immunosuppression of colorectal cancer. As it happens, the shortcomings of radiotherapy, chemotherapy and biotherapy can be made up for. These finished products from Chinese herbs, which are legal for sale and use, can be directly used on patients with tu-mors.

Accordingly, this research is aimed to study the revers-ing effects of different traditional Chinese antitumor med-icines on tumor immunosuppression caused by colorectal cancer, to analyze their correlated molecules, and ulti-mately to seek an antitumor compounded prescription that can effectively reverse tumor immunosuppression of colorectal cancer. This research may lead to a turning point in the wide-spread clinical application of promising traditional Chinese medicines as antitumor drugs with multiple targets, and will provide experimental evidence for guiding the appropriate application of traditional Chi-nese antitumor medicines in tumor therapy.


Cell lines and animalsThe murine colorectal tumor cell line (Colon26) was

gifted from Professor Bao-en Shan (Tumor Research Centre, Fourth Hospital of Hebei Medical University, Shijiazhuang, China). The murine T-cell lymphoma cell line (YAC-1) was stocked by our laboratory (Department of Training, Bethune Military Medical College, Shijia-zhuang, China).

BALB/c mice, equal numbers of male and female, weighing 18–22 g (Grade II, Certificate No. 04-056, pur-chased from the Center for Experimental Studies, Hebei Province, China), were used in preparation of murine splenocytes for analysis of T lymphocytes and NK im-munosuppressions. All procedures involving animals and their care were approved by the Ethics Committee of the Heibei Medical University, China, and were in ac-cordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All the mice were raised in a specific pathogen-free and laminar flow isolated hoods room with controlled temperature and hu-midity. Animals were free to drink boiled water.

ReagentsThe medium used for culture was RPMI-1640 (GIBCO,

USA), supplemented with 10% fetal calf serum, penicil-lin (100 000 U/L) and streptomycin (100 mg/L). Concana-valin A (ConA), DMSO and 3-{4,5-dimethyl-2-thiazolyl}-2,5–diphenyl-2H-tetrazolium (MTT) were produced by Sigma Biotechnology Co., USA. Anti-CD3ε-FITC (CD3 FITC) and anti-IL-2Rα-FITC were purchased from EB Biotechnology, USA, and anti-CD3ζ-PE (CD247 PE) was from Santa Cruz Biotechnology, USA. The ABC-ELISA quantitative kits of mouse TGF-β1, VEGF, IL-4, IL-6 and IL-10 were from R&D Systems, USA. The ELISA quan-titative kit of PGE2 was from Sigma Biotechnology Co., USA.

The following six injections of traditional Chinese medicines were evaluated: Arsenious acid (AS), produced by YIDA Pharmacology Limited Company, Haerbin, China (No. 20040201, 10 mg/10mL/ampule); Ligustra-zine hydrochloride (LHC), produced by YONGKANG Pharmacology Limited Company, Beijing, China (No. H11020960, 40 mg/2 mL/ampule); Astragalus mongholi-cus bge (AMB), produced by DIAOJIUHONG Pharmacol-ogy Limited Company, Chengdu, China (No. Z51021776, 20 g/10 mL/ampule); Matrine N-oxide (MOX), produced by TIANJIN Biochemistry Pharmacology Limited Com-pany, Tianjin, China (No. H12020506, 0.2 g/2 mL/am-pule); Polyporus umbellatus polysaccharide (PUPS), pro-duced by ZHENGDATIANQING Pharmacology Limited Company of Jiangsu Province, Lianyungang, China [No. ZZ-5458-SU(1999)-185701, 20 mg/2 mL/ampule]; and


Artesunate (ART), produced by NANYAO Pharmacology Limited Company, Guilin, China (No. H10930195, 60 mg per ampule), which was dissolved in 1 mL 5% NaHCO3 before administration.

Proliferation assay to determine the initial concentration and cultivated duration of Colon26 cells

Colon26 cells (initial concentrations of 2.5 × 107 /L, 2 × 108 /L and 1.6 × 109 /L, respectively) were inoculated in 96-well plates (200 μL/well) and flasks (10 mL) for 12 h, 24 h, 36 h, 48 h, 60 h and 72 h, respectively. After incu-bation at 37 ˚C in a humidified 5% CO2 atmosphere, the proliferation of cells in 96-well plates were measured by MTT analysis, and the absorbances at 490 nm (A490) were recorded by an ELISA plate reader (Type of Mk353, Ther-moLabsystems, Finland). Meanwhile, cells in flasks were harvested, the rates of adherence were observed under microscope, the percentages of live cells were counted by trypan blue staining, and the values of PH in medium were measured by test papers. Finally, the initial concen-tration and cultivated duration of Colon26 cells shown with good proliferation curve, higher rates of adherence, higher percentage of live cells and steady PH in medi-um were determined to be used in the following experi-ments.

Colon26 cell viability assay to determine the concentrations of traditional Chinese medicines

Colon26 cells in a logarithmic growth phase were used in 96-well plates with final concentrations of 2 × 108 cells/L (40 000 per well). Six traditional Chinese medi-cines were respectively supplemented in a range of con-centrations (final concentrations shown in Table 1). The control wells without medicine were set synchronously. After incubation for 48 h at 37 ℃ in a humidified 5% CO2 atmosphere, the proliferation of Colon26 was measured by MTT analysis, and the values of A490 were recorded. Finally, the traditional Chinese medicines at the highest concentrations that did not decrease the proliferation of Colon26 were used in the following experiments.

Preparation of supernatants from Colon26 cells pretreated with traditional Chinese medicines

Colon26 cells were suspended at the final concentra-tion of 2 × 108 cells/L in culture flasks, with or without a traditional Chinese medicine. After being cultured for 48 h, Colon26 cells were gathered as cells pretreated with different medicines (AS-C, LHC-C, AMB-C, MOX-C, PUPS-C or ART-C) and corresponding control cells un-treated with medicines (Control-C). After being washed twice to wipe off any remaining medicine, the cells were resuspended at a concentration of 2 × 108 cells/L and recultivated for another 48 hours. Cells were then harvested as the first recultivated cells (AS-C1, LHC-C1, AMB-C1, MOX-C1, PUPS-C1, ART-C1 and Control-C1), and the supernatants from the first recultivated cells (AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1, ART-S1 and Control-S1) were gathered. Once again, after being washed twice, cells were resuspended and recultivated for 48 hours. The Colon26 cells were then harvested as the second recultivated cells (AS-C2, LHC-C2, AMB-C2, MOX-C2, PUPS-C2, ART-C2 and Control-C2), and the supernatants from the second recultivated cells (AS-S2, LHC-S2, AMB-S2, MOX-S2, PUPS-S2, ART-S2 and Con-trol-S2) were gathered. All of the cells were counted, and their viability was assessed by trypan blue staining.

Analysis of growth of indicator cells after cocultivation with supernatant from Colon26 cells

The murine splenocytes or YAC-1 were cocultivated in triplicate with the supernatants of Control-S1 and Control-S2 (100 μL/well) for 72 h or 8 h, respectively. A control containing only cells was set up. A490 was mea-sured by MTT analysis.

Effect of traditional Chinese medicines on inhibition of NK killing activity

The murine splenocytes were suspended as effectors (E, 2 × 1010 cells/L), and the YAC-1 were suspended as target cells (T, 5 × 108 cells/L). In 96-well plates, each well of experimental group (E + T, E : T = 40 : 1) contained E (50 μL), T (50 μL) and supernatant (100 μL). Meanwhile, the control of normal killing without any supernatants,

Medicines Concentrations of seven dilutions1 2 3 4 5 6 7

AS 10.00 8.00 4.00 2.00 1.00 0.50 0.25LHC 4000.00 1000.00 250.00 62.50 12.00 6.00 3.00AMB 1000.00 500.00 250.00 125.00 62.50 31.25 15.63MOX 9.60 4.80 2.40 1.20 0.60 0.30 0.15PUPS 4000.00 2000.00 1000.00 500.00 250.00 125.00 62.50ART 200.00 100.00 50.00 25.00 12.50 6.25 3.13Note: mg/L for AS, LHC, PUPS and ART; g/L for AMB and MOX

Table 1 Concentrations of traditional Chinese medicines used for test


E controls and T controls were set up in triplicate. After being incubated for 8 h, all of A490 were detected by MTT method, and the NK killing activity were calculated as follows: the rate of NK killing (%) = [1 – (AE+T – AE) / AT] × 100%, and the inhibitory rate of NK killing (%) = [(Rate of normal killing of Control – Rate of killing of experimental groups) / Rate of normal killing of Control] × 100%.

Effect of traditional Chinese medicines on inhibition of lymphocyte transformation

The murine splenocytes (1 × 1010 cells/L) were sus-pended in 96-well plates in triplicate, with each well of the experimental group containing 50 μL splenocytes, 50 μL ConA (20 mg/L) and 100 μL supernatant. Meanwhile, the control of normal transformation without superna-tant was set up, also in triplicate. After being incubated for 72 h, all of A490 were measured by MTT method, and the inhibitory rate of transformation = [(A of normal con-trol – A of experimental group) / A of normal control] × 100%.

Effects of traditional Chinese medicines on inhibition of IL-2Rα, CD3ε and ζ chain expressions in murine splenocytes

The murine splenocytes (1 × 1010 cells/L) were incubat-ed with 0.25 mL ConA (20 mg/L) and 0.5 mL supernatant in culture flasks for 48 h. Meanwhile, the control with-

out any supernatant was set up. Then, 1 × 106 cells were taken out and incubated with anti-IL-2Rα-FITC (10 μL) or anti-CD3ε-FITC (10 μL) and anti-CD3ζ-PE (10 μL) in the dark at room temperature for 20 minutes. After cen-trifugation, the cells were washed and resuspended with 0.5 mL PBS. Finally, the percentages of IL-2Rα+, CD3ε+ζ+ and CD3ε–ζ+ cells were measured by FCM (FACS Van-tuge, BD, USA), and the inhibitory rate = [(Percentage of control – Percentage of experimental group) / Percent-age of control] × 100%. The murine isotypes of unrelated IgG labeling were taken as negative controls by the same fluorescence.

Effects of traditional Chinese medicines on immunosuppressive molecules secreted by Colon26 tumor cells

The concentrations of six immunosuppressive mol-ecules in the supernatants were measured by quantitative ELISA kit according to the manufacturer’s instructions. Based on standard curves, the concentrations of TGF-β1, VEGF, IL-4, IL-6, IL-10 and PGE2 in the supernatants from Colon26 tumor cells were examined.

Statistical analysisThe data were expressed as the mean ± SD. A one-

sided statistical analysis was conducted using SPSS 13.0 software. The F test (ANOVA), q test, t test and linear relation were employed appropriately. The multianaly-sis of correlativity was used to ascertain the correlation between the changes in immunosuppressive molecules secretion and immunosuppression of Colon26 cells after pretreatment with each traditional Chinese medicine. A P value < 0.05 was considered significant.

Results

The initial concentration and cultivated duration of Colon26 cells

As shown in Table 2 and Fig. 1, with the initial con-centration of 2.5 × 107 /L, cells grew too slowly to lay over the bottom of flasks till cultivated over 72 h, showing that this concentration was lower for experiments. With the initial concentration of 1.6 × 109 /L, cells laid over com-

Table 2 The growth of Colon26 cells with different initial concentrations

Time2.5 × 107 /L 2 × 108 /L 1.6 × 109 /L

A490Percentage of

live cellPH of

medium A490Percentage of

live cellPH of

medium A490Percentage of

live cellPH of

medium12 h 0.031 ± 0.005 100% 7.0 0.155 ± 0.017 100% 7.0 0.905 ± 0.030 100% 7.024 h 0.058 ± 0.008 100% 7.0 0.513 ± 0.026 100% 7.0 1.097 ± 0.058 96% 7.036 h 0.103 ± 0.012 100% 7.0 0.782 ± 0.020 100% 7.0 1.282 ± 0.043 90% 6.048 h 0.283 ± 0.003 100% 7.0 1.126 ± 0.031 99% 7.0 0.862 ± 0.048 85% 5.060 h 0.565 ± 0.007 100% 7.0 0.943 ± 0.080 90% 6.5 0.823 ± 0.042 83% 5.072 h 0.935 ± 0.011 100% 7.0 0.854 ± 0.045 82% 5.0 0.452 ± 0.023 46% 4.0

Fig. 1 The growth of Colon26 cells with different initial concentrations measured by MTT analysis


pletely when cultivated for 12 h, its percentage of live cells was reduced greatly at 24 h along with a large amount of non-adherent cells and decreased PH in medium, shown that this concentration was higher for experiments. With the initial concentration of 2 × 108 /L, the proliferation curve revealed good trend of growth. Satisfied rate of ad-herence, higher percentage of live cells and steady PH in

medium were observed at 48 h. Therefore, Colon26 cells with initial concentration of 2 × 108 /L and cultivated du-ration of 48 h were used in the following experiments.

The concentrations of traditional Chinese medicines used in experiments

As shown in Table 3, compared with controls of Co-lon26 untreated with any medicine, the highest concen-trations of the traditional Chinese medicines that did not change proliferation of Colon26 were 0.5 mg/L AS, 6 mg/L LHC, 250 g/L AMB, 0.3 g/L MOX, 125 mg/L PUPS and 6.25 mg/L ART. As shown in Table 4, the live cell counts and A490 of recultivated Colon26 cells pretreated with tra-ditional Chinese medicines had no significant changes compared with that of controls (all P > 0.05).

Effect of supernatants on growth of indicator cells in immune function analysis

As shown in Table 5, no changes in murine splenocyte and YAC-1 cell proliferation were observed after co-in-cubation with supernatants of Colon 26 tumor cells when compared with controls (all P > 0.05).

Contrastive analysis of the down-regulating effects of different antitumor traditional Chinese medicines on immunosuppression caused by Colon26 tumor cells

As shown in Tables 6 and 7, the supernatants of the Colon26 cells steadily suppressed immune functions of NK and T lymphocytes (Control-S1 vs. Control-S2, all P

Table 3 Effects of traditional Chinese medicines at different concentrations on proliferation of Colon26 tumor cells (n = 10)

Medicines A490 of Colon26’s proliferation1 2 3 4 5 6 7

AS 0.440 ± 0.050** 0.518 ± 0.068** 0.790 ± 0.105** 0.840 ± 0.068** 0.920 ± 0.073** 1.059 ± 0.080 1.052 ± 0.093LHC 0.073 ± 0.013** 0.675 ± 0.060** 0.833 ± 0.075** 0.919 ± 0.083** 0.949 ± 0.100* 1.063 ± 0.092 1.055 ± 0.105AMB 0.337 ± 0.033** 0.793 ± 0.129** 1.030 ± 0.076 1.014 ± 0.081 1.015 ± 0.076 1.025 ± 0.074 1.056 ± 0.067MOX 0.078 ± 0.009** 0.317 ± 0.051** 0.521 ± 0.061** 0.804 ± 0.059** 0.895 ± 0.068** 1.034 ± 0.067 1.027 ± 0.068PUPS 0.765 ± 0.066** 0.931 ± 0.082** 0.952 ± 0.097** 0.998 ± 0.089** 1.017 ± 0.094* 1.065 ± 0.091 1.095 ± 0.115ART 0.218 ± 0.040** 0.607 ± 0.064** 0.890 ± 0.099** 0.976 ± 0.088** 1.031 ± 0.098* 1.060 ± 0.094 1.084 ± 0.108Note: Compared with control (1.084 ± 0.111), * P < 0.05, ** P < 0.01. 1–7 were the concentrations of the six traditional Chinese medicines shown in Table 1

Table 4 Growth of Colon26 cells pretreated with or without traditional Chinese medicines (n = 3)Groups Number of live cells (× 108 /L) F testControl-C 4.49 ± 0.20Control-C1 4.53 ± 0.15Control-C2 4.71 ± 0.19AS-C 4.75 ± 0.08 F = 1.236, P = 0.328AS-C1 4.77 ± 0.17AS-C2 4.59 ± 0.23LHC-C 4.40 ± 0.14 F = 2.137, P = 0.130LHC-C1 4.51 ± 0.16LHC-C2 4.75 ± 0.05AMB-C 4.57 ± 0.25 F = 1.525, P = 0.259AMB-C1 4.41 ± 0.14AMB-C2 4.73 ± 0.07MOX-C 4.56 ± 0.41 F = 0.390, P = 0.846MOX-C1 4.64 ± 0.32MOX-C2 4.71 ± 0.14PUPS-C 4.48 ± 0.20 F = 1.680, P = 0.214PUPS-C1 4.42 ± 0.15PUPS-C2 4.72 ± 0.06ART-C 4.87 ± 0.29 F = 2.707, P = 0.073ART-C1 4.38 ± 0.18ART-C2 4.95 ± 0.21Note 1: AS-C, LHC-C, AMB-C, MOX-C, PUPS-C and ART-C were Co-lon26 cells treated with six traditional Chinese medicines; Control-C was Colon26 cells treated without any medicines. Note 2: AS-C1, LHC-C1, AMB-C1, MOX-C1, PUPS-C1 and ART-C1 were first recultivated Colon26 cells pretreated with six traditional Chinese medicines; Control-C1 was first recultivated Colon26 cells pretreated without any medicines. Note 3: AS-C2, LHC-C2, AMB-C2, MOX-C2, PUPS-C2 and ART-C2 were second recultivated Colon26 cells pretreated with six traditional Chinese medicines; Control-C2 was second recultivated Colon26 cells pretreated without any medicines.

Table 5 Effect of Colon26 supernatants on proliferation of indicator cells used in immune experiments (A490, n = 3)

Supernatants Proliferation of YAC-1 Proliferation of murine splenocyte

Control-S1 0.480 ± 0.041 0.240 ± 0.035Control-S2 0.483 ± 0.037 0.236 ± 0.024Control 0.478 ± 0.048 0.242 ± 0.039Note 1: Control was group treated without any supernatant of Colon26. Note 2: Control-S1 was the supernatant from first recultivated Colon26 cells pretreated without any medicines. Note 3: Control-S2 was the supernatant from second recultivated Colon26 cells pretreated without any medicines.


> 0.05). The inhibitory rates of NK killing and CD3ε–ζ+ expression were approximately 30%, and those of T lym-phocyte transformation and expression of IL-2Rα and CD3ε+ζ+ were close to 70%. The down-regulating effects of different traditional Chinese medicines on immuno-

suppression caused by Colon26 were shown significantly. The expressions of CD3ε and ζ chain by FCM were shown in Fig. 2.

The results of ANOVA and q test analysis on NK im-munosuppression were as follows: (1) There were differ-

Table 6 Effects of supernatants from re-cultivated Colon26 after treated with traditional Chinese medicines on murine splenocytes’ immunofunc-tionsGroups NK killing (%) Transformation (A490) IL-2Rα (%) CD3ε+ζ+ (%) CD3ε–ζ+ (%)Control 73.38 ± 9.32 0.65 ± 0.05 21.01 ± 1.13 14.41 ± 2.21 6.27 ± 0.73Control-S1 52.33 ± 8.36◇ 0.21 ± 0.06◇ 6.39 ± 1.08◇◇◇ 4.59 ± 0.31◇◇ 4.81 ± 0.18◇

Control-S2 53.98 ± 7.21◇ 0.28 ± 0.06◇ 6.75 ± 0.35◇◇◇ 4.28 ± 0.10◇◇ 4.72 ± 0.51◇

AS-S1 60.45 ± 11.78 0.31 ± 0.10** 17.80 ± 5.21* 10.82 ± 0.51*** 4.46 ± 0.39AS-S2 59.53 ± 14.83 0.27 ± 0.09# 16.83 ± 5.10△ 4.62 ± 0.34## 4.32 ± 0.31LHC-S1 71.62 ± 9.96** 0.31 ± 0.13* 12.25 ± 2.30* 13.58 ± 0.72*** 6.21 ± 0.77*LHC-S2 71.66 ± 12.26△△ 0.22 ± 0.09# 12.68 ± 3.46△ 13.11 ± 2.29△△ 5.21 ± 0.16#△

AMB-S1 71.27 ± 14.32** 0.42 ± 0.08*** 6.51 ± 1.79 15.19 ± 1.28*** 5.62 ± 0.33*AMB-S2 71.52 ± 13.93△△ 0.39 ± 0.08△△ 10.08 ± 2.56#△ 15.95 ± 2.58△△ 5.43 ± 0.08△

MOX-S1 48.02 ± 11.80 0.27 ± 0.05* 9.03 ± 1.47* 13.95 ± 2.70* 7.95 ± 0.29***◇MOX-S2 49.16 ± 10.22 0.23 ± 0.06#△ 7.29 ± 0.44# 4.27 ± 0.41## 4.79 ± 0.33##

PUPS-S1 58.54 ± 5.81 0.45 ± 0.13*** 8.75 ± 1.73* 9.20 ± 0.52*** 6.02 ± 0.10***PUPS-S2 54.32 ± 5.67 0.47 ± 0.14△△△ 6.59 ± 0.40# 8.98 ± 0.67△△△ 4.36 ± 0.08##

ART-S1 58.22 ± 4.52 0.43 ± 0.14*** 15.43 ± 2.56** 13.81 ± 1.76*** 5.45 ± 0.26*ART-S2 55.03 ± 5.72 0.54 ± 0.15#△△△ 18.26 ± 3.53#△△ 12.74 ± 0.83#△△△ 4.67 ± 0.25#

Note 1: Compared with control, ◇: P < 0.05; ◇◇: P < 0.001; ◇◇◇: P < 0.0005. Compared with control-S1, *: P < 0.05; **: P < 0.001; ***: P < 0.0005. Compared with AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1, and ART-S1, respectively, #: P < 0.05; ##: P < 0.0005. Compared with control-S2, Δ: P < 0.05; ΔΔ: P < 0.001; ΔΔΔ: P < 0.0005. Note 2: Control was the group treated without any supernatant of Colon26 cells.Note 3: AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1 and ART-S1 were the supernatants from first recultivated Colon26 cells pretreated with six tradi-tional Chinese medicines; Control-S1 was first recultivated Colon26 cells pretreated without any medicines.Note 4: AS-S2, LHC-S2, AMB-S2, MOX-S2, PUPS-S2 and ART-S2 were the supernatants from second recultivated Colon26 cells pretreated with six traditional Chinese medicines; Control-S2 was the supernatants from second recultivated Colon26 cells pretreated without any medicines.

Table 7 Effects of traditional Chinese medicines on immunosuppressions caused by Colon26 tumor cells (n = 10, %)

Groups Inhibitory rates of immune functions (%)NK killing Transformation IL-2Rα CD3ε+ζ+ CD3ε–ζ+

Control-S1 30.19 ± 6.09 67.11 ± 8.22 69.60 ± 5.15 68.15 ± 2.17 23.34 ± 2.86Control-S2 27.80 ± 7.80 65.98 ± 6.90 67.89 ± 1.66 70.27 ± 5.56 24.77 ± 8.14AS-S1 17.61 ± 6.05* 57.06 ± 12.38* 30.90 ± 8.15** 24.89 ± 3.57** 28.87 ± 6.28AS-S2 25.20 ± 6.89# 61.97 ± 11.97# 35.76 ± 10.91△△ 67.92 ± 2.33## 25.84 ± 10.27LHC-S1 8.74 ± 5.53** 57.86 ± 11.33* 40.44 ± 5.71* 5.74 ± 1.65** 1.54 ± 0.78**LHC-S2 8.80 ± 4.82△△ 69.57 ± 12.75# 39.64 ± 8.53△ 9.25 ± 0.91#△△△ 16.90 ± 2.57##△

AMB-S1 4.10 ± 2.01** 33.56 ± 12.71* 69.00 ± 8.52 –5.75 ± 1.60*** 10.31 ± 5.20*AMB-S2 3.68 ± 1.96△△ 36.40 ± 13.84△ 0.45 ± 4.43#△ –10.89 ± 2.89#△△△ 14.83 ± 1.27△

MOX-S1 27.56 ± 11.01 54.98 ± 8.30* 57.34 ± 2.57* 3.39 ± 2.24** –26.74 ± 4.56***MOX-S2 27.67 ± 13.45 63.86 ± 9.78## 65.30 ± 3.99# 70.28 ± 2.87## 23.60 ± 5.26##

PUPS-S1 12.32 ± 5.59* 28.36 ± 9.63** 58.34 ± 6.15* 36.18 ± 3.63* 4.04 ± 1.52**PUPS-S2 21.32 ± 8.21# 30.52 ± 1.24△△ 67.97 ± 3.01# 37.79 ± 9.00△ 30.71 ± 4.68##

ART-S1 15.67 ± 6.54* 34.45 ± 5.84* 28.21 ± 6.52** 4.57 ± 1.79*** 13.03 ± 4.08*ART-S2 20.30 ± 6.63# 12.96 ± 5.12##△△ 13.52 ± 6.16#△△ 14.25 ± 6.39#△△△ 25.57 ± 3.96#

Note 1: Compared with control-S1, *: P < 0.05; **: P < 0.001; ***: P < 0.0005. Compared with AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1, and ART-S1, respectively, #: P < 0.05; ##: P < 0.0005. Compared with Control-S2 , Δ: P < 0.05; ΔΔ: P < 0.001; ΔΔΔ: P < 0.0005. Note 2: AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1 and ART-S1 were the supernatants from first recultivated Colon26 cells pretreated with six tradi-tional Chinese medicines; Control-S1 was first recultivated Colon26 cells pretreated without any medicines. Note 3: AS-S2, LHC-S2, AMB-S2, MOX-S2, PUPS-S2 and ART-S2 were the supernatants from second recultivated Colon26 cells pretreated with six traditional Chinese medicines; Control-S2 was the supernatants from second recultivated Colon26 cells pretreated without any medicines.


ences among the effects of the six medicines on inhibition of NK killing activity (F = 9.047, P = 0.000). The down-regulating effects of AS, PUPS and ART on inhibition of NK killing showed no differences (all P > 0.05, inhibi-tory rates were approximately 50%), those of LHC and AMB showed no differences (P > 0.05, inhibitory rates were approximately 75%), and those of AMB were the strongest and longest (inhibitory rates were 86.76%). (2) There were differences among the effects of the six medi-cines on inhibition of CD3ε–ζ+ expression (F = 26.162, P = 0.000). The initial decreasing levels of suppressed CD3ε–ζ+

expression caused by LHC and PUPS showed no differ-ence (P > 0.05, decreasing approximately 80%–90%), those caused by AMB and ART showed no difference (P > 0.05, decreasing approximately 40%–50%), and those caused by MOX were significantly different compared with others (all P = 0.000, inhibition was completely re-versed and the elevated expression was significant, but transient). The decreasing effect of LHC was the strongest and longest.

The results of ANOVA and q test analysis on T cell immunosuppression were as follows: (1) Differences

Fig. 2 Effects of traditional Chinese medicines on the suppressed expression of CD3ε (CD3 FITC) and ζ chains (CD247 PE) caused by Colon26 tumor cells


among the effects of the six medicines on inhibition of T cell transformation were observed (F = 23.686, P = 0.000). The decreasing levels of inhibited transformation caused by AS, LHC and MOX showed no difference (P > 0.05, decreasing approximately 15% and then rapidly return-ing to the level of control), and those caused by AMB, PUPS and ART showed no difference (P > 0.05, decreas-ing approximately 50% and lasting longer). (2) Differenc-es among the effects of the six medicines on inhibition of IL-2Rα expression were observed (F = 8.222, P = 0.001). The decreasing levels of suppressed IL-2Rα expression caused by AS, LHC and ART showed no difference (P > 0.05, decreasing approximately 50% and lasting longer), those caused by MOX and PUPS showed no difference (P > 0.05, decreasing approximately 15% and then rap-idly returning to the level of control), and those caused by ART were the strongest. (3) Differences among the ef-fects of the six medicines on inhibition of CD3ε+ζ+ expres-sion existed (F = 22.000, P = 0.000). The initial decreasing levels caused by AS and PUPS showed no difference (P > 0.05, decreasing approximately 50%–60%), those caused by LHC, AMB, MOX and ART showed no difference (P > 0.05, inhibition was reversed almost completely and the elevated expression was significant), and the decreasing effect of AMB was the strongest.

Contrastive analysis of the effects of different antitumor traditional Chinese medicines on immunosuppressive molecules secreted by Colon26 tumor cells

As shown in Table 8, all of the six immunosuppressive

molecules existed steadily in the supernatants of Colon26, in which the concentration of TGF-β1 was the highest at (1295.23 ± 29.85) pg/mL, and all the others was about 10–30 pg/mL. The results of ANOVA and q test analy-sis on immunosuppressive molecules were as follows: (1) Differences were observed among the effects of the six medicines on TGF-β1 (F = 425.699, P = 0.000). The de-creasing levels caused by AS, MOX and ART showed no differences (all P > 0.05, decreasing approximately 25%), those caused by LHC, AMB and PUPS showed differences (all P = 0.000, decreasing approximately 45%, 50% and 33%, respectively), and the decreasing effect of AMB was the strongest. (2) Differences were observed among the effects of the six medicines on IL-6 (F = 11.852, P = 0.000). No changes or up-regulating effects on IL-6 were found after pretreatment with AMB, MOX, PUPS or ART, and approximately 5% decreasing effects were shown af-ter pretreatment with AS and LHC in different phases. (3) Differences were shown among the effects of the six medicines on IL-10 (F = 3.587, P = 0.023). Up-regulat-ing effects on IL-10 were found after pretreatment with PUPS, and approximately 5% decreasing effects were shown after pretreatment with the other five medicines. The decreasing effect of AS was the strongest. (4) No dif-ferences existed among the effects on VEGF (F = 0.598, P = 0.727), IL-4 (F = 2.559, P = 0.069) and PGE2 (F = 2.587, P = 0.063).

Analysis of the correlation between the changes in immunosuppressive molecules secretion and immunosuppression of Colon26

Table 8 Concentrations of immunosuppressive molecules secreted by Colon26 after pretreated with traditional Chinese medicines (n = 3, pg/mL)Groups TGF-β1 VEGF IL-4 IL-6 IL-10 PGE2

Control-S1 1295.23 ± 29.85 29.18 ± 1.90 27.70 ± 0.70 26.55 ± 0.60 29.52 ± 2.50 9.69 ± 0.42Control-S2 1266.05 ± 19.90 29.30 ± 1.80 27.65 ± 1.70 26.63 ± 1.00 29.63 ± 1.90 9.71 ± 0.44AS-S1 996.30 ± 17.38*** 28.25 ± 0.14 27.01 ± 0.13* 26.22 ± 0.18 27.35 ± 0.11* 26.33 ± 0.25**AS-S2 1026.50 ± 18.94##Δ 30.16 ± 0.17 27.54 ± 0.10# 25.56 ± 0.09#Δ 27.70 ± 0.23Δ 8.91 ± 0.20##Δ

LHC-S1 725.50 ± 6.26** 28.56 ± 2.11 28.12 ± 0.22 25.24 ± 0.78* 28.23 ± 0.45* 27.04 ± 0.66**LHC-S2 984.32 ± 8.36##ΔΔ 29.23 ± 0.86 27.47 ± 0.56 26.58 ± 0.34# 27.49 ± 0.42#Δ 25.78 ± 0.25#ΔΔ

AMB-S1 639.74 ± 4.77*** 29.70 ± 0.86 27.73 ± 0.58 28.69 ± 0.37* 27.99 ± 0.61* 24.88 ± 0.16**AMB-S2 560.91 ± 5.48##ΔΔ 30.59 ± 1.09 28.47 ± 0.52 27.03 ± 0.46# 29.68 ± 0.30# 19.37 ± 0.11#ΔΔ

MOX-S1 988.26 ± 35.73** 29.31 ± 2.04 28.54 ± 0.52 26.38 ± 0.59 28.21 ± 0.64* 21.48 ± 0.20**MOX-S2 627.50 ± 6.32##ΔΔΔ 33.48 ± 1.43#Δ 29.46 ± 0.63 26.49 ± 0.37 28.13 ± 0.51Δ 12.19 ± 0.18#Δ

PUPS-S1 869.99 ± 5.30** 28.09 ± 1.27 27.90 ± 0.14 26.81 ± 0.66 30.04 ± 0.45 28.06 ± 0.32**PUPS-S2 949.22 ± 8.06###ΔΔ 30.48 ± 0.67#Δ 29.98 ± 0.34#Δ 29.65 ± 0.40#Δ 31.85 ± 0.88#Δ 17.47 ± 0.14#Δ

ART-S1 1015.77 ± 6.81** 28.57 ± 1.21 28.17 ± 0.60 26.26 ± 0.62 27.75 ± 0.35* 33.62 ± 0.61***ART-S2 687.94 ± 6.74##ΔΔ 29.60 ± 0.92 31.28 ± 0.26##Δ 29.12 ± 0.54#Δ 28.10 ± 0.25Δ 21.68 ± 0.32#ΔΔ

Note 1: Compared with control-S1, *: P < 0.05; **: P < 0.001; ***: P < 0.0005. Compared with AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1, and ART-S1, respectively, #: P < 0.05; ##: P < 0.001; ###: P < 0.0005. Compared with control-S2, Δ: P < 0.05; ΔΔ: P < 0.001; ΔΔΔ: P < 0.0005. Note 2: AS-S1, LHC-S1, AMB-S1, MOX-S1, PUPS-S1 and ART-S1 were the supernatants from first recultivated Colon26 cells pretreated with six tradi-tional Chinese medicines; Control-S1 was first recultivated Colon26 cells pretreated without any medicines.Note 3: AS-S2, LHC-S2, AMB-S2, MOX-S2, PUPS-S2 and ART-S2 were the supernatants from second recultivated Colon26 cells pretreated with six traditional Chinese medicines; Control-S2 was second recultivated Colon26 cells pretreated without any medicine.


cells after pretreatment with traditional Chinese medicines

(1) A positive correlation was found between the sup-pression of inhibition of NK killing and decreasing secre-tion of TGF-β1 from Colon26 tumor cells after pretreat-ment with AS, AMB, PUPS and ART (r = 0.996, P = 0.029; r = 0.999, P = 0.014; r = 0.998, P = 0.020; r = 0.998, P = 0.019, respectively). A correlation was also found be-tween the suppression of inhibition of NK killing and decreasing secretion of TGF-β1 and IL-10 from Colon26 after pretreatment with LHC (r = 0.994, P = 0.036; r = 0.937, P = 0.031, respectively).

(2) A positive correlation was shown between the sup-pression of inhibition of transformation and decreasing secretion of TGF-β1 from Colon26 after pretreatment with AS, AMB, PUPS and ART (r = 0.999, P = 0.009; r = 0.999, P = 0.014; r = 1.000, P = 0.001; r = 1.000, P = 0.001, respectively), and between the suppression of inhibition of transformation and decreasing secretion of TGF-β1 and IL-10 after pretreatment with LHC (r = 0.998, P = 0.021; r = 0.956, P = 0.022, respectively).

(3) A positive correlation was shown between the sup-pression of inhibition of IL-2Rα expression and decreas-ing secretion of TGF-β1 after pretreatment with AS, LHC, MOX, PUPS and ART (r = 0.999, P = 0.016; r = 1.000, P = 0.002; r = 0.999, P = 0.014; r = 0.997, P = 0.025; r = 0.998, P = 0.019, respectively).

(4) A positive correlation was shown between the sup-pression of inhibition of CD3ε+ζ+ expression and decreas-ing secretion of TGF-β1 after pretreatment with AS, AMB, MOX, PUPS and ART (r = 0.992, P = 0.041; r = 0.997, P = 0.024; r = 0.998, P = 0.020; r = 0.993, P = 0.037; r = 0.992, P = 0.039, respectively), and between the suppression of inhibition of CD3ε+ζ+ expression and decreasing secretion of TGF-β1 and IL-10 after pretreatment with LHC (r = 0.993, P = 0.036; r = 0.999, P = 0.013, respectively).

(5) A positive correlation was shown between the sup-pression of inhibition of CD3ε–ζ+ expression and decreas-ing secretion of IL-10 after pretreatment with AMB and MOX (r = 0.995, P = 0.019; r = 0.993, P = 0.024, respec-tively).

Discussion

It is well known that cell mediated immunity is most dominant in tumor immunity, including adaptive immu-nity monitored by T lymphocytes and innate immunity monitored by NK cells. Through the secretion of immu-nosuppressive molecules, colorectal tumor cells can im-pede the differentiation, proliferation and activation of immunocytes, blocking their pathways of signal transduc-tion. The immunosuppression of NK and T lymphocytes is prominent and common [25, 26]. Functional T lympho-cytes can be activated in vitro by certain stimulators, such

as ConA, where their activation signals are transduced by CD3ε and ζ chains, and subsequently activated T cells ex-press the IL-2Rα chain. Usually, the killing activity of NK is measured by cytotoxicity on sensitive target cells such as YAC-1, and its activated signals are also transduced by ζ chain [27]. Accordingly, transformation induced by ConA and the expression of IL-2Rα and CD3ε+ζ+ on mu-rine splenocytes were examined for the immune function of T cells, and the NK cytotoxicity and CD3ε–ζ+ expres-sion on murine splenocytes were measured for the im-mune function of NK. These five immune functions can be used to show the effects of traditional Chinese medi-cines on colorectal tumor immunosuppression of NK and T lymphocytes.

In the experimental design, it was considered that tra-ditional Chinese medicines could impact the prolifera-tion and function of indicator cells in analysis of immune function and the tumor cells, so the supernatants from recultivated Colon26 tumor cells that were cocultured with traditional Chinese medicine first and then washed off completely were used in this study. As results shown, no changes were found in the proliferation of murine splenocytes and YAC-1 after being cocultured with the supernatant of unpretreated Colon26 cells. This indicates that the effects of traditional Chinese medicines on the immunosuppression of Colon26 cells under this experi-mental design are ensured, and not due to the changed proliferation of indicator cells.

Similarly, the immunosuppression is attributed to the immunosuppressive molecules secreted by tumor cells that exist in the supernatant, and the secretion of immu-nosuppressive molecules is determined directly by tumor cell proliferation. For this reason, it is necessary to make sure that the concentrations of the six traditional Chinese medicines used in our experiments would not impede the proliferation of Colon26 cell. Meanwhile, after pretreat-ment by traditional Chinese medicines with determined concentrations for 48 h, the tumor cells were washed to clean off any remaining medicine before being reculti-vated for another 48 h twice. Compared with a control that was not pretreated, neither the counts nor A490 of the recultivated tumor cells showed any significant changes. This indicates that the changed immunosuppressive mol-ecules in supernatants and the down-regulated tumor im-munosuppression are actually caused by traditional Chi-nese medicines rather than by an altered proliferation of Colon 26 tumor cells.

It has been shown that colorectal tumor cells can exert remarkable immunosuppression in which the inhibitory rate of T cell proliferation, activation and signal transduc-tion is close to 70%, and in which the inhibition of NK killing and its signal transduction of activation mediated by ζ chain is approximately 30%. Colon26 tumor cells can secrete immunosuppressive molecules, of which the


concentration of TGF-β1 is the highest at (1295 ± 29.85) pg/mL. After pretreatment with six traditional Chinese medicines, reduced immunosuppressive molecules secre-tion from Colon26 and down-regulated tumor immuno-suppression with different kinds, level and duration were observed, allowing the antitumor activity of NK and T cells to resume and the immunosuppression from tumor cells to be eliminated partly or completely.

In detail, the patterns of the diversiform, pleiotropic and overlapping effects on down-regulated immunosup-pression and immunosuppressive molecules were shown. Different type, level and duration of down-regulated im-munosuppression and immunosuppressive molecules se-cretion indicated that there were various sensitivities to different traditional Chinese medicines. It was possibly caused by the different active ingredients of medicines via different correlated sites or pathways on the same kind of tumor cells. The similar down-regulating effects of dif-ferent traditional Chinese medicines on tumor immuno-suppression and immunosuppressive molecules secretion may be dependent on the similar effects of the different active ingredients. Based on multianalysis of correlativ-ity, for the down-regulating effect on the inhibition of NK killing and T lymphocytes’ transformation resulted by Colon26, the correlated molecules of AS, AMB, PUPS and ART were TGF-β1, and the correlated molecules of LHC were TGF-β1 and IL-10. For the down-regulating effect on the inhibition of IL-2Rα expression, the corre-lated molecules of AS, LHC, MOX, PUPS and ART were TGF-β1. For the down-regulating effect on the inhibi-tion of CD3ε+ζ+ expression, the correlated molecules of AS, AMB, MOX, PUPS and ART were TGF-β1, and the correlated molecules of LHC were TGF-β1 and IL-10. For the down-regulating effect on the inhibition of CD3ε–ζ+ expression, the correlated molecules of AMB and MOX were IL-10. Accordingly, reducing tumor immunosup-pression of NK and T lymphocytes through the down-regulation of immunosuppressive molecules secreted by tumor cells could be one of the novel antitumor mecha-nisms of traditional Chinese medicines.

Considering the pathogenesis of carcinoma based on traditional medicine, such as the ‘obstruction of collater-als by heat and toxic materials’, ‘stagnation of vital energy and blood stasis’, ‘condensation of phlegm and wet’ and ‘deficiency of vital energy’, traditional Chinese medicines can exert antitumor effects through different processes to modify and reverse these disadvantages. Of the six antitu-mor traditional Chinese medicines used in our study, AS belongs to the category of ‘treating the toxicity with poi-sonous agents’, MOX and ART belong to the category of ‘heat-clearing’, LHC belongs to the category of ‘activating blood circulation to dissipate blood stasis’, PUPS belongs to the categories of ‘softening hardness and dispersing accumulation’ and ‘dispelling dampness and eliminating

sputum’ and AMB belongs to the category of ‘supporting the vital energy and removing the accumulated evil’. It was shown that five of the antitumor traditional Chinese medicines, except for MOX, reduced the inhibition of NK killing activity, of which AMB had the strongest reduc-ing effect (approximately 86.76%) and the longest dura-tion. Five of the antitumor traditional Chinese medicines, except for AS, reduced the inhibition of NK CD3ε–ζ+ ex-pression, of which MOX had the strongest reducing effect (reversed completely and the elevated expression was sig-nificant) with transient duration, and LHC had a decreas-ing effect of 93.40% with longer duration. The level of the down-regulating effects of AMB, PUPS and ART on suppressed transformation of T lymphocytes were stron-ger (approximately 50%) and longer than other three medicines. The down-regulating effect of ART on sup-pressed IL-2Rα expression of T cells was the strongest (approximately 50%) with a longer duration time. The down-regulating effect of AMB on decreased CD3ε+ζ+ expression of T cells was the strongest and was reversed completely, and the elevated expression was significant. TGF-β1 and IL-10 were the main correlated molecules of the traditional Chinese medicines on immunosuppres-sion of colorectal tumor. The decreasing effect of AMB on TGF-β1 was the strongest (55.75%) and that of AS on IL-10 was the strongest (7.35%). Accordingly, it should be taken into consideration in clinics to use an antitumor compounded prescription of ART combined with AMB and LHC. By this method, it is expected that the disad-vantages on efficiency of treatment caused by colorectal tumor immunosuppression should be avoided. Further-more, whether or not other compounded prescriptions that consist of same categories of ‘clearing away heat and toxic material’ combined with ‘strengthening healthy & eliminating pathogens’ and ‘promoting blood circulation to remove blood stasis’ also can exert similar effects, are expected to be proved. And animal experiments in vi-vo should be performed to provide further evidences of these reversing effects on tumor immunosuppression, by which the therapeutic potentials of this compound pre-scription will be revealed.

All of these six traditional Chinese medicines have been authorized, produced and sold legally. Although many antitumor activities were focused on [15–22], and their different down-regulating effect on tumor immuno-suppressions has not been contrastively reported till now. A ‘dialectic idea’ is a pivot in traditional medicine: based on the comprehensive analysis and evaluation of illness and patient immunity, the antitumor multitarget treat-ment design and individualized treatment were advo-cated. Compared with other remedial medicines used for patients with tumors (especially chemotherapeutic medi-cines), traditional Chinese medicines used for treatment or adjuvant treatment have shown therapeutic potential


because of their multi-targets, safety, and high efficiency. In clinics, we propose that the antitumor traditional Chi-nese medicines with different targets be selected perti-nently and relevantly, according to the types and level of tumor immunosuppression combined with the mecha-nisms and the development of the tumor. Meanwhile, it should match treatment with the pattern of reversing ef-fect on immunosuppression of the tumor patients, which aligns with the idea of an individualized and combined multitarget treatment design. It is expected to avoid the blindness on the usage of antitumor traditional Chinese medicines and to elevate the therapeutic efficiency.

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B-cell specific moloney leukemia virus insertion site 1 gene (Bmi-1) which belongs to the Polycomb gene family plays an important role in the growth and proliferation of cells as well as the self-renewal of stem cells. Studies have confirmed that it is abnormally expressed in mul-tiple human tumors such as leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, etc, and is associated with tumor metastasis and extension [1–3]. It has also been demonstrated that Bmi-1 is essential for the self-renewal of leukemia hematopoietic stem cells and the prolifera-tion of stem/progenitor cells [4, 5]. Chronic granulocytic leukemia (CGL) is a kind of malignant proliferating dis-ease which is derived from marrow pluripotent hemato-poietic stem cells. Over-expression of Bmi-1 gene in CGL has been largely reported [6]. Then, whether Bmi-1 can be used as a target in treatment of leukemia requires further exploration. Therefore, in the present study, the effects of

Bmi-1 on the proliferation, apoptosis and aging of T cells were observed through inhibiting Bmi-1 expression in T Lymphocytic leukemia Jurkat cells via RNAi technology.


Main reagentsThe pRNAT-U6.2 was purchased from GeneScript

(USA). BglII, HindIII and T4 DNA ligase were purchased from Promega (USA). LipofectAmineTM2000, G418, Trizol and RT-PCR Kit were purchased from Invitrogen (USA). Bmi-1 (sc-10745) and β-actin antibodies were purchased from Santa Cruz, USA. T Lymphocytic leukemia Jurkat cell line was purchased from the cell bank of Chinese Academy of Science in Shanghai. RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco (USA). The β-galactosidase staining kit was from Cell Biolabs, USA.

Down-regulation of Bmi-1 by RNA interference in Jurkat cellsShangen Zheng1, Qibin Jing2, Yaqiong Zheng1, Yinjuan Ding1, Qianchuan Huang1, Guoqiang Zhao3

1 Department of Clinical Transfusion, Wuhan General Hospital of Guangzhou Millitary Command, Wuhan 430070, China2 Health Care Agency of Air Force Early Institute , Wuhan 430019, China3 Department of Microbiology, Basic Medical Science School, Zhengzhou University, Zhengzhou 450000, China

Received: 1 August 2012 / Revised: 28 September 2012 / Accepted: 25 December 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of our study was to investigate the effects of down-regulation Bmi-1 by RNA interference (RNAi) in T Lymphocytic leukemia Jurkat cells. Methods: Two complementary oligonucleotide strands were synthesized based on the siRNA sequence targeting Bmi-1 gene. After annealing, siRNA strands were recombined into the pRNAT-U6.2 vector, and then DNA sequencing was carried out following transformation and amplification. The recombinant was transfected into Jurkat cells with liposomes. Positive colonies were obtained through G418 selection. The mRNA and protein expressions of Bmi-1 were detected by RT-PCR and Western-blot, respectively. Effects of Bmi-1 silence on cell proliferation, cell cycle and cell aging of Jurkat cells were detected by MTT assay, flow cytometry, colony formation assay and SA-β-Gal staining, respectively. Results: The siRNA recombinant targeting Bmi-1 gene was successfully constructed. All three siRNA recombinants could significantly inhibit the expression of Bmi-1. The siRNA targeting 825nt-843nt (GACCAGACCACTACT GAAT) has the strongest inhibitory effect on Bmi-1 expression, with almost complete inhibition on Bmi-1 mRNA and protein expressions. Compared with the non-transfection group and the empty vector group, growth velocity and colony formation ability were significantly decreased, while the proportion of cells in G1 phase and the percentage of senile cells were signifi-cantly increased in highly transfected group (P < 0.05). Conclusion: Down-regulation Bmi-1 by RNA interference (RNAi) could significantly inhibit the growth of Jurkat cells in vitro.

Key words Bmi-1; siRNA; Jurkat cells

Correspondence to: Shanggen Zheng. Email: [email protected]


Screening of siRNA sequence targeting Bmi-1Human Bmi-1 gene sequence (NM_005180) was

scanned and siRNA sequences were designed by the system of http://www.ambion.com/techlib/misc/siRNA_tools.html (Ambion). Three siRNA sequences with 19 ba-sic radicals including 808nt-826nt (GATGTCCAAGTTCA CAAGA), 825nt-843nt (GACCAGACCACTACTGAAT) and 1227nt-1245nt (GGAGGAACCTTTAAAGGAT) were eventually designed after BLAST homology analysis. Three pairs of hairpin-like DNA oligonucleotide single strands (808F and 808R; 825F and 825R; 1227F and 1227R) were synthesized, respectively. BamHI and XhoI restric-tion enzymes were affiliated on the two terminals. The siRNAs were synthesized by Shanghai Sangon Biological Engineering Technology＆Sevices Co, Ltd. (China).

808F 5’-GATCC GATGTCCAAGTTCACAAGA TTC AAGAGA TCTTGTGAACTTGGACATC TTTTTT C-3’; 808R 5’-TCGAG AAAAAA GATGTCCAAGTTCACAA GA TCTCTTGAA TCTTGTGAACTTGGACATC G-3’; 825F 5’-GATCC GACCAGACCACTACTGAAT TTCA AGAGA ATTCAGTAGTGGTCTGGTC TTTTTT C-3’; 825R 5’-TCGAG AAAAAA GACCAGACCACTACT GAAT TCTCTTGAA ATTCAGTAGTGGTCTGGTC G-3’; 1227F 5’-GATCC GGAGGAACCTTTAAAGGAT TTCA AGAGA ATCCTTTAAAGGTTCCTCC TTTTTT C-3’; 1227R 5’-TCGAG AAAAAA GGAGGAACCTTTAAAGG AT TCTCTTGAA ATCCTTTAAAGGTTCCTCC G-3’.

Construction of siRNA recombinant targeting Bmi-1

Double strands DNAs (siBmi808, siBmi825 and siB-mi1227) were generated from three pairs of hairpin-like DNA oligonucleotide single strands (808F and 808R; 825F and 825R; 1227F and 1227R). Then the DNAs were li-gated with the pRNAT-U6.2 vector. The ligation sys-tem including 5 μL of ligation buffer, 1 μL of stick linear pRNAT-U6.2 vector, 1 μL of T4 ligase, 3 μL of annealing products (siBmi808, siBmi825 and siBmi1227) reacted at 4 ℃ overnight. The ligation product was transformed into the competence DH5α, and single colonies were selected for plasmid preparation and DNA sequence analysis. The siRNA recombinants targeting Bmi-1 including pRNAT-U6.2-siBmi808, pRNAT-U6.2-siBmi825 and pRNAT-U6.2-siBmi1227 were obtained.

Cell transfection and screeningJurkat cells were maintained in the RPMI-1640 me-

dium containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin in 37 ℃ incubator with 5% CO2. Cells in log phase were washed with serum-free and an-tibiotic-free RPMI 1640 medium for three times. Trans-fection was carried out according to the instruction of LipofectamineTM2000. Cells which were transfected with pRNAT-U6.2-si Bmi 808, pRNAT-U6.2-si Bmi 825 and

pRNAT-U6.2-siBmi 1227 served as group 1, 2 and 3. Cells which were transfected with the pRNAT-U6.2 empty vec-tor served as the control group, and Jurkat cells without transfection served as the blank control. Six hours after transfection, cells were continuously maintained in RPMI 1640 medium containing 10% FBS for 48 h. The medium was replaced by the RPMI 1640 medium containing 800 μg/mL G418 and 10% FBS. After all the cells in the con-trol group died, the concentration of G418 was changed to 300 μg/mL for consecutive incubation for 2 weeks. The resistant colonies were obtained for detection.

RT-PCRTotal RNA was extracted with Trizol. cDNA was syn-

thesized from RNA according to the instruction. The primers for Bmi-1 were shown as follows: P1 5’-GGAGA CCAGCAAGTATTGTCC-3’; P2 5’-GACCATTCCTTCTC CAGGTAT-3’. The size of the fragment was 517 bp. β-actin served as the inner control. The proliferation prod-uct was separated with 1.5% agarose gel containing 0.5 μg/mL EB. The gray scale ratio of Bmi-1 to β-actin was calculated as the relative expression of Bmi-1.

Western-blot Whole cell lysate was prepared with the sonication

method, and the protein concentration was detected. The whole cell lysate was separated on SDS-PAGE and transferred onto a PVDF membrane with electro-trans-membrane method. The membrane was blocked with 5% nonfat dried milk for 6 h, and then probed with 1:200 polyclonal rabbit anti-human (Bmi-1) antibody at 4 ℃ overnight. Then, the membrane was incubated with 1 : 1000 goat anti-rabbit IgG second antibody for 1 h. Bmi-1 protein band on PVDF membrane was visualized with the luminescent technique.

MTT assayCells in log phase were collected and seeded at 104

cells/well in a 96-well plate, with 200 μL medium in each well. The cells were incubated at a 37 ℃ incubator with 5% CO2 for 1–5 days. Twenty μL of MTT solution (5 mg/mL) was added into each well. After 4 hours of incubation at 37 ℃, cell medium was removed and 150 μL of DMSO was added into each well. The crystal was fully dissolved through 10 min shaking. The absorbance readings (A) for each well were carried out at 570 nm using a microplate reader. The growth curve was drawn with the time as the abscissa axis and the A value as the longitudinal axis.

Colony formation assayCells were divided into five groups. 1 × 103 cells were

seeded onto 4 g/L agarose gel for 2 weeks of incubation. After incubation, cells were fixed with methanol and stained with Giemsa. Cell colonies were counted under


light microscope.

Cell cycle detection 1 × 106 cells were washed with PBS twice and then

fixed with 70% ethanol. Cells were stained with 1 mL PI (100 mg/L), and detected by flow cytometry.

Aging cell stainingThe transfected cells in each group were seeded into

a 6-well plate. Twenty-four hours after cell adherence, cells were stained with cell aging β-galactosidase staining kit (Cell Biolabs, USA) according to the instruction. Five fields of vision were randomly selected for photograph, and the percentage of aging cells under each field of vi-sion was counted.

Statistical analysisAll data were analyzed with SPSS 13.0 software. Stu-

dent t test was adopted for the comparison of matched groups, with α = 0.05 as the standard of test.

Results

Construction of siRNA recombinant targeting Bmi-1

Three annealing products of hairpin-like single strand DNA (siBmi808, siBmi825 and siBmi1227) were ligated with pRNAT-U6.2 double sticking plasmids. The ligation product was transformed into DH5α, and then spread on the Ampicillin-LB plate. Single colonies were selected, and DNA sequencing was performed. The positive recom-binants with correct sequences (pRNAT-U6.2-siBmi808, pRNAT-U6.2-siBmi825 and pRNAT-U6.2-siBmi1227) were obtained (Fig. 1).

Inhibition of Bmi-1 expression detected by RT-PCR and Western-blot

The mRNA and protein expressions of Bmi-1 were in-hibited in Jurket cells of Group 1, 2 and 3, which were transfected with the pRNAT-U6.2-siBmi808, pRNAT-U6.2-siBmi825 and pRNAT-U6.2 -siBmi1227, respective-ly. Group 2 had the highest inhibitory effect. There was high Bmi-1 expression at mRNA and protein levels in two control groups (empty vector group and blank control group), suggesting that the constructed siRNA could ef-fectively inhibit Bmi-1 expression in Jurket cells (Fig. 2).

MTT assayDuring the first 1–5 days, the OD values of cells in the

empty vector group and the blank control group were not significantly different as compared with group 1 and group 3 (P > 0.05). OD values of cells at the 4th day and 5th day in group 2 were significantly decreased as com-pared with the two control groups (P < 0.05), suggesting

that high inhibition of Bmi-1 in Jurkat cells could inhibit the growth velocity of the cells (Fig. 3).

Colony formation assayThe average cell colonies 14 days after culture were

seen in Table 1. The average cell colonies in group 2 were significantly decreased as compared with two control groups (empty vector group and blank control group) (P < 0.05), while the average cell colonies in Group and 3 were not significantly different as compared with two control groups (Fig. 4), suggesting that high inhibition of Bmi-1 in Jurkat cells could decrease the colony formation ability.

Aging cells stainingThe results of aging cells staining were shown in Table

1. The average cell aging rates of group 1, 2 and 3 were significantly increased as compared with two control groups (P < 0.05), with the highest average cell aging rate in group 2, suggesting that inhibitory Bmi-1 expression could increase the aging of Jurkat cells, and the cell aging was associated with the inhibition level of Bmi-1.

Cell cycle assayThe cell cycle data were shown in Table 2. The results

showed that the proportion of G1 cells in three experi-mental groups was significantly increased as compared with the control group, while the proportion of cells in S phase was significantly decreased (P < 0.05).

Discussion

Human Bmi-1 gene which locates in 10p11.23 consists of 10 exons, encoding a nuclear protein containing 326 amino acids. Jacobs et al confirmed that INK4a/ARF was the downstream regulatory site of Bmi-1 gene, which was directly and negatively regulated by Bmi-1, thereby af-fecting cell proliferation and aging in vitro and in vivo [7]. It has been shown that the hematopoietic stem cells of Bmi-1 mouse embryonic stem cells were normal, while the hematopoietic stem cells of Bmi-1 mouse embryon-ic stem cells after birth were significantly reduced. The transplanted embryonic stem cells and bone marrow from Bmi-1-/- mouse can only transiently maintain the hema-topoietic function. No self-renewal of hematopoietic stem cells was detected in Bmi-1 mice [8], which suggests that Bmi-1 is essential for maintenance of self-renewal of he-matopoietic stem cells. In acute myeloid leukemia (AML), a small group of tumor blastoid cells are considered as leu-kemia stem cells which have self-renewal ability, and can start and maintain a long-term number of leukemia cells [9]. Lessard et al discovered that the number of Bmi-1-/- mouse leukemic cells was significantly decreased after 10 days of culture, and the cells arrested in G1 phase, and the


apoptotic cells were increased, suggesting that Bmi-1 had a decisive effect on the on the proliferation and leukemo-genic ability of leukemia stem cells [5].

Therefore, whether Bmi-1 can be used as a target for the inhibition of leukemic cells is still controversial. It has been demonstrated that the interference of Bmi-1 gene could inhibit the growth of leukemia cells, while others showed ineffective results. However, in the pres-ent study, three siRNA recombinants targeting Bmi-1 (pRNAT-U6.2-siBmi808, pRNAT-U6.2-siBmi825 and Fig. 4 Comparison of average cell colonies among groups. * P < 0.05

Fig. 2 Bmi-1 expressions detected by RT-PCR and Western-blot. (a) RT-PCR; (b) Western-blot. Cells in group 1–3 were transfected with pRNAT-U6.2-siBmi808, pRNAT-U6.2-siBmi825 and pRNAT-U6.2-siB-mi1227. Cells in group 4 were transfected with pRNAT-U6.2 empty vector. Group 5 was the blank control group; (c) Comparison of Bmi-1 mRNA expressions in five groups. Compared with two control groups, Bmi-1 ex-pressions in group 1 and group 3 were significantly decreased (* P < 0.05), and Bmi-1 expression in group 2 was highly significantly decreased (** P < 0.01)

Fig. 3 Cell growth curve in each group

Fig. 1 Sequencing results of recombinant. (a) pRNAT-U6.2-siBmi808; (b) pRNAT-U6.2-siBmi825; (c) pRNAT-U6.2-siBmi1227. The sequences of three recombinants were completely consistent with the corresponding designed sequences


pRNAT-U6.2-siBmi1227) were successfully constructed. Among them, pRNAT-U6.2-siBmi825 showed the stron-gest inhibitory effect which can almost completely inhib-it Bmi-1 expression in transfected Jurkat cells. MTT assay and colony formation assay showed that low inhibition of Bmi-1 expression in Jurkat cells (experimental group 1, experimental group 3) has no significantly effect on the cell growth rate and colony formation ability, while high inhibition of Bmi-1 expression in Jurkat cells (experimen-tal group 2) could significantly inhibit the cell growth rate and colony formation ability. Cell aging staining and flow cytometry experiments showed that all different levels inhibition of Bmi-1 in Jurkat cells could increase the pro-portion of G1 phase cells and decrease the proportion of S phase cells, and increase cell aging. Moreover, the cell aging rate was associated with and inhibition degree of Bmi-1 gene. Therefore, we considered that there maybe existed two reasons resulting in the inconsistent results: (1) different cell lines; (2) different Bmi-1 expression lev-els of the interfered cells.

In conclusion, siRNA recombinant (pRNAT-U6.2-siBmi825) targeting the 825nt-843nt (GACCAGACCAC

TACTGAAT) sequence of Bmi-1 which has high silence effect on Bmi-1 expression of Jurkat cells was success-fully constructed in our study. The results showed that significant inhibition of Bmi-1 expression could suppress cell growth and colony formation ability of Jurkat cells. Moreover, both the proportion of cells in G1 phase and the percentage of the aging cells were significantly in-creased.

References

Jiang L, Li J, Song L. Bmi-1, stem cells and cancer. Acta Biochim Biophys Sin (Shanghai), 2009, 41: 527–534. Raaphorst FM. Self-renewal of hematopoietic and leukemic stem cells: a central role for the Polycomb-group gene Bmi-1. Trends Im-munol, 2003, 24: 522–524. Park IK, Morrison SJ, Clarke MF. Bmi1, stem cells, and senescence regulation. J Clin Invest, 2004, 113: 175–179. Park IK, He Y, Lin F, et al. Differential gene expression profiling of adult murine hematopoietic stem cells. Blood, 2002, 99: 488–498. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature, 2003, 423: 255–260. Guney I, Wu S, Sedivy JM. Reduced c-Myc signaling triggers telo-mere-independent senescence by regulating Bmi-1 and p16 (INK4a). Proc Natl Acad Sci U S A, 2006, 103: 3645–3650. Jacobs JJ, Kieboom K, Marino S, et al. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature, 1999, 397: 164–168.Park IK, Qian D, Kiel M, et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature, 2003, 423: 302–305. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med, 1997, 3: 730–737.

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Table 1 Results of colony formation assay and aging cells staining

Groups Plasmid n Colony formation assayAverage colonies (piece)

Aging cells staining Average cell aging rate in the field of vision (%)

1 pRNAT-U6.2-siBmi808 6 71.8 ± 8.7 17.3 ± 2.1*2 pRNAT-U6.2-siBmi825 6 48.3 ± 6.1* 22.5 ± 4.3**3 pRNAT-U6.2-siBmi1227 6 73.7 ± 9.8 19.3 ± 2.8*Empty vector pRNAT-U6.2 6 81.9 ± 12.5 13.1 ± 1.7Blank control – 5 87.2 ± 13.7 11.9 ± 1.1* Compared with two control groups, with significant difference (P < 0.05); ** Compared with two control groups, with highly significant difference (P < 0.01)

Table 2 Cell cycle distribution of each group (%, χ ± s)Groups G0/G1 S G2/M1 60.2 ± 4.3* 32.9 ± 2.1* 6.9 ± 0.42 62.1 ± 4.1* 31.4 ± 2.4* 6.5 ± 0.43 58.2 ± 3.7* 32.6 ± 2.1* 9.2 ± 0.6Empty vector 48.5 ± 3.5 42.1 ± 2.8 9.4 ± 0.5Blank control 47.9 ± 3.1 44.5 ± 2.7 7.6 ± 0.5* Compared with two control groups, with significant difference (P < 0.05)


A dynamic equilibrium of histone acetyltransferase (HAT) and histone deacetylase (HDAC) functions con-trol the level of acetylated histones in nuclear chroma-tin. Removal of the acetyl group from histones by histone deacetylases causes chromatin compaction and inhibi-tion of gene expression [1]. While a microbial metabolite, trichostatin A (TSA), is a potent reversible inhibitor of mammalian histone deacetylases [2–10]. It can inhibit can-cer cell growth in vitro and in vivo, reverse oncogene-transformed cell morphology, and enhance cell differen-tiation [11].

Nowadays, the pharmacodynamic study about TSA focuses on the same density of cultured cells, which has been to a higher level. However, the study of TSA acting on cultured cell of different densities was not reported. In this paper, we would discover that the therapeutical effect was significantly different when TSA was used to treat cultured cell of different densities.

Now, many researchers apply spectral technique to study the biologic molecular or cellular state, which re-flect the change of molecular configuration and compo-nent in cells. And then, researchers could find out what

arouses spectrum change through spectrum analysis [12].In this work, 0.2 µM TSA was applied to act on human

cervical carcinoma cells (HeLa cells) of different densi-ties. Then, the cycle arrest effect and UV absorption spec-trum of cell were investigated, which provided support to analyze the effect of TSA on cancer cell. At the same time, this study would discover some important relation-ship between cycle arrest effect and UV absorption spec-trum of cell.


Cell culture and TSA treatment HeLa cells grew in Roswell Park Memorial Institute

(RPMI) 1640 culture medium containing 10% (v/v) fetal calf serum and were incubated at 37 ℃ in a humidified atmosphere of 5% CO2.

The abbreviation of Trichostatin A (Sigma, USA) is TSA. TSA was dissolved in dimethyl sulphoxide (DMSO) to 50 µM and stored at –20 ℃. When the experiment be-gan, the culture medium was replaced with culture me-dium containing TSA (0.2 µM) [13].

To evaluate the effects of the TSA on cancer cell, cells were planted in 50 mL tissue culture flasks (surface area 25.0 cm2). Three groups of A, B, C were arranged before experiment, 16 × 104, 8 × 104, 4 × 104 /mL HeLa cells were

Cell-cycle-dependent variation in UV absorption spectrum of Hela cells treated with Trichostatin A*Fengqiu Zhang1, Xiaoxia Wang1, Zhanguo Yang2

1 Physical Engineering College, Zhengzhou University, Zhengzhou 450052, China2 Henan Province Institute of Metrology, Zhengzhou 450008, China

Received: 19 July 2012 / Revised: 7 September 2012 / Accepted: 25 October 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of our study was to discovery the different cell cycle arrest effect after different densities HeLa cells treated with Trichostatin A (TSA). In addition, this study would find some important relationship between cycle arrest effect and UV absorption spectrum of cell. Methods: 0.2 µM TSA was applied to act on HeLa cells of different density. Then, the cycle arrest effect and UV absorption spectrum of cells were investigated, which provide support to analyze the effect of TSA on cancer cells. Results: Cell cycle arrest effect in G0/G1 of the lower density cells was more obvious than that in other groups. The other discovery in this work was that the cellular UV absorption value was higher when the density of cultured cell was lower. Conclusion: This experiment would guide the clinical study on early or late stage cancer patients in the future. On the other hand, this work indicates when cells were arrested in G0/G1 phase, the cellular absorption value increased at the same time, so UV absorption spectrum could characterize the change of cell cycle.

Key words Trichostatin A; HeLa cells; cell cycle; UV absorption spectrum

Correspondence to: Fengqiu Zhang. Email: [email protected]* Supported by a grant from the Education Office of Henan Province in China (No. 12A140013).


planted to each group respectively. In this work, each group was repeated for three times. After 24 h, the cul-ture medium was replaced with culture medium contain-ing TSA (0.2 µM). Then, cells in each group would be di-gested and counted after incubation for additional 24 h.

Cell cycle analysisHeLa cells treated with TSA were harvested by tryp-

sin, and washed twice with PBS. The cells were fixed overnight with 70% cold ethanol, and then stained with PI solution consisting of 45 µg/mL PI, 10 µg/mL RNase A, and 0.1% Triton X-100. After one-hour incubation at room temperature in the dark, the cells were detected by a FACSVantage SE flow cytometer (equipped with a 488-nm argon laser), and the data was analyzed on ModFit software [14].

Spectrophotometer and experimental conditions

Japanese SHIMADZU spectrophotometer (UV3101), deuterium lamp stimulated, and scanned range 190 nm–400 nm. The control pool and sample pool were both made of quartz, which were implanted 3 mL PBS to cali-brate baseline. Then, the control pool was not moved, the harvested cells were transfered in PBS to 5 × 104/mL, 3 mL cellular liquid was implanted in sample pool for spec-trum analysis.

Statistical analysisData were compared each other using the two-tailed

t-test, A P value less than 0.05 is considered statistically significant. Data were expressed as means ± SE derived from three independent experiments.

Results

Proliferation inhibitory effect of HeLa cells treated with TSA

The cells treated with TSA were harvested by trypsin, and then those cells were counted. Time was marked as the horizontal axis, and inhibitory rate of cellular pro-liferation was marked as longitudinal axis to plot graph with origin software. Inhibitory rate = (1 – Cell number in the experimental group / Cell number in the control group) × 100%.

The results showed the inhibitory rate was distinct when the density of cultured cells was different (Fig. 1). The inhibitory rate of cellular proliferation was higher when the density of cells was lower.

TSA induces cell cycle arrest The somatic cell cycle was divided into an interphase,

designated for cellular growth and DNA synthesis, and a mitotic phase, in which a single cell divides into two

daughter cells. Interphase was further subdivided into two gap phases (G1 and G2) separated by a phase of DNA synthesis (S phase) [15].

Experimental results indicated that TSA could induced G0/G1 phase arrest and concomitant decrease in the number of cells in S phase when the density of cultured cell was lower (Fig. 2). Moreover, the difference was sta-tistically significant（P < 0.05）between C group and A group, B group. Namely, this effect of cell cycle arrest was better when the cellular density was lower.

TSA induced absorption spectrum change of HeLa cells

Fig. 3 was the absorption spectrum graph of different density cells treated with TSA for 24 h. There were char-acteristic absorption peak at 202 nm and 275 nm nearby respectively. The cell number was 5 × 104/mL in each sample pool when the cell spectrum was selected, the ab-sorption intensity changed clearly, which was regular re-markably. The cellular absorption value increased when the density of cultured cells was lower.

Fig. 3 showed the absorption value of cells in C group was greater than that in A group or B group. Moreover, the difference was statistically significant (P < 0.05) at 202 nm nearby (Fig. 4). The absorption value of cells in C group was still greater than that in A or B group at 275 nm nearby, but the difference was not obvious (Fig. 5).

Discussion

Cancer is the cell-cycle disease. Invariably, compo-nents of the cell cycle are deregulated in cancer result-ing in uncontrolled cellular proliferation. The cell cycle consists of four distinct stages: two gap-phases (G1 and G2), where RNA and protein syntheses occur, an S-phase, where DNA synthesis and replication occur, and an M-phase, where the cell undergoes mitosis and divides into two daughter cells [16].

At a checkpoint in G1, these signals culminate in a mo-lecular mechanism that allows only a binary decision–to either commit to the mitotic cell cycle and enter S-phase for regular DNA replication, or to undergo cycle arrest [17, 18].

In this work, TSA (0.2 µM) was applied to act on cul-tured cells of different densities. Results indicated that TSA had the inhibitory effect of cell proliferation and arrested cell cycle in G0/G1, cell number in S decreased (Fig. 1, 2). In addition, the effect of cell cycle arrest in the lower density group was more obvious than that in other groups.

The other discovery in this work was that cellular ab-sorption spectrum changed in different groups, and the cellular absorption value was higher when the density of cultured cell was lower (Fig. 3), especially at C group.


Through further analysis, we could find the difference was statistically significant (P < 0.05) at 202 nm nearby (Fig. 4), but the difference was not obvious at 275 nm nearby (Fig. 5).

The absorption value at 202 nm and 275 nm should root from aromatic amino acid. The absorption value at 202 nm is the characteristic spectral band (ethylenic

band), and it has large absorption intensity, it includes E1 band and E2 band. The absorption value at 275 nm should be benzenoid band (B band). Both of the B band and E band is the spectral band of aromatic compounds, but the spectral peak at 275 nm was faintish [19].

The cellular absorption spectrum reflects the change of biomolecules in cell. In this study, the change trend of cellular spectrum was same with the change of cell cycle. The effect of cell cycle arrest was more obvious when the density of cultured cell was lower. Similarly, cellu-

Fig. 1 Proliferation inhibitory rate of HeLa cells after treated for 24 h with 0.2 µM TSA. A, B, C groups were arranged before experiment, 16 × 104, 8 × 104, 4 × 104/mL HeLa cells were planted to each group respec-tively

Fig. 2 Cell cycle analysis of HeLa cells after treated for 24 h with 0.2 µM TSA. * P < 0.05; A, B, C groups were arranged before experiment, 16 × 104, 8 × 104, 4 × 104/mL HeLa cells were planted to each group respectively

Fig. 3 The UV absorption spectrum of different density cells treated with 0.2 µM TSA for 24 h

Fig. 4 The absorption value of different density cells at 202 nm nearby. * P < 0.05; A, B, C groups were arranged before experiment, 16 × 104, 8 × 104, 4 × 104/mL HeLa cells were planted to each group respectively

Fig. 5 The absorption value of different density cells at 275 nm nearby. A, B, C groups were arranged before experiment, 16 × 104, 8 × 104, 4 × 104/mL HeLa cells were planted to each group respectively


lar absorption intensity was larger when the density of cultured cell was lower. Those results indicate that UV spectral technique could characterize the change of cell cycle. When cells were arrested in G0/G1 phase, the cel-lular absorption value increased too.

We know cellular replication is regulated in an orderly fashion from G1 to S. The G0/G1 phase is very important in cellular proliferation, it is the prophase of DNA syn-thesis, and it prepares for DNA synthesis. The metabolism in G0/G1 phase is active, RNA and protein would synthe-size in this phase. In this study, TSA inhibited cellular proliferation with the manner of G0/G1 arrest, so cells could not enter S phase to synthesize DNA. The RNA and protein synthesis in G0/G1 phase were characterized by absorption spectrum. Protein was increasing; aromatic amino acid was increasing too, absorption intensity was larger and larger accordingly.

In this study, we could find there were precise fit be-tween cycle arrest and absorption spectrum analysis. Cel-lular cycle analysis is the common method in pharmaco-dynamic study, but there are many unfavorable factors in experiment. Firstly, time of cycle analysis is longer, and operating is more troublesome; secondly, the reagent of cycle analysis should be purchased; lastly, this cycle analysis depends on flow cytometry, which exists only in laboratories with better condition. In another word, not all researchers are able to carry out cellular cycle analysis. However, spectrophotometer is the common analytical instrument, which exists in almost all the laboratories. Researchers need not buy any operating reagent, and UV absorption spectrum could be carried out immediately after cells are harvested. So we are looking forward the absorption spectrum to be a new path of pharmacody-namic study in the future, which could save lots of time and money.

The other important contribution of this experiment was that the effect was very different when TSA was used to act on different density cells. So this experiment would guide the clinical study on early or late stage cancer pa-tients in the future.

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From October 12th–14th, 2012, the 8th Conference of Chinese Cancer Rehabilitation and Palliative Care was held in Qingdao. As a part of World Hospice and Pallia-tive Care Day 2012 in China, the conference focused on advanced cancer patients’ quality of life and the multi-disciplinary cooperation to improve their survival.

The conference was hosted by Cancer Reha-bilitation and Palliative Care Committee, China (CRPC), and jointly un-dertaken by Shandong Cancer Rehabilitation and Palliative Care Com-mittee, cancer branch of Qingdao Medical Associ-ation and Affiliated Hos-pital of Medical College, Qingdao University. More than 1000 cancer experts and clinicians from Mainland China, Hongkong and Taiwan regions, USA, England and Belgium, participated in the conference. The topics of the conference covered widely, including cancer pain, supportive care, Chinese tradition-al medicine, nursery, social work and clinical research.

Professor Jinming Yu, a member of the Chinese Acade-my and the director of Shandong Cancer Hospital, Profes-sor Ying Wang, the deputy secretary-general of Chinese Anti-Cancer Association, Professor Shiying Yu, the chair-men of CRPC, Professor Xingsheng Wang, the director of Affiliated Hospital of Medical College, Qingdao Univer-sity, Mayor Xin Luan from Qingdao Government, took part in the opening ceremony. The ceremony was hosted by Professor Jun Liang, the director of Cancer Center, Af-filiated Hospital of Medical College, Qingdao University.

The highlights of the opening cer-emony focused on the three excellent speeches given by foreign experts. Pro-fessor Sheila Payne, the chairman of European Association of Palliative Care (EAPC), gave a speech titled as “Infor-mation and palliative care needs for pa-

tients with advanced cancer”. Her speech included four parts: introduction to EAPC; the core factors of palliative care; the education frame of palliative care; the practice of palliative care. Professor Bart Morlion, the chair-man of Belgian branch of International Association for the Study Pain (IASP), gave a speech titled as “trends in cancer pain management WHO stepladder: a modern vi-sion”. His speeches include six parts: cancer pain; the gen-eral points; WHO stepladder; critical evaluation; the new concepts of guidelines; the titration of opioid drugs; the advantages of oxycodone and the multi-model strategy. Professor Xin Wang, from MD Anderson, gave a speech on “Clinical & Translational Research on Symptom and Palliative Care: Training and Application”. Her speeches included four parts: pragmatic training realities faced by academic clinical investigators in the United States; ap-

Treat advanced cancer patient with care, strengthen multi-disciplinary cooperation–The 8th Conference of Chinese Cancer Rehabilitation and Palliative Care was held in Qingdao

Author/ Photo: Yi Cheng, from Tongji Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Received: 6 November 2012 / Revised: 20 November 2012 / Accepted: 25 November 2012© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Activities Report


method of palliative care–psychodrama. It allowed pa-tients and their relatives put themselves in other’s posi-tion, experience other’s emotion and feeling, and thus improve behavior and habits.

At the end of the conference, 10 excellent papers were elected out. And Professor Shukui Qin, the secretary of CSCO, received the flag of CRPC. The 9th conference would be hold in Nanjing, 2013.

proaches for faculty to successfully conduct clinical or translational studies in symptom control and palliative care; example from symptom studies; encouragement to participate in initiatives in the area of symptom and pal-liative care research.

Further information and concepts about rehabilitation and palliative care revealed. Twenty-four more speeches draw participants’ attention. Especially, Professor Jinlin You, from Taiwan Nanhua University, showed a new


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1. Arms and scope Chinese-German Journal of Clinical Oncology is an international professional academic periodical on oncology, being co-edited by China and Germany. The Journal, with the authors from around world, es-pecially from China, is dominated in introducing the clinical experience of diagnosis and treatment as well as leading scientific research achievement in the tumor domain, in addition to report basic theory researches that help instruct the clinical practice of oncology and closely connect with the discipline. Manuscripts submitted for publication must contain a statement to the effect that all human studies have been reviewed by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in an appropriate version of the 1964 Declaration of Helsinki. It should also be stated clearly in the text that all persons gave their informed consent prior to their inclusion in the study. Details that might disclose the identity of the subjects under study should be omitted. The editors reserve the right to reject manuscripts that do not comply with the above-mentioned require-ments. The author will be held responsible for false statements or for failure to fulfill the above-mentioned requirements.

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5. Science communicationManaging directors: DONG Weiguo, XIA JunTongji Hospital, Jie Fang Da Dao 1095430030 Wuhan, ChinaTel.: +86-27-83662630, Fax: +86-27-83662645Email: [email protected]

6. ProductionEditorial Office of Chinese-German Journal of Clinical Oncology430030 Wuhan, ChinaAddress for courier, express, and registered mail:Jie Fang Da Dao 1095430030 Wuhan, ChinaTel.: +86-27-83662630, Fax: +86-27-83662645Email: [email protected]: Editorial Office of Chinese-German Journal of Clinical OncologyPrinter: Changjiang Spatial Information Technology Engineering Co., Ltd. (Wuhan) Hangce Information Cartorgraphy Printing Filial, Wuhan, ChinaPrinted in People’s Republic of China

Ownership and copyright© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg

名誉主编

W.-W.Höpker 吴孟超孙燕

主编

陈安民于世英 Anthony D.Ho

常务副主编

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副主编

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目次

683 DNA修复基因BRCA1和DNA-PKcs在放射治疗中有很大的

潜能

689 肿瘤转移的几种理论模型及其意义

691 MicroRNA在人类结肠癌中的研究进展

694 SPECT/CT同机融合显像和CT在估测口腔恶性肿瘤侵犯

下颌骨范围的应用价值

699 408例70岁以上老年肺癌患者的预后因素分析

705 STAT3、CEA在人肺腺癌细胞A594中的相关性研究

710 光子束和电子束在早期乳腺癌患者放疗瘤床加量计划的

剂量学研究

716 乳腺癌患者外周血中SBEM和hMAM的个体化检测意义

721 抗瘤中药制剂对结直肠癌T及NK细胞免疫抑制的体外逆转

作用

732 RNAi抑制Bmi-1基因表达对Jurkat细胞的影响

737 HeLa细胞经曲古菌素A处理后紫外光谱的细胞周期依赖性

变化

741 关注晚期患者，强化多学科协作

—青岛举办第八届中国癌症康复与姑息医学大会

743 本刊投稿须知

704 本刊2013年征稿启事

本期责任编辑王叶宁夏军陈静吴强本期排版编辑王文革

中德临床肿瘤学杂志Chinese-German Journal of Clinical Oncology

月刊 1984年9月创刊 2002年3月改刊第11卷第12期 2012年12月25日出版

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