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Chinese Journal of Natural Medicines 2014, 12(8): 0607−0612doi: 10.3724/SP.J.1009.2014.00607

Chinese Journal of Natural Medicines

Inhibitory activities of Lignum Sappan extractives on growth and growth-related signaling of tumor cells

ZHANG Qing, LIU Jing-Li, QI Xiao-Man, QI Chun-Ting, YU Qiang*

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

Available online 20 August 2014

[ABSTRACT] AIM: To investigate the active constituents of Lignum Sappan (Caesalpinia sappan L.) on growth-related signaling and cell mitosis. METHOD: The influence of the ethyl acetate (EtOAc) extract of Lignum Sappan and its constituents on growth-related signaling were evaluated by a luciferase assay in cells stably- transfected with NF-κB, STAT1, or STAT3 responsive luciferase reporter plas-mid. The inhibitory effect on the cell cycle was determined by flow cytometric analysis. The anti-tumor activities were assessed in vitro and in vivo. RESULTS: The EtOAc extract of Lignum Sappan had inhibitory activities on growth-related signaling and cell mitosis. Three major active compounds were sappanchalcone, brazilin, and butein. Sappanchalcone blocked cell cycle progression in the G2/M phase, brazilin inhibited TNFα/NF-κB signaling, while butein inhibited IL-6/STAT3 signaling, as well as TNFα/NF-κB signaling. The three compounds all demonstrated cytotoxic activities against human tumor cells in vitro. In a S180 tumor cell-bearing mice model, the anti-tumor efficacy of the EtOAc extract was better than the individual compounds acting alone. CONCLUSION: These results indicate that Lignum Sappan contains multiple active compounds with different antitumor activi-ties, which act synergistically to enhance their anti-tumor effects. The EtOAc extract of Lignum Sappan may be better than indi-vidual active constituent as a novel medicine for the treatment of cancer.

[KEY WORDS] Caesalpinia sappan L.; Lignum Sappan; Anti-tumor activity; Cell cycle blocker; Signaling pathway inhibitor; Syn-ergy

[CLC Number] R965 [Document code] A [Article ID] 2095-6975(2014)08-0607-06

Introduction

Lignum Sappan is derived from the heartwood of Caes-alpinia sappan L. and is used as dyestuff or in tradisional medicines. Studies of Lignum Sappan have revealed several pharmacological activities, including anti-inflammatory [1-2], anti-allergic [3], antifungal [4], antiviral [5], antioxidation [6-7], and vasorelaxant activities [8]. In addition, Lignum Sappan has also been used in traditional Eastern medicine as an anti-tumor agent. Kim et al. reported that the chloroform ex-tract of Lignum Sappan induced cell death and growth inhibi-

[Received on] 21-May-2013 [Research funding] This project was supported by the China Na-tional Natural Science Foundation Grant (Nos. 91129701; 81102848; 31129004) and the China National Science and Technology 973 grant (No. 2012CB910704). [*Corresponding author] YU Qiang, Prof., Tel: 86-21-50801790, Fax: 86-21-50800306, E-mail: qyu@sibs.ac.cn These authors have no conflict of interest to declare.

tion in oral cancer cells and increased levels of the cell cycle regulatory proteins p53 and p21 [9]. They identified isoliq-uiritigenin 2'-methyl ether from the chloroform extract that induced growth arrest and apoptosis in oral cancer cells through heme oxygenase-1 [10].

Increasing evidence has shown that inflammatory re-sponses and cancer development are closely related [11-12]. It has been estimated that 25% of all human cancers in adults result from chronic inflammation [13]. For example, inflam-matory bowel disease predisposes to colon cancer and hepati-tis predisposes to liver cancer. Molecular and cellular path-ways, which connect inflammation and cancer have emerged as attractive targets for prevention and therapy [14-17]. Tran-scriptional factors of the nuclear factor-κB (NF-κB) and sig-nal transducers and activators of transcription (STATs) are ubiquitously expressed and control numerous physiological processes, including development, differentiation, immunity, metabolism, and cancer. Both NF-κB and STATs are rapidly activated in response to various stimuli, including stresses and cytokines, although they are regulated by entirely different

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signaling mechanisms. Once activated, NF-κB and STATs control the expression of anti-apoptotic, pro-proliferative, and immune response genes.

The extracts of Lignum Sappan have anti-inflammation and anti-tumor activities. Sappanchalcone and brazilin are anti-inflammation constituents in Lignum Sappan [1-2]. It was found in these laboratories that the EtOAc extract of Lignum Sappan had inhibitory activities on growth-related signaling and mitosis. In this report, three active compounds (sap-panchalcone, brazilin, and butein) have been identified from the EtOAc extract of Lignum Sappan, their inhibitory effects on cell growth and growth-related signaling evaluated, and the antitumor activities of the EtOAc extract and individual active compound in vitro and in vivo were examined.

Materials and Methods

Plant material Lignum Sappan (Batch number: 101118) was purchased

from Shanghai Yang-he-tang Chinese Herbal Medicine Com-pany Ltd and identified by LIU Jing-Li. A voucher specimen of these materials was deposited for reference in Shanghai Institute of Materia Medica (AP0721). Extraction and isolation

The dried heartwood was crushed into a powder, and the powder (100 g) was extracted with 95% EtOH (reflux, 1 h, × 3) to give an EtOH extract (13.05 g). The extract was parti-tioned between EtOAc and water to give an EtOAc-soluble fraction (9.269 g). The EtOAc-soluble fraction was subjected to silica gel (200−300 mesh, 310 g) column chromatography eluting with CHCl3-MeOH [100 : 0 (2 L), 100 : 1 (3 L), 50 : 1 (1.5 L), 25 : 1 (7.5 L), 15 : 1 (2.5 L), 10 : 1 (3 L), and 0 : 100 (1 L)] to give 126 tubes. Similar fractions were identified by TLC assay and combined to give sixteen fractions: Fr. 1-16. Fr. 10 (120.9 mg) was subjected to silica gel (200−300 mesh，16 g) column chromatography (eluent: petroleum ether: ace-tone, 3 : 1, 1 L) to give forty two sub-fractions (Fr. A1−A42). The combined Fr. A21−A26 eluent was evaporated under reduced pressure to give a yellow compound, sappanchalcone (97%, 11.1 mg). Fr. 11−13 (883 mg) was subjected to silica gel (200−300 mesh, 60 g) column chromatography (eluent: petroleum ether: ethyl acetate, 4:3, 1.5 L) to give eighty three sub-fractions (Fr. B1−B83). The combined Fr. B45−B53 was evaporated under reduced pressure to give a red compound, brazilin (98%, 43.7 mg).

Sappanchalcone: Molecular formula: C16H14O5; Uv λmax (MeOH): 361, 250 nm; 1H NMR (400 MHz, CD3OD) δ: 7.58 (1H, d, J = 8.4 Hz), 7.50 (1H, d, J = 15.8 Hz), 7.36 (1H, d, J = 15.8 Hz), 7.11 (1H，d，J = 2.0 Hz), 6.98 (1H，dd，J = 8.2, 2.0 Hz), 6.79 (1H，d, J = 8.3 Hz), 6.51 (1H，d, J = 2.3 Hz), 6.45 (1H, dd, J = 8.5, 2.1 Hz), 3.88 (3H, s); 13C NMR (100 MHz, CD3OD) δ: 193.6 (C=O), 164.9 (C-2’), 162.9 (C-4’), 150.0 (C-4), 147.3 (C-3), 145.0 (C-β), 134.2 (C-6’), 129.1 (C-1), 125.6 (C-6), 123.8 (C-α), 122.2 (C-1’), 117.1 (C-5), 115.7 (C-2), 109.4 (C-5’),

100.6 (C-3’), 56.6 (OCH3). Brazilin: Molecular formula: C16H14O5; UV λmax (MeOH): 200,

286 nm; 1H NMR (400 MHz, CD3OD) δ: 7.18 (1H，d，J = 8.4 Hz), 6.47 (1H，dd，J = 8.4，2.4 Hz), 6.29 (1H，d，J = 2.8 Hz), 6.70 (1H，s), 6.60 (1H，s), 3.68 (1H，d，J = 11.6 Hz), 3.92 (1H，dd，J = 11.2，1.2 Hz), 3.96 (1H，s), 2.76 (1H，d，J = 15.6 Hz), 3.02 (1H，d，J = 15.6 Hz); 13C NMR (100 MHz, CD3OD) δ: 156.7 (C-4a), 154.5 (C-3), 144.5 (C-9), 144.1 (C-10), 136.2 (C-11a), 131.1 (C-1), 130.1 (C-7a), 114.3 (C-1a), 111.7 (C-8), 111.2 (C-11), 108.8 (C-2), 103.1 (C-4), 76.9 (C-6a), 69.6 (C-6), 49.8 (C-12), 39.2 (C-7). Cell lines and reagents

Gifts of 293/NF-κB cells, HepG2/STAT1, and HepG2/ STAT3 cells were provided by Prof. FU Xin-Yuan (National University of Singapore, Singapore). The 293/NF-κB cells were 293 cells stably transfected with a NF-κB responsive firefly lu-ciferase reporter plasmid. HepG2/STAT1 or HepG2/STAT3 cells were HepG2 cells stably transfected with a STAT1 or STAT3-responsive firefly luciferase reporter plasmid. Human tumor cell lines HepG2, H522, and COLO 205 were obtained from the American Type Culture Collection. The murine sarcoma S180 cell line was purchased from Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China. TNFα, IFNγ, interleukin-6, DMSO, taxol (97%), butein (98%) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). The luciferase assay sys-tem was purchased from Promega (Madison, WI, USA). Animals

Male Balb/c mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China. Animals were housed in a controlled conditions of temperature (23 °C ± 2 °C), humidity (50% ± 5%), and a 12 h light/dark cycle. The animals had free access to sterile food and water. All the protocols of the animal experiments were approved by the Ethics Committee of Shanghai Institute of Materia Medica, and the research complied with the Guide for the Care and Use of Laboratory Animals. Luciferase assay

The 293/NF-κB cells (2 × 104 per well) were seeded into 96-well cell culture microplates (Corning, New York, NY, USA) and allowed to grow for 48 h and then treated with samples for 1 h, followed by stimulation with 25 ng·mL−1 TNF-α for 5.5 h. Luciferase activity was deter-mined using the Promega luciferase kits according to the manufacturer’s instruction. HepG2/STAT1 or HepG2/STAT3 cells were stimulated with 100 U·mL−1 IFNγ or 10 ng·mL−1 interleukin-6. The cell number was counted at seeding and was controlled by equal seeding. All luciferase assay experiments were repeated at least three times to minimize the difference caused by cell number. Flow cytometry analysis

HGC-27 cells were incubated with samples for 24 h. The cells were harvested and washed with PBS, and re-suspended in ice-cold 75% ethanol (1 mL). After being left to stand

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overnight, cell pellets were collected by centrifugation, re-suspended in PBS (500µL, 50 µg·mL−1 RNase in PBS), and incubated at 37 °C for 30 min. Then propidium iodide solu-tion (25 µL, 25µg·mL−1) was added, and the mixture was allowed to stand in ice for 1 h. Fluorescence emitted from the propidium iodide-DNA complex was quantitated after excita-tion of the fluorescent dye by FAC-Scan cytometry. Taxol was used as a positive control in this experiment. MTT assay

About 5 000 cells of HepG2, H522, COLO 205, or S180 per well were seeded into 96-well plates. Twenty-four h later, cells were treated with vehicle control (DMSO) or samples for 72 h. The MTT assay was performed by adding MTT solution (20 μL, 5 mg·mL−1) to each well and incubated for 3 h at 37 °C. The supernatant was aspirated, and the MTT- for-mazan crystals formed by metabolically viable cells were dissolved in DMSO (150 mL). The absorbance was measured by a microplate reader at a wavelength of 570 nm. IC50 values (50% concentration of inhibition) were determined through non-linear regression analysis. In vivo tumor model

BALB/c mice were inoculated subcutaneously with 2 × 106 S180 cells/0.2 mL PBS. After 24 h, the mice were ran-domly assigned to five groups (n = 8, excluding taxol group): (a) model, (b) taxol (10 mg·kg−1), (c) brazilin (400 mg·kg−1), (d) sappanchalcone (200 mg·kg−1) and (e) EtOAc extract (200 mg·kg−1). Each mouse received either vehicle or samples intraperitoneally every day for a total of seven doses. Mice were sacrificed eight days after cell inoculation, and the tu-mors were surgically removed for measurement. Statistical analysis

Data are presented as x ± s. A one-way ANOVA determined

whether the results had statistical significance. In some cases, Student's t test was used for comparing two groups. The level of statistical significance was set at P < 0.05.

Results

Effects of the EtOAc extract on cell mitosis and signal trans-duction

Lignum Sappan was first extracted with refluxing 95% EtOH, The EtOH extract was fractionated with EtOAc to yield EtOAc-soluble fraction. The content of brazilin and sappanchalcone in the EtOAc extract was 18.82% and 1.98% respectively by HPLC methods. A preliminary evaluation revealed that the EtOAc extract of Lignum Sappan had in-hibitory activities on cell mitosis and growth-related signaling pathway. The EtOAc extract interfered with cell mitosis. The cell population was blocked at 56.62% in G2/M phase 24 h after they were exposed to the EtOAc extract at 50 µg·mL−1 (Fig. 2). The EtOAc extract also inhibited the IL-6-induced STAT3 and TNFα-induced NF-κB luciferase activities. The IC50 values were (6.50 ± 0.07) µg·mL−1 and (15.21 ± 0.16) µg·mL−1, respectively. It did not obviously influence IFNγ/STAT1 cell signal transduction at the same concentra-tion (data not shown). Three active constituents of the EtOAc extract are identified

In order to identify the active constituents of the EtOAc extract from Lignum Sappan, further isolation was performed. Two active compounds were isolated and identified as sap-panchalcone and brazilin (Fig. 1) by comparing physico-chemical and spectroscopic data with literature values [18-19]. Another active constituent, butein, was reported to be isolated from the EtOAc fraction of Lignum Sappan [20]. It was pur-chased from Sigma Aldrich, and its activities examined.

Fig. 1 Structures of the active constituents identified from the EtOAc extract of Lignum Sappan

Sappanchalcone arrests cells at G2/M phase Sappanchalcone induced gradual accumulation of rounded

cells, which resemble the appearance of mitotic cells. The effects of sappanchalcone on cell cycle were therefore examined. Sap-panchalcone (10 µg·mL−1) induced G2/M arrest (97% block) after 24 h of sample exposure (Fig. 2). Brazilin and butein inhibit cell signal transduction.

The active constituents from the EtOAc extract were then evaluated for activity on TNFα/NF-κB and IL-6/STAT3 cell signal transductions. Among the isolated compounds, brazilin

effectively inhibited the TNFα-induced expression of the NF-κB-responsive promoter reporter gene in 293 cells with an IC50 of (21.50 ± 0.18) µg·mL−1 (Table 1). Butein effectively suppressed the IL-6-induced expression of the STAT3- responsive promoter reporter gene in HepG2 cells with an IC50 of (5.47 ± 0.10) µg·mL−1, as well as the TNFα/ NF-κB signal transduction (Table 1). Sappanchalcone, brazilin, and butein inhibit growth of human tumor cells.

The cytotoxic effects of sappanchalcone, brazilin, and

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Fig. 2 Effects of the EtOAc extract and sappanchalcone on cell cycle distribution. HGC-27 cells were incubated with indicated concentration of samples for 24 h. The cells were then fixed and stained with PI, and analyzed by flow cytometry

Table 1 Effects of the EtOAc extract and compounds on signaling pathway and on growth of tumor cells (IC50) ( x ± s)

Sample IL-6/STAT3 TNFα/NF-κB S180 HepG2 H522 COLO 205

EtOAc extract (μg·mL−1) 6.50 ± 0.07 15.21 ± 0.16 12.22 ± 0.06 13.51 ± 0.12 12.78 ± 0.16 8.81 ± 0.07

Sappanchalcone (μg·mL−1) > 30 > 30 6.63 ± 0.05 0.91 ± 0.18 1.31 ± 0.16 21.76 ± 0.10

Brazilin (μg·mL−1) > 30 21.50 ± 0.18 2.56 ± 0.17 11.91 ± 0.19 3.70 ± 0.09 6.47 ± 0.13

Butein (μg·mL−1) 5.47 ± 0.10 15.07 ± 0.10 4.60 ± 0.16 1.78 ± 0.13 10.40 ± 0.14 3.95 ± 0.08

Taxol (μg·mL−1) N.D. N.D. 0.13 ± 0.02 0.20 ± 0.03 2.00 ± 0.04 0.05 ± 0.03N.D., not detected.

butein were examined using human tumor cell lines of different tissue origins, including the hepatocellular car-cinoma cell line HepG2, the lung adenocarcinoma cell line H522, and the colorectal adenocarcinoma cell line COLO 205. The data indicated that the EtOAc extract showed similar cytotoxic effects on different tumor cells, but the three compounds exhibited differential antiproliferative activities (Table 1). Effects of the EtOAc extract and single compound on tumor growth in vivo

To test the antitumor effects in vivo, a mouse xenograft

model bearing S180 mouse sarcoma cells was used. The EtOAc extract and its active constituents significantly inhib-ited S180 growth both in vitro (Table 1) and in vivo (Fig. 3). The in vivo tumor inhibition rate was 64.84% for the EtOAc extract, 39.22% for brazilin, and 57.61% for sappanchalcone respectively. The body weight of the mice was also decreased in the EtOAc extract and sappanchalcone treated groups (Fig. 3A). One animal died on the fourth day of the treatment of sappanchalcone. Taxol was used as the positive control. These data suggest the EtOAc extract has better antitumor effects than the individual active constituents from Lignum Sappan.

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Fig. 3 Effects of the EtOAc extract and individual active constituents on S180 tumor cell growth in vivo. (A) The body weights of S180 bearing mice were measured after treatment with samples. (B) S180-bearing mice were sacrificed eight days after cell inoculation, and the tumors were surgically removed for measurement. ***P < 0.001, sappanchalcone, brazilin, or the EtOAc extract vs model group; ##P < 0.01, brazilin vs the EtOAc extract; (C) S180 tumor tissues

Discussion

Lignum Sappan has been used in traditional Chinese medicine to treat cancer. These studies found that the EtOAc extract of Lignum Sappan inhibited growth-related signaling and mitosis of tumor cells. The chemical constituents were isolated and their effects on cell cycle and cell signal trans-duction analyzed. Three active constituents from the EtOAc extract were identified: sappanchalcone blocked cell cycle progression in the G2/M phase, brazilin inhibited TNFα-induced NF-κB-luciferase activities, and butein inhib-ited the IL-6-induced STAT3-luciferase activities, as well as TNFα/NF-κB signal transduction. From the aspect of chemi-cal structure, sappanchalcone and butein belong to the chro-mane-type compounds, and brazilin belongs to brazilin-type compounds. Isoliquiritigenin-2'-methyl ether, which has been reported to induce apoptosis in oral cancer cells through heme oxygenase-1 [10], belongs to the chromane-type compounds.

These results indicate that Lignum Sappan contains different types of compounds with distinct cytotoxic activities, and their molecular targets may be different.

Sappanchalcone or brazilin, as anti-inflammatory con-stituents from Lignum Sappan, could inhibit the growth of human tumor cells. In these studies, brazilin inhibited the TNFα/NF-κB signaling. Sappanchalcone, as a biosynthetic precursor of brazilin [21], did not show inhibitory effects on TNFα/NF-κB signal transduction. It has been reported that brazilin inhibits lipopolysaccharide-induced DNA binding activity of NF-κB and AP-1 in RAW 264.7 macrophage cells [22]. Butein is another active constituent identified from Lig-num Sappan. It suppresses many protein activities. Such as NF-κB, cyclin D1, c-Myc, STAT3, ICAM-1, EGFR, and JNK, etc. [23-24]. In the luciferase assay, butein inhibited IL-6/STAT3 and TNFα/NF-κB signal transduction. NF-κB and STAT3 play key roles in many physiological processes, such as innate and adaptive immune responses, cell proliferation, cell death, and inflammation. It has become clear that aberrant regulation of the NF-κB and STAT3 signaling pathways that control its activity are involved in cancer development and progression, as well as in resistance to chemo- and radiotherapy [25-29]. NF-κB and STAT3 are a critical link between inflammation and cancer [14-15, 17]. Much effort has been put into strategies to inhibit NF-κB and STAT3 activation for the treatment of in-flammation and cancer [29-32]. Therefore, brazilin and butein would be beneficial for immunotherapy of transplantation, autoimmune, and allergic diseases, as well as for cancer treatment.

In these studies, it was found that the anti-proliferative effects of EtOAc extract were as effective as that of sap-panchalcone in vivo, but that sappanchalcone caused the death of mice. The anti-proliferative effect of brazilin was better than that of the extract in vitro, but the tumor inhibitory rate of brazilin was lower than that of the extract in vivo, because of the fast metabolism of brazilin (data not shown). It has been reported that the inhibition rate of butein on hepato-cellular carcinoma is 53.90% after mice receive an intrap-eritoneal injection of 2 mg·kg−1 butein for three consecu-tive weeks [24]. But the EtOAc extract contains much less than 1% butein. Therefore, the EtOAc extract is superior to the individual active constituents as an antitumor drug, considering both efficacy and safety. The extract contains many active constituents with different antitumor activities. These active constituents may synergistically enhance antitumor effects by targeting different proteins. Some other constituents in the extract may also influence the metabolism of individual antitumor constituents. Further studies are needed to systematically explore these possi-bilities. In summary, these findings provide evidence that sap-panchalcone, brazilin, and butein isolated from Lignum Sappan heartwood inhibit the growth of human tumor cells. Sap-panchalcone are cell cycle blockers, while brazilin and butein

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are signal transduction inhibitors. The data also suggest that the EtOAc extract of Lignum Sappan may be better than the indi-vidual active constituent as a novel treatment for cancer.

References

[1] Sasaki Y, Hosokawa T, Nagai M, et al. In vitro study for inhibi-tion of NO production about constituents of Sappan Lignum [J]. Biol Pharm Bull, 2007, 30 (1): 193-196.

[2] Washiyama M, Sasaki Y, Hosokawa T, et al. Anti-inflammatory constituents of Sappan Lignum [J]. Biol Pharm Bull, 2009, 32 (5): 941-944.

[3] Yodsaoue O, Cheenpracha S, Karalai C, et al. Anti-allergic activity of principles from the roots and heartwood of Caes-alpinia sappan on antigen-induced beta-hexosaminidase release [J]. Phytother Res, 2009, 23 (7): 1028-1031.

[4] Niranjan Reddy VL, Ravikanth VV, Jansi Lakshmi VV, et al. Inhibitory activity of homoisoflavonoids from Caesalpinia sappan against Beauveria bassiana [J]. Fitoterapia, 2003, 74 (6): 600-602.

[5] Liu AL, Shu SH, Qin HL, et al. In vitro anti-influenza viral activities of constituents from Caesalpinia sappan [J]. Planta Med, 2009, 75 (4): 337-339.

[6] Badami S, Moorkoth S, Rai SR, et al. Antioxidant activity of Caesalpinia sappan heartwood [J]. Biol Pharm Bull, 2003, 26 (11): 1534-1537.

[7] Lee MJ, Lee HS, Kim H, et al. Antioxidant properties of ben-zylchroman derivatives from Caesalpinia sappan L. against oxidative stress evaluated in vitro [J]. J Enzyme Inhib Med Chem, 2010, 25 (5): 608-614.

[8] Sasaki Y, Suzuki M, Matsumoto T, et al. Vasorelaxant activity of Sappan Lignum constituents and extracts on rat aorta and mesenteric artery [J]. Biol Pharm Bull, 2010, 33 (9): 1555-1560.

[9] Kim EC, Hwang YS, Lee HJ, et al. Caesalpinia sappan induces cell death by increasing the expression of p53 and p21WAF1/ CIP1 in head and neck cancer cells [J]. Am J Chin Med, 2005, 33 (3): 405-414.

[10] Lee YM, Jeong GS, Lim HD, et al. Isoliquiritigenin 2'-methyl ether induces growth inhibition and apoptosis in oral cancer cells via heme oxygenase-1 [J]. Toxicol In Vitro, 2010, 24 (3): 776-782.

[11] Grivennikov SI, Karin M. Inflammation and oncogenesis: a vicious connection [J]. Curr Opin Genet Dev, 2010, 20(1): 65-71.

[12] Reuter S, Gupta SC, Chaturvedi MM, et al. Oxidative stress, inflammation, and cancer: how are they linked? [J]. Free Radic Biol Med, 2010, 49 (11): 1603-1616.

[13] Wu Y, Zhou BP. Inflammation: a driving force speeds cancer metastasis [J]. Cell Cycle, 2009, 8 (20): 3267-3273.

[14] Grivennikov SI, Karin M. Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk in cancer [J]. Cytokine Growth Factor Rev, 2010, 21(1): 11-19.

[15] Pensa S, Watson CJ, Poli V. Stat3 and the inflammation/acute phase response in involution and breast cancer [J]. J Mammary Gland Biol Neoplasia, 2009, 14 (2): 121-129.

[16] Aggarwal BB, Vijayalekshmi RV, Sung B. Targeting in-flammatory pathways for prevention and therapy of cancer: short-term friend, long-term foe [J]. Clin Cancer Res, 2009, 15 (2): 425-430.

[17] Bollrath J, Greten FR. IKK/NF-kappaB and STAT3 pathways: central signalling hubs in inflammation-mediated tumour promotion and metastasis [J]. EMBO Rep, 2009, 10 (12): 1314-1319.

[18] Namikoshi M, Nakata H, Nuno M, et al. Homoisoflavonoids and related compounds III. Phenolic constituents of Caesalpinia japonica Sieb. et Zucc [J]. Chem Pharm Bull, 1987, 35 (9): 3568-3575.

[19] Nguyen MT, Awale S, Tezuka Y, et al. Xanthine oxidase inhibi-tors from the heartwood of Vietnamese Caesalpinia sappan [J]. Chem Pharm Bull (Tokyo), 2005, 53 (8): 984-988.

[20] Chen YP, Liu L, Zhou YH, et al. Chemical constituents from Sappan Lignum [J]. J Chin Pharm Sci, 2008, 17 (1): 82-86.

[21] Nagai M, Nagumo S, Eguchi I, et al. Sappanchalcone from Caesalpinia sappan L., the proposed biosynthetic precursor of brazilin [J]. Yakugaku Zasshi, 1984, 104 (9): 935-938.

[22] Bae IK, Min HY, Han AR, et al. Suppression of lipopolysac-charide-induced expression of inducible nitric oxide synthase by brazilin in RAW 264.7 macrophage cells [J]. Eur J Pharma-col, 2005, 513 (3): 237-242.

[23] Yadav VR, Prasad S, Sung B, et al. The role of chalcones in suppression of NF-κB-mediated inflammation and cancer [J]. Int Immunopharmacol, 2011; 11 (3): 295-309.

[24] Rajendran P, Ong TH, Chen L, et al. Suppression of signal transducer and activator of transcription 3 activation by butein inhibits growth of human hepatocellular carcinoma in vivo [J]. Clin Cancer Res, 2011, 17 (6): 1425-1439.

[25] Bromberg JF, Wrzeszczynska MH, Devgan G, et al. Stat3 as an oncogene [J]. Cell, 1999, 98 (3): 295-303

[26] Devarajan E, Huang S. STAT3 as a central regulator of tumor metastases [J]. Curr Mol Med, 2009, 9 (5): 626-633

[27] Wu Y, Zhou BP. TNF-alpha/NF-kappaB/Snail pathway in can-cer cell migration and invasion [J]. Br J Cancer, 2010, 102 (4): 639-644.

[28] Wang S, Liu Z, Wang L, et al.NF-kappaB signaling pathway, inflammation and colorectal cancer [J]. Cell Mol Immunol, 2009, 6 (5): 327-334.

[29] Baud V, Karin M. Is NF-kappaB a good target for cancer ther-apy? Hopes and pitfalls [J]. Nat Rev Drug Discov, 2009, 8 (1): 33-40.

[30] Tas SW, Vervoordeldonk MJ, Tak PP. Gene therapy targeting nuclear factor-kappaB: towards clinical application in inflam-matory diseases and cancer [J]. Curr Gene Ther, 2009, 9 (3): 160-170.

[31] Lin Y, Bai L, Chen W, et al. The NF-kappaB activation pathways, emerging molecular targets for cancer prevention and therapy [J]. Expert Opin Ther Targets, 2010, 14 (1): 45-55.

[32] Turkson J. STAT proteins as novel targets for cancer drug discovery [J]. Expert Opin Ther Targets, 2004, 8 (5): 409-422.

Cite this article as: ZHANG Qing, LIU Jing-Li, QI Xiao-Man, QI Chun-Ting, YU Qiang. Inhibitory activities of Lignum Sappan extractives on growth and growth-related signaling of tumor cells [J]. Chinese Journal of Natural Medicines, 2014, 12(8): 607-612.

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