Toxicology in Vitro 24 (2010) 142–147

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Toxicology in Vitro

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Methylanthraquinone from Hedyotis diffusa WILLD induces Ca2+-mediatedapoptosis in human breast cancer cells

Zheng Liu *, Ming Liu, Miao Liu, Jianchun LiSchool of Life Sciences and Biopharmaceuticals, Shenyang Pharmaceutical University, Shenyang 110016, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 June 2009Accepted 12 August 2009Available online 15 August 2009

Keywords:Hedyotis diffusa WILLDApoptosisCaspaseCalpainCytochrome c[Ca2+]i

0887-2333/$ - see front matter � 2009 Published bydoi:10.1016/j.tiv.2009.08.002

* Corresponding author. Tel.: +86 24 23986283; faxE-mail address: liu306608@sohu.com.cn (Z. Liu).

Methylanthraquinone from Hedyotis diffusa WILLD exhibited potent anticancer activity in many kinds ofcancer cells. However, the exact mechanism and signaling pathway involved in methylanthraquinone-induced apoptosis have not been fully elucidated. Therefore, we explored the mechanisms of methylan-thraquinone-mediated apoptosis in MCF-7 human breast cancer cells. When MCF-7 cells were co-incu-bated with methylanthraquinone, the percentage of apoptotic cell and S phase of cell cycle wasmarkedly increased. In addition, a rise in intracellular calcium levels, phosphorylation of JNK and activa-tion of calpain were found in MCF-7 cells after exposure to methylanthraquinone. With the methylan-thraquinone-mediated reduction of mitochondrial membrane potential, cytochrome c was releasedfrom mitochondria to cytosol. Moreover, methylanthraquinone strongly induced cleavage of caspase-4,caspase-9 and caspase-7 in MCF-7 cells. These results suggested that methylanthraquinone from Hedyotisdiffusa WILLD induced MCF-7 cells apoptosis via Ca2+/calpain/caspase-4 pathway.

� 2009 Published by Elsevier Ltd.

1. Introduction

The use of herbal intervention is widespread in all regions of thedeveloping world and is rapidly growing in developed countries(Yan et al., 2006). Medicinal plants are widely used in the treat-ment of various cancers in many Asian countries and are recog-nized as an attractive alternative to surgical therapy andradiotherapy (Xie et al., 2009). Hedyotis diffusa WILLD has beenknown as a traditional Chinese medicine (TCM) for a long time,and widely applied in the treatment of inflammations such asappendicitis, urethritis, and bronchitis, due to its antibacterialactivity (Ahmad et al., 2005; Lin et al., 2002; Shan et al., 1999). Re-cently, this herb has gained increasingly attention to its usage as anantitumor herb, such as therapy in liver, lung, colon, brain, pan-creas and other cancers (Fang et al., 2004). Up to now, three majorclasses of this herb compounds, including triterpenes, polysaccha-ride and anthraquinones, have been reported as bioactive com-pounds from this herb (Ahmad et al., 2005; Li et al., 2008).

In spite of the extensive use of herbal therapies, there is insuf-ficient scientific evidence validating their efficacy and safety. Thus,basic research aimed at elucidating the underlying antitumormechanisms of Hedyotis diffusa WILLD is very important for theuse of this herbal medicine. Recently, scientists have focused onthe potential role of extracts of TCM for cancer treatment. Shi et

Elsevier Ltd.

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al. reported that two anthraquinones from Hedyotis diffusa WILLDinduced HepG2 cell apoptosis via caspase-3 activation (Shi et al.,2008). However, the molecular mechanism is still equivocal up tonow. The main aim of this work was to study the possible apopto-tic mechanism of methylanthraquinone, which was extracted fromHedyotis diffusa WILLD, in MCF-7 cells. The MCF-7 cells are oftenused in studies of apoptosis and are well known for its uniquecharacteristic of being deficient in caspase-3 (Kugawa et al.,2004). In addition, breast cancer cells are susceptible to generationof a sustained Ca2+ response (Sergeev, 2004).

2. Materials and methods

2.1. Materials

The human breast cancer MCF-7 cell line was purchased fromShanghai institutes for biological science, Chinese academy of sci-ences (Shanghai, China). DMEM and fetal calf serum (FCS) werepurchased from Gibco (Invitrogen Co., CA, USA). Primary antibodiesagainst caspase-7, caspase-9, cytochrome c, anti-phospho-JNK, cal-pain I large subunit (l-type) antibody and peroxidase-conjugatedgoat antimouse or antirabbit secondary antibody were purchasedfrom Cell signaling technology (Beverly, MA, USA). Primary anti-body against caspase-4 was purchased from Calbiochem (USA).Fluo-3/AM, rhodamine123 (Rh123), acridine orange (AO), ethylenedibromide (EB) and propidium iodide (PI) were purchased fromSigma (St. Louis, MO, USA). Methylanthraquinone was purchasedfrom Hean technology company (Shanghai, China) and its structure

http://dx.doi.org/10.1016/j.tiv.2009.08.002mailto:liu306608@sohu.com.cnhttp://www.sciencedirect.com/science/journal/08872333http://www.elsevier.com/locate/toxinvit

Fig. 1. The structure of methylanthraquinone.

Z. Liu et al. / Toxicology in Vitro 24 (2010) 142–147 143

was given in Fig. 1. All other chemicals used in the experimentswere commercial products of reagent grade.

2.2. Cell culture and cytotoxicity assay

MCF-7 cells were cultured in DMEM supplemented with 10%heat-inactivated FCS, 100 units/ml penicillin/streptomycin, as wellas 2 mM glutamine. In addition, insulin (10 lg/ml) was added inMCF-7 cells culture medium. All cells were incubated at 37 �C ina humidified atmosphere of 5% CO2. The exponentially growingMCF-7 cells were seeded into 24-well flat-bottomed plates at adensity of 5 � 105/mL. After 12 h, the cells were treated with theindicated concentrations of methylanthraquinone. The cells werecollected by trypsinization at different treated-time points (d1,d2, d3, d4 and d5) and counted by a Thoma hemocytometer(Shanghai, China) using the trypan blue dye exclusion method forcell viability.

2.3. Intracellular free calcium analysis

Intracellular free calcium ([Ca2+]i) was measured using Ca2+

indicator Fluo-3/AM as previously described (Burchiel et al.,2000). Briefly, MCF-7 cells, which treated with methylanthraqui-none (30 lM) for 12, 24, or 48 h, respectively, were harvestedand approximately 1 � 106 cells per sample were loaded with2 lM Fluo-3/AM for 1 h. After incubated, 200 ll RPMI-Hepes med-ium was added to achieve 2 � 106 cells/ml suspension. Immedi-ately before analysis of cells for changes in intracellular Ca2+,20 ll PI (50 lg/ml) solution was added to each cell sample. MCF-7 cells were then analyzed for the green Fluo-3 emission and thered PI fluorescence using FACScan flow cytometry (Becton Dickin-son, USA) following excitation with an argon laser. Winlist 4.0 soft-ware was used for fluorescence analysis.

2.4. Quantitation of apoptotic cells

To determine whether cytotoxicity-induced by methylanthra-quinone was due to apoptosis, cell apoptosis was determined byPI staining, PI/Annexin V-FITC staining and AO/EB staining. Afterexposed to methylanthraquinone (30 lM) for 12, 24, or 48 h,MCF-7 cells were collected and treated with different fluoro-chrome. For PI staining, cells were fixed with ice-cold 70% ethanolat �20 �C overnight, and then analyzed using FACScan flow cytom-etry after treatment with RNase A (50 lg/ml) at room temperaturefor 30 min and PI (100 lg/ml) staining for 15 min. Secondly, cellapoptosis was evaluated using AnnexinV-FITC and PI double stain-ing (Jin et al., 2006). After harvested, MCF-7 cells were stainedaccording to manufacturer’s instruction and then analyzed usingFACScan flow cytometry. Lastly, cell apoptosis was observed byfluorescence microscopy (Leica, Germany) using AO/EB staining.Briefly, 1 ll of a stock solution (100 lg/mL AO and EB) was addedto 25 ll of cell suspension. EB-negative cells with nuclear shrink-age, blebbing, and apoptotic bodies were counted as apoptotic cell.The percentage of apoptotic cells was calculated after observing atotal of 300 cells (Wang et al., 2007).

2.5. Assessment the change of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was measured usingflow cytometry with Rh123 and PI double stain (Ren et al., 2006).About 1 � 106 cells, which treated methylanthraquinone (30 lM)for 12, 24, or 48 h, were harvested and incubated with Rh123(10 lg/ml) at 37 �C for 30 min, then PI (10 lg/ml) was added andincubated for 5 min. The percentages of Rh123�/PI+ and Rh123�/PI� presented the effective collapsed MMP.

2.6. Western blots

MCF-7 cells, treated with methylanthraquinone (30 lM) for 12,24, or 48 h, were harvested and washed with PBS. Cytosolic andmitochondrial fractions were prepared as previous described (Xieet al., 2007). The detection of cytochrome c in cytoplasm and themitochondria fractions was analyzed by Western blot. Total cellu-lar protein was isolated using the protein extraction buffer (con-taining 150 mM NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA, 0.1%Triton-100, 5% glycerol and 2% SDS). Protein concentrations weredetermined using the protein assay kit according to manufacturer’sinstruction (Biyuntian, Jiangsu, China). Equal amounts of proteins(50 lg/lane) were fractionated using 8–12% SDS–PAGE and trans-ferred to PVDF membranes. The membranes were incubated withprimary antibodies against calpain, caspase-9, caspase-4, cas-pase-7, as well as cytochrome c (1:5000). After washed with PBS,the membranes were incubated with peroxidase-conjugated goatantimouse or antirabbit secondary antibody (1:3000), followedby enhanced chemiluminescence staining through the enhancedchemiluminescence system. Actin was used to normalize for pro-tein loading (Huang et al., 2004).

2.7. Data analysis

All data were presented as mean ± SD and analyzed using Stu-dent’s t test or analysis of variance (ANOVA) followed by q test.

3. Results

3.1. Effects of methylanthraquinone on cell proliferation

To determine whether methylanthraquinone had antitumor ef-fects in vitro, we examined the cytotoxic effects on MCF-7 cells bythe trypan blue exclusion assay. The data demonstrated that meth-ylanthraquinone exhibited a dose- and time-dependent growthinhibition effects (Fig. 2). The values of EC50 are 18.62 ± 2.71 and42.19 ± 3.84 lM for 24 and 48 h, respectively.

3.2. Change in [Ca2+]i following methylanthraquinone treatment

We used Fluo-3 and PI double staining to investigate anychange in [Ca2+]i following methylanthraquinone treatment ofMCF-7 cells. In Fig. 3A, The percentages of top right quadrantsand bottom right quadrants presented the effective increase of[Ca2+]i. The results indicated that methylanthraquinone-induced[Ca2+]i increase in a time-dependent manner in MCF-7 cells. Fur-thermore, calpain was activated after methylanthraquinone treat-ment in a time-dependent manner (Fig. 3B).

3.3. Effect of methylanthraquinone on cell apoptosis and cell cycleperturbation

To determine whether observed methylanthraquinone-induceddecrease in growth rate was due to cell apoptosis, PI staining wasperformed. The sub-G1 peak and the accumulation of cells in the

Fig. 2. Antiproliferation effect of methylanthraquinone on MCF-7 cells by trypanblue dye exclusion assay, (�x� s; and n = 3).

144 Z. Liu et al. / Toxicology in Vitro 24 (2010) 142–147

G0/G1 phase in a time-dependent manner were observed after theMCF-7 cells treatment with 30 lM methylanthraquinone (Fig. 4A).To examine whether the sub-G1 peak was due to apoptosis, thespecific apoptosis assay was performed using Annexin V-FITCand PI staining. Annexin V-FITC positive and PI negative cells (bot-tom right quadrants) represent early-apoptosis cells and AnnexinV-FITC positive and PI positive cells (top right quadrants) representlate-apoptosis cells. Methylanthraquinone treatment led to a sig-nificant increase in the percentage of cells that were positive forAnnexin V and/or PI. An increase in the percentage of early and lateapoptotic cells was observed at the all three time-point in MCF-7cells in a time-dependent manner (Fig. 4B). For further authentica-tion cell apoptosis, especially Annexin V-FITC positive and PI posi-tive cells were due to apoptosis, we observed cell morphous byfluorescence microscopy using AO/EB staining. Apoptotic cellswere stained by AO and showed densely green yellow or fragment,however, necrotic cells were stained by EB and showed cardinalred. Our results demonstrated that many cells were stained byAO and showed typical apoptotic character, while a few cells werestained by EB, suggesting cytotoxicity-induced by methylanthra-quinone was due apoptosis (Fig. 4C).

3.4. Effect of methylanthraquinone on mitochondrial disruption

The disruption of mitochondrial integrity is one of the consider-able events for cell apoptosis. To assess whether the methylanthra-

Fig. 3. Methylanthraquinone (30 lM) induces intracellular Ca2+ concentration ([Ca2+]i) inand PI double staining. The percentages of top right quadrants and bottom right quaexpression of calpain in MCF-7 cells treated with methylanthraquinone (30 lM) by We

quinone affects the function of mitochondria, the MMP wasanalyzed by FACS can flow cytometry. In Fig. 5A, the bottom leftquadrant represented the percentage of Rh123-/PI- cells, top leftrepresented the percentage of Rh123�/PI+ cells and bottom rightrepresented the percentage of Rh123+/PI- cells, top right repre-sented the percentage of Rh123+/PI+ cells. The percentage ofRh123�/PI� and Rh123�/PI+ cells increased in a time-dependentmanner in MCF-7 cells after treatment with methylanthraquinone.Furthermore, a drop of MMP is usually accompanied with releaseof cytochrome c from mitochondria to cytoplasm and our dataauthenticated this theory (Fig. 5B). These results indicated thatmethylanthraquinone-induced mitochondria damage and MMPloss.

3.5. Effect of methylanthraquinone on apoptosis-related proteins

Several apoptosis-associated proteins such as caspase and Bcl-2family members have been shown to play critical roles in Ca2+-mediated apoptosis. To determine whether these proteins are in-volved in the mediation of methylanthraquinone-induced cellapoptosis in MCF-7 cells, we examined their expression by Wes-tern blot. Our data demonstrated methylanthraquinone-inducedcaspase-4, caspase-9 and caspase-7 activation in MCF-7 cells. Inaddition, we also found that methylanthraquinone-induced c-JunNH2-terminal kinase (JNK) phosphorylation, Bcl-2 protein expres-sion downregulation and Bax upregulation (Fig. 6).

4. Discussion

Potent antitumor activity of many anthraquinone derivativeshave been demonstrated, such as adriamycin, mitoxantone, whichhas led to numerous synthetic or extract from herbs studies on thetumoricidal mechanism of these derivatives (Lai et al., 2009; Wanget al., 2008). Due to the carcinogenicity of some anthraquinones,the study was gradually focused on some herbs which containanthraquinones, because these herbs were applied for a long timeand no obvious carcinogenicity, such as Rhubarb (Doi et al., 2005;Huang et al., 2007). The induction of tumor cells apoptosis is a ma-jor strategy for antitumor drugs studies. Apoptosis can be triggeredby several stimuli and is controlled by three major pathways,namely the mitochondrial pathway, membrane death receptorpathway and Ca2+-mediated endoplasmic reticulum pathway(Sun and Peng, 2009). Flow cytometry analysis demonstrated thatmethylanthraquinone markedly induced MCF-7 cells apoptosisand G0/G1 phase cell cycle arrest, suggesting the growth inhibitionof methylanthraquinone is due to apoptosis. These effects were

crease and calpain activation in MCF-7 cells. A, [Ca2+]i is detected using Fluo-3/AMdrants presented the effective increase of [Ca2+]i (�x� s and n = 3). B, The proteinstern blot analysis. Actin protein is blotted as control.

Fig. 4. Methylanthraquinone (30 lM) induces MCF-7 cells apoptosis and cell cycle arrest. (A) Cell cycle is detected using PI staining. The experiment is performed thrice andthe result is similar. (B) Cell apoptosis is detected using Annexin V-FITC and PI double staining. Bottom right quadrants represent early-apoptosis cells and top right quadrantsrepresent late-apoptosis cells. (C) Cell apoptosis was observed by fluorescence microscopy using AO/EB staining (200�).

Fig. 5. Methylanthraquinone (30 lM) induces MMP MCF-7 cells decrease and cytochrome c release. (A) MMP is detected using PI and Rh123 double staining. The percentageof bottom left quadrant and top left quadrant represent MMP decrease (�x� s and n = 3). (B) The protein expression of cytochrome c in MCF-7 cells treated withmethylanthraquinone (30 lM) by Western blot analysis. Actin protein is blotted as control.

Z. Liu et al. / Toxicology in Vitro 24 (2010) 142–147 145

consistent with rhein which is an anthraquinone extractive fromRhubarb (Hsia et al., 2009).

Recent studies identify the ER as a third subcellular compart-ment implicated in apoptotic execution. Accumulation of mis-folded proteins and changes in Ca2+ homeostasis in ER result inER stress and lead to cell apoptosis (Chiang et al., 2005). Interest-ingly, anthraquinone derivatives often induce tumor cells apopto-

sis via Ca2+-mediated endoplasmic reticulum pathway and manyanti-tumorous herbs contain anthraquinone component (Linet al., 2007, 2009). The increase of intracellular [Ca2+]i inducesapoptosis in various cancer models (Orrenius et al., 2003; Sergeev,2004b). In present investigation, we reported for the first time thatmethylanthraquinone, an extractive from Hedyotis diffusa WILLD,could induce [Ca2+]i sustained increase in MCF-7 cells. Calpain is

Fig. 6. The expression of apoptosis-related proteins in MCF-7 cells treated withmethylanthraquinone (30 lM) for 12, 24 or 48 h by Western blot analysis. Equalamounts (50 lg/lane) of cellular protein are fractionated on 8–12% SDS–PAGE gelsand transferred to PVDF membranes as described in Section 2. Actin protein isblotted as a control.

146 Z. Liu et al. / Toxicology in Vitro 24 (2010) 142–147

an intracellular cysteine protease that modulates Ca2+-dependentapoptosis (Garcia et al., 2005). Calpain-mediated proteolysis repre-sents a major pathway of post-translational modification thatinfluences various aspects of cell physiology including apoptosis,migration and proliferation (Selvakumar et al., 2006). Our data alsoimplied that this apoptosis required activation of the Ca2+-depen-dent l-calpain in MCF-7 cells.

The regulation of ER calcium has been reported to be a controlpoint in ER and mitochondrial cross-talk apoptotic signal pathway.Excessive Ca2+ accumulation within the mitochondria is one of theprimary causes for mitochondrial permeability transition and MMPlose (Garcia et al., 2005). Mitochondria are one of the most suscep-tible organelles to apoptotic stimulus and have a crucial role in theapoptotic signaling. The loss of MMP induces cytochrome c releasefrom mitochondria to cytoplasm, which leads to the activation ofcaspase-9 and downstream cleavage of caspase-3. The release ofcytochrome c from mitochondria can be lethal to cells, so it is agood indicator which suggesting mitochondrial damage (Xueet al., 2003). Previous study indicated that anthraquinones whichwere extracted from Hedyotis diffusa WILLD could induce MMP lossand caspase-3 activation in HepG2 cell (Shi et al., 2008). However,whether caspase-3 activation is essential to anthraquinone-in-duced cell apoptosis is unknown, so we selected MCF-7 cell linefor further investigation. The MCF-7 cell line is often used in stud-ies of apoptosis and is well known for its unique characteristic ofbeing deficient in caspase-3 (Delaney et al., 2007). Our data dem-onstrated that anthraquinone-induced MMP loss, cytochrome c re-lease from mitochondria to cytoplasm and caspase-9 activation inMCF-7 cells. Moreover, caspase-7 was activated in MCF-7 cells,indicating the function of caspase-3 was partly substituted by cas-pase-7. Human caspase-4 has been reported to be localized in theER and to be cleaved in cells treated with ER stress agents (Pelletieret al., 2006). Our results showed that methylanthraquinone-in-duced caspase-4 activation in MCF-7 cells, indicating that cas-pase-4 was also involved in methylanthraquinone-mediatedapoptosis.

In addition, methylanthraquinone also induced JNK phosphory-lation. Several studies have indicated that JNK can mediate apopto-sis through various mechanisms, including regulation of Bcl-2family proteins (Sung et al., 2008). So we assessed the expressionof Bcl-2 and Bax proteins. Bcl-2 is an important element in mito-chondria-mediated apoptosis for preventing cytochrome c release

from the mitochondria. In contrast, Bax can induce the release ofcytochrome c from the mitochondria (Liu et al., 2006). The presentreport revealed that methylanthraquinone-induced apoptosis wascompanied by an increased expression of Bax as well as a reducedprotein level of Bcl-2 in MCF-7 cells.

In summary, the present results suggested that methylanthra-quinone-induced apoptosis via Ca2+/calpain/caspase-4 pathway inMCF-7 cells. In addition, the Bcl-2 family was also involved inthe regulation of methylanthraquinone-mediated apoptosis.

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Methylanthraquinone from Hedyotis diffusa WILLD induces Ca2+-mediated apoptosis in human breast cancer cellsIntroductionMaterials and methodsMaterialsCell culture and cytotoxicity assayIntracellular free calcium analysisQuantitation of apoptotic cellsAssessment the change of mitochondrial membrane potentialWestern blotsData analysis

ResultsEffects of methylanthraquinone on cell proliferationChange in [Ca2+]i following methylanthraquinone treatmentEffect of methylanthraquinone on cell apoptosis and cell cycle perturbationEffect of methylanthraquinone on mitochondrial disruptionEffect of methylanthraquinone on apoptosis-related proteins

DiscussionReferences

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