Marchantin C, a macrocyclic bisbibenzyl, induces apoptosisof human glioma A172 cells

Yan-Qiu Shi, Yong-Xiang Liao, Xian-Jun Qu, Hui-Qing Yuan, Song Li,Jian-Bo Qu, Hong-Xiang Lou *

Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University,44 Wen Hua Xi Road, Jinan, Shandong 250012, China

Received 7 October 2007; received in revised form 2 December 2007; accepted 3 December 2007

Abstract

Macrocyclic bisbibenzyls, a class of characteristic components derived from liverworts, are attracting more and moreattention because of their wide range of biological significance, including anti-bacterial, anti-fungus, anti-oxidation andcytotoxicity. Herein, we investigated the pro-apoptotic effect of marchantin C on human glioma A 172 cells. The resultsdemonstrated that marchantin C conferred dose-dependent inhibitory effects onto cell growth, viability and colony forma-tion ability of A 172 cells. Morphological observation and DNA laddering assay showed that marchantin C-treated A172cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation, the appearance of membrane-enclosedapoptotic bodies and DNA laddering fragment. Moreover, flow cytometric detection of phosphatidylserine externalizationindicated that marchantin C-induced apoptosis occurred in a dose-dependent manner. RT-PCR and western blot assayfurther substantiated that marchantin C, as a promising pro-apoptotic agent, had strong effects to induce A172 cell apop-tosis, suggesting that the action was achieved through up-regulating Bax and down-regulating Bcl-2.! 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Macrocyclic bisbibenzyl; Marchantin C; Apoptosis; Human glioma cells

1. Introduction

Apoptosis, as a physiological mode of cell death,can be triggered by a variety of extrinsic and intrin-sic signals [1]. It is also most frequently taken as animportant chemotherapy to combat cancer cells [2].Therefore, searching for agents that can trigger

apoptosis of tumor cells has become an attractivestrategy in anti-cancer drug discovery [3].

Natural products with diverse bioactivities andstructures are becoming an important source ofnovel agents with pharmaceutical potential [4]. Agood many of polyphenols, such as resveratrol[5], curcumin [6] and (!)-epigallocatechin [7] exhi-bit anti-proliferative activities towards cancer cellsvia induction of apoptosis. Macrocyclic bisbibenz-yls, a class of secondary metabolites producedexclusively in liverworts, is a family of phenoliccompounds belonging to stilbenoids [8]. These

0304-3835/$ - see front matter ! 2007 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.canlet.2007.12.014

* Corresponding author. Tel.: +86 531 88382012; fax: +86 53188382019.

E-mail address: louhongxiang@sdu.edu.cn (H.-X. Lou).

Available online at www.sciencedirect.com

Cancer Letters 262 (2008) 173–182

www.elsevier.com/locate/canlet

compounds arise biogenetically from lunularicacid or lunularin, which is widely distributed inleafy and thalloid liverworts [9,10]. Many of themsuch as marchantin A have been demonstrated topossess a wide range of biological activities includ-ing cytotoxicity, anti-bacterial and anti-fungalactivity as well as inhibitory effects on cyclooxy-genase, calmodulin and 5-lipoxygenase [11,12].Our previous reports have confirmed that march-antin C (Fig. 1) isolated from liverworts Marchan-tia polymorpha L., Ptagiochasm intermedium L.and Asterella angusta [13] exhibited potential anti-fungal activity toward common clinical pathogenicfungus Candida albicans [14]. In the present study,we examined the inhibitory effect of marchantin Con human glioma A172 cells and investigated itsrole in apoptosis induction at both cellular andmolecular levels. It was confirmed that apoptosisinduced by marchantin C in A172 cells may beimplemented by stimulating the expression of pro-apoptosis protein Bax and depressing the expressionof sup-apoptosis protein Bcl-2.

2. Materials and methods

2.1. Chemicals

The structure of marchantin C, isolated from A. angus-ta was identified by interpretation of spectral data (MS,1H NMR, 13C NMR, 2D NMR) as described previously(Fig. 1). The compound was dissolved in dimethylsulfox-ide (DMSO) at 10 mM as stock solution and dilutedaccording to experimental requirements when used.

2.2. Cell line and cell culture

Human glioma A172 cells, obtained from the Ameri-can Type Culture Collection (ATCC), were cultured inDMEM supplemented with 10% fetal bovine serum,100 U/mL of penicillin and 100 lg/mL of streptomycin.Cells were cultured at 37 "C in a humidified atmosphereof 5% CO2.

2.3. MTT assay

MTT is now widely used to quantitate cell prolifera-tion and cytotoxicity [15]. A172 cells (1 " 104 per well)were seeded into 96-well plates. After 24 h incubation,the cells were treated with marchantin C of varying con-centrations (2, 4, 8, 16 and 32 lM) for the required timeperiod. After removing the medium, incubate cells with20 lL MTT (5 mg/mL, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; Sigma, St. Louis, MO,USA) for 4 h. Light absorbance of the solution was mea-sured at 570 nm on a plate reader (Bio-rad, USA). Tripli-cate experiments were conducted in each test.

2.4. Clonogenic assay

Effects of marchantin C on the proliferation ability ofsingle A172 cell was assessed by the colony formationassay which was performed as described by reportedmethod [16]. Briefly, growing cells (1 " 103 cells per well)were seeded into 6-well plates. After 24 h of incubation,feed different doses of marchantin C to cells and continueculture in complete medium for two weeks. After that,formed colonies were stained with 0.5% crystal violetand counted under the microscope (Olympus, Japan).An aggregate composed of more than 50 cells was recog-nized as a colony.

2.5. Cell viability analysis by trypan blue exclusion

Aliquots (5 lL each) of the cell suspension (5 " 104

cells/mL) were mixed with 45 mL of 0.4% trypan bluestaining solution. The number of viable cells (unstained)versus dead cells (blue stained) was determined by ahemacytometer.

2.6. Staining of suspension cells with Hoechst 33258

One hundred microliters of cell suspension (3 " 105

cells/mL) was incubated with 1 lL of Hoechst 33258(1 mg/mL in ddH2O) for 10 min. The cells were appliedto cytospin chambers and centrifuged at 500 rpm for2 min. After air-drying, put glass coverslips over cellsand make them visualized under UV excitation andphotographed using a fluorescent microscope (Leica

O

OH

OH

O

Fig. 1. Structure of marchantin C.

174 Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182

Microsystems Holdings GmbH, Germany). Cells werescored apoptotic if the nuclei presented chromatin con-densation, marginalization or nuclear beading.

2.7. Electron microscopy observation

About 5 " 105 cells, after maintaining in completemedium in the presence or absence of 8 lg/mL marchan-tin C for 24 h, were fixed with 4% glutaraldehyde for 1 hat room temperature. After 3000 rpm of centrifugation,postfixation was carried out in 1% OsO4 in 0.15 M phos-phate buffer for 1 h, followed by a rapid wash in the samebuffer. Specimen dehydration was performed by meansalcohol-water solutions of varying concentrations (50%,70%, 95%, 100%, 15–20 min each) and final treatmentwith 100% propylene oxide for 30 min. Araldite embed-ding started with a 1:1 mixture of propylene oxide:araldite(v/v) for 1 h, followed by 1:3 mixture overnight at roomtemperature.

After an additional treatment in undiluted resin for1 h, polymerization was performed at 60 "C for 3–4 days.Sectioning was generally proceeded by the analysis ofsemithin sections, stained at 40 "C with 1% toluidine blue.Stained thin sections were located on copper grids. Obser-vation under conventional transmission electron micro-scope (JEM-1200EX, Japan) was generally performed at60 KV.

2.8. DNA fragmentation analysis by agarose gelelectrophoresis

DNA ladder quantification was done by apoptoticDNA ladder kit (Roche applied Science, Germany). Gli-oma A 172 cells were lysed on ice for 30 min using lysisbuffer (10 mM Tris–HCl, 10 mM urea, 6 M guanidine–HCl, 20% Triton X-100), and the lysate was treated withRNase A (50 lg/mL) and proteinase K (100 lg/mL).DNA was precipitated and electrophoresed on 1% aga-rose gel for fragmentation analysis. After staining withethidium bromide, the gel was photographed under aUV transilluminator (Tanon, China).

2.9. Flow cytometric analysis

Annexin V–FITC/PI apoptosis detection kit (Becton–Dickinson, USA) was applied to apoptosis detection.Cells (5 " 105 per well) were seeded into 6-well platesand then treated with varying concentrations of marchan-tin C as mentioned above. After 24 h incubation, the cellswere washed twice with ice-cold PBS (0.01 M, pH7.2).Load 100 lL of each cell sample and centrifuge at 200 gfor 5 min. Remove the supernatant and resuspend cellsin 100 lL of Annexin V/FITC according to manufac-turer’s instruction. Cell apoptosis was analyzed on aFACScan flow cytometry (Becton–Dickinson, USA).

Annexin V-positive, PI-negative cells were scored asapoptotic. Double-stained cells were considered either asnecrotic or as late apoptotic [17].

2.10. RT-PCR analysis

Reverse transcriptase-polymerase chain reaction (RT-PCR) assay was carried out to analyze the mRNA levelof Bax and Bcl-2. Total RNA was extracted using theRNAeasy kit according to the manufacturer’s instructions(Bioecon Biotec Co., LTA., China). The purity of RNAwas checked by OD260/280 of RNA samples (>1.8). cDNAwas synthesized through reverse transcription using M-MLV Reverse Transcriptase and Oligo (dT) primer.PCR was performed using gene-specific primers for Bcl-2 and Bax. GAPDH gene was co-amplified to serve asan internal control. Corresponding primer sequencesand PCR condition were designed as: Bcl-2 from Gen-Bank NM 000633 (sense: 50-AGCTGCACCTGACGCCCTT-30 and antisense: 50-CAGCCAGGAGAAATCAAACAGAGG-30; product size of 250 bp, Tm = 56.5 "C),Bax from GenBank L22473, 5 (sense: 50-CCAAGAAGCTGAGCGAGTGTCTC-30 nd ntisense: 50-AGTTGCCGTCTGCAAACATGTCA-30; product size of 230 bp;Tm = 55.5 "C), GAPDH from GenBank NG003019(sense: 50-GACAACGGCTCCGGCATGTGCA-30 andantisense: 50-TGAGGATGCCTCTCTTGCTCTG-30,product size of 530 bp; Tm = 60 "C).

PCR products were specified on 2% agarose gels con-taining 0.5 lg/ml of ethidium bromide and photographedunder a UV transilluminator.

2.11. Western blot analysis

The expression level of Bax and Bcl-2 proteins wasevaluated through Western blot analysis. Cells(3 " 106) were lysed in 100 lL of lysis buffer throughthree freeze–thaw cycles between !80 "C and 37 "C.Total protein was determined using Bradford method.Equal amounts of protein in the cell extracts were frac-tionated by 12% SDS–PAGE and then electrotrans-ferred onto nitrocellulose membranes. After blockingwith TBST buffer (20 mM Tris-buffered saline and0.5% Tween) containing 5% nonfat dry milk for 1 hat room temperature, the membranes were incubatedwith specific primary antibodies (Santa Cruz Biotech-nology, Inc., USA) prepared in TBST buffer containing5% nonfat dry milk at room temperature for 2 h, whichfollowed by washing for three times and reaction withHRP-conjugated secondary antibodies (Santa Cruz Bio-technology, Inc., USA) for 1 h at room temperature.After washed with TBST buffer for three times, the pro-teins on the membrane were detected by chemilumines-cence agents (ECL, Amersham). The data is expressedas the relative density of the protein normalized tob-actin, averaged over three experiments.

Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182 175

2.12. Statistical analysis

Statistical significance was determined by the Student’stwo-tailed t-test. P < 0.05 was considered as statistical sig-nificant. Each test was conducted at least for three times.

3. Results

3.1. Anti-glioma activity

To determine the inhibitory effect of marchantin C onhuman glioma A172 cell growth, MTT assay was firstlyperformed. As shown in Fig. 2, marchantin C led to aremarkable viability inhibition of A172 cells in dose-and time-dependent manners. However, A172 cellsshowed different sensitivity to varying concentrations ofmarchantin C, with higher susceptibility to increasedmarchantin C (16, 32 lM). Incubation with marchantinC ranging from 2 to 4 lM did not show obvious inhibitionto A172 cell proliferation.

In this study, Trypan Blue Exclusion analysis was alsoused to assess the effect of marchantin C on cell viability.Therefore, growth inhibition induced by marchantin Cwas further verified by staining with trypan blue (datanot shown).

In addition, we evaluated the effect of marchantin C onreproductive potential of single cell by colony formationassay. As shown in Fig. 3, marchantin C treatment couldresult in a concentration-dependant inhibitory effect onthe colony formation ability of cells, Colony formationin the presence of 2, 4, 8 and 16 lM of marchantin Cwas inhibited by 2.8%, 28.3%, 56.7% and 95.6%, respec-tively, which suggested a strong anti-proliferation ofmarchantin C on human glioma cells.

3.2. Marchantin C induced carcinoma cells apoptosis

To examine the effect of marchantin C on cell mor-phology during cell death, morphological changes ofuntreated cells (equivalent amount of DMSO) and cellstreated with 8, 12 and 16 lM of marchantin C for 24 hwere observed by fluorescent microscopy after Hoechst33258 staining. The results showed that cells treated withmarchantin C exhibited evident apoptosis characteristicsincluding nuclear fragmentation, chromatin compaction(Fig. 4a), cell shrinkage and membrane integrity loss ordeformation of late apoptotic appearance. This observa-tion demonstrated that marchantin C induced apoptosisin A172 cells.

Additionally, the result of electron microscope(Fig. 4b) also showed that apoptotic nuclei display a typ-ical morphology comprising segregation or condensationof chromatin adjacent to the nuclear envelope oftenaccompanied by nuclear convolution and fragmentationin membrane-enclosed bodies.

We then analyzed the chromosomal DNA fragmenta-tion by agarose gel electrophoresis. As shown in Fig. 5,A172 cells chromatin underwent internucleosomal cleav-age during 24 h treatment with 8, 12 and 16 lM of march-antin C, as demonstrated by the characteristic ladderpattern formed by DNA migration.

To further quantify apoptosis induced by marchantinC, A172 cells were stained with Annexin V–FITC andpropidium iodide (PI), and subsequently analyzed by flow

Fig. 2. Dose- and time-dependent cytotoxicity of marchantin Cto A172 cells. Cells were treated with 0, 2, 4, 8, 16 and 32 lMmarchantin C, respectively, for 24, 48 and 72 h. Inhibition ratewas expressed as cell death versus reference (cell viabil-ity = 100%). Each data is expressed as the mean ± SD obtainedfrom triplicate experiments.

Fig. 3. Inhibition effects of marchantin C on colony formationability of A172 cells. Cells were treated with 0, 2, 4, 8 and 16 lMmarchantin C for 2 weeks. Formed colonies were counted undermicroscope after 0.5% crystal violet staining. An aggregateconsists of more than 50 cells was counted as a colony. Inhibitionrate was calculated by comparing colony number of treated cellswith those of untreated one. Each data is expressed as themean ± SD obtained from triplicate experiments (*P < 0.01).

176 Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182

Fig. 4. Morphology observation of marchantin C induced apoptosis in glioma A172 cell. (a) Fluorescent micrographs of marchantin Ctreated and untreated A172 cells after Hoechst 33258 staining. A172 cells treated with 0, 8, 12 and 16 lM marchantin C for 24 h, weresubjected to Hoechst 33258 staining and then viewed under a fluorescent microscope at magnification of 400". (A), control; (B–D)represent 8, 12 and 16 lM marchantin C treated A172 cells, respectively. Arrows indicate apoptotic characteristics of treated cells. (b)Transmission electron microscope micrographs of marchantin C treated A172 cells. Cells treated with 16 lM marchantin C for 24 h wereviewed under transmission electron microscope at 60 KV and magnification of 4000". (A), control; (B), 16 lMmarchantin C treated A172cell. Results are representative of triplicate experiments.

Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182 177

cytometry. The Annexin V assay evaluated phospholipidsturnover from the inner to the outer lipid layer of theplasma membrane, an event typically associated withapoptosis. As indicated by flow cytometry analysis(Fig. 6), the proportion of Annexin V-staining cells wasincreased in marchantin C-treated cells. After 24 h oftreatment, the percentage of Annexin V-positive cellswas 0.13% for control (0.2% DMSO), 16.5% for 8 lMmarchantin C and 21.3% for 12 lM and 32.4% for16 lM marchantin C, respectively. A172 cells treated withmarchantin C exhibited a significant increase in the num-ber of apoptotic cells in a dose-dependent way.

3.3. Increase of Bax: Bcl-2 ratio in the marchantin c-induced apoptosis cells exhibited Bax activation and Bcl-2suppression

To investigate effect of marchantin C on expressionlevels of Bcl-2 and Bax, we measured mRNA levels ofthe two proteins by RT-PCR analysis with GAPDH asinternal control to ensure that equal amounts of geneswere loaded in each lane. The result showed that mRNAlevel of Bcl-2 was decreased and companied by notableincrease of Bax in A172 cells treated with 8, 12 and16 lM of marchantin C for 24 h. (Fig. 7a).

Western blot analysis was used to examine alternationsin Bcl-2 expression with b-actin as internal control. Alter-nations in the expression levels of Bax and Bcl-2 proteinsoccurred after marchantin C treatment. As shown inFig. 7b, after 24 h exposure to 8, 12 and 16 lM of march-antin C, the expression of Bax was up-regulated in a dose-dependent manner, while Bcl-2 expression was down-reg-ulated in treated A172 cells, Therefore, marchantin Ctreatment resulted in a remarkable concentration-depen-dent increase of the Bax/Bcl-2 ratio that triggered apopto-sis (Fig. 7c). This result not only supported the notion thatmarchantin C induced apoptosis, but also suggested thatthe apoptosis of A172 cells treated with marchantin Cpartly functioned by modulating the expression of Baxand Bcl-2 proteins.

4. Discussion

With increasing applications of the well knownnatural anti-cancer drugs extracted from plants,such as paclitaxel [18], adriamycin [19], etoposide[20], and camptothecin [21] in cancer chemother-apy, exploring anti-cancer agents from naturalplants is becoming a winsome strategy in anti-can-cer drug development and chemotherapy study.Marchantin-type of bisbibenzyls, a class of macro-cyclic phenolic compounds from liverworts, struc-turally consisted of two biphenyl bonds linked bydiaryl ether bonds and phenolic hydroxyl groupsin different number [22]. Studies have showed thatthis class of natural products exerted strong anti-oxidant capability to scavenge free radicals andinhibitory effect on inducible NOS in macrophagesbecause of the presence of phenolic hydroxylgroups and double biphenyl bonds [23,24]. Previ-ously, marchantin C was certified to generate sig-nificant cytotoxicity against P-388 leukaemia celllines with IC50 of 8 lg/mL as well as the anti-microbial activity against the Gram-positive bacte-rium Bacillus subtilis [22,25]. Although marchantinC exhibited cytotoxicity to a number of tumor celllines such as A172, K562, HepG2, MX-1 andHeLa (data not shown), herein we chose A172 asa model to study its apoptotic mechanism basedon its lowest IC50 value when exposed to marchan-tin C. In the present study, we first assessed thegrowth inhibitory effect of macrocyclic bisbibenzylmarchantin C on human glioma A172 cells usingMTT assay, colony formation assay and trypanblue exclusion test. As shown in Figs. 2 and 3,marchantin C treatment gave rise to significantcytotoxicity to A172 cells in a dose-dependentmanner. To explain the mode in which marchantin

Fig. 5. Marchantin C induced DNA fragmentation in gliomaA172 cells. A172 cells were treated with 0, 8, 12 and 16 lMmarchantin C for 24 h separately. Chromosomal DNA wasisolated and analyzed on 1% agarose gel electrophoresis. Resultsare representative of triplicate experiments.

178 Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182

C attacked tumor cells, we further examined mor-phological changes of A172 cells in the absenceand presence of marchantin C by Hoechst 33258staining and transmission electron microscopeobservation. The results showed obvious apoptosischaracteristics as viewed in Fig. 4. In addition,chromosome DNA in A172 cells presented ladderfragments after exposure to various doses ofmarchantin C, which was shown in Fig. 5. Thequantitative analysis of the phosphatidylserineexternalization through Annexin V/FITC and PIstaining indicated that the percentage of AnnexinV/FITC and PI double positive cells at the lateapoptosis, was increased in a concentration-depen-dent manner after 24 h treatment of marchantin C(Fig. 6). These results confirmed that marchantin Cevoked apoptosis in A172 cells.

To further investigate the possible mechanism ofaction by which marchantin C induced apoptosis,

we evaluated the expression level of Bcl-2 and Baxthrough RT-PCR analysis and Western blot assay.The results showed that the expression of Bax inA172 cells was significantly up-regulated by march-antin C in a dose-dependent manner, thus providinga possible explanation for the observed apoptosis inthe experiment. On the other hand, Bcl-2, an anti-apoptotic factor, was decreased in marchantin Ctreated A172 cells. This result suggested that march-antin C-induced apoptosis in A172 cells is partlymediated by the Bcl-2 pathway.

Apoptosis, as a regulable biological mode of celldeath, included two major types of pathways,namely, the death-receptor-mediated extrinsic path-way and the mitochondria-dependent intrinsic path-way [26,27]. Bcl-2 family proteins, as criticalcheckpoints, play important roles in controllingthe mitochondria-dependent intrinsic pathway [28].So far more than 20 members of Bcl-2 family have

Fig. 6. Flow cytometry analysis of marchantin C induced apoptosis. Cells treated with 0, 8, 12 and 16 lM marchantin C, respectively, for24 h. (A), control; (B–D) represent 8, 12 and 16 lM marchantin C treated A172 cells, respectively. Results are representative of triplicateexperiments.

Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182 179

been identified in human including sup-apoptosisproteins (such as Bcl-2, Bcl-xL) and pro-apoptosisproteins (such as Bax, Bak) [29]. A large amountof evidences have shown that invalid therapeuticoutcomes were mostly correlated with overexpres-sion of anti-apoptotic proteins, especially Bcl-2, invarious malignant tumors [30]. Anti-cancer effectsof many currently available chemotherapeuticsagents may be inhibited by up-regulating Bcl-2expression to block the apoptotic pathway [30].Thereby, antagonizing the function of Bcl-2 maybe a useful strategy for restoring normal apoptoticprocesses in cancer cells, resulting in the sensitiza-tion of cancer cells to chemotherapy. On the otherhand, Bax, as a pro-apoptosis member of the Bcl-2 family, was shown to constitute a requisite gate-way to the mitochondria-dependent pathway ofapoptosis [31]. Thus, restoring the sensitivity of can-

cer cells to anti-tumor agents can also be carried outby up-regulating Bax expression [32]. Bcl-2 and Baxproteins, as two major members of the Bcl-2 family,may form heterodimer complex to cause mutualneutralization of their functions which resulting inapoptosis triggering [33]. Therefore, the balancebetween the expression levels of Bcl-2 and Bax iscritical in determining the fate of cells, survival ordeath (Fig. 7c).

In summary, we believed that the marchantin C-inhibited proliferation of cancer cells was achievedmainly via triggering apoptosis rather than necrosis.It was verified that the A172 cell apoptosis inducedby marchantin C associated with increased expres-sion of Bax and decreased expression of Bcl-2,which indicated that marchantin C, as a pro-apop-totic agent, may find its potential application in can-cer chemotherapy.

Fig. 7. Apoptosis-related protein Bax and Bcl-2 regulation by marchantin C in treated A172 cells. (a) RT-PCR analysis of transcriptionlevel of Bax and Bcl-2 in with 0, 8, 12 and 16 lM marchantin C treated A172 cells for 24 h. Total RNA was extracted from cells treatedwith marchantin C for 24 h. RT-PCR products were analyzed on 2% agarose gel electrophoresis with GAPDH as internal control. (b)Western blot analysis of expression level of Bax and Bcl-2 in marchantin C treated A172 cells with b-actin as internal control. Cells treatedwith 0, 8, 12 and 16 lM marchantin C for 24 h, respectively, were lysed and lysates were subjected to Western blot assay. Results are fromone experiment representative of triplicate experiments. (c) Western blot results were quantitated by densitometric analysis of proteinbands. Data are expressed as mean ± SD of three separate experiments.

180 Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182

Acknowledgements

This project was supported by National NaturalScience Foundation of China (No. 30271537 and30730109) and Shandong Provincial Foundationfor Scientific Research (No. 2006GG1102023 and2005GG3202107).

Appendix A. Supplementary data

Supplementary data associated with thisarticle can be found, in the online version, atdoi:10.1016/j.canlet.2007.12.014.

References

[1] B. Fadeel, S. Orrenius, Apoptosis: a basic biologicalphenomenon with wide-ranging implications in humandisease, J. Intern. Med. 258 (2005) 479–517.

[2] W. Hu, J.J. Kavanagh, Anticancer therapy targeting theapoptotic pathway, Lancet Oncol. 4 (2003) 721–729.

[3] J.C. Reed, Apoptosis-targeted therapies for cancer, CancerCell 3 (2003) 17–22.

[4] D.J. Newman, G.M. Cragg, K.M. Snader, The influence ofnatural products upon drug discovery, Nat. Prod. Rep. 17(2000) 215–234.

[5] P.R. van Ginkel, D. Sareen, L. Subramanian, Q. Walker,S.R. Darjatmoko, M.J. Lindstrom, A. Kulkarni, D.M.Albert, A.S. Polans, Resveratrol inhibits tumor growth ofhuman neuroblastoma and mediates apoptosis by directlytargeting mitochondria, Clin. Cancer Res. 13 (2007) 5162–5169.

[6] J.J. Johnson, H. Mukhtar, Curcumin for chemopreventionof colon cancer, Cancer Lett. 255 (2007) 170–181.

[7] V. Noe, S. Penuelas, R.M. Lamuela-Raventos, J. Permanyer,C.J. Ciudad, M. Izquierdo-Pulido, Epicatechin and a cocoapolyphenolic extract modulate gene expression in humanCaco-2 cells, J. Nutr. 134 (2004) 2509–2516.

[8] C. Schwartner, C. Michel, K. Stettmaier, H. Wagner, W.Bors, Marchantins and related polyphenols from liverwort:physico-chemical studies of their radical-scavenging proper-ties, Free Radic. Biol. Med. 20 (1996) 237–244.

[9] S. Friederich, M. Rueffer, Y. Asakawa, M.H. Zenk, Cyto-chromes P-450 catalyse the formation of marchantins A andC in Marchantia polymorpha, Phytochemistry 52 (1999)1195–1202.

[10] S. Friederich, U.H. Maier, B. Deus-Neumann, Y. Asakawa,M.H. Zenk, Biosynthesis of cyclic bis(bibenzyls) in March-antia polymorpha, Phytochemistry 50 (1999) 589–598.

[11] Y. Asakawa, Chemical constituents of the bryophytes, in:W. Herz, W.B. Kirby, R.E. Moore, W. Steglich, Ch. Tamm(Eds.), Progress in the Chemistry of Organic Natural.Products, Springer, Vienna, 1995, pp. 1–562.

[12] Y. Asakawa, Phytochemistry of bryophytes, in: J. Romeo(Ed.), Phytochemicals in Human Health Protection, Nutri-tion, and Plant Defense, Kluwer Academic/Plenum Publish-ers, New York, 1999, pp. 319–342.

[13] J. Xing, C.F. Xie, J.B. Qu, H.F. Guo, B.B. Lv, H.X. Lou,Rapid screening for bisbibenzyls in bryophyte crude extracts

using liquid chromatography/tandem mass spectrometry,Rapid Commun. Mass Spectrom. 21 (2007) 2467–2476.

[14] C. Niu, J.B. Qu, H.X. Lou, Antifungal bis[bibenzyls] fromthe Chinese liverwort Marchantia polymorpha L, Chem.Biodivers 3 (2006) 34–40.

[15] X.J. Qu, J.L. Yang, P.J. Russell, D. Goldstein, Changes inepidermal growth factor receptor expression in humanbladder cancer cell lines following interferon-alpha treat-ment, J. Urol. 172 (2004) 733–738.

[16] Haseeb Ahsan, Shannon Reagan-Shaw, Jorien Breur, NihalAhmad, Sanguinarine induces apoptosis of human pancreaticcarcinoma AsPC-1 and BxPC-3 cells via modulations in Bcl-2family proteins, Cancer Lett. 249 (2007) 198–208.

[17] G. Wang, H. Chen, M. Huang, N. Wang, J. Zhang, Y.Zhang, et al., Methyl protodioscin induces G2/M cell cyclearrest and apoptosis in HepG2 liver cancer cells, CancerLett. 241 (2006) 102–109.

[18] N. Matsuhashi, M. Saio, A. Matsuo, Y. Sugiyama, S. Saji,Apoptosis induced by 5-fluorouracil, cisplatin and paclitaxelare associated with p53 gene status in gastric cancer cell lines,Int. J. Oncol. 26 (2005) 1563–1567.

[19] M. Hirao, K. Fujitani, T. Tsujinaka, Phase I study of thecombination of nedaplatin, adriamycin and 5-fluorouracilfor treatment of advanced esophageal cancer, Dis. Esopha-gus 17 (2004) 247–250.

[20] E.L. Baldwin, N. Osheroff, Etoposide, topoisomerase II andcancer, Curr. Med. Chem. Anticancer Agents 5 (2005) 363–372.

[21] K. Legarza, L.X. Yang, New molecular mechanisms ofaction of camptothecin-type drugs, Anticancer Res. 26(2006) 3301–3305.

[22] Y. Asakawa, M. Toyota, M. Tori, T. Hashimoto, Chemicalstructures of macrocyclic bis(bibenzyls) isolated from liver-worts (Hepaticae), Spectroscopy 14 (2000) 149–175.

[23] Y. Asakawa, Recent advances in phytochemistry of bryo-phyte sacetogenins, terpenoids and bis(bibenzyl)s fromselected Japanese, Taiwanese, New Zealand, Argentineanand European liverworts, Phytochemistry 56 (2001) 297–312.

[24] L. Harinantenaina, D.N. Quang, N. Takeshi, T. Hashimoto,C. Kohchi, G. Soma, Y. Asakawa, Bis(bibenzyls) fromliverworts inhibit lipopolysaccharide-induced inducible NOSin RAW 264.7 cells: a study of structure–activity relation-ships and molecular mechanism, J. Nat. Prod. 68 (2005)1779–1781.

[25] J. Scher, E.J. Burgess, S. Lorimer, N.B. Perry, A cytotoxicsesquiterpene and unprecedented sesquiterpene-bisbibenzylcompounds from the liverwort Schistochila glaucescens,Tetrahedron 58 (2002) 7875–7882.

[26] A.H. Wyllie, Apoptosis: an overview, Br. Med. Bull. 53(1997) 451–465.

[27] A. Ashkenazi, V.M. Dixit, Death receptors: signaling andmodulation, Science 281 (1998) 1305–1308.

[28] G. Kroemer, The proto-oncogene Bcl-2 and its role inregulating apoptosis, Nat. Med. 3 (1997) 614–620.

[29] B. Antonsson, J.C. Matinou, The Bcl-2 protein family, Exp.Cell. Res. 256 (2000) 50–57.

[30] J.C. Reed, Double identity for proteins of the Bcl-2 family,Nature 387 (1997) 773–776.

[31] M.C. Wei, W.X. Zong, E.H. Cheng, T. Lindsten, V.Panoutsakopoulou, A.J. Ross, et al., Proapoptotic BAXand BAK: a requisite gateway to mitochondrial dysfunctionand death, Science 292 (2001) 727–730.

Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182 181

[32] C.L. Zhang, L.J. Wu, S. Tashiro, S. Onodera, T. Ikejima,Oridonin induces apoptosis of HeLa cells via alteringexpression of Bcl-2/Bax and activating caspase-3/ICADpathway, Acta Pharmacol. Sin. 25 (2004) 691–698.

[33] S. Nascimento Pde, A.A. Ornellas, M.M. Campos, M.A.Scheiner, W. Fiedler, G. Alves, Bax and bcl-2 imbalance andHPB infection in penile tumors and adjacent tissues, Prog.Urol. 14 (2004) 353–359.

182 Y.-Q. Shi et al. / Cancer Letters 262 (2008) 173–182