Bee venom induces apoptosis through intracellular Ca2+-modulated intrinsic death pathway in human bladder cancer cells

Original Article: Laboratory Investigationiju_2876 61..70

Bee venom induces apoptosis through intracellularCa2+-modulated intrinsic death pathway in human bladdercancer cellsSiu-Wan Ip,1 Yung-Lin Chu,1 Chun-Shu Yu,5 Po-Yuan Chen,2 Heng-Chien Ho,3 Jai-Sing Yang,4

Hui-Ying Huang,1 Fu-Shin Chueh,6 Tung-Yuan Lai8,* and Jing-Gung Chung2,7,*Departments of 1Nutrition, 2Biological Science and Technology, 3Biochemistry and 4Pharmacology, and 5School of Pharmacy, ChinaMedical University, Departments of 6Health and Nutrition Biotechnology and 7Biotechnology, Asia University, Taichung, and8Department of Chinese Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

Abbreviations & AcronymsAIF = apoptosis-inducingfactorBV = bee venomEndo G = endonuclease GER = endoplasmicreticulum

Correspondence: Jing-GungChung Ph.D., Department ofBiological Science andTechnology, China MedicalUniversity, No. 91,Hsueh-Shih Road, Taichung40402, Taiwan. Email:[email protected]*These authors contributedequally to this work.

Received 3 June 2011;accepted 19 September 2011.Online publication 11December 2011

Objectives: To focus on bee venom-induced apoptosis in human bladder cancerTSGH-8301 cells and to investigate its signaling pathway to ascertain whether intracel-lular calcium iron (Ca2+) is involved in this effect.Methods: Bee venom-induced cytotoxic effects, productions of reactive oxygenspecies and Ca2+ and the level of mitochondrial membrane potential (DYm) were ana-lyzed by flow cytometry. Apoptosis-associated proteins were examined by Western blotanalysis and confocal laser microscopy.Results: Bee venom-induced cell morphological changes and decreased cell viabilitythrough the induction of apoptosis in TSGH-8301 cell were found. Bee venom promotedthe protein levels of Bax, caspase-9, caspase-3 and endonuclease G. The enhancementsof endoplasmic reticulum stress-related protein levels were shown in bee venom-provoked apoptosis of TSGH-8301 cells. Bee venom promoted the activities of caspase-3,caspase-8, and caspase-9, increased Ca2+ release and decreased the level of DYm.Co-localization of immunofluorescence analysis showed the releases of endonuclease Gand apoptosis-inducing factor trafficking to nuclei for bee venom-mediated apoptosis.The images revealed evidence of nuclear condensation and formation of apoptotic bodiesby 4′,6-diamidino-2-phenylindole staining and DNA gel electrophoresis showed the DNAfragmentation in TSGH-8301 cells.Conclusions: Bee venom treatment induces both caspase-dependent and caspase-independent apoptotic death through intracellular Ca2+-modulated intrinsic deathpathway in TSGH-8301 cells.

Key words: apoptosis, bee venom, endoplasmic reticulum stress, human bladdercancer TSGH-8301 cells, intracellular Ca2+ release.

Introduction

Bladder cancer is a significant problem worldwide and a leading cause of death.1 In theUSA, approximately 14 336 people died of bladder cancer and nearly 70 980 new cases ofbladder cancer were diagnosed in 2009.2 In Taiwan, 3.3 individuals per 100 000 die annuallyfrom bladder cancer, based on the 2009 report from the Department of Health, Taiwan.Therapy for bladder cancer, including surgery, radiotherapy, chemotherapy and radiotherapywith chemotherapies are usually applied,3,4 but these treatments are still inadequate. Numer-ous studies thus focus on the development of novel and effective chemotherapeutic agentsthat could effectively inhibit bladder cancer.

Bee venom (BV), a traditional medicine, has been used to treat arthritis, rheumatism,back pain, cancerous tumors, and skin diseases.5 It contains many active components,including melittin, phospholipase A2, apamin, adolapin and mast cell-degranulating pep-tide.6 BV has a wide variety of pharmaceutical properties such as immune system func-tions.7 It has been reported to travel along the neural pathways from the spine to various

International Journal of Urology (2012) 19, 61–70 doi: 10.1111/j.1442-2042.2011.02876.x

© 2011 The Japanese Urological Association 61

trigger points and injured areas to help repair nerve damageand restore mobility.8,9 S.c. apipuncture therapy of BVreduces the visceral nociceptive effects.10,11 BV also has thecapacity to kill cancer cells or induce apoptotic celldeath.12–14 It has been reported that the promise of thisremedy exists with live honeybees, which make tumors dis-appear by killing cancer cells.15 Furthermore, BV treatmentprotected ethanol-induced hepatocyte apoptosis through theregulation of Bcl-2 family with the subsequent inactivationof the caspase and poly-(ADP-ribose) polymerase (PARP).16

Our earlier studies also showed that BV-induced apoptosisin human breast cancer13 and cervical12 cells via amitochondria-dependent pathway. Recently, BV andmelittin-inhibited prostate cancer cells in vitro and in vivohave been reported, and these effects may be related tonuclear factor kappa-light-chain-enhancer of activated Bcells/caspase signal mediated induction of apoptotic celldeath.14 However, there is no available information to showthat BV-induced cytotoxic effects on human bladder cancercells. The present study investigated that BV-induced cyto-toxic effects and found out its possible molecular mecha-nism in TSGH-8301 human bladder cancer cells.

Methods

Chemicals and reagents

BV, dimethyl sulfoxide (DMSO), propidium iodide (PI),tris(hydroxymethyl)aminomethane hydrochloric acid (Tris-HCl), triton X-100 and penicillin-streptomycin werepurchased from Sigma Aldrich (St Louis, MO, USA).Bis-N,N,N′,N′-tetraacetic acid-AM and fluorescent probes2′, 7′-dichlorofluorescin diacetate, Fluo-3/AM and 3,3-dihexyloxacarbocyanine iodide (DiOC6) were obtained fromInvitrogen Life Technologies (Carlsbad, CA, USA). Anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-caspase-4and anti-PARP were obtained from Cell Signaling Technol-ogy (Danvers, MA, USA). Antibodies for Bcl-X, Bax, Bid,Endo G, X-linked inhibitor of apoptosis protein (XIAP),PARP, Fas, Fas ligand, TRAIL, PERK, ATF-6b IRE-1aGRP78, GADD153 and XBP-1 were purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA). Caspase-3,-8 and -9 substrates (PhiPhiLux-G1D1 for caspase-3,CaspaLux 8-L1D2 for caspase-8 and CaspaLux-M1D2 forcaspase-9) were purchased from OncoImmunin (Gaithers-burg, MD, USA). Materials and chemicals for electrophore-sis were purchased from Bio-Rad (Hercules, CA, USA).Cell culture dishes were purchased from Falcon (San Diego,CA, USA). RPMI 1640 medium, fetal bovine serum (FBS),L-glutamine and penicillin-streptomycin were obtainedfrom Invitrogen Life Technologies.

Cell culture

The human bladder cancer TSGH-8301 cell line was pur-chased from the Food Industry Research and Development

Institute (Hsinchu, Taiwan). The cells were placed into75 cm2 tissue culture flasks in RPMI 1640 medium supple-mented with 10% FBS, 2 mmol/L-glutamine, 100 units/mLpenicillin and 100 mg/mL streptomycin, and grown underhumidified 5% water and 95% air grown at 37°C and oneatmosphere.17 Subconfluent cells (80%) were passaged witha solution containing 0.25% trypsin and 0.02% ethylenedi-amine tetra-acetic acid (EDTA) (Invitrogen).

Determinations of cell morphologicalchanges and viability

TSGH-8301 cells in a 12-well plate at a density of 2 ¥ 105

cells/well was treated with 0, 1.25, 2.5, 5, 7.5 and 10 mg/mLof BV for 24 and 48 h. The cell morphological changes weredirectly examined and photographed in each well under aphase-contrast microscope at 200 ¥ magnification. The per-centage of viable cells was determined by using a PI exclu-sion method. Cells were collected, re-suspended in PBScontaining 4 mg/mL PI and then analyzed by a flow cytom-eter (Becton Dickinson FACSCalibur, Franklin Lakes, NJ,USA) as previously described.17,18 The cell viability wascalculated as the ratio of BV-treated cells to 1% DMSOvehicle-control cells.

Comet assay

TSGH-8301 cells (1 ¥ 105 cells) were treated with BV (0, 2.5,7.5 and 10 mg/mL) for 48 h. In brief, the cells were embeddedonto a 0.5% low melting point agar (Sigma Aldrich Corp.),and then immersed in an ice-cold cell lysis solution(2.5 mol/L sodium chloride [NaCl] mmol/L Tris-HCl,100 mmol/L sodium sulfide [Na2] EDTA and 1% (v/v) TritonX-100, adjusted to pH 10 with sodium hydroxide [NaOH]) at4°C for 1 h as described elsewhere.19 The slides were dena-tured with 300 mmol/L NaOH, 1 mmol/L Na2 EDTA atpH 13 at 4°C for 20 min and electrophoresis was carried outat 25 V, 300 mA for 25 min. Thereafter, the slides wereflooded with 0.4 mol/L Tris-HCl at pH 7.5 at 4°C for 15 minand then dried in methanol (Sigma Aldrich) for 5 min. Cel-lular DNA was stained with 4 mg/mL PI and examined undera fluorescence microscope as described previously.20,21

Recorded images were analyzed using the CometScore soft-ware available as freeware (TriTek., Sumerduck, VA, USA).

Determination of DNA fragmentation

Approximately 1 ¥ 106 cells/well of TSGH-8301 cells in6-well plates were exposed to 0, 2.5, 5, 7.5 and 10 mg/mL ofBV for 48 h. Total DNA was isolated from each sampleusing a DNA purification kit (Genemark Technology,Tainan, Taiwan) and resolved in an 1.8% agarose gel con-taining 0.3 mg/mL ethidium bromide (EtBr, Sigma Aldrich)in a 0.5X TBE buffer (0.045 mol/L Tris, 0.045 mol/L boric

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62 © 2011 The Japanese Urological Association

acid , 1 mmol/L Na2 EDTA acid, pH 8.3) after electrophore-sis for 45 min. The DNA bands were visualized, examined,and photographed as described previously.22

4�,6-diamidino-2-phenylindole(DAPI) staining

Approximately 2 ¥ 105 cells/well of TSGH-8301 cells in12-well plates were treated with 0, 2.5, 7.5 and 10 mg/mL ofBV and grown in 5% water and 95% air at 37°C for 48 h,while adding only DMSO (solvent) for the control regimen.The cells in each treatment and control were stained usingDAPI dye, and examined and photographed under fluores-cence microscopy as described elsewhere.23,24

Measurements of caspase-3, caspase-8 andcaspase-9 activities

Cells at a density of 2 ¥ 105 cells plated onto 12-well plateswere the conditions under which BV was individually addedto each well at final concentration of 10 mg/mL for 0, 1, 6,12, 24 or 48 h. The cells were trypsinized, centrifuged fromeach treatment and thereafter collected, washed twice withPBS and re-suspended in 50 mL of 10 mmol/L substratesolution (PhiPhiLux-G1D1 for caspase-3, CaspaLux 8-L1D2

for caspase-8 and CaspaLux-M1D2 for caspase-9) (OncoIm-munin, Gaithersburg, MD, USA), before being incubated at37°C for 60 min. The cells were washed again by PBS andanalyzed by flow cytometry as previously described.21,24

Determination of apoptotic-associatedproteins by Western blotting

TSGH-8301 cells were plated at a density of 1 ¥ 106 cells on6-well plates and exposed to 10 mg/mL of BV for 6, 12, 24and 48 h to examine the various protein levels correlated withapoptosis and endoplasmic reticulum (ER) stress signaling.The cells were harvested and washed with cold PBS beforebeing lysed in the PRO-PREP protein extraction solution(iNtRON Biotechnology, Seongnam, Gyeonggi-Do, Korea)and the total protein was collected from TSGH-8301 cellsbefore determining the levels of Bcl-X, Bax, Bid, caspase-9and caspase-3, Endo G, XIAP, PARP, Fas, Fas ligand,TRAIL,caspase-8, PERK, ATF-6b, ATF-6a, IRE-1a, GRP78,GADD153, XBP-1 and caspase-4. About 40 mg protein fromeach sample was resolved over 12% sodium dodecylsulfatepolyacrylamide gel electrophoresis and transferred toa polyvinylidene fluoride-nitrocellulose membrane sheet(Immobilon-P, Millipore, Bedford, MA, USA) and detectedby an Immobilon Western Chemiluminescent HRP substratekit (Millipore) according to the manufacturer’s instructions.The blot was soaked in blocking buffer (5% non-fat drymik/0.05% Tween 20 in 20 mmol/L Tris buffered saline atpH 7.6) at room temperature for 1 h then incubated with

individual monoclonal antibodies in a blocking buffer at 4°Covernight, followed by secondary antibody horseradish per-oxidase conjugate and then bands were detected usingChemiDoc XRS Plus (Bio-Rad, Hercules, CA, USA).18,25

Measurements of intracellular calcium iron(Ca2+) release and DYm in TSGH-8301 cells

Approximately 2 ¥ 105 cells/well of TSGH 8301 cells in12-well plates were pretreated with or without 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)(a Ca2+ chelator, Invitrogen) and exposed to 10 mg/mL ofBV for 0, 1, 3, 6, 12 and 24 h to determine the level of Ca2+

release, DYm and cell viability. Cells from controls andtreatments were harvested by centrifugation and werewashed twice by PBS. The cells were then re-suspended in500 mL of Fluo-3/AM (2.5 mg/mL) for Ca2+ and 500 mL ofDiOC6 (1 mmol/L) for DYm at 37°C in a darkroom for30 min. Consequently, the cells were analyzed immediatelyby flow cytometry as previously described.23,24,26

Immuno-staining and confocallaser microscopy

An assay to confirm whether the protein translocated frommitochondria to nuclei cells was performed as describedpreviously.22,24 Briefly, about 5 ¥ 104 cells/well of TSGH-8301 cells plated on 4-well chamber slides were treated withor without BAPTA and then treated without (control) or with10 mg/mL BV for 24 h. The cells were washed, then fixedwith 4% formaldehyde in PBS for 15 min, and permeabilizedwith 0.3%Triton-X 100 in PBS for 1 h containing blocking ofnon-specific binding sites using 2% BSA.Then the fixed cellswere incubated with anti-human Endo G and anti-humanAIFantibodies (1:100 dilution, respectively) overnight. The cellswere washed twice with PBS followed by staining with asecondary antibody (fluorescein isothiocianate-conjugatedgoat anti-mouse immunoglobulin G at 1:100 dilution) for40 min, followed by PI staining before the photomicrographswere taken by using a Leica TCS SP2 Confocal SpectralMicroscope (Leica Microsystems Heidelberg, Mannheim,Germany) as previously described.22,24

Statistical analyses

The difference between the control and BV-treated groupswere analyzed using the Student’s t-test, a probability ofP < 0.05 being considered significant.

Results

Effects of BV on cell morphological changesand percentage of viable TSGH-8301 cells

In order to examine the effects of BV on cell morphologicalchanges and the percentage of viable TSGH-8301 cells, the

Bee venom induces apoptosis in cancer


cells were treated with various doses of BV for 24 h andexamined by a phase-contrast microscope. The percentageof viable cells was measured by a flow cytometric assay asshown in Figure 1. The results indicate that BV-inducedmorphological changes occur in a dose-dependent effect(Fig. 1) and BV also decreases the percentage of viable cellsby about 12–65% at treatment between 1.25–10 mg/mL.These effects are both dose-dependent and time-dependent(Fig. 2).

Effects of BV on apoptosis were examinedby comet assay, DNA gel electrophoresisand DAPI staining in TSGH-8301 cells

Flow cytometric analysis already showed the sub-gap 1group presented in TSGH-8301 cells after exposure to BV(data not shown) and these findings are needed to re-confirmthe apoptotic cell death. Cells were treated with variousdoses (2.5, 5, 7.5 and 10 mg/mL) of BV for 48 h. As shownin Figure 3, these assays demonstrated that BV-inducedDNA damage was examined by comet assay (Fig. 3a) andDAPI staining (Fig. 3c) when compared with controlsample. Also, DNA fragmentation was examined by DNAgel electrophoresis (Fig. 3b) in TSGH-8301 cells. Takentogether, we concluded that BV-decreased the percentage ofviable TSGH-8301 cells and is mediated through DNAdamage and induction of apoptotic death.

Effects of BV on the activities of caspase-3,caspase-8 and caspase-9 in TSGH-8301 cells

To investigate whether caspase-3, caspase-8 and caspase-9are involved in BV-induced apoptosis, the activity ofcaspases were detected by using PhiPhiLux-G1D1 for

o μg/mL 1.25 μg/mL 2.5 μg/mL

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Fig. 1 The effects of bee venom (BV) on cell morphologicalchanges in TSGH-8301 cells. The cells were treated withvarious doses of BV for 24 and 48 h. The cells were examinedand photographed by a phase-contrast microscope and werecollected by centrifugation.

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Fig. 2 The effects of bee venom (BV) on the percentage oftotal viable TSGH-8301 cells. The cells were exposed to variousconcentrations of BV for 24 and 48 h and then were collectedby centrifugation. The viable cells were measured by flowcytometric assay. Each point is mean � SD of three experi-ments. *Significantly different from the control at P < 0.05.( ) 24 h; ( ) 48 h.

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Fig. 3 The effects of bee venom (BV) were examined on theoccurrence of apoptosis by (a) comet assay, (b) DNA gel elec-trophoresis and (c) 4′,6-diamidino-2-phenylindole staining inTSGH-8301 cells. Cells were incubated with various concentra-tions of BV for 48 h. *Significantly different from the control atP < 0.05.

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caspase-3, CaspaLux 8-L1D2 for caspase-8 and CaspaLux-M1D2 for caspase-9, respectively. The results presented inFigure 4 indicated the various caspases in BV-induceda rapid rise in caspase-3, caspase-8 and caspase-9activities. Also, we suggest that BV-provoked apoptosis isdone through caspase-dependent pathways in TSGH-8301cells.

Effects of BV on proteins levels inTSGH-8301 cells

In order to investigate whether apoptotic proteins areinvolved in BV-induced cell death, apoptosis-associatedproteins were measured by Western blotting. Results areshown in Figure 5 which indicated that the protein level ofBcl-X and Bid (Fig. 5a), XIAP (Fig. 5b) in TSGH-8301cells after treatment with BV were down-regulated, but theprotein levels of Bax (Fig. 5a), active form of caspase-8,caspase-3 and caspase-9, Endo G, PARP, Fas, Fas ligand,

TRAIL (Fig. 5b) were upregulated in BV-treated cells,which might lead to cell apoptosis. Moreover, BV alsoincreased the levels of PERK, ATF-6a, ATF-6b, IRE-1a,GRP78, GADD153, XBP-1 and caspase-4 (Fig. 5c) andthese are correlated with the ER stress signaling. Based onthese results, we suggest that BV-induced apoptosis aremediated through both the caspase-dependent andindependent death pathway and ER stress in TSGH-8301cells.

Effects of BAPTA on BV on Ca2+ release andDYm levels and percentage of viableTSGH-8301 cells

Cells after pretreatment with or without BAPTA wereexposed to BV for 24 h and harvested to examine the levelsof Ca2+ and DYm and the percentage of viable cells. Theseresults can be seen in Figure 6a–c. Significant changes inCa2+ release were observed from 1 h and up to 3 h following

Fig. 4 The effects of bee venom (BV)on the activities of (a) caspase-3, (b)caspase-8 and (c) caspase-9 in TSGH-8301 cells. About 2 ¥ 105 cells/well ofTSGH-8301 cells in 12-well plates weretreated with or without BV for 24 h. *Sig-nificantly different from the control andBV treatment at P < 0.05.

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the treatment of TSGH-8301 cells (Fig. 6a). BAPTA mightprotect against BV-induced cell death in TSGH-8301 cells(Fig. 6b). Also, BAPTA increased the protein levels ofcaspase-3, caspase-9, Bc1-XS, Bcl-XL and GADD153 whencompared to the BV alone treatment sample (Fig. 6c). BVsignificantly decreased the level of DYm in TSGH-8301cells and BAPTA is able to increase the BV-triggered loss ofDYm in TSGH-8301 cells (Fig. 6d). Based on these obser-vations, the loss of DYm involved intracellular Ca2+ afterBV exposure and the influence of cell viability in TSGH-8301 cells. Therefore, we suggest that intracellular Ca2+

might play a vital factor in BV-induced apoptosis and ERstress in TSGH-8301 cells.

Effects of BV on Endo G and AIF nucleartranslocation in TSGH-8301 cells

BV-treated TSGH-8301 cells pretreated with or withoutBAPTA were harvested to react with AIF and Endo G anti-bodies separately and PI staining, which were measuredwith confocal laser microscope. The results are shown inFigure 7, which indicate that BV treatment for 24 hincreased the levels of Endo G (Fig. 7a) and AIF (Fig. 7b),and BAPTA significantly decreased the levels of Endo G andAIF (Fig. 7c) in TSGH-8301 cells.

Discussion

Bladder cancer is one of the most common malignancies inthe world.27 Unfortunately, there are limited treatmentoptions for this disease, and its frequency is still high.28 Ourstudy is the first report to show that BV inhibits the growthof a human bladder cancer cell line, TSGH-8301 in vitro.BV-induced morphological changes decreased viable cellsand underwent apoptosis in a dose-dependent and time-dependent manner in TSGH-8301 cells. These observationswere verified by morphological changes of the nucleus(Fig. 1), DNA fragmentation (Fig. 3b), and DNA condensa-tion (Fig. 3c).

In order to further understand the molecular mechanismsinvolved in apoptosis caused by BV, the expression of theapoptosis-related proteins such as Fas, Bax, caspase-3 andcaspase-9 and changes in DYm were assessed in TSGH-8301 cells. It is well known that the ratio of Bax/Bcl-2 playsan important role in apoptosis. A high ratio of Bax to Bcl-2caused DYm loss, and Apaf-1, procaspase-9, cytochrome cand AIF proteins were released from the mitochondria tocytosol or nuclei and then caused apoptosis.29 Our resultsalso show that BV decreased the level of DYm (Fig. 5d) andincreased the level of cytochrome c release (data not show)in TSGH-8301 cells, which may be involved in the changes

Fig. 5 The effects of bee venom (BV)on the levels of apoptosis-associatedproteins levels in TSGH-8301 cells on (a)Bcl-X, Bax and Bid , (b) caspase-9 andcaspase-3, endonuclease G (Endo G),X-linked inhibitor of apoptosis protein(XIAP), poly-(ADP-ribose) polymerase(PARP), Fas, Fas ligand , TRAIL andcaspase-8 and (c) PERK, ATF-6b, IRE-1a,GRP78, GADD153, XBP-1 and caspase-4examined by Western blotting. Cells(1 ¥ 106/well) were treated with10 mg/mL of BV for 0, 6, 12, 24 and 48 h,and then total protein was prepared andinvestigated. The evaluation of the asso-ciated protein levels was carried out byWestern blotting.

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of Bax and Bcl-2 protein levels. The results from Westernblotting also showed that BV promoted the level of Bax(Fig. 6a). Figure 6 also shows that BV increased the nucleartranslocation of AIF and Endo G in TSGH-8301 cells.Therefore, we also suggest that BV-mediated apoptosis ofTSGH-8301 cells occurs via a mitochondria-dependentpathway.

It was reported that the mitochondrial pathway wasinvolved in the efflux of Apaf-1, procaspase-9 and cyto-chrome c from mitochondria to cytoplasm, leading to theactivation of caspase-3, caspase-6, and caspase-7.30,31 Inaddition, our findings also show that activation ofcaspase-9, caspase-3, and caspase-8 after the TSGH-8301cells were treated with BV. These occurrences of mitochon-drial apoptotic events play an important role inBV-mediated apoptosis. Our results show that BVincreased cleaved caspase-3, caspase-8 and caspase-9(Fig. 4b) and activity (Fig. 3) in TSGH-8301 cells. More-

over, the increase of cleaved caspase-9 led to the subse-quent activation of downstream caspase-3, an apoptoticexecutioner (Fig. 4b). The Western blot analysis showedthat BV inhibited the protein level of the XIAP (Fig. 4b). Itwas reported that the IAP family of proteins includecIAP-1, cIAP-2, and XIAP and that surviving that interac-tion and inhibiting the activity of processed caspases canimpede the apoptotic process.32,33 It was also reported thatcaspase-9 and caspase-3 are IAP targets, whereas caspase-8is not.34 Our results show that a pan-caspase inhibitor,Z-VAD-FMK, prevents apoptosis induction in BV-treatedTSGH-8301 cells (data not shown).

It is reported that intracellular Ca2+ interacts with ERstress and undergoes mitochondrial membrane depolariza-tion during cell apoptosis.35 ER stress-mediated apoptosiswas triggered by an accumulation of unfolded proteinresponse (UPR) and increasing the hallmarks of proteinlevels, including GADD153, GRP78 and caspase-4

Fig. 6 The effects of bee venom (BV) onthe production of intracellular calciumiron (Ca2+) and mitochondrial membranepotential (DYm) levels in TSGH-8301cells. The cells were pretreated withor without 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA) (Ca2+ chelator), and then incu-bated with 10 mg/mL of BV for varioustime periods. The cells were stained indi-vidually by (a) Flou-3/AM dye for Ca2+ and(b) 3,3-dihexyloxacarbocyanine iodidefor DYm and then examined and quanti-fied by flow cytometry. The cells weredetermined for the TSGH-8301 (c) viablecells and (d) protein levels. *Differsbetween BV and control. P < 0.05.

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1.00 0.72 0.52 0.72

1.00 0.61 1.31 0.82

1.00 1.01 1.00 1.01

Caspase-9

GADD153

Caspase-3 35 kDa

Bcl-XS

Bcl-XL

100

120

DYm

(%) 80

60

40

20

0–BV (10 μg/mL) – + +

–BAPTA

(10 μmol/L)+ – +

*



signals.36–38 The results (shown in Fig. 4c) indicate that theincreased protein levels of PERK, ATF-6a, ATF-6b, IRE-1a, GRP78, GADD153, XBP-1 and caspase-4 in BV-treatedTSGH-8301 cells were performed by Western blotting,finally leading to apoptotic cell death. The previous study byTu et al. reported that BV quickly induced calcium fluctua-tion and mitochondrial dysfunction, leading to the release ofAIF and Endo G in the nuclei in human melanoma A2058cells and this effect is achieved through Ca2+-dependent butcaspase-independent apoptotic signaling;39 Importantly, ourfindings are in agreement with the report,39 showing that BVstimulated intracellular Ca2+ release after 1 h exposure inTSGH-8301 cells (Fig. 5a). Alternatively, our study alsofound that pretreatment with BAPTA, a Ca2+ chelator inBV-treated TSGH-8301 cells, is able to increase cell viabil-ity (Fig. 5b), alter the protein abundance (caspase-3,caspase-9, Bcl-XS, Bcl-XL and GADD153) (Fig. 5c) andprevent loss of DYm (Fig. 5d). These results suggest thatBV-induced apoptosis is mediated through intracellularCa2+-modulated ER stress and the intrinsic death pathway inTSGH-8301 cells.

As shown in Figure 8, our results propose a model ofBV-induced apoptosis in human bladder cancer TSGH-8301cells. BV also induced the release of reactive oxygen speciesdata not shown) and Ca2+ and ER stress-mediated apoptoticdeath and it is further shown that adaptor proteins (Fas/CD95) act as a receptor for BV that promote the activationof initiator caspases such as caspase-8 and caspase-9 and

Endo G

Control

10 μg/mLBV

Control

Control

10 μg/mLBV

10 μg/mLBV

10 μg/mL BV+BAPTA (10 μM)

-FITC

AIF-FITC

AIF-FITC PI MERGE

Endo G-FITC PI MERGE

10 μm 10 μm 10 μm

10 μm 10 μm 10 μm

10 μm 10 μm 10 μm

10 μm 10 μm 10 μm

20 μm 20 μm 20 μm

20 μm 20 μm 20 μm

(a)

(b)

(c)

Fig. 7 The effects of bee venom (BV) on endonuclease G(Endo G) and apoptosis-inducing factor (AIF) nuclear transloca-tions in TSGH-8301 cells. Cells (5 ¥ 104 cells/well; 4-wellchamber slides) pretreated without or with 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BATPA)were incubated with 10 mg/mL of BV for 24 h. The cells werefixed and stained with primary antibodies. (a) Endo G. (b) AIF. (c)The levels of Endo G and AIF showing control, BV 10 mg/mL andBV 10 mg/mL+BAPTA 10 mmol/L, which were labeled with sec-ondary antibodies fluorescein isothiocyanate (FITC) and theproteins were detected by a confocal laser microscopicsystem. The nuclei were stained by propidium iodide (PI). Theareas of co-localization between AIF expressions and nucleiwere merged.

BAPTA

MitochondriaBcl-XL

Bax

Bid

UPR

ER stress

DNA damage

Bee venom

FasL TRAILFas

ROSCa2+ releaseDYm

Caspase-8

Caspase-9AIFEndo G

Genome digestion

XIAP

ApoptosisCaspase-3

Cytochrome c

Fig. 8 Proposed models for the molecular mechanisms ofbee venom (BV)-induced apoptosis in human bladder cancerTSGH-8301 cells. BV-induced endoplasmic reticulum (ER) stress(upregulation of GADD153) promotes the production of reac-tive oxygen species (ROS), intracellular calcium iron (Ca2+) andreduced mitochondrial membrane potential (DYm) level,leading to caspase-9 and caspase-3 activation before causingapoptosis in TSGH-8301 cells. XIAP, X-linked inhibitor of apop-tosis protein.

S-W IP ET AL.


activate effector caspase such as caspase-3 in TSGH-8301cells. Therefore, we demonstrated that BV-induced apopto-sis occurs through multiple pathways in human bladdercancer TSGH-8301 cells.

Acknowledgments

This work was supported by China Medical University(CMU99-ASIA-23) and in part by Taiwan Department ofHealth Clinical Trial and Research Center of Excellence(DOH100-TD-B-111–004).

Conflict of interest

None declared.

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Bee venom induces apoptosis through intracellular Ca2+-modulated intrinsic death pathway in human bladder cancer cells

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