Cell Biology International 33 (2009) 1165e1172www.elsevier.com/locate/cellbi

Targeting apoptosis in the hormone- and drug-resistant prostate cancercell line, DU-145, by gossypol/zoledronic acid combination

U.A. Sanli a, G. Gorumlu a, C. Erten a, M.K. Gul a, E. Cengiz a, Y. Kucukzeybek a,B. Karaca a, H. Atmaca b, S. Uzunoglu b, B. Karabulut a, R. Uslu a,*

a Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, 35100 Bornova, Izmir, Turkeyb Section of Molecular Biology, Department of Biology, Faculty of Science and Arts, Celal Bayar University, 45140 Muradiye, Manisa, Turkey

Received 10 February 2009; revised 11 June 2009; accepted 17 August 2009

Abstract

Possible synergistic cytotoxic and apoptotic effects of gossypol with zoledronic acid on DU-145 cells were explored, along with the rationalebehind any observed synergism due to the different apoptotic proteins involved. XTT cell proliferation assay was used to assess the cytotoxicity,and DNA fragmentation and caspase 3/7 activity were measured to verify apoptosis. Human Apoptosis Array was used to evaluate apoptoticproteins. The synergistic cytotoxic combination treatment had a versatile effect on apoptotic proteins, through inhibition of anti-apoptoticproteins (including cIAP-1, cIAP-2, survivin, livin, claspin, p53, p21, PON-2 and heat shock proteins) and concurrently the induction ofpro-apoptotic proteins (Bad, Bax, Fas, FADD, cleaved caspase-3 and p27). Both drugs had a minimal toxicity profile comparing to cytotoxicagents. Combination treatments targeting many pivotal apoptosis-related proteins may be a rationale option for treatment of prostate cancer.� 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Gossypol; Zoledronic acid; Synergy; Apoptosis; DU-145; Prostate

1. Introduction

Prostate cancer is the most common cancer in the Westernworld after skin cancer and the third most common death-causing disease in men (Jemal et al., 2008). Althoughdocetaxel chemotherapy has become the first-line standard ofcare for hormone-refractory prostate cancer (HRPC) based onthe results of 2 large randomized trials, PSA responses rarelyexceed 50% and median survival is <20 months (Beer et al.,2003). Some problems are also encountered during docetaxeltreatment, including serious side effects in most of thepatients. Other cytotoxic chemotherapy or radiotherapymodalities do not show any significant improvement in patientcondition due to the high recurrence of apoptosis-resistance inHRPC (Koivisto et al., 1998; Pilat et al., 1998). Thus, there is

* Corresponding author. Tel.: þ90 232 390 39 06; fax: þ90 232 374 73 21.

E-mail address: ruchan.uslu@ege.edu.tr (R. Uslu).

1065-6995/$ - see front matter � 2009 International Federation for Cell Biology.

doi:10.1016/j.cellbi.2009.08.006

no standard therapy available for the treatment of thehormone-independent stage of prostate cancer (Feldman andFeldman, 2001). This has led to exploration of novel alterna-tive therapeutic strategies such as using different cytotoxicagents, combination of androgen blockades and coapplicationof certain agents which may have the potential to increasesensitivity of cancer cells to chemotherapy and radiotherapy.In this regard, many phytochemicals, including gossypol (GP),with its diversified pharmacologic properties, have shownpromising results in inhibiting prostate cancer cells in vitro(Huang et al., 2006; Xu et al., 2005).

GP is a yellowish compound extracted from cotton plant(Gossypium species). In the late 1960s after several observa-tions were made on GP’s antifertility action in Chinese men,it attracted attention of many investigators looking at themechanisms responsible for this property (Coutinho, 2002).It was first demonstrated by Tuszynski and Cossu (1984) thatGP has anticancer effects against several tumor cell lines, themost sensitive of which are melanoma and colon carcinoma

Published by Elsevier Ltd. All rights reserved.

Abbreviations

HRPC hormone-refractory prostate cancerDMSO dimethyl sulfoxideXTT sodium 30-[1-(phenylaminocarbonyl)-3,4-tetra-

zolium]-bis (4-methoxy-6-nitro)CI combination indexCoI confidence indexIC50 50% inhibitory concentrationcIAP inhibitor of apoptosis proteinPON-2 paraoxonase-2FADD Fas-associated death domainHSP heat shock proteinGP gossypolZA zoledronic acid

1166 U.A. Sanli et al. / Cell Biology International 33 (2009) 1165e1172

cells. Although the data about the molecular mechanismsinduced and/or inhibited in GP exposed tumor cells is limited,GP was shown to induce apoptosis through inhibition of theanti-apoptotic Bcl-2 family members, and loss of mitochon-drial membrane potential with activation of caspase-3(Zhang et al., 2007; Mohammmad et al., 2005). But furtherapoptosis-related mechanisms need to be elucidated.

Bisphosphonates are widely used for the treatment of bonemetastases in prostate cancer. However, several in vitro studieshave also demonstrated anti-proliferative and cytostatic effectsof different bisphosphonates on prostate cancer cells (Fro-migue et al., 2000; Heikkila et al., 2002; Green, 2003). Thenitrogen-containing bisphosphonate, zoledronic acid (ZA), isthe most potent member of this family. Recent studies havefocused on the use of ZA as an anti-cancer agent (Dumonet al., 2004).

Since both of the agents have known anti-proliferativeeffects on human prostate carcinoma cells, and also minimalside effects, we investigated the possible synergistic effects ofGP in combination with ZA in the hormone- and drug-resistanthuman prostate cancer cell line, DU-145. This line is androgenreceptor negative, and drug resistant. It is an ideal model tostudy the effects and mechanisms of various anti-canceragents, as it is an aggressive model of metastatic humanprostate cancers. Furthermore, we investigated the mechanisticrationale for the observed synergistic effect of the combinationtreatment, which showed inhibition of many apoptosis-relatedproteins that are the most likely targets in cancer cells.

2. Materials and methods

2.1. Cell lines and reagents

Human DU-145 prostate cancer cell lines were obtainedfrom ICLC (Genova, Italy). The cells were grown as mono-layers in adherent cell lines and were routinely cultured inRPMI 1640 supplemented with 10% heat-inactivated fetalbovine serum, 1% L-glutamine, 1% penicillinestreptomycin in

75 cm2 polystyrene flasks (Corning Life Sciences, UK) andmaintained at 37 �C in a humidified air atmosphere with 5%CO2. Growth and morphology were monitored and cells werepassaged when they had reached 90% confluence. Cell culturesupplies were obtained from Biological Industries (KibbutzBeit Haemek, Israel). GP (>98% purity) was obtained fromSigma Chemical Co. (St. Louis, MO, USA). A stock solutionof GP (10 mM) was prepared in dimethyl sulphoxide(DMSO). ZA was a generous gift from Novartis Pharmaceu-ticals Inc (Basel, Switzerland). A stock solution of ZA wasprepared at a 10 mM in distilled water and aliquots werestored at �20 �C. Final dilutions were made immediatelybefore use, and new stock solutions were prepared for eachexperiment. DMSO concentration in the assay was <0.1%,which had no cytotoxic effect on the tumor cells. All otherchemicals, unless mentioned, were purchased from Sigma.

2.2. XTT viability assay

For the viability assay, after verifying cell viability usingthe trypan blue dye-exclusion test in a Cellometer automaticcell counter (Nexcelom Inc., Lawrence, MA, USA), cellswere seeded at 104/well in 200 mL into 96-well flat-bottommicrotitre plates with or without different concentrations ofthe drugs. Plates were incubated at 37 �C in a 5% CO2

incubator for the indicated periods. At the end of incubation,100 mL of tetrazolium salt (XTT, Roche Applied Science,Mannheim, Germany) was added to each well, and plateswere incubated at 37 �C for a further 4 h. Absorbance wasmeasured at 450 nm against a reference wavelength at650 nm using a microplate reader (DTX 880 MultimodeReader, Beckman Coulter, Fullerton, CA, USA). The meanof triplicate experiments for each dose was used to calculatethe 50% inhibitory concentration (IC50) and the combinationindex (CI) values.

2.3. Evaluation of apoptosis by DNA fragmentationanalysis

Apoptosis was measured with a Cell Death DetectionELISA Plus Kit (Roche Applied Science, Germany) accordingto the manufacturer’s instructions. The relative amounts ofmono- and oligo-nucleosomes generated from the apoptoticcells were quantified using monoclonal antibodies directedagainst DNA and histones by ELISA. Briefly, the cytoplasmicfraction of the untreated control, GP-, ZA- and combination-treated cells were transferred onto a streptavidin-coated plateand incubated for 2 h at room temperature with a mixture ofperoxidase conjugated anti-DNA and biotin labelled antihi-stone. The plate was washed thoroughly, incubated with2,29-azino-di-[3-ethylbenzthiazolinesulphonate] diammoniumsalt, and the absorbance was measured at 405 nm witha reference wavelength at 490 nm (DTX 880 MultimodeReader, Beckman Coulter, Fullerton, CA, USA).

Apoptosis was also confirmed by the Caspase-Glo 3/7Assay (Promega, Madison, WI, USA) according to the man-ufacturer’s instructions.

1167U.A. Sanli et al. / Cell Biology International 33 (2009) 1165e1172

2.4. Expression levels of apoptosis-related proteins

To determine the effect of combined treatment with GPand ZA on the expression levels of apoptosis-related multi-proteins in DU-145 cells, a R&D Human Apoptosis ArrayKit (R&D Systems, UK) was used according to the instruc-tion’s manual. Lysates from untreated controls and cells thatwere exposed to drugs were used. The list of proteinsassessed is shown in Table 1. The principle of the methodwas comprised of a nitrocellulose membrane that was coatedwith specific antibodies for each protein, forming an array.After blocking the membrane, the sample was added andincubated at room temperature. Protein detection wascompleted by incubation with a biotinylated antibody fol-lowed by horseradish peroxidase-conjugated streptavidin.The signals were detected using chemiluminescence in theKodak� Gel Logic 1500 imaging system (CarestreamMolecular Imaging, Newhaven, CT, USA). The spots werequantified by a computer-assisted system for image analysis(Koadarray� 2.6 software). Normalized intensities werecalculated from each array by first subtracting the localbackground from each spot and then normalizing by theaverage intensity of the arrays. The data were corrected forthe cell protein content of each well. The relative expressionlevel of each protein was calculated according to both spotpixel mean and a confidence index (CoI) of 0e100 assignedto the each spot by the Koadarray algorithms. The spot pixelmean value represents the background-subtracted totalintensity of each spot. The spot intensity is then given by thetotal of all the background-subtracted values within the spotarea. Pixels determined by Koadarray� as part of an artefactwere excluded. CoI is based on several variables, includingspot shape, intensity and homogeneity. A value of >50(�SD) indicates a reliable spot. Changes in protein expres-sion after exposure with the drugs were expressed as thefactor of decrease or increase.

2.5. Statistical analysis

All experiments were conducted in triplicate and the resultsexpressed as the mean� SD, with differences assessed

Table 1

List of apoptosis-related proteins in Human Apoptosis Array Kit, R&D

Systems�.

Bad TRAIL R1/DR4 PON-2

Bax TRAIL R2/DR5 p21/CIPI/CDNKIA

Bcl-2 FADD p27/Kip1

Bcl-x Fas/TNFSF6 Phospho-p53 (S15)

Pro-Caspase-3 HIF-1 alpha Phospho-p53 (S46)

Cleaved Caspase-3 HO-1/HMOX1/HSP32 Phospho-p53 (S392)

Catalase HSP 32 Phospho-Rad17 (S635)

cIAP-1 HSP 27 SMAC/Diablo

cIAP-2 HSP60 Survivin

Claspin HSP70 TNF RI/TNFRSFIA

Clusterin HTRA2/Omi XIAP

Cytochrome c Livin

statistically p values determined by Student’s t-test. Themedian dose-effect analysis was used to assess the interactionbetween agents. Determination of the synergistic vs additivevs antagonistic cytotoxic effects of the combined treatment ofcells with GP and ZA were assessed by Biosoft CalcuSynprogram (Ferguson, MO, USA). CI was used to expresssynergism (CI< 1), additive effect (CI¼ 1), or antagonism(CI> 1) (Chou and Talalay, 1984).

3. Results

3.1. Effects of GP and ZA on growth of human DU-145prostate carcinoma cells, in vitro

To evaluate the effects of GP and ZA on the growth ofhuman prostate cancer cells, cells were exposed to increasingconcentrations of GP and ZA (5e100 mM) for 24, 48 and 72 h.Both GP and ZA decreased cell proliferation in a time- anddose-dependent manner (data not shown). The highestcytotoxicity was observed at 72 h. The XTT test was used tocalculate IC50 value for each agent. The data showed that therewere also 12, 39, and 76% reduction in cell proliferation at5, 10, and 20 mM GP, respectively (Fig. 1A). The IC50 value ofGP on DU-145 cells was 10 mM.

DU-145 cells exposed to 10, 40, and 80 mM ZA showed 4,17, and 30% reduction in cell proliferation, respectively, andthe IC50 value of ZA was 90 mM (Fig. 1B).

Fig. 1. Effects of GP (1A) and ZA (1B) on the growth of DU-145 cells. The

IC50 concentration of gossypol was determined by XTT assay for each cell line

as described. The XTT assays were performed using triplicate samples in at

least 3 independent experiments.

Table 2

Combination index values of gossypol or ZA alone and their combination on

1168 U.A. Sanli et al. / Cell Biology International 33 (2009) 1165e1172

3.2. Synergistic effects of GP and ZA in human DU-145prostate cancer cells

growth inhibition of DU-145 cells. Combination index (CI) values were

calculated from the XTT cell proliferation assays, according to CalcuSyn�

software. The CI was used to express synergism (CI< 1), additive effect

(CI¼ 1), or antagonism (CI> 1), where CI< 0.5 represents strong synergism.

Concentration of drugs CI value Interpretation

Gossypol (5 mM)þ Zoledronic acid (5 mM) 0002 Strong synergism

Gossypol (10 mM)þ Zoledronic acid (5 mM) 0005 Strong synergism

p< 0.05.

After showing the cytotoxic effects of GP and ZA givenindividually to cells, we then examined the possible syner-gistic/additive effects of their combination. For this, we chosedifferent doses of GP and ZA which were less than the IC50

values for each drug. 5 mM GP and 5 mM ZA alone and theircombination resulted in 4, 12, and 77% reduction in cellproliferation in DU-145 cells (Fig. 2). The graph clearlydemonstrates the synergism between GP and ZA on prostatecancer cells.

We also calculated the combination index (CI) values forthese agents from the XTT cell proliferation assay by using theCalcuSyn programme. Strong synergistic effects of differentdose combinations were determined (Table 2).

3.3. Effect of sequential treatment

Since the previous findings demonstrated that tumor cellswith GP and ZA resulted in significant synergy at 72 h, weexamined the effect of sequential treatment of DU-145 cellswith either GP or ZA treatment alone before treatment withthe second agent. Pretreatment of tumor cells with GP for36 h, washing and then treating for an additional 36 h with ZAalso resulted in synergistic cytotoxicity. Also, pretreatment oftumor cells with ZA for 36 h and wash followed by treatmentfor an additional 36 h with GP resulted in synergistic cyto-toxicity (data not shown). Thus, significant synergistic cyto-toxicity occurs regardless of which agent is applied first.

3.4. Apoptotic effects of GP and ZA alone or incombination in human DU-145 prostate cancer cells

To induce apoptosis of prostate carcinoma cells by GP andZA alone or in combination, DU-145 cells were exposed toincreasing concentrations of drugs for 72 h and the levels ofmono- and oligo-nucleosome fragments were quantified usingCell Death Detection Kit. DNA fragmentation was synergistic

Fig. 2. Effects of the combination treatment of both agents on the growth of

DU-145 cells. Cytotoxicity was determined by the XTT assay after 72 h

culture. The XTT assays were performed using triplicate samples in at least 3

independent experiments.

(Fig. 3). The doses chosen for determination of apoptosis werestrongly synergistic as calculated by the CalcuSyn pro-gramme. The increase in DNA fragmentation was 1.06 or 1.96fold, for 5 mM ZA or 5 mM GP alone, individually andrespectively, and a massive 13.3 fold increase for the combi-nation treatment compared to untreated controls (Fig. 3).

3.5. Increase in apoptosis through caspase 3/7 enzymeactivity in GP and ZA alone or in combination in humanDU-145 prostate cancer cells

Caspases are commonly referred to as hangmen ofapoptosis. To evaluate whether caspases have a role in GP-and ZA-induced apoptosis, Caspase-Glo 3/7 Assay was usedto detect the any in enzyme activity. DU-145 cells wereexposed to different drug concentrations of GP and ZA.There were 2.83 and 5.34 fold increases in caspase 3/7enzyme activity in 5 mM GP or 5 mM ZA applied singly toDU-145 cells, respectively, compared to untreated controls,whereas combination of both resulted in a 22 fold increaseenzyme activity (Fig. 4). This clearly demonstrates thesynergistic apoptosis-inducing effect of GP and ZA treatmentin DU-145 cell lines.

3.6. Changes in apoptosis-related protein expression ofDU-145 cells exposed to a combination of GP and ZA

The data for apoptosis-related proteins after treatment witheither GP or ZA alone or the combination on DU-145 cells are

Fig. 3. Apoptotic effects of gossypol and ZA in combination or either agent

alone in DU-145 cells as evident from DNA fragmentation analysis. Combi-

nation treatment significantly enhanced apoptosis by DNA fragmentation.

Fig. 4. Relative luminescence unit (RLU) changes in caspase 3/7 enzyme

activity in gossypol and ZA combination, or either agent alone in DU-145

cells. The synergistic effect on the induction of apoptosis is clearly seen.

1169U.A. Sanli et al. / Cell Biology International 33 (2009) 1165e1172

given as fold changes in Table 3. Proteins were analyzedwhich had >1.5-fold increase in expression or >1.5-folddecrease in expression when compared with untreated control.

Combination treatment with GP (5 mM) and ZA (5 mM)resulted in significant changes in some of the importantapoptosis-related proteins ( p< 0.05, Table 3). Amongapoptosis inductive proteins, Bad, Bax and FADD, Fas/TNFS6, cleaved caspase-3 and p27/Kip1 were up-regulated( p< 0.05); these proteins are considered to be the mediatorsof apoptosis in cancer cells. Among the proteins known toinhibit apoptosis, some which play pivotal role as anti-apoptotic elements of the apoptotic machinery were inhibitedby the combination treatment ( p< 0.05). For example, fromthe members of the inhibitor apoptosis family (IAPs), cIAP-1,cIAP-2, survivin (BIRC5) and livin (BIRC7) were

Table 3

Spot pixel (SP) mean values of apoptosis-related proteins after treatment with GP a

protein was calculated according to both spot pixel mean values� standard deviatio

Koadarray� algorithms.

Protein S.P. Mean

value of P. Control

S.P. M

value

Bad 46.5� 1.69 50.5�Bax 23.3� 2.1 26.4�Cleaved caspase-3 24.5� 0.7 34.6�Claspin 24.4� 0.35 24.4�PON-2 40.2� 1.69 35.5�p21/CIPI/CDNKIA 41.9� 1.4 25.4�p27/Kip1 17.5� 0.7 25.9�Phospho-p53 (S15) 120.3� 0.84 95.4�cIAP-1 68.9� 2.8 59.6�cIAP-2 24.5� 0.7 21.8�Survivin 21.5� 0.7 18.5�Livin 25.8� 1.69 23.7�HSP27 116.5� 0.7 78.4�HSP60 40.2� 0.84 26.6�HSP32 47.0� 2.8 39.5�FADD 16.0� 0.7 24.8�Fas/TNFSF6 45.3� 1.06 48.8�

considerably inhibited (Table 4). Of the other anti-apoptoticmolecules, p53, which has a unique role in tumor progressionand drug resistance, is also diminished by the combinationtreatment as compared to the untreated control cells. Inaddition, members of the heat shock protein family (HSPs) econsidered as targets for the treatment in cancer e includingHSP 27, 32 and 60 were significantly reduced with GP and ZAin DU-145 cells. Claspin and PON-2 (paraoxonase-2), whichalso have anti-apoptotic and cell protective activity, werereduced by combination treatment.

4. Discussion

This data provides the evidence that treatment of hormone-and drug-resistant prostate cancer cells with a combination ofGP and ZA results in a significant synergistic cytotoxicactivity and apoptosis. This effect was observed in a dose- andtime-dependent manner.

Apoptosis is one of the major goals of cancer treatmentwhich is characterized by the cell shrinkage, blebbing of theplasma membrane, and chromatin condensation that areassociated with cleavage of DNA into ladders (Chen et al.,1996; Yu et al., 1999). Despite responses to some effectivetherapeutic approaches, decreased ability to undergo apoptosisas a result of acquired drug resistance can occur in many typesof human malignancies, including prostate cancer (Barry et al.,1990; Hoffman and Liebermann, 1994). Therefore furtherdevelopment in combining different anti-cancer agents thatcan induce or enhance apoptosis seems to be a promisingstrategy. In addition, since prostate cancer is mainly a diseaseof elderly men, the wide spectrum toxic side effects of cyto-toxic agents used for the treatment is an another limitation indaily oncologic practice. The combination of GP and ZA, bothof which have quite potent inhibitory effects on the

nd ZA alone, or the combination of both. The relative expression level of each

n (SD) and a confidence index from 0 to 100 assigned to the each spot by the

ean

of GP

S.P. Mean

value of ZA

S.P. Mean

value of combination

0.7 48.6� 0.84 84.5� 0.7

1.69 47.8� 0.7 87.3� 1.06

1.06 32.2� 1.69 86.4� 0.7

3.5 24.4� 0.7 13.4� 1.06

0.7 38.8� 1.4 22.8� 0.84

3.8 37.8� 1.2 12.8� 1.2

2.1 23.4� 1.2 80.5� 1.2

2.1 118.4� 0.7 32.4� 1.4

0.84 67.9� 0.7 39.6� 2.4

2.8 21.4� 2.8 15.5� 1.69

0.7 20.8� 1.4 5.3� 1.06

3.8 23.6� 2.1 14.9� 2.8

3.8 80.4� 3.8 24.5� 1.2

2.4 39.8� 2.8 21.5� 1.4

0.7 44.5� 1.69 14.6� 2.1

1.4 18.8� 1.06 78.5� 1.06

2.8 52.8� 0.7 106.1� 0.7

Table 4

Fold changes of apoptosis-related proteins after treatment with GP and ZA

alone or the combination of both. Proteins were analyzed which had at least

a 1.5-fold increase or decrease in expression compared with untreated control.

Protein Fold changes in protein levels

GP ZA Combination

Bad þ1.1 þ1.1 þ1.8

Bax þ1.1 þ2.1 þ3.8

Cleaved caspase-3 þ1.4 þ1.3 þ3.5

Claspin þ1.0 þ1.0 �1.8

PON-2 þ1.1 þ1.0 �1.8

p21/CIPI/CDNKIA þ1.6 þ1.1 �3.3

p27/Kip1 þ1.5 þ1.3 þ4.6

Phospho-p53 (S15) �1.3 �1.0 �3.7

cIAP-1 �1.2 �1.0 �1.7

cIAP-2 �1.1 �1.1 �1.6

Survivin �1.1 �1.0 �4.0

Livin �1.1 �1.1 �1.7

HSP27 �1.5 �1.5 �4.8

HSP60 �1.5 �1.0 �1.8

HSP32 �1.2 �1.1 �3.2

FADD þ1.5 þ1.2 þ4.9

Fas/TNFSF6 þ1.1 þ1.2 þ2.3

1170 U.A. Sanli et al. / Cell Biology International 33 (2009) 1165e1172

proliferation of cancer cells with very low toxic side effectscompared with conventional chemotherapeutics, might be oneof the solutions in the treatment of prostate cancer.

The study of apoptosis reveals that many oncogenes, tumorsuppressor genes and apoptosis-related proteins are involvedin this complex cell regulation system (Ashkenazi and Dixit,1999). While searching for the underlying mechanism ofenhanced cytotoxicity and apoptosis with the combinationtreatment, many pivotal proteins were found to be linked to theinduction of apoptosis in DU-145 prostate cancer cells by theprotein array method. The fold changes in apoptosis-relatedproteins after the exposure to the combination treatment werestatistically significant, thus indicating the synergistic activityof both drugs ( p< 0.05). Since apoptosis-related proteins areemerging targets in recent years, inhibition/upregulation ofthese proteins by GP/ZA treatment might open a new avenuefor the treatment of hormone- and drug refractory prostatecancer.

Bcl-2 proteins are the cornerstone proteins in response toapoptosis in many types of cancer cells. While some of theseproteins (e.g. bcl-2 and bcl-xL) are antiapoptotic, the others(including Bad, Bax and Bid) are pro-apoptotic (Huang et al.,2003; Lin et al., 2007). When there is an excess of pro-apoptotic proteins, cells are more sensitive to apoptosis. In ourcombination treatment procedures, both Bad and Bax wereinduced by 1.8 and 3.75 fold, respectively, compared tountreated controls, which clearly indicates induction ofapoptosis in DU-145 cells by the GP/ZA combination.Although the exact underlying mechanism of strong synergyachieved by this combination remains to be fully elucidated,there are some literature clues showing that the both drugsexert their antiapoptotic effect through the Bcl-2 familyproteins. This may be one of the main routes of the apoptoticmachinery that is an important intersection for both drugs in

their synergism (Karabulut et al., 2009; Caraglia et al., 2007;Zhang et al., 2007).

Within the cell, apoptosis activation occurs mainly throughseparate but related extrinsic and intrinsic apoptosis signalingpathways, both of which are potential targets for cancertreatment (Lee et al., 2008). The extrinsic pathway is activatedwhen apoptosis inducing ligands, such as Fas, bind to the cellsurface pro-apoptotic receptors and trigger caspase cascade.This binding results in cell death inducing signaling complex,which has an adaptor protein, Fas-associated death domain(FADD); consequently this complex activates an effectorcaspase, such as caspase-3, 6 and 7 (Johnstone et al., 2002).Thus, by upregulating these pro-apoptotic proteins, Fas, FADDand caspase-3, with GP-ZA treatment, induction of apoptosisis immediately triggered as a result of this targeted therapeuticapproach (Table 4).

Cyclin-dependent kinases (CDKs), together with cyclinsand their regulatory subunits, govern cell-cycle progression ineukaryotic cells. The synergistic combination of GPeZAresulted in activation and upregulation of p27/Kip1 in DU-145cells. p27/Kip1 is a member of a family of CDK inhibitors(CDIs) that bind to cyclin/CDK complexes and arrest celldivision. There is considerable evidence that p27/Kip1 playsan important role in multiple fundamental cellular processes,including cell proliferation, cell differentiation, and apoptosis(Sgambato et al., 2000).

Of the proteins that are significantly inhibited by thecombination treatment, p53 is of unique importance. Previousstudies demonstrated that both p53-dependent and p53-independent apoptosis occur in cancer cells in response tomany apoptosis inducers (Chao et al., 2000; Vogelstein et al.,2000). As a result, p53 is being pursued as a target for cancertherapy with novel therapeutic chemicals. The diverse phos-phorylation sites of p53 seem to play different roles in cellularstress. Phosphorylation of p53 at serine-15 and serine-20 sitescan activate and stabilize it; however, the phosphorylation ofp53 at serine-46 induces apoptosis (Liu et al., 2008). Inaddition, numerous p53-dependent target genes that play rolesas downstream effectors of p53 function in the cell have beenidentified. As an example, cyclin-dependent kinase inhibitorp21/Waf 1 is a direct p53 target and inhibition of it results incell cycle arrest (El-Deiry et al., 1993). We found bothphospho-p53 serine-15 and p21/Waf 1 proteins were signifi-cantly inhibited by the drug combination, which suggests thatthe combination treatment is a strong inducer of apoptosis inDU-145 cells.

Heat shock proteins (HSPs) have recently been identified astargets in cancer treatment. HSPs play important roles infolding, intracellular localization, and degradation of cellularproteins, but the cellular role of HSPs in cancers is notcompletely understood. However, some reports suggest thatparticularly high HSP 27, 32, 70 and 90 expression levels areassociated with a poor prognosis and drug resistance forcertain cancers, including carcinomas of the stomach, liver,and prostate, and also osteosarcomas (Kondo et al., 2007;Glaessgen et al., 2008; Banerji, 2009). Inhibition of theseproteins is associated with good response in cancer cells.

1171U.A. Sanli et al. / Cell Biology International 33 (2009) 1165e1172

Combination treatment did result in inhibition of HSPs 27, 32and 60 in prostate cancer cells (Table 4).

Survivin is a unique anti-apoptotic protein, being thesmallest member of the inhibitor apoptosis (IAP) genefamily. Survivin protein levels decrease 4 fold after thecombination treatment compared to untreated cells. Manystudies suggested that survivin (BIRC5) plays a major role incontrol of apoptosis and proliferation (Altieri, 2003; Li,2003). In addition, several studies have shown that survivin(BIRC5) is an important marker of an unfavourable course ofthe disease, indicating poor survival (Li, 2003; Adida et al.,1998). This property of survivin (BIRC5) expression isspecific to cancer cells and makes it a novel therapeutictarget for the effective treatment of cancer. In addition sur-vivin, another IAP family protein, livin (BIRC7), expressionis also diminished, a finding consistent with other reports;these have shown that downregulation of livin expressionincreases the rate of apoptotic cell death, reduces tumorgrowth potential, and sensitizes tumor cells to chemothera-peutic drugs (Wang et al., 2008).

5. Conclusions

Our results revealed that GP significantly enhances theanti-tumor activity of ZA in hormone- and drug-resistantprostate cancer cells, DU-145, by a synergistic manner. In thelast years, the understanding of apoptosis has provided thebasis for novel targeted therapies that can induce death incancer cells or sensitize them to cytotoxic agents. These novelagents include those targeting the pathways and proteins thatcontrol the apoptotic machinery are ideal drug models forcancer treatment. GP/ZA combination which target manypivotal apoptosis-related proteins in prostate cancer cells maybe one of the rationale options for hormone- and drug-resistantprostate cancer. However, further studies are needed in orderto elucidate cause and effect relationships between theseprotein alterations and treatment outcome. Since both agentshave minimal toxicity profile comparing to cytotoxic chemo-therapeutic agents, this combination may be good a candidatefor treatment of HRPC which is seen commonly in elderlypopulation.

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