European Journal of Pharmacology 641 (2010) 35–40

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology

Pamidronate inhibits antiapoptotic bcl-2 expression through inhibition of themevalonate pathway in prostate cancer PC-3 cells

Kazuhiro Iguchi, Yoshiki Tatsuda, Shigeyuki Usui, Kazuyuki Hirano ⁎Laboratory of Pharmaceutics, Gifu Pharmaceutical University, 1-25-4, Daigaku-nishi, Gifu, Gifu 501-1196, Japan

⁎ Corresponding author. Tel.: +81 58 230 8118; fax:E-mail address: hirano@gifu-pu.ac.jp (K. Hirano).

0014-2999/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ejphar.2010.05.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 February 2010Received in revised form 14 April 2010Accepted 6 May 2010Available online 25 May 2010

Keywords:Bcl-2PamidronateBisphosphonateProstate cancerMevalonate pathwayRap1

Bisphosphonates are expected to be efficacious to prevent the growth of metastatic cancer in bone tissue.Bone metastases often occur in patients with various cancers, such as breast, lung and prostate cancer. Bcl-2is a potent antiapoptotic protein and its expression is known to be closely related to its function. In thisstudy, to investigate the effect of bisphosphonates on cancer cells, we focused on bcl-2 expression inbisphosphonate-treated prostate cancer cells. First, we observed that bcl-2 mRNA expression in PC-3 wassignificantly inhibited to 12% of the control level by treatment with 100 μM pamidronate for 12 h. Inhibitionwas seen in cells treated with nitrogen-containing bisphosphonates, which have the ability to inhibitisoprenoid biosynthesis via the mevalonate pathway, but not in non-nitrogen-containing etidronate.Simultaneous treatment with geranylgeraniol, an intermediate of the mevalonate pathway, significantlyblocked inhibition by pamidronate, and treatment with geranylgeranyl transferase inhibitor GGTI-286 alsosuppressed bcl-2 mRNA expression. Furthermore, pamidronate inhibited the translocation of Rap1 protein tothe membrane fraction, suggesting that a change in posttranslational modification of Rap1 occurred intreated cells. Finally, knockdown of Rap1 by siRNA resulted in the inhibition of bcl-2 expression. Theseresults strongly indicate that bcl-2 reduction in bisphosphonate-treated PC-3 cells is dependent on inhibitionof the mevalonate pathway. The inhibitory effect of bisphosphonates on bcl-2 expression shown in prostatecancer cell line should be tested in animal experiments and clinical studies.

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1. Introduction

Bisphosphonates are pyrophosphate analogues, which show highaffinity for the hydroxyapatite of bone tissue, and are used effectivelyin the treatment of metabolic bone diseases including osteoporosis,Paget's disease and hypercalcemia associated with malignant tumor.Bisphophonates also reduce skeletal-related events, such as patho-logical fracture and bone pain, in cancer patients with bonemetastasis(Fleisch, 2002; Fleisch et al., 1969). In addition, clinical evidence hasaccumulated showing that bisphosphonates suppress bonemetastasisand finally prolong survival in cancer patients, especially those withbreast cancer. It was recently reported that zoledronic acid plusendocrine therapy improves disease-free survival in patients withestrogen-sensitive breast cancer (Gnant et al., 2009). Besides breastcancer, the beneficial effect of bisphosphonates in patients withprostate cancer is also highly expected since prostate cancer oftenmetastasizes to bone tissue, and clinical studies also support thetherapeutic benefits of bisphosphonate treatment in prostate cancerpatients (Asahi et al., 2003; Smith et al., 2001).

There are several proposed anticancer mechanisms of bispho-sphonates. For instance, direct actions on cancer cells, such as theinhibition of cell growth and invasion, are believed to be importantmechanisms. (Boissier et al., 2000; Iguchi et al., 2006b, 2007; Lee et al.,2001; Nishida et al., 2003; Virtanen et al., 2002). In addition,bisphosphonates reduce the expression of several genes related totumor cell functions (Asahi et al., 2006; Iguchi et al., 2006a; Valenti etal., 2006). In some cases, the action of bisphosphonates is explained bythe inhibition of enzymes in the mevalonate pathway, resulting in adecrease in the formation of the isoprenoid lipids farnesyl pyrophos-phate and geranylgeranyl pyrophosphate (Iguchi et al., 2006a,b, 2007;Oades et al., 2003; Virtanen et al., 2002). Isoprenoid lipids arenecessary for membrane localization of intracellular proteins, partic-ularly the small GTP-binding proteins Ras, Rho, Rac, and Rap, whichare involved in a number of signaling pathways (Walker and Olson,2005).

Bcl-2, which protects cells from death induced by several externalor internal factors, is a key regulator of the apoptotic pathway (Yip andReed, 2008). In hormone-refractory prostate cancer, a progressive andfatal form of the disease, bcl-2 is often overexpressed (Colombel et al.,1993; McDonnell et al., 1992). Overexpression of bcl-2 in prostatecancer decreases apoptosis induction by antitumor drugs in vitro andin vivo, and the poor prognosis in hormone-refractory prostate canceris thus considered to be associated with bcl-2 overexpression, and

36 K. Iguchi et al. / European Journal of Pharmacology 641 (2010) 35–40

targeting bcl-2 is a promising approach to treat advanced prostatecancer (Kang and Reynolds, 2009; Wang et al., 2008). In fact, a clinicalstudy has shown a tumor-suppressive effect of R-(-)-gossypol aceticacid (AT-101), a smallmolecule interactingwith bcl-2 familymembers(Liu et al., 2009).

There are several reports regarding the inhibitory effect on bcl-2expression by bisphosphonates (Asahi et al., 2006; Oades et al., 2003;Senaratne et al., 2000). In this study, to elucidate the molecularmechanism underlying the inhibition of bcl-2 expression by bispho-sphonates, we examined whether the inhibition of bcl-2 expressionwould be mediated by the inhibition of isoprenoid lipid formation.

2. Materials and methods

2.1. Materials

Pamidronate, alendronate, risedronate and etidronate were pur-chased from LKT laboratories (St. Paul, MN, USA). Methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and N-4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl-(L)-leucine methyl ester (GGTI-286) werefrom Calbiochem (La Jolla, CA, USA). Farnesol and geranylgeraniolwere from Sigma Aldrich (St. Louis, MO, USA) and MP Biomedicals(Aurora, OH,USA), respectively. Docetaxel andpacritaxelwere obtainedfrom Toronto Research Chemicals (North York, ON, Canada) and SigmaAldrich, respectively. Anti-bcl-2 mouse monoclonal and anti-glycer-aldehydes-3-phosphate dehydrogenase (GAPDH) goat polyclonal anti-bodies were from Sigma Aldrich and Santa Cruz Biotechnology (SantaCruz, CA, USA), respectively. Anti-Ras, anti-Rho, and anti-Rap1 mousemonoclonal antibodies were purchased from BD Bioscience (FranklinLakes, NJ, USA). Rap1 small interfering RNA (siRNA) and control siRNAwere obtained from Santa Cruz Biotechnology. Human PC-3 prostaticcarcinoma cellswere from theAmerican Type Culture Collection (ATCC:Manassas, VA, USA). All other chemicals were of analytical grade.

2.2. Cell culture

Human prostate PC-3 cells were maintained in RPMI-1640medium containing 10% fetal bovin serum (FBS) and antibioticsunder a humidified atmosphere with 5% CO2 at 37 °C.

2.3. RNA isolation, reverse transcription-PCR (RT-PCR) and quantativeRT-PCR

Total RNAwas extracted using TRIzol reagent (Invitrogen, Carlsbad,CA, USA) and then first-strand complementary DNA was synthesizedfrom 5 μg total RNA using SuperScript III (Invitrogen), as describedpreviously (Iguchi et al., 2007). PCR was performed under thefollowing conditions with specific primer pairs: 25 cycles of 1 min at94 °C, 1 min at 60 °C, and 1 min at 72 °C for Rap1a; 19 cycles of 30 s at94 °C, 30 s at 60 °C, and 1 min at 72 °C for GAPDH. After PCR, thereaction products were resolved on 1.75% agarose gels and visualizedwith ethidium bromide.

Real-time monitoring of PCR reactions was performed using theiCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories,Hercules, CA, USA) with the SYBR Premix Ex Taq (Takara, Shiga,Japan). At the end of PCR, dissociation curve analysis was performedto examine the specificity of the PCR product. The GAPDH house-keeping gene was used for normalization of bcl-2 mRNA expression.PCR was performed under the following conditions: 35 cycles of 5 s at95 °C and 20 s at 63 °C, for bcl-2; 35 cycles of 10 s at 95 °C and 20 s at60 °C for GAPDH.

The primers used in this studywere 5′-AGTGCCAGCATTCCAGACTT-3′and 5′-TTTCTCCAGGGACTTGTGC-3′ for RT-PCR analysis of Rap1a, 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and 5′-CATGTGGGCCAT-GAGGTCCACCA-3′ for RT-PCR analysis of GAPDH, 5′-CTGCGAA-

GAACCTTGTGTGA-3′ and 5′-TGTCCCTACCAACCAGAAGG-3′ for real-timeRT-PCR analysis of bcl-2, 5′-CCAGCAAGAGCACAAGAGGA-3′ and 5′-GCAACTGTGAGGAGGGGAGA-3′ for real-time RT-PCR analysis of GAPDH.

2.4. Preparation of cell lysate and western blot analysis

Western blotting was performed as described previously (Tatsudaet al., 2010), with minor modifications. For detection of bcl-2 in totalcell lysate, cells were harvested with cold phosphate-buffered saline(PBS), and lysed by five cycles of freezing and thawing. For detectionof small G proteins, subcellular fractionation was performed using aProteoExtract Subcellular Proteome Extraction Kit (Calbiochem). Theprotein concentration of each sample was quantified by the Bradfordassay and 10 μg sample was separated by SDS-PAGE (12.5%) andtransferred to a polyvinylidene fluoride membrane. After beingblocked with 1% bovine serum albumin in PBS/0.1% Tween-20 for1 h at room temperature, the membrane was incubated with the firstantibody overnight at 4 °C. After repeated washing, the membranewas incubated with the secondary antibody, conjugated horseradishperoxidase, for 2 h at room temperature. Immunoreactive bands weredetected using an ECL system or ECLplus system (GE Healthcare, LittleChalfont, Buckinghamshire, UK).

2.5. siRNA transfection

Rap1 siRNA (sc-36384) or control siRNA (sc-36869) was trans-fected at 100 nM concentration with Lipofectamine 2000 (Invitrogen)into PC-3 cells cultured in serum free RPMI-1640. After 20 min, themediumwas removed and fresh medium supplemented with 10% FBSwithout antibiotics was added. The transfected cells were incubatedfor 7 days and total RNA was isolated.

2.6. Cell viability assay

Cell growth was evaluated by measuring fluorescence intensity inthe presence of alamar blue (Wako, Osaka, Japan), using a modifiedprotocol described elsewhere (Iguchi et al., 2007). Briefly, cells wereseeded in 96-well plates (Sumilon, Tokyo, Japan) at a density of6×102 cells/well in culture medium, incubated overnight, and pre-treated with or without 100 μM bisphosphonate for 24 h. After pre-treatment, the medium was replaced with fresh medium containing0.2 or 2 nM paclitaxel or docetaxel and further incubated for 72 h.Alamar blue solution was then added to the medium and the plateswere incubated for 1 h. Fluorescence intensity was measured using aPOLARstar Galaxy (Millipore, Bedford, MA, USA) with excitation andemission wavelengths at 544 nm and 612 nm, respectively.

2.7. Statistical analysis

The significance of differences between multiple groups wasdetermined by one-way analysis of variance followed by the Dunnetttest. The significance of differences between two groups was assessedby unpaired Student's t-test. A P-value less than 0.05 was consideredsignificant.

3. Results

3.1. Inhibition of bcl-2 expression by bisphosphonate in PC-3 cells

Firstly, we examined the effect of pamidronate on bcl-2 mRNAexpression by real-time RT-PCR in prostate cancer PC-3 cells. The cellswere incubated with the indicated concentrations of pamidronate for24 h, or with 100 μM pamidronate for various periods. As shown inFig. 1A and B, bcl-2 expression was significantly decreased to about12% of the control after 24 h of 100 μM pamidronate treatment andthe decrease occurred in a time-dependent manner. Furthermore, the

Fig. 1. Effect of pamidronate on bcl-2 expression in PC-3 cells. (A) Cells were treatedwith the indicated concentrations of pamidronate for 24 h. (B) Cells were treated with100 μM pamidronate for the indicated times. After incubation, total RNA was isolatedand subjected to real-time RT-PCR analysis. The results were normalized for GAPDHmRNA levels. **Pb0.01, ***Pb0.001 vs. control. (C) Cells were treated with 100 μMpamidronate for 4 days. After incubation, the cells were collected with PBS and lysed byfive cycles of freezing and thawing, and then the lysates were subjected to Westernblotting using anti-bcl-2 or anti-GAPDH antibody.

37K. Iguchi et al. / European Journal of Pharmacology 641 (2010) 35–40

protein expression of bcl-2 was also inhibited by pamidronate after4 days of treatment.

3.2. Inhibition of bcl-2 expression by pamidronate is mediated by itsinhibitory effect of protein prenylation

To determine the mechanism behind the decrease in bcl-2expression by pamidronate, we examined the effect of calciumchelators, ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), onbcl-2 expression in PC-3 cells since bisphosphonates chelate divalentcations. As shown in Fig. 2A, 100 μM or 200 μM EDTA or EGTA had nosignificant effects on bcl-2 expression, suggesting that bcl-2 inhibitionby pamidronate was not caused by the chelation of divalent cations.

Next we focused on the inhibitory effect of themevalonate pathwayby bisphosphonates. Nitrogen-containing bisphosphonates inhibitfarnesyl pyrophosphate synthase or geranylgeranyl pyrophosphatesynthase in the mevalonate pathway, resulting in the inhibition ofisoprenoid formation and isoprenoid transfer to protein (Fleisch, 2002;Russell et al., 2007). To determinewhether inhibition of themevalonatepathway is involved in the inhibition of bcl-2 expression by pamidro-nate, we examined the effect of nitrogen-containing bisphosphonates,precursors of isoprenoid intermediates, and isoprenoid transferaseinhibitors. Fig. 2B shows that the decrease in bcl-2 expression wasobserved in cells treated with nitrogen-containing bisphosphonate,

pamidronate, alendronate and risedronate, but not non-nitrogen-containing etidronate. As shown in Fig. 2C, coincubation withpamidronate and geranylgeraniol (precursor of geranylgeranyl pyro-phosphate) blocked the inhibition of bcl-2 expression by pamidronate.Moreover, a geranylgeranyl transferase inhibitor, GGTI-286, significant-ly suppressed bcl-2 expression (Fig. 2D). Coincubationwith a precursorof farnesyl pyrophosphate, farnesol, and a farnesyl transferase inhibitor,FTI-277, showed no effect on bcl-2 mRNA expression. These resultssuggest that pamidronate-induced bcl-2 suppression is associated withthe inhibition of geranylgeranyl pyrophosphate synthesis in themevalonate pathway.

3.3. Inhibition of bcl-2 expression by pamidronate is involved inRap1 signaling

Small G proteins, such as Ras, Rho, and Rap, are modifiedposttranslationally by isoprenoid lipids and then activated (Walkerand Olson, 2005). Since the depletion of intracellular geranylgeranylpyrophosphate inhibits the posttranslational activation of small Gprotein, we examined the activation status of Ras, Rho and Rap1 inpamidronate-treated PC-3 cells. Small G proteins translocate fromcytosol to membrane when they are posttranslationally modified byisoprenoids, so the activation status can be estimated by comparingthe levels of small G protein in cytosol and membrane fractions.Western blot analysis showed that Rap1 was detected mainly in themembrane fraction in control cells, whereas the level of Rap1 in themembrane fraction was decreased in response to pamidronatetreatment and that in the cytosolic fraction was increased (Fig. 3A).The level of Rho in the cytosolic fraction seemed to slightly increase inresponse to pamidronate treatment. Therewere no significant changesin the level of Ras between control and treated cells. These resultssuggest that pamidronate treatment mainly affects Rap1 posttransla-tional modification in PC-3 cells. In addition, no significant transloca-tion of Rap1, Ras, and Rho was observed in non-nitrogen-containingetidronate-treated PC-3 cells, implying that the translocation of Rap1by pamidronate resulted from inhibition of the mevalonate pathway(Fig. 3A).

We next performed a knockdown experiment using Rap1 siRNA toexamine the involvement of Rap1 signaling in bcl-2 expression.Knockdown of Rap1 was confirmed by RT-PCR analysis (Fig. 3B).Fig. 3C shows that bcl-2 mRNA expression in Rap1 siRNA transfectedPC-3 cells was decreased to about 20% of that of control cells. Theseresults indicate that the inhibition of bcl-2 expression in pamidronate-treated PC-3 cells was caused by the inhibition of Rap1 activation.

3.4. Bisphosphonate increases cytotoxicity of antitumor drugs

Bcl-2 is an important antiapoptotic molecule, and its expressionwas decreased in bisphosphonate-treated cells. Bcl-2 prevents celldeath induced by apoptosis-inducing agents, including chemothera-peutic drugs (Yip and Reed, 2008). Hence, we tested whetherpretreatment with bisphosphonate would affect the viability ofdocetaxel or paclitaxel-treated PC-3 cells. As shown in Fig. 4A, theviability of docetaxel-treated PC-3 cells pretreated with alendronatewas significantly lower than that of cells not pretreated withalendronate. The viability of paclitaxel-treated PC-3 cells pretreatedwith alendronate was also observed to be more potent than controlcells. A similar pattern was seen in cells pretreated with risedronate(Fig. 4B). Since the cytotoxic effect of pamidronate on PC-3 cells after4 days was severe, we could not evaluate the combination effect ofpamidronate and the drugs on cell viability.

4. Discussion

In this study, we found that the inhibition of antiapoptotic bcl-2expression by bisphosphonates was mediated through inhibition of

Fig. 2. Involvement of themevalonate pathway in pamidronate-induced bcl-2mRNA reduction in PC-3 cells. (A) Cells were treated with EDTA or EGTA for 24 h. (B) Cells were treatedwith 100 μMof various bisphosphonates for 24 h. (C) Cells were treated with 20 μM farnesol or geranylgeraniol with or without 100 μMpamidronate for 24 h. (D) Cells were treatedwith 20 μM FTI-277 or GGTI-286 for 24 h. After incubation, total RNA was isolated and subjected to real-time RT-PCR analysis. The results were normalized for GAPDH mRNA levels.*Pb0.05, **Pb0.01, ***Pb0.001 vs. control, †Pb0.05 vs. pamidronate.

38 K. Iguchi et al. / European Journal of Pharmacology 641 (2010) 35–40

the mevalonate pathway in prostate cancer PC-3 cells. Nitrogen-containing bisphosphonates act as inhibitors of the mevalonatepathway, resulting in the inhibition of protein prenylation (Fleisch,2002; Russell et al., 2007). Pamidronate showed that Rap1, a small Gprotein, translocated to the cytosol from the membrane in PC-3 cells.

Fig. 3. Regulation of Bcl-2mRNA expression by Rap1 in PC-3 cells. (A) Cells were treatedwith 100 μM pamidronate (left panels) or etidronate (right panels) for 24 h. Cytosoland membrane fractions were prepared and 10 μg samples were subjected to Westernblotting using anti-Rap1, Ras, or Rho antibody. (B, C) Cells were treated with 100 nMRap1 siRNA oligonucleotide for 7 days. After incubation, total RNA was isolated andsubjected to RT-PCR analysis (B, duplicate samples are shown) or real-time RT-PCRanalysis (C). The results were normalized for GAPDHmRNA levels. **Pb0.01 vs. control.

Fig. 4. Effect of anticancer drugs on cell viability with or without bisphosphonates in PC-3 cells. Cells were pretreated with or without (A) alendronate and (B) risedronate for24 h. After incubation, the cells were treated with docetaxel or paclitaxel for 72 h. Cellviability was examined by alamar blue assay. Values are the means±S.D. from fivedifferent incubations. *Pb0.05, ***Pb0.001 vs. control, †Pb0.05 vs. pamidronate.

39K. Iguchi et al. / European Journal of Pharmacology 641 (2010) 35–40

Moreover, siRNAmediated-knockdown of Rap1 led to the reduction ofbcl-2 mRNA expression. These findings suggest that the inhibition ofbcl-2 expression by bisphosphonates is involved in the inhibitoryeffect of Rap1 activity.

There are several reports demonstrating the increased inhibitoryeffect of bisphosphonates on the growth of cancer cells treated withtaxanes (docetaxel and paclitaxel) (Fabbri et al., 2008; Kim et al.,2005; Lu et al., 2008). We also observed an increased cytotoxic effectby the combination of these drugs in PC-3 cells. Previous studies havesuggested mechanisms underlying the cytotoxic effect of bispho-sphonates; for example, (i) nitrogen-containing bisphosphonatesinduced changes in cell cycle distribution in several cancer cell lines(Lee et al., 2001; Ory et al., 2007), (ii) bisphosphonates modulate cellsurvival pathways, the MEK/ERK and PI3K/Akt pathways, in leukemiaHL-60, osteosarcoma MG-63, and prostate cancer LNCaP cells (Fabbriet al., 2008; Inoue et al., 2005; Nishida et al., 2003), and (iii)bisphosphonates inhibit antiapoptotic bcl-2 expression, as in thepresent study. The mechanism of cytotoxicity by docetaxel andpaclitaxel is explained by the inhibition of cell cycle progression in theG2/M phase by promoting tubulin polymerization and microtubulestabilization; therefore, taxane and bisphosphonates are likely to havedifferent mechanisms of antitumor activity. We found that bcl-2expression was suppressed by treatment with nitrogen-containingbisphosphonates in PC-3 cells, although it is not clear whether theincreased cytotoxic effect of taxane on cell viability was directlyinvolved in the downregulation of bcl-2 by bisphosphonates. Theresults of the combination of bisphosphonates and docetaxel on celldeath are interesting to improve prostate cancer treatment sincedocetaxel is well-tolerated and clinically active in patients withadvanced prostate cancer (Bradley and Hussain, 2008).

We performed experimentswith bisphosphonate at a concentrationof 100 μM. The concentration of pamidronate in serum is significantlylower than that used in this studyafter a 90-mgdosevia 2-hour infusion,which is the standard of care for cancer patients. However, because ofthe high affinity of bisphosphonate for bone hydroxyapatite, theconcentration at the site of bone resorption stimulated by tumor cellsis considered to be much higher than in serum, and the concentrationthere is speculated to reach 0.8 mM (Sato et al., 1991). Therefore,prostate cancer cellsmetastasized to bone are expected to be exposed tosufficiently high concentrations of bisphosphonate.

An earlier study suggested that bisphosphonate-mediated bcl-2downregulation is caused by modification of the p53 phosphorylationstatus (Ory et al., 2007). The experiment showed that zoledronic acidinhibited the phosphorylation of p53, leading to inhibition of bcl-2expression in osteosarcomaSRGA cells, which express functionalwild-type p53, while the reduction was not seen in osteosarcoma MG-63cells and Saos-2 cells with mutated or null p53. In our study, we haveshown the effect on bcl-2 expression in p53-null PC-3 cells, implyingthat p53 phosphorylation does not explain the effect of pamidronateon bcl-2 expression in PC-3 cells. In addition, pamidronate suppressedbcl-2 mRNA expression in DU145 cells, which have mutated p53,suggesting again that there are other mechanisms responsible for thesuppression of bcl-2 expression by bisphosphonates (data not shown).Here we have revealed that the regulation of bcl-2 expression iscontrolled by Rap1 activity, and this is the first report showing thatRap1 signaling is involved in bcl-2 expression.

There are two conceivable mechanisms by which Rap1 inactiva-tion reduces bcl-2 mRNA expression. (i) Bcl-2 expression is reportedto be regulated by the ERK and Akt pathways (Pugazhenthi et al.,2000; Wang et al., 2006), which are activated by Rap1 signaling(Raaijmakers and Bos, 2009; Wang et al., 2001); therefore, Rap1suppression may inhibit the ERK and Akt pathways, in turn reducingbcl-2 expression. (ii) Rap1 is known to antagonize Ras function viacompetitive interference. Inhibition of Ras farnesylation is found tolead to the induction of bcl-2 expression (Mazzocca et al., 2003);hence, Rap1 inactivation may result in Ras activation, and conse-

quently decrease bcl-2 expression. In the present study, we did notperform any experiments to verify the mechanisms described above.Testing the models therefore remains a future issue.

We found that bcl-2 mRNA expression was decreased afterpamidronate treatment for 12 h, while the decrease in the proteinlevel of bcl-2 was observed 4 days after pamidronate treatment. Theresult was consistent with that of previous reports using differentcells; the decrease in bcl-2 protein expression by 100 μMpamidronatewas observed 3 to 4 days after treatment in breast cancer MDA-MB-231 cells (Senaratne et al., 2000). Furthermore, a similar result wasshown earlier with a different stimulus, namely, the decrease in themRNA level of bcl-2 by 4-hydroxytamoxifen was as early as 24 h aftertreatment, while the protein level remained unchanged for 96 h inlymphoma EL4 sublines (Taylor et al., 1996). The protein level of bcl-2is known to be regulated by several stimuli through posttranslationalmodifications such as phosphorylation and ubiquitination, butwithout stimuli, the turnover of bcl-2 protein is relatively long(Azad et al., 2006; Li et al., 2004). Taken together, the time lagbetween the changes in protein andmRNA expression of bcl-2 may beexplained by the turnover rate of bcl-2 protein.

The effects of bisphosphonates on bcl-2 expression were mainlyexamined using a PC-3 prostate cancer cell line, but the effect on theexpression is not restricted to a single cell line. Besides PC-3 cells, wealso found that bcl-2 expression was significantly decreased afterpamidronate treatment in prostate cancer DU145 cells (data notshown). In addition, Lu et al. reported that zoledronate inhibited bcl-2expression in an animal model (Lu et al., 2008). It would beinteresting to investigate the effects of bisphosphonates on anti-apoptotic bcl-2 expression in prostate cancer patients.

5. Conclusions

In summary, we have shown that bcl-2 expression is inhibited bybisphosphonates in prostate cancer cells. The inhibition of bcl-2expression by bisphosphonates is thought to be mediated through theinhibition of geranylgeranyl pyrophosphate synthesis via the meva-lonate pathway. Increased expression of antiapoptotic bcl-2 protein isrelated to the progression of various types of cancer; bcl-2 inhibitionis therefore believed to be an attractive target for cancer intervention(Kang and Reynolds, 2009). Our study showed that combinationtreatment with bisphosphonates and taxane resulted in increasedcytotoxicity in PC-3 cells. These findings concerning the inhibitoryeffect of bisphosphonates on bcl-2 expression may support their usefor the treatment of prostate cancer.

Acknowledgement

This study was supported by a grant-in-aid for young scientists(no. 18791130) from the Ministry of Education, Culture, Sports,Science, and Technology of Japan.

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