Zoledronic acid in combination with serine/threonine phosphatase inhibitors induces enhanced cytotoxicity and apoptosis in hormone-refractory prostate cancer cell lines by decreasing the activities of PP1 and PP2A

Authors

  • Yalcin Cirak,

    1. Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Bornova, Izmir
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  • Umut Varol,

    1. Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Bornova, Izmir
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  • Harika Atmaca,

    1. Section of Molecular Biology, Department of Biology, Faculty of Science and Arts, Celal Bayar University, Muradiye, Manisa, Turkey
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  • Asli Kisim,

    1. Section of Molecular Biology, Department of Biology, Faculty of Science and Arts, Celal Bayar University, Muradiye, Manisa, Turkey
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  • Canfeza Sezgin,

    1. Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Bornova, Izmir
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  • Bulent Karabulut,

    1. Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Bornova, Izmir
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  • Selim Uzunoglu,

    1. Section of Molecular Biology, Department of Biology, Faculty of Science and Arts, Celal Bayar University, Muradiye, Manisa, Turkey
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  • Ruchan Uslu,

    1. Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Bornova, Izmir
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  • Burcak Karaca

    Corresponding author
    1. Division of Medical Oncology, Tulay Aktas Oncology Hospital, School of Medicine, Ege University, Bornova, Izmir
      Burcak Karaca, Ege University School of Medicine, Tulay Aktas Oncology Hospital, 35100 Bornova, Izmir, Turkey. e-mail: burcak.karaca@ege.edu.tr; karacaburcak@hotmail.com
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Burcak Karaca, Ege University School of Medicine, Tulay Aktas Oncology Hospital, 35100 Bornova, Izmir, Turkey. e-mail: burcak.karaca@ege.edu.tr; karacaburcak@hotmail.com

Abstract

What's known on the subject? and What does the study add?

Prostate cancer is the second most common cancer diagnosed among elderly men. Current standard of care with surgery, chemotherapy or radiation in prostate cancer patients are of limited efficacy, especially in the androgen refractory state of the disease, and unfortunately metastatic disease remains incurable. Skeletal metastases are the most common site for metastases for prostate cancer and bisphosphonates have been widely used for the treatment of morbidity due to skeletal related events. Zoledronic acid (ZA) is the most potent member of the nitrogen containing new generation bisphosphonate (N-BPs) family. Okadaic acid (OA) and Calyculin A (CA) are the most commonly used inhibitors of PP1 and 2A. OA, extracted from common black sponge Halachondria okaddai is a potent inhibitor of protein phosphatases, PP1 and PP2A, and CA was isolated from another marine sponge, Discodermia calyx. Therapies based on combinations of chemotherapeutics with phosphatase inhibitors that target signaling pathways within the cell with different mechanisms of action, may be useful for increasing therapeutic effect and also diminish toxic side effects by decreasing the doses of conventional chemotherapeutics.

Although clinically well known, the in vitro effects of ZA on cancer cells and the underlying mechanisms are not well elucidated. In our previous studies, we have already shown anticancer effect of ZA in hormone-and drug refractory prostate cancer cells, PC-3 and DU-145. In addition to this, we have also shown that this anticancer effect may be augmented with some cytotoxic agents in prostate cancer. Now, in our present study, we have investigated whether ZA induced growth inhibition and apoptosis in PC-3 and DU-145 may be enhanced by the combination with CA or OA, through inhibition of serine/threonine phosphatases in prostate cancer cells. Both ZA/CA and ZA/OA combinations inhibited the cell viability of hormone-and drug refractory prostate cancer cells at in vivo achievable therapeutic concentrations. Moreover, a potentiation of the apoptotic effects of the combinations was also observed in the same experimental conditions. This is the first report of a synergistic combination of ZA with phosphatase inhibitors CA and OA which inhibits cell viability and induces apoptosis in human hormone and drug refractory prostate cancer cells.

OBJECTIVES

  • • To investigate if the cytotoxic and apoptotic effect of zoledronic acid (ZA) can be enhanced by the addition of the serine/threonine protein phosphatase inhibitors calyculin A (CA) and okadaic acid (OA) in hormone and drug refractory prostate cancer cells, PC-3 and DU-145.
  • • To discover the effect of these combination treatments on phosphatase 1 (PP1) and PP2A protein expression levels in prostate cancer cells.

MATERIALS AND METHODS

  • • An XTT cell viability assay was used to determine cytotoxicity.
  • • Apoptosis was evaluated by enzyme-linked immunosorbent assay (ELISA) using a Cell Death Detection ELISA Plus Kit and verified by measuring caspase 3/7 enzyme activity.
  • • The PP1 and PP2A enzyme activities were evaluated by serine/threonine phosphatase ELISA and expression levels of PP1 and PP2A proteins were then re-assessed by Western blot analysis.

RESULTS

  • • Combination of ZA with either CA or OA showed synergistic cytotoxicity and apoptosis compared with any agent alone in both PC-3 and DU-145 prostate cancer cells.
  • • The combination of ZA with phosphatase inhibitors resulted in enhanced suppression of both PP1 and PP2A enzyme activity and protein levels, which was more overt with the ZA/CA combination.

CONCLUSION

  • • Results from our study increase the translational potential of our in vitro findings and offer the basic rationale for the design of new combinatory strategies with ZA and phosphatase inhibitors for the treatment of prostate cancer, which may become resistant to conventional therapy.
Abbreviations
BP

bisphosphonate

ZA

zoledronic acid

N-BP

new bisphosphonate

FPP

farnesyl pyrophosphatase

GGPP

geranylgeranyl pyrophosphate

PP

phosphatase

OA

okadaic acid

CA

calyculin A

INTRODUCTION

Prostate cancer is the second most common cancer diagnosed among elderly men and is the third underlying cause of cancer death in the western world [1]. Current standards of care with surgery, chemotherapy or radiation in prostate cancer patients are of limited efficacy, especially in the androgen refractory state of the disease, and unfortunately metastatic disease remains incurable [2,3].

The bone is the most common site for metastases of prostate cancer and bisphosphonates (BPs) have been widely used for the treatment of morbidity due to skeletally related events and also serve as a primary therapeutic option for malignant hypercalcaemia [4]. However, far beyond their effects on skeletally related events, BPs have also been shown to exert a direct cytotoxic action on different types of tumour cells in vitro, including prostate cancer cell lines [5–7]. Zoledronic acid (ZA) is the most potent member of the nitrogen-containing new-generation bisphosphonate (N-BP) family. The therapeutic efficacy of N-BPs can be traced to their specificity to bind and inhibit farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway [8,9]. Blockade of the enzymatic activity of FPPS by N-BP depletes the formation of intermediate isoprenoids, such as farnesyl pyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) in the mevalonate pathway, which are needed for post-translational prenylation of a variety of GTPases, such as Rap, Rho and Ras [10,11]. These GTPases play important roles in the survival pathways. Hence, disruption of these proteins induces a series of changes leading to cell death and apoptosis, and these effects have been suggested to underlie the cytotoxic effects of N-BPs, including ZA [12–14].

The protein serine/threonine phosphatases control the phosphorylation of numerous proteins, such as GTPases, involved in cell signalling and are crucial for cell growth and survival [15]. Mammalian protein phosphatases have been classified into the phosphatase 1 (PP1), PP2A, PP2B and PP2C families based on their biochemical properties. Among its numerous members, PP1 and PP2A are considered the two major enzymes because of their ubiquitous abundance and broad specificity [16]. PP1 and PP2A together account for more than 90% of all serine/threonine phosphatase activity in mammalian cells. As PP1 and PP2A are involved in numerous cellular processes and several signalling pathways that are important for cell proliferation and apoptosis, targeting of phosphatase activity is accepted as an attractive strategy for the treatment of cancer.

Okadaic acid (OA) and calyculin A (CA) are the most commonly used inhibitors of PP1 and 2A. Okadaic acid, extracted from the common black sponge Halachondria okaddai, is a potent inhibitor of PP1 and PP2A, and CA was isolated from another marine sponge, Discodermia calyx[17]. These inhibitors are useful in delineating the mechanisms underlying the proliferation and survival properties of cancer cells related to protein phosphorylation/dephosphorylation reactions.

This study aimed to investigate if the cytotoxic and apoptotic effect of ZA could be enhanced by the addition of the serine/threonine protein phosphatase inhibitors CA and OA in hormone and drug refractory prostate cancer cells, PC-3 and DU-145; and to discover the effect of these combination treatments on PP1 and PP2A protein expression levels in prostate cancer cells.

MATERIALS AND METHODS

Human prostate cancer cells (PC-3 and DU-145) were obtained from ICLC (Genova, Italy). The cells were grown as monolayer adherent cell lines and were routinely cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, 1% penicillin–streptomycin in 75-cm2 polystyrene flasks (Corning Life Sciences, Tewksbury, MA, USA) and maintained at 37 °C in a humidified atmosphere with 5% CO2. Growth and morphology were monitored and cells were passaged when they had reached 90% confluence. Cell culture supplies were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). ZA was a generous gift from Novartis Pharmaceuticals Inc. (Basel, Switzerland) and was prepared in distilled water. CA and OA were obtained from Tocris Bioscience (Bristol, UK) and prepared in dimethyl sulphoxide. The final dilutions were made immediately before use, and new stock solutions were made for each experiment. The dimethyl sulphoxide concentration in the assay did not exceed 0.1% and was not cytotoxic to the tumour cells. PP1 and PP2A antibodies were obtained from Abcam (Cambridge, UK). All other chemicals, unless mentioned, were purchased from Sigma Chemical Co. (St Louis, MO, USA).

After verifying cell viability using a trypan blue dye exclusion test by Cellometer automatic cell counter (Nexcelom Inc., Lawrence, MA, USA), cells were seeded at approximately 1 × 104 cells/well in a final volume of 200 µlL in 96-well flat-bottom microtitre plates. After an overnight incubation, cells were treated with ZA, CA or OA alone, or with ZA in combination with CA or OA. Plates were incubated at 37 °C in a 5% CO2 incubator for the indicated time periods. At the end of incubation, 100 µL of XTT (Roche Applied Science, Mannheim, Germany) was added to each well, and plates were incubated at 37 °C for an additional 4 h. Absorbance was measured at 450 nm against a reference wavelength at 650 nm using a microplate reader (Beckman Coulter, DTX 880 Multimode Reader; Fullerton, CA, USA). The mean of triplicate experiments for each dose was used to calculate the median inhibition concentration (IC50) and the combination index.

Apoptosis was evaluated by ELISA using Cell Death Detection ELISA Plus Kit (Roche Applied Science, Mannheim, Germany) according to the manual instructions. The relative amounts of mono- and oligonucleosomes generated from the apoptotic cells were quantified using monoclonal antibodies directed against DNA and histones by ELISA. Briefly, cytoplasmic fraction of the untreated controls, and of the ZA, CA or OA alone, or ZA in combination with CA or OA treated cells were transferred onto a streptavidin-coated plate and incubated for 2 h at room temperature with a mixture of peroxidase conjugated anti-DNA and biotin-labelled antihistone. The plate was washed thoroughly and incubated with 2,29-azino-di-[3-ethylbenzthiazolinesulfonate] diammonium salt; then absorbance was measured at 405 nm with a reference wavelength at 490 nm (Beckman Coulter, DTX 880 Multimode Reader). Detection of apoptosis was verified by measuring caspase 3/7 enzyme activity.

For the phosphatase activity assay DU-145 and PC-3 cells (4 × 106) were washed three times with ice-cold Tris-buffered saline (pH 7.4) and then disrupted in 500 µL M-PER Reagent. The lysates were immediately centrifuged at 14 000g for 10 min and the supernatants of the cells were transferred immediately into ultracentrifuge tubes. The activity of PP1 and PP2A was assayed by the serine/threonine phosphatase system (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. The enzyme activity was measured by a non-radioactive method based on the determination of free phosphate with colour development. Absorbance was measured at 630 nm with a plate reader (Beckman Coulter, DTX 880 Multimode Reader).

For Western blot analysis PC-3 and DU-145 cells were grown for 72 h in the absence or presence of ZA, CA and OA alone, ZA in combination with CA and OA for 72 h at 37 °C. To prepare cell lysates for Western blot analysis, cell pellets were lysed in buffer containing 20 mm Tris(tris(hydroxymethyl)aminomethane)-HCl (pH 8.0), 137 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm Na3VO4, 25 mm glycerophosphate and phosphatase inhibitor cocktail 1 (Sigma). After centrifugation at 14 000g for 15 min, protein concentrations were quantified in duplicate by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were separated by SDS–PAGE and transferred to polyvinylidene difluoride Immobilon-P membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% non-fat dry milk prepared in Tris-buffered saline containing 0.1% Tween 20 (TBST0.1) at room temperature for 1 h. The membrane was then incubated with primary antibodies (PP1 and PP2A monoclonal antibodies: ab52619 and ab32104, respectively, Abcam, UK) at room temperature for 1 h. Dilutions of primary antibodies were prepared according to the manufacturer's instructions. After several washes in TBST, membranes were incubated with appropriate secondary antibodies (1:2000 dilution; Millipore Upstate USA, Charlottesville, VA, USA) at room temperature for 1 h. The protein bands recognized by the antibodies were visualized by the Kodak Gel Logic 1500 Imaging System and quantified by ImageJ software (NIH, Bethesda, MD, USA).

All experiments were conducted in triplicate and the results are expressed as the mean ±sd, with differences assessed statistically and P values determined by Student's t test. P < 0.05 was accepted as significant. The median dose–effect analysis by Chou and Talalay [18] was used to assess the interaction between agents. Determinations of the synergistic versus additive versus antagonistic cytotoxic effects of the combination of ZA with CA or OA were assessed using Biosoft CalcuSyn software (Ferguson, MO, USA). The combination index was used to express additive effect (CI = 1), antagonism (CI > 1), synergism (CI < 1), and strong synergism (CI < 0.5).

RESULTS

A time-dependent and dose-dependent decrease in the viability of PC-3 and DU-145 cells was observed in response to ZA and the serine/threonine protein phosphatase inhibitors CA and OA. To evaluate the cytotoxic effects of ZA, CA or OA alone on the viability of human androgen and drug refractory prostate cancer cells, PC-3 and DU-145 were exposed to increasing concentrations of ZA (20–120 µm), CA (0.5–10 nm) and OA (1–120 nm) for 24, 48 and 72 h, and an XTT cell proliferation assay was performed. Highest cytotoxicity was observed at 72 h and IC50 values of ZA in PC-3 and DU-145 cells were found to be 85.4 and 90.9 µm, respectively. Moreover, there was a time-dependent and dose-dependent cytotoxic effect by ZA or each inhibitor alone (Fig. 1A–C). For PC-3 cells, IC50 values of CA and OA were found to be 4.3 nm and 56.6 nm at 72 h, respectively. For DU-145 cells, IC50 values were found to be 11.2 nm and 108 nm at 72 h, respectively (Fig. 1B,C).

Figure 1.

Effects of zoledronic acid (ZA) (A), calyculin A (CA) (B) and okadaic acid (OA) (C) on the viability of PC-3 and DU-145 prostate cancer cells. Cytotoxicity was determined by the XTT cell viability test at 72 h. The results are expressed as the mean of three different experiments (P < 0.05).

The combination of ZA with either CA or OA showed synergistic cytotoxicity compared with any agent alone in both PC-3 and DU-145 prostate cancer cells. To study the possible synergistic/additive effects of ZA in combination with CA or OA, PC-3 and DU-145 cells were exposed to different concentrations of each agent alone, and in combination for 24, 48 and 72 h (data not shown). The synergism or additivity was evaluated by combination index values calculated using Calcusyn 2.0 software. Combination experiments showed strong synergistic cytotoxicity with both of the phosphatase inhibitors in hormone and drug refractory prostate cancer cells, PC-3 and DU-145, at 72 h, compared with any agent alone. For example 60 µm ZA and 40 nm OA alone resulted in 42% and 37% decreases, respectively, but their combined treatment resulted in 88% decrease in the viability of PC-3 cells. Use of 80 µm ZA and 2.5 nm CA alone resulted in 56% and 19% decreases, respectively, but their combined treatment resulted in an 88% decrease in the viability of PC-3 cells (Fig. 2A). Used alone, 40 µm ZA and 80 nm OA showed 18% and 52% decreases respectively, but their combination resulted in 79% decrease in the viability of DU-145 cells. Moreover 60 µm ZA and 10 nm CA alone showed 20% and 58% decreases, respectively, but their combination resulted in a 95% decrease in the viability of DU-145 cells (Fig. 2B). All of the concentrations for each combination that were found to be synergistic/strongly synergistic in PC-3 and DU-145 cells are presented in Table 1.

Figure 2.

Effects of combination treatment using zoledronic acid (ZA) with calyculin A (CA) on the viability of PC-3 (A) and DU-145 (B) cells. Cytotoxicity was determined by the XTT cell viability test at 72 h. The results are expressed as the mean of three different experiments (P < 0.05).

Table 1. Combination index values of zoledronic acid in combination with CA or OA on the viability of PC-3 and DU-145 cells
PC-3DU-145
Concentration of drugsCI valueInterpretationConcentration of drugsCI valueInterpretation
  1. CA, calyculin A; OA, okadaic acid. ZA, zoledronic acid. Combination index (CI) values were calculated from the XTT cell viability assays.

ZA (60 µm) + CA (2,5 nm)0.62SynergismZA (40 µm) + CA (10 nm)0.28Strong synergism
ZA (80 µm) + CA (1 nm)0.37Strong synergismZA (60 µm) + CA (5 nm)0.28Strong synergism
ZA (80 µm) + CA (2,5 nm)0.33Strong synergismZA (60 µm) + CA (10 nm)0.09Very strong synergism
ZA (60 µm) + OA (40 nm)0.149Strong synergismZA (40 µm) + OA (80 nm)0.227Strong synergism
ZA (40 µm) + OA (40 nm)0.168Strong synergismZA (40 µm) + OA (40 nm)0.319Strong synergism

The combination of ZA with either CA or OA induced DNA fragmentation and caspase 3/7 enzyme activity in a synergistic manner compared with any agent alone in DU-145 and PC-3 cells. To examine the induction of apoptosis in response to ZA in combination with CA or OA in prostate cancer cells, two different methods were used: DNA fragmentation analysis and caspase 3/7 enzyme activity assay. Our results clearly showed that ZA, CA and OA alone induced apoptosis in a dose-dependent manner. In PC-3 cells exposed to 60 µm ZA or 40 nm OA alone, 7-fold or 5.6-fold increases in DNA fragmentation, respectively, were recorded but a 13.2-fold increase was found in the combined treatment compared with untreated controls (P < 0.05). When 80 µm ZA or 2.5 nm CA alone were used there were 7.6-fold or 4-fold increases in DNA fragmentation in the exposed PC-3 cells, respectively, but their combined treatment resulted in a 15.2-fold increase in DNA fragmentation compared with untreated controls (P < 0.05) (Fig. 3A).

Figure 3.

Effects of combination treatment using zoledronic acid (ZA) with calyculin A (CA) on the % changes in DNA fragmentation of PC-3 (A) and DU-145 (B) cells show apoptosis. The bar graphs values represent the means ±sd of three experiments (P < 0.05).

There were 2-fold or 3.4-fold increases in DNA fragmentation in 40 µm ZA or 80 nm OA alone exposed in DU-145 cells respectively, but a 6.8-fold increase was observed in the combined treatment compared with untreated controls (P < 0.05). There were 3.7-fold or 6.4-fold increases in DNA fragmentation in 60 µm ZA or 10 nm CA exposed in DU-145 cells respectively, whereas the combination treatment resulted in an 11.6-fold increase in DNA fragmentation compared with untreated controls (P < 0.05) (Fig. 3B).

Caspase 3/7 activity experiments showed that there were 7.5-fold or 8.9-fold increases in activity in PC-3 cells treated with 60 µm ZA or 40 nm OA alone, respectively, but the combined treatment resulted in a 17.7-fold increase in caspase 3/7 activity compared with untreated controls (P < 0.05). There were 2.8-fold or 2.6-fold increases in the caspase 3/7 activity between 80 µm ZA or 2.5 nm CA alone treated and untreated PC-3 cells, respectively. However, the combination treatment resulted in a 12.7-fold increase in caspase 3/7 activity (P < 0.05).

There were 2.4-fold or 5.2-fold increases in activity in DU-145 cells treated with 40 µm ZA or 80 nm OA alone, respectively, whereas the combined treatment resulted in a 12-fold increase in caspase 3/7 activity compared with untreated controls (P < 0.05). There were 6.7-fold or 6.2-fold increases in caspase 3/7 activity in DU-145 cells exposed to 60 µm ZA or 10 nm CA alone, respectively, whereas the combination treatment resulted in a 15.5-fold increase in caspase 3/7 activity compared with untreated controls (P < 0.05) (data not shown).

For the initial characterization of phosphatase activity in both PC-3 and DU-145 cells, the serine/threonine phosphatase system was used. Results for each fraction were given as pmol P/min/µg protein as the mean of at least three separate experiments. There was a gradual decrease in both PP1 and PP2A activities during the cytotoxicity process in both PC-3 and DU-145 cells when treated with ZA, CA or OA alone and in combination-treated cells compared with the untreated cells. Moreover, the combination of ZA with phosphatase inhibitors resulted in an enhanced decrease in PP1 and PP2A activities, which was more overt with the ZA/CA combination (Fig. 4).

Figure 4.

Zoledronic acid (ZA) caused inhibition of phosphatase 1 (PP1) and PP2A phosphatase activity in PC3 and DU145 cells. The suppression was enhanced with the addition of either calyculin A (CA) or okadaic acid (OA) to ZA in both cancer cell lines. Cells were treated with increasing concentrations of ZA (20–120 µm), for 72 h. Then, cell lysates were assayed for PP1-specific and PP2A-specific phosphatase activity by enzyme-linked immunosorbent assay.

The PP1 and PP2A protein expression levels were further analysed by Western blot analysis. The cells were incubated with IC50 and IC75 doses of ZA for 72 h and the bands were normalized to β-actin and control. Expression levels of both PP1 and PP2A enzymes were decreased by exposure to ZA for 72 h. Moreover, co-treatment of ZA with CA or OA resulted in decreases in the levels of PP1 and PP2A compared with treatment with these agents alone and with untreated controls (Figs 5 and 6).

Figure 5.

Western blot analyses of phosphatase 1 (PP1) and PP2A expression after zoledronic acid (ZA) alone or in combination with calyculin A (CA) or okadaic acid (OA) in PC3 and DU145 cells. Exponentially growing cell cultures were treated with these agents alone or in combination; cytosolic cell lysates were prepared from cells at the indicated time subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted with PP1 and PP2A antibodies. Representative data from several independent experiments are shown.

Figure 6.

Quantification of Western blot analysis of phosphatase 1 (PP1) and PP2A expression after zoledronic acid (ZA) in combination with calyculin A (CA) or okadaic acid (OA) in PC3 and DU145 cells by ImageJ software.

DISCUSSION

The phosphatases PP1 and PP2A control the phosphorylation of numerous proteins involved in cell signalling and are important regulators of cell growth [15]. It has long been recognized that both CA and OA exert their cellular effects by binding and inhibiting type 1 and 2A serine/threonine protein phosphatases [17]. PP1 and PP2A differ significantly in their sensitivity to phosphatase inhibitors in vitro[19,20]. Whereas CA has nearly equivalent inhibitory activities against PP1 and PP2A, OA has 100-fold greater selectivity for PP2A over PP1. As the activities of phosphatases are needed for the control of cell signalling cascades, there is great therapeutic potential in targeting these enzymes. Therapies based on combinations of chemotherapeutics with phosphatase inhibitors that target signalling pathways within the cell with different mechanisms of action, may be useful for increasing therapeutic effect and may also diminish toxic side effects by decreasing the doses of conventional chemotherapeutics.

Zoledronic acid has been used in the management of bone metastases of various cancers and has become a treatment modality for malignant bone lesions in patients with several tumours [21]. A direct antitumour effect of BPs has been shown in several human cancer cells including multiple myeloma, breast, prostate and pancreatic cancer cells [22–24]. In addition, ZA has been reported to cooperate with different anti-cancer agents (e.g. docetaxel, imatinib, doxorubicin, ifosfamide, gemcitabine and cisplatin) in affecting cancer cells in vitro or in vivo[25–28]. The mevalonate pathway is a biosynthetic pathway responsible for the production of cholesterol and isoprenoid lipids, particularly the farnesyl- and geranylpyrophosphates which are required for the prenylation of small GTP proteins that play crucial roles in signalling pathways controlling cell growth. Inhibition of the mevalonate pathway has been proposed as being the fundamental molecular mechanism of many of the observed anti-cancer effects of N-BPs, such as ZA, both in vitro and in vivo[12]. After FPPS inhibition by N-BPs, the pathway is blocked and inhibition of protein prenylation occurs as a result of the lack of FPP and GGPP enzymes. The prenylation process is mandatory for the function of several proteins involved in key cellular processes such as proliferation, differentiation, adhesion and carcinogenesis [25].

Although clinically well known, the in vitro effects of ZA on cancer cells and the underlying mechanisms are not well elucidated. In our previous studies, the anti-cancer effect of ZA has been shown in the hormone and drug refractory prostate cancer cells PC-3 and DU-145. In addition to this, the anti-cancer effect can be augmented with some cytotoxic agents in prostate cancer [28]. Now, in the present study, we investigated whether the ZA-induced growth inhibition and apoptosis in PC-3 and DU-145 could be enhanced by the combination of ZA with CA or OA, through inhibition of serine/threonine phosphatases in prostate cancer cells. Results showed that ZA in combination with phosphatase inhibitors had a strongly synergistic cytotoxic effect, which was detected with the Calcusyn software. Both ZA/CA and ZA/OA combinations inhibited the cell viability of hormone and drug refractory prostate cancer cells at in-vivo-achievable therapeutic concentrations. These data clearly suggest that inhibition of geranylgeranylation and farnesylation of small GTP-binding proteins are important underlying mechanisms of the anti-proliferative effects of ZA in prostate cancer cells. Moreover, potentiation of the apoptotic effects of the combinations was also observed in the same experimental conditions.

There are several reports that serine/threonine protein phosphatase inhibitors were synergistically cytotoxic when used in combination with chemotherapeutics. Saydam et al. [29] showed that CA and OA augmented the interferon-α2b-induced cytotoxicity in K562 cells. In another study, the combination of the PP1 and PP2A inhibitor microcystin with 5-fluorauracil showed strong synergism on colon adenocarcinoma cells [30]. Our data also show that ZA suppressed PP1 and PP2A enzyme activities as well as decreased protein levels in both prostate cancer cell lines. Hence, inhibition of serine/threonine phosphatase activity may be one of the main routes of ZA-induced anti-cancer effect in prostate cancer cells and this effect is synergistically augmented by the addition of CA or OA.

This is the first report of a synergistic combination of ZA with the phosphatase inhibitors CA and OA, which inhibits cell viability and induces apoptosis in human hormone and drug refractory prostate cancer cells. As prostate cancer is mainly a disease of elderly men, new therapeutic strategies with fewer side effects than cytotoxic treatment are being researched. The results from our study present the translational potential of our in vitro findings and offer a basic rationale for the design of new combinatory strategies with ZA and phosphatase inhibitors for the treatment of prostate cancer, which may develop resistance to conventional therapy.

SOURCE OF FUNDING

Funding provided by Burcak Karaca.

CONFLICT OF INTEREST

None declared.

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