Zoledronic Acid Effects Interleukin-6 Expression in Hormone-Independent Prostate Cancer Cell Lines

Objective: To investigate the inhibitory effects of zoledronic acid (ZA) on tumor related growth factor IL-6 in hormone resistant prostate cancer cell lines. The association between apoptosis and IL-6 inhibition was also assessed. Materials and Methods: PC-3 and DU145 cell lines were treated with different concentrations of ZA (1-100μM) at various intervals (24-72 h.). The cell viability was investigated by XTT assay and apoptotic effect was evaluated by cell death Results: PC-3 and DU145 cell lines were sensitive to ZA mediated cytotoxicity in a doseand time-dependent manner. of IL-6 secretion was associated with increased apoptosis in DU145 cells (p = 0.002), there was no similar association for Conclusion: ZA exhibited a timeand dose-dependent apoptotic effect on PC-3 and DU145 prostate cancer cell lines and this effect was associated with inhibited secretion of IL-6 in DU145 cell line.


INTRODUCTION
Prostate cancer is most common among elderly men, and in 2007, the estimated number of the newly diagnosed prostate cancer cases was 218.890 in USA (1).Although local curative treatment strategies disease, androgen deprivation therapy represents the standard treatment in patients with metastatic prostate resistant prostate cancer and progression is inevitable Investigative Urology Investigative Urology during androgen deprivation treatment.Unfortunately, no any other effective and curative alternative treatment has been reported for these patients.
The new treatment modalities are primarily focused on growth factors that stimulate the proliferation of prostate cancer cells.Interleukin-6 (IL-6) is a growth factor for prostate cancer cells and its high serum levels are known to be directly associated with clinical prognosis of the disease (2,3).It has also been shown that IL-6 signaling pathway is active and up-IL-6 signaling pathway is actively used in metastatic prostate cancers and hormone independent prostate cancer cell lines.
Several clinical trials have already demonprostate cancer patients (5,6).The growth of metastases may be inhibited by modifying the bone microenvironment using bisphosphonates.They also exert direct cytotoxic and apoptotic effects on a variety of human tumor cell lines including myeloma, breast cancer and prostate cancer (7-9).Zoledronic acid (ZA) is the most potent nitrogen containing bisphosphonate compound.It has been shown to inhibit cell growth and induce apoptosis in (10).Current evidence on the effects of ZA suggests that it is a potential chemotherapeutic agent for the treatment of prostate cancer, either as monotherapy or in combination.Despite the overwhelming in vitro studies investigating the anti-tumor activity of the combined use of ZA with different chemotherapeutics, the molecular targets and mechanisms of ZA in tumor cells remains a subject of debate.
We hypothesize that ZA may exert its antitumor effect by inhibiting the tumor related growth factor IL-6. Considering the potential role of IL-6 in the growth regulation of PC-3 and DU145 cell lines, the present study was planned to investigate the relationship between the anti-tumor activity of ZA and IL-6 secretion in these cells under in vitro conditions.

MATERIALS AND METHODS
Chemicals -Cell culture supplies were obtained from Biological Industries (Kibbutz Beit Haemek, Israel).Zoledronic acid was a generous gift from The stock solution of zoledronic acid was prepared at a concentration of 1 mM in distilled water and aliquots were stored at -20 o C. All other chemicals, unless otherwise mentioned, were purchased from Sigma Chemical Co (USA).
Cell lines and culture -The androgen-refractory prostate cancer cell lines, PC-3 and DU145, were preferentially used since they secrete IL-6 and actively use IL-6 signaling pathway for growth promoting effects (11) and to maintain resistance to chemotherapy.These cell lines were kindly provided by Dr. Levent Turkeri from Marmara University, Istanbul, Turkey.PC-3 and DU145, adherent cell lines were cultured in lin-streptomycin.All cell cultures were incubated at 2 Cell viability assay -The effects of different concentrations of zoledronic acid (1,10,30,60,90, and 100μM) on PC-3 and DU145 cell lines were evaluated cation of cell viability by tryptan blue exclusion test, cells were plated on a 96-well plate in 200μL culture medium at a concentration of 10 4 cells/well.At 24, 48 and 72 hours of incubation, a 50μL of XTT labeling mixture was added to each well.The optical density was measured at 450 nm with a reference wavelength at 650 nm using a microplate reader (Beckman Coulcytotoxicity was calculated as follows: % Cystotoxicity = 1 _ A of experimental well x 100 A of positive control well where A is the absorbance. Evaluation of apoptosis -The Cell Death Detection many) was used to detect mono-oligonucleosomes of apoptosis after zoledronic acid induced cell death.and zoledronic acid treated cells were transferred to a streptavidin-coated plate supplied by the manufacturer.A mixture of Anti-histone-biotine and Antifor 2 hours.The complex was then simultaneously conjugated to form an immune complex on the plate, which then was read for optical density at 405 nm with a reference wavelength at 490 nm.The enrichment of mono-oligonucleosomes in cell lysates was calculated as absorbance of zoledronic acid treated cells/absorbance of untreated controls.
Caspase 3/7 activity assay -The Caspase-Glo 3/7 assay (Promega, Madison, WI) was used to measure caspase 3/7 activity, according to the manufacturer's instructions.PC-3 cells were plated on a 96-well plate in 100μL culture medium at a concentration of 10 4 cells/well.After incubation with increasing concentrations of zoledronic acid, 100μL of Caspase-Glo 3/7 reagent was added to each well.Then the mixture was incubated for one hour at room temperature and the luminescence of each sample was measured using a plate-reading luminometer (Beck-Determination of interleukin-6 secretion zoledronic acid treated PC-3 and DU145 cells by using Human IL-6 ELISA Kit (Biosource International Inc., California, USA).The cells were plated on 24well plates at a concentration of 10 5 cells per well and incubated for 24, 48 and 72 hours with increasing concentrations of zoledronic acid (1-100μM).Supernatants were collected for all culture conditions and analyzed for IL-6 levels using a standard ELISA kit according to manufacturer's instructions.Standard IL-6 standards provided by kit.IL-6 levels in zoledronic acid treated cells were recalculated based on the IL-6 levels from untreated control cells at the end of treatment in order to compensate the differences due to cell number.The decrease in IL-6 levels was controls and DU-145 treated cells were isolated by Science, Mannheim, Germany).Primers and probes man Interleukin-6).The procedure was carried out as a single step method for reverse transcription from Light Cycler apparatus was used with the following sequence: denaturation at 95ºC for 10 minutes, then LightCycler (1.5) Instrument.
Statistical analyses -All experiments were set up in triplicate and the results were expressed as mean (version 5) (San Diego, CA, USA) was used for the analysis of data and graphic presentations.Student's

RESULTS
The cytotoxic and apoptotic effects of zoledronic acid -PC-3 and DU145 cell lines were sensitive to ZA mediated cytotoxicity; the maximum cytotoxicity was achieved at 72 hour with 100μM concentration of ZA.The cytotoxicity was proportional with the increasing concentrations of ZA for both cell lines and the difference from untreated controls was statistically time-and dose-dependent apoptosis in both cell lines.Data for PC-3 cell line regarding apoptosis is given in Figure -1.For PC-3 cell line, Caspase 3/7 activity was IL-6 secretion as detected in the supernatants of PC-3 and DU145 cells -Incubation of PC-3 and DU145 cells with increasing concentrations of ZA ure-3 and 4).This effect was detected with the lowest dose and at the earliest time points.A difference in terms of dose-dependent inhibition of IL-6 secretion between two cell lines could only be observed at 72 hours.The lowest level of IL-6 secretion was achieved at 24 hours for PC-3 cells.
For DU-145 cells, a four-fold decrease in IL-6 secretion was found in ZA treated cells with 60 controls.However, for PC-3 cells, IL-6 secretion was only halved with the same concentrations and IL-6 Interestingly, for PC-3 cell line, there was no association between the degree of apoptosis and inhibition of IL-6 secretion following ZA treatment, (p = 0.347).In contrast , for DU145 cells, the inhibition of IL-6 secretion was correlated with the degree of apoptosis (p = 0.002).

Figure 2 -Concentration dependent caspase 3/7 activity in PC-3 cell line following exposure to zoledronic acid. PC-3 cell line was treated with increasing concentrations of zoledronic acid for 72 hours and then the levels of caspase 3/7 was measured using caspase-
formed in DU-145 cells, in order to examine whether the reduction of IL-6 levels is associated with a de-treated with 30 and 90μM ZA, compared to untreated

COMMENTS
apoptosis in PC-3 and DU145 prostate cancer cell lines in a dose-and time-dependent manner.However, PC-3 and DU145 cell lines.In addition, two cell lines differed in terms of IL-6 secretion.The degree of apoptosis was not related to the level of the inhibition of IL-6 secretion for PC-3 cells, which also secrete other hand, the level of reduction in IL-6 secretion was correlated with the degree of ZA induced apoptosis in DU145 cell lines.Based on this data, it might be speculated that anti-tumoral effects of ZA could also be mediated by IL-6 and related signaling pathways in prostate cancer cells.
It has been well-documented that IL-6 is a multifunctional cytokine that plays an important role in the regulation of hematopoiesis, immune response, ment (12).It is produced by different cells including lymphoid or non-lymphoid cells and malignant tissues (13).All prostate cells including normal prostate epithelia, cells originated from benign prostatic hyperplasia and malignant prostate cancer are shown to be capable of secreting IL-6 in cell cultures (14).Furthermore, increased secretion of IL-6 ligand and its receptors in serum has been reported for all stages of prostate cancer including hormone refractory pa- tients (14,15).Also, clinical prognosis of prostate cancer is directly affected by serum IL-6 levels (15) and IL-6 plays an important role for the development of resistance to chemotherapeutics used in prostate cancer (16).Moreover, exogenous administration of IL-6 has been shown to inhibit doxorubicin-induced apoptosis in PC-3 cells (17).
In vitro studies demonstrated an increase in the proliferation of prostate cancer cells with IL-6 stimulation (18,19) and a decrease in growth rate of androgen insensitive PC-3 and DU145 cell lines treated with anti-IL-6 antibodies (16).These results suggest that the combined use of anticancer agents with drugs resulting in an inhibition of IL-6 expres-ticularly in patients with hormone refractory prostate cancer.
ed regarding the stimulatory/inhibitory effects of IL-6 on the proliferation on various prostate cancer cell lines (11,20,21).These differences may be attributed to a several reasons related to IL-6 signaling pathway.Firstly, there are membranous and soluble forms of IL-6 ligand and its receptor, which are strictly regulated.Secondly, gp130, the signal transducer of IL-6 on the membrane, can be activated by various growth factors.Thirdly, activated gp130, either simultaneously or preferentially, triggers three intracellular pathways by the alteration of intracellular domain.IL-6 signaling is mediated by JAK-STAT, ras-raf-MAPK and PI3K-Akt signaling.It has been suggested that one or two alternative pathways are preferentially more active in different cell lines.IL-6 can also be up-and down regulated by autocrine or paracrine effects and feed-back mechanism (11,20,22,23).Its expression is regulated by several transcription factors such as intracellular signaling pathways of IL-6 also regulate these transcription factors.
Zoledronic acid may affect a some molecules in signal transduction pathways including cell proliferation process (ras-raf-MAPK), tumor suppressor genes, apoptotic pathways, cell cycle proteins and posttranslational processes.Since ZA affects the binding of ras proteins to the membrane via protein prenylation (10), it might indirectly inhibit cell proliferation.In a recent study by Cavarretta et al., the effect of IL-6 was shown to be mediated by oncogene Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family in prostate cell line (24).The association between ZA treatment and IL-6 secretion may also be regulated by Mcl-1 expression.
Several authors have previously suggested that ZA cannot induce apoptosis (9,25).Such an inconsistency might be explained by the differences in ZA concentrations (25) and treatment durations (9).The present study indicates that a longer treatment period with higher concentrations of ZA is necessary to induce apoptosis.Interestingly, when bisphosphonates are combined with other common The synergic cytotoxic effect of ZA has previously been detected on prostate cancer cells (26,27).
Few studies investigated the relation between IL-6 expression/secretion and ZA treatment.A decreased IL-6 expression has been reported after ZA treatment in bone marrow stromal cells under in vitro -ZA infusion has been demonstrated in cancer patients with fever (30).Although the disagreements between the studies may be explained by the variations of in vivo and in vitro conditions, all of these observations clearly points out that IL-6 has an important role in the processes related to both bone microenvironment and metastases in prostate cancer (31).The present study shows a correlation between the degree of ZA induced apoptosis and the inhibition of IL-6 secretion, implying that the apoptotic effect of ZA is associated with IL-6 and related pathways.Exogenous administration of IL-6 do not interfere the anticancer actions of ZA on PC-3 cells, which supports the above-mentioned association (17).
tations: either the reduction of IL-6 secretion itself induces the apoptotic process or it may be the outcome of ZA induced apoptosis in a dose dependent between the decrease in IL-6 expression and the degree of apoptotic process, it could be suggested that ZA directly inhibits the autocrine mechanisms of IL-6 expression.
It would also be worth mentioning that ZA may indirectly induce apoptotic mechanisms through affecting signal transduction pathways on the upstream region of apoptotic pathway.This probability may explain the reduction of IL-6 secretion with increased apoptosis, which was observed after ZA treatment in our study.Therefore, it can be suggested that ZA not only directly induces apoptotic pathways, but also indirectly affects one or more signal transduction molecules located on upstream region, which cause the apoptosis in PC-3 and DU145 cell lines.For these reasons, it is necessary to determine the target molecules that play key roles on the effects of ZA.

CONCLUSION
The present in vitro study shows a time-and dose-dependent apoptotic effect of ZA on both PC-3 and DU145 prostate cancer cell lines, which correlates with an inhibitory effect on IL-6 expression in DU145 cells.Additional research is required to further elucidate the activity of IL-6 and its role in the pathogenesis of advanced prostate cancer at cellular and molecular levels.Also, further studies are required to investigate the down regulation of oncogene Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, which is regulated directly by IL-6 in ZA treated cells (24).The inhibition of IL-6 with anti-IL-6 antibody sensitizes androgen-independent prostate cancer cells to chemotherapeutic agents in vitro (32); thus, treatment modalities targeting IL-6 may have multiple advantages in prostate cancer patients who conventional treatment alternatives (18).

Figure 3 -
Figure 3 -viable cell/well was treated with increasing concentrations of zoledronic acid.IL-6 levels were measured after 48 and 72 hours by ELISA in the supernatants of zoledronic acid treated

Figure 4 -
Figure 4viable cell/well was treated with increasing concentrations of zoledronic acid.IL-6 levels were measured after 48 and 72 hours by ELISA in the supernatants of zoledronic acid Figure 5 -