An investigation of cytotoxic and apoptotic effect of zoledronic acid and Gossypol in ovarian cancer cell line (OVCAR-3)

Ovarian cancer remains the most common cause of death from a gynecologic malignancy. Epithelial ovarian cancer comprises 90% of all ovarian cancers. Most patients with this disease have advanced stage disease (ie, stage III?IV) at the time of diagnosis, which is associated with a substantially poorer prognosis than its early-stage counterparts. The `gold standard? for optimal chemotherapeutic response in women with ovarian cancer is combinational or sequential chemoterapy treatment. One of the biggest problem in the treatment of recurrent ovarian cancer is multidrug resistance. Therefore, investigation of new combinational drug treatments is of vital importance in ovarian cancer cell models, such as OVCAR-3. Gossypol (GP) extracted from cotton plant (Gossypium species), is a new drug candidate with antitumor properties. Zoledronic acid (ZA) is a third generation bisphosphonate and it?s antiproliferative and apoptotic properties were studied in several cancer cell lines.Our first objective is to determine the cytotoxic and apoptotic effects of gossypol and zoledronic acid in OVCAR-3 cells. Secondly, to find out the synergistic cytotoxic and apoptotic effect of gossypol and zoledronic acid by combining them in non-constant and constant ratios. Thirdly, to make contribution indirectly to treatment options of recurrent ovarian cancer.The human ovarian cancer cell line OVCAR-3 was cultured in RPMI 1640 and their viability monitored in certain intervals by trypan blue dye exclusion assay. Stock solutions of gossypol and zoledronic acid were prepared and new aliquots were used in each experiment. 104 cells in per well were treated with increasing concentrations of gossypol and zoledronic acid alone and in combination for 24, 48, 72 hours. The IC50 doses were determined by drawing dose-response curves after 72 h drug treatment. Drug-mediated cytotoxicity was determined by XTT colorimetric assay and apoptosis was detected by using ELISA based Cell Death Detection Kit. Apoptotic data were confirmed by using Caspase-Glo 3/7 Assay. All assays were set up in triplicate and the results were expressed as the mean + SD. The synergism in drug combinations was assessed by CalcuSyn software based on median effect equation and combination index methods.IC50 values for ZA and GP were determined as 14 and 25 µM, respectively. The most cytotoxic effect of gossypol was obtained in 80 µM dose at 72 hours, while the most cytotoxic effect of zoledronic acid was in 50 µM at 72 hours. Both gossypol and zoledronic acid inhibits cell proliferation in dose and time dependent manner.ZA and GP in combination was synergistic in following ratios and concentrations; 1:104 (2,5 nM ZA+25 µM GP), 1:3 (5 µM ZA+15 µM GP). CI values for ZA-GP combination were calculated as 0,285, 0,163 respectively in above concentrations. These lowest CI values were indicator of strong synergism. The same CI values were not maintained when the ZA-GP in synergistic constant ratio were tested in fold decreasing concentrations. The percentage of relative apoptosis at 72 hours was compared between single drugs and combination treatment. These apoptotic data were also confirmed by significant increase in caspase 3/7 enzyme activity in combined treatments.This study is the first investigation of cytotoxic and apoptotic effects of gossypol and ZA-GP combination on human OVCAR-3 ovarian cancer cells. For the first time, it is shown that ZA-GP combination treatment has significant cytotoxic and apoptotic effects on OVCAR-3 cells. Based on these preliminary data, ZA-GP combination is a new promising combination therapy for recurrent ovarian cancer. So, new studies are needed to explore the effects of ZA-GP combination on several ovarian cancer cells.Key words: Apoptotic effect, Cytotoxicity, Gossypol, Human OVCAR-3 Ovarian Cancer Cell, Synergism, Zoledronic Acid.