ΔΔCT = ΔCT (drugs treated) – ΔCT (control) for RNA samples. ΔCT is the log2 difference in CT between the target gene and endogenous controls by #GSK461364 supplier randurls[1|1|,|CHEM1|]# subtracting the average CT of controls from each replicate. The fold change for each treated sample relative to the control sample = 2-ΔΔCT. Statistical analysis All experiments were conducted in triplicate and the results expressed as the mean ± (sd), with differences assessed statistically p values determined by Student’s t- test. p < 0.05 was accepted as significant. Median dose effect analysis, a measure of synergism or antagonism, was determined by the method of Chou and Talalay, using their computer program (Biosoft CalcuSyn,

Ferguson, MO, USA) to assess drug interaction. We chose this method because it takes into account both the potency of each drug or combination of drugs and the shape of dose-effect curve. CalcuSyn software which is based on this method was used to calculate the CI. Synergy, additivity and antagonism were defined as CI < 1, CI = 1, CI > 1, respectively, where CI ≤ 0.5 characterizes strong synergy. For this analysis, concentrations of ATRA and zoledronic acid were chosen as clinically achievable concentrations and below the IC50 values [22]. Results Effect of either single ATRA or zoledronic acid on the viability of OVCAR-3 and MDAH-2774

cells To evaluate the effects of ATRA on the viability of human ovarian cancer cells, OVCAR-3 and MDAH-2774 cells were exposed to increasing concentrations of ATRA (40 to 140 nM) for 24, 48 and 72 h, and XTT cell viability assay was performed.

selleck screening library ATRA decreased cell viability in a time- and dose dependent manner both in OVCAR-3 and MDAH-2774 cells (data not shown). As shown in figure 1, there were 20-, 41-, and 73% decrease in cell Acyl CoA dehydrogenase viability of OVCAR-3 cells exposed to 40-, 100-, and 120 nM of ATRA, respectively, when compared to untreated controls at 72 h (p < 0.05). In addition, there were there were 28-, 49.5-, and 58% decrease in cell viability of MDAH-2774 cells exposed to 40-, 100-, and 120 nM of ATRA, respectively, when compared to untreated controls at 72 h (figure 1) (p < 0.05). Highest cytotoxicity was observed at 72 h and IC50 values of ATRA were calculated from cell proliferation plots and found to be 85 and 82 nM in OVCAR-3 and MDAH-2774 cells, respectively. Figure 1 Effect of ATRA on viability of OVCAR-3 and MDAH-2774 cells at 72 h in culture. The data represent the mean of three different experiments (p < 0.05). We also examined the effect of zoledronic acid on OVCAR-3 and MDAH-2774 cells. Cells were exposed to increasing concentrations of zoledronic acid (2.5- to 40 μM) for 24, 48 and 72 h. There were 18-, 26-, and 60% decreases in cell viability of OVCAR-3 cells exposed to 5-, 10-, and 20 μM of zoledronic acid, respectively, when compared to untreated controls at 72 h (figure 2) (p < 0.05).