Preclinical findings in our laboratory have shown that the antiprogestin RU-38486 (RU) inhibits ovarian cancer cell growth blocking the cell cycle at the G1-S phase transition, up-regulating cyclin dependent kinase (Cdk) inhibitors p21cip1 and p27kip1, and inhibiting Cdk2 activity (Clin Cancer Res 2007, 13, 3370-3379). The goal of this study was to assess whether two antiprogestins structurally related to RU, named ORG-31710 (ORG) and CDB-2914 (CDB), also have growth inhibition effects on ovarian cancer cells. Two ovarian cancer cell lines of different genetic backgrounds, OV2008 (p53 wt) and SK-OV-3 (p53 null), were exposed to varying concentrations of RU, ORG or CDB for 72 h, and cell number and viability, and distribution within the cell cycle were assessed by micro-cytometry using exclusion fluorochromes and propidium iodide, respectively. For each dose-response curve, the amount of drug needed to achieve 50% growth inhibition (IC50) was calculated utilizing drug interaction software. RU, ORG, and CDB significantly inhibited growth in both OV2008 and SK-OV-3 cells with a potency of RU (IC50 ~ 10 μM) > ORG (IC50 ~ 18 μM) > CDB (IC50 ~ 25 μM). The three steroids growth arrested the cells without killing them at lower concentrations (i.e. cytostatic effect), but were cytotoxic at higher concentrations as shown by the decline in cell viability and the increased percentage of cells with sub-diploid DNA content. In a second set of experiments, cells were exposed to a cytostatic dose of the drugs for 48 h, and protein extracts were obtained to assess the expression of the cell cycle related proteins p21cip1, p27kip1, Cdk2, and cyclin E by Western blot, and the activity of Cdk2 via an in vitro kinase assay using histone H1 as substrate. The growth arrest induced by the lower concentrations of the steroids was associated with increased nuclear levels of p21cip1 and p27kip1, decreased nuclear abundances of cyclin E and Cdk2, and decreased nuclear Cdk2 activity. Further studies on the lethality of the compounds at the higher concentrations tested revealed the induction of apoptotic cell death as supported by the presence of fragmented DNA in agarose gel electrophoresis and the morphological features of apoptosis in DAPI-stained cells. Altogether these data demonstrate that ORG and CDB have similar effects than RU in growth inhibiting ovarian cancer cells. The growth inhibition effects of RU, ORG, and CDB are associated with marked inhibition of Cdk2, a key enzyme driving progression of the cell cycle in ovarian cancer, whereas the lethality induced by higher concentrations of the steroids is associated with cell death by apoptosis. Due to their cytostatic and cytotoxic properties these steroid compounds originally designed for contraceptive purposes emerge as attractive new agents for ovarian cancer therapy. Supported by NIH grant CA121991.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA