Introduction: Cisplatin is an effective and widely used chemotherapeutic agent against various human cancers, including ovarian cancer. However, the major limitation of cisplatin treatment is the development of resistance in previously responsive ovarian cancers. Hence, there is a need to develop new drugs that are effective in treating the drug-resistant tumors. We studied the efficacy of a novel di-arylidenyl piperidin-4-one derivative (EF-24) and its mechanisms for inducing cytotoxicity in cisplatin resistant (CR) ovarian cancer cells. The specific Aim of this study was to evaluate the effect of EF-24 induction of PTEN (phosphatase and tensin homologue deleted on chromosome ten) expression, and the resulting synergistic suppression of CR cell growth and apoptosis through down-regulation of the Akt pathway. Methods: G2/M cell-cycle arrest was examined by FACS analysis. G2/M cell-cycle associated proteins p53, p21, p27, MDM2, cycB1, expression of cleaved caspases, poly ADP-ribose polymerase (PARP), FasL, PTEN, p53, and phosphorylation of Akt were examined using Western blot. PTEN, pAktSer473, and Thr308 levels were determined by immunoprecipitation. An ubiquitination assay was used to examine the inhibition of PTEN degradation. The PTEN over-expression experiments were performed using a wild-type PTEN cDNA. Further, we verified the role of PTEN by using PTEN siRNA transfection. Results: A 75% reduction in the number of cells after 2 days demonstrated that 2 μM, EF-24 significantly inhibited the proliferation of the CR cells. Cleavages of caspase-8, caspase-3, and PARP within 24 hours treatment were clear induction of apoptosis. Of interest, EF-24 induced a membranous FasL expression in the CR cells. Consistent with the increase in FasL expression, a marked attenuation in the phosphorylation of Akt was identified within 6 hours in the EF-24-treated cells. EF-24 activated PTEN expression in CR cells, which in turn was shown to antagonize PI3K/Akt signaling. The upregulation of PTEN was, at least in part, due to an accelerated inhibition of ubiquitin-dependent PTEN degradation. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt phosphorylation at Ser473 and Thr308. This resulted in decreased apoptosis and increased cell survival. In addition, we confirmed that the over-expression of PTEN further increased apoptosis. Conclusions: We have shown, forthe first time, that EF-24 induces G2/M cell-cycle arrest and apoptosis by the induction of PTEN expression in CR human ovarian cancer cells. The upregulation of PTEN inhibited Akt and MDM2 which enhanced the level of p53, thereby inducing G2/M arrest and apoptosis. Therefore, EF24 appears to have a potential therapeutic role in human ovarian cancer through constitutive activation of PTEN

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA