Anti-tumor effects of chemotherapeutics are enhanced by genistein in pancreatic cancer.

5412

Purpose: Currently standard therapies for pancreatic cancer consist of administering cytotoxic chemotherapeutic agents or irradiation treatment alone, or in combination. Unfortunately the therapeutic benefit remains modest due to high degree of inherent and acquired chemoresistance. We recently reported that genistein augments pancreatic tumor sensitivity to gemcitabine due to the inactivation of Akt and NF-κB signalling (Cancer Res. 2005; 65:9064-72). In order to further dissect the molecular pathway, we used MDA Panc-28 and stably transfected MDA Panc-28/IκBαM [phosphorylation defective IκBα (S32, 36A) that blocks NF-κB activation] pancreatic cancer cells exposed to genistein with and without gemcitabine, cisplatin or oxaliplatin. Method: MDA Panc-28 and MDA Panc-28/IκBαM cells were cultured in complete DMEM media with and without genistein (0-100μM) for 72 hrs for initial studies. Subsequently chemosensitivity towards three drugs- gemcitabine, cisplatin and oxaliplatin was also assessed in vitro using MTT, and apoptosis was assessed by quantifying cytoplasmic histone-DNA fragments using Cell Death Detection ELISA kit upon pretreatment with 25μM genistein (48 hrs) followed by gemcitabine, cisplatin or oxaliplatin (72 hrs). The molecular mechanism for enhanced chemosensitivity was investigated by performing Western immunoblot for pAkt, Bcl-2, Bcl-xL, and PARP. Additionally, gel shift assay was performed to elucidate the role of NF-κB transcription factor in suppressing apoptosis and attenuating the effect of chemotherapeutic agents by genistein. Results: The MDA Panc-28/IκBαM cells without NF-κB expression was found relatively more sensitive to genistein compared to parental MDA Panc-28 cells.Genistein pretreatment also significantly enhanced the anti-proliferative and pro-apoptotic effect of gemcitabine, cisplatin and oxaliplatin in NF-κB null cells relative to untreated control and the parental MDA Panc-28 cells. Several NF-κB downstream target molecules such as Bcl-xL, Bcl-2 and cyclin D1, survivin and MMP-9 were differentially affected by genistein pretreatment and combination treatments, which appears to be due to down regulation of NF-κB activity. Conclusion: Our results strongly suggest that chemosensitization of human pancreatic cancer cells is due to inhibition of NF-κB and Akt signaling, and thus it could form the basis for rational drug designing or utilization for the treatment of pancreatic tumors with better outcome.