Genistein induced anticancer effects on pancreatic cancer cell lines involves mitochondrial apoptosis, G0/G1cell cycle arrest and regulation of STAT3 signalling pathway

Background: Genistein is a natural flavonoid that has been reported to exhibit anticancer effects against different types of cancers which include, but are not limited to, breast and oral squamous cell carcinoma. Purpose: The present study was designed to evaluate the anticancer effects of the natural flavonoid genistein against pancreatic cancer cell lines and to explore the underlying mechanism. Methods: Antiproliferative activity was investigated by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining. DNA damage was assessed by comet assay. Reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) were determined by flow cytometry. Cell migration was examined by wound healing assay. Protien expressions were determined by western blotting. Results: Antiproliferative assay revealed that genistein reduced the cell viability of pancreatic cancer cells in a dose dependent manner with an IC50 of 20 and 25 mu M against Mia-PaCa2 and PANC-1 cancer cell lines respectively. However, its antiproliferative effects were less pronounced against non-cancerous pancreatic ductal epithelial cell line (H6C7) as evident from the IC50 of 120 mu M. Genistein induced significant morphological changes in pancreatic cancer cells and triggered cell cycle arrest in G(0)/G(1) phase. DAPI staining and flow cytometric analysis revealed that genistein induced apoptosis in a dose dependent manner through generation of substantial amounts of ROS and reduction of MMP. However, treatment of the pancreatic cancer with genistein and ascorbic acid could abrogate the effects of genistein on cell viability. Protien expression analysis revealed that genistein upregulated cytosolic cytochrome c, Bax, cleaved Caspase-3 and cleaved caspase-9 expressions with concomitant downregulation of Bcl-2 expression. Moreover, genistein inhibited the phosphorylation of signal transducer and activator of transcription STAT3 proteins and downregulated the expression of survivin, cyclin D1 and ALDH1A1 in Mia-PaCa2 cells in a dose dependent manner. Interestingly, genistein could inhibit the cell migration potential of the Mia-PaCa2 cells which was further associated with the downregulation of metalloproteinases (MPP-2 and MPP-9). Conclusion: Taken together, we propose that genistein exerts anticancer activity in pancreatic cancer cells through induction of ROS mediated mitochondrial apoptosis, cell cycle arrest and regulation of STAT3 and may therefore prove beneficial in the management of pancreatic cancers cancer.