Phloretin and Hesperetin are bioflavonoid belongs to two different classes, and are reported to show potent inhibitory effects on cell proliferation in different cell lines. However, very few studies conducted to check the effect of combination of bioflavonoid on the different cell lines. This bioflavonoid may play essential roles in inhibition of tumor progression in DU-145 cell line. Therefore, we investigated the expression of different pro-apoptotic and anti-apoptotic protein after treatment of DU-145 cells with phloretin and hesperetin alone and in combination in order to compare the effects of these flavonoids in cancer. We also performed an experiment to find out the effect of these flavonoids on the cell morphology, viability and proliferation on DU-145 cells.

Our data indicate that after 72 hour treatment with the hesperetin shows more potent inhibitory effect than 24 hours of treatment. Almost same results we found with the Phloretin treatment up 72 hours, but phloretin shows more potent inhibitory effect than hespereitn. The combination of hesperetin and phloretin is also shows more potent inhibitory effect on cell proliferation. To find out the possible mechanism for cell death we also carried out western blot analysis and we checked the expression of pro-apoptotic proteins BAD, Cyt c and Apaf-1. We also checked the Bcl-2 and Bcl-xl anti-apoptotic protein expressions. And we confirmed the occurrence of apoptosis by performing DNA fragmentation experiments in response to bioflavonoid treatment.

In summary, these studies demonstrate that hesperetin and phloretin alone and in combination of this bioflavonoid significantly inhibit ell proliferation by inducing apoptosis by mitochondrial Cyt c pathway. These studies also show that combination of bioflavonoid is more effective at 48 hours when compared to each bioflavonoid alone. Further studies must be performed to investigate the combination of bioflavonoid response at 72 hours and thee possible reason for their ineffectiveness.