Introduction: Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL) is a member of the tumor necrosis factor superfamily. TRAIL, a valuable agent for cancer therapy, induces apoptosis in the majority of cancer cells, but development of resistance limits its clinical use in many malignancies including prostate cancer. We have demonstrated that Tetrandrine derivatives (TET), promote apoptosis in PCa cells in vitro and in vivo. In the present study, we investigated the ability of TET to sensitize tumor cells to TRAIL. We also investigated the role of two-death receptors- DR4 and DR5 in overcoming TRAIL resistance in prostate cancer cells.

Methods: Prostate cancer cell lines LNCaP and LNCaP derived C4-2b were used for this study. Cell lines were treated with TET and TRAIL alone or in various combinations of these two for different time points. mRNA levels were quantitated via Real Time-PCR and changes in protein expression were analyzed by Western Blot. Cytotoxicity was evaluated using the Crystal violet and MTT survival assay. Cells were transfected with Lentiviral-shRNA to generate stable DR4 and DR5 single and double knockdown cells. Apoptosis assay was done using BD Pharmingen FITC Annexin-V Apoptosis Detection kit I.

Results: LNCaP and C4-2b cells did not show a significant response following exposure to TRAIL. Exposure of LNCaP and C4-2b cells to TET resulted in increased mRNA and protein levels of death receptors DR4, DR5. Pretreatment of both cell lines with TET for 12h, followed by TRAIL treatment for another 24h increased the apoptosis inducing potential of TRAIL in these cells. Exposure of TET treated cells in vitro to soluble h-TRAIL in combination with TET resulted in synergistic apoptosis response. shRNA targeting DR4 and DR5 significantly reduced the levels of DR4 and DR5 protein. There was a reduced expression of DR4 and DR5 in the knock down cells in response to TET in time-dependent and concentration-dependent manner. In comparison to scrambled cells, DR4, DR5 single knockdown and DR4/DR5 double-knock down cells showed reduced sensitivity to TRAIL and TET. These cells were less apoptotic as compared to scrambled LNCaP cells as revealed by our apoptosis assay data. Taken together these data suggest that TET induced sensitization of PCa cells to TRAIL is mediated in part by DR4/DR5.

Conclusion: Our results suggest that TET induces apoptosis in part by inducing the expression of DR4 and DR5, and provide the proof-of-principle for a novel therapeutic strategy to sensitize cancer cells to apoptosis.

Acknowledgement: Financial support from following Sources is gratefully acknowledged: (VA Merit Award 1BX001258; NCI RO1-CA161880); Carroll W. Feist Endowed Chair Funds, FWCC support and Chair commitment LSUHSC-Shreveport).

Citation Format: Gauri Shishodia, Sweaty Koul, Qin Dong, Sergey Slepenkov, Hari K. Koul. Tetrandrine promotes prostate cancer cell apoptosis in part by up-regulation of death receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 253.