Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5 through the ROS-JNK-CHOP Signaling Axis in Human Ovarian Carcinoma Cell Lines

Abstract

Tanshinone IIA (TIIA) extracted from Salvia miltiorrhiza has been shown to possess antitumor and TRAIL-sensitizing activity. The involvement of DRS in the mechanism whereby TIIA exerts its effects is unknown. This study aimed to explore the mechanism underlying TIIA augmentation of TRAIL-induced cell death in ovarian carcinoma cells. Cell viability was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of relating signaling proteins. Transcriptional activation was explored by a dual-luciferase reporter assay. We found that TIIA sensitized human ovarian carcinoma cells to TRAIL-induced extrinsic apoptosis. Combined treatment with subtoxic concentrations of TIIA and TRAIL was more effective than single treatments with respect to cytotoxicity, donogenic inhibition, and the induction of caspase-8 and PARP activity in ovarian carcinoma cell lines TOV-21G and SKOV3. TIIA induced DRS protein and mRNA expression in a concentration-dependent manner. DR5/Fc treatment markedly suppressed the TRAIL cytotoxicity enhanced by TIIA. These results indicate that DRS plays an essential role in TIIA-induced TRAIL sensitization and that induction of DRS by TIIA is mediated through the up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP gene expression by shRNA attenuated DRS up-regulation and rescued cell viability under the treatment of TIIA-TRAIL combination. TILA. promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DRS expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DRS. In addition, the quenching of ROS using NAC prevented the induction of JNK phosphorylation and CHOP induction. Furthermore, inhibition of ROS by NAC significantly attenuated TRAIL sensitization by TIIA. Taken together, these data suggest that TIIA enhances TRAIL-induced apoptosis by upregulating DRS receptors through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cells.