SH003 Induces DR5-Mediated Caspase-Dependent Apoptosis of NSCLC Through Inhibition of AKT Survival Pathway

Abstract

Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is well known to selectively induce apoptotic cell death in cancer cells, not in normal cells, with death receptors (DRs)—DR4 and DR5. In consequence of this specialty, this cytokine and its receptors are considered for candidates of target therapy in clinic. SH003, a new traditional medicine-based polyherbal preparation, consists of Astragalus membranaceus (Am), Angelica gigas (Ag) and Trichosanthes kirilowii Maximowicz (Tk). In this study, we investigated whether SH003 can induce apoptosis through DRs in non-small-cell lung cancer (NSCLC) cells. Methods Cell proliferation and cytotoxicity were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenic assay, and, trypan blue exclusion staining, protein expression by western blot analysis, and apoptosis by fluorescence-activated cell sorting analysis. Results We found that SH003-induced apoptosis in NSCLC cells through several mechanisms. First of all, MTT and colony formation assay confirmed the growth-inhibitory effect of SH003 in H460 cells. Second, SH003 upregulated the expression of DR4 and DR5. Third, it activated caspase-8, caspase-7, and caspase-3 cascades, which are essential for DR-mediated extrinsic apoptosis. The effect of SH003-induced apoptosis was significantly abolished by inhibition of caspases enzymes. And also, SH003 cleaved caspse-9. Fourth, SH003 reduced AKT kinase phosphorylation, and overexpression of AKT abrogated the caspase-dependent apoptosis by SH003. Fifth, SH003 inactivated ERK, but, constitutive ERK expression did not completely reduce SH003-mediated growth inhibition and apoptosis. Conclusions SH003 potentiates caspase-dependent apoptosis of NSCLC through the upregulation of DRs, activation of caspase cascades and downregulation of AKT cell survival pathways.