Role of Gasdermin E in the Biogenesis of Apoptotic Cell-Derived Exosomes.

The gasdermins are a family of pore-forming proteins that has recently been suggested to play a central role in pyroptosis. In this study, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic human HeLa and HEK293 cells, GSDMA, GSDMC, GSDMD, and GSDME increased the release of apoptotic exosomes. GSDMB and DFNB59, in contrast, negatively affected the release of apoptotic exosomes. GSDME at its full-length and cleaved forms was localized in the exosomes and exosomal membrane. Full-length and cleaved forms of GSDME are suggested to increase Ca2+ influx to the cytosol through endosomal pores and thus increase the biogenesis of apoptotic exosomes. In addition, the GSDME-mediated biogenesis of apoptotic exosomes depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins, that is, annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of apoptotic exosomes begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that GSDME-bearing tumors released apoptotic exosomes to induce inflammatory responses in the in vivo mouse 4T1 orthotropic model of BALB/c breast cancer. The data indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

Upregulation of DR5 and Downregulation of Survivin by IITZ-01, Lysosomotropic Autophagy Inhibitor, Potentiates TRAIL-Mediated Apoptosis in Renal Cancer Cells via Ubiquitin-Proteasome Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

Arylquin 1, a potent Par-4 secretagogue, induces lysosomal membrane permeabilization-mediated non-apoptotic cell death in cancer cells.

Arylquin 1, a small-molecule prostate-apoptosis-response-4 (Par-4) secretagogue, targets vimentin to induce Par-4 secretion. Secreted Par-4 binds to its receptor, 78-kDa glucose-regulated protein (GRP78), on the cancer cell surface and induces apoptosis. In the present study, we investigated the molecular mechanisms of arylquin 1 in cancer cell death. Arylquin 1 induces morphological changes (cell body shrinkage and cell detachment) and decreases cell viability in various cancer cells. Arylquin 1-induced cell death is not inhibited by apoptosis inhibitors (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitors (necrostatin-1), and paraptosis inhibitors. Furthermore, arylquin 1 significantly induces reactive oxygen species levels, but antioxidants [N-acetyl-l-cysteine and glutathione ethyl ester] do not inhibit arylquin 1-induced cell death. Furthermore, Par-4 knock-down by small interfering RNA confers no effect on cytotoxicity in arylquin 1-treated cells. Interestingly, arylquin 1 induces lysosomal membrane permeabilization (LMP), and cathepsin inhibitors and overexpression of 70-kDa heat shock protein (HSP70) markedly prevent arylquin 1-induced cell death. Therefore, our results suggest that arylquin 1 induces non-apoptotic cell death in cancer cells through the induction of LMP.

Elucidation for modulation of death receptor (DR) 5 to strengthen apoptotic signals in cancer cells.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis via death receptor (DR) 4 or DR5 preferentially in cancer cells, and not in normal cells with relatively high decoy receptor expression. However, multiple mechanisms in cancer cells induce resistance to DRs-mediated apoptosis. Therefore, understanding of molecular mechanisms for resistance to DRs-mediated apoptosis can find the strategy to increase sensitivity. Although multiple proteins are involved in resistance to DRs-mediated apoptosis, we focus on modulation of DR5 to overcome resistance. Here, we discuss regulation of DR5 expression or activation by epigenetic modification, transcription factor at the transcriptional levels, micro RNA and RNA-binding proteins at the post-transcriptional levels, and ubiquitination and glycosylation at the post-translational levels. In addition, we also mention about relationship between localization of DR5 and death signaling activation. The purpose of this review is to help understand relationship between regulatory mechanisms of DR5 and resistance to TRAIL or DRs-targeted agonist monoclonal antibodies, and to develop innovative anti-cancer therapies through regulation of DR5 signaling.

Cepharanthine Enhances TRAIL-Mediated Apoptosis Through STAMBPL1-Mediated Downregulation of Survivin Expression in Renal Carcinoma Cells.

Cepharanthine (CEP) is a natural plant alkaloid, and has anti-inflammatory, antineoplastic, antioxidative and anticancer properties. In this study, we investigated whether CEP could sensitize renal carcinoma Caki cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. CEP alone and TRAIL alone had no effect on apoptosis. However, combined CEP and TRAIL treatment markedly enhanced apoptotic cell death in cancer cells, but not in normal cells. CEP induced downregulation of survivin and cellular-FLICE inhibitory protein (c-FLIP) expression at post-translational levels. Ectopic expression of survivin blocked apoptosis by combined treatment with CEP plus TRAIL, but not in c-FLIP overexpression. Interestingly, CEP induced survivin downregulation through downregulation of deubiquitin protein of STAM-binding protein-like 1 (STAMBPL1). Overexpression of STAMBPL1 markedly recovered CEP-mediated survivin downregulation. Taken together, our study suggests that CEP sensitizes TRAIL-mediated apoptosis through downregulation of survivin expression at the post-translational levels in renal carcinoma cells.