Emerging results suggest that ceramides with different fatty acid chain lengths might perform distinct functions in the regulation of tumor growth and therapy. ER is definitely a critical organelle in the induction of apoptosis and is responsible for intracellular Ca2+ storage. More important, the ER provides a topologically unique membranous network for protein modifications such as glycosylation, disulfide relationship formation, and appropriate protein folding and assembly. Thus, a significant quantity of ER resident proteins either sequester Ca2+ or function as molecular chaperones to monitor appropriate protein folding. Therefore, failure of this machinery to collapse newly synthesized ER client proteins, or perturbation of the ER-Ca2+ equilibrium, presents unique dangers to the cell, which disrupts normal cellular functions and is termed as ER stress (10, 11). A complex homeostatic mechanism, known as the unfolded protein response (UPR), offers evolved to minimize ER stress by increasing the protein folding capacity of ER, reducing the pace of secretory protein synthesis, and/or increasing the chaperone capacity in cells (10, 11). Therefore, both transcriptional and translational signals play a role in UPR. This concerted and complex UPR is definitely mediated through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), pancreatic ER kinase (PKR)-like ER kinase (PERK), and activating transcription element 6 (ATF6; refs. 12,13,14). In resting cells, all three ER stress proteins are taken care of in an inactive state by their association with the ER chaperone GRP78 (also known as Bip) (15). On build up of unfolded proteins, GRP78 dissociates from these stress receptors, leading to their activation, which causes the UPR. Consequently, UPR is essentially a prosurvival response to reduce the build up of unfolded proteins, or protein aggregates, and to restore normal ER homeostasis. However, if protein unfolding or aggregation is definitely prolonged and cannot Oleanolic Acid be resolved, then UPR signaling switches from prosurvival to proapoptosis mode (16, 17). It is known that signaling through PERK, IRE1, and ATF6 can result in apoptosis during prolonged ER stress, with an activation of their downstream focuses on, primarily the CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), JNK, and Bcl-2 family proteins, which are involved in the commitment phase of ER-stress-mediated apoptosis (18,19,20,21). Although CHOP generally induces gene manifestation, it down-regulates the transcription of Bcl-2 (22,23,24), leading to cell death. Recently, the functions of ceramides in the induction of ER stress and apoptosis have been reported in various human malignancy cells (25, 26). However, the mechanisms by which and test was used. Ideals of < 0.05 were considered significant. RESULTS Knockdown of CerS6 using siRNAs induces apoptosis in HNSCC cells Our earlier studies (3, 27, 28) have suggested the activation of caspase 3 and caspase 9 in these cells Oleanolic Acid (Fig. 1siRNA induces ER-stress-mediated apoptosis We next investigated how knockdown of CerS6 induces apoptosis. We IgM Isotype Control antibody (FITC) hypothesized that inhibition of CerS6 manifestation and C16-ceramide generation might mediate ER stress, leading to apoptosis in these cells. The possible involvement of CerS6/C16-ceramide in ER stress was prompted by several lines of evidence: synthesis of ceramide happens in the ER; consequently, attenuation of Oleanolic Acid the generation of a major ER residing protein and/or lipid molecule (C16-ceramide) might impact ER homeostasis; and manifestation of wt-CerS6. the mutant CerS6 in the safety of ER stress and apoptosis mediated by siRNA against CerS6 were examined. Treatment with CerS6 siRNA down-regulated Oleanolic Acid the manifestation of the endogenous protein significantly (90%), as compared with Oleanolic Acid scrambled siRNA-treated cells (Fig. 3selective induction of the ATF6/CHOP arm of UPR To determine the mechanisms by which down-regulation of C16-ceramide induces ER stress, activation of the 3 arms of the ER-stress-response pathways was examined after knockdown of CerS6. Manifestation levels of ATF6, IRE1, or ATF4 were down-regulated using siRNAs, compared with controls. Down-regulation of the genes was confirmed by RT-PCR (Fig. 5activation of CHOP and induces loss of mitochondrial membrane potential. the activation of the ATF6/CHOP axis selectively, which then results in decreased manifestation of Bcl-2,.