The Bcl-2 family of proteins comprises well characterized regulators of apoptosis, consisting of anti-apoptotic members and pro-apoptotic members. Pro-apoptotic members possessing BH1, BH2, and BH3 domains (such as Bax and Bak) act as a gateway for a variety of apoptotic signals. Bax is normally localized to the cytoplasm in an inactive form. In response to apoptotic stimuli, Bax translocates to the mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c, but it is still largely unknown how the mitochondrial translocation and proapoptotic activity of Bax is regulated. Here we report that cytoplasmic protein 14-3-3 binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism, as well as through direct cleavage of 14-3-3 by caspases. Unlike Bad, the interaction with 14-3-3 is not dependent on the phosphorylation of Bax. In isolated mitochondria, we found that 14-3-3 inhibited the integration of Bax and Bax-induced cytochrome c release. Bax-induced apoptosis was inhibited by overexpression of either 14-3-3 or its mutant (which lacked the ability to bind to various phosphorylated targets but still bound to Bax), whereas overexpression of 14-3-3 was unable to inhibit apoptosis induced by a Bax mutant that did not bind to 14-3-3. These findings indicate that 14-3-3 plays a crucial role in negatively regulating the activity of Bax.

Nitric oxide (NO) is implicated in apoptosis and has both cytotoxic and cytoprotective effects. Exogenous NO induced the death of PC12 and HeLa cells via a process showing features of both apoptosis and necrosis, with chromatin condensation, nuclear compaction, and mitochondrial swelling. Activation of caspases was not observed during NO-induced cell death. In addition, cell death was not inhibited by peptide caspase inhibitors or by expression of p35, a baculovirus-encoded caspase inhibitor, indicating that NO-induced cell death was independent of caspases. NO-induced cell death was enhanced by Bax expression in a caspase-independent manner and prevented by the anti-cell death protein Bcl-2. Although Bcl-2 has previously been shown to prevent cell death by inhibiting caspase activation, these results indicate that it can also prevent cell death via a caspase-independent mechanism.Nitric oxide (NO) 1 is enzymatically generated from L-arginine by constitutive or inducible NO synthase, and has a number of physiological roles, including smooth muscle relaxation, and neurotransmission (1, 2). NO has also been implicated in a variety of pathological phenomena, such as septic shock, ␤-cell destruction, and transplant rejection (1, 2). Some of these pathological events are closely related to apoptotic cell death (3,4). In many studies, NO has been shown to induce apoptosis, although the precise mechanism involved is still unclear (5-7). In contrast, some investigators have suggested that NO also has the ability to prevent cell death (8 -10), which seems to be mediated by the inhibition of caspases (8), common mediators of apoptosis (11). So far, more than 10 caspases have been identified in mammals. Apoptosis is also regulated by Bcl-2 family proteins, including anti-apoptotic proteins such as Bcl-2 and Bcl-x L , and pro-apoptotic proteins such as Bax and Bak (12). Accumulating evidence suggests that Bcl-2 acts upstream of caspase activation to prevent apoptosis (13,14).In this study, we analyzed NO-induced cell death, particularly focusing on the role of caspases as well as the influence of apoptosis-regulating molecules such as Bcl-2 family proteins. EXPERIMENTAL PROCEDURESReagents-Caspase inhibitors and substrates were purchased from Peptide Inc. (Minoh, Japan). Other chemicals were purchased from Wako Chemical Co. (Tokyo, Japan).Cell Lines and Transfection-HeLa cells, a human cervical carcinoma-derived cell line, and PC12 cells, a rat pheochromocytoma cell line, were maintained in RPMI 1640 culture medium, as described elsewhere (15). A stable transfectant of PC12 cells expressing mouse Bax (designated as PC12-Bax) was obtained by infecting retrovirus that was produced from the packaging cell line ⌿2 transfected with the retroviral vector pBC140 (15) containing mouse bax cDNA. A stable transfectant of PC12 cells expressing human Bcl-2 (designated as PC12-Bcl-2) was obtained by transfecting the pUC-CAGGS vector bearing the human bcl-2 cDNA using electroporation (13). Empty vector-transduced cells were used as the...

Purpose: The treatment of cancer with oncolytic viruses primarily depends on the selective viral replication in cancer cells. However, a replication-incompetent hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) suppresses the growth of human cancer cells as effectively as replicationcompetent live HVJ without producing toxic effects in nonmalignant cells. Here, we analyze the molecular mechanism of the oncolytic activity of HVJ-E.Experimental Design: The molecules responsible for HVJ-E-induced cancer cell death were elucidated in prostate cancer cell lines, and the effect of HVJ-E on orthotopic prostate cancers was evaluated in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice.Results: The liposome-mediated transfer of viral RNA genome fragments from HVJ-E suppressed the viability of prostate cancer cells but not the viability of the noncancerous prostate epithelium. Knockdown experiments using siRNAs showed that the cancer cell-selective killing induced by HVJ-E was mediated by retinoic acid-inducible gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). Downstream of the RIG-I/MAVS pathway, both TNF-related apoptosis-inducing ligand (TRAIL) and Noxa were upregulated by HVJ-E in the castration-resistant prostate cancer cell line PC3 but not in the noncancerous prostate epithelial cell line PNT2. TRAIL siRNA but not Noxa siRNA significantly inhibited HVJ-E-induced cell death in PC3 cells. However, Noxa siRNA effectively suppressed HVJ-E-induced cell death in DU145 cells, another castration-resistant prostate cancer cell line, in which Noxa but not TRAIL was upregulated by HVJ-E. Furthermore, the orthotopic prostate cancers were dramatically eradicated in immunodeficient mice injected with HVJ-E.Conclusion: The RIG-I/MAVS signaling pathway represents an attractive target for cancer therapy.

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The administration of rikkunshito resulted in symptomatic relief and improved gastric emptying in profoundly handicapped patients with delayed gastric emptying.