Hsp72 inhibits Fas-mediated apoptosis upstream of the mitochondria in type II cells

Heat shock protein 72 (Hsp72) inhibits apoptosis induced by some stresses that trigger the intrinsic apoptosis pathway. However, with the exception of TNFalpha-induced apoptosis, a role for Hsp72 in modulating the extrinsic pathway of apoptosis has not been clearly established. In this study, it was demonstrated that Hsp72 could inhibit Fas-mediated apoptosis of type II CCRF-CEM cells, but not type I SW480 or CH1 cells. Similar results were obtained when Fas ligand or an agonistic Fas antibody initiated the Fas apoptosis pathway. In CCRF-CEM cells, Hsp72 inhibited mitochondrial membrane depolarization and cytochrome c release but did not alter surface Fas expression or processing of caspase-8 and Bid, indicating that Hsp72 acts upstream of the mitochondria to inhibit Fas-mediated apoptosis. Thus, the ability of Hsp72 to inhibit Fas-mediated apoptosis is limited to type II cells where involvement of the intrinsic pathway is required for efficient effector caspase activation.     

Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

Hsp105 (Hsp105alpha and Hsp105beta), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105alpha regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105alpha or Hsp105beta by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105alpha or Hsp105beta. In addition, we found that overexpression of Hsp105alpha or Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105alpha or Hsp105beta. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells.     

Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1

Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome.     

Ku70 suppresses the apoptotic translocation of Bax to mitochondria

Bax induces mitochondrial-dependent cell death signals in mammalian cells. However, the mechanism of how Bax is kept inactive has remained unclear. Yeast-based functional screening of Bax inhibitors from mammalian cDNA libraries identified Ku70 as a new Bax suppressor. Bax-mediated apoptosis was suppressed by overexpression of Ku70 in mammalian cells, but enhanced by downregulation of Ku70. We found that Ku70 interacts with Bax, and that the carboxyl terminus of Ku70 and the amino terminus of Bax are required for this interaction. Bax is known to translocate from the cytosol to mitochondria when cells receive apoptotic stimuli. We found that Ku70 blocks the mitochondrial translocation of Bax. These results suggest that in addition to its previously recognized DNA repair activity in the nucleus, Ku70 has a cytoprotective function in the cytosol that controls the localization of Bax.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria

Increasing autophagy is beneficial for curing hepatocellular carcinoma (HCC). Damage-regulated autophagy modulator (DRAM) was recently reported to induce apoptosis by mediating autophagy. However, the effects of DRAM-mediated autophagy on apoptosis in HCC cells remain unclear. In this study, normal hepatocytes (7702) and HCC cell lines (HepG2, Hep3B and Huh7) were starved for 48 h. Starvation induced apoptosis and autophagy in all cell lines. We determined that starvation also induced DRAM expression and DRAM-mediated autophagy in both normal hepatocytes and HCC cells. However, DRAM-mediated autophagy was involved in apoptosis in normal hepatocytes but not in HCC cells, suggesting that DRAM-mediated autophagy fails to induce apoptosis in hepatoma in response to starvation. Immunoblot and immunofluorescence assays demonstrated that DRAM translocated to mitochondria and induced mitophagy, which led to apoptosis in 7702 cells. In HCC cells, starvation also activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which blocks the translocation of DRAM to mitochondria through the binding of p-AKT to DRAM in the cytoplasm. Inactivation of the PI3K/AKT pathway rescued DRAM translocation to mitochondria; subsequently, mitochondrial DRAM induced apoptosis in HCC cells by mediating mitophagy. Our findings open new avenues for the investigation of the mechanisms of DRAM-mediated autophagy and suggest that promoting DRAM-mediated autophagy together with PI3K/AKT inhibition might be more effective for autophagy-based therapy in hepatoma.     

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.     

Stabilization and translocation of p53 to mitochondria is linked to Bax translocation to mitochondria in simvastatin-induced apoptosis

Statins are cholesterol-lowing drugs with pleiotropic effects including cytotoxicity to cancer cells. In this study, we investigated the signaling pathways leading to apoptosis by simvastatin. Simvastatin induced cardinal features of apoptosis including increased DNA fragmentation, disruption of mitochondrial membrane potential (MMP), and increased caspase-3 activity by depleting isoprenoids in MethA fibrosarcoma cells. Interestingly, the simvastatin-induced apoptosis was accompanied by p53 stabilization involving Mdm2 degradation. The apoptosis was ameliorated in p53 knockdown clones of MethA cells as well as p53^−/− HCT116 cells. The stabilized p53 protein translocated to mitochondria with Bax, and cytochrome c was released into cytosol. Moreover, knockdown or deficiency of p53 expression reduced both Bax translocation to mitochondria and MMP disruption in simvastatin-induced apoptosis. Taken together, these all indicate that stabilization and translocation of p53 to mitochondria is involved in Bax translocation to mitochondria in simvastatin-induced apoptosis.     

Acute acidification or amiloride treatment suppresses the ability of Hsp70 to inhibit heat-induced apoptosis

Inhibition of stress-induced apoptosis by the molecular chaperone protein Hsp70 is a contributing factor in tumorigenesis and suppression of this ability could increase the effectiveness of anti-tumor therapy. Tumor cells exist in an acidic environment and acute acidification can sensitize tumor cells to heat-induced cell death. However, the ability of Hsp70 to prevent apoptosis under these conditions has not been examined. The effect of acute acidification on heat-induced apoptosis was examined in a human T-cell line with tetracycline-regulated Hsp70 expression. Apoptosis was inhibited in cells exposed to hyperthermia in acidic media when examined 6 h after the heat stress, but resumed if cells were returned to physiological pH during this recovery period. Long-term proliferation assays showed that acute acidification sensitized cells to heat-induced apoptosis. Hsp70 expressing cells were also sensitized and this was correlated with a reduced ability to suppress the activation of JNK (c-jun N-terminal kinase), Bax and caspase-3. Further sensitization could be achieved with the NHE1 (Na⁺/H⁺ exchanger) inhibitor HMA (5-(N, N-hexamethylene) amiloride), which potentiated JNK activation in heat-shocked cells. These results demonstrate that the ability of Hsp70 to suppress apoptosis is compromised when cells are exposed to hyperthermia in an acidic environment, which is correlated with an impaired ability to inhibit JNK activation.     

The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (alpha9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.     

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis

It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 μg/ml) and UV light (50 J/cm²), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone, and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV lightmediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.     

Inhibition of the JNK/Bim pathway by Hsp70 prevents Bax activation in UV-induced apoptosis

Here we studied the mechanism by which heat shock protein 70 (Hsp70) prevents Bax activation during ultraviolet (UV)-induced apoptosis. UV treatment led to c-Jun N-terminal kinase (JNK) phosphorylation, Bim redistribution and subsequent Bax activation. Bim depletion caused a smaller reduction in apoptosis than that by JNK inhibition, indicating that Bim activation is not entirely responsible for induction of apoptosis and other mechanisms are involved. Hsp70 knockdown resulted in high levels of activated JNK and Bax, while Hsp70 overexpression inhibited these processes. These findings demonstrate that Hsp70 prevented Bax activation via inhibiting the JNK/Bim pathway. Simultaneously, increased binding of Hsp70 to Bax was observed. Collectively, our results for the first time demonstrate that Hsp70 prevents Bax activation both by inhibiting the JNK/Bim pathway and by interacting with Bax in UV-induced apoptosis.     

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.     

Lithium inhibits aluminum-induced apoptosis in rabbit hippocampus,by preventing cytochrome c translocation,Bcl-2 decrease,Bax elevation and caspase-3 activation

A variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis-regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti-apoptotic proteins Bcl-2 and Bcl-X(L), increases levels of the pro-apoptotic Bax, activates caspase-3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum-induced translocation of cytochrome c, and up-regulates Bcl-2 and Bcl-X(L,) down-regulates Bax, abolishes caspase-3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis-controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis-regulatory proteins present in the subcellular organelles of rabbit brain.     

LPLI inhibits apoptosis upstream of Bax translocation via a GSK‐3β‐inactivation mechanism

Low‐power laser irradiation (LPLI), a non‐damage physical therapy, which has been used clinically for decades of years, is shown to promote cell proliferation and prevent apoptosis. However, the underlying mechanisms that LPLI prevents cell apoptosis remain undefined. In this study, based on real‐time single‐cell analysis, we demonstrated for the first time that LPLI inhibits staurosporine (STS)‐induced cell apoptosis by inactivating the GSK‐3β/Bax pathway. LPLI could inhibit the activation of GSK‐3β, Bax, and caspase‐3 induced by STS. In the searching for the mechanism, we found that, LPLI can activate Akt, which was consistence with our former research, even in the presence of STS. In this anti‐apoptotic process, the interaction between Akt and GSK‐3β increased gradually, indicating Akt interacts with and inactivates GSK‐3β directly. Conversely, LPLI decreased the interaction between GSK‐3β and Bax, with the suppression of Bax translocation to mitochondria, suggesting LPLI inhibits Bax translocation through inactivating GSK‐3β. These results were further confirmed by the experiments of co‐immunoprecipitation. Wortmannin, an inhibitor of phosphatidylinositol 3′‐OH kinase (PI3K), potently suppressed the activation of Akt and subsequent anti‐apoptotic processes induced by LPLI. Taken together, we conclude that LPLI protects against STS‐induced apoptosis upstream of Bax translocation via the PI3K/Akt/GSK‐3β pathway. These findings raise the possibility of LPLI as a promising therapy for neuron‐degeneration disease induced by GSK‐3β. J. Cell. Physiol. 224:218–228, 2010 © 2010 Wiley‐Liss, Inc.     

NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

ABSTRACT: BACKGROUND: Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV), the matrix (M) protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV.FindingIn order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10 % at 48 h post-infection). In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells. CONCLUSION: Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s) upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.     

Protein unfolding by mitochondria. The Hsp70 import motor

Protein unfolding is a key step in the import of some proteins into mitochondria and chloroplasts and in the degradation of regulatory proteins by ATP-dependent proteases. In contrast to protein folding, the reverse process has remained largely uninvestigated until now. This review discusses recent discoveries on the mechanism of protein unfolding during translocation into mitochondria. The mitochondria can actively unfold preproteins by unraveling them from the N-terminus. The central component of the mitochondrial import motor, the matrix heat shock protein 70, functions by both pulling and holding the preproteins.     

Clusterin inhibits apoptosis by interacting with activated Bax

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.