Effects of short‐chain acyl‐CoA dehydrogenase on cardiomyocyte apoptosis

Short‐chain acyl‐CoA dehydrogenase (SCAD), a key enzyme of fatty acid β‐oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert‐butyl hydroperoxide (tBHP)‐induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down‐regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA‐1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl‐2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP‐activated protein kinase α (p‐AMPKα) and Peroxisome proliferator‐activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways.     

AT‐101 (R‐(−)‐gossypol acetic acid) enhances the effectiveness of androgen deprivation therapy in the VCaP prostate cancer model

Prostate cancer remains a leading cause of cancer death in American men. Androgen deprivation therapy (ADT) is the most common treatment for advanced prostate cancer patients; however, ADT fails in nearly all cases resulting in castration resistant or androgen‐insensitive (AI) disease. In many cases, this progression results from dysregulation of the pro‐survival Bcl‐2 family proteins. Inhibition of pro‐survival Bcl‐2 family proteins, therefore, may be an effective strategy to delay the onset of AI disease. Gossypol, a small molecule inhibitor of pro‐survival Bcl‐2 family proteins, has been demonstrated to inhibit AI prostate cancer growth. The apoptotic effect of gossypol, however, has been demonstrated to be attenuated by the presence of androgen in a prostate cancer xenograft mouse model (Vertebral Cancer of Prostate [VCaP]) treated with AT‐101 (R‐(?)‐gossypol acetic acid). This study was undertaken to better understand the in vitro effects of androgen receptor (AR) on AT‐101‐induced apoptosis. VCaP cells treated with AT‐101 demonstrated an increase in apoptosis and downregulation of Bcl‐2 pro‐survival proteins. Upon AR activation in combination with AT‐101 treatment, apoptosis is reduced, cell survival increases, and caspase activation is attenuated. Akt and X inhibitor of apoptosis (XIAP) are downregulated in the presence of AT‐101, and AR stimulation rescues protein expression. Combination treatment of bicalutamide and AT‐101 increases apoptosis by reducing the expression of these pro‐survival proteins. These data suggest that combination therapy of AT‐101 and ADT may further delay the onset of AI disease, resulting in prolonged progression‐free survival of prostate cancer patients. J. Cell. Biochem. 110: 1187–1194, 2010. Published 2010 Wiley‐Liss, Inc.     

IBRDC2, an IBR‐type E3 ubiquitin ligase,is a regulatory factor for Bax and apoptosis activation

Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax^?/? cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.     

BBR induces apoptosis in HepG2 cell through an Akt‐ASK1‐ROS‐p38MAPKs‐linked cascade

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase‐3. The expressions of Bcl‐2 protein and pro‐caspase‐3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR‐induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR‐induced apoptosis effects via inhibition of Bax activation and Bcl‐2 inactivation. BBR‐induced, dose‐dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1‐mediated activation of JNK and p38 pathways. J. Cell. Biochem. 109: 329–338, 2010. © 2009 Wiley‐Liss, Inc.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Sex differences in the level of Bcl‐2 family proteins and caspase‐3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal rats

In developing rats, sex differences in the number of apoptotic cells are found in the central division of the medial preoptic nucleus (MPNc), which is a significant component of the sexually dimorphic nucleus of the preoptic area, and in the anteroventral periventricular nucleus (AVPV). Specifically, male rats have more apoptotic cells in the developing AVPV, whereas females have more apoptotic cells in the developing MPNc. To determine the mechanisms for the sex differences in apoptosis in these nuclei, we compared the expression of the Bcl‐2 family members and active caspase‐3 in postnatal female and male rats. Western blot analyses for the Bcl‐2 family proteins were performed using preoptic tissues isolated from the brain on postnatal day (PD) 1 (day of birth) or on PD8. In the AVPV‐containing tissues of PD1 rats, there were significant sex differences in the level of Bcl‐2 (female > male) and Bax (female < male) proteins, but not of Bcl‐xL or Bad proteins. In the MPNc‐containing tissues of PD8 rats, there were significant sex differences in the protein levels for Bcl‐2 (female < male), Bax (female > male), and Bad (female < male), but not for Bcl‐xL. Immunohistochemical analyses showed significant sex differences in the number of active caspase‐3‐immunoreactive cells in the AVPV on PD1 (female < male) and in the MPNc on PD8 (female > male). We further found that active caspase‐3‐immunoreactive cells of the AVPV and MPNc were immunoreactive for NeuN, a neuronal marker. These results suggest that there are sex differences in the induction of apoptosis via the mitochondrial pathway during development of the AVPV and MPNc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

The protective effects of grape seed procyanidin B2 against asporin mediates glycated low‐density lipoprotein induced‐cardiomyocyte apoptosis and fibrosis

The progression of diabetic cardiomyopathy is related to cardiomyocyte dysfunction and apoptosis. Our previous studies showed that asporin (ASPN) was significantly increased in the myocardium of db/db mice through proteomics, and grape seed procyanidin B2 (GSPB2) significantly inhibited the expression of ASPN in the heart of db/db mice. We report here that ASPN played a critical role in glycated low‐density lipoproteins (gly‐LDL) induced‐cardiomyocyte apoptosis. We found that gly‐LDL upregulated ASPN expression. ASPN increased H9C2 cardiomyocyte apoptosis with down‐regulation of Bcl‐2, upregulation of transforming growth factor‐β1, Bax, collagen III, fibronectin, and phosphorylation of smad2 and smad3. However, GSPB2 treatment reversed ASPN‐induced impairments in H9C2 cardiomyocytes. These results provide evidence for the cardioprotective action of GSPB2 against ASPN injury, and thus suggest a new target for fighting against diabetic cardiomyopathy.     

Pro‐apoptotic activity of mBNIP‐21 depends on its BNIP‐2 and Cdc42GAP homology (BCH) domain and is enhanced by coxsackievirus B3 infection

Our previous study reported that mouse BNIP‐21 (mBNIP‐21) induces apoptosis through a mitochondria‐dependent pathway. To map the functional domains of mBNIP‐21, we performed mutational analyses and demonstrated that the BNIP‐2 and Cdc42GAP homology (BCH) domain is required for apoptosis induction by mBNIP‐21 targeting the mitochondria and inducing cytochrome c release. This pro‐apoptotic activity was enhanced by coxsackievirus infection. However, deletion of the Bcl‐2 homology 3 (BH3)‐like domain, a well‐known cell ‘death domain’ in proapoptotic Bcl‐2 family proteins, did not affect the activity of mBNIP‐21. These data were further supported by transfection of a mouse Bax (mBax) mutant, whose BH3 was replaced by the mBNIP‐21 BH3‐like domain. This replacement significantly reduced the pro‐apoptotic activity of mBax. We also found that the predicted calcium binding domain has no contribution to the mBNIP‐21‐induced apoptosis. Further mapping of the motifs of BCH domain demonstrated that deletion of the hydrophobic motif proximal to the C‐terminal of the BCH significantly reduced its proapoptotic activity. These findings suggest that mBNIP‐21, as a member of the BNIP subgroup of the Bcl‐2‐related proteins, functions without need of BH3 but its BCH domain is critical for its activity in inducing cell elongation, membrane protrusions and apoptotic cell death.     

Cyclooxygenase‐2 over‐expression inhibits liver apoptosis induced by hyperglycemia

Increased expression of COX‐2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX‐2 protects hepatocytes from several pro‐apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX‐2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX‐2 (hCOX‐2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post‐injection, Wt diabetic animals showed a decrease in PI3K activity and P‐Akt levels, an increase of P‐JNK, P‐p38, pro‐apoptotic Bad and Bax, release of cytochrome c and activities of caspases‐3 and ‐9, leading to an increased apoptotic index. This situation was improved in diabetic COX‐2 Tg. In addition, SID COX‐2 Tg showed increased expression of anti‐apoptotic Mcl‐1 and XIAP. Pro‐apoptotic state in the liver of diabetic animals was improved by over‐expression of COX‐2. We also analyzed the roles of high glucose‐induced apoptosis and hCOX‐2 in vitro. Non‐transfected and hCOX‐2‐transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non‐transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX‐2‐transfected cells was suppressed by addition of DFU (COX‐2 selective inhibitor), and mimicked by addition of PGE₂ in non‐transfected cells. Taken together, these results demonstrate that hyperglycemia‐induced hepatic apoptosis is protected by hCOX‐2 expression. J. Cell. Biochem. 114: 669–680, 2013. © 2012 Wiley Periodicals, Inc.     

Up‐Regulation and Redistribution of Bax in Ultraviolet B‐Irradiated Melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0–30 mJ/cm²). The levels of p53, Bax, and Bcl‐2, determined by immunoblotting, revealed a dose‐dependent increase in p53 and Bax, but the level of Bcl‐2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl‐2. These data suggest that the high constitutional level of Bcl‐2 may protect melanocytes from UVB‐induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Knockdown of Mtfp1 can minimize doxorubicin cardiotoxicity by inhibiting Dnm1l‐mediated mitochondrial fission

The long‐term usage of doxorubicin (DOX) is largely limited due to the development of severe cardiomyopathy. Many studies indicate that DOX‐induced cardiac injury is related to reactive oxygen species generation and ultimate activation of apoptosis. The role of novel mitochondrial fission protein 1 (Mtfp1) in DOX‐induced cardiotoxicity remains elusive. Here, we report the pro‐mitochondrial fission and pro‐apoptotic roles of Mtfp1 in DOX‐induced cardiotoxicity. DOX up‐regulates the Mtfp1 expression in HL‐1 cardiac myocytes. Knockdown of Mtfp1 prevents cardiac myocyte from undergoing mitochondrial fission, and subsequently reduces the DOX‐induced apoptosis by preventing dynamin 1‐like (Dnm1l) accumulation in mitochondria. In contrast, when Mtfp1 is overexpressed, a suboptimal dose of DOX can induce a significant percentage of cells to undergo mitochondrial fission and apoptosis. These data suggest that knocking down of Mtfp1 can minimize the cardiomyocytes loss in DOX‐induced cardiotoxicity. Thus, the regulation of Mtfp1 expression could be a novel therapeutic approach in chemotherapy‐induced cardiotoxicity.     

Induced pluripotent stem cell‐conditional medium inhibits H9C2 cardiomyocytes apoptosis via autophagy flux and Wnt/β‐catenin pathway

Induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) could improve cell viability in many types of cells and may be a better alternative for the treatment of myocardial infarction. This study aimed to examine the influence of iPS‐CM on anti‐apoptosis and the proliferation of H9C2 cardiomyocytes and investigate the underlying mechanisms. H9C2 cardiomyocytes were exposed to 200 μmol/L hydrogen peroxide (H₂O₂) for 24 hours with or without pre‐treatment with iPS‐CM. The ratio of apoptotic cells, the loss of mitochondrial membrane potential (△Ψm) and the levels of intracellular reactive oxygen species were analysed by flow cytometric analysis. The expression levels of BCL‐2 and BAX proteins were analysed by Western blot. Cell proliferation was assessed using cell cycle and EdU staining assays. To study cell senescence, senescence‐associated β‐galactosidase (SA‐β‐gal) staining was conducted. The levels of malondialdehyde, superoxide dismutase and glutathione were also quantified using commercially available enzymatic kits. The results showed that iPS‐CM containing basic fibroblast growth factor significantly reduced H₂O₂‐induced H9C2 cardiomyocyte apoptosis by activating the autophagy flux pathway, promoted cardiomyocyte proliferation by up‐regulating the Wnt/β‐catenin pathway and inhibited oxidative stress and cell senescence. In conclusion, iPS‐CM effectively enhanced the cell viability of H9C2 cardiomyocytes and could potentially be used to inhibit cardiomyocytes apoptosis to treat myocardial infarction in the future.     

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.     

p53 signalling controls cell cycle arrest and caspase‐independent apoptosis in macrophages infected with pathogenic Leptospira species

Pathogenic Leptospira species, the causative agents of leptospirosis, have been shown to induce macrophage apoptosis through caspase‐independent, mitochondrion‐related apoptosis inducing factor (AIF) and endonuclease G (EndoG), but the signalling pathway leading to AIF/EndoG‐based macrophage apoptosis remains unknown. Here we show that infection of Leptospira interrogans caused a rapid increase in reactive oxygen species (ROS), DNA damage, and intranuclear foci of 53BP1 and phosphorylation of H2AX (two DNAdamage indicators) in wild‐type p53‐containing mouse macrophages and p53‐deficient human macrophages. Most leptospire‐infected cells stayed at the G₁ phase, whereas depletion or inhibition of p53 caused a decrease of the G₁‐phase cells and the early apoptotic ratios. Infection with spirochaetes stimulated a persistent activation of p53 and an early activation of Akt through phosphorylation. The intranuclear translocation of p53, increased expression of p53‐dependent p21^Cip1/WAF1 and pro‐apoptotic Bcl‐2 family proteins (Bax, Noxa and Puma), release of AIF and EndoG from mitochondria, and membrane translocation of Fas occurred during leptospire‐induced macrophage apoptosis. Thus, our study demonstrated that ROS production and DNA damage‐dependent p53‐Bax/Noxa/Puma‐AIF/EndoG signalling mediates the leptospire‐induced cell cycle arrest and caspase‐independent apoptosis of macrophages.     

Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance

Akt is a pro‐survival kinase frequently activated in human cancers and is associated with more aggressive tumors that resist therapy. Here, we connect Akt pathway activation to reduced sensitivity to chemotherapy via Akt phosphorylation of Bax at residue S184, one of the pro‐apoptotic Bcl‐2 family proteins required for cells to undergo apoptosis. We show that phosphorylation by Akt converts the pro‐apoptotic protein Bax into an anti‐apoptotic protein. Mechanistically, we show that phosphorylation (i) enables Bax binding to pro‐apoptotic BH3 proteins in solution, and (ii) prevents Bax inserting into mitochondria. Together, these alterations promote resistance to apoptotic stimuli by sequestering pro‐apoptotic activator BH3 proteins. Bax phosphorylation correlates with cellular resistance to BH3 mimetics in primary ovarian cancer cells. Further, analysis of the TCGA database reveals that 98% of cancer patients with increased BAX levels also have an upregulated Akt pathway, compared to 47% of patients with unchanged or decreased BAX levels. These results suggest that in patients, increased phosphorylated anti‐apoptotic Bax promotes resistance of cancer cells to inherent and drug‐induced apoptosis.     

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFα in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFα, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFα, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFα release and caspase expression.