Heme oxygenase-1 gene transfer inhibits angiotensin II-mediated rat cardiac myocyte apoptosis but not hypertrophy

Cardiac myocyte apoptosis underlies the pathophysiology of cardiomyopathy, and plays a critical role in the transition from myocardial hypertrophy to heart failure. Angiotensin II (Ang II) induces cardiac myocyte apoptosis and hypertrophy which contribute to heart failure possibly through enhanced oxidative stress; however, the mechanisms underlying the activation of both pathways and their interactions remain unclear. In the present study, we have investigated whether overexpression of the antioxidant protein heme oxygenase-1 (HO-1) protects against apoptosis and hypertrophy in cultured rat cardiac myocytes treated with Ang II. Our findings demonstrate that Ang II (100 nM, 24 h) alone upregulates HO-1 expression and induces both myocyte hypertrophy and apoptosis, assessed by measuring terminal deoxynucleotidyltransferase dUTP nick-end labelling (TUNEL) staining, caspase-3 activity and mitochondrial membrane potential. Ang II elicited apoptosis was augmented in the presence of tin protoporphyrin, an inhibitor of HO activity, while HO-1 gene transfer to myocytes attenuated Ang II-mediated apoptosis but not hypertrophy. Adenoviral overexpression of HO-1 was accompanied by a significant increase in Ang II induced phosphorylation of Akt, however, Ang II-mediated p38 mitogen activated protein kinase (MAPK) phosphorylation was attenuated. Inhibition of phosphotidylinositol-3-kinase enhanced myocyte apoptosis elicited by Ang II, however, p38MAPK inhibition had no effect, suggesting that overexpression of HO-1 protects myocytes via augmented Akt activation and not through modulation of p38MAPK activation. Our findings identify the signalling pathways by which HO-1 gene transfer protects against apoptosis and suggest that overexpression of HO-1 in cardiomyopathies may delay the transition from myocyte hypertrophy to heart failure.     

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathway

Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2.     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.     

Syk is required for the activation of Akt survival pathway in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H(2)O(2). In Syk-deficient DT40 cells treated with low doses of H(2)O(2) (10-100 microm), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H(2)O(2), a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway. Furthermore, viability of Syk-deficient cells, after exposure to H(2)O(2), was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.     

MiR-28 inhibits cardiomyocyte survival through suppressing PDK1/Akt/mTOR signaling

MicroRNAs play critical roles in regulating cell survival under multiple pathological conditions of heart diseases. Oxidative stress-induced apoptosis contributes greatly to heart ischemia-reperfusion injury. Herein, we describe a novel regulatory role of miR-28 on the survival of cardiomyocytes. We show that miR-28 was upregulated in cardiomyocytes treated with hydrogen peroxide (H₂O₂). MiR-28 gain of function sensitized cell apoptosis, whereas miR-28 loss of function partially rescued cell apoptosis induced by H₂O₂. Importantly, we observed a significant reduction in Akt/mammalian target of rapamycin (mTOR) signaling activity after miR-28 treatment. Luciferase activity assay and western blot analysis both revealed that, phosphoinositide-dependent kinase-1 (PDK1), which is critical for Akt activation, was directly and negatively modulated by miR-28. Our results therefore indicate that miR-28 regulates oxidative stress-induced cell apoptosis in heart muscle cells, which possibly involves a PDK1/Akt/mTOR-dependent mechanism. MIR-28 could serve as a critical therapeutic target to diminish oxidative stress-induced cell death in the heart.     

Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells

Hsp105alpha and Hsp105beta are major heat shock proteins in mammalian cells that belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has opposite effects on stress-induced apoptosis depending on the cell type. However, it is not fully understood how Hsp105 regulates stress-induced apoptosis. In this study, we examined how Hsp105alpha and Hsp105beta regulate H2O2-induced apoptosis by using HeLa cells in which expression of Hsp105alpha or Hsp105beta was regulated using doxycycline. Overexpression of Hsp105alpha and Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria in H2O2-treated cells. Furthermore, both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) were activated by treatment with H2O2, and the activation of both kinases was suppressed by overexpression of Hsp105alpha and Hsp105beta. However, H2O2-induced apoptosis was suppressed by treatment with a potent inhibitor of p38 MAPK, SB202190, but not a JNK inhibitor, SP600125. These findings suggest that Hsp105alpha and Hsp105beta suppress H2O2-induced apoptosis by suppression of p38 MAPK signaling, one of the essential pathways for apoptosis.     

Proteasome inhibition represses unfolded protein response and Nox4, sensitizing vascular cells to endoplasmic reticulum stress-induced death

BACKGROUND: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. CONCLUSION/RELEVANCE: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.     

Epidermal growth factor receptor-dependent,NF-kappaB-independent activation of the phosphatidylinositol 3-kinase/Akt pathway inhibits ultraviolet irradiation-induced caspases-3, -8, and -9 in human keratinocytes

Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB pathways function to promote cellular survival following stress. Recent evidence indicates that the anti-apoptotic activity of these two pathways may be functionally dependent. Ultraviolet (UV) irradiation causes oxidative stress, which can lead to apoptotic cell death. Human skin cells (keratinocytes) are commonly exposed to UV irradiation from the sun. We have investigated activation of the PI3K/Akt and NF-kappaB pathways and their roles in protecting human keratinocytes (KCs) from UV irradiation-induced apoptosis. This activation of PI3K preceded increased levels (3-fold) of active/phosphorylated Akt. UV (50 mJ/cm2 from UVB source) irradiation caused rapid recruitment of PI3K to the epidermal growth factor receptor (EGFR). Pretreatment of KCs with EGFR inhibitor PD169540 abolished UV-induced Akt activation/phosphorylation, as did the PI3K inhibitors LY294002 or wortmannin. This inhibition of Akt activation was associated with a 3-4-fold increase of UV-induced apoptosis, as measured by flow cytometry and DNA fragmentation ELISA. In contrast to Akt, UV irradiation did not detectably increase nuclear localization of NF-kappaB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-kappaB activation by adenovirus-mediated overexpression of dominant negative IKK-beta or IkappaB-alpha did not increase UV-induced apoptosis. However, adenovirus-mediated overexpression of constitutively active Akt completely blocked UV-induced apoptosis observed with PI3K inhibition by LY294002, whereas adenovirus mediated overexpression of dominant negative Akt increased UV-induced apoptosis by 2-fold. Inhibition of UV-induced activation of Akt increased release of mitochondrial cytochrome c 3.5-fold, and caused appearance of active forms of caspase-9, caspase-8, and caspase-3. Constitutively active Akt abolished UV-induced cytochrome c release and activation of caspases-9, -8, and -3. These data demonstrate that PI3K/Akt is essential for protecting human KCs against UV-induced apoptosis, whereas NF-kappaB pathway provides little, if any, protective role.     

Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells

Oxidative stress activates various signal transduction pathways, including Jun N-terminal kinase (JNK) and its substrates, that induce apoptosis. We reported here the role of angiopoietin-1 (Ang1), which is a prosurvival factor in endothelial cells, during endothelial cell damage induced by oxidative stress. Hydrogen peroxide (H2O2) increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2-induced apoptosis and concomitant JNK phosphorylation. The inhibition of H2O2-induced JNK phosphorylation was reversed by inhibitors of phosphatidylinositol (PI) 3-kinase and dominant-negative Akt, and constitutively active-Akt attenuated JNK phosphorylation without Ang1. These data suggested that Ang1-dependent Akt phosphorylation through PI 3-kinase leads to the inhibition of JNK phosphorylation. H2O2-induced phosphorylation of SAPK/Erk kinase (SEK1) at Thr261, which is an upstream regulator of JNK, was also attenuated by Ang1-dependent activation of the PI 3-kinase/Akt pathway. In addition, Ang1 induced SEK1 phosphorylation at Ser80, suggesting the existence of an additional signal transduction pathway through which Ang1 attenuates JNK phosphorylation. These results demonstrated that Ang1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI 3-kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.     

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart

microRNAs (miRs) have emerged as critical modulators of various physiological processes including stem cell differentiation. Indeed, miR-1 has been reported to play an integral role in the regulation of cardiac muscle progenitor cell differentiation. However, whether overexpression of miR-1 in embryonic stem (ES) cells (miR-1-ES cells) will enhance cardiac myocyte differentiation following transplantation into the infarcted myocardium is unknown. In the present study, myocardial infarction (MI) was produced in C57BL/6 mice by left anterior descending artery ligation. miR-1-ES cells, ES cells, or culture medium (control) was transplanted into the border zone of the infarcted heart, and 2 wk post-MI, cardiac myocyte differentiation, adverse ventricular remodeling, and cardiac function were assessed. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared with ES cells. Assessment of apoptosis revealed that overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-AKT and inhibition of caspase-3, phosphatase and tensin homolog, and superoxide production. A significant reduction in interstitial and vascular fibrosis was quantified in miR-1-ES cell and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1-ES cell and ES cell groups was observed. Finally, mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls (P < 0.05). Our data suggest miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI, ultimately giving rise to enhanced cardiac repair, regeneration, and function.     

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-alpha via MAPK and PI3K/Akt signaling pathways

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-alpha induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-alpha. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-alpha-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-alpha. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.     

Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo

Oxidants such as H(2)O(2) can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H(2)O(2) also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H(2)O(2) causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H(2)O(2) can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H(2)O(2) of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H(2)O(2) to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H(2)O(2) is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H(2)O(2) to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H(2)O(2) acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H(2)O(2) can induce caspase-9 and -3 activation and cause apoptosis.