Titanium dioxide nanoparticles induce apoptosis through the JNK/p38-caspase-8-Bid pathway in phytohemagglutinin-stimulated human lymphocytes

We investigated the signaling pathways underlying nano-TiO₂-induced apoptosis in cultured human lymphocytes. Nano-TiO₂ increased the proportion of sub-G1 cells, activated caspase-9 and caspase-3, and induced caspase-3-mediated PARP cleavage. Nano-TiO₂ also induced loss of mitochondrial membrane potential, which suggests that nano-TiO₂ induces apoptosis via a mitochondrial pathway. A time-sequence analysis of the induction of apoptosis by nano-TiO₂ revealed that nano-TiO₂ triggered apoptosis through caspase-8/Bid activation. We also observed that inhibition of caspase-8 by z-IETD-fmk suppressed the caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and apoptosis. Nano-TiO₂ activated two MAPKs, p38 and JNK. In addition, the selective p38 inhibitor SB203580 and selective JNK inhibitor SP600125 suppressed nano-TiO₂-induced apoptosis and caspase-8 activation to moderate and significant extents, respectively. Knockdown of protein levels of JNK1 and p38 using an RNA interference technique also suppressed caspase-8 activation. Our results suggest that nano-TiO₂-induced apoptosis is mediated by the p38/JNK pathway and the caspase-8-dependent Bid pathway in human lymphocytes.     

Daxx deletion mutant (amino acids 501-625)-induced apoptosis occurs through the JNK/p38-Bax-dependent mitochondrial pathway

Death-associated protein (Daxx) deletion mutant (aa 501-625) has been known to be an inducer of apoptosis. In this study, we observed that the Bax-dependent mitochondrial death signaling pathway plays an important role in Daxx501-625-induced apoptosis. Daxx fragment-induced activation of caspase-9 and -3 was mediated through the apoptosis signal-regulating kinase 1 (ASK1)-MEK-c-Jun-N-terminal kinase (JNK)/p38-Bax pathway. By overexpressing JNK-binding domain (JBD) of JIP1, a JNK-inhibitory protein, and treatment with SB203580, a specific p38 inhibitor, DU-145 cells were made resistant to Daxx501-625-induced apoptosis. Capase-3 deficiency, Bax deficiency, or overexpression of a dominant-negative caspase-9 mutant prevented apoptosis, even though the Daxx501-625 fragment still activated the ASK1-MEK-MAPK pathway. Interestingly, Daxx501-625-induced Bcl-2 interacting domain (Bid) cleavage was suppressed in the dominant-negative caspase-9 mutant cells, whereas Bim was still phosphorylated in these cells. These results suggest that cleavage of Bid occurs downstream of caspase-9 activation. In contrast, phosphorylation of Bim is upstream of caspase-9 activation. Taken together, our results suggest that Daxx501-625-induced apoptosis is mediated through the ASK1-MEK-JNK/p38-Bim-Bax-dependent caspase pathway.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. However, the intracellular death signaling mechanisms by which Cin inhibits tumor cell growth are poorly understood. In this study, we investigated the effect of mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the stress-responsive MAPK pathway induced by Cin in PLC/PRF/5 cells. Trypan blue staining assay indicated that Cin was cytotoxic to PLC/PRF/5 cells. Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and accumulation of sub-G1 peak. It down-regulated the Bcl-2 and Mcl-1 expression, and up-regulated Bax protein in a time-response manner. Treatment with 1 microM Cin resulted in an activation of caspase-8 and cleavage of Bid to its truncated form in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin-induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK. These results conclude that Cin triggers apoptosis in PLC/PRF/5 cells could be through the activation of pro-apoptotic Bcl-2 family (Bax and Bid) proteins and MAPK signaling pathway.     

Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome c and caspases

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH-SY5Y neuroblastoma cells demonstrated by activation of caspase-9 and caspase-3, cleavage of poly(ADP-ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen-activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA-induced cell death as well as against caspase-9 and caspase-3 activation. In addition, the antioxidant N-acetyl-L-cysteine (NAC) effectively blocks DA-induced p38 kinase activation, caspase-9 and caspase-3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA-induced cell death as well as activation of p38 kinase and caspase-3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase-9 and caspase-3 activation.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.     

Downregulation of p38 kinase pathway by cAMP response element-binding protein protects HL-60 cells from iron chelator-induced apoptosis

The signaling mechanisms that control apoptotic events evoked by iron chelators are largely unknown. We found that cAMP response element-binding protein (CREB) is cleaved during iron chelator deferoxamine (DFO)-induced apoptosis, and that the cleavage is largely prevented by the cell-permeable analog of cAMP, dibutyryl-cAMP (dbcAMP), a known CREB activator. In addition, dbcAMP profoundly reduced DFO-induced apoptosis along with significant suppression of caspase-3 and -8 activation and inhibition of loss of mitochondrial potential. These results led us to investigate whether CREB activation is functionally connected with the MAPK family members because we previously demonstrated that p38 kinase is involved in iron chelator-induced apoptosis of HL-60 cells. dbcAMP by itself rapidly induced CREB phosphorylation but dramatically inhibited DFO-induced phosphorylation of all three MAPK family members. However, disruption of CREB expression by antisense oligodeoxyribonucleotide (AS-ODN) only restored p38 kinase activation, and simultaneously attenuated dbcAMP-induced protection of HL-60 cells from DFO-induced cell death. Conversely, inhibition of p38 kinase activity by SB203580 significantly reduced DFO-induced CREB cleavage as well as apoptosis, indicating a cross-talk between CREB and p38 kinase. Collectively, these results demonstrate that cAMP-dependent CREB activation plays an important role in protecting HL-60 cells from iron chelator-induced apoptosis, presumably through downregulation of p38 kinase.     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Apoptosis of macrophages induced by Trichomonas vaginalis through the phosphorylation of p38 mitogen-activated protein kinase that locates at downstream of mitochondria-dependent caspase activation

Trichomonas vaginalis, a flagellated protozoan parasite, is the causative organism of trichomoniasis. We have recently demonstrated that T. vaginalis induces apoptotic cell death via a Bcl-x(L)-dependent pathway in RAW264.7 macrophages. In this study, we attempted to characterize in detail the signaling cascades resulting in T. vaginalis-induced macrophage apoptosis, focusing particularly on mitochondrial changes and the role of p38 mitogen-activated protein kinase (p38 MAPK) activation. We found that T. vaginalis induced mitochondrial changes including the release of cytochrome c and the serial activation of caspases, leading to the activation of p38 MAPK in macrophages. These biochemical changes culminated in the apoptosis of the host cells. Caspase inhibitors induced a significant inhibition of T. vaginalis-induced nuclear damage, as well as the activation of p38 MAPK. Treatment with the p38 MAPK inhibitor, SB203580, or the overexpression of kinase-inactive p38 MAPK, induced an attenuation of T. vaginalis-induced apoptosis but not cytochrome c release, the activation of caspase-9 and caspase-3, or PARP cleavage. Furthermore, SB203580 treatment to human macrophages consistently blocked T. vaginalis-induced apoptosis. Collectively, our findings indicate that p38 MAPK signaling cascade is requisite to apoptosis of T. vaginalis-infected macrophage, and this apoptotic process occurs via the phosphorylation of p38 MAPK, which is located downstream of mitochondria-dependent caspase activation, conferring insight into the plausible molecular mechanism of T. vaginalis-immune evasion from macrophage attack.     

Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.     

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFalpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways

Treatment with the anti-leukemic drug arsenic trioxide (As(2)O(3), 1-4 microM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFalpha. As(2)O(3) plus TNFalpha increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIP(L) expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIP(L) down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As(2)O(3) does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFalpha-provoked stimulation of p65-NF-kappaB translocation to the nucleus and the increase in NF-kappaB binding activity. Treatments with TNFalpha alone or with As(2)O(3) plus TNFalpha cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As(2)O(3) plus TNFalpha-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As(2)O(3) to TNFalpha-induced apoptosis in promonocytic leukemia cells is an Akt/NF-kappaB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

p38 inhibitor SB203580 sensitizes the resveratrol-induced apoptosis in human lung adenocarcinoma (A549) cells

Based on our recent findings that resveratrol, a natural plant polyphenol found in red grape skins as well as other food products, induces apoptosis via a caspase-independent intrinsic pathway in human lung adenocarcinoma cells, this study is designed to explore whether SB203580, a p38 inhibitor, potentiates the resveratrol-induced apoptosis of human lung adenocarcinoma (A549) cells. We found that pretreatment with SB203580 enhanced the resveratrol-induced apoptosis by accelerating the intrinsic apoptotic pathway including Bax activation, loss of mitochondrial membrane potential, and activation of both caspase-9 and -3. Although treatment with resveratrol alone did not induce caspase-8 activation, cotreatment with both SB203580 and resveratrol not only enhanced FasL cleavage but also activated caspase-8, indicating that the extrinsic apoptotic pathway may be involved in the synergistic effect. Collectively, we for the first time demonstrate that SB203580 synergistically enhances the resveratrol-induced apoptosis by accelerating Bax-mediated intrinsic pathway and initiating extrinsic pathway, suggesting a possible alternative therapeutic strategy for human lung cancer.     

Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells

We previously demonstrated that the phytosphingosine-induced apoptosis was accompanied by the concomitant induction of both the caspase-8-mediated and mitochondrial activation-mediated apoptosis pathways. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in the activation of these two distinct cell death pathways induced by phytosphingosine in human cancer cells. Phytosphingosine caused strong induction of caspase-8 activity and caspase-independent Bax translocation to the mitochondria. A rapid decrease of phosphorylated ERK1/2 and a marked increase of p38 MAPK phosphorylation were observed within 10 min after phytosphingosine treatment. Activation of ERK1/2 by pretreatment with phorbol 12-myristate 13-acetate or forced expression of ERK1/2 attenuated phytosphingosine-induced caspase-8 activation. However, Bax translocation and caspase-9 activation was unaffected, indicating that down-regulation of the ERK activity is specifically required for the phytosphingosine-induced caspase-8-dependent cell death pathway. On the other hand, treatment with SB203580, a p38 MAPK-specific inhibitor, or expression of a dominant negative form of p38 MAPK suppressed phytosphingosine-induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation but did not affect caspase-8 activation, indicating that activation of p38 MAPK is involved in the mitochondrial activation-mediated cell death pathway. Our results suggest that phytosphingosine can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, enhancing the understanding of the molecular mechanisms utilized by naturally occurring metabolites to regulate cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both our understanding of cell death events and development of cancer therapeutic agents.     

Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells

Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 microM) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38(MAPK)) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38(MAPK)-specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H(2)O(2) accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38(MAPK) phosphorylation. In a similar manner, p38(MAPK) phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38(MAPK) activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells.     

Recombinant tissue metalloproteinase inhibitor-3 protein induces apoptosis of murine osteoblast MC3T3-E1

Tissue inhibitor of metalloproteinases (TIMPs) plays an essential role in the regulation of bone metabolism. Here we report that recombinant tissue metalloproteinase inhibitor-3 (TIMP-3) protein induces the apoptosis of MC3T3-E1 osteoblasts. Cell apoptosis was detected by sandwich-enzyme-immunoassay. Fas and Fasl protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-8. The phosphorylation of JNK, p38 and ERK1/2 was examined by Western blot analysis. The ELISA suggested that TIMP-3 promoted MC3T3-E1 cell apoptosis. TIMP-3 treatment induced the expression of Fas and Fasl proteins, and the activation of caspase-8 and caspase-3. TIMP-3 treatment induced p38 and ERK phosphorylation. SB203580 and PD98059, the inhibitor of p38 and ERK, respectively, abolished the TIMP-3 effect on osteoblast apoptosis. In conclusion, the signal pathway through which TIMP-3 induces MC3T3-E1 cell apoptosis, mediated by Fas and involves the p38 and ERK signal transduction pathways.     

Mild thermotolerance induced at 40°C protects HeLa cells against activation of death receptor-mediated apoptosis by hydrogen peroxide

Preexposure to mild temperatures such as 40°C induces thermotolerance, whereby cells resist subsequent exposure to a toxic insult. This study investigates the protective effect of mild thermotolerance (3h, 40°C) against activation of death receptor-mediated apoptosis by H(2)O(2) in HeLa cells. H(2)O(2) (5-50μM) caused rapid activation (1-3h) of the Fas death receptor pathway of apoptosis, which was evident by up-regulation of the death ligand FasL and recruitment of the adaptor protein Fas-associated death domain to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, t-Bid translocation to mitochondria, and caspase-9 activation. These changes were all diminished in thermotolerant cells. Mild thermotolerance also protected cells against cytotoxicity from H(2)O(2) as well as execution-phase events of apoptosis such as caspase-3 activation and chromatin condensation. The antioxidant polyethylene glycol-catalase abolished FasL induction and caspase-8 activation due to H(2)O(2). FasL up-regulation; activation of caspases-8, -2, -9, and -3; and chromatin condensation were decreased by the p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in the activation of death receptor-mediated apoptosis by H(2)O(2). This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against induction of apoptosis by oxidative stress.     

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.     

Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.