Amylin-induced cytotoxicity is associated with activation of caspase-3 and MAP kinases

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.     

Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.     

Bcl-xL prevents staurosporine-induced hepatocyte apoptosis by restoring protein kinase B/mitogen-activated protein kinase activity and mitochondria integrity

Our study reports that staurosporine induces apoptosis in cultured rat hepatocytes in a dose- and time-dependent fashion. Staurosporine induced apparent cleavage of caspase-8, caspase-9, and caspase-3. The release of cytochrome c from mitochondria, and Bid activation were also detected in staurosporine-treated primary hepatocytes. These results suggest that mitochondria-mediated cell death signaling may be involved in staurosporine-induced hepatocyte apoptosis. Bcl-x(L) overexpression protected from "loss of" mitochondrial transmembrane potential and prevented staurosporine-induced caspase-3 and caspase-8 cleavage. Overexpression of constitutively active ERK and PKB inhibited staurosporine-induced caspase-3 activation and hepatocyte death. PI3K inhibitor (LY294002) and ERK inhibitor (PD98059) significantly reversed the protective effects of Bcl-x(L) on staurosporine-induced hepatocyte death. Our data suggest that Bcl-x(L) prevents staurosporine-induced hepatocyte apoptosis by modulating protein kinase B and p44/42 mitogen-activated protein kinase activity and disrupts mitochondria death signaling.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

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.     

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.     

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.     

p38 mitogen-activated protein kinase mediates bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide

p38 mitogen-activated protein kinase is activated and involved in cleavage of caspase-3 during apoptosis induced by a number of stimuli. However, the signaling events triggered by p38 that result in caspase-3 activation are still unknown. In human leukemia cells, two reactive oxygen species, singlet oxygen and hydrogen peroxide (H(2)O(2)), selectively stimulated the phosphorylation of p38. Preincubation of cells with SB203580, a specific inhibitor of p38, dose dependently inhibited DNA fragmentation induced by singlet oxygen but not by H(2)O(2). Protection from apoptosis by SB203580 correlated with inhibition of caspase-3, and several events that are associated with caspase-3 activation, including Bid cleavage, decrease in mitochondrial transmembrane potential and release of cytochrome c from mitochondria, whereas caspase-8 cleavage was not affected by this inhibitor. In contrast, blockade of caspase-8 with Ile-Glu-Thr-Asp-fluoromethyl ketone is sufficient to prevent formation of DNA fragments and to inhibit all the above signaling events, with exception of p38 phosphorylation, in both singlet oxygen- and H(2)O(2)-treated cells. These data suggest that caspase-3 activation is regulated through redundant signaling pathways that involve p38 and caspase-8 acting upstream of Bid during singlet oxygen-induced apoptosis, whereas the activation of caspase-3 by H(2)O(2) is only governed by a caspase-8-mediated apoptotic pathway.     

Effect of Dicycloplatin,a Novel Platinum Chemotherapeutical Drug,on Inhibiting Cell Growth and Inducing Cell Apoptosis

Dicycloplatin, a new supramolecular platinum-based antitumor drug, has been approved by the State Food and Administration (SFDA) of China. In this study, we investigated the anticancer activity of dicycloplatin in cancer cells and signaling pathways involved in dicycloplatin-induced apoptosis. Dicycloplatin inhibited the proliferation of cancer cells and increased the percentage of apoptosis in a concentration-dependent manner. Besides, some apoptosis related events were observed after treatment with dicycloplatin, including increase of reactive oxygen species (ROS), collapse of mitochondrial membrane potential (Δψm), release of cytochrome c from the mitochondria to the cytosol, upregulation of p53, which were accompanied by activation of caspase-9, caspase-3, caspase-8, and poly (ADP-ribose) polymerase cleavage in a concentration-dependent manner. The role of apoptosis in dicycloplatin-mediated cell death was further confirmed by the concomitant treatment with caspase-8 or caspase-9 inhibitors, which inhibited apoptosis and PARP cleavage. Intracellular glutathione (GSH) was also found to inhibit the cytotoxic effect of dicycloplatin. In conclusion, these findings suggest that dicycloplatin induces apoptosis through ROS stress-mediated death receptor pathway and mitochondrial pathway which is similar to carboplatin.     

p38 MAP kinase regulates benzo(a)pyrene-induced apoptosis through the regulation of p53 activation

Polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), are widespread in the environment and cause untoward effects, including carcinogenesis, in mammalian cells. However, the molecular mechanism of apoptosis by BaP is remained to be elusive. Pharmacological inhibition of p38 kinase markedly inhibited the BaP-induced cytotoxicity, which was proven as apoptosis characterized by an increase in sub-G(0)/G(1) fraction of DNA content, ladder-pattern fragmentation of genomic DNA, and catalytic activation of caspase-3 with PARP cleavage. Our data also demonstrated that activation of caspase-3 was accompanied with activation of caspase-9 and mitochondrial dysfunction, which was also apparently suppressed by pretreatment with p38 kinase inhibitors. Also, pharmacological inhibition of p38 markedly inhibited the phosphorylation, accumulated expression, and transactivation activity of p53 in BaP-treated cells. Adenoviral overexpression of human p53 (wild-type) further augmented in increase of PARP cleavage and the sub-G(0)/G(1) fraction of DNA content. Furthermore, p53 mediated apoptotic activity in BaP-treated cells was inhibited by p38 kinase inhibitor. The current data collectively indicate that BaP induces apoptosis of Hepa1c1c7 cells via activation of p53-related signaling, which was, in part, regulated by p38 kinase.     

Activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphostin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

Sulindac-derived reactive oxygen species induce apoptosis of human multiple myeloma cells via p38 mitogen activated protein kinase-induced mitochondrial dysfunction

Non-steroidal anti-inflammatory drugs are well known to induce apoptosis of cancer cells independent of their ability to inhibit cyclooxygenase-2, but the molecular mechanism for this effect has not yet been fully elucidated. The purpose of this study was to elucidate the potential signaling components underlying sulindac-induced apoptosis in human multiple myeloma (MM) cells. We found that sulindac induces apoptosis by promoting ROS generation, accompanied by opening of mitochondrial permeability transition pores, release of cytochrome c and apoptosis inducing factor from mitochondria, followed by caspase activation. Bcl-2 cleavage and down-regulation of the inhibitor of apoptosis proteins (IAPs) family including cIAP-1/2, XIAP, and survivin, occurred downstream of ROS production during sulindac-induced apoptosis. Forced expression of survivin and Bcl-2 blocked sulindac-induced apoptosis. Most importantly, sulindac-derived ROS activated p38 mitogen-activated protein kinase and p53. SB203580, a p38 mitogen-activated protein kinase inhibitor, and RNA inhibition of p53 inhibited the sulindac-induced apoptosis. Furthermore, p53, Bax, and Bak accumulated in mitochondria during sulindac-induced apoptosis. All of these events were significantly suppressed by SB203580. Our results demonstrate a novel mechanism of sulindac-induced apoptosis in human MM cells, namely, accumulation of p53, Bax, and Bak in mitochondria mediated by p38 MAPK activation downstream of ROS production.     

p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis

The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro-caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol.     

Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin metalloproteinase activity. We previously reported that overexpression of TIMP-3 inhibits MMPs and induces apoptotic cell death in a variety of cell types and demonstrated that apoptosis is mediated through the N terminus of TIMP-3, which harbors the MMP inhibitory domain. However, little is known about the mechanisms underlying TIMP-3-induced apoptosis. Here we demonstrate that overexpression of TIMP-3 induced activation of initiator caspase-8 and -9 and promoted caspase-mediated cleavage of the death substrates poly(ADP-ribose) polymerase and focal adhesion kinase. Furthermore, TIMP-3 induced mitochondrial activation as demonstrated by loss of mitochondrial membrane potential and release of cytochrome c. Intervention studies demonstrated that overexpression of Bcl-2, the anti-apoptotic mitochondrial membrane protein, or CrmA, a viral serpin inhibitor of caspase-8, completely inhibited TIMP-3-induced apoptosis. Furthermore, a dominant-negative Fas-associated death domain mutant inhibited TIMP-3-induced death substrate cleavage and apoptotic death. Taken together, these results indicate that TIMP-3 overexpression induces a type II apoptotic pathway initiated via a Fas-associated death domain-dependent mechanism.     

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.     

Caspase-3: Its potential involvement in Cr(III)-induced apoptosis of lymphocytes

In this study, we have examined the role of caspase-3 in apoptosis of lymphocytes induced by the chromium(III) complexes viz. tris-(1,10-phenanthroline)chromium(III) chloride (Cr(III)-phen) and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] perchlorate (Cr(III)-salprn). Evidence for caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in lymphocytes exposed to Cr(III) complexes is revealed through Western blotting analysis. Blocking the activity of caspase-3 with z-DEVD-fmk, prevents apoptosis as evidenced through [3H]-thymidine incorporation, DNA fragmentation assay and measurement of sub-G1 cells by flow cytometry. Pretreatment of lymphocytes with free radical scavengers completely attenuates the activity of caspase-3 suggesting that reactive oxygen species (ROS) are upstream activators of caspase-3. Preincubation of lymphocytes with PP2, a selective Src-family tyrosine kinase inhibitor, abolishes the activation of caspase-3 indicating that Src-family tyrosine kinases viz. p56lck, p59fyn and p53/56lyn are mediators of caspase-3 activation during Cr(III) exposure. Collectively, our findings support a plausible mechanism in which Cr(III) mediates ROS generation that precedes the up-regulation of p56lck, p59fyn and p53/56lyn which eventually activates caspase-3 to promote apoptotic cell death of lymphocytes. To our knowledge, this is the first report suggesting the importance of Src-family tyrosine kinases for the activation of caspase-3 in metal-induced apoptotic cell death.     

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.     

Dopamine-induced toxicity is synergistically potentiated by simultaneous HSP-90 and Akt inhibition in oligodendrocyte progenitors

Oligodendrocyte progenitors are highly susceptible to various insults. Their limited antioxidant defenses and high levels of apoptotic factors, such as Bax and pro-caspase-3 contribute to their sensitivity. We previously showed that dopamine (DA) is toxic to oligodendrocyte progenitors by inducing superoxide generation, lowering glutathione levels and promoting apoptosis through caspase-3 activation. In contrast, factors that contribute to cell survival and defense against dopamine (DA) toxicity are less studied. Here, we explored the role of two molecules which play important roles in cell survival, namely the heat shock protein 90 (HSP-90) and the protein kinase Akt, using the selective inhibitors, 17-AAG and Akt inhibitor III, respectively. The HSP-90 inhibitor caused a decrease in P-Akt level, induced caspase-3 activation, increased nuclear condensation and caused a loss in cell viability. Furthermore, 17-AAG potentiated DA-induced apoptosis by enhancing caspase-3 activation. In addition, the Akt inhibitor alone exacerbated DA toxicity and in combination with 17-AAG caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation. Together, these results indicate that HSP-90 is essential for oligodendrocyte progenitor survival. Both HSP-90 and Akt play important roles in concert in the defense against DA-induced apoptosis.