Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56 in human Jurkat T cells

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of caspase-12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by overexpression of Bcl-xL. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132-induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, Δψm loss, and cleavage of procaspase-9 (47 kDa) to active form (35 kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G₁ peak, Bak activation, and Δψm loss were markedly reduced by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and mitochondria-dependent activation of caspase cascade were more significant in p56^lck-stable transfectant JCaM1.6/lck than in p56^lck-deficient JCaM1.6/vector. The cytotoxicity of MG132 toward p56^lck-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56^lck enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.     

Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56lck in human Jurkat T cells

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of caspase-12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by overexpression of Bcl-xL. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132-induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, Δψm loss, and cleavage of procaspase-9 (47kDa) to active form (35kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G(1) peak, Bak activation, and Δψm loss were markedly reduced by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and mitochondria-dependent activation of caspase cascade were more significant in p56(lck)-stable transfectant JCaM1.6/lck than in p56(lck)-deficient JCaM1.6/vector. The cytotoxicity of MG132 toward p56(lck)-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56(lck) enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.     

Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 microM) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.     

17Alpha-estradiol arrests cell cycle progression at G2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

A pharmacological dose (2.5-10 μM) of 17α-estradiol (17α-E₂) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17α-E₂ was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G₂/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17α-E₂-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G₂/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17α-E₂-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G₁/S boundary, 17α-E₂ failed to induce the G₂/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17α-E₂ toward Jurkat T cells is attributable to apoptosis mainly induced in G₂/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.     

Irciniastatin A induces JNK activation that is involved in caspase-8-dependent apoptosis via the mitochondrial pathway

Irciniastatin A (ISA)/psymberin, a pederin-type natural product isolated from marine sponge, exhibits extremely potent and selective cytotoxicity against certain human cancer cell lines, but its molecular target and cytotoxic mechanisms are still unknown. Here we show that ISA is a potent inhibitor of protein translation, and induces apoptosis accompanied with activation of the stress-activated protein kinases via the mitochondrial pathway in human leukemia Jurkat cells. ISA potently inhibited protein translation, and induced a slow but prolonged activation of the stress-activated protein kinases, JNK and p38, at between 1h and 6h after treatment. In Bcl-x(L)-transfected cells, the activation of JNK and p38 by ISA was shortened. The same results were obtained in the cells treated with N-acetyl-L-cysteine, suggesting that the prolonged activation of JNK and p38 by ISA is mediated by reactive oxygen species generated from mitochondria. ISA strongly induced apoptosis, which was partially suppressed by the JNK inhibitor SP600125, but not by the p38 inhibitor SB202190. Apoptosis induction by ISA was partially reduced, but not suppressed by SP600125 in caspase-8-deficient Jurkat cells. These results suggest that ISA activates stress-activated kinases by a mitochondria-mediated mechanism, and that activation of JNK is required for caspase-8-dependent apoptosis.     

Mechanism underlying cytotoxicity of thialysine, lysine analog, toward human acute leukemia Jurkat T cells

We first report the mechanism for the inhibitory effect of the lysine analog, thialysine on human acute leukemia Jurkat T cells. When Jurkat T cells were treated with thialysine (0.32-2.5 mM), apoptotic cell death along with several biochemical events such as mitochondrial cytochrome c release, caspase-9 activation, caspase-3 activation, degradation of poly (ADP-ribose) polymerase, and DNA fragmentation was induced in a dose- and time-dependent manner. However, these thialysine-induced apoptotic events were significantly abrogated by an ectopic expression of Bcl-xL, which is known to block mitochondrial cytochrome c release. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, also suppressed thialysine-induced apoptotic events. Comparison of the thialysine-induced alterations in the cell cycle distribution between Jurkat T cells transfected with Bcl-xL gene (J/Bcl-xL) and Jurkat T cells transfected with vector (J/Neo) revealed that the apoptotic cells were mainly derived from the cells accumulated in S and G2/M phases following thialysine treatment. The interruption of cell cycle progression in the presence of thialysine was accompanied by a significant decline in the protein level of cdk4, cdk6, cdc2, cyclin A, cyclin B1, and cyclin E. These results demonstrate that the cytotoxic activity of thialysine toward Jurkat T cells is attributable to not only apoptotic cell death mediated by a mitochondria-dependent death signaling pathway, but also interruption of cell cycle progression by a massive down-regulation in the level of cdks and cyclins.     

Acetoxycycloheximide (E-73) rapidly induces apoptosis mediated by the release of cytochrome c via activation of c-Jun N-terminal kinase

Cycloheximide (CHX) is an inhibitor of protein synthesis and commonly used to modulate death receptor-mediated apoptosis or to induce apoptosis in a number of normal and transformed cells. In this study we show that a close structural derivative of CHX, acetoxycycloheximide (E-73) induced rapid processing of procaspases and subsequent apoptosis with much higher efficacy than CHX in human leukemia Jurkat T cells. E-73 induced the release of cytochrome c from mitochondria even in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone. The Bcl-2 family protein Bcl-x(L) suppressed cytochrome c release as well as processing of procaspases-3, -8, and -9 in E-73-treated cells. In Jurkat T cells transfected with the caspase-8 modulator FLIP(L), E-73 still induced activation of procaspase-3 and subsequent apoptosis, suggesting that the caspase-8 activity is dispensable for apoptosis. In contrast to CHX, E-73 drastically induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Inhibitory profiles of small-molecular kinase inhibitors revealed that JNK activation was critical for induction of cytochrome c release in E-73-induced apoptosis. Thus, our present results demonstrate that E-73, unlike CHX, induces strong activation of the JNK pathway and triggers rapid apoptosis mediated by the release of cytochrome c.     

Arsenic induces reactive oxygen species-caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways

Arsenic (As), a well-known high toxic metal, is an important environmental and industrial contaminant, and it induces oxidative stress, which causes many adverse health effects and diseases in humans, particularly in inorganic As (iAs) more harmful than organic As. Recently, epidemiological studies have suggested a possible relationship between iAs exposure and neurodegenerative disease development. However, the toxicological effects and underlying mechanisms of iAs-induced neuronal cell injuries are mostly unknown. The present study demonstrated that iAs significantly decreased cell viability and induced apoptosis in Neuro-2a cells. iAs also increased oxidative stress damage (production of malondialdehyde (MDA) and ROS, and reduction of Nrf2 and thioredoxin protein expression) and induced several features of mitochondria-dependent apoptotic signals, including: mitochondrial dysfunction, the activations of PARP and caspase cascades, and the increase in caspase-3 activity. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these iAs-induced responses. iAs also increased the phosphorylation of JNK and ERK1/2, but did not that p38-MAPK, in treated Neuro-2a cells. NAC and the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) abrogated iAs-induced cell cytotoxicity, caspase-3/-7 activity, and JNK and ERK1/2 activation. Additionally, exposure of Neuro-2a cells to iAs triggered endoplasmic reticulum (ER) stress identified through several key molecules (GRP 78, CHOP, XBP-1, and caspase-12), which was prevented by NAC. Transfection with GRP 78- and CHOP-specific si-RNA dramatically suppressed GRP 78 and CHOP expression, respectively, and attenuated the activations of caspase-12, -7, and -3 in iAs-exposed cells. Therefore, these results indicate that iAs induces ROS causing neuronal cell death via both JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-triggered apoptosis pathways.     

Beta-sitosterol induces anti-proliferation and apoptosis in human leukemic U937 cells through activation of caspase-3 and induction of Bax/Bcl-2 ratio

Beta-sitosterol is the main dietary phytosterol found in plants and has been shown to inhibit proliferation and induce apoptosis in human solid tumors such as colon and breast cancers. However, the mechanism by which beta-sitosterol induces apoptosis is not completely understood in leukemic cells. This study investigated the mechanism of apoptosis induced by beta-sitosterol in human leukemic U937 cells. beta-Sitosterol induced cytotoxicity and apoptosis in U937 cells in a concentration dependent manner, as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis, and flow cytometry analysis. The increase in apoptosis induced by beta-sitosterol was associated with down-regulation of Bcl-2, degradation of poly-(ADP-ribose) polymerase (PARP) and phospholipase C (PLC)-gamma1 protein, and activation of caspase-3. beta-Sitosterol induced apoptosis was not associated with changes in the expression of Bcl-xL, Bax, or inhibitor of apoptosis proteins (IAPs). z-DEVD-fmk, a caspase-3 specific inhibitor, blocked caspase-3 activation and PARP degradation, and significantly attenuated beta-sitosterol-induced apoptosis. This suggests that caspase-3 activation is partially essential for beta-sitosterol-induced apoptosis. Bcl-2 overexpression also significantly blocked caspase-3 activation and the decrease in PARP cleavage by beta-sitosterol, and effectively attenuated the apoptotic response to beta-sitosterol. These results show that beta-sitosterol potently induces apoptosis in U937 cells and that beta-sitosterol-induced apoptosis is related to the selective activation of caspase-3 and induction of Bax/Bcl-2 ratio.     

Arachidonic acid-induced apoptosis of human neuroblastoma SK-N-SH cells is mediated through mitochondrial alteration elicited by ROS and Ca-evoked activation of p38α MAPK and JNK1

Arachidonic acid (AA)-induced apoptosis of human neuroblastoma SK-N-SH cells was characteristic of elevation of intracellular Ca²⁺ concentration ([Ca²⁺]i), ROS generation, activation of 38 MAPK and JNK and loss of mitochondrial membrane potential (ΔΨm). Subsequent modulation of Bcl-2 family members and cytochrome c release accompanied with activation of caspase-9 and -3 were involved in the death of SK-N-SH cells. BAPTA-AM (Ca²⁺ chelator) pretreatment rescued viability of AA-treated cells through abolishing phosphorylation of p38 MAPK and JNK, ΔΨm loss and ROS generation. N-Acetylcysteine (ROS scavenger) pretreatment reduced the dissipation of ΔΨm, but insignificantly affected AA-induced p38 MAPK and JNK activation. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) attenuated mitochondrial depolarization, degradation of Bcl-2/Bcl-xL, and mitochondrial translocation of Bax. Transfection of specific siRNA proved that p38α MAPK and JNK1 were involved in modulating Bcl-2 family proteins. Taken together, our data suggest that the cytotoxicity of AA toward SK-N-SH cells is mediated through mitochondria-dependent death pathway, eliciting by AA-induced ROS generation and Ca²⁺-evoked activation of p38α MAPK and JNK1.     

Chromene induces apoptosis via caspase-3 activation in human leukemia HL-60 cells

In this study, the potent anti-tumor effects of brown algae on human leukemia HL-60 cells were investigated. The Sargassum siliquastrum extract among the 14 species of brown algae exhibited profound growth inhibitory effect on HL-60 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, therefore, S. siliquastrum was selected for use in further experiments. The highest inhibitory activity of S. siliquastrum on HL-60 cells was detected in the chloroform fraction, and the active compound was identified as a kind of chromene, sargachromanol E (SE). SE treatment showed significant growth inhibitory effects on HL-60 cells in a dose-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies, fragmented DNA ladder, and the accumulation of DNA in the sub-G₁ phase of cell cycle. SE induced apoptosis was accompanied by downregulation of Bcl-xL, upregulation of Bax, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited cell cytotoxicity, apoptotic characteristics such as apoptotic bodies, sub-G₁ DNA content, and cleavage of PARP induced by SE. These results suggest that SE exerts its growth inhibitory effects on HL-60 cells through caspase-3-mediated induction of apoptosis. Therefore, SE offers promising chemotherapeuric potential to prevent cancers such as human leukemia.     

4-hydroxynonenal induces apoptosis via caspase-3 activation and cytochrome c release

We investigated the mechanism by which 4-hydroxynonenal (HNE), a major aldehydic product of lipid peroxidation, induces apoptosis in tumor cells. Treatment of human colorectal carcinoma (RKO) cells with HNE-induced poly-ADP-ribose-polymerase (PARP) cleavage and DNA fragmentation in a dose- and time-dependent manner. The induction of PARP cleavage and DNA fragmentation paralleled caspase-2, -3, -8, and -9 activation. Pretreatment of cells with an inhibitor of caspase-3, z-DEVD-fmk, or a broad spectrum caspase inhibitor, z-VAD-fmk, abolished caspase activation and subsequent PARP cleavage. Constitutive expression of high levels of Bcl-2 protected cells from HNE-mediated apoptosis. In addition, Bcl-2 overexpression inhibited cytochrome c release from mitochondria and subsequent caspase-2, -3, and -9 activation. These findings demonstrate that HNE triggers apoptotic cell death through a mitochondrion-dependent pathway involving cytochrome c release and caspase activation. Bcl-2 overexpression protected cells from HNE-induced apoptosis through inhibition of cytochrome c release.     

Mitogenic activity of <Emphasis Type="Italic">Artocarpus lingnanensis</Emphasis> lectin and its apoptosis induction in Jurkat T cells

Lectins are a class of carbohydrate-binding proteins or glycoproteins and used in the purification and characterization of glycoproteins according to their specificity to carbohydrates. In the present study, the mitogenic activity of Artocarpus lingnanensis lectin (ALL) and its apoptosis induction in Jurkat T cells were explored. MTT assay revealed strong mitogenic potential of ALL. Meanwhile, the anti-cancer activity of ALL was also explored using the human leukemic Jurkat T cell line. ALL exhibited strong binding affinity (97%) to the cell membrane, which could be effectively inhibited by N-acetyl-d-galactosaminide (NAD). ALL induced time- and dose-dependent growth inhibition in Jurkat T cells. ALL could induce morphologic change and increase the hypodiploid cell population with the decreased population of S and G2/M phases. The induction of phosphatidylserine externalization and PARP cleavage further confirmed its apoptosis-inducing activity due to the activation of caspase-8 and -9. The inhibition of caspase-9 but not caspase-8 could rescue ALL-induced apoptotic cells. Further studies showed that ALL enhanced the cleavage of Bid, the release of cytochrome C, the depolarization of mitochondria and the activation of caspase-3. ALL downregulated the expression of Bcl-xl and Bcl-2 without impact on Bax and Bad. In addition, the activation of p38/JNK MAPK signaling pathways was observed to be a requisite for ALL apoptotic activity. In contrast, ALL could not induce apoptosis of normal T cells. These findings present the differential effect of ALL on Jurkat and normal T lymphocytes, suggesting its therapeutic value in leukemia.     

Induction of apoptosis in HT-29 colon cancer cells by crude saponin from Platycodi Radix

This study examined the apoptotic effects of crude saponins acquired from the roots of Platycodon grandiflorum (SPR) in HT-29 human colon cancer cells.SPR decreased HT-29 cell proliferation in dose- and time-dependent manners by inducing apoptosis via DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. The apoptosis induced by SPR was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. SPR stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. SPR increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. SPR also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that SPR inhibits HT-29 cell proliferation by inducing apoptosis, which may be mediated via both caspase-dependent and -independent pathways.     

Camalexin induces apoptosis in T-leukemia Jurkat cells by increased concentration of reactive oxygen species and activation of caspase-8 and caspase-9

Camalexin, a major indole phytoalexin of Arabidopsis thaliana, accumulates in various cruciferous plants in response to environmental stress and reportedly displays antimicrobial activities against various plant pathogens. However, its cytotoxicity against eukaryotic cells and potential as a prospective drug for human diseases has been examined only in a limited context. Our data demonstrate the time- and concentration-dependent cytotoxicity of camalexin on human T-leukemia Jurkat cells in the micromolar range, and the lower potency of cytotoxic effects on human lymphoblasts and primary fibroblasts. Cytotoxicity of camalexin is enhanced by the glutathione-depleting agent buthionine sulfoximine and completely blocked by pan-caspase inhibitor Z-VAD-FMK. Treatment of Jurkat cells with camalexin resulted in activation of caspase-8, caspase-9, caspases-3/7, and apoptosis that was detected by the presence of a sub-G1 population of cells, externalization of phosphatidyl serine and decreased mitochondrial membrane potential. Staining with 2',7'-dichlorodihydrofluorescein diacetate and dihydroethidium bromide displayed increased concentration of reactive oxygen species (ROS) early in camalexin-treated Jurkat cells, prior to the onset of apoptosis, while staining with MitoSOX(?) dye identified mitochondria as a source of increased ROS. Our data suggest that this phytochemical, which has a wide range of predicted pharmacological activities, induces apoptosis in Jurkat leukemia cells through increased ROS followed by dissipation of mitochondrial membrane potential and execution of caspase-9- and caspase-8-initiated apoptosis. This is, to the best of our knowledge, the first report on antileukemic activity and mode of action of this unique indole phytoalexin.     

Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury

Methylmercury (MeHg) is well-known for causing irreversible damage in the central nervous system as well as a risk factor for inducing neuronal degeneration. However, the molecular mechanisms of MeHg-induced neurotoxicity remain unclear. Here, we investigated the effects and possible mechanisms of MeHg in the mouse cerebrum (in vivo) and in cultured Neuro-2a cells (in vitro). In vivo study showed that the levels of LPO in the plasma and cerebral cortex significantly increased after administration of MeHg (50 μg/kg/day) for 7 consecutive weeks. MeHg could also decrease glutathione level and increase the expressions of caspase-3, -7, and -9, accompanied by Bcl-2 down-regulation and up-regulation of Bax, Bak, and p53. Moreover, treatment of Neuro-2a cells with MeHg significantly reduced cell viability, increased oxidative stress damage, and induced several features of mitochondria-dependent apoptotic signals, including increased sub-G1 hypodiploids, mitochondrial dysfunctions, and the activation of PARP, and caspase cascades. These MeHg-induced apoptotic-related signals could be remarkably reversed by antioxidant NAC. MeHg also increased the phosphorylation of ERK1/2 and p38, but not JNK. Pharmacological inhibitors NAC, PD98059, and SB203580 attenuated MeHg-induced cytotoxicity, ERK1/2 and p38 activation, MMP loss, and caspase-3 activation in Neuro-2a cells. Taken together, these results suggest that the signals of ROS-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic pathways that are involved in MeHg-induced neurotoxicity.     

Etoposide induces pancreatic β-cells cytotoxicity via the JNK/ERK/GSK-3 signaling-mediated mitochondria-dependent apoptosis pathway

Etoposide is widely used in the treatment of the different types of tumors such as pancreatic cancer. However, etoposide also causes several unwanted side-effects in normal viable cells, including pancreatic β-cells, which are vulnerable to chemical-induced injuries, and the molecular mechanisms underlying etoposide-induced apoptosis are still unclear. Here, the results showed that in RIN-m5F cells (a β-cell-derived cell line), the number of viable cells was significantly decreased after 24 h of etoposide treatment and underwent mitochondria-dependent apoptotic signals accompanied by mitochondrial dysfunction, and increases in the population of sub-G1 hypodiploid cells and apoptotic cells, caspase-3 activity, and the activation of caspase cascades. Etoposide also increased the phosphorylation levels of glycogen synthase kinase (GSK)-3α/β in treated RIN-m5F cells. Pretreatment with LiCl, a GSK-3 inhibitor, prevented etoposide-induced mitochondria-dependent apoptosis and GSK-3 protein phosphorylation in RIN-m5F cells. Furthermore, exposure of the cells to etoposide induced the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK)1/2 but not p38-MAPK, which was suppressed by the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059), respectively. Additionally, pretreatment with both SP600125 and PD98059 effectively suppressed etoposide-induced β-cell cytotoxicity, apoptosis, and GSK-3 protein phosphorylation; however, LiCl did not reverse JNK and ERK1/2 phosphorylation. Taken together, these results suggest that etoposide is capable of causing cytotoxicity on pancreatic β-cells by inducing apoptosis through the JNK/ERK-mediated GSK-3 downstream-triggered mitochondria-dependent signaling pathway.     

Diallyl disulfide induces apoptosis in human colon cancer cell line (COLO 205) through the induction of reactive oxygen species,endoplasmic reticulum stress,caspases casade and mitochondrial-dependent pathways

In this study, we investigated the effects of DADS on human colon cancer cell line COLO 205 on cell cycle arrest and apoptosis in vitro. After 24 h treatment of COLO 205 cells with DADS, the dose- and time-dependent decreases of viable cells were observed and the IC₅₀ was 22.47 μM. The decreased percentages of viable cells are associated with the production of ROS. Treatment of COLO 205 cells with DADS resulted in G2/M phase arrest and apoptosis occurrence through the mitochondrial-pathway (Bcl-2, Bcl-xL down-regulation and Bak, Bax up-regulation). DADS increased cyclin B, cdc25c-ser-216-9 and Wee1 but did not affect CDK1 protein and gene expression within 24 h of treatment. DADS-induced apoptosis was examined and confirmed by DAPI staining and DNA fragmentation assay. DADS promoted caspase-3, -8 and -9 activity and induced apoptosis were accompanied by increasing the levels of Fas, phospho-Ask1 and -JNK, p53 and decreasing the mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-9 and -3. The COLO 205 cells were pre-treated with JNK inhibitor before leading to decrease the percentage of apoptosis which was induced by DADS. Inhibition of caspase-3 activation blocked DADS-induced apoptosis on COLO 205 cells.     

Diallyl trisulfide-induced apoptosis of bladder cancer cells is caspase-dependent and regulated by PI3K/Akt and JNK pathways

Diallyl trisulfide (DATS) is one of the major organosulfur components of garlic (Allium sativum L.), which inhibits the proliferation of various cancer cells, but the exact mechanisms of this action in human bladder cancer cells still remain largely unresolved. In this study, we investigated how DATS induces apoptosis in T24 human bladder cancer cells in vitro. Treatment of T24 cells with DATS resulted in potent anti-proliferative activity. Additionally, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, DATS reduced the expression of anti-apoptotic Bcl-2 and Bcl-xL, and inhibitor of apoptosis protein family proteins, but the expression of pro-apoptotic Bax and death receptor-related proteins was increased compared with the controls. DATS also activated caspase-8 and -9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly-ADP-ribose polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation through treatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in DATS-treated T24 cells. The study further investigated the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the apoptotic effect of DATS, and showed that DATS deactivates Akt. Additionally, DATS activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38 MAPK, in T24 cells. Unlike ERK, JNK inhibitors reversed DATS-induced apoptosis and growth inhibition; however, inhibition of PI3K/Akt notably enhanced the apoptotic action of DATS. The results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways, which is mediated through the blocking of PI3K/Akt and the activation of the JNK pathway.