Gypenosides induce apoptosis in human hepatoma Huh-7 cells through a calcium/reactive oxygen species-dependent mitochondrial pathway

We have previously reported that gypenosides induce apoptosis in human hepatocarcinoma Huh-7 cells through a mitochondria-dependent caspase-9 activation cascade. In order to further explore the critical events leading to apoptosis in gypenosides-treated cells, the following effects of gypenosides on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MPT), and the subcellular distribution of Bcl-2 and Bax. We show that gypenosides-induced apoptosis was accompanied by the generation of intracellular ROS, disruption of MPT, and inactivation of ERK, as well as an increase in mitochondrial Bax and a decrease of mitochondrial Bcl-2 levels. Ectopic expression of Bcl-2 or treatment with furosemide attenuated gypenosides-triggered apoptosis. Treatment with ATA caused a drastic prevention of apoptosis and the gypenosides-mediated mitochondrial Bcl-2 decrease and Bax increase, but failed to inhibit ROS generation and MPT dysfunction. Incubation with antioxidants significantly inhibited gypenosides-mediated ROS generation, ERK inactivation, MPT and apoptosis. Moreover, an increase of the intracellular calcium ion (Ca(2+)) concentration rapidly occurred in gypenosides-treated Huh-7 cells. Buffering of the intracellular Ca(2+) increase with a Ca(2+) chelator BAMTA/AM blocked the gypenosides-elicited ERK inactivation, ROS generation, Bcl-2/Bax redistribution, mitochondrial dysfunction, and apoptosis. Based on these results, we propose that the rise in intracellular Ca(2+) concentration plays a pivotal role in the initiation of gypenosides-triggered apoptotic death.     

Organotins induce apoptosis by disturbance of [Ca(2+)](i) and mitochondrial activity, causing oxidative stress and activation of caspases in rat thymocytes

Di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) cause thymus atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of thymotoxicity by these chemicals. In vitro, a similar concentration-dependency has been observed. The purpose of the present research was to investigate the mechanisms underlying DNA fragmentation induced by these organotin compounds in freshly isolated rat thymocytes. As previously shown for TBTC, DBTC is also able to significantly increase intracellular Ca(2+) level ([Ca(2+)](i)). The rise in [Ca(2+)](i), already evident 5 min after treatment, was followed by a dose- and time-dependent generation of reactive oxygen species (ROS) at the mitochondrial level. Simultaneously, organotins induced the release of cytochrome c from the mitochondrial membrane into the cytosol. ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca(2+) chelator, or rotenone, an inhibitor of the electron entry from complex I to ubiquinone, indicating the important role of Ca(2+) and mitochondria during these early intracellular events. Furthermore, we demonstrated that rotenone prevents apoptosis induced by 3 microM DBTC or TBTC and, in addition, that both BAPTA and Z-DEVD FMK (mainly a caspase-3 inhibitor) decreased apoptosis by DBTC (already shown for TBTC). Taken together these data show the apoptotic pathway followed by organotin compounds starts with an increase of [Ca(2+)](i), then continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.     

Cadmium-induced apoptosis in murine macrophages is antagonized by antioxidants and caspase inhibitors

Cadmium is a toxic heavy metal that accumulates in the environment and is commonly found in cigarette smoke and industrial effluents. This study was designed to determine the role of reactive oxygen species (ROS) generation, and its antagonism by antioxidants, in cadmium-mediated cell signaling and apoptosis in murine macrophage cultures. Cadmium-generated ROS production was observed in J774A.1 cells at 6 h, reverting to control levels at 16 and 24 h. The ROS production was concentration related between 20 and 500 microM cadmium. Activation of caspase-3 was observed at 8 h and DNA fragmentation at 16 h in the presence of 20 microM cadmium, suggesting that caspase-3 activation is a prior step to DNA fragmentation in cadmium-induced apoptosis. Inhibitors of caspase-3, -8, -9, and a general caspase inhibitor suppressed cadmium-induced caspase-3 activation and apoptosis indicating the importance of caspase-3 in cadmium-induced toxicity in these cells. Protection against the oxidative stress with N-acetylcysteine (NAC) and silymarin (an antioxidant flavonoid) blocked cadmium-induced apoptosis. Pretreatment of cells with NAC and silymarin prevented cadmium-induced cell injury, including growth arrest, mitochondrial impairment, and necrosis, and reduced the cadmium-elevated intracellular calcium ([Ca2+]i), suggesting that the oxidative stress is a source of increased [Ca2+]i. NAC inhibited cadmium-induced activation of mitogen-activated protein kinases, the c-Jun NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK). However, silymarin provided only a partial protection for JNK activation, and only at the low concentration did it inhibit cadmium-induced ERK activation. Inhibition of caspase-3 protected oxidative stress produced by cadmium, suggesting that the activation of caspase-3 also contributes to generation of reactive oxygen species (ROS). Results emphasized the role of ROS, Ca2+ and mitogen-activated protein kinases in cadmium-induced cytotoxicity in murine macrophages.     

Concentration dependence of the mechanisms of tributyltin-induced apoptosis.

Tributyltin chloride (TBT), an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide, and component of antifouling paints. In this study, we investigated the concentration dependence of the mechanisms of tributyltin cytotoxicity in PC12 cells. Exposure of PC12 cells to both 500 nM and 2 microM tributyltin increased the number of cells showing nuclear fragmentation, a typical apoptotic feature, and activated caspase-3. The peak Ca(2+) concentration in 2 microM tributyltin-treated cells was higher than that in 500 nM tributyltin-treated cells. The intracellular Ca(2+) increase induced by 2 microM tributyltin was mediated by Ca(2+) release from both inositol 1,4,5-trisphosphate receptor and ryanodine receptor, while the Ca(2+) increase induced by 500 nM tributyltin was mediated through the voltage-dependent calcium channel (VDCC). Next, we investigated whether the mechanisms leading to cell death after Ca(2+) increase were different. Reactive oxygen species (ROS) were involved only in 2 microM tributyltin-induced cell death, while c-jun N-terminal kinase (JNK) mediated only 500 nM tributyltin-induced toxicity. Thus, caspase-dependent apoptosis caused by 2 microM tributyltin was mediated by a large Ca(2+) increase via inositol 1,4,5-trisphosphate receptor and ryanodine receptor, followed by generation of ROS. Apoptosis caused by 500 nM tributyltin was mediated by a moderate Ca(2+) increase through the VDCC, followed by phosphorylation of JNK. These results suggest that apoptosis by TBT is induced via distinct pathways depending on the TBT concentration, and we showed a rare example that upstream mechanisms of apoptosis are distinct depending on strength of toxic insult.     

Nicotine attenuates beta-amyloid peptide-induced neurotoxicity, free radical and calcium accumulation in hippocampal neuronal cultures

1. Recent studies indicate that neuronal loss in Alzheimer's disease (AD) is accompanied by the deposition of beta-amyloid protein (A beta) in senile plaques. Nicotine as a major component of cigarette smoke has been suggested to have a protective effect for neurons against A beta neurotoxicity. 2. Our present study demonstrates that nicotine protected cultured hippocampal neurons against the A beta-induced apoptosis. Nicotine effectively inhibits apoptosis in hippocampal cultures caused by A beta(25-35) or A beta(1-40) treatment and increase of caspase activity induced by A beta(25-35) or A beta(1-40). 3. Measurements of cellular oxidation and intracellular free Ca(2+) showed that nicotine suppressed A beta-induced accumulation of free radical and increase of intracellular free Ca(2+). 4. Cholinergic antagonist mecamylamine inhibited nicotine-induced protection against A beta-induced caspase-3 activation and ROS accumulation. 5. The data show that the protection of nicotine is partly via nicotinic receptors. Our results suggest that nicotine may be beneficial in retarding the neurodegenerative diseases such as AD.     

Signal transduction of p53-independent apoptotic pathway induced by hexavalent chromium in U937 cells

It has been reported that the hexavalent chromium compound (Cr(VI)) can induce both p53-dependent and p53-independent apoptosis. While a considerable amount of information is available on the p53-dependent pathway, only little is known about the p53-independent pathway. To elucidate the p53-independent mechanism, the roles of the Ca(2+)-calpain- and mitochondria-caspase-dependent pathways in apoptosis induced by Cr(VI) were investigated. When human lymphoma U937 cells, p53 mutated cells, were treated with 20 microM Cr(VI) for 24 h, nuclear morphological changes and DNA fragmentation were observed. Production of hydroxyl radicals revealed by electron paramagnetic resonance (EPR)-spin trapping, and increase of intracellular calcium ion concentration monitored by digital imaging were also observed in Cr(VI)-treated cells. An intracellular Ca(2+) chelator, BAPTA-AM, and calpain inhibitors suppressed the Cr(VI)-induced DNA fragmentation. The number of cells showing low mitochondrial membrane potential (MMP), high level of superoxide anion radicals (O(2)(-)), and high activity of caspase-3, which are indicators of mitochondria-caspase-dependent pathway, increased significantly in Cr(VI)-treated cells. An antioxidant, N-acetyl-l-cysteine (NAC), decreased DNA fragmentation and inhibited the changes in MMP, O(2)(-) formation, and activation of caspase-3 induced by Cr(VI). No increase of the expressions of Fas and phosphorylated JNK was observed after Cr(VI) treatment. Cell cycle analysis revealed that the fraction of G2/M phase tended to increase after 24 h of treatment, suggesting that Cr(VI)-induced apoptosis is related to the G2 block. These results indicate that Ca(2+)-calpain- and mitochondria-caspase-dependent pathways play significant roles in the Cr(VI)-induced apoptosis via the G2 block, which are independent of JNK and Fas activation. The inhibition of apoptosis and all its signal transductions by NAC suggests that intracellular reactive oxygen species (ROS) are important for both pathways in Cr(VI)-induced apoptosis of U937 cell.     

Neuroprotective effects of phlorotannins isolated from a brown alga, Ecklonia cava, against H2O2-induced oxidative stress in murine hippocampal HT22 cells

Exposure of neurons to hydrogen peroxide (H(2)O(2)) results in oxidative stress and the activation of a cascade of intracellular toxic events resulting in oxidation, lipid peroxidation, and Ca(2+) elevation, ultimately resulting in cell death. In this study, we attempted to characterize the neuroprotective effects of phlorotannins isolated from Ecklonia cava, including phloroglucinol, eckol, triphloroethol A, eckstolonol, and dieckol, against H(2)O(2)-induced cell damage in murine hippocampus neuronal (HT22) cells. We measured the reactive oxygen species (ROS) and lipid peroxidation levels and evaluated the resultant cell death and alterations in Ca(2+)-concentrations. All phlorotannins were to scavenge intracellular ROS and repress ROS accumulation, thus preventing lipid peroxidation. Consquently, all phlorotannins reduced H(2)O(2)-induced cell death in HT22 cells. Moreover, phlorotannins inhibited H(2)O(2)-induced Ca(2+) release. This study provides a new useful strategy for preventing neuronal H(2)O(2)-induced oxidative stress.     

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.     

Cadmium-induced apoptosis in human normal liver L-02 cells by acting on mitochondria and regulating Ca(2+) signals

Cadmium is a well-known toxic compound for the liver. It has been demonstrated to induce hepatotoxicity partly via apoptosis, but no uniform mechanism of apoptosis has so far been proposed. This study was first to determine whether cadmium-induced apoptosis in L-02 cells, second to observe the mechanism of cadmium-induced apoptosis. Studies of morphology, DNA fragmentation and apoptotic rate demonstrated that 60μM cadmium induced apoptosis with strong effects on cell viability. A concomitant time-dependent decrease of Bcl-2 and mitochondrial transmembrane potential (ΔΨ(m)) was observed. Subsequently, increase of caspase-3 activity and release of mitochondrial AIF were detected. However, cell pretreatment with a broad-specificity caspase inhibitor (Z-Asp) did not abolish apoptosis. These data demonstrated that the apoptotic events involved a mitochondria-mediated apoptotic pathway but not necessarily caspase-dependent signaling. On the other hand, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of cadmium-exposed cells had significant increases and the Bapta-AM, a well-known calcium chelator, pretreatment partially blocked cadmium-induced apoptosis, indicating that the elevation of [Ca(2+)](i) may play an important role in the apoptosis. Together, these results support the notion that cadmium-induced hepatotoxicity is comparable to effects in L-02 by inducing apoptotic pathways on the basis of acting on mitochondria and regulating Ca(2+) signals.     

Induction of apoptosis by 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol from Isaria japonica Yasuda through intracellular reactive oxygen species formation and caspase-3 activation in human leukemia HL-60 cells.

Recently we have reported that the trichothecene mycotoxin 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) from the fruiting bodies of Isaria japonica Yasuda is a potent inducer of apoptosis in human promyelocytic HL-60 cells. The present study aims to characterize the molecular events leading to AETD-induced apoptosis in HL-60 cells. The percentage of apoptotic cells (annexin-V-positive cell population) increased dose- and time-dependently after AETD exposure. Apoptosis of HL-60 cells by AETD was associated with the formation of intracellular reactive oxygen species (ROS), the depletion of intracellular glutathione (GSH) and the activation of caspase-3. Pretreating the cells with the antioxidant N-acetyl-L-cystein (NAC) and the caspase-3 inhibitor Z-DEVD-fmk abrogated AETD-induced apoptosis and caspase-3 activation. NAC blocked intracellular ROS formation and GSH depletion, but Z-DEVD-fmk did not. These results indicate that AETD induces apoptosis in HL-60 cells by causing intracellular ROS formation and GSH depletion followed by the downstream event of caspase-3 activation.     

Mechanism of apoptosis induced by diazoxide,a K<Superscript>+</Superscript> channel opener,in HepG2 Human hepatoma cells

The effect of diazoxide, a K+ channel opener, on apoptotic cell death was investigated in HepG2 human hepatoblastoma cells. Diazoxide induced apoptosis in a dose-dependent manner and this was evaluated by flow cytometric assays of annexin-V binding and hypodiploid nuclei stained with propidium iodide. Diazoxide did not alter intracellular K+ concentration, and various inhibitors of K+ channels had no influence on the diazoxide-induced apoptosis; this implies that K+ channels activated by diazoxide may be absent in the HepG2 cells. However, diazoxide induced a rapid and sustained increase in intracellular Ca(2+) concentration, and this was completely inhibited by the extracellular Ca(2+) chelation with EGTA, but not by blockers of intracellular Ca(2+) release (dantrolene and TMB-8). This result indicated that the diazoxide-induced increase of intracellular Ca(2+) might be due to the activation of a Ca(2+) influx pathway. Diazoxide-induced Ca(2+) influx was not significantly inhibited by either voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), or by inhibitors of Na+, Ca(2+)-exchanger (bepridil and benzamil), but it was inhibited by flufenamic acid (FA), a Ca(2+)-permeable nonselective cation channel blocker. A quantitative analysis of apoptosis by flow cytometry revealed that a treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the diazoxide-induced apoptosis. Taken together, these results suggest that the observed diazoxide-induced apoptosis in the HepG2 cells may result from a Ca(2+) influx through the activation of Ca(2+)-permeable non-selective cation channels. These results are very significant, and they lead us to further suggest that diazoxide may be valuable for the therapeutic intervention of human hepatomas.     

Ca2+ influx mediates apoptosis induced by 4-aminopyridine, a K+ channel blocker, in HepG2 human hepatoblastoma cells

Apoptosis appears to be implicated in the pathogenesis and therapeutic applications of cancer. In this study we investigated the induction of apoptosis by 4-aminopyridine (4-AP), a K(+) channel blocker, and its mechanism in HepG2 human hepatoblastoma cells. 4-AP reduced cell viability and induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, 4-AP induced a sustained increase in intracellular Ca(2+) concentration, which was completely inhibited by the extracellular Ca(2+) chelation with EGTA. 4-AP also induced Mn(2+) influx, indicating that the 4-AP-induced increased intracellular Ca(2+) levels were due to activation of Ca(2+) influx pathway. 4-AP also depolarized membrane potential that was measured by using di-O-C(5)(3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca(2+) influx was significantly inhibited not by voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), but by flufenamic acid (FA), a known nonselective cation channel blocker. Quantitative analysis of apoptosis by the flow cytometry revealed that treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the 4-AP-induced apoptosis. Taken together, these results suggest that the observed 4-AP-induced apoptosis in the HepG2 cells may result from Ca(2+) influx through the activation of voltage-sensitive Ca(2+)-permeable non-selective cation channels. These results further suggest that membrane potential change by modulation of K(+) channel activity may be involved in the mechanism of apoptosis in human hepatoma cells.     

Critical role of calcium overloading in cadmium-induced apoptosis in mouse thymocytes

Cadmium (Cd) is a well-known environmental carcinogen and immunotoxin. Currently the direct cytotoxic effects of Cd on thymocytes are largely unexplored. The main objective of the present study was to investigate the apoptogenic property of Cd and the mechanisms involved, using primary cultured mouse thymocytes as a model. Cd-induced apoptosis in thymocytes was studied by TdT-mediated dUTP nick end-labeling assay and DNA gel electrophoresis. The results showed that Cd was able to cause apoptosis in mouse thymocytes in a time- and dose-dependent manner. Moreover, Cd exposure led to a rapid and sustained intracellular calcium (Ca2+) elevation, followed by caspase-3 activation and PARP cleavage, all of which preceded the characteristic DNA fragmentation. BAPTA-AM, a specific intracellular Ca2+ chelator, abolished Cd-induced Ca2+ overloading and subsequently inhibited caspase-3 activation, PARP cleavage, and apoptosis.It is believed that intracellular Ca2+ elevation may trigger caspase-3 activation either through mitochondria or through activation of Ca2+-dependent protease in Cd-treated thymocytes. Results from this study thus provide new information for a better understanding of the immunotoxic and immunomodulatory effects of Cd.     

meso-Dihydroguaiaretic acid attenuates the neurotoxic effect of staurosporine in primary rat cortical cultures

The effect of meso-dihydroguaiaretic acid (MDGA) on the staurosporine-induced neuronal apoptosis and its potential mechanism were investigated using primary cultures of rat cortical cells as an assay system. Treatment of MDGA at the concentrations of 0.1, 1.0 and 10 microM significantly protected neuronal cells against Staurosporine-induced apoptosis. The neuroprotective activity of MDGA was the most potent at the concentration of 1.0 microM and was not increased at higher concentration. MDGA reduced apoptotic characteristics induced by STS; MDGA reduced the condensed nuclei in staurosporine-injured rat cortical cells. MDGA diminished the calcium influx that accompanies the staurosporine-induced apoptosis, and inhibited the subsequent overproduction of reactive oxygen species and peroxide to the level of control cells. It also preserved cellular activity of superoxide dismutase, an antioxidative enzyme reduced by staurosporine insult. In addition, MDGA significantly inhibited caspase-3/7 activation and cytochrome c release. Taken together, these results suggested that MDGA protected neuronal cells against staurosporine-induced apoptosis through the inhibition of Ca(2+) influx, cellular oxidation, cytochrome c release and caspase-3/7 activation.     

Identification of molecular signatures predicting the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs)

There are multiple lines of evidence showing that environmental toxicants including pesticides may contribute to neuronal cell death. Fipronil (FPN) is a phenylpyrazole insecticide that acts on insect GABA receptors. Although the action of FPN is restricted to insect neuronal or muscular transmitter systems, a few studies have assessed the effects of this neurotoxicant on neuronal cell death distinct from an insect. To determine the mechanisms underlying FPN-induced neuronal cell death, we evaluated the ability of this chemical to induce oxidative stress and studied the involvement of mitogen activated protein kinases (MAPKs) in FPN-induced apoptosis stress in human neuroblastoma SH-SY5Y (SH-SY5Y) cells. Exposure of SH-SY5Y cells to FPN led to the production of reactive oxygen species (ROS) and apoptotic cell death via activation of caspase-9 and caspase-3. Interestingly, the antioxidant, N-acetyl-cysteine (NAC) attenuated apoptotic cell death and ROS production induced by FPN. These results indicated that oxidative stress plays a central role in FPN-induced cytotoxicity. Mitochondrial complex I activity was also inhibited by FPN treatment. These finding indicate that FPN triggers intrinsic apoptosis via the mitochondrial signaling pathway that is initiated by the generation of ROS. Furthermore, FPN treatment induced phosphorylation of MAPK members. Activation of these protein kinases by FPN was involved in the onset of apoptosis as inhibitors specific to these kinases protect against FPN-induced cell death as well as ROS generation. Our data indicate that FPN-induced apoptosis is mediated primarily by the generation of ROS and activation of MAPK members followed by activation of the intrinsic apoptotic pathway.     

Cell injury and its protection in astrocytes

Incubation of cultured astrocytes in Ca(2+)-containing medium after exposure to Ca(2+)-free medium causes Ca2+ influx followed by delayed cell death. Here, we summarize the mechanisms underlying the Ca(2+)-mediated injury of cultured astrocytes and the protective effects of drugs against Ca(2+)-reperfusion injury. Our results show that Ca(2+)-reperfusion injury of astrocytes appears to be mediated by apoptosis as evidenced by DNA fragmentation and nuclear condensation. Calpain, reactive oxygen species (ROS) production, calcineurin, caspase-3, and NF-kappa B activation are involved in Ca(2+)-reperfusion injury. Several drugs including T-588 and idebenone protect astrocytes against Ca(2+)-reperfusion injury. The protective effect of idebenone is mediated by nerve growth factor production, whereas that of T-588 is mediated mainly by the mitogen-activated protein/extracellular signal-regulated kinase signal cascade.     

The role of mitochondria-mediated intrinsic death pathway in gingerdione derivative I6-induced neuronal apoptosis

Neuronal death induced by I6 displayed apoptotic characteristics but the precise mechanism has not been fully elucidated. In the present studies, I6 at 24 h after intraperitoneal administration significantly decreased the density of surviving neurons and increased caspase-3 activity in frontal cortex, suggesting that peripherally administered I6 may cross BBB to induce CNS toxicity. In rat embryonic primary cortical cells, I6-induced reduction of mitochondrial viability and neuronal apoptosis was inhibited by vitamin E. In addition, I6-induced reactive oxygen species (ROS) caused the disruption of mitochondria membrane potential (MMP), the release of cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), resulting in activation of mitochondrial-mediated intrinsic death pathway. Pre-treatment with antioxidant vitamin E or N-acetylcysteine (NAC) completely abolished the I6-induced generation of ROS, loss of MMP, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of PARP. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler, significantly reduced I6-induced neuronal death as well as caspase-3 activation and PARP cleavage. These results suggest that I6 induces neuronal death by promoting intracellular ROS production to cause a loss of MMP that result in release of cytochrome c and activation of mitochondria-mediated intrinsic death pathway.