大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

A phospholipase c-dependent intracellular ca2+ release pathway mediates the capsaicin-induced apoptosis in HepG2 human hepatoma cells

The effect of capsaicin on apoptotic cell death was investigated in HepG2 human hepatoma cells. Capsaicin induced apoptosis in time- and dose-dependent manners. Capsaicin induced a rapid and sustained increase in intracellular Ca2+ concentration, and BAPTA, an intracellular Ca2+ chelator, significantly inhibited capsaicin-induced apoptosis. The capsaicin-induced increase in the intracellular Ca2+ and apoptosis were not significantly affected by the extracellular Ca2+ chelation with EGTA, whereas blockers of intracellular Ca2+ release (dantrolene) and phospholipase C inhibitors, U-73122 and manoalide, profoundly reduced the capsaicin effects. Interestingly, treatment with the vanilloid receptor antagonist, capsazepine, did not inhibit either the increased capsaicin-induced Ca2+ or apoptosis. Collectively, these results suggest that the capsaicin-induced apoptosis in the HepG2 cells may result from the activation of a PLC-dependent intracellular Ca2+ release pathway, and it is further suggested that capsaicin may be valuable for the therapeutic intervention of human hepatomas.     

Nonylphenol induces apoptosis in rat testicular Sertoli cells via endoplasmic reticulum stress

Nonylphenol (NP) is a widely distributed environment contaminant and has been documented to disrupt testicular development and decrease male fertility. Amongst possible targets of this compound are testicular Sertoli cells, which play a crucial role in supporting and nourishing sperm cells. In the present study, we found that NP treatment could cause dramatic morphological changes as well as decreased cell viability of Sertoli cells, while the following Annexin V–PI staining demonstrated that NP treatment led to increased proportion of cell apoptosis, which was evidenced again by the detection of condensation and marginal changes of chromatins using Hoechst staining and transmission microscopy observation. In addition, increased intracellular Ca²⁺ levels and changes of endoplasmic reticulum (ER) ultrastructure were also observed in NP-treated groups, indicating the action of NP on ER. The subsequent data showed that the expressions of ER-stress signaling targeted genes GRP78 and gadd153 were elevated, suggesting the activation of ER-stress signal pathway. Furthermore, the detection of ER-stress related proteins by western blotting revealed that the expression of gadd153 was upregulated by NP, whereas the expressions of GRP78 and ERp57 were both first upregulated and then inhibited. Taken together, it is suggested that NP can induce ER stress in Sertoli cells, which may plays an important role in the induction of apoptosis.     

The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells

Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca²⁺ levels ([Ca²⁺]_i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca²⁺]_i rises which were reduced by removing extracellular Ca²⁺. Eugenol-induced [Ca²⁺]_i rises were not altered by store-operated Ca²⁺ channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca²⁺]_i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca²⁺]_i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca²⁺]_i rises by inducing PLC-dependent release of Ca²⁺ from the endoplasmic reticulum and caused Ca²⁺ influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway.     

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.     

Ketoconazole-induced JNK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

This study examined the effect of ketoconazole on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca²⁺ levels in MG63 osteosarcoma cells. Ketoconazole at 20–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that ketoconazole induced phosphorylation of ERK and JNK, but not p38, MAPKs. Ketoconazole-induced cell death and apoptosis were partially reversed by the selective JNK inhibitor SP600125, but not by the selective ERK inhibitor PD98059, suggesting that ketoconazole’s cytotoxic action was via JNK, but not via ERK and p38 MAPKs. Ketoconazole at a concentration of 100 μM induced [Ca²⁺]_i increases. Chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) totally inhibited ketoconazole-induced [Ca²⁺]_i increases without reversing ketoconazole-induced cell death. Collectively, in MG63 cells, ketoconazole induced cell death and apoptosis via evoking JNK phosphorylation in a Ca²⁺-independent manner.     

Organic solvent-induced proximal tubular cell apoptosis via caspase-9 activation

Long-term exposure to solvents is associated with apoptosis, which is implicated in the development and progression of tubulo-interstitial fibrosis and chronic renal failure. In our previous study, we demonstrated that toluene and p-xylene as the most commonly used organic solvents induced proximal tubular cells apoptosis. This study was conducted to assess the apoptotic pathway of toluene and p-xylene induced proximal tubular apoptosis. This was assessed by measuring the caspase-9 activity LLC-PK1 cells exposed to both compounds. A model of proximal tubular cell (LLC-PK1) cytotoxicity exposed to 1 mM of either p-xylene or toluene was compared to untreated control for caspase-9 activity and Bax/Bcl-2 protein level. Furthermore, DNA fragmentation in the presence of caspase-9 inhibitor (Z-LEHD-FMK) in a dose-dependent manner was assessed. Both compounds induced caspase-9 activity, which was accompanied by up-regulation of Bax, whereas Bcl-2 level did not change. DNA fragmentation induced by both solvents was inhibited by caspase-9 inhibitor in dose-dependent manner. This data suggest that p-xylene or toluene induces nephrotoxicity via mitochondrial caspase-9 pathway. This mechanism involves up-regulation of the apoptotic protein, Bax.     

Arsenic trioxide induces the apoptosis in bone marrow mesenchymal stem cells by intracellular calcium signal and caspase-3 pathways

It was previously reported that excessive arsenic trioxide would produce cardiovascular toxicity. Bone marrow mesenchymal stem cells (BMSCs) have been shown to play a supporting role in cardiovascular functions. The increasing apoptosis of BMSCs commonly would promote the development of cardiovascular diseases. Thus we hypothesize that arsenic trioxide caused apoptosis in BMSCs, which provided a better understanding of arsenic toxicity in hearts. The present study was designed to investigate the proapoptotic effects of arsenic trioxide on BMSCs and explore the mechanism underlying arsenic trioxide-induced BMSCs apoptosis. We demonstrate that arsenic trioxide significantly inhibited survival ratios of BMSCs in a concentration-dependent and time-dependent manner. The Annexin V/PI staining and terminal deoxynucleotidyl transferasemediated dUTP nick-end labelling (TUNEL) assay also showed that arsenic trioxide markedly induced the apoptosis of BMSCs. The caspase-3 activity was obviously enhanced in the presence of arsenic trioxide in a concentration-dependent manner in BMSCs. Additionally, arsenic trioxide caused the increase of intracellular free calcium ([Ca²⁺]_i) in rat BMSCs. BAPTA pretreatment may attenuate the apoptosis of BMSCs induced by arsenic trioxide. Taken together, arsenic trioxide could inhibit the proliferation and induce the apoptosis of BMSCs by modulating intracellular [Ca²⁺]_i, and activating the caspase-3 activity.     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

Catalytic activity-independent pathway is involved in phospholipase A2-induced apoptotic death of human leukemia U937 cells via Ca-mediated p38 MAPK activation and mitochondrial depolarization

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A₂ (PLA₂), the present study is conducted to explore whether PLA₂ induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA₂) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA₂. It was found that BPB–PLA₂-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB–PLA₂, elevation of intracellular Ca²⁺ levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca²⁺ chelator) and nifedipine (L-type Ca²⁺ channel blocker) abrogated Ca²⁺ increase and p38 MAPK activation, and rescued viability of BPB–PLA₂-treated U937 cells. BPB–PLA₂-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA₂ mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA₂ activity, they induced death of U937 cells. The signaling pathway of PLA₂ mutants in inducing cell death was indistinguishable from that of BPB–PLA₂. Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA₂-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca²⁺-mediated p38 MAPK activation.     

Piperazine designer drugs induce toxicity in cardiomyoblast h9c2 cells through mitochondrial impairment

Abuse of synthetic drugs is widespread among young people worldwide. In this context, piperazine derived drugs recently appeared in the recreational drug market. Clinical studies and case-reports describe sympathomimetic effects including hypertension, tachycardia, and increased heart rate. Our aim was to investigate the cytotoxicity of N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(4-methoxyphenyl) piperazine (MeOPP), and 1-(3,4-methylenedioxybenzyl) piperazine (MDBP) in the H9c2 rat cardiac cell line. Complete cytotoxicity curves were obtained at a 0–20 mM concentration range after 24 h incubations with each drug. The EC₅₀ values (μM) were 343.9, 59.6, 570.1, and 702.5 for BZP, TFMPP, MeOPP, and MDBP, respectively. There was no change in oxidative stress markers. However, a decrease in total GSH content was noted for MDBP, probably due to metabolic conjugation reactions. All drugs caused significant decreases in intracellular ATP, accompanied by increased intracellular calcium levels and a decrease in mitochondrial membrane potential that seems to involve the mitochondrial permeability transition pore. The cell death mode revealed early apoptotic cells and high number of cells undergoing secondary necrosis. Among the tested drugs, TFMPP seems to be the most potent cytotoxic compound. Overall, piperazine designer drugs are potentially cardiotoxic and support concerns on risks associated with the intake of these drugs.     

Anandamide-induced Ca2+ elevation leading to p38 MAPK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

The effect of anandamide on human osteoblasts is unclear. This study examined the effect of anandamide on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca2+ levels in MG63 osteosarcoma cells. Anandamide at 50-200 microM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that anandamide induced expression of ERK, JNK and p38 MAPK. Anandamide-induced cell death and apoptosis were reversed by SB203580, but not by PD98059 and SP600125, suggesting that anandamide's action was via p38 MAPK, but not via ERK and JNK. Anandamide at 1-100 microM induced [Ca2+]i increases. Removal of extracellular Ca2+ decreased the anandamide response, indicating that anandamide induced Ca2+ influx and Ca2+ release. Chelation of intracellular Ca2+ with BAPTA reversed anandamide-induced cell death and p38 MAPK phosphorylation. Collectively, in MG63 cells, anandamide induced [Ca2+]i increases which evoked p38 MAPK phosphorylation. This p38 MAPK phosphorylation subsequently activated caspase-3 leading to apoptosis.     

T-2 toxin initially activates caspase-2 and induces apoptosis in U937 cells

T-2 toxin, which belongs to a group of mycotoxins synthesized by Fusarium fungi that are widely encountered as natural contaminants, induced apoptosis with distinct morphological and biological features in U937 cells. The concentration of more than 10nM T-2 toxin affected cell viability, induced nuclear and DNA fragmentation and caspase-3 activation. Caspase-2, -3, -8, and -9 were activated during T-2 toxin-induced apoptosis. T-2 toxin neither inhibited mitochondrial respiratory chain complexes I-IV in isolated mitochondria nor decreased ATP levels in U937 cells. Both enzyme activity assay and Western blot analysis revealed that T-2 toxin activated caspase-2 earlier than caspase-3, -8, and -9. Caspase-2 inhibitor (VDVAD-CHO/fmk) and caspase-8 inhibitor (IETD-CHO/fmk) completely blocked the T-2 toxin-induced process of procaspase-3, while caspase-9 inhibitor (LEHD-CHO/fmk) did so less effectively. Caspase-2 inhibitor entirely blocked T-2 toxin-induced caspase-8, and -9 activation. These results clearly indicate that activation of caspase-2 is essential to T-2 toxin-induced apoptosis and that apoptotic signals are mainly transmitted via caspase-8 and caspase-3 rather than mitochondrial pathway.     

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.     

Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca concentration

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca²⁺ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.     

Atorvastatin induces mitochondrial dysfunction and cell apoptosis in HepG2 cells via inhibition of the Nrf2 pathway

Atorvastatin (ATO) is a 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor widely used to treat hypercholesterolemia. However, clinical application is limited by potential hepatotoxicity. Nuclear factor‐erythroid 2‐related factor 2 (Nrf2) is a master regulator of cellular antioxidants, and oxidative stress is implicated in statin‐induced liver injury. This study investigated mechanisms of ATO‐induced hepatotoxicity and potential mitigation by Nrf2 signaling. ATO reduced Nrf2 and antioxidant enzyme superoxide dismutase‐2 (SOD2) expression in human hepatocarcinoma HepG2 cells. ATO also induced concentration‐dependent HepG2 cell toxicity, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential (MMP) and cellular adenosine triphosphate (ATP). Further, ATO induced mitochondria‐dependent apoptosis as indicated by increased Bax/Bcl‐2 ratio, cleaved caspase‐3, mitochondrial cytochrome c release and Annexin V‐fluorescein isothiocyanate/propidium iodide staining. Tert‐butylhydroquinone enhanced Nrf2 and SOD2 expression, and partially reversed ATO‐induced cytotoxicity, ROS accumulation, MMP reduction, ATP depletion and mitochondria‐dependent apoptosis. In conclusion, the present study demonstrates that ATO induces mitochondrial dysfunction and cell apoptosis in HepG2 cells, at least in part, via inhibition of the Nrf2 pathway. Nrf2 pathway activation is a potential prevention for ATO‐induced liver injury.     

Tacrine induces apoptosis through lysosome- and mitochondria-dependent pathway in HepG2 cells

Tacrine (THA) is a competitive inhibitor of cholinesterase. Administration of THA for the treatment of Alzheimer’s disease results in a reversible hepatotoxicity in 30–50% of patients, as indicated by elevated alanine aminotransferase levels. However, the intracellular mechanisms have not yet been elucidated. In our previous study, we found that THA induced cytotoxicity and mitochondria dysfunction by ROS generation and 8-OHdG formation in mitochondrial DNA in HepG2 cells. In this study, the mechanism underlying was further investigated. Our results demonstrated that THA induced dose-dependent apoptosis with cytochrome c release and activation of caspase-3. THA-induced apoptosis was inhibited by treating cells with a ROS inhibitor, YCG063. In addition, we observed that THA led to an early lysosomal membrane permeabilization and release of cathepsin B. Pretreatment with CA-074Me, a specific cathepsin B inhibitor resulted in a significant but not complete decrease in tacrine-induced apoptosis. These data suggest that tacrine-induced cell apoptosis involves both mitochondrial damage and lysosomal membrane destabilization, and ROS is the critical factor that integrates tacrine-induced mitochondrial and lysosomal death pathways.     

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.