Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells

The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18beta-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 muM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18beta-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Protective Effect of 1-Methylated β-Carbolines Against 3-Morpholinosydnonimine-Induced Mitochondrial Damage and Cell Viability Loss in PC12 Cells

The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.     

Bradykinin enhances reactive oxygen species generation, mitochondrial injury, and cell death induced by ATP depletion--a role of the phospholipase C-Ca(2+) pathway

This study aimed to study the effect of bradykinin on reactive oxygen species (ROS) generation, mitochondrial injury, and cell death induced by ATP depletion in cell culture. Renal tubular cells were subjected to ATP depletion. Cell death was evaluated with LDH release, sub-G0/G1 fraction, Hoechst staining, and annexin V binding assay. ROS generation, mitochondrial membrane potential (DeltaPsi(m)), and intramitochondrial calcium were evaluated with flow cytometry. Translocation of cytochrome c and activation of apoptotic protein were analyzed with cell fractionating and Western blotting. Intracellular calcium was measured with a spectrofluorometer. Bradykinin enhanced cellular LDH release, apoptosis, generation of superoxide, and hydrogen peroxide induced by ATP depletion. Bradykinin also enhanced the loss of DeltaPsi(m), translocation of cytochrome c into cytosol, and activation of apoptotic protein. The intracellular/mitochondrial calcium was higher in bradykinin-treated cells. All these effects were reversed by coadministration with bradykinin B2 receptor (B2R) antagonist. Besides, blocking the phospholipase C (PLC) could reverse the synergistic effect of bradykinin with ATP depletion on ROS generation, mitochondrial damage, accumulation of intracellular/mitochondrial calcium, and apoptosis. Activation of B2R aggravates ROS generation, mitochondrial damage, and cell death induced by ATP depletion. These effects may act through the PLC-Ca(2+) signaling pathway.     

Glycyrrhizin Prevents 7-Ketocholesterol Toxicity Against Differentiated PC12 Cells by Suppressing Mitochondrial Membrane Permeability Change

Defects in mitochondrial function participate in the induction of neuronal cell injury. In neurodegenerative conditions, oxidative products of cholesterol are elevated and oxysterols seem to be implicated in neuronal cell death. The present work was designed to study the inhibitory effect of licorice compounds glycyrrhizin and 18β-glycyrrhetinic acid against the toxicity of 7-ketocholesterol in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, loss of the mitochondrial transmembrane potential, increase in the cytosolic Bax and cytochrome c levels, caspase-3 activation and cell death in differentiated PC12 cells. Glycyrrhizin and 18β-glycyrrhetinic acid prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death. The results obtained show that glycyrrhizin and 18β-glycyrrhetinic acid may prevent the 7-ketocholesterol-induced neuronal cell damage by suppressing changes in the mitochondrial membrane permeability.     

Modality of Cell Death Induced by Foscan®-Based Photodynamic Treatment in Human Colon Adenocarcinoma Cell Line HT29

Apoptosis induced by photodynamic therapy (PDT) is considered to be an important factor defining the treatment outcome. Nevertheless, the relevance of apoptotic events in overall cell death should be established for every given photosensitizer. The present study addresses the contribution of Foscan-(meta-tetra(hydroxyphenyl)chlorine; mTHPC) photosensitized apoptosis in overall cell death in a model of cultured HT29 adenocarcinoma cells. Early events of cell death were assessed by the evaluation of mitochondrial response to mTHPC-mediated PDT, cytochrome c release and membrane depolarization. Apoptosis was measured through the activity of caspase-3 and the binding of the fluorescent conjugate Ca2+-dependent protein Annexin-V on membrane externalized phosphatidylserine at 2, 4, and 24 h post-PDT. Immediately after mTHPC-PDT, from 28 to 57% cells exhibited cytochrome c release concomitantly with mitochondrial membrane depolarization for light doses inducing more than 90% overall cell death. The maximum of caspase-3 activation (12-fold more than control) was reached 24 h after irradiation at fluence inducing 90% cell death (LD(90)). The corresponding measurement of apoptotic cells (12% of Annexin-V bound cells) confirmed the mild and delayed apoptotic response of HT29 cells to mTHPC-PDT.     

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.     

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Tyrosine Kinase Inhibitor AG126 Reduces 7-Ketocholesterol-Induced Cell Death by Suppressing Mitochondria-Mediated Apoptotic Process

The preventive effect of tyrosine kinase inhibitor AG126 against the 7-ketocholesterol toxicity was investigated in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, the mitochondrial membrane permeability changes, the formation of reactive oxygen species and the depletion of GSH, which leads to cell death in differentiated PC12 cells. Tyrphostin AG126 significantly attenuated the 7-ketocholesterol-induced decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic pro-apoptotic Bax levels, mitochondrial membrane potential loss, cytochrome c release and subsequent caspase-3 activation. The inhibitory effect of tyrphostin AG126 may be supported by the inhibitory effect on another oxysterol 25-hydroxycholesterol-induced cell death. The results show that tyrphostin AG126 may prevent the 7-ketocholesterol toxicity by suppressing the mitochondrial membrane permeability change that leads to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and the depletion of GSH.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Kynurenic acid attenuates MPP(+)-induced dopaminergic neuronal cell death via a Bax-mediated mitochondrial pathway

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults. However, the molecular mechanism of this protective effect has not been identified. In this study, we examined the protective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP(+)), the best-characterized toxin inducing pathological changes resembling Parkinson's disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells. Pre-treatment of KYNA attenuated MPP(+)-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP(+)-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse of mitochondrial membrane potential (DeltaPsi(m)), release of cytochrome c from mitochondria into the cytoplasm, and increases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Our results indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrial function in MPP(+)-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.     

18β-Glycyrrhetinic acid potentiates Hsp90 inhibition-induced apoptosis in human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathway

The Hsp90 inhibition has been shown to induce apoptosis in various cancer cells. The licorice compounds may enhance the anti-cancer drug effect. However, effect of the licorice compounds on the Hsp90 inhibition-induced apoptosis in ovarian cancer cells has not been studied. To assess the ability of 18β-glycyrrhetinic acid to promote apoptosis, we examined whether 18β-glycyrrhetinic acid potentiated the Hsp90 inhibitor-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Radicicol and geldanamycin induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels, an increase in Bax levels, the mitochondrial transmembrane potential loss, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1, and an increase in the tumor suppressor p53 levels. 18β-Glycyrrhetinic acid enhanced Hsp90 inhibitor-induced apoptosis-related protein activation, nuclear damage, and cell death. The results suggest that 18β-glycyrrhetinic acid may potentiate the Hsp90 inhibition-induced apoptosis in ovarian carcinoma cell lines via the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated cell death pathway, leading to activation of caspases. Combination of Hsp90 inhibitors and 18β-glycyrrhetinic acid may confer a benefit in the treatment of epithelial ovarian adenocarcinoma.     

Elevated skeletal muscle apoptotic signaling following glutathione depletion

Oxidative stress has a well-established role in numerous intracellular signaling pathways, including apoptosis. Glutathione is an important cellular antioxidant and is the most abundant low molecular weight thiol in the cell. Although previous work has shown a link between glutathione and apoptosis, this relationship has not been defined in skeletal muscle. The present investigation examined the effect of glutathione depletion on skeletal muscle apoptotic signaling, and mitochondrial apoptotic-susceptibility. Administration of l-buthionine-[S,R]-sulfoximine (BSO; 30 mM in drinking water for 10 days) caused glutathione depletion in whole muscle and isolated mitochondria, as well as elevated muscle catalase protein content and reactive oxygen species (ROS) generation. Glutathione depletion was associated with elevated DNA fragmentation, mitochondrial Bax levels, Poly(ADP-ribose) polymerase (PARP) cleavage, and calpain activity; however, caspase-3, -8, and -9 activity were not altered. BSO administration was also associated with higher cytosolic and nuclear protein levels of apoptosis-inducing factor (AIF), but not cytochrome c, second mitochondria-derived activator of caspase (Smac), or endonuclease G (EndoG). In addition, isolated mitochondria from BSO animals demonstrated significantly lower membrane potential, increased Ca²⁺-induced permeability transition pore opening, and greater basal and ROS-induced AIF and cytochrome c release. These results demonstrate that glutathione depletion in skeletal muscle increases caspase-independent signaling, as well as augments mitochondrial-associated apoptotic events to subsequent cell death stimuli.     

Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells.

Recent studies have demonstrated that induction of apoptosis is related to the cell growth inhibition potential of Salvia Miltiorrhiza (SM), a traditional herbal medicine. In the present study, we further explore the mechanistic pathway involved in SM-induced apoptosis in human hepatoma HepG2 cells. A rapid decline of intracellular glutathione (GSH) and protein thiol content was found in SM-treated cells. Moreover. SM exposure resulted in mitochondrial dysfunction as demonstrated by: (i) the onset of mitochondrial permeability transition (MPT); (ii) the disruption of mitochondrial membrane potential (MMP); and (iii) the release of cytochrome c from mitochondria into the cytosol. Subsequently, elevated level of intracellular reactive oxygen species (ROS) was observed prior to the onset of DNA fragmentation. However, no caspase-3 cleavage was observed throughout the whole period of SM treatment, while a caspase-3-independent poly(ADP-ribose) polymerase (PARP) cleavage was noted at the late stage in SM-induced apoptosis. Pretreatment of cells with N-acetylcysteine (NAC), the GSH synthesis precursor, conferred complete protection against MMP loss, ROS generation and apoptosis induced by SM. MPT inhibitors, cyclosporin A plus trifluoperazine, partially restored intracellular GSH content, and reduced SM-induced ROS formation and subsequently inhibited cell death. Moreover, antioxidants NAC, deferoxamine and catalase had little effect on GSH depletion and mitochondrial dysfunction, yet still were able to completely protect cells from SM-induced apoptosis. Taken together, our results suggest that SM deplete intracellular thiols, which, in turn, causes MPT and subsequent increase in ROS generation, and eventually apoptotic cell death.     

Potentiation of tumor necrosis factor-alpha-induced cell death by rottlerin through a cytochrome-C-independent pathway

The protein kinase C (PKC) signal transduction pathway negatively regulates receptor-initiated cell death. In HeLa cells, tumor necrosis factor-alpha (TNF)-mediated cell death involved mitochondria and was blocked by the overexpression of Bcl-2. The PKC-specific inhibitor bisindolylmaleimide and the PKCdelta inhibitor rottlerin enhanced TNF-induced cell death. We have investigated if potentiation of TNF-induced cell death by rottlerin involved amplification of the mitochondrial pathway. TNF induced cleavage of the proapoptotic protein Bid and release of mitochondrial cytochrome c. Rottlerin enhanced activation of caspase-8 and cleavage of Bid. It also enhanced activation of caspase-9 but it did not increase cytochrome c in the cytosol. It, however, increased release of mitochondrial apoptosis-inducing factor (AIF) to the cytosol. Overexpression of Bcl-2 prevented release of both cytochrome c and AIF to the cytosol. Prolonged exposure (> or =6 h) of HeLa cells to rottlerin and TNF decreased the level of cytochrome c but not of AIF in the cytosol. These results suggest that rottlerin activates a cytochrome-c-independent cell death pathway to potentiate cell death by TNF.     

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X_L protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.     

Cytochrome c is dispensable for fas-induced caspase activation and apoptosis.

Cytochrome c is thought to play an important role in the initiation of apoptosis following its release from mitochondria. It is controversial whether such release is also involved in caspase activation and apoptotic cell death after ligation of the cell surface molecule Fas. We addressed this issue by investigating cells from the human cell lines Jurkat and SKW6 which had been treated with the inhibitor of the mitochondrial F0/F1-ATPase, oligomycin. Oligomycin-treatment led, over a wide range of concentrations, to ATP-depletion and, at similar concentrations, abrogated the appearance of caspase-3-like activity caused by stauroporine. Electroporation of cytochrome c protein into intact cells induced caspase activation in both cell lines and significant nuclear apoptosis in Jurkat cells. In ATP-depleted cells, electroporation of cytochrome c induced neither caspase activation nor nuclear fragmentation. Fas-induced caspase activation and nuclear apoptosis, however, were unaffected by the depletion of ATP. Thus, cytochrome c is unlikely to be an important factor in Fas-induced cell death.     

Cudrania tricuspidata Stem Extract Induces Apoptosis via the Extrinsic Pathway in SiHa Cervical Cancer Cells

The focus of this study is the anti-cancer effects of Cudrania tricuspidata stem (CTS) extract on cervical cancer cells. The effect of CTS on cell viability was investigated in HPV-positive cervical cancer cells and HaCaT human normal keratinocytes. CTS showed significant dose-dependent cytotoxic effects in cervical cancer cells. However, there was no cytotoxic effect of CTS on HaCaT keratinocytes at concentrations of 0.125–0.5 mg/mL. Based on this cytotoxic effect, we demonstrated that CTS induced apoptosis by down-regulating the E6 and E7 viral oncogenes. Apoptosis was detected by DAPI staining, annexin V-FITC/PI staining, cell cycle analysis, western blotting, RT-PCR, and JC-1 staining in SiHa cervical cancer cells. The mRNA expression levels of extrinsic pathway molecules such as Fas, death receptor 5 (DR5), and TNF-related apoptosis-inducing ligand (TRAIL) were increased by CTS. Furthermore, CTS treatment activated caspase-3/caspase-8 and cleavage of poly (ADP-ribose) polymerase (PARP). However, the mitochondrial membrane potential and expression levels of intrinsic pathway molecules such as Bcl-2, Bcl-xL, Bax, and cytochrome C were not modulated by CTS. Taken together, these results indicate that CTS induced apoptosis by activating the extrinsic pathway, but not the intrinsic pathway, in SiHa cervical cancer cells. These results suggest that CTS can be used as a modulating agent in cervical cancer.     

Involvement of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis.

In the present study, we studied the molecular mechanism underlying cell death induced by a cancer chemoprotective compound benzyl isothiocyanate (BITC). The cytotoxic effect of BITC was examined in rat liver epithelial RL34 cells. Apoptosis was induced when the cells were treated with 20 mum BITC, characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane and caspase-3 activation, whereas no caspase activation and propidium iodide incorporation into cell were detected with 50 mum BITC that induced necrosis. The mitochondrial death pathway was suggested to be involved in BITC-induced apoptosis because the treatment of cells with BITC-induced caspase-9-dependent apoptosis and mitochondrial transmembrane potential (Delta Psi m) alteration. We demonstrated here for the first time that BITC directly modifies mitochondrial functions, including inhibition of respiration, mitochondrial swelling, and release of cytochrome c. Moreover, glutathione depletion by diethyl maleate significantly accelerated BITC-triggered apoptosis, suggesting the involvement of a redox-dependent mechanism. This was also implicated by the observations that intracellular accumulation of reactive oxygen species, including superoxide (O(2)) and hydroperoxides (HPOs), was indeed detected in the cells treated with BITC and that the intracellular HPO level was significantly attenuated by pretreatment with N-acetylcysteine. The treatment with a pharmacological scavenger of O(2), Tiron, also diminished the HPO formation by approximately 80%, suggesting that most of the HPOs were H(2)O(2) derived from the dismutation of O(2). These results suggest that BITC induces apoptosis through a mitochondrial redox-sensitive mechanism.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.