Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.     

Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells

Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS‐induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild‐type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin‐binding protein F1‐disrupted mutant SAM1‐infected cells. In Bcl‐2‐overexpressing HeLa cells (HBD98‐2‐4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98‐2‐4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS‐infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.     

Hispidulin Induces Apoptosis Through Mitochondrial Dysfunction and Inhibition of P13k/Akt Signalling Pathway in HepG2 Cancer Cells

Hispidulin is a flavonoid compound which is an active ingredient in a number of traditional Chinese medicinal herbs. However, it’s therapeutic activity remains poorly understood. The present study investigated the pro-apoptotic effects and mechanism by which Hispidulin induces apoptosis in human hepatoblastoma cancer (HepG2) cells. The results showed that Hispidulin induced cell death in a dose- and time-dependent manner in HepG2 cells whereas no toxic reaction was observed in normal human liver cells at indicated concentration. This study also demonstrated that Hispidulin induces apoptosis through mitochondrial dysfunction, which is characterized by decreased Bcl-2/Bax ratio, disrupted mitochondrial membrane potential and increased release of cytochrome C and activated capase-3. Our results also showed that mitochondrial dysfunction was triggered by Hispidulin-induced excessive ROS generation. Hispidulin also significantly inhibited Akt activation. ROS inhibitor NAC abrogated the inhibitory effect of Hispidulin on P13k/Akt signalling pathway and the proapoptotic effect in HepG2 cells. Our results demonstrate for the first time that Hispidulin induces apoptosis in HepG2 cells and suggested that the pro-apoptotic effect of Hispidulin was mediated through mitochondrial dysfunction and inhibition of P13k/Akt signalling pathway. Since no toxic effect was observed when normal liver cells were treated with Hispidulin, Hispidulin may have the potential to be used as therapeutic for liver cancer.     

Glucocorticoids Suppress Mitochondrial Oxidant Production via Upregulation of Uncoupling Protein 2 in Hyperglycemic Endothelial Cells

Diabetic complications are the leading cause of morbidity and mortality in diabetic patients. Elevated blood glucose contributes to the development of endothelial and vascular dysfunction, and, consequently, to diabetic micro- and macrovascular complications, because it increases the mitochondrial proton gradient and mitochondrial oxidant production. Therapeutic approaches designed to counteract glucose-induced mitochondrial reactive oxygen species (ROS) production in the vasculature are expected to show efficacy against all diabetic complications, but direct pharmacological targeting (scavenging) of mitochondrial oxidants remains challenging due to the high reactivity of some of these oxidant species. In a recent study, we have conducted a medium-throughput cell-based screening of a focused library of well-annotated pharmacologically active compounds and identified glucocorticoids as inhibitors of mitochondrial superoxide production in microvascular endothelial cells exposed to elevated extracellular glucose. The goal of the current study was to investigate the mechanism of glucocorticoids'' action. Our findings show that glucocorticoids induce the expression of the mitochondrial UCP2 protein and decrease the mitochondrial potential. UCP2 silencing prevents the protective effect of the glucocorticoids on ROS production. UCP2 induction also increases the oxygen consumption and the “proton leak” in microvascular endothelial cells. Furthermore, glutamine supplementation augments the effect of glucocorticoids via further enhancing the expression of UCP2 at the translational level. We conclude that UCP2 induction represents a novel experimental therapeutic intervention in diabetic vascular complications. While direct repurposing of glucocorticoids may not be possible for the therapy of diabetic complications due to their significant side effects that develop during chronic administration, the UCP2 pathway may be therapeutically targetable by other, glucocorticoid-independent pharmacological means.     

Mitochondrial reactive oxygen species affect sensitivity to curcumin-induced apoptosis

Curcumin exhibits anticancer activity in vivo and triggers tumor cell apoptosis in vivo and in vitro. Several in vitro studies suggest that curcumin-induced apoptosis is associated with reactive oxygen species (ROS) production and/or oxidative stress in transformed cells. This study compared and contrasted the effects of curcumin on human skin cancer cells and their respiration-deficient (rho0) clones to characterize the prospective oxidative stress signaling responsible for initiating apoptosis. Curcumin promoted a dose-and time-dependent G2/M cell cycle arrest and/or apoptosis in COLO 16 cells. Apoptosis induction in COLO 16 cells was associated with DNA fragmentation, cell shrinkage, the externalization of cell membrane phosphatidylserine, and mitochondrial disruption, which were preceded by an increase in intracellular ROS production. Pharmacologically lowering the mitochondrial bioenergetic capacity, as well as the constitutive ROS levels, in COLO 16 cells suppressed the cytotoxic effects of curcumin. Correspondingly, the rho0 counterparts of COLO 16 cells were markedly resistant to ROS production, mitochondrial disruption, and DNA fragmentation following curcumin exposure. These observations implied that the diminution of mitochondrial ROS production protected cells against the cytotoxic effects of curcumin, and support the notion that mitochondrial respiration and redox tone are pivotal determinants in apoptosis signaling by curcumin in human skin cancer cells.     

Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis

Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.     

Protein kinase C-ERK1/2 signal pathway switches glucose depletion-induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by macrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N-acetyl-L-cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase-9 and caspase-3 activation, indicating a critical role of ROS in determination of GD-induced cell death mode. We demonstrate that protein kinase C-dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD-induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD-induced degradation of copper zinc SOD. Thus, these results suggest that GD-induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis-to-apoptosis switch.     

Notoginsenoside R1 attenuates oxidative stress-induced osteoblast dysfunction through JNK signalling pathway

Oxidative stress (OS)-induced mitochondrial damage and the subsequent osteoblast dysfunction contributes to the initiation and progression of osteoporosis. Notoginsenoside R1 (NGR1), isolated from Panax notoginseng, has potent antioxidant effects and has been widely used in traditional Chinese medicine. This study aimed to investigate the protective property and mechanism of NGR1 on oxidative-damaged osteoblast. Osteoblastic MC3T3-E1 cells were pretreated with NGR1 24 h before hydrogen peroxide administration simulating OS attack. Cell viability, apoptosis rate, osteogenic activity and markers of mitochondrial function were examined. The role of C-Jun N-terminal kinase (JNK) signalling pathway on oxidative injured osteoblast and mitochondrial function was also detected. Our data indicate that NGR1 (25 μM) could reduce apoptosis as well as restore osteoblast viability and osteogenic differentiation. NGR1 also reduced OS-induced mitochondrial ROS and restored mitochondrial membrane potential, adenosine triphosphate production and mitochondrial DNA copy number. NGR1 could block JNK pathway and antagonize the destructive effects of OS. JNK inhibitor (SP600125) mimicked the protective effects of NGR1while JNK agonist (Anisomycin) abolished it. These data indicated that NGR1 could significantly attenuate OS-induced mitochondrial damage and restore osteogenic differentiation of osteoblast via suppressing JNK signalling pathway activation, thus becoming a promising agent in treating osteoporosis.     

Oxidative stress and mitochondrial dysfunction play a role in myelodysplastic syndrome development,diagnosis, and prognosis: A pilot study

Abstract

The imbalance between reactive oxygen species (ROS) production and their elimination by antioxidants leads to oxidative stress. Depending on their concentration, ROS can trigger apoptosis or stimulate cell proliferation. We hypothesized that oxidative stress and mitochondrial dysfunction may participate not only in apoptosis detected in some myelodysplastic syndrome (MDS) patients, but also in increasing proliferation in other patients. We investigated the involvement of oxidative stress and mitochondrial dysfunction in MDS pathogenesis, as well as assessed their diagnostic and prognostic values. Intracellular peroxides, superoxide, superoxide/peroxides ratio, reduced glutathione (GSH), and mitochondrial membrane potential (Δψ_mit) levels were analyzed in bone marrow cells from 27 MDS patients and 12 controls, by flow cytometry. We observed that all bone marrow cell types from MDS patients had increased intracellular peroxide levels and decreased GSH content, compared with control cells. Moreover, oxidative stress levels were MDS subtype— and risk group—dependent. Low-risk patients had the highest ROS levels, which can be related with their high apoptosis; and intermediate-2-risk patients had high Δψ_mit that may be associated with their proliferative potential. GSH levels were negatively correlated with transfusion dependency, and peroxide levels were positively correlated with serum ferritin level. GSH content proved to be an accurate parameter to discriminate patients from controls. Finally, patients with high ROS or low GSH levels, as well as high superoxide/peroxides ratio had lower overall survival. Our results suggest that oxidative stress and mitochondrial dysfunction are involved in MDS development, and that oxidative stress parameters may constitute novel diagnosis and/or prognosis biomarkers for MDS.     

Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated low-density lipoprotein

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD⁺/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.     

High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria

Tungstate (W) is recognized as an agent of environmental pollution and a substitute to depleted uranium. According to some preliminary studies, tungstate toxicity is related to the formation of reactive oxygen species (ROS) under abnormal pathological conditions. The kidneys and liver are the main tungstate accumulation sites and important targets of tungstate toxicity. Since the mitochondrion is the main ROS production site, we evaluated the mechanistic toxicity of tungstate in isolated mitochondria for the first time, following a two‐step ultracentrifugation method. Our findings demonstrated that tungstate‐induced mitochondrial dysfunction is related to the increased formation of ROS, lipid peroxidation, and potential membrane collapse, correlated with the amelioration of adenosine triphosphate and glutathione contents. The present study indicated that mitochondrial dysfunction was associated with disruptive effects on the mitochondrial respiratory chain and opening of mitochondrial permeability transition (MPT) pores, which is correlated with cytochrome c release. Our findings suggest that high concentrations of tungstate (2 mM)‐favored MPT pore opening in the inner membranes of liver and kidney mitochondria of rats. Besides, the results indicated higher tungstate susceptibility in the kidneys, compared with the liver.     

Different influences on mitochondrial function,oxidative stress and cytotoxicity of antibiotics on primary human neuron and cell lines

Although antibiotics are generally well tolerated, their toxic effects on the central nervous system have been gained attention. In this study, we systematically investigated the neuron toxicity of antibiotics from six different classes. We show that clinically relevant concentrations of metronidazole, tigecycline, azithromycin and clindamycin but not ampicillin or sulfamethoxazole induce apoptosis of human primary neuron cells and lines. Notably, tigecycline, azithromycin and clindamycin cause neuron cell oxidative damage whereas metronidazole has no effect on reactive oxygen species (ROS) production, suggesting that metronidazole induces neuron death via ROS‐independent mechanism. Tigecycline, azithromycin and clindamycin induce mitochondrial dysfunctions via targeting different mitochondrial respiratory complexes, leading to mitochondrial membrane potential disruption and energy crisis. The deleterious effects of antibiotics are reversed by pretreatment of neuron cells with antioxidant. Our work highlights the different influences of antibiotics on mitochondrial dysfunction, oxidative damage and cytotoxicity in neuron cells. We also provide a strategy to prevent the neurotoxicity.     

Reactive oxygen species in choline deficiency-induced apoptosis in rat hepatocytes

Choline deficiency (CD) is involved in hepatocellular carcinoma and CD-induced apoptosis may be implicated in cellular malignant transformation. In this report, we studied the effects of choline deficiency on generation of reactive oxygen species (ROS) using the fluorescent probe dichlorodihydrofluorescein diacetate and the possible role of ROS on CD-induced apoptosis in cultured CWSV-1 cells, an immortalized rat hepatocyte. This cell line is reported to become tumorigenic by step-wise culturing in lower levels of choline. Our data demonstrate that CD induces a time- and dose-dependent increase in ROS in CWSV-1 cells. The increase in ROS production may be related to dysfunction of the mitochondrial respiratory chain. Our data also demonstrated that ROS generation occurred before CD-induced apoptosis, suggesting ROS may play a key role in signaling CD-induced apoptosis in CWSV-1 cells.     

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.     

The presence of Estrogen Receptor β modulates the response of breast cancer cells to therapeutic agents

Breast cancer is a leading cause of death for women. The estrogen receptors (ERs) ratio is important in the maintenance of mitochondrial redox status, and higher levels of ERβ increases mitochondrial functionality, decreasing ROS production. Our aim was to determine the interaction between the ERα/ERβ ratio and the response to cytotoxic treatments such as cisplatin (CDDP), paclitaxel (PTX) and tamoxifen (TAM). Cell viability, apoptosis, autophagy, ROS production, mitochondrial membrane potential, mitochondrial mass and mitochondrial functionality were analyzed in MCF-7 (high ERα/ERβ ratio) and T47D (low ERα/ERβ ratio) breast cancer cell lines. Cell viability decreased more in MCF-7 when treated with CDDP and PTX. Apoptosis was less activated after cytotoxic treatments in T47D than in MCF-7 cells. Nevertheless, autophagy was increased more in CDDP-treated MCF-7, but less in TAM-treated cells than in T47D. CDDP treatment produced a raise in mitochondrial mass in MCF-7, as well as the citochrome c oxidase (COX) and ATP synthase protein levels, however significantly reduced COX activity. In CDDP-treated cells, the overexpression of ERβ in MCF-7 caused a reduction in apoptosis, autophagy and ROS production, leading to higher cell survival; and the silencing of ERβ in T47D cells promoted the opposite effects. In TAM-treated cells, ERβ-overexpression led to less cell viability by an increment in autophagy; and the partial knockdown of ERβ in T47D triggered an increase in ROS production and apoptosis, leading to cell death. In conclusion, ERβ expression plays an important role in the response of cancer cells to cytotoxic agents, especially for cisplatin treatment.     

TNF-alpha/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

Previously we have shown that both Rac1 and c-Jun NH(2)-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-alpha/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-alpha-induced apoptosis. In this study, we showed that TNF-alpha/CHX-induced ROS production and hydrogen peroxide (H(2)O(2))-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-alpha-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-alpha-induced mitochondrial ROS production. Moreover, TNF-alpha-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-alpha-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-alpha-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.     

Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4 mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca²⁺) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca²⁺ and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine (PS) externalization, nuclear condensation, and DNA fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca²⁺ and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections.     

Intracellular glutathione depletion and reactive oxygen species generation are important in α-hederin-induced apoptosis of P388 cells

alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.