Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.     

N-acetylcysteine improves in vitro the function of macrophages from mice with endotoxin-induced oxidative stress

Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells cause the oxidative stress involved in septic shock induced by endotoxin. This oxidative stress can be controlled to a certain degree by antioxidants, which is specially important for a type of immune cell, i.e. the phagocyte, that uses ROS to kill microorganisms and needs antioxidants in order to support its functions. In a previous study we have observed changes in several functions of peritoneal macrophages from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. N-acetylcysteine (NAC) is a thiolic antioxidant that improves the immune response, and we have observed that when administered intraperitoneally (150 mg/kg) at 30 min after LPS injection it counteracts the effects of LPS on macrophages and lymphocytes. In the present work, we have studied the in vitro effect of several concentrations of NAC (0.001, 0.01, 0.1, 1 and 2.5 mM) on the following functions: adherence to substrate, chemotaxis, ingestion of particles, ROS production and the release of tumor necrosis factor (TNFalpha) of peritoneal macrophages from BALB/c mice at 2, 4,12 and 24 h after LPS injection. The results show that the administration of NAC (especially at 0.1 mM) decreases raised adherence, ingestion, ROS production and TNFalpha levels in macrophages from animals injected with LPS, bringing these functions to values near those of control animals. These effects which seem to be linked to a modulation of NF-kappaB, suggest that the improvement of immune functions observed in previous work after injection of NAC to animals with endotoxic shock could be due to a direct action of this thiol antioxidant on immune cells.     

Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages

The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.     

Lycopene prevents 7-ketocholesterol-induced oxidative stress,cell cycle arrest and apoptosis in human macrophages

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10–25 μM) alone and in combination with lycopene (0.5–2 μM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, β-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than β-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.     

TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-α signaling and ROS production

Background

Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a key regulator of signal cascades of TNF-α receptor and TLR4, and can induce NF-κB activation for preventing cell apoptosis and eliciting inflammation response.

Results

TAK1 inhibitor (TAKI) can decrease the cell viability of murine bone marrow-derived macrophages (BMDM), RAW264.7 and BV-2 cells, but not dermal microvascular endothelial cells, normal human epidermal keratinocytes, THP-1 monocytes, human retinal pigment epithelial cells, microglia CHME3 cells, and some cancer cell lines (CL1.0, HeLa and HCT116). In BMDM, TAKI-induced caspase activation and cell apoptosis were enhanced by lipopolysaccharide (LPS). Moreover, TAKI treatment increased the cytosolic and mitochondrial reactive oxygen species (ROS) production, and ROS scavengers NAC and BHA can inhibit cell death caused by TAKI. In addition, RIP1 inhibitor (necrostatin-1) can protect cells against TAKI-induced mitochondrial ROS production and cell apoptosis. We also observed the mitochondrial membrane potential loss after TAKI treatment and deterioration of oxygen consumption upon combination with LPS. Notably TNF-α neutralization antibody and inhibitor enbrel can decrease the cell death caused by TAKI.

Conclusions

TAKI-induced cytotoxicity is cell context specific, and apoptosis observed in macrophages is dependent on the constitutive autocrine action of TNF-α for RIP1 activation and ROS production.     

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.     

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.     

N-Acetyl Cysteine Depletes Reactive Oxygen Species and Prevents Dental Monomer-Induced Intrinsic Mitochondrial Apoptosis In Vitro in Human Dental Pulp Cells

Purpose

To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC) on this process.

Methods

Human dental pulp cells (hDPCs) were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.

Results

Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS) and depletion of glutathione (GSH), differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI) staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.

Conclusions

Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis.     

Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress.

Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNF alpha production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNF alpha and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.     

Essential role of STAT1 in caspase-independent cell death of activated macrophages through the p38 mitogen-activated protein kinase/STAT1/reactive oxygen species pathway

Unlike other immune cells, activation of macrophages by stimulating agents, such as lipopolysaccharide (LPS), confers significant resistance to many apoptotic stimuli, but the underlying mechanism of this phenomenon remains largely unknown. Here, we demonstrate that LPS-induced early caspase activation is essential for macrophage survival because blocking caspase activation with a pancaspase inhibitor (zVAD [benzyloxycarbonyl-Val-Ala-Asp]) rapidly induced death of activated macrophages. This type of death process by zVAD/LPS was principally mediated by intracellular generation of superoxide. STAT1 knockout macrophages demonstrated profoundly decreased superoxide production and were resistant to treatment with zVAD/LPS, indicating the crucial involvement of STAT1 in macrophage death by zVAD/LPS. STAT1 level and activity were reciprocally regulated by caspase activation and were associated with cell death. Activation of STAT1 was critically dependent upon serine phosphorylation induced by p38 mitogen-activated protein kinase (MAPK) because a p38 MAPK inhibitor nullified STAT1 serine phosphorylation, reactive oxygen species (ROS) production, and macrophage death by zVAD/LPS. Conversely, p38 MAPK activation was dependent upon superoxide and was also nullified in STAT1 knockout macrophages, probably due to impaired generation of superoxide. Our findings collectively indicate that STAT1 signaling modulates intracellular oxidative stress in activated macrophages through a positive-feedback mechanism involving the p38 MAPK/STAT1/ROS pathway, which is interrupted by caspase activation. Furthermore, our study may provide significant insights in regards to the unanticipated critical role of STAT1 in the caspase-independent death pathway.     

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.     

Induction of apoptosis by sodium selenite in human acute promyelocytic leukemia NB4 cells: involvement of oxidative stress and mitochondria.

The mechanisms involved in the anti-carcinogenic activity of selenium remained to be elucidated. In the present study, we examined sodium selenite induced apoptosis and oxidative stress in human acute promyelocytic leukemia cell lines (NB4). Cell growth and viability were assessed by trypan blue exclusion and cell counting; apoptosis by DNA electrophoresis and analysis of intracellular DNA contents; reactive oxygen species and reduced glutathione in the cell were measured by lucigenin dependent chemoluminescent (CL) test and spectrophotometer; mitochondrial transmembrane potential was measured by flow cytometry. Sodium selenite could inhibit the growth and induce apoptosis of NB4 cells. Sodium selenite could increase the production of reactive oxygen species (ROS) in NB4 cells and decrease the level of intracellular reduced glutathione, but caused no change in the activity of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx). Sodium selenite enhanced the collapse of mitochondrial transmembrane potential (MTP), in parallel with the production of ROS. Finally antioxidant N-acetylcysteine (NAC) could inhibit the ROS production, MTP collapse and apoptosis in NB4 cells. Our results suggested that sodium selenite could induce apoptosis of NB4 cells through mitochondrial change mediated by production of reactive oxygen species within the cells.     

Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress

Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNFα production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNFα and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.     

A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages

Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages.     

Serum deprivation-induced HepG2 cell death is potentiated by CYP2E1

Induction of oxidative stress plays a key role in serum deprivation-induced apoptosis. CYP2E1 plays an important role in toxicity of many chemicals and ethanol and produces oxidant stress. We investigated whether CYP2E1 expression can sensitize HepG2 cells to toxicity as a consequence of serum deprivation. The models used were HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells which do not express CYP2E1. E47 cells showed greater growth inhibition and enhanced cell death after serum deprivation, as compared to the C34 cells. DNA ladder and flow cytometry assays indicated that apoptosis occurred at earlier times after serum deprivation in E47 than C34 cells. Serum withdrawal-induced E47 cell death could be rescued by antioxidants, the mitochondrial permeability transition inhibitor cyclosporine A, z-DEVD-fmk, and a CYP2E1 inhibitor 4-methylpyrazole. Increased production of reactive oxygen species (ROS) and lipid peroxidation occurred in E47 cells after serum deprivation, and there was a corresponding decline in the E47 cell mitochondrial membrane potential and reduced glutathione (GSH) levels. We propose that the mechanism of this serum withdrawal plus CYP2E1 toxicity involves increased production of intracellular ROS, lipid peroxidation, and decline of GSH levels, which results in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of serum deprivation-induced cell death by CYP2E1 may contribute to the sensitivity of the liver to alcohol-induced ischemia and growth factor deprivation.     

Metformin prevents methylglyoxal-induced apoptosis by suppressing oxidative stress in vitro and in vivo

Methylglyoxal (MGO) is an active metabolite of glucose and plays a prominent role in the pathogenesis of diabetic vascular complications, including endothelial cell apoptosis induced by oxidative stress. Metformin (MET), a widely prescribed antidiabetic agent, appears to reduce excessive reactive oxygen species (ROS) generation and limit cell apoptosis. However, the molecular mechanisms underlying this process are still not fully elucidated. We reported here that MET prevents MGO-induced apoptosis by suppressing oxidative stress in vitro and in vivo. Protein expression and protein phosphorylation were investigated using western blotting, ELISA, and immunohistochemical staining, respectively. Cell viability and apoptosis were assessed by the MTT assay, TUNEL staining, and Annexin V-FITC and propidium iodide double staining. ROS generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Our results revealed that MET prevented MGO-induced HUVEC apoptosis, inhibited apoptosis-associated biochemical changes such as loss of MMP, the elevation of the Bax/Bcl-2 ratio, and activation of cleaved caspase-3, and attenuated MGO-induced mitochondrial morphological alterations in a dose-dependent manner. MET pretreatment also significantly suppressed MGO-stimulated ROS production, increased signaling through the ROS-mediated PI3K/Akt and Nrf2/HO-1 pathways, and markedly elevated the levels of its downstream antioxidants. Finally, similar results were obtained in vivo, and we demonstrated that MET prevented MGO-induced oxidative damage, apoptosis, and inflammation. As expected, MET reversed MGO-induced downregulation of Nrf2 and p-Akt. In addition, a PI3K inhibitor (LY-294002) and a Nrf2 inhibitor (ML385) observably attenuated the protective effects of MET on MGO-induced apoptosis and ROS generation by inhibiting the Nrf2/HO-1 pathways, while a ROS scavenger (NAC) and a permeability transition pores inhibitor (CsA) completely reversed these effects. Collectively, these findings broaden our understanding of the mechanism by which MET regulates apoptosis induced by MGO under oxidative stress conditions, with important implications regarding the potential application of MET for the treatment of diabetic vascular complications.Subject terms: Apoptosis, Stress signalling     

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.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨ_m), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨ_m loss, and apoptosis were all significantly reduced in TLR4^?/? macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.