Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.     

The decrease of mitochondrial NADH dehydrogenease and drug induced apoptosis in doxorubicin resistant A431 cells

Doxorubicin (DOX) resistant A10A cells derived from human squamous carcinoma A431 cells were found to exhibit a smaller degree of apoptosis after DOX treatment as compared to their parent cells. Induction of reactive oxygen species (ROS) formation and mitochondrial depolarization by DOX were more pronounced in the parent cells than in the A10A cells. The fact that catalase suppressed the DOX effect on ROS induction, mitochondrial depolarization and apoptosis in both cell lines suggests an involvement of ROS in the DOX-induced apoptosis. To investigate the underlying mechanisms for DOX resistance in A10A cells, RT-PCR based differential display was used. One of the clones, which was down-regulated in the A10A cells, had sequence homology with part of the mitochondrial NADH dehydrogenase III (ND3) gene. NADH dehydrogenase plays an important role in generating ROS during DOX treatment. The results indicate that down-regulation of ND3 may at least in part contribute to the mechanism for A10A cells resistant to DOX-induced apoptosis.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

Mitochondrial ROS‐induced ERK1/2 activation and HSF2‐mediated AT1R upregulation are required for doxorubicin‐induced cardiotoxicity

Doxorubicin (DOX), one useful chemotherapeutic agent, is limited in clinical use because of its serious cardiotoxicity. Growing evidence suggests that angiotensin receptor blockers (ARBs) have cardioprotective effects in DOX‐induced cardiomyopathy. However, the detailed mechanisms underlying the action of ARBs on the prevention of DOX‐induced cardiomyocyte cell death have yet to be investigated. Our results showed that angiotensin II receptor type I (AT₁R) plays a critical role in DOX‐induced cardiomyocyte apoptosis. We found that MAPK signaling pathways, especially ERK1/2, participated in modulating AT₁R gene expression through DOX‐induced mitochondrial ROS release. These results showed that several potential heat shock binding elements (HSE), which can be recognized by heat shock factors (HSFs), located at the AT₁R promoter region. HSF2 markedly translocated from the cytoplasm to the nucleus when cardiomyocytes were damaged by DOX. Furthermore, the DNA binding activity of HSF2 was enhanced by DOX via deSUMOylation. Overexpression of HSF2 enhanced DOX‐induced cardiomyocyte cell death as well. Taken together, we found that DOX induced mitochondrial ROS release to activate ERK‐mediated HSF2 nuclear translocation and AT₁R upregulation causing DOX‐damaged heart failure in vitro and in vivo.     

Chemotherapy-induced acute vascular injury involves intracellular generation of ROS via activation of the acid sphingomyelinase pathway

Several categories of chemotherapy confer substantial risk for late-term vascular morbidity and mortality. In the present study, we aimed to investigate the mechanism of acute chemotherapy-induced vascular injury in normal tissues. Specifically, we looked at activation of the acid sphingomyelinase (ASMase)/ceramide pathway, which leads to generation of reactive oxygen species (ROS) and induction of oxidative stress that may result in vascular injury.In particular, we focused on two distinct drugs, doxorubicin (DOX) and cisplatin (CIS) and their effects on normal endothelial cells. In vitro, DOX resulted in increased ASMase activity, intra-cellular ROS production and induction of apoptosis. CIS treatment generated significantly reduced effects in endothelial cells. In-vivo, murine femoral arterial blood flow was measured in real-time, during and after DOX or CIS administration, using fluorescence optical imaging system. While DOX caused constriction of small vessels and disintegration of large vessels' wall, CIS induced minor vascular changes in arterial blood flow, correlating with the in vitro findings. These results demonstrate that DOX induces acute vascular injury by increased ROS production, via activation of ASMase/ceramide pathway, while CIS increases ROS production and its immediate extracellular translocation, without causing detectable acute vascular injury. Our findings may potentially lead to the development of new strategies to prevent long-term cardiovascular morbidity in cancer survivors.     

熊去氧胆酸对阿霉素诱导的H9c2心肌细胞的影响及机制研究

目的:探讨熊去氧胆酸(UDCA)对阿霉素(DOX)诱导的H9c2心肌细胞损伤的影响及机制.方法:体外培养H9c2细胞,1 μMDOX和不同浓度UDCA处理H9c2,CCK-8法测定细胞活力;实时定量聚合酶链反应检测心肌细胞凋亡分子Bax及炎症因子IL-1β、IL-6的表达;Western blotting检测UDCA对...     

Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction

Mitochondria are the most important sensor for apoptosis. Extracellular adenosine is well reported to induce apoptosis of tumor cells. Here we found that extracellular adenosine suppresses the cell growth by induction of apoptosis in BEL-7404 liver cancer cells, and identified a novel mechanism that extracellular adenosine triggers apoptosis by increasing Reactive Oxygen Species (ROS) production and mitochondrial membrane dysfunction in the cells. We observed that adenosine increases ROS production, activates c-Caspase-8 and -9 and Caspase effectors, c-Caspase-3 and c-PARP, induces accumulation of apoptosis regulator Bak, decreases Bcl-xL and Mcl-1, and causes the mitochondrial membrane dysfunction and the release of DIABLO, Cytochrome C, and AIF from mitochondria to cytoplasm in the cells; ROS inhibitor, NAC significantly reduces adenosine-induced ROS production; it also shows the same degree of blocking adenosine-induced loss of mitochondrial membrane potential (MMP) and apoptosis. Our study first observed that adenosine increases ROS production in tumor cells and identified the positive feedback loop for ROS-mediated mitochondrial membrane dysfunction which amplifies the death signals in the cells. Our findings indicated ROS production and mitochondrial dysfunction play a key role in adenosine-induced apoptosis of 7404 cells.     

The influence of calcium and reactive oxygen species on influenza virus-induced apoptosis

In previous studies we observed that influenza A and B viruses induce apoptosis in Madin-Darby canine kidney (MDCK) cells and that this apoptosis is blocked by expression of bcl-2. Using a well-characterized, highly virulent, avian influenza virus, A/Turkey/Ontario/7732/66 (H5N9) (Ty/Ont), we sought to better understand this system. We investigated the influence of two cellular factors that are known to function in other models of apoptosis inhibited by bcl-2, calcium (Ca(2+)) and reactive oxygen species (ROS). Although Ca(2+) chelators generally inhibit apoptosis, treatment of MDCK cells with either an extracellular chelator, EGTA, or an intracellular chelator, BAPTAAM, induced apoptosis instead and enhanced Ty/Ont-induced apoptosis. Conversely, treatment with an ionophore, ionomycin, blocked the viral-induced apoptosis. In terms of ROS, neither treatment with antioxidants, N(2) flushing to induce hypoxia, nor nigericin (a compound which, like bcl-2, stabilizes the mitochondrial membrane potential against the effects of ROS and subsequent Ca(2+) dysregulation) were able to block Ty/Ont-induced apoptosis. Therefore, it is likely that ROS play little, if any, role in influenza-induced apoptosis in MDCK cells and the influence of Ca(2+) appears to be opposite to that in the majority of other more classical models of apoptosis.     

Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and -independent apoptosis in L929 cells

Evidence that curcumin may have anticancer activities has renewed interest in its potential to prevent and treat disease. In this study, we show that curcumin-mediated rapid generation of reactive oxygen species (ROS) leads to apoptosis by modulating different apoptotic pathways in mouse fibroblast L929 cells. We show for the first time that curcumin-induced rapid ROS generation causes the release of apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus, hence, leading to caspase 3-independent apoptosis. However, our studies also show that curcumin induces the release of cytochrome c from mitochondria, causing activation of caspase 3, and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Furthermore, curcumin-induced ROS generation leads to the induction of the proapoptotic protein p53 and its effector protein p21 and down-regulation of cell cycle regulatory proteins such as Rb and cyclin D1 and D3. Both glutathione (GSH) and N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of curcumin-induced ROS generation, AIF release from mitochondria, and caspase activation. Additionally, pretreatment of L929 cells with these antioxidants completely blocked the induction of p53-dependent p21 accumulation. In conclusion, our data show that in addition to caspase 3 activation, curcumin-induced rapid ROS generation leads to AIF release, and the activation of the caspase-independent apoptotic pathway.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Identification of a novel mechanism for reversal of doxorubicin-induced chemotherapy resistance by TXNIP in triple-negative breast cancer via promoting reactive oxygen-mediated DNA damage

Given that triple-negative breast cancer (TNBC) lacks specific receptors (estrogen and progesterone receptors and human epidermal growth factor receptor 2) and cannot be treated with endocrine therapy, chemotherapy has remained the mainstay of treatment. Drug resistance is reportedly the main obstacle to the clinical use of doxorubicin (DOX) in this patient population. Accordingly, screening molecules related to chemoresistance and studying their specific mechanisms has clinical significance for improving the efficacy of chemotherapy in TNBC patients. Thioredoxin-interacting protein (TXNIP) is a metabolism-related protein that plays a tumor suppressor role in various malignant tumors; however, the specific role of TXNIP in tumor chemoresistance has not been reported. In the present study, we explored the potential molecular mechanism of TXNIP in the chemoresistance of TNBC for the first time. The results showed that TXNIP inhibited the proliferation of TNBC drug-resistant cells and promoted apoptosis in vitro and in vivo. Furthermore, TXNIP promoted the synthesis of reactive oxygen species (ROS) and the accumulation of DNA damage caused by DOX and increased γ-H2AX levels in a time and dose-dependent manner. Moreover, ROS scavenger pretreatment could block DNA damage induced by TXNIP and restore the resistance of TNBC resistant cells to DOX to a certain extent. In addition, we found that the small molecule c-Myc inhibitor 10058-F4 promoted TXNIP expression, increased ROS synthesis in cells, and could enhance the cytotoxicity of chemotherapy drugs in vitro and in vivo when combined with DOX. These results indicated that c-Myc inhibitor 10058-F4 could induce TXNIP upregulation in TNBC drug-resistant cells, and the upregulated TXNIP increased the accumulation of ROS-dependent DNA damage, thereby decreasing chemotherapy resistance of TNBC. Our findings reveal a new mechanism of mediating drug resistance and provide a new drug combination strategy to overcome DOX resistance in TNBC.Subject terms: Breast cancer, Cancer therapeutic resistance     

Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis

In the past, investigators have successfully used iron chelators to mitigate the cardiotoxicity of doxorubicin (DOX), a widely used anticancer drug that induces reactive oxygen species (ROS), oxidative damage, and apoptosis. Although intracellular iron plays a critical role in initiating DOX-induced apoptosis, the molecular mechanism(s) that link iron, ROS, and apoptosis are still unknown. In this study, we demonstrate that apoptosis results from the exposure of bovine aortic endothelial cells to DOX and that the apoptotic cell death is accompanied by a significant increase in cellular iron ((55)Fe) uptake and activation of iron regulatory protein-1. Furthermore, DOX-induced iron uptake was shown to be mediated by the transferrin receptor (TfR)-dependent mechanism. Treatment with the anti-TfR antibody (IgA class) dramatically inhibited DOX-induced apoptosis, iron uptake, and intracellular oxidant formation as measured by fluorescence using dichlorodihydrofluorescein. Treatment with cell-permeable iron chelators and ROS scavengers inhibited DOX-induced cellular (55)Fe uptake, ROS formation, and apoptosis. Based on these findings, we conclude that DOX-induced iron signaling is regulated by the cell surface TfR expression, intracellular oxidant levels, and iron regulatory proteins. The implications of TfR-dependent iron transport in oxidant-induced apoptosis in endothelial cells are discussed.     

Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Noxa expression through the formation of S-nitrosohomocysteine

Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.     

A high-fat, refined-carbohydrate diet affects renal NO synthase protein expression and salt sensitivity.

Chronic consumption of a high-fat, refined-carbohydrate (HFS) diet causes hypertension. In an earlier study, we found increased nitric oxide (NO) inactivation by reactive oxygen species (ROS) and functional NO deficiency in this model. Given the critical role of NO in renal sodium handling, we hypothesized that diet-induced hypertension may be associated with salt sensitivity. Female Fischer rats were fed an HFS or a standard low-fat, complex-carbohydrate (LFCC) rat chow diet starting at 2 mo of age for 2 yr. Arterial blood pressure, renal neuronal NO synthase (nNOS), endothelial NO synthase (eNOS), and inducible NO synthase (iNOS) protein and nitrotyrosine abundance (a marker of NO inactivation by ROS), and urinary NO metabolite excretion were measured. To assess salt sensitivity, the blood pressure response to a high-salt (4%) diet for 1 wk was determined. After 2 yr, renal nNOS and urinary NO metabolite excretion were significantly depressed, whereas arterial pressure, eNOS, iNOS, and nitrotyrosine were elevated in the HFS group but remained virtually unchanged in the LFCC group. Consumption of the high-salt diet resulted in a significant rise in arterial pressure in the HFS, but not in the LFCC, group. Thus chronic consumption of an HFS diet results in hypertension and salt sensitivity, which may be in part due to a combination of ROS-mediated NO inactivation and depressed renal nNOS protein expression.