Autophagy induction as an efficient strategy to eradicate tumors

The understanding of the mechanisms of cell-death execution and the role that they play in different diseases opens new therapeutic strategies. Currently, increasing evidence indicates that autophagy is a frequent cell-death mechanism, so the question arises: Could autophagy stimulation be considered an antitumor therapy? Several autophagy inducers have been used as anticancer agents and, although complete tumor eradication has not been demonstrated, the antitumor effect is very promising. We have recently demonstrated that strong autophagy stimulation mediated by the combined generation of cyanide and oxidative stress could efficiently suppress tumor growth in an aggressive brain cancer model such as glioblastoma. We have used the plant enzyme linamarase, which metabolizes the innocuous substrate linamarin to generate cyanide in a continuous and controlled way inducing mitochondrial fragmentation. Glucose oxidase addition induces oxidative stress that increases cell vacuolization. The combination of both insults favors mitochondrial engulfment by vacuoles accelerating cell death that is mediated by autophagy.     

Therapy mediated by mitophagy abrogates tumor progression

Autophagy is mainly a cellular recycling process that promotes survival, but it can also cause cell death if cell injury persists. The role of mitophagy in tumorigenesis remains uncertain. Other cell death types, such as apoptosis or necrosis, are often altered during tumor development and therefore are not ideal targets to generate efficient antitumor therapies. We have used the system linamarase/linamarin/glucose oxidase (lis/lin/GO) to eliminate tumor cells. This therapeutic strategy is based on the combination of cyanide and oxidative stress to abrogate tumor growth. After severe mitochondrial insult by lis/lin/GO, the electron transport chain is blocked and hydrogen peroxide production increased. This triggers a degradative phase of these damaged organelles inducing mitophagy that finally leads to cell death. This death process depends on the vacuole generation, BNIp3 and the formation of autolysosomes. Importantly, evasion of apoptosis is known to result in resistance to anti-cancer therapies but this inhibition also augments sensitivity to autophagy, which could be used to promote tumor regression. We explored the potential of this powerful mitophagy-inducing system in vitro and in vivo to eradicate human malignant tumors.     

Therapy mediated by mitophagy abrogates tumor progression

Autophagy is mainly a cellular recycling process that promotes survival, but it can also cause cell death if cell injury persists. The role of mitophagy in tumorigenesis remains uncertain. Other cell death types, such as apoptosis or necrosis, are often altered during tumor development and therefore are not ideal targets to generate efficient antitumor therapies. We have used the system linamarase/linamarin/glucose oxidase (lis/lin/GO) to eliminate tumor cells. This therapeutic strategy is based on the combination of cyanide and oxidative stress to abrogate tumor growth. After severe mitochondrial insult by lis/lin/GO, the electron transport chain is blocked and hydrogen peroxide production increased. This triggers a degradative phase of these damaged organelles inducing mitophagy that finally leads to cell death. This death process depends on the vacuole generation, BNIp3 and the formation of autolysosomes. Importantly, evasion of apoptosis is known to result in resistance to anti-cancer therapies but this inhibition also augments sensitivity to autophagy, which could be used to promote tumor regression. We explored the potential of this powerful mitophagy-inducing system in vitro and in vivo to eradicate human malignant tumors.     

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling

Reactive oxygen and nitrogen species change cellular responses through diverse mechanisms that are now being defined. At low levels, they are signalling molecules, and at high levels, they damage organelles, particularly the mitochondria. Oxidative damage and the associated mitochondrial dysfunction may result in energy depletion, accumulation of cytotoxic mediators and cell death. Understanding the interface between stress adaptation and cell death then is important for understanding redox biology and disease pathogenesis. Recent studies have found that one major sensor of redox signalling at this switch in cellular responses is autophagy. Autophagic activities are mediated by a complex molecular machinery including more than 30 Atg (AuTophaGy-related) proteins and 50 lysosomal hydrolases. Autophagosomes form membrane structures, sequester damaged, oxidized or dysfunctional intracellular components and organelles, and direct them to the lysosomes for degradation. This autophagic process is the sole known mechanism for mitochondrial turnover. It has been speculated that dysfunction of autophagy may result in abnormal mitochondrial function and oxidative or nitrative stress. Emerging investigations have provided new understanding of how autophagy of mitochondria (also known as mitophagy) is controlled, and the impact of autophagic dysfunction on cellular oxidative stress. The present review highlights recent studies on redox signalling in the regulation of autophagy, in the context of the basic mechanisms of mitophagy. Furthermore, we discuss the impact of autophagy on mitochondrial function and accumulation of reactive species. This is particularly relevant to degenerative diseases in which oxidative stress occurs over time, and dysfunction in both the mitochondrial and autophagic pathways play a role.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

ZX-29, a novel ALK inhibitor,induces apoptosis via ER stress in ALK rearrangement NSCLC cells and overcomes cell resistance caused by an ALK mutation

Although anaplastic lymphoma kinase (ALK) inhibitors have good clinical efficacy, the inevitable development of drug resistance is the most common obstacle to their clinical application. There is an urgent need to develop more effective and selective ALK inhibitors to overcome the problem of drug resistance. Here, we screened a series of ALK inhibitors and found that ZX-29 displayed potent cytotoxic activity against ALK rearrangement non-small cell lung cancer (NSCLC) NCI-H2228 cells. Then, we investigated the antitumor effects of ZX-29. We demonstrated that ZX-29 time- and dose-dependently inhibited the viability of NCI-H2228 cells, induced cell cycle arrest in the G1 phase, and then they subsequently progressed into cell death. The type of cell death induced by ZX-29 was apoptosis through endoplasmic reticulum (ER) stress. Interestingly, ZX-29 induced protective autophagy, and inhibiting autophagy could enhance the antitumor effect of ZX-29. Furthermore, ZX-29 suppressed tumor growth in a mouse xenograft model. More importantly, ZX-29 could overcome the drug resistance caused by the ALK G1202R mutation. In conclusion, we demonstrated that ZX-29 showed excellent anti-ALK rearrangement NSCLC activity in vitro and in vivo and overcame the drug resistance caused by an ALK mutation. Therefore, ZX-29 is a promising antitumor drug targeting ALK rearrangement or ALK G1202R mutation NSCLC.     

Compensatory mechanisms and the type of injury determine the fate of cells with impaired macroautophagy

The relationship between the degradative process of autophagy and cellular death pathways remains unclear. Macroautophagy may potentially function to prevent or promote cell death, and both effects have been reported in studies of cells with a block in macroautophagy. To better delineate the function of macroautophagy in cell death, we contrasted the responses to death stimuli in wild-type and atg5^-/- murine embryonic fibroblasts. We have reported that a knockout of the critical macroautophagy gene ATG5 sensitizes cells to death receptor ligand-induced death from Fas and tumor necrosis factor-α. Death occurs by caspase-dependent apoptosis resulting from activation of the mitochondrial death pathway. In contrast, atg5^-/- cells are more resistant to death induced by oxidative stress from menadione or UV light. This resistance was associated with an up-regulation of chaperone-mediated autophagy. Inhibition of this form of autophagy sensitizes cells to death from menadione, suggesting that the compensatory up-regulation of chaperone-mediated autophagy, and not the loss of macroautophagy, prevents death from menadione. These findings demonstrate that the effects of a loss of macroautophagy on the cellular death response differ depending on the mechanism of cellular injury and the compensatory changes in other forms of autophagy.

Addendum to: Wang Y, Singh R, Massey AC, Kane SS, Kaushik S, Grant T, Xiang Y, Cuervo AM, Czaja MJ. Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus. J Biol Chem 2008; 283:4766-77.     

L-carnitine protects C2C12 cells against mitochondrial superoxide overproduction and cell death

AIM To identify and characterize the protective effect that L-carnitine exerted against an oxidative stress in C2C12 cells.METHODS Myoblastic C2C12 cells were treated with menadione, a vitamin K analog that engenders oxidative stress, and the protective effect of L-carnitine(a nutrient involved in fatty acid metabolism and the control of the oxidative process), was assessed by monitoring various parameters related to the oxidative stress, autophagy and cell death. RESULTS Associated with its physiological function, a muscle cell metabolism is highly dependent on oxygen and may produce reactive oxygen species(ROS), especially under pathological conditions. High levels of ROS are known to induce injuries in cell structure as they interact at many levels in cell function. In C2C12 cells, a treatment with menadione induced a loss of transmembrane mitochondrial potential, an increase in mitochondrial production of ROS; it also induces autophagy and was able to provoke cell death. Pre-treatment of the cells with L-carnitine reduced ROS production, diminished autophagy and protected C2C12 cells against menadione-induced deleterious effects. CONCLUSION In conclusion, L-carnitine limits the oxidative stress in these cells and prevents cell death.     

To die or to live: the dual role of poly(ADP-ribose) polymerase-1 in autophagy and necrosis under oxidative stress and DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1), activated by DNA strand breaks, participates in the DNA repair process physiologically. Excessive activation of PARP-1 mediates necrotic cell death under the status of oxidative stress and DNA damage. However, it remains elusive whether and how PARP-1 activation is involved in autophagy and what is the function of PARP-1-mediated autophagy under oxidative stress and DNA damage. We recently demonstrate that hydrogen peroxide (H₂O₂) induces autophagy through a novel autophagy signalling mechanism linking PARP-1 activation to the LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway. Furthermore, PARP-1-mediated autophagy plays a cytoprotective role in H₂O₂-induced necrotic cell death as suppression of autophagy greatly sensitizes H2O2-induced cell death. Our study thus identifies a novel function of PARP-1 in mediating autophagy and it appears that PAPR-1 possesses a dual role in modulating necrosis and autophagy under oxidative stress and DNA damage: on the one hand, overactivation of PARP-1 leads to ATP depletion and necrotic cell death; on the other hand, PARP-1 activation promotes autophagy via the LKB1-AMPK-mTOR pathway to enhance cell survival. The cellular decision of life or death depends on the balance between autophagy and necrosis mediated by these two distinct pathways.     

RAGE regulates autophagy and apoptosis following oxidative injury

The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes including diabetes, inflammation, and cancer. Compared with apoptosis ("programmed cell death"), autophagy is a genetically programmed, evolutionarily conserved cell survival process that degrades long-lived cellular proteins and organelles ("programmed cell survival"). Recently we reported that RAGE is an important regulator of oxidative stress in pancreatic cancer cells. Upregulation of RAGE expression by the nuclear factor (NF)-κB pathway decreases reactive oxygen species (ROS)-induced oxidative injury. In contrast, suppression of RAGE expression increases pancreatic tumor cell sensitivity to oxidative injury. Furthermore, RAGE is a positive regulator of autophagy, and negative regulator of apoptosis during oxidative stress. These findings provide insight into how crosstalk between apoptosis and autophagy is mediated via ROS signaling with a process involving RAGE.     

Geraniol Protects Against the Protein and Oxidative Stress Induced by Rotenone in an In Vitro Model of Parkinson’s Disease

Dysfunction of autophagy, mitochondrial dynamics and endoplasmic reticulum (ER) stress are currently considered as major contributing factors in the pathogenesis of Parkinson’s disease (PD). Accumulation of oxidatively damaged cytoplasmic organelles and unfolded proteins in the lumen of the ER causes ER stress and it is associated with dopaminergic cell death in PD. Rotenone is a pesticide that selectively kills dopaminergic neurons by a variety of mechanism, has been implicated in PD. Geraniol (GE; 3,7-dimethylocta-trans-2,6-dien-1-ol) is an acyclic monoterpene alcohol occurring in the essential oils of several aromatic plants. In this study, we investigated the protective effect of GE on rotenone-induced mitochondrial dysfunction dependent oxidative stress leads to cell death in SK-N-SH cells. In addition, we assessed the involvement of GE on rotenone-induced dysfunction in autophagy machinery via α-synuclein accumulation induced ER stress. We found that pre-treatment of GE enhanced cell viability, ameliorated intracellular redox, preserved mitochondrial membrane potential and improves the level of mitochondrial complex-1 in rotenone treated SK-N-SH cells. Furthermore, GE diminishes autophagy flux by reduced autophagy markers, and decreases ER stress by reducing α-synuclein expression in SK-N-SH cells. Our results demonstrate that GE possess its neuroprotective effect via reduced rotenone-induced oxidative stress by enhanced antioxidant status and maintain mitochondrial function. Furthermore, GE reduced ER stress and improved autophagy flux in the neuroblastomal SK-N-SH cells. The present study could suggest that GE a novel therapeutic avenue for clinical intervention in neurodegenerative diseases especially for PD.     

A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress

Under oxidative stress, poly(ADP-ribose) polymerase-1 (PARP-1) is activated and contributes to necrotic cell death through ATP depletion. On the other hand, oxidative stress is known to stimulate autophagy, and autophagy may act as either a cell death or cell survival mechanism. This study aims to explore the regulatory role of PARP-1 in oxidative stress-mediated autophagy and necrotic cell death. Here, we first show that hydrogen peroxide (H(2)O(2)) induces necrotic cell death in Bax-/- Bak-/- mouse embryonic fibroblasts through a mechanism involving PARP-1 activation and ATP depletion. Next, we provide evidence that autophagy is activated in cells exposed to H(2)O(2). More importantly, we identify a novel autophagy signaling mechanism linking PARP-1 to the serine/threonine protein kinase LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, leading to stimulation of autophagy. Finally, we demonstrate that autophagy plays a cytoprotective role in H(2)O(2)-induced necrotic cell death, as suppression of autophagy by knockdown of autophagy-related gene ATG5 or ATG7 greatly sensitizes H(2)O(2)-induced cell death. Taken together, these findings demonstrate a novel function of PARP-1: promotion of autophagy through the LKB1-AMPK-mTOR pathway to enhance cell survival in cells under oxidative stress.     

Mito-Tempol and Dexrazoxane Exhibit Cardioprotective and Chemotherapeutic Effects through Specific Protein Oxidation and Autophagy in a Syngeneic Breast Tumor Preclinical Model

Several front-line chemotherapeutics cause mitochondria-derived, oxidative stress-mediated cardiotoxicity. Iron chelators and other antioxidants have not completely succeeded in mitigating this effect. One hindrance to the development of cardioprotectants is the lack of physiologically-relevant animal models to simultaneously study antitumor activity and cardioprotection. Therefore, we optimized a syngeneic rat model and examined the mechanisms by which oxidative stress affects outcome. Immune-competent spontaneously hypertensive rats (SHRs) were implanted with passaged, SHR-derived, breast tumor cell line, SST-2. Tumor growth and cytokine responses (IL-1A, MCP-1, TNF-α) were observed for two weeks post-implantation. To demonstrate the utility of the SHR/SST-2 model for monitoring both anticancer efficacy and cardiotoxicity, we tested cardiotoxic doxorubicin alone and in combination with an established cardioprotectant, dexrazoxane, or a nitroxide conjugated to a triphenylphosphonium cation, Mito-Tempol (4) [Mito-T (4)]. As predicted, tumor reduction and cardiomyopathy were demonstrated by doxorubicin. We confirmed mitochondrial accumulation of Mito-T (4) in tumor and cardiac tissue. Dexrazoxane and Mito-T (4) ameliorated doxorubicin-induced cardiomyopathy without altering the antitumor activity. Both agents increased the pro-survival autophagy marker LC3-II and decreased the apoptosis marker caspase-3 in the heart, independently and in combination with doxorubicin. Histopathology and transmission electron microscopy demonstrated apoptosis, autophagy, and necrosis corresponding to cytotoxicity in the tumor and cardioprotection in the heart. Changes in serum levels of 8-oxo-dG-modified DNA and total protein carbonylation corresponded to cardioprotective activity. Finally, 2D-electrophoresis/mass spectrometry identified specific serum proteins oxidized under cardiotoxic conditions. Our results demonstrate the utility of the SHR/SST-2 model and the potential of mitochondrially-directed agents to mitigate oxidative stress-induced cardiotoxicity. Our findings also emphasize the novel role of specific protein oxidation markers and autophagic mechanisms for cardioprotection.     

Compensatory mechanisms and the type of injury determine the fate of cells with impaired macroautophagy

The relationship between the degradative process of autophagy and cellular death pathways remains unclear. Macroautophagy may potentially function to prevent or promote cell death, and both effects have been reported in studies of cells with a block in macroautophagy. To better delineate the function of macroautophagy in cell death, we contrasted the responses to death stimuli in wild-type and atg5(-/-) murine embryonic fibroblasts. We have reported that a knockout of the critical macroautophagy gene ATG5 sensitizes cells to death receptor ligand-induced death from Fas and tumor necrosis factor-alpha. Death occurs by caspase-dependent apoptosis resulting from activation of the mitochondrial death pathway. In contrast, atg5(-/-) cells are more resistant to death induced by oxidative stress from menadione or UV light. This resistance was associated with an upregulation of chaperone-mediated autophagy. Inhibition of this form of autophagy sensitizes cells to death from menadione, suggesting that the compensatory upregulation of chaperone-mediated autophagy, and not the loss of macroautophagy, prevents death from menadione. These findings demonstrate that the effects of a loss of macroautophagy on the cellular death response differ depending on the mechanism of cellular injury and the compensatory changes in other forms of autophagy.     

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia–reperfusion injury in hypertrophic heart

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91^phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3DeltaTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3DeltaTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.     

Role of mitochondrial oxidative stress in the apoptosis induced by diospyrin diethylether in human breast carcinoma (MCF-7) cells

Mitochondria and associated oxidative stress have been shown to play critical roles in apoptotic death induced by various stress agents. Previously, we reported the antitumor property of diospyrin (D1), a plant-derived bisnaphthoquinonoid, and its diethylether derivative (D7), which was found to cause apoptotic death in human cancer cell lines. The present study aims to explore the relevant mechanism of apoptosis involving generation of cellular reactive oxygen species (ROS) by D7 in human breast carcinoma (MCF-7) cells. It was found that while D7 inhibited the proliferation of tumor cells, the associated apoptosis induced by D7 was prevented by treating the cells with N-acetyl-L: -cysteine (NAC), an antioxidant, and cyclosporine A (CsA), an inhibitor of mitochondrial permeability transition (MPT). Experiments using suitable inhibitors also demonstrated that D7 could alter the electron flow in mitochondrial electron transport chain by affecting target(s) between complex I and complex III, and indicated the probable site of D7-induced generation of ROS. These results were further supported by confocal microscopic observation on changes in mitochondrial organization and shape in cells treated with D7. Taken together, the results of our study clearly suggested that the apoptosis induced by D7 would involve alteration of MPT, cardiolipin peroxidation, migration of Bax from cytosol to mitochondria, decreased expression of Bcl-2, and release of cytochrome c, indicating oxidative mechanism at the mitochondrial level in the tumor cells.