Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase gene enhances selenite-induced apoptosis

Abstract

Selenium has been shown to play a chemopreventive role in human cancer, presumably by inducing tumour cell apoptosis. Selenite is thought to induce oxidative stress by the generation of the superoxide anion and catalysing the oxidation of thiol groups. It has previously been reported that control of the mitochondrial redox balance is a primary function of mitochon-drial NADP⁺-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. When investigating whether IDPm would be a vulnerable target of selenite, the loss of enzyme activity was observed. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm and enhanced cells’ susceptibility of selenite-induced apoptosis, as indicated by morphological evidence of apoptosis, DNA fragmentation and the modulation of mitochondrial function and apoptotic marker proteins. These results suggest that IDPm siRNA sensitizes HeLa cells to selenite-induced apoptotic cell death, presumably through the perturbation of the cellular redox status.     

Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances heat shock-induced apoptosis

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. In this report, we demonstrate that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by heat shock. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of heat shock-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by heat shock and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer therapy.     

Attenuated mitochondrial NADP+-dependent isocitrate dehydrogenase activity enhances EGCG-induced apoptosis

(−)-Epigallocatechin-3-gallate (EGCG), a well-known chemopreventive factor, induces cancer cells undergoing apoptosis. Over the last several years, we have shown that the mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems. Here, we show that EGCG induced the inactivation of IDPm as a purified enzyme and in cultured cancer cells in a dose- and time-dependent manner. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. In addition, transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly attenuated the activity of IDPm and substantially enhanced EGCG-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation, and the modulation of mitochondrial function and apoptotic marker proteins. Taken together, our results suggest that the suppression of IDPm activity resulted in the disruption of cellular redox balance and subsequently exacerbates EGCG-induced apoptotic cell death in HeLa cells. These results might have implications for developing an effective combination modality in cancer treatment.     

Knockdown of sensitive to apoptosis gene by small interfering RNA enhances the sensitivity of PC3 cells toward actinomycin D and etoposide

Abstract

Actinomycin D and etoposide induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Sensitive to apoptosis gene (SAG) protein, a redox inducible zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger. The present report show that knockdown of SAG expression in PC3 cells greatly enhances apoptosis induced by actinomycin D and etoposide. Transfection of human prostate cancer PC3 cells with SAG small interfering RNA (siRNA) markedly decreased the expression of SAG, enhancing the susceptibility of actinomycin D- and etoposide-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that SAG may play an important role in regulating the apoptosis induced by actinomycin D and etoposide and the sensitizing effect of SAG siRNA on the apoptotic cell death of PC3 cells offers the possibility of developing a modifier of cancer chemotherapy.     

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells. [BMB Reports 2014; 47(4): 209-214]     

Small interfering RNA-mediated silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase enhances the sensitivity of HeLa cells toward tumor necrosis factor-alpha and anticancer drugs

Tumor necrosis factor-alpha (TNF-alpha) and several anticancer drugs induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In the present report, we show that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by TNF-alpha and anticancer drugs. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of anticancer agent-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by TNF-alpha and anticancer drugs and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

Regulation of high glucose-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.     

Regulation of ethanol-induced toxicity by mitochondrial NADP-dependent isocitrate dehydrogenase

Chronic alcohol administration has been known to increase peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. We previously reported that control of the mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) through to supply NADPH for antioxidant systems. In the present study, we demonstrate that modulation of IDPm expression in HepG2 cells regulates ethanol-induced toxicity. We observed the significantly enhanced protection to cell killing, lipid peroxidation, protein oxidation, oxidative DNA damage, and decrease in generation of intracellular reactive oxygen species and reactive nitrogen species in IDPm-overexpressed cells compared to control cells upon exposure to ethanol. In contrast, transfection of HepG2 cells with IDPm short interfering RNA markedly decreased the expression of IDPm, modulating cellular redox status and subsequently enhancing the susceptibility of ethanol-induced toxicity. These studies support the hypothesis that IDPm plays an important role in regulating the toxicity induced by ethanol presumably through maintaining the cellular redox status.     

Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.     

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase

Singlet oxygen ( 1 O 2 ) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.     

Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.     

RNA interference targeting cytosolic NADP+-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome.     

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.     

Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.     

Cellular defense against heat shock-induced oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of IDPm against heat shock in HEK293 cells, an embryonic kidney cell line, was investigated in control and cells transfected with the cDNA for IDPm, where IDPm activity was 6–7 fold higher than that in the control cells carrying the vector alone. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in control cells as compared to HEK293 cells in which IDPm was over-expressed. We also observed the significant difference in the cellular redox status reflected by the endogenous production of reactive oxygen species, NADPH pool and GSH recycling between two cells. The results suggest that IDPm plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of reactive oxygen species.     

Modulation of NADP+-dependent isocitrate dehydrogenase in aging

Abstract

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP⁺-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46–48 population doubling level (PDL) and then gradually decreased at later PDL. 2′,7′-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.     

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP₊-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Inactivation of mitochondrial NADP+-dependent isocitrate dehydrogenase by hypochlorous acid

Myeoloperoxidase catalyses the formation of hypochlorous acid (HOCl) via reaction of H(2)O(2) with Cl(-) ion. Although HOCl is known to play a major role in the human immune system by killing bacteria and other invading pathogens, excessive generation of this oxidant is known to cause damage to tissue. Recently, it was demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. This study investigated whether the IDPm would be a vulnerable target of HOCl as a purified enzyme and in intact cells. Loss of enzyme activity was observed and the inactivation of IDPm was reversed by thiols. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly enhanced HOCl-induced oxidative damage to cells. The HOCl-mediated damage to IDPm may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.