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.     

Sensitive to apoptosis gene protein regulates ionizing radiation-induced apoptosis

Organisms exposed to ionizing radiation (IR) undergo increases in the production of reactive oxygen species (ROS), which are determinant components in the induction of apoptosis. Sensitive to apoptosis gene (SAG) encodes a redox-inducible and apoptosis-protective antioxidant protein. This report demonstrates that the modulation of SAG expression in cultured cells regulates IR-induced apoptosis. A protective role for SAG against IR-induced apoptosis was found in U937 cells transfected with SAG cDNA. A significant decrease in the endogenous production of ROS was also observed in SAG over-expressing cells, compared to control cells, exposed to 2 Gy γ-irradiation. These results suggest that SAG plays an important role in regulating IR-induced apoptosis, presumably by maintaining the cellular redox status. Because SAG is over-expressed in many human cancers, targeting SAG expression may have therapeutic value in cancer treatment. Transfection of the pancreatic cancer cell line PC3 with SAG small interfering RNA markedly attenuated the expression of SAG, augmenting their susceptibility to IR-induced apoptosis. The knockdown of SAG expression by RNA interference combined with radiotherapy may be a potential method for radiosensitization.     

Oxalomalate, a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase, regulates 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. 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) by supplying NADPH for antioxidant systems. The protective role of ICDH against heat shock-induced apoptosis in U937 cells was investigated in the control and the cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to heat shock, there was a distinct difference between the control cells and the cells pre-treated with 3mM oxalomalate for 3h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. The oxalomalate pre-treated cells showed significant enhancement of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to the control cells upon exposure to heat shock. This study indicates that ICDH may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.     

Regulation of nitric oxide-induced apoptosis by sensitive to apoptosis gene protein

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Nitric oxide (NO), a radical species produced by many types of cells, is known to play a critical role in many regulatory processes, yet it may also participate in collateral reactions at higher concentrations, leading to cellular oxidative stress. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates NO-induced apoptosis. When we examined the protective role of SAG against NO-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed the significant decrease in the endogenous production of ROS and oxidative DNA damage in SAG-overexpressed cells compared to control cells upon exposure to NO. These results suggest that SAG plays an important protective role in NO-induced apoptosis, presumably, through regulating the cellular redox status.     

Mitochondrial autophagy protects against heat shock-induced apoptosis through reducing cytosolic cytochrome c release and downstream caspase-3 activation

Autophagy is an evolutionarily conserved process for bulk degradation of cytoplasmic components, including large molecules and organelles. It can either help to enhance or to resist apoptosis, depending on the circumstances. The mechanism of how autophagy impacts apoptosis and the subsequent cellular events upon heat shock remains unclear. In this study, we demonstrate for the first time that mitochondrial membrane permeability transition (MPT)-sensitive mitochondrial autophagy can protect against heat-induced apoptosis through reduction of cytosolic cytochrome c release and downstream caspase-3 activation. With confocal microscopy, it was revealed that as autophagosomes increased, mitochondrial content was mass decreased after heat shock. Detailed analysis shows that a single swelling mitochondrion could be entrapped into autophagosome. The depolarization of mitochondria preceded the mitochondrial loss, and both could be abolished by MPT inhibitor cyclosporine (CsA). In addition, along with the decrease of mitochondrial content, the level of total cytochrome c was also reduced, resulting in a reduction of its release to cytoplasm. When heat shock was combined with 3-methyladenine (3-MA), an inhibitor of autophagy, the mitochondrial loss and the reduction of total cytochrome c were both inhibited, and then caspase-3 activation and cell apoptosis were increased. Thus, it is reasonable to believe that, heat shock-induced cellular events can be modulated by controlling autophagy, and this may represent a novel approach to enhance the efficacy of hyperthermia.     

Human sensitive to apoptosis gene protein inhibits peroxynitrite-induced DNA damage

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. The present study was undertaken to test the hypothesis that human SAG protects from DNA damage induced by peroxynitrite, a potent physiological inorganic toxin. The present study has shown that SAG significantly inhibits single strand breaks in supercoiled plasmid DNA induced by synthesized peroxynitrite (ONOO(-)) and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion. The formation of 8-hydroxy-2(')-deoxyguanosine in calf thymus DNA by peroxynitrite and SIN-1 was also significantly inhibited by SAG. The protective effect on peroxynitrite-mediated DNA damage was completely abolished by the reaction of SAG with N-ethylmaleimide, a chemical modification agent for the sulfhydryl group of proteins. These observations suggested that the sulfhydryl group of cysteines in SAG could react directly with peroxynitrite to prevent DNA damage.     

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]     

The effect of dietary lipid manipulation on murine splenic lymphocytes apoptosis and heat shock protein over expression

In this study, we kept BALB/c mice on a hyperlipidic diet for 120 days and then assessed the predisposition to apoptosis and the appearance of heat shock protein (Hsp) on splenic lymphocytes. By immunoblot analysis, bands corresponding to Hsp 60 and Hsp 70 in cells from mice kept on a saturated fatty acid diet showed a greater expression already after 1 month while two other bands, which correspond to Hsp 25 and Hsp 27, were slightly present after 1 month of treatment. In cells from mice kept on a diet rich in unsaturated fatty acid, there was a marked expression of Hsp 25 and Hsp 27 after only 30 days of treatment, which was maintained constant for up to 4 months; while for bands corresponding to Hsp 60 and Hsp 70, a significant minor signal was only detectable after 2-4 months from the beginning of the treatment. Splenic lymphocytes from animals kept on a lipidic diet containing saturated fatty acids were more susceptible to death by apoptosis, while cells of animals treated with unsaturated fatty acid were shown to be more resistant.     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

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.     

Caspases activation in hyperthermia-induced stimulation of TRAIL apoptosis

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.     

Regulation of protein turnover by heat shock proteins

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin–proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.     

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.     

Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells

It is known that small glutamine-rich TPR-containing protein (SGT) is the member of TPR motif family. However, the biological functions of SGT remain unclear. In this paper, we report that SGT plays a role in apoptotic signaling. Ectopic expression of SGT enhances DNA fragment and nucleus breakage after the induction of apoptosis. Increasing mRNA level of SGT is also observed in 7721 cells undergoing apoptosis, knockdown the expression of endogenous SGT contributes to the decrease of apoptosis of 7721 cells. Deletion analysis reveals that TPR domain is critical to pro-apoptotic function of SGT. Furthermore, we demonstrated that the PARP cleavage and cytochrome c release are enhanced when SGT is overexpressed in 7721 cells during apoptosis. Collectively, our results indicate that SGT is a new pro-apoptotic factor.     

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.     

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.     

A rubrerythrin-like oxidative stress protein of Clostridium acetobutylicum is encoded by a duplicated gene and identical to the heat shock protein Hsp21

Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with proteins predicted to be encoded by the genome of this bacterium revealed that this stress protein is encoded by two almost identical open reading frames CAC3597 and CAC3598. These genes encode a rubrerythrin-like protein with the rubredoxin-like FeS4 domain at the N-terminus and the ferritin-like diiron domain (rubrerythrin domain) at the C-terminus. Thus, the order of the two putative functional domains is reversed compared to "normal" rubrerythrins. This protein is proposed to be involved in the oxidative stress response of strict anaerobic bacteria. Northern blot analysis indicated that hsp21 is induced by heat and oxidative stress (air, H2O2). Hsp21 of C. acetobutylicum can be considered as a "reverse" rubrerythrin and a role of this stress protein, which is conserved among clostridia and other strict anaerobic bacteria, in the heat and oxidative stress response is proposed.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.     

Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoicacid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves.