Electron transport chain activity is a predictor and target for venetoclax sensitivity in multiple myeloma.

The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the 'primed' state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy.

The Differential Expression of Mitochondrial Function-Associated Proteins and Antioxidant Enzymes during Bovine Herpesvirus 1 Infection: A Potential Mechanism for Virus Infection-Induced Oxidative Mitochondrial Dysfunction.

Reactive oxidative species (ROS) are important inflammatory mediators. Electrons escaping from the mitochondrial electron transport chain (ETC) during oxidative phosphorylation (OXPHOS) in the mitochondrial respiratory chain (RC) complexes contribute to ROS production. The cellular antioxidant enzymes are important for maintaining ROS release at the physiological levels. It has been reported that BoHV-1 infection induces overproduction of ROS and oxidative mitochondrial dysfunction in cell cultures. In this study, we found that chemical interruption of RC complexes by TTFA (an inhibitor of RC complex II), NaN3 (an inhibitor of RC complex IV), and oligomycin A (an inhibitor of ATP synthase) consistently decreased virus productive infection, suggesting that the integral processes of RC complexes are important for the virus replication. The virus infection significantly increased the expression of subunit SDHB (succinate dehydrogenase) and MTCO1 (cytochrome c oxidase subunit I), critical components of RC complexes II and IV, respectively. The expression of antioxidant enzymes including superoxide dismutase 1 (SOD1), SOD2, catalase (CAT), and glutathione peroxidase 4 (GPX4) was differentially affected following the virus infection. The protein TFAM (transcription factor A, mitochondrial) stimulated by either nuclear respiratory factor 1 (NRF1) or NRF2 is a key regulator of mitochondrial biogenesis. Interestingly, the virus infection at the late stage (at 16 h after infection) stimulated TFAM expression but decreased the levels of both NRF1 and NRF2, indicating that virus infection activated TFAM signaling independent of either NRF1 or NRF2. Overall, this study provided evidence that BoHV-1 infection altered the expression of molecules associated with RC complexes, antioxidant enzymes, and mitochondrial biogenesis-related signaling NRF1/NRF2/TFAM, which correlated with the previous report that virus infection induces ROS overproduction and mitochondrial dysfunction.

Detection of hydrogen peroxide production in the isolated rat lung using Amplex red.

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H2O2) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H2O2 using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR's highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O2) concentration of ventilation gas on lung rate of H2O2 release into the perfusate. Baseline lung rate of H2O2 release was 8.45 ± 0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O2 in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O2-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H2O2, and that most of acute hyperoxia-enhanced lung rate of H2O2 release is from nonmitochondrial rather than mitochondrial sources.

Targeting succinate:ubiquinone reductase potentiates the efficacy of anticancer therapy.

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.

Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation.

Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.

Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II.

Melanogenesis is a highly conserved process of cytophotoprotection from UV radiation present in many species. Although both mitochondrial function and UV radiation insults are well-documented promoters of increased cellular stress, their individual molecular relationships with skin pigmentation have not been clearly resolved. This study provides evidence for a direct relationship between cellular melanin content, superoxide flux, and mitochondrial function at complex II. Direct and significant correlation between increased pigmentation and complex II turnover was observed in genetically different melanoma cell lines of varied basal pigmentation states (P < 0.01). The same trend was also observed when comparing genetically identical cell cultures with increasing levels of induced pigmentation (P < 0.005). The observation of increased steady-state levels of the catalytic complex II succinate dehydrogenase subunit A alongside hyperpigmentation suggested coregulation of activity and pigment production (P < 0.01). The study also presents novel evidence for a relationship between hyperpigmentation and increased superoxide-generating capacity at complex II. By amperometrically monitoring superoxide flux from differently pigmented FM55 melanocytes and their isolated mitochondria, a dynamic and responsive relationship between pigmentation, complex II function, and intracellular superoxide generation was observed (P < 0.005). The data support hyperpigmentation as a protective antioxidant mechanism in response to complex II-mediated reactive oxygen species generation.

Homocysteine induces the expression of C-reactive protein via NMDAr-ROS-MAPK-NF-κB signal pathway in rat vascular smooth muscle cells.

OBJECTIVE: Homocysteine (Hcy) is known as an independent risk factor for atherosclerosis. C-reactive protein (CRP) directly participates in initiation and progression of atherosclerosis. However, there is no direct evidence to demonstrate pro-inflammatory effect of Hcy on vascular smooth muscle cells (VSMCs) through CRP. In the present study, we examined the effect of Hcy on CRP expression and investigated the related mechanism in VSMCs. METHODS AND RESULTS: Protein expression and secretion were detected by Western blot and ELISA, respectively. mRNA expression was detected by RT-PCR. Superoxide anion was detected by lucigenin chemiluminometry and the immunofluorescence staining was observed by a fluorescence microscope. The results revealed that Hcy significantly induced mRNA and protein expressions of CRP in VSMCs both in vitro and in vivo, and anti-IL-1ß or anti-IL-6 neutralizing antibody alone or in combination partially reduced Hcy-induced CRP expression. Hcy increased the expression of NR1 subunit of N-methyl-d-aspartate receptor (NMDAr), and MK-801 alleviated Hcy-induced CRP expression in VSMCs. Further studies showed that Hcy-stimulated superoxide anion generation in VSMCs. Nevertheless, pretreatment of the cells with MK-801, TTFA and DPI significantly reduced Hcy-stimulated superoxide anion generation, and antioxidant NAC decreased Hcy-induced CRP expression in VSMCs. Additionally, PD98059, SB205380 or PDTC antagonized Hcy-induced CRP expression, and MK-801, NAC, PD98059 or SB205380 inhibited Hcy-activated phosphorylations of ERK1/2 and p38. CONCLUSION: The present study demonstrates that Hcy is able to initiate an inflammatory response in VSMCs by stimulating CRP production, which is mediated through NMDAr-ROS-ERK1/2/p38-NF-κB signal pathway. These findings provide new evidence for a role of Hcy in pathogenesis of atherosclerosis.

Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase.

Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH-ubiquinone oxidoreductase (Na(+)-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na(+)-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na(+)-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na(+)-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na(+)-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na(+)-NQR orthologs.

Angiotensin II induces C-reactive protein expression via AT1-ROS-MAPK-NF-κB signal pathway in hepatocytes.

BACKGROUND: C-reactive protein (CRP) participates in development of inflammatory diseases. Hepatocytes are a major contributor of circulating CRP. Although angiotensin II (Ang II) is known to evoke inflammatory response, it remains unknown whether Ang II induces CRP expression in hepatocytes. The present study observed effect of Ang II on CRP expression and the related signal pathway in hepatocytes. METHODS: mRNA and protein expressions in human hepatocytes were determined with RT-PCR and Western blot respectively. Reactive oxygen species (ROS) was measured using a fluorescence probe. CRP in liver and serum of rats was determined by immunohistochemistry and ELISA respectively. RESULTS: Ang II induced mRNA and protein expression of CRP in hepatocytes and increased CRP production in liver and CRP level in serum. Losartan reduced Ang II- induced CRP expression in hepatocytes. Losartan and thenoyltrifluoroacetone decreased Ang II-stimulated ROS production. N-acetylcysteine antagonized Ang II-induced CRP expression. Losartan and N-acetylcysteine inhibited Ang II-activated ERK1/2. Unlike ERK1/2, only losartan inhibited Ang II-activated JNK. Furthermore, pyrrolidine dithiocarbamate abolished Ang II-induced CRP expression. CONCLUSION: Ang II has ability to induce CRP expression in hepatocytes in vitro and in vivo through AT1 receptor followed by ROS, MAPK and NF-κB signal pathway.

Malonate inhibits virulence gene expression in Vibrio cholerae.

We previously found that inhibition of the TCA cycle, either through mutations or chemical inhibition, increased toxT transcription in Vibrio cholerae. In this study, we found that the addition of malonate, an inhibitor of succinate dehydrogenase (SDH), decreased toxT transcription in V. cholerae, an observation inconsistent with the previous pattern observed. Unlike another SDH inhibitor, 2-thenoyltrifluoroacetone (TTFA), which increased toxT transcription and slightly inhibited V. cholerae growth, malonate inhibited toxT transcription in both the wild-type strain and TCA cycle mutants, suggesting malonate-mediated inhibition of virulence gene expression is independent to TCA cycle activity. Addition of malonate also inhibited ctxB and tcpA expressions but did not affect aphA, aphB, tcpP and toxR expressions. Malonate inhibited cholera toxin (CT) production in both V. cholerae classical biotype strains O395N1 and CA401, and El Tor biotype strain, N16961. Consistent with previous reports, we confirmed that these strains of V. cholerae did not utilize malonate as a primary carbon source. However, we found that the addition of malonate to the growth medium stimulated V. cholerae growth. All together, these results suggest that metabolizing malonate as a nutrient source negatively affects virulence gene expression in V. cholerae.

Reactive oxygen species are generated by the respiratory complex II--evidence for lack of contribution of the reverse electron flow in complex I.

Succinate-driven oxidation via complex II (CII) may have a significant contribution towards the high rates of production of reactive oxygen species (ROS) by mitochondria. Here, we show that the CII Q site inhibitor thenoyltrifluoroacetone (TTFA) blocks succinate + rotenone-driven ROS production, whereas the complex III (CIII) Qo inhibitor stigmatellin has no effect, indicating that CII, not CIII, is the ROS-producing site. The complex I (CI) inhibitor rotenone partially reduces the ROS production driven by high succinate levels (5 mm), which is commonly interpreted as being due to inhibition of a reverse electron flow from CII to CI. However, experimental evidence presented here contradicts the model of reverse electron flow. First, ROS levels produced using succinate + rotenone were significantly higher than those produced using glutamate + malate + rotenone. Second, in tumor mitochondria, succinate-driven ROS production was significantly increased (not decreased) by rotenone. Third, in liver mitochondria, rotenone had no effects on succinate-driven ROS production. Fourth, using isolated heart or hepatoma (AS-30D) mitochondria, the CII Qp anti-cancer drug mitochondrially targeted vitamin E succinate (MitoVES) induced elevated ROS production in the presence of low levels of succinate(0.5 mm), but rotenone had no effect. Using sub-mitochondrial particles, the Cu-based anti-cancer drug Casiopeina II-gly enhanced succinate-driven ROS production. Thus, the present results are inconsistent with and question the interpretation of reverse electron flow from CII to CI and the rotenone effect on ROS production supported by succinate oxidation. Instead, a thermodynamically more favorable explanation is that, in the absence of CIII or complex IV (CIV) inhibitors (which, when added, facilitate reverse electron flow by inducing accumulation of ubiquinol, the CI product), the CII redox centers are the major source of succinate-driven ROS production.

Two thiophenes compounds are partial peroxisome proliferator-activated receptor α/γ dual agonists.

In our previous study, two synthetic thiophenes such as IMB-05 and IMB-15 were found as peroxisome proliferator-activated receptor gamma (PPARγ) agonists and exhibited beneficial effects on glucose tolerance of diabetic mice in vivo. In the present study, their effect on the transactivity of other nuclear receptors was further investigated. IMB-05 and IMB-15 could not only activated PPARγ but also efficiently activate PPARα in GAL4-hPPARα/γ (ligand binding domain (LBD)) chimeric receptor assay and PPAR response element (PPRE)-luc reporter gene assay with EC(50) values of 1.8-5.2 µM, whereas no activity was observed in other nuclear receptor assays. In addition, the maximal efficacy of IMB-05 and IMB-15 in activating PPARα/γ was approximately 30% of that observed with Wy14643 and rosiglitazone. These data indicate that the two thiophene derivatives are novel class of partial PPARα/γ dual agonists, which may be the mechanism underlying their regulatory effects on glucose homeostasis.

The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels.

Inactivation of the TCA cycle enzyme, fumarate hydratase (FH), drives a metabolic shift to aerobic glycolysis in FH-deficient kidney tumors and cell lines from patients with hereditary leiomyomatosis renal cell cancer (HLRCC), resulting in decreased levels of AMP-activated kinase (AMPK) and p53 tumor suppressor, and activation of the anabolic factors, acetyl-CoA carboxylase and ribosomal protein S6. Reduced AMPK levels lead to diminished expression of the DMT1 iron transporter, and the resulting cytosolic iron deficiency activates the iron regulatory proteins, IRP1 and IRP2, and increases expression of the hypoxia inducible factor HIF-1α, but not HIF-2α. Silencing of HIF-1α or activation of AMPK diminishes invasive activities, indicating that alterations of HIF-1α and AMPK contribute to the oncogenic growth of FH-deficient cells.

A common mechanism links differently acting complex II inhibitors to cardioprotection: modulation of mitochondrial reactive oxygen species production.

In this study, we have analyzed the effect of different cardioprotective complex II inhibitors on the mitochondrial production of reactive oxygen species (ROS) because ROS seem to be essential for signaling during preconditioning to prevent ischemia/reperfusion injury. Despite different binding sites and concentrations required for half-maximal inhibition-ranging from nanomolar for the Q site inhibitor atpenin A5 to millimolar for the succinate analog malonate-all inhibitors modulated ROS production in the same ambivalent fashion: they promoted the generation of superoxide at the Q(o) site of complex III under conditions of "oxidant-induced reduction" but attenuated ROS generated at complex I due to reverse electron transfer. All inhibitors showed these ambivalent effects independent of the presence of K(+). These findings suggest a direct modulation of mitochondrial ROS generation during cardioprotection via complex II inhibition and question the recently proposed role of complex II as a regulatory component of the putative mitochondrial K(ATP) channel.

Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction.

The formation of reactive oxygen species (ROS) and the change of the intracellular pH (pH(i)) are common phenomena during apoptosis. How they are interconnected, however, is poorly understood. Here we show that numerous anticancer drugs and cytokines such as Fas ligand and tumour necrosis factor α provoke intracellular acidification and cause the formation of mitochondrial ROS. In parallel, we found that the succinate:ubiquinone oxidoreductase (SQR) activity of the mitochondrial respiratory complex II is specifically impaired without affecting the second enzymatic activity of this complex as a succinate dehydrogenase (SDH). Only in this configuration is complex II an apoptosis mediator and generates superoxides for cell death. This is achieved by the pH(i) decline that leads to the specific dissociation of the SDHA/SDHB subunits, which encompass the SDH activity, from the membrane-bound components of complex II that are required for the SQR activity.

Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through mitochondrial superoxide overproduction in adipocytes.

Hypoadiponectinemia and hyperresistinemia may be important in mediating signals from adipocytes to insulin-sensitive tissue and vasculature. However, the mechanism that mediates the aberrant production of adipokines remains poorly understood. In this study, we have investigated the effect of intermittent high glucose on the expression of adiponectin and resistin, and the production of 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine in the adipocytes, either in the presence or in the absence of Mn(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) or thenoyltrifluoroacetone (TTFA). 3T3-L1 adipocytes were incubated for 72 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, 5 mmol/l alternating with 20 mmol/l glucose, with or without MnTBAP and TTFA. We measured the expression of resistin and adiponectin. The production of nitrotyrosine and 8-OHdG as oxidative stress parameter was measured. Both constant and intermittent high glucose significantly suppressed the expression and secretion of adiponectin, and increased expression and secretion of resistin in mature adipocytes compared to normal glucose conditions. However, these effects were significantly greater under intermittent high glucose conditions compared to constant high glucose. The levels of nitrotyrosine and 8-OHdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA reversed the aberrant production of adiponectin and resistin, as well as overproduction of nitrotyrosine and 8-OHdG in adipocytes induced by constant or intermittent high glucose. Intermittent high glucose exacerbates the aberrant production of adiponectin and resistin through reactive oxygen species overproduction at the mitochondrial transport chain level in adipocytes, indicating that glycemic variability has important pathological effects on the secretion of adipokines.

Inhibition of succinate dehydrogenase dysregulates histone modification in mammalian cells.

Remodelling of mitochondrial metabolism is a hallmark of cancer. Mutations in the genes encoding succinate dehydrogenase (SDH), a key Krebs cycle component, are associated with hereditary predisposition to pheochromocytoma and paraganglioma, through mechanisms which are largely unknown. Recently, the jumonji-domain histone demethylases have emerged as a novel family of 2-oxoglutarate-dependent chromatin modifiers with credible functions in tumourigenesis. Using pharmacological and siRNA methodologies we show that increased methylation of histone H3 is a general consequence of SDH loss-of-function in cultured mammalian cells and can be reversed by overexpression of the JMJD3 histone demethylase. ChIP analysis revealed that the core promoter of IGFBP7, which encodes a secreted protein upregulated after loss of SDHB, showed decreased occupancy by H3K27me3 in the absence of SDH. Finally, we provide the first evidence that the chief (type I) cell is the major methylated histone-immunoreactive constituent of paraganglioma. These results support the notion that loss of mitochondrial function alters epigenetic processes and might provide a signature methylation mark for paraganglioma.

Downregulation of gap junction expression and function by endoplasmic reticulum stress.

Gap junctional intercellular communication (GJIC) plays a critical role in the control of multiple cell behavior as well as in the maintenance of tissue and organ homeostasis. However, mechanisms involved in the regulation of gap junctions (GJs) have not been fully understood. Given endoplasmic reticulum (ER) stress and dysfunction of GJs coexist in several pathological situations, we asked whether GJs could be regulated by ER stress. Incubation of mesangial cells with ER stress-inducing agents (thapsigargin, tunicamycin, and AB(5) subtilase cytotoxin) resulted in a decrease in connexin 43 (Cx43) expression at both protein and mRNA levels. This was accompanied by a loss of GJIC, as evidenced by the reduced numbers of dye-coupled cells after single cell microinjection or scrape loading dye transfer. Further studies demonstrated that ER stress significantly inhibited the promoter activity of the Cx43 gene, reduced [(35)S]-methionine incorporation into Cx43 protein and accelerated degradation of Cx43. ER stress also decreased the Cx43 protein levels in several different cell types, including human umbilical vein endothelial cells, mouse-derived renin-secreting cells and human hepatoma cells. Furthermore, induction of ER stress by hypoxic chemicals thenoyltrifluoroacetone and cobalt chloride was found to be associated with a reduction in Cx43. Our findings thus reveal a close link between ER stress and GJs. ER stress may represent a novel mechanism underlying the altered GJs in a variety of pathological situations.

Mitochondrial dysfunction by complex II inhibition delays overall cell cycle progression via reactive oxygen species production.

Mitochondrial complex II defect has recently been implicated in cellular senescence and in the ageing process of which a critical phenotype is retardation and arrest of cellular growth. However, the underlying mechanisms of how complex II defect affects cellular growth, remain unclear. In this study, we investigated the effect of complex II inhibition using a subcytotoxic dose (400 microM) of 2-thenoyltrifluoroacetone (TTFA), a conventional complex II inhibitor, on cell cycle progression. TTFA (400 microM) directly decreased KCN-sensitive cellular respiration rate to 67% of control and disrupted the mitochondrial membrane potential. In contrast to other respiratory inhibitors such as rotenone, antimycin A, and oligomycin, TTFA prolonged the duration of each phase of the cell cycle (G1, S, and G2/M) equally, thereby delaying overall cell cycle progression. This delay was accompanied by a biphasic increase of reactive oxygen species (ROS) and concurrent glutathione oxidation, in addition to a slight decrease in the cellular ATP level. Finally, the delay in cell cycle progression caused by TTFA was proved to be mainly due to ROS overproduction and subsequent oxidative stress, as evidenced by its reversal following pretreatment with antioxidants. Taken together, these results suggest that an overall delay in cell cycle progression due to complex II defects may contribute to ageing and degenerative diseases via inhibition of cellular growth and proliferation without arrest at any specific phase of the cell cycle.

Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species.

Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induces cell death through generating reactive oxygen species (ROS). The role of mETC inhibitors in autophagy-induced cell death is unknown. Herein, we determined that inhibitors of complex I (rotenone) and complex II (TTFA) induce cell death and autophagy in the transformed cell line HEK 293, and in cancer cell lines U87 and HeLa. Blocking the expression of autophagic genes (beclin 1 and ATG5) by siRNAs or using the autophagy inhibitor 3-methyladenine (3-MA) decreased cell death that was induced by rotenone or TTFA. Rotenone and TTFA induce ROS production, and the ROS scavenger tiron decreased autophagy and cell death induced by rotenone and TTFA. Overexpression of manganese-superoxide dismutase (SOD2) in HeLa cells decreased autophagy and cell death induced by rotenone and TTFA. Furthermore, blocking SOD2 expression by siRNA in HeLa cells increased ROS generation, autophagy and cell death induced by rotenone and TTFA. Rotenone- and TTFA-induced ROS generation was not affected by 3-MA, or by beclin 1 and ATG5 siRNAs. By contrast, treatment of non-transformed primary mouse astrocytes with rotenone or TTFA failed to significantly increase levels of ROS or autophagy. These results indicate that targeting mETC complex I and II selectively induces autophagic cell death through a ROS-mediated mechanism.