Inhibition Of Respiratory Complex Research Articles

Abstract B070: Myc synthetic lethality as a therapeutic strategy for resistance: Insights into the targetability of mitochondrial complex I in Myc-high TNBC

Abstract Patients with advanced triple-negative breast cancer (TNBC) frequently develop chemoresistance, emphasizing an urgent need for novel therapeutic strategies able to eradicate treatment resistant tumor cells. Recent studies have highlighted MYC as a common oncogenic alteration and a driver of stem-like TNBC phenotype, which commonly features high mitochondrial respiratory activity instated through OXPHOS system and chemoresistance. Interestingly, emerging evidence suggests that residual tumors from chemo treatment remain susceptible to OXPHOS inhibition through inhibition of respiratory complex I. Our previous studies have revealed a specific MYC-dependent and tumor suppressive synthetic lethal action for combination of metformin and BH3 mimetics in cells, ex vivo and in vivo models of aggressive breast cancer (PMID: 30728358). Metformin is a systemic and local metabolic modulator, which acts as a weak complex I inhibitor and promoter of AMPK activation. Here, we have explored the role of MYC in OXPHOS regulation, to address the question if metformin specifically and lethally targets MYC-modulated OXPHOS in MYC-high TNBC. We found that a selective c-MYC inhibitor (EN4) leads to a significant decrease in complex I activity despite only a modest drop in complex I mass in endogenously MYC-high TNBC cell lines. Conversely, engineered MYC overexpression in these cells upregulated complex I activity. Our current work explores transcriptomic, proteomic, and functional approaches to the mechanisms through which MYC regulates complex I activity, structure, and dynamics, ultimately hoping to define a biological rationale for therapeutic targeting of OXPHOS in Myc-high TNBC. Citation Format: Antti O Hiltunen, Linda J Id, Juha Klefström. Myc synthetic lethality as a therapeutic strategy for resistance: Insights into the targetability of mitochondrial complex I in Myc-high TNBC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B070.

Exploring ND-011992, a quinazoline-type inhibitor targeting quinone reductases and quinol oxidases

Bacterial energy metabolism has become a promising target for next-generation tuberculosis chemotherapy. One strategy to hamper ATP production is to inhibit the respiratory oxidases. The respiratory chain of Mycobacterium tuberculosis comprises a cytochrome bcc:aa3 and a cytochrome bd ubiquinol oxidase that require a combined approach to block their activity. A quinazoline-type compound called ND-011992 has previously been reported to ineffectively inhibit bd oxidases, but to act bactericidal in combination with inhibitors of cytochrome bcc:aa3 oxidase. Due to the structural similarity of ND-011992 to quinazoline-type inhibitors of respiratory complex I, we suspected that this compound is also capable of blocking other respiratory chain complexes. Here, we synthesized ND-011992 and a bromine derivative to study their effect on the respiratory chain complexes of Escherichia coli. And indeed, ND-011992 was found to inhibit respiratory complex I and bo3 oxidase in addition to bd-I and bd-II oxidases. The IC50 values are all in the low micromolar range, with inhibition of complex I providing the lowest value with an IC50 of 0.12 µM. Thus, ND-011992 acts on both, quinone reductases and quinol oxidases and could be very well suited to regulate the activity of the entire respiratory chain.

Inhibition of mitochondrial transcription by the neurotoxin MPP+

The neurotoxin MPP+ triggers cell death of dopamine neurons and induces Parkinson's disease symptoms in mice and men, but the immediate transcriptional response to this neurotoxin has not been studied. We therefore treated human SH-SY5Y cells with a low dose (0.1 mM) of MPP+ and measured the effect on nascent transcription by precision run-on sequencing (PRO-seq). We found that transcription of the mitochondrial genome was significantly reduced already after 30 min, whereas nuclear gene transcription was unaffected. Inhibition of respiratory complex I by MPP+ led to reduced ATP production, that may explain the diminished activity of mitochondrial RNA polymerase. Our results show that MPP+ has a direct effect on mitochondrial function and transcription, and that other gene expression or epigenetic changes induced by this neurotoxin are secondary effects that reflect a cellular adaptation program.

NDUFS3 knockout cancer cells and molecular docking reveal specificity and mode of action of anti-cancer respiratory complex I inhibitors.

Inhibition of respiratory complex I (CI) is becoming a promising anti-cancer strategy, encouraging the design and the use of inhibitors, whose mechanism of action, efficacy and specificity remain elusive. As CI is a central player of cellular bioenergetics, a finely tuned dosing of targeting drugs is required to avoid side effects. We compared the specificity and mode of action of CI inhibitors metformin, BAY 87-2243 and EVP 4593 using cancer cell models devoid of CI. Here we show that both BAY 87-2243 and EVP 4593 were selective, while the antiproliferative effects of metformin were considerably independent from CI inhibition. Molecular docking predictions indicated that the high efficiency of BAY 87-2243 and EVP 4593 may derive from the tight network of bonds in the quinone binding pocket, although in different sites. Most of the amino acids involved in such interactions are conserved across species and only rarely found mutated in human. Our data make a case for caution when referring to metformin as a CI-targeting compound, and highlight the need for dosage optimization and careful evaluation of molecular interactions between inhibitors and the holoenzyme.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

VDAC1 as a target in cisplatin anti-tumor activity through promoting mitochondria fusion

Cisplatin is one of the most effective anti-cancer drugs, but its efficacy is limited by the development of resistance. Previous studies have shown that mitochondria play critical roles in cisplatin cytotoxicity, however, the exact mechanism of mitochondria involved in cisplatin sensitivity has not been clarified. In this study, cisplatin triggered mitochondrial oxidative stress and the decrease of mitochondria membrane potential in human cervical cancer cells. Then we screened a series of mitochondrial relevant inhibitors, including mitochondrial mPTP inhibitors DIDS and CsA, and mitochondrial respiratory complex inhibitors Rot and TTFA. Among these, only DIDS, as the inhibitor of mitochondrial outer membrane protein VDAC1, showed strong antagonism against cisplatin toxicity. DIDS mitigated cisplatin-induced MFN1-dependent mitochondrial fusion, mitochondrial dysfunction and oxidative damage. These findings demonstrated that VDAC1 may serve as a potential therapeutic target in the increase sensitivity of cisplatin, which provides an attractive pharmacological therapy to improve the effectiveness of chemotherapy.

Does Metformin Satisfy as an Option for Host-Directed Therapy in COVID-19?

Metformin is a dimethyl biguanide known for its anti-hyperglycemic effects since 1922 and, thus, has been used in the treatment of Type 2 Diabetes Mellitus. Apart from this, its function as an anti-inflammatory, anti-oxidant, anti-thrombotic, and anti-microbial agent has also been studied largely. Its effect via inhibition of respiratory complex 1 and glycerol-3-phosphate dehydrogenase (GPD2) in the mitochondria and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and activation of Adenosine Mono Phosphate dependent kinase (AMPK) in the cytoplasm are its already known mechanisms of action. With the newer proposed effects on endosome/ lysosome regulation via Na+/H+ exchangers and V-ATPase, thereby affecting autophagy, coupled with its use in ARDS owing to its immunomodulatory effects and anti-viral action, the use of metformin against the novel Coronavirus is hypothesized.

Senescent Cell Depletion Through Targeting BCL-Family Proteins and Mitochondria.

Senescent cells with replicative arrest can be generated during genotoxic, oxidative, and oncogenic stress. Long-term retention of senescent cells in the body, which is attributed to highly expressed BCL-family proteins, chronically damages tissues mainly through a senescence-associated secretory phenotype (SASP). It has been documented that accumulation of senescent cells contributes to chronic diseases and aging-related diseases. Despite the fact that no unique marker is available to identify senescent cells, increased p16INK4a expression has long been used as an in vitro and in vivo marker of senescent cells. We reviewed five existing p16INK4a reporter mouse models to detect, isolate, and deplete senescent cells. Senescent cells express high levels of anti-apoptotic and pro-apoptotic genes compared to normal cells. Thus, disrupting the balance between anti-apoptotic and pro-apoptotic gene expression, such as ABT-263 and ABT-737, can activate the apoptotic signaling pathway and remove senescent cells. Mitochondrial abnormalities in senescent cells were also discussed, for example mitochondrial DNA mutation accumulation, dysfunctional mitophagy, and mitochondrial unfolded protein response (mtUPR). The mitochondrial-targeted tamoxifen, MitoTam, can efficiently remove senescent cells due to its inhibition of respiratory complex I and low expression of adenine nucleotide translocase-2 (ANT2) in senescent cells. Therefore, senescent cells can be removed by various strategies, which delays chronic and aging-related diseases and enhances lifespan and healthy conditions in the body.

Different Effects of Metformin and A769662 on Sodium Iodate-Induced Cytotoxicity in Retinal Pigment Epithelial Cells: Distinct Actions on Mitochondrial Fission and Respiration.

Oxidative stress-associated retinal pigment epithelium (RPE) cell death is critically implicated in the pathogenesis of visual dysfunction and blindness of retinal degenerative diseases. Sodium iodate (NaIO3) is an oxidative retinotoxin and causes RPE damage. Previously, we found that NaIO3 can induce human ARPE-19 cell death via inducing mitochondrial fission and mitochondrial dysfunction. Although metformin has been demonstrated to benefit several diseases possibly via AMP-activated protein kinase (AMPK) activation, it remains unknown how AMPK affects retinopathy in NaIO3 model. Therefore, in this study, we compared the effects of metformin and AMPK activator A769662 on NaIO3-induced cellular stress and toxicity. We found that A769662 can protect cells against NaIO3-induced cytotoxicity, while metformin exerts an enhancement in cell death. The mitochondrial reactive oxygen species (ROS) production as well as mitochondrial membrane potential loss induced by NaIO3 were not altered by both agents. In addition, NaIO3-induced cytosolic ROS production, possibly from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and counteracting cell death, was not altered by A769662 and metformin. Notably, NaIO3-induced mitochondrial fission and inhibition of mitochondrial respiration for ATP turnover were reversed by A769662 but not by metformin. In agreement with the changes on mitochondrial morphology, the ERK-Akt signal axis dependent Drp-1 phosphorylation at S616 (an index of mitochondrial fission) under NaIO3 treatment was blocked by A769662, but not by metformin. In summary, NaIO3-induced cell death in ARPE cells primarily comes from mitochondrial dysfunction due to dramatic fission and inhibition of mitochondrial respiration. AMPK activation can exert a protection by restoring mitochondrial respiration and inhibition of ERK/Akt/Drp-1 phosphorylation, leading to a reduction in mitochondrial fission. However, inhibition of respiratory complex I by metformin might deteriorate mitochondrial dysfunction and cell death under NaIO3 stress.

OPB-51602, a Direct Inhibitor of STAT3, Impairs Growth and Viability of Tumor Cells Through Inhibition of Respiratory Complex I

STAT3 is a transcription factor involved in numerous cellular activities, including inflammation, proliferation, and survival, but it also plays a non-transcriptional role in modulating mitochondrial metabolism. Given its diverse functions in human cancer, it is an emerging therapeutic target. Here we show that OPB-51602, a small molecule inhibitor of STAT3, is highly toxic in a STAT3- dependent manner. Specifically, drug toxicity depends on STAT3 mitochondrial functions as tumor cells expressing only a mitochondrially-restricted form of STAT3 are highly sensitive to the compound, while STAT3-null cells are protected. OPB-51602 treatment abrogated complex I activity and led to mitophagy, actin skeletal rearrangements, and apoptosis. Cells undergoing reduced oxidative phosphorylation or expressing the NDI1 NADH dehydrogenase enzyme from Saccharomyces cerevisiae that bypasses mammalian complex I, were resistant to OPB-51602 toxicity. These results show that targeting mitochondrial STAT3 function causes a synthetic lethality involving complex I inhibition that could be exploited for cancer chemotherapy.

Primary cilia mediate mitochondrial stress responses to promote dopamine neuron survival in a Parkinson\u2019s disease model

A primary cilium is an antenna-like structure on the cell surface that plays a crucial role in sensory perception and signal transduction. Mitochondria, the ‘powerhouse’ of the cell, control cell survival, and death. The cellular ability to remove dysfunctional mitochondria through mitophagy is important for cell survival. We show here that mitochondrial stress, caused by respiratory complex inhibitors and excessive fission, robustly stimulates ciliogenesis in different types of cells including neuronal cells. Mitochondrial stress-induced ciliogenesis is mediated by mitochondrial reactive oxygen species generation, subsequent activation of AMP-activated protein kinase and autophagy. Conversely, abrogation of ciliogenesis compromises mitochondrial stress-induced autophagy, leading to enhanced cell death. In mice, treatment with mitochondrial toxin, MPTP elicits ciliary elongation and autophagy in the substantia nigra dopamine neurons. Blockade of cilia formation in these neurons attenuates MPTP-induced autophagy but facilitates dopamine neuronal loss and motor disability. Our findings demonstrate the important role of primary cilia in cellular pro-survival responses during mitochondrial stress.

Respiratory complex II in mitochondrial dysfunction-mediated cytotoxicity: Insight from cadmium

In the present work we studied action of several inhibitors of respiratory complex II (CII) of mitochondrial electron transport chain, namely malonate and thenoyltrifluoroacetone (TTFA) on Cd2+-induced toxicity and cell mortality, using two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D and isolated rat liver mitochondria (RLM). It was shown that malonate, an endogenous competitive inhibitor of dicarboxylate-binding site of CII, restored in part RLM respiratory function disturbed by Cd2+. In particular, malonate increased both phosphorylating and maximally uncoupled respiration rates in KCl medium in the presence of CI substrates as well as palliated changes in basal and resting state respiration rates produced by the heavy metal on the mitochondria energized by CI or CII substrates. Notably, malonate enhanced Cd2+-induced swelling of the mitochondria energized by CI substrates in KCl and, in a much lesser extent and at higher [Cd2+], in sucrose media but did not influence on the Cd2+ effects in NaCl medium. Besides, malonate did not affect swelling in sucrose media of RLM energized by CIV substrates under using of Cd2+ or Ca2+ whereas it strongly increased the mitochondrial swelling produced by selenite. In addition, malonate produced some protection against Cd2+-promoted necrotic death of AS-30D and PC12 cells and reduced intracellular reactive oxygen species (ROS) formation evoked by Cd2+ in PC12 cells. Importantly, TTFA, an irreversible competitive inhibitor of Q-binding site of CII, per se induced apoptosis of AS-30D cells which was inhibited by co-treatment with Cd2+ as well as decreased the Cd2+-enhanced intracellular ROS formation. In turn, decylubiquinone (dUb) at low μM concentrations did not protect AS-30D cells against the Cd2+-induced necrosis and enhanced the Cd2+-induced apoptosis of the cells. High μM concentrations of dUb were highly toxic for the cells. As consequence, the findings give new evidence indicative of critical involvement of CII in mechanism(s) of Cd2+-produced cytotoxicity and support the notion on CII as a perspective pharmacological target in mitochondria dysfunction-mediated conditions and diseases.

5-Hydroxy-1,4-naphthalenedione exerts anticancer effects on glioma cells through interaction with the mitochondrial electron transport chain

Survival of patients with glioblastoma remains within the range of several months despite advances in therapeutic options. We have already shown that 5-hydroxy-1,4-naphthalenedione (juglone) exerts antiproliferative, anti-invasive, and cytotoxic effects on glioma C6 cells. Here, we further revealed that juglone is relatively selective to glioma cells as compared to the normal glial culture, and investigated its mechanisms of action. The incubation of glioma C6 cells with juglone generated high levels of reactive oxygen species (ROS). The produced ROS accounted for the anticancer effect of juglone as antioxidant N-acetylcysteine reduced both cytotoxic and antiproliferative activities of juglone. Furthermore, high resolution respirometry revealed that juglone decreased oxygen consumption mainly by affecting pyruvate/malate- and glutamate/malate-induced mitochondrial respiration. The inhibition of respiratory complex I by amytal decreased juglone-triggered generation of ROS and diminished its anticancer activity. Thus, our results indicate that juglone generates ROS through interaction with respiratory complex I in glioma C6 cells, and, in turn, induces the anticancer effects.

Determinants of Anti-Cancer Effect of Mitochondrial Electron Transport Chain Inhibitors: Bioenergetic Profile and Metabolic Flexibility of Cancer Cells.

Recent evidence highlights that energy requirements of cancer cells vary greatly from normal cells and they exhibit different metabolic phenotypes with variable participation of both glycolysis and oxidative phosphorylation (OXPHOS). Interestingly, mitochondrial electron transport chain (ETC) has been identified as an essential component in bioenergetics, biosynthesis and redox control during proliferation and metastasis of cancer cells. This dependence converts ETC of cancer cells in a promising target to design small molecules with anti-cancer actions. Several small molecules have been described as ETC inhibitors with different consequences on mitochondrial bioenergetics, viability and proliferation of cancer cells, when the substrate availability is controlled to favor either the glycolytic or OXPHOS pathway. These ETC inhibitors can be grouped as 1) inhibitors of a respiratory complex (e.g. rotenoids, vanilloids, alkaloids, biguanides and polyphenols), 2) inhibitors of several respiratory complexes (e.g. capsaicin, ME-344 and epigallocatechin-3 gallate) and 3) inhibitors of ETC activity (e.g. elesclomol and VLX600). Although pharmacological ETC inhibition may produce cell death and a decrease of proliferation of cancer cells, factors such as degree of inhibition of ETC activity by small molecules, bioenergetic profile and metabolic flexibility of different cancer types or subpopulations of cells in a particular cancer type, can affect the impact of the anti-cancer actions. Particularly interesting are the adaptive mechanisms induced by ETC inhibition, such as induction of glutamine-dependent reductive carboxylation, which may offer a strategy to sensitize cancer cells to inhibitors of glutamine metabolism.

All trans retinoic acid depresses the content and activity of the mitochondrial ATP synthase in human keratinocytes

Proteomic analysis shows that treatment of keratinocytes cultures with all trans retinoic acid (ATRA), under condition in which it inhibits cell growth, results in marked decrease of the level of the F1-β subunit of the catalytic sector of the mitochondrial FoF1 ATP synthase complex. Enzymatic analysis shows in ATRA-treated keratinocytes a consistent depression of the ATPase activity, with decreased olygomycin sensitivity, indicating an overall alteration of the ATP synthase complex. These findings, together with the previously reported inhibition of respiratory complex I, show that depression of the activity of oxidative phosphorylation enzymes is involved in the cell growth inhibitory action of ATRA.

Toxicity of lithium on isolated heart mitochondria and cardiomyocyte: A justification for its cardiotoxic adverse effect.

Mitochondria play an important role in myocardial tissue homeostasis; therefore, deterioration in mitochondrial function will eventually lead to cardiomyocyte and endothelial cell death and consequently cardiovascular dysfunction. Lithium (Li+ ) is an effective drug for bipolar disorder with known cardiotoxic side effects. This study was designed to investigate the effects of Li+ on mitochondria and cardiomyocytes isolated from the heart of Wistar rat. Results revealed that Li+ induced a concentration- and time-dependent rise in mitochondrial ROS formation, inhibition of respiratory complexes (II), mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release in rat heart mitochondria and also induced Caspase 3 activation through mitochondrial pathway, decline of ATP and lipid peroxidation in rat cardiomyocytes. These results indicate that the cardiotoxic effects of Li+ were initiated from mitochondrial dysfunction and oxidative stress, which finally ends in cytochrome c release and cell death signaling heart cardiomyocytes.

Neuronal Hemoglobin Expression and Its Relevance to Multiple Sclerosis Neuropathology.

Multiple sclerosis (MS) is characterized by demyelination and progressive neurological disability. Previous studies have reported defects to mitochondria in MS including decreased expression of nuclear encoded electron transport chain subunit genes and inhibition of respiratory complexes. We previously reported increased levels of the hemoglobin β subunit (Hbb) in mitochondrial fractions isolated from postmortem MS cortex compared to controls. In the present study, we performed immunohistochemistry to determine the distribution of Hbb in postmortem MS cortex and identified proteins which interact with Hbb by liquid chromatography tandem mass spectrometry (LC-MS/MS). We found that Hbb was enriched in pyramidal neurons in internal layers of the cortex and interacts with subunits of ATP synthase, histones, and a histone lysine demethylase. We also found that Hbb is present in the nucleus and that expression of Hbb in SH-SY5Y neuroblastoma cells increased trimethylation of histone H3 on lysine 4 (H3K4me3), a histone mark that regulates cellular metabolism. These data suggest that Hbb may be a part of a mechanism linking neuronal energetics with epigenetic changes to histones in the nucleus and may provide neuroprotection in MS by supporting neuronal metabolism.

Alcohol hangover: impairments in behavior and bioenergetics in central nervous system

Alcohol hangover (AH) is defined as the temporary state after alcohol binge-like drinking, starting when EtOH is absent in plasma. Results from our laboratory have shown behavioral impairments and mitochondrial dysfunction in an experimental model of AH in mice. Our model consisted in a single i.p. injection of EtOH (3.8 g/kg BW) or saline solution in male and female mice, sacrificing the animals 6 hours after injection. Motor and affective behavior together with mitochondrial function and free radical production were evaluated in brain cortex and cerebellum during AH. Results showed that hangover animals exhibited a significant reduction in neuromuscular coordination, motor strength and locomotion together with a loss of gait stability and walking deficiencies. Moreover, an increment in anxiety-like behavior together with fear-related phenotype and depression signs were observed. In relation to bioenergetics metabolism, AH induced a reduction in oxygen uptake, inhibition of respiratory complexes, changes in mitochondrial membrane permeability, decrease in transmembrane potential, increase in O2 •- and H2O2 production and impairment in nitric oxide metabolism. All together our data suggest that the physiopathological state of AH involves behavioral impairments and mitochondrial dysfunction in mouse brain cortex and cerebellum showing the long lasting effects of acute EtOH exposure in CNS.

Current topics on inhibitors of respiratory complex I

There are a variety of chemicals which regulate the functions of bacterial and mitochondrial complex I. Some of them, such as rotenone and piericidin A, have been indispensable molecular tools in mechanistic studies on complex I. A large amount of experimental data characterizing the actions of complex I inhibitors has been accumulated so far. Recent X-ray crystallographic structural models of entire complex I may be helpful to carefully interpret this data. We herein focused on recent hot topics on complex I inhibitors and the subjects closely connected to these inhibitors, which may provide useful information not only on the structural and functional aspects of complex I, but also on drug design targeting this enzyme. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

Mitochondrial Respiratory Dysfunction Induces Claudin-1 Expression via Reactive Oxygen Species-mediated Heat Shock Factor 1 Activation, Leading to Hepatoma Cell Invasiveness

Although mitochondrial dysfunction has been implicated in tumor metastasis, it is unclear how it regulates tumor cell aggressiveness. We have reported previously that human hepatoma cells harboring mitochondrial defects have high tumor cell invasion activity via increased claudin-1 (Cln-1) expression. In this study, we demonstrated that mitochondrial respiratory defects induced Cln-1 transcription via reactive oxygen species (ROS)-mediated heat shock factor 1 (HSF1) activation, which contributed to hepatoma invasiveness. We first confirmed the inverse relationship between mitochondrial defects and Cln-1 induction in SNU hepatoma cells and hepatocellular carcinoma tissues. We then examined five different respiratory complex inhibitors, and complex I inhibition by rotenone most effectively induced Cln-1 at the transcriptional level. Rotenone increased both mitochondrial and cytosolic ROS. In addition, rotenone-induced Cln-1 expression was attenuated by N-acetylcysteine, an antioxidant, and exogenous H2O2 treatment was enough to increase Cln-1 transcription, implying the involvement of ROS. Next we found that ROS-mediated HSF1 activation via hyperphosphorylation was the key event for Cln-1 transcription. Moreover, the Cln-1 promoter region (from -529 to +53) possesses several HSF1 binding elements, and this region showed increased promoter activity and HSF1 binding affinity in response to rotenone treatment. Finally, we demonstrated that the invasion activity of SNU449 cells, which harbor mitochondrial defects, was blocked by siRNA-mediated HSF1 knockdown. Taken together, these results indicate that mitochondrial respiratory defects enhance Cln-1-mediated hepatoma cell invasiveness via mitochondrial ROS-mediated HSF1 activation, presenting a potential role for HSF1 as a novel mitochondrial retrograde signal-responsive transcription factor to control hepatoma cell invasiveness.