Green tea catechins EGCG and ECG enhance the fitness and lifespan of Caenorhabditis elegans by complex I inhibition.

Green tea catechins are associated with a delay in aging. We have designed the current study to investigate the impact and to unveil the target of the most abundant green tea catechins, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). Experiments were performed in Caenorhabditis elegans to analyze cellular metabolism, ROS homeostasis, stress resistance, physical exercise capacity, health- and lifespan, and the underlying signaling pathways. Besides, we examined the impact of EGCG and ECG in isolated murine mitochondria. A concentration of 2.5 µM EGCG and ECG enhanced health- and lifespan as well as stress resistance in C. elegans. Catechins hampered mitochondrial respiration in C. elegans after 6-12 h and the activity of complex I in isolated rodent mitochondria. The impaired mitochondrial respiration was accompanied by a transient drop in ATP production and a temporary increase in ROS levels in C. elegans. After 24 h, mitochondrial respiration and ATP levels got restored, and ROS levels even dropped below control conditions. The lifespan increases induced by EGCG and ECG were dependent on AAK-2/AMPK and SIR-2.1/SIRT1, as well as on PMK-1/p38 MAPK, SKN-1/NRF2, and DAF-16/FOXO. Long-term effects included significantly diminished fat content and enhanced SOD and CAT activities, required for the positive impact of catechins on lifespan. In summary, complex I inhibition by EGCG and ECG induced a transient drop in cellular ATP levels and a temporary ROS burst, resulting in SKN-1 and DAF-16 activation. Through adaptative responses, catechins reduced fat content, enhanced ROS defense, and improved healthspan in the long term.

Partial inhibition of mitochondrial complex I ameliorates Alzheimer's disease pathology and cognition in APP/PS1 female mice.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

ITGB2-mediated metabolic switch in CAFs promotes OSCC proliferation by oxidation of NADH in mitochondrial oxidative phosphorylation system.

Objectives: Integrins, the coordinator of extracellular and intracellular signaling, are often found to be aberrant in tumors and can reshape the tumor microenvironment. Although previous studies showed that integrin beta 2 (ITGB2) is important for host defense, its expression profile and role in tumors, especially in cancer associated fibroblasts (CAFs) are still unknown. Methods: Immunofluorescence stain and fluorescence activated cell sorting were used to analyze the ITGB2 expression profile in oral squamous cell carcinoma (OSCC). RT-PCR and western blot were used to compare ITGB2 expression in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs). Clinical data and function-based experiments were used to investigate the promoting tumor growth ability of ITGB2 expressing CAFs. Enhanced glycolysis activity was identified by using bioinformatics analyses and GC/MS assays. MCT1 knockdown OSCC cell lines were constructed to explore the pro-proliferative mechanisms of ITGB2 expressing CAFs in multiple in vitro and in vivo assays. Results: We found that CAFs exhibited significantly higher ITGB2 expression than the matched NFs. In addition, higher ITGB2 expression in CAFs was correlated with higher TNM stages and more Ki67+ tumor cells, indicating its ability to promote OSCC proliferation. Further, co-culture assay demonstrated that ITGB2-mediated lactate release in CAFs promoted OSCC cell proliferation. Mechanically, ITGB2 regulated PI3K/AKT/mTOR pathways to enhance glycolysis activity in CAFs. Accordingly, lactate derived from ITGB2-expressing CAFs was absorbed and metabolized in OSCC to generate NADH, which was then oxidized in the mitochondrial oxidative phosphorylation system (OXPHOS) to produce ATP. Notably, inhibiting the OXPHOS system with metformin delayed the proliferative capacity of OSCC cells cultured in the ITGB2-expressing CAFs medium. Conclusions: Our study uncovered the ITGB2high pro-tumoral CAFs that activated the PI3K/AKT/mTOR axis to promote tumor proliferation in OSCC by NADH oxidation in the mitochondrial oxidative phosphorylation system.

Functional interactions between complex I and complex II with nNOS in regulating cardiac mitochondrial activity in sham and hypertensive rat hearts.

Nitric oxide (NO) affects mitochondrial activity through its interactions with complexes. Here, we investigated regulations of complex I (C-I) and complex II (C-II) by neuronal NO synthase (nNOS) in the presence of fatty acid supplementation and the impact on left ventricular (LV) mitochondrial activity from sham and angiotensin II (Ang-II)-induced hypertensive (HTN) rats. Our results showed that nNOS protein was expressed in sham and HTN LV mitochondrial enriched fraction. In sham, oxygen consumption rate (OCR) and intracellular ATP were increased by palmitic acid (PA) or palmitoyl-carnitine (PC). nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC), did not affect OCR or cellular ATP increment by PA or PC. However, SMTC increased OCR with PA + malonate (a C-II inhibitor), but not with PA + rotenone (a C-I inhibitor), indicating that nNOS attenuates C-I with fatty acid supplementation. Indeed, SMTC increased C-I activity but not that of C-II. Conversely, nNOS-derived NO was increased by rotenone + PA in LV myocytes. In HTN, PC increased the activity of C-I but reduced that of C-II, consequently OCR was reduced. SMTC increased both C-I and C-II activities with PC, resulted in OCR enhancement in the mitochondria. Notably, SMTC increased OCR only with rotenone, suggesting that nNOS modulates C-II-mediated OCR in HTN. nNOS-derived NO was partially reduced by malonate + PA. Taken together, nNOS attenuates C-I-mediated mitochondrial OCR in the presence of fatty acid in sham and C-I modulates nNOS activity. In HTN, nNOS attenuates C-I and C-II activities whereas interactions between nNOS and C-II maintain mitochondrial activity.

NDUFAB1 confers cardio-protection by enhancing mitochondrial bioenergetics through coordination of respiratory complex and supercomplex assembly.

The impairment of mitochondrial bioenergetics, often coupled with exaggerated reactive oxygen species (ROS) production, is a fundamental disease mechanism in organs with a high demand for energy, including the heart. Building a more robust and safer cellular powerhouse holds the promise for protecting these organs in stressful conditions. Here, we demonstrate that NADH:ubiquinone oxidoreductase subunit AB1 (NDUFAB1), also known as mitochondrial acyl carrier protein, acts as a powerful cardio-protector by conferring greater capacity and efficiency of mitochondrial energy metabolism. In particular, NDUFAB1 not only serves as a complex I subunit, but also coordinates the assembly of respiratory complexes I, II, and III, and supercomplexes, through regulating iron-sulfur biosynthesis and complex I subunit stability. Cardiac-specific deletion of Ndufab1 in mice caused defective bioenergetics and elevated ROS levels, leading to progressive dilated cardiomyopathy and eventual heart failure and sudden death. Overexpression of Ndufab1 effectively enhanced mitochondrial bioenergetics while limiting ROS production and protected the heart against ischemia-reperfusion injury. Together, our findings identify that NDUFAB1 is a crucial regulator of mitochondrial energy and ROS metabolism through coordinating the assembly of respiratory complexes and supercomplexes, and thus provide a potential therapeutic target for the prevention and treatment of heart failure.

Mitochondrial Complex I Inhibition by Metformin Limits Reperfusion Injury.

Transient, reversible blockade of complex I during early reperfusion after ischemia limits cardiac injury. We studied the cardioprotection of high dose of metformin in cultured cells and mouse hearts via the novel mechanism of acute downregulation of complex I. The effect of high dose of metformin on complex I activity was studied in isolated heart mitochondria and cultured H9c2 cells. Protection with metformin was evaluated in H9c2 cells at reoxygenation and at early reperfusion in isolated perfused mouse hearts and in vivo regional ischemia reperfusion. Acute, high-dose metformin treatment inhibited complex I in ischemia-damaged mitochondria and in H9c2 cells following hypoxia. Accompanying the complex I modulation, high-dose metformin at reoxygenation decreased death in H9c2 cells. Acute treatment with high-dose metformin at the end of ischemia reduced infarct size following ischemia reperfusion in vitro and in vivo, including in the AMP kinase-dead mouse. Metformin treatment during early reperfusion improved mitochondrial calcium retention capacity, indicating decreased permeability transition pore (MPTP) opening. Acute, high-dose metformin therapy decreased cardiac injury through inhibition of complex I accompanied by attenuation of MPTP opening. Moreover, in contrast to chronic metformin treatment, protection by acute, high-dose metformin is independent of AMP-activated protein kinase activation. Thus, a single, high-dose metformin treatment at reperfusion reduces cardiac injury via modulation of complex I.

Inducing cancer indolence by targeting mitochondrial Complex I is potentiated by blocking macrophage-mediated adaptive responses.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.

Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future.

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

Regulation of bioenergetics through dual inhibition of aldehyde dehydrogenase and mitochondrial complex I suppresses glioblastoma tumorspheres.

Background: Targeted approaches for treating glioblastoma (GBM) attempted to date have consistently failed, highlighting the imperative for treatment strategies that operate on different mechanistic principles. Bioenergetics deprivation has emerged as an effective therapeutic approach for various tumors. We have previously found that cancer cells preferentially utilize cytosolic NADH supplied by aldehyde dehydrogenase (ALDH) for ATP production through oxidative phosphorylation (OxPhos). This study is aimed at examining therapeutic responses and underlying mechanisms of dual inhibition of ALDH and OxPhos against GBM. Methods: For inhibition of ALDH and OxPhos, the corresponding inhibitors, gossypol and phenformin were used. Biological functions, including ATP levels, stemness, invasiveness, and viability, were evaluated in GBM tumorspheres (TSs). Gene expression profiles were analyzed using microarray data. In vivo anticancer efficacy was examined in a mouse orthotopic xenograft model. Results: Combined treatment of GBM TSs with gossypol and phenformin significantly reduced ATP levels, stemness, invasiveness, and cell viability. Consistently, this therapy substantially decreased expression of genes associated with stemness, mesenchymal transition, and invasion in GBM TSs. Supplementation of ATP using malate abrogated these effects, whereas knockdown of ALDH1L1 mimicked them, suggesting that disruption of ALDH-mediated ATP production is a key mechanism of this therapeutic combination. In vivo efficacy confirmed remarkable therapeutic responses to combined treatment with gossypol and phenformin. Conclusion: Our findings suggest that dual inhibition of tumor bioenergetics is a novel and effective strategy for the treatment of GBM.

The lignan manassantin is a potent and specific inhibitor of mitochondrial complex I and bioenergetic activity in mammals.

Dineolignans manassantin A and B from the plant Saururus cernuus are used in traditional medicine to manage a wide range of ailments such as edema, jaundice, and gonorrhea. Cell-based studies have identified several molecular target candidates of manassantin including NF-κB, MAPK, STAT3, and hypoxia-inducible factor 1α (HIF-1α). It is unclear whether or how these structurally diverse proteins or pathways mediate any of the medical benefits of manassantin in vivo Moreover, it has recently been reported that manassantin causes developmental arrest in zebrafish by inhibiting the mitochondrial complex I, but it is unknown whether manassantin inhibits mitochondrial respiration in intact mammalian cells and live animals. Here, we present direct evidence that manassantin potently and specifically inhibits the mitochondrial complex I and bioenergetic activity in mammalian systems. Manassantin had no effect on complex II- or complex IV-mediated respiration. Of note, it decreased NADH-ubiquinone reductase activity but not the activity of NADH-ferricyanide reductase. Treatment with manassantin reduced cellular ATP levels and concomitantly stimulated AMP-activated protein kinase in vitro and in vivo As an adaptive response to manassantin-induced bioenergetic deficiency, mammalian cells up-regulated aerobic glycolysis, a process mediated by AMP-activated protein kinase (AMPK) independently of HIF-1α. Together these results demonstrate a biologically important activity of manassantin in the control of complex I-mediated respiration and its profound effects on oxygen utilization, energy homeostasis, and glucose metabolism in mammalian cells.

Deguelin and Its Role in Chronic Diseases.

Deguelin is one of four major naturally occurring rotenoids isolated from root extracts and is best recognized as a NADH: ubiquinone oxidoreductase (complex I) inhibitor, resulting in significant alterations in mitochondrial function. Deguelin has also been implicated as a regulator of apoptosis through signaling pathways, such as the (PI3K)/Akt pathway, as well as an initiator of cell cycle arrest. Consequently, this compound has accrued great interest as a potential chemopreventive and chemotherapeutic. Additionally, deguelin exposure has been linked to Parkinson's disease (PD). PD is a neurodegenerative disorder, characterized by a substantial loss of dopaminergic neurons in the substantia nigra, as well the manifestation of symptoms such as bradykinesia, rigidity, and rest tremor. While exploring the genetic impact of PD is imperative, environmental factors, such as exposure to pesticides, herbicides, and insecticides, have also been connected to the development of PD. The etiology and pathogenesis of PD are yet to be fully understood and elucidated, but mitochondrial dysfunction is gaining recognition as a molecular hallmark of PD. In fact, deguelin has been reported to elicit PD-like symptoms (degeneration of the dopaminergic pathway) in rats administered with deguelin (6 mg/kg/day for 6 days), possibly through the inhibition of mitochondrial complex I. Further research investigating the mechanisms by which deguelin inhibits central cellular processes is essential in order to advance any prospective research addressing potential applications and risks of deguelin.

Targeting respiratory complex I to prevent the Warburg effect.

In the last 10 years, studies of energetic metabolism in different tumors clearly indicate that the definition of Warburg effect, i.e. the glycolytic shift cells undergo upon transformation, ought to be revisited considering the metabolic plasticity of cancer cells. In fact, recent findings show that the shift from glycolysis to re-established oxidative metabolism is required for certain steps of tumor progression, suggesting that mitochondrial function and, in particular, respiratory complex I are crucial for metabolic and hypoxic adaptation. Based on these evidences, complex I can be considered a lethality target for potential anticancer strategies. In conclusion, in this mini review we summarize and discuss why it is not paradoxical to develop pharmacological and genome editing approaches to target complex I as novel adjuvant therapies for cancer treatment. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors.

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.

The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics.

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression.

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.

Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition.

PURPOSE: Previous experiments have demonstrated that short-term hyperglycemia in rats renders the retina resistant to subsequent metabolic insults. The present study aimed to elucidate putative mechanisms involved in this protective response. METHODS: Retinal cultures comprising neurons and glia were treated with the mitochondrial complex I inhibitor, rotenone, at a range of concentrations, for up to 24 hours. In some cases, glucose or the alternative energy substrates, pyruvate or lactate, and/or inhibitors of glycolysis or the pentose phosphate pathway (PPP) were also applied. Cell viability was assessed using complementary techniques: immunocytochemistry, immunoblotting, cytotoxicity assay, and TUNEL. Cellular levels of ATP, reactive oxygen species (ROS), and nicotinamide adenine dinucleotide phosphate (NAD[P]H) were also assessed. RESULTS: Rotenone caused the preferential loss of neurons from retinal cultures in a concentration-dependent manner; glial cells were also affected, but only at a higher concentrations (10 µM). Cell loss was by apoptosis, and was preceded by a reduction of both cellular ATP and NAD(P)H levels and an increase in the production of ROS. Glucose counteracted the detrimental effects of rotenone. This involved a reduction in ROS levels and an increase in the cellular ATP/NAD(P)H ratio. The protective effect of glucose was partially reversed by either PPP or glycolysis inhibition. CONCLUSIONS: Glucose rescued cultured rat retinal cells from rotenone-induced toxicity. Glucose acted via both the PPP and the glycolytic pathway, maintaining cellular ATP and NAD(P)H levels and reducing ROS production. These data have implications for treatment of retinal diseases that involve metabolic compromise to neurons.

Inhibition of mitochondrial complex I by various non-steroidal anti-inflammatory drugs and its protection by quercetin via a coenzyme Q-like action.

Mitochondrial dysfunction plays a major role in the development of oxidative stress and cytotoxicity induced by non-steroidal anti-inflammatory drugs (NSAIDs). A major objective of the present study was to investigate whether in vitro the NSAIDs, aspirin, indomethacin, diclofenac, piroxicam and ibuprofen, which feature different chemical structures, are able to inhibit mitochondrial complex I. All NSAIDs were effective inhibitors when added both, directly to mitochondria isolated from rat duodenum epithelium (50 µM) or to Caco-2 cells (250 µM). In the former system, complex I inhibition was concentration-dependent and susceptible to competition and reversion by the addition of coenzyme Q (32.5-520 µM). Based on reports suggesting a potential gastro-protective activity of quercetin, the ability of this flavonoid to protect isolated mitochondria against NSAIDs-induced complex I inhibition was evaluated. Low micromolar concentrations of quercetin (1-20 µM) protected against such inhibition, in a concentration dependent manner. In the case of aspirin, quercetin (5 µM) increased the IC50 by 10-fold. In addition, the present study shows that quercetin (5-10 µM) can behave as a "coenzyme Q-mimetic" molecule, allowing a normal electron flow along the whole electron transporting chain (complexes I, II, III and IV). The exposed findings reveal that complex I inhibition is a common deleterious effect of NSAIDs at the mitochondrial level, and that such effect is, for all tested agents, susceptible to be prevented by quercetin. Data provided here supports the contention that the protective action of quercetin resides on its, here for first time-shown, ability to behave as a coenzyme Q-like molecule.

Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target.

The antitumor agent 11ß (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11ß against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11ß; flow cytometry studies showed that 11ß exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11ß inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11ß blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11ß enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11ß, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11ß, which supplements conventional DNA adduct formation to promote cancer cell death.

Celastrol targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent cytotoxicity in tumor cells.

BACKGROUND: Celastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity. METHODS: The downregulation of heat shock protein 90 (HSP90) client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK), and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS) was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP) and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC) complexes. RESULTS: Celastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC), an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP). Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 µM celastrol in the absence and presence of NAC. Moreover, the inhibition of MRC complex I activity preceded ROS accumulation in H1299 cells after celastrol treatment. CONCLUSION: We identified ROS as the key intermediate for celastrol-induced cytotoxicity. JNK was activated by celastrol-induced ROS accumulation and then initiated mitochondrial-mediated apoptosis. Celastrol induced the downregulation of HSP90 client proteins through ROS accumulation and facilitated ROS accumulation by inhibiting MRC complex I activity. These results identify a novel target for celastrol-induced anticancer activity and define its mode of action.

Berberine as a promising safe anti-cancer agent - is there a role for mitochondria?

Metabolic regulation is largely dependent on mitochondria, which play an important role in energy homeostasis. Imbalance between energy intake and expenditure leads to mitochondrial dysfunction, characterized by a reduced ratio of energy production (ATP production) to respiration. Due to the role of mitochondrial factors/events in several apoptotic pathways, the possibility of targeting that organelle in the tumor cell, leading to its elimination is very attractive, although the safety issue is problematic. Berberine, a benzyl-tetra isoquinoline alkaloid extracted from plants of the Berberidaceae family, has been extensively used for many centuries, especially in the traditional Chinese and Native American medicine. Several evidences suggest that berberine possesses several therapeutic uses, including anti-tumoral activity. The present review supplies evidence that berberine is a safe anti-cancer agent, exerting several effects on mitochondria, including inhibition of mitochondrial Complex I and interaction with the adenine nucleotide translocator which can explain several of the described effects on tumor cells.