Therapeutic Targeting of Mitochondrial Plasticity and Redox Control to Overcome Cancer Chemoresistance.

Significance: Mitochondria are subcellular organelles performing essential metabolic functions contributing to cellular bioenergetics and regulation of cell growth or death. The basic mitochondrial function in fulfilling the need for cell growth and vitality is evidenced whereby cancer cells with depleted mitochondrial DNA (rho zero, p0 cells) no longer form tumors until newly recruited mitochondria are internalized into the rho zero cells. Herein lies the absolute dependency on mitochondria for tumor growth. Hence, mitochondria are key regulators of cell death (by apoptosis, necroptosis, or other forms of cell death) and are, therefore, important targets for anticancer therapy. Recent Advances: Mitochondrial plasticity regulating their state of fusion or fission is key to the chemoresistance properties of cancer cells by promoting pro-survival pathways, enabling the mitochondria to mitigate against the cellular stresses and extreme conditions within the tumor microenvironment caused by chemotherapy, hypoxia, or oxidative stress. Critical Issues: This review discusses many characteristics of mitochondria, the processes and pathways controlling the dynamic changes occurring in the morphology of mitochondria, the roles of reactive oxygen species, and their relationship with mitochondrial fission or fusion. It also examines the relationship of redox to mitophagy when mitochondria become compromised and its effect on cancer cell survival, stemness, and the changes accompanying malignant progression from primary tumors to metastatic disease. Future Directions: A challenging question that arises is whether the changes in mitochondrial dynamics and their regulation can provide opportunities for improving drug targeting during cancer treatment and enhancing survival outcomes. Antioxid. Redox Signal. 39, 591-619.

Targeting the redox imbalance in mitochondria: A novel mode for cancer therapy.

Changes in reactive oxygen species (ROS) levels affect many aspects of cell behavior. During carcinogenesis, moderate ROS production modifies gene expression to alter cell function, elevating metabolic activity and ROS. To avoid extreme ROS-activated death, cancer cells increase antioxidative capacity, regulating sustained ROS levels that promote growth. Anticancer therapies are exploring inducing supranormal, cytotoxic oxidative stress levels either inhibiting antioxidative capacity or promoting excess ROS to selectively destroy cancer cells, triggering mechanisms such as apoptosis, autophagy, necrosis, or ferroptosis. This review exemplifies pro-oxidants (natural/synthetic/repurposed drugs) and their clinical significance as cancer therapies providing revolutionary approaches.

Tea tree oil extract causes mitochondrial superoxide production and apoptosis as an anticancer agent, promoting tumor infiltrating neutrophils cytotoxic for breast cancer to induce tumor regression.

The antitumor activity of the tea tree oil (TTO) derived product, Melaleuca Alternifolia Concentrate (MAC) was characterized mechanistically at the molecular and cellular level. MAC was analyzed for its anticancer activity against human prostate (LNCaP) and breast (MCF-7) cancer cell lines growing in vitro. MAC (0.02-0.06% v/v) dose-dependently induced the intrinsic (mitochondrial) apoptotic pathway in both the LNCaP and MCF-7 cell lines, involving increased mitochondrial superoxide production, loss of mitochondrial membrane potential (MMP), caspase 3/7 activation, as well as the presence of TUNEL+ and cleaved-PARP+ cell populations. At concentrations of 0.01-0.04% v/v, MAC caused cell cycle arrest in the G0/1-phase, as well as autophagy. The in vivo anticancer actions of MAC were examined as a treatment in the FVB/N c-Neu murine model for spontaneously arising breast cancers. Intratumoral MAC injections (1-4% v/v) significantly suppressed tumor progression in a dose-dependent manner and was associated with greater levels of tumor infiltrating neutrophils exhibiting anticancer cytotoxic activity. Induction of breast cancer cell death by MAC via the mitochondrial apoptotic pathway was also replicated occurring in tumors treated in vivo. In conclusion, our data highlights the potential for the Melaleuca-derived MAC product inducing anticancer neutrophil influx, supporting its application as a novel therapeutic agent.

Phase I/II parallel double-blind randomized controlled clinical trial of perispinal etanercept for chronic stroke: improved mobility and pain alleviation.

Background: Previous open-label studies showed that chronic post-stroke pain could be abated by treatment with perispinal etanercept, although these benefits were questioned. A randomized double-blind placebo controlled clinical trial was conducted to test perispinal etanercept for chronic post-stroke pain.Research design and methods: Participants received two treatments, either perispinal etanercept (active) or saline (control). Primary outcomes were the differences in daily pain levels between groups analyzed by SPSS.Results: On the 0-100 points visual analog scale, perispinal etanercept reduced mean levels for worst and average daily pain from baseline after two treatments by 19.5 - 24 points (p < 0.05), and pain alleviation was maintained in the etanercept group, with no significant change in the control group. Thirty percent of etanercept participants had near complete pain abatement after first treatment. Goniometry of the paretic arm showed improved mean shoulder rotation by 55 degrees in active forward flexion for the etanercept group (p = 0.003) only.Conclusions: Perispinal etanercept can provide significant and ongoing benefits for the chronic post-stroke management of pain and greater shoulder flexion by the paretic arm. Effects are rapid and highly significant, supporting direct action on brain function.Trial registration: ACTRN12615001377527 and Universal Trial Number U1111-1174-3242.

Repurposing drugs as pro-oxidant redox modifiers to eliminate cancer stem cells and improve the treatment of advanced stage cancers.

Over the last decade, three major advances have contributed in improving the response rates against cancer including, immunotherapy; greater understanding of the molecular, biochemical, and cellular mechanisms in carcinogenesis thereby providing drug targets; and identification of reliable biomarkers for early detection to facilitate the earlier stage treatment of disease. However, no single universal cancer cure has yet been found, although combinations from the above areas have steadily improved survival outcomes. Hence, chemotherapy remains a key component in the oncologist's arsenal for cancer therapy, despite frequent development of drug resistance and more aggressive cancers with onset of advanced stage metastases. The focus here is to explore the repurposing of old drugs that cause pro-oxidative overload to overcome onset of resistance to chemotherapy and enhance chemotherapeutic responses, particularly against metastatic cancer. Excellent examples of US Food and Drug Administration approved drugs suitable for repurposing are the potent and specific thioreductase inhibitor auranofin and the nonsteroidal anti-inflammatory drug, celecoxib. Recently, both drugs were shown to selectively target and kill metastatic cancer cells and cancer stem cells (CSCs), predominantly by promoting excessive mitochondrial reactive oxygen species. Thus, targeting intracellular redox systems of advanced stage metastatic cancer cells and CSCs can promote an overload of pro-oxidative stress to activate the intrinsic pathway for programmed cell death. It is envisaged that more clinical studies will incorporate longer term use of repurposed drugs, such as auranofin or celecoxib, to target redox systems in cancer cells as part of common practice postcancer diagnosis, providing enhanced chemotherapeutic responses and increased cancer survival.

Increased All-Cause Mortality by Antipsychotic Drugs: Updated Review and Meta-Analysis in Dementia and General Mental Health Care.

It is almost ten years since the Banerjee 2009 report established that inappropriate prescribing of antipsychotics in the elderly was occurring in the UK and such patients had an 85% increased risk of adverse events and greater mortality. This report was a critical analysis addressing the outcomes of treatment practices for dementia in UK patients and globally, aimed at reducing prescribing of antipsychotic drugs for dementia. Since 2009, many significant studies worldwide (including several more recent large retrospective studies) provide more extensive longitudinal data for the adverse impacts of antipsychotic drugs in dementia. We have used the data in these studies including from over 380,000 dementia patients, with 85,069 prescribed antipsychotic agents as well as from 359,235 non-dementia antipsychotic drug users to provide an up-dated meta-analysis. This is the first meta-analysis to include evidence from general mental health studies showing that antipsychotic drugs precipitate excessive mortality across the spectrum. Prescribing of antipsychotic drugs for dementia or for other mental health care should be avoided and alternative means sought for handling behavioral disorders of such patients.

Novel STAT binding elements mediate IL-6 regulation of MMP-1 and MMP-3.

Dynamic remodelling of the extracellular matrix (ECM) is a key feature of cancer progression. Enzymes that modify the ECM, such as matrix metalloproteinases (MMPs), have long been recognised as important targets of anticancer therapy. Inflammatory cytokines are known to play a key role in regulating protease expression in cancer. Here we describe the identification of gamma-activated site (GAS)-like, signal transducer and activator of transcription (STAT) binding elements (SBEs) within the proximal promoters of the MMP-1 and MMP-3 genes, which in association with AP-1 components (c-Fos or Jun), bind STAT-1 in a homodimer like complex (HDLC). We further demonstrate that MMP expression and binding of this complex to SBEs can either be enhanced by interleukin (IL)-6, or reduced by interferon gamma (IFN-γ), and that IL-6 regulation of MMPs is not STAT-3 dependent. Collectively, this data adds to existing understanding of the mechanism underlying cytokine regulation of MMP expression via STAT-1, and increases our understanding of the links between inflammation and malignancy in colon cancer.

The in vitro and in vivo antiviral properties of combined monoterpene alcohols against West Nile virus infection.

West Nile Virus (WNV) is a mosquito-borne flavivirus that can cause neuroinvasive disease in humans and animals for which no therapies are currently available. We studied an established combination of monoterpene alcohols (CMA) derived from Melaleuca alternifolia, against WNV infection. The in vitro results show that CMA exhibits virucidal activity, as well as reduces the viral titres and percentage of infected cells. The antiviral mechanism of action of CMA was studied. We found that CMA did not alter the intracellular pH, neither induced apoptosis, but did induce cell cycle arrest in the G0/G1-phase although that was not the antiviral mechanism. Furthermore, we tested CMA in vivo using IRF 3(-)(/)(-)/7(-/-)mice and it was found that CMA treatment significantly delayed morbidity due to WNV infection, reduced the loss of body weight and reduced the viral titres in brain. These findings suggest that CMA could be a therapeutic agent against WNV infection.

Redox state influence on human galectin-1 function.

Intracellular and extracellular functions of human galectin-1 are influenced by its redox surroundings due to the presence of six cysteines within its amino acid sequence. Galectin-1 recognises intracellular-membrane-anchored Ras proteins that act as molecular switches regulating multiple signal transduction pathways. Human tumours frequently express Ras proteins that have become continuously activated due to point mutations, and this typically leads to deregulation of tumour cell growth, angiogenesis and invasion of metastatic cancer cells. Of significance is that galectin-1 preferably recognises H-Ras, one of the human Ras isoforms, and in particular galectin-1 recognition of the H-Ras farnesyl moiety is paramount to H-Ras membrane anchorage, a prerequisite step for H-Ras-mediated signal transduction regulating normal cell growth and malignant transformation. Herein the impact of the redox state on galectin-1's ability to interact with farnesyl analogues is explored. We demonstrate for the first time that reduced galectin-1 directly binds farnesyl and does so in a carbohydrate-independent manner. A K28T mutation abolishes farnesyl recognition by reduced dimeric galectin-1 whilst its carbohydrate-binding activity is retained, thus demonstrating the presence of an independent region on galectin-1 pertaining to growth inhibitory activity. Intriguingly, oxidised galectin-1 also recognises farnesyl, the biological implication of this novel finding is yet to be elucidated. Further, the redox effect on galectin-1 extracellular function was investigated and we discover that oxidised galectin-1 demonstrates a protective effect upon acute lymphoblastic leukaemia cells challenged by oxidative stress.

Immunomodulatory activity of Melaleuca alternifolia concentrate (MAC): inhibition of LPS-induced NF-κB activation and cytokine production in myeloid cell lines.

Melaleuca alternifolia concentrate (MAC) is a mixture predominantly composed of monoterpenoids and sesquiterpenes, refined from the essential oil of the tea tree by removing up to 99% of the more toxic, hydrophobic monoterpenes. MAC was examined here for its immunomodulatory effects on the human THP1 and murine RAW264.7 myeloid leukemic cell lines as models for macrophage-like cells. Firstly, MAC levels were determined that did not affect either the survival or proliferation of these cell lines in vitro. Next, the levels of lipopolysaccharide (LPS)-induced production of cytokines (IL-6, TNFα, IL-10, GM-CSF, IFNγ and IL-3) were examined from the myeloid cell lines using multiplex assays. Many of the LPS-inducible cytokines produced by either cell lines could be significantly inhibited by MAC. Closer examination of the mechanism of action of MAC showed that it inhibited the LPS-induced activation of IκB phosphorylation and nuclear factor (NF)-κB signalling and translocation, inhibiting iNOS protein expression and NO production. These results demonstrate that MAC exerts its immunomodulatory effects by inhibiting NF-κB signalling activation and levels of cytokine production by macrophage-like cell lines.

Development of a potent melanoma vaccine capable of stimulating CD8(+) T-cells independently of dendritic cells in a mouse model.

At present, there are no vaccines approved for the prevention or treatment of malignant melanoma, despite the amount of time and resources that has been invested. In this study, we aimed to develop a self-contained vaccine capable of directly stimulating anticancer CD8(+) T-cell immune responses. To achieve this, three whole-cell melanoma vaccines were developed expressing 4-1BBL or B7.1 T-cell co-stimulatory molecules individually or in combination. The ability of engineered vaccine cell lines to stimulate potent anticancer immune responses in C57BL/6 mice was assessed. Mice vaccinated with cells overexpressing both 4-1BBL and B7.1 (B16-F10-4-1BBL-B7.1-IFNγ/ß anticancer vaccine) displayed the greatest increases in CD8(+) T-cell populations (1.9-fold increase versus control within spleens), which were efficiently activated following antigenic stimulation, resulting in a 10.7-fold increase in cancer cell cytotoxicity relative to control. The enhanced immune responses in B16-F10-4-1BBL-B7.1-IFNγ/ß-vaccinated mice translated into highly efficient rejection of live tumour burdens and conferred long-term protection against repeated tumour challenges, which were likely due to enhanced effector memory T-cell populations. Similar results were observed when dendritic cell (DC)-deficient LTα(-/-) mice were treated with the B16-F10-4-1BBL-B7.1-IFNγ/ß anticancer vaccine, suggesting that the vaccine can directly stimulate CD8(+) T-cell responses in the context of severely reduced DCs. This study shows that the B16-F10-4-1BBL-B7.1-IFNγ/ß anticancer vaccine acted as a highly effective antigen-presenting cell and is likely to be able to directly stimulate CD8(+) T-cells, without requiring co-stimulatory signals from either CD4(+) T-cells or DCs, and warrants translation of this technology into the clinical setting.

Who controls the ATP supply in cancer cells? Biochemistry lessons to understand cancer energy metabolism.

Applying basic biochemical principles, this review analyzes data that contrasts with the Warburg hypothesis that glycolysis is the exclusive ATP provider in cancer cells. Although disregarded for many years, there is increasing experimental evidence demonstrating that oxidative phosphorylation (OxPhos) makes a significant contribution to ATP supply in many cancer cell types and under a variety of conditions. Substrates oxidized by normal mitochondria such as amino acids and fatty acids are also avidly consumed by cancer cells. In this regard, the proposal that cancer cells metabolize glutamine for anabolic purposes without the need for a functional respiratory chain and OxPhos is analyzed considering thermodynamic and kinetic aspects for the reductive carboxylation of 2-oxoglutarate catalyzed by isocitrate dehydrogenase. In addition, metabolic control analysis (MCA) studies applied to energy metabolism of cancer cells are reevaluated. Regardless of the experimental/environmental conditions and the rate of lactate production, the flux-control of cancer glycolysis is robust in the sense that it involves the same steps: glucose transport, hexokinase, hexosephosphate isomerase and glycogen degradation, all at the beginning of the pathway; these steps together with phosphofructokinase 1 also control glycolysis in normal cells. The respiratory chain complexes exert significantly higher flux-control on OxPhos in cancer cells than in normal cells. Thus, determination of the contribution of each pathway to ATP supply and/or the flux-control distribution of both pathways in cancer cells is necessary in order to identify differences from normal cells which may lead to the design of rational alternative therapies that selectively target cancer energy metabolism.

Indoleamine-2,3-dioxygenase elevated in tumor-initiating cells is suppressed by mitocans.

Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs, with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms, depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans, represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES), suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II, involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein.

Human T cell leukemia virus type I tax-induced IκB-ζ modulates tax-dependent and tax-independent gene expression in T cells.

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL) and various inflammatory disorders including HTLV-I-associated myelopathy/tropical spastic paraparesis. HTLV-I oncoprotein Tax is known to cause permanent activation of many cellular transcription factors including nuclear factor-κB (NF-κB), cyclic adenosine 3',5'-monophosphate response element-binding protein, and activator protein 1 (AP-1). Here, we show that NF-κB-binding cofactor inhibitor of NF-κB-ζ (IκB-ζ) is constitutively expressed in HTLV-I-infected T cell lines and ATL cells, and Tax transactivates the IκB-ζ gene, mainly through NF-κB. Microarray analysis of IκB-ζ-expressing uninfected T cells demonstrated that IκB-ζ induced the expression of NF-κB. and interferon-regulatory genes such as B cell CLL/lymphoma 3 (Bcl3), guanylate-binding protein 1, and signal transducer and activator of transcription 1. The transcriptional activation domain, nuclear localization signal, and NF-κB-binding domain of IκB-ζ were required for Bcl3 induction, and IκB-ζ synergistically enhanced Tax-induced Bcl3 transactivation in an NF-κB-dependent manner. Interestingly, IκB-ζ inhibited Tax-induced NF-κB, AP-1 activation, and HTLV-I transcription. Furthermore, IκB-ζ interacted with Tax in vitro and this interaction was also observed in an HTLV-I-transformed T cell line. These results suggest that IκB-ζ modulates Tax-dependent and Tax-independent gene transcription in T cells. The function of IκB-ζ may be of significance in ATL genesis and pathogenesis of HTLV-I-associated diseases.

HIF expression and the role of hypoxic microenvironments within primary tumours as protective sites driving cancer stem cell renewal and metastatic progression.

Hypoxic microenvironments frequently exist in many solid tumours with oxygen levels fluctuating temporally and spatially from normoxia to hypoxia. The response to hypoxia in human cells is mainly regulated by hypoxia-inducible factors (HIFs), a family of transcription factors which orchestrate signalling events leading to angiogenesis and tumorigenesis. Several events conspire together to lead to the stabilization of HIF-α, commonly expressed in many cancer cell types. These events can result from low oxygen tensions occurring within the expanding tumour mass to produce hypoxic microenvironments or from mutations whereby the HIFs cause changes in expression of genes involved in several cellular functions. Hypoxia-mediated HIF-α regulation has gained significant prominence in tumour biology over recent years, and the hypoxic microenvironments have been shown to facilitate and trigger major molecular and immunological processes necessary to drive the progression of tumours to malignancy. More recently, it has been realized that the hypoxic microenvironments also play significant roles in shielding tumour cells from immune attack by promoting immune suppression. In addition, the hypoxic microenvironment promotes many other oncogenic events, such as the metabolic reconfiguration of tumour cells, neovascularization, epithelial to mesenchymal transition (EMT), and cancer stem cell renewal and accumulation. This article reviews the molecular mechanisms underlying tumour hypoxia and their pro-tumour contributions, such as immune suppression, development of nascent and more permeable tumour vasculature, selective cancer stem cell renewal, accumulation, mobilization and promotion of EMT leading to tumour cell metastasis.

REST negatively and ISGF3 positively regulate the human STAT1 gene in melanoma.

STAT1 plays a pivotal role in signal transduction and transcriptional activation in response to type I and II IFNs. Regulation of STAT1 expression has significant consequences in human cancer cells, where STAT1 deficiencies have been associated with cellular resistance to type I IFN. Distinct promoter, enhancer, and repressor regions have previously been described in the regulatory part of the human STAT1 gene extending as far as the second intron. A putative IFN-stimulated response element sequence in the STAT1 promoter is inducible by type I IFN and binds the IFN-α/ß-induced complex, ISGF3. Together with the previously characterized IRF-E/GAS/IRF-E (IGI) motif, these positive regulatory elements provide a means for intracellular amplification of STAT1 expression, which is necessary for increasing cell responsiveness to the IFNs. In contrast, the transcriptional repressor REST binds to an RE-1 element in the STAT1 repressor region and in doing so represses transcription from the STAT1 gene regulatory region in melanoma cells lines. Repression significantly decreased in a REST-null cell line. Altering REST function from a transcriptional repressor into an activator as REST-VP16 increased expression from RE-1-targeted reporters. RNA expression of 65 melanoma cell lines by microarray and selected lines with known IFN responsiveness showed significant inverse correlations between STAT1/REST that were related to cellular responses to IFN. Thus REST, through the intronic RE-1 element, provides a means for downregulating STAT1 expression, affecting melanoma responsiveness to IFN. Intracellular levels of REST may be a useful marker to test for IFN resistance and as a novel therapeutic target in IFN-resistant melanomas.

Classification of mitocans, anti-cancer drugs acting on mitochondria.

Mitochondria have emerged as an intriguing target for anti-cancer drugs, inherent to vast majority if not all types of tumours. Drugs that target mitochondria and exert anti-cancer activity have become a focus of recent research due to their great clinical potential (which has not been harnessed thus far). The exceptional potential of mitochondria as a target for anti-cancer agents has been reinforced by the discouraging finding that even tumours of the same type from individual patients differ in a number of mutations. This is consistent with the idea of personalised therapy, an elusive goal at this stage, in line with the notion that tumours are unlikely to be treated by agents that target only a single gene or a single pathway. This endows mitochondria, an invariant target present in all tumours, with an exceptional momentum. This train of thoughts inspired us to define a class of anti-cancer drugs acting by way of mitochondrial 'destabilisation', termed 'mitocans'. In this communication, we define mitocans (many of which have been known for a long time) and classify them into several classes based on their molecular mode of action. We chose the targets that are of major importance from the point of view of their role in mitochondrial destabilisation by small compounds, some of which are now trialled as anti-cancer agents. The classification starts with targets at the surface of mitochondria and ending up with those in the mitochondrial matrix. The purpose of this review is to present in a concise manner the classification of compounds that hold a considerable promise as potential anti-cancer drugs.

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.