Functional linkage between mitochondrial electron transport, glycolysis, and AMP-activated protein kinase signaling underlying cancer cell survival

Functional linkage between mitochondrial electron transport, glycolysis, and AMP-activated protein kinase signaling underlying cancer cell survival | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional linkage between mitochondrial electron transport, glycolysis, and AMP-activated protein kinase signaling underlying cancer cell survival Momoko Uchida, Masato Higugrashi, Hidetsugu Nakagawa, Fumihiro Ishikawa, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8003013/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Mitochondrial metabolism has emerged as a potential target for cancer therapy because of its essential role in cell proliferation and survival beyond ATP production. However, translation into clinical practice is challenging owing to several limitations, including the difficulty in distinguishing cancerous mitochondria from noncancerous mitochondria. The heterogeneity of cancerous mitochondria further complicates this effort. Herein, we focused on cancerous mitochondria exhibiting low DNA/RNA levels and respiratory function compared with those exhibited by normal cells and determined the effect of low mitochondrial respiratory activity on cancer cells. Interestingly, mitochondria low-type (mt-Low) cancer cells derived from hepatocellular carcinoma were selectively and highly sensitive to electron transport chain (ETC) inhibition. Specifically, cells died under the treatment with the ETC inhibitors, rotenone and antimycin A, at lower doses compared with cells exhibiting normal respiratory activity (mt-Normal), although ATP levels were sustained in both types of cells under these conditions. In mt-Normal cells, glycolysis increased and AMP-activated protein kinase was activated upon ETC inhibition, which critically contributed toward cell survival. However, mt-Low cells could not induce these responses, which resulted in cell death. Based on these results, therapeutics targeting respiratory function have emerged as promising precision medicines for mt-Low cancers. Similar to conventional ETC inhibitors, small interfering RNAs targeting core subunits of respiratory complex I or III were effective in inhibiting cell proliferation (complex I and III) and survival (complex I) of mt-Low cancer cells, encouraging pionerring anticancer approaches using the next-generation modality. respiratory chain complex mitochondrial electron transport glycolysis AMP-activated protein kinase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Studies on cancer metabolism, which have been inspired by Otto Warburg’s discovery of aerobic glycolysis, have substantially progressed over the several decades to further elucidate the metabolic phenotype of cancer cells [ 1 , 2 ]. The molecular mechanisms explaining how cancer cells reprogram metabolism to cope with the increased anabolic demands for incessant and durable cell proliferation are more understandable [ 1 – 5 ]. Accordingly, our view on mitochondrial metabolism in cancer cells has also evolved from the original concept that misinterpreted aerobic glycolysis as a consequence of irreversible mitochondrial respiratory defects in cancer cells. Mitochondria in most tumors are intact and actively performing oxidative phosphorylation (OXPHOS) [ 1 , 6 , 7 ]. Furthermore, mitochondrial respiration is indispensable for cell proliferation. Thus, cell proliferation is impeded by pharmacological or genetic inhibition of electron transport chain (ETC), specifically, respiratory complex I and III [ 8 – 12 ]. From a conventional perspectives, ETC activity likely affects cell proliferation through ATP generation and/or reactive oxygen species (ROS) release; however, because of the diverse roles of ETC beyond ATP generation, including metabolite production through TCA cycle flux and pyrimidine synthesis pathway for macromolecule biosynthesis, alternatives are also possible. Recent studies have highlighted the importance of ETC in the production of electron acceptors, which fulfill a specific metabolic requirement to support cell proliferation. For example, de novo synthesis of aspartate, a key biosynthetic precursor of proteins, and purine and pyrimidine nucleotides, is critically dependent on NAD + regeneration by ETC complex I [ 10 , 13 ]. Recently, we found that NAD + regenerated by complex I regulates p21 Cip1 expression and cancer cell proliferation through SIRT activity [ 14 ]. Ubiquinol oxidation to ubiquinone by ETC complex III is also required for tumor growth [ 9 ]. Evidence supporting the role for ETC in cell proliferation has led to attempts that target ETC for cancer therapy; however, the translation of various ETC inhibitors into clinical practice has been challenging. Thus far, clinical trials have failed because of setbacks, such as dose-limiting toxicity, lack of potency and specificity, even when preclinical results were promising [ 15 – 19 ]. These failures have emphasized the importance of discriminating between noncancerous and cancerous mitochondria, or identifying and focusing on specific phenotypes of cancerous mitochondria and targeting their distinct vulnerabilities for the development of safe and effective mitochondrial-based therapies. According to a recent mitochondrial phenotyping study, the expression/function of the OXPHOS complex is consistently lower in cancer tissues compared with matched normal tissues across several cancer types [ 20 ]. Genome-wide analyses of mitochondrial DNA (mtDNA) and mitochondrial RNA (mtRNA) showed a significant decrease in mtDNA copy number on average in 7 of 15 cancer types and a concomitant decrease in mtRNA levels [ 21 ]. These results suggest that the sizeable proportion of cancerous mitochondria exhibit a functionally lower mtDNA/RNA/OXPHOS phenotype compared with their normal counterparts in numerous types of cancers. This difference can be potentially exploited to develop mitochondrial medicines that can differentiate between cancerous and normal mitochondria as well as specifically target cancer cells with lower mitochondrial activity. Our recent study of hepatocellular carcinoma (HCC) cell lines revealed that over half (4 of 7) of them exhibited a mt-Low phenotype with lower mtDNA/RNA and membrane potential compared with non-malignant hepatocytes [ 22 ], which agreed with the previous studies. Similarly, 61% (17 of 28) of in vivo HCC tissues were mt-Low cases [ 22 ]. In this study, we characterized the metabolic traits of these HCC cell lines, especially focusing on mt-Low cells. Interestingly, mt-Low HCC cells are highly and selectively sensitive to ETC inhibition. Mechanistically, the sensitivity of mt-Low cancer cells to ETC inhibition results from the loss of a sufficient functional linkage between ETC, glycolysis, and AMP-activated protein kinase (AMPK) signaling. Materials and methods Materials Antimycin A (AMA) and rotenone (Rot) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Sigma-Aldrich, Inc. (St Louis, MO, USA), respectively. Staurosporine and 2-Deoxy-D-glucose were purchased from Wako Pure Chemical Corporation (Osaka, Japan). Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin A (OligoA) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Compound C and metformin were purchased from Merck KGaA (Darmstadt, Germany) and Enzo Life Sciences, Inc. (Farmingdale, NY, USA), respectively. AICAR and A-769622 (A-7) were purchased from FUJIFILM and Adooq Bioscience LLC (Irvine, CA, USA), respectively. Cell culture The cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) or the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan), and maintained in their respective media for subculture. For the experiments, the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Shimadzu Diagnostics Corp., Tokyo, Japan) supplemented with 10% fetal bovine serum to standardize the nutrient supply. Cell proliferation and viability assay The cells were seeded at a density of 5 × 10 4 or 1 × 10 5 cells in 12-well plate. Total and viable cell numbers were counted with a Countess II Automated Cell Counter (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell viability was assessed by performing trypan blue staining. Measurements of lactate and ATP levels Cells (1.5 × 10 5 or 2 × 10 5 ) were seeded into a single well of a 12-well plate. Lactate released into the culture medium was measured using the Lactate Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The culture supernatant was collected and diluted 10-fold with water and analyzed. The working solution was incubated with the supernatant for 30 min at 37°C, and the absorbance was measured at 450 nm using a Varioskan Lux (Thermo Fisher Scientific Inc.) microplate reader. The remaining cells were lysed, and intracellular ATP levels were measured using an ATP determination kit (Thermo Fisher Scientific, Inc.) as described previously [ 23 ]. Measurement of glucose consumption Cells (0.5 × 10 5 or 1 × 10 5 ) were plated in a single well of a 12-well plate. The following day, the cells were treated with or without reagents after aliquots of the supernatant were collected (sup 0 h). After 12 h, the supernatants (i.e., medium; sup 12 h) were collected from each well, and the cells were counted. Glucose concentrations in the sup (0 h) and (12 h) were measured using the Glucose Assay Kit-WST (Dojindo Laboratories) based on the manufacturer’s instructions, and glucose consumption (µmol/h) was calculated from the difference in glucose levels between sup (0 h) and sup (12 h), normalized with 10⁶ cells. LDH release assay Lactate dehydrogenase (LDH) activity retained in cells and released into the culture media was determined using the LDH-Cytotoxic Test wako (Wako Pure Chemical Industries, Ltd.) according to manufacturer’s instructions. Briefly, 0.5 × 10 5 or 1 × 10 5 cells were seeded into a single well of a 12-well plate and treated with the reagent the following day. After 24–72 h, the medium was centrifuged, and the resulting supernatant and pellets were collected. The remaining cells in each well were solubilized with phosphate buffer solution (PBS) containing 0.2% Tween-20 together with the cells pelleted from the medium. Equivalent aliquots of the supernatant and cell lysate were incubated with the coloring solution after clearing by centrifugation, and the absorbance at 570 nm was measured using a Varioskan Lux (Thermo Fisher Scientific, Inc.). Fresh medium and PBS/0.2% Tween were used as a control for the supernatants and cell lysates, respectively. LDH release was calculated as the ratio of LDH activity in the medium to the total LDH activity. Western blotting Western blotting was performed as previously described [ 22 ]. The primary antibodies used are listed in Table S1 . The specific bands were quantified using ImageJ software (version 1.53 k, National Institutes of Health, Bethesda, MD, USA) to compare densities. RNA interference Small interfering RNA (siRNA) (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands). Transfection of the siRNA was achieved using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Inc.) as described previously [ 14 ]. Statistical analysis All results are reported as the mean ± standard deviation with * P < 0.05, ** P < 0.01, and *** P < 0.001. Comparisons between two groups were analyzed using a two-tailed Student’s t -test. For multiple comparisons, the data were analyzed using one-way ANOVA with Bonferroni’s post hoc correction to identify datasets that differed from the control data. A p-value of < 0.05 was considered significant. Results Selective and high sensitivity of mt-Low HCC cells to ETC inhibition We previously categorized seven HCC cell lines into an mt-Normal or mt-Low phenotype based on normal or low mtDNA/RNA content and membrane potential compared with primary hepatocytes [ 22 ]. Oxygen consumption rates confirmed that the respiratory activities were higher and lower in mt-Normal cells and mt-Low cells, respectively (Fig. S1 ). We further characterized the metabolic properties of the two groups (mt-Normal cells; HepG2, JHH-1, and JHH-6, mt-Low cells; HLF, HuH-7, JHH-2, and JHH-4). Specifically, we disrupted respiratory activity using the established ETC inhibitors, AMA and Rot, which inhibit complexes III and I, respectively, and observed the response of the mt-Normal and mt-Low cells. In the experiments, all cell lines were cultured in DMEM throughout the study to standardize the nutrient supply. The mt-Normal cells were largely tolerable to inhibitor treatment within the concentration range used in the experiments (IC50 of AMA and Rot are 16 nM and 56 nM, respectively [ 24 ]). However, the inhibitors, especially AMA, were remarkably cytotoxic to the mt-Low cells, except JHH-2, at lower doses, where the majority of the mt-Normal cells were alive (Fig. 1 A). Therefore, the sensitivity to the ETC inhibition was markedly different between the two groups. However, the sensitivities to FCCP and OligA, which interfere with ATP synthesis at the final step of OXPHOS, were indistinguishable between the two groups and independent of respiratory levels of the cells (Fig. 1 B). Moreover, they were comparably tolerable to FCCP and OligA treatment over the concentration range used in this study (IC50 of FCCP and OligA; 4.8–8.5 µM and 0.21–3.8 µM, respectively [ 25 , 26 ]). Sensitivity to staurosporine (IC50; 0.3 µM [ 27 ]), a representative toxic substance, was also independent of the groups (Fig. 1 B). Collectively, the results indicated the selective and high sensitivity of mt-Low HCC cells to ETC inhibition. The sensitivity to ETC inhibition is dependent upon a variety of cellular processes that sustain energy homeostasis. Because a major role of ETC is to support ATP synthesis through OXPHOS, the ATP synthetic potential of cells is the most likely determinant of sensitivity to ETC inhibition. However, this was unlikely because ATP levels were sustained for several hours following AMA treatment in mt-Normal and mt-Low cells (Fig. S2 A), possibly because of metabolic plasticity as described below. Moreover, in contrast to the intolerance toward ETC inhibition, the mt-Low and mt-Normal cells were tolerant to direct ATP synthesis inhibition by FCCP and OligA (Fig. 1 B), suggesting that they are equally adaptable for the inhibition of OXPHOS-driven ATP synthesis. Cellular antioxidant capacity is likely an alternative determinant of sensitivity to ETC inhibition. The inhibition of ETC by Rot and AMA accompanies ROS production [ 28 , 29 ]. Accordingly, cells require a sufficient antioxidant defense during ETC inhibition. Therefore, we quantified total glutathione (GSH) levels as a major antioxidant with the ratio of GSH/glutathione disulfide. Both parameters varied among the HCC cell lines at steady-state levels and during AMA treatment, but appeared unrelated to the sensitivity to ETC inhibition or mt-Normal/Low status (Fig. S2 B-C). Among cellular signaling, PI3K/AKT pathway plays an important role in cell survival and is frequently implicated in resistance to toxic substances [ 30 , 31 ]. This may be relevant to atypical resistant phenotypes of JHH-2 to these inhibitors (Fig. 1 ). As shown in Fig. S2 D, AKT was distinctly activated in JHH-2. In conclusion, the sensitivity of the mt-Low HCC cells to ETC inhibition was independent of ATP synthetic potential, antioxidant defense capacity, and PI3K/AKT survival signaling, except JHH-2. Differences in glycolysis availability between mt-Normal and mt-Low HCC cells To gain insight into determinants of sensitivity to ETC inhibition in the mt-Low cells, we compared fundamental metabolic characteristics, glycolytic/mitochondrial ATP synthetic capacity and glycolysis availability, between mt-Normal and mt-Low HCC cells. We first evaluated glycolytic/mitochondrial ATP synthetic capacity by disrupting the two synthetic pathways using 2-deoxy-D-glucose (2-DG) or OligA, which inhibit OXPHOS and glycolytic ATP synthesis, respectively. Consistent with the above observation of sustained ATP levels under ETC inhibition (Fig. S2 A), the results indicated that the cancer cells in both groups are highly metabolically plastic and flexibly generate ATP through glycolysis or OXPHOS in response to the availability of these two pathways (Fig. 2 A). Alternatively, a surplus quantity of ATP may be normally reserved in cells [ 32 ]. Conversely, glycolysis availabilities, which are evaluated by measuring lactate release from cells in the presence of OligA, were different between the two groups. During OligA treatment, in which OXPHOS is inhibited, cells generally enhance glycolysis by mobilizing their reserve capacity for glycolysis to compensate for OXPHOS, releasing more lactate into the medium. Accordingly, the amount of released lactate in the presence of OligA reflects a maximum level of glycolysis availability of cells, and the difference from the control conditions without OligA treatment corresponds to the reserve capacity for glycolysis. In this study, lactate release was significantly increased during OligA treatment in mt-Normal HepG2 and JHH-1cells (Fig. 2 B); however, the release was only marginally increased in mt-Low cells, such as HLF and JHH-4, under these conditions, suggesting that mt-Low cancer cells lack a sufficient reserve capacity for glycolysis or are mechanistically defective in upregulating glycolysis. In summary, the mt-Normal and mt-Low cells were similarly able to synthesize ATP by glycolytic and mitochondrial pathways, but differed in the upregulation of glycolysis during ETC inhibition. Upregulation of glycolysis is important for cell survival during limited ETC activity Based on the above results, the difference between mt-Normal and mt-Low cells with regard to sensitivity for ETC inhibition may be attributed to their difference in glycolysis availability. Consistent with this idea, the mt-Normal cells, which are resistant to ETC inhibition, exhibited enhanced glycolysis during ETC inhibition with AMA (Fig. 3 A), concomitant with increased glucose consumption (Fig. 3 B). However, glycolysis and glucose consumption were only slightly enhanced in mt-Low cells under these conditions (Fig. 3 A, B). These results suggested that if glycolysis were sufficiently upregulated, mt-Low cells could resist ETC inhibition, similar to mt-Normal cells. In fact, the mt-Low cells became tolerant of AMA treatment (Fig. 3 C; viability) when glycolysis was enhanced following glucose supplementation into the medium (Fig. 3 C; lactate assay). Conversely, glycolysis inhibition with 2-DG markedly decreased cell survival (Fig. 3 D). These results highlight the importance of upregulating glycolysis for cell survival during limited ETC activity. In addition to glucose, supplementation with other biosynthetic precursors or intermediates was also effective at mitigating cell death induced by ETC inhibition. Pyruvate and uridine significantly decreased LDH release and cell death at supra-physiological concentrations during ETC inhibition by Rot treatment (Fig. 3 E). When ETC was inhibited by AMA, cell death was partially relieved by pyruvate supplementation (Fig. 3 F). These results suggest that metabolic pathways driven by glucose and other metabolites substitute for ETC function, which is important for cell survival. Activation of AMP-activated protein kinase underlies cell survival during limited ETC activity AMPK is an energy-sensing kinase, which is activated by various metabolic stresses that lower cellular energy levels, and has various roles in cellular energy homeostasis [ 33 ]. In the HCC cell lines, an active form of AMPK phosphorylated at threonine 172 was observed at various levels during ETC inhibition (Fig. 4 A-B). Interestingly, the activation levels of AMPK correlated with the resistance of the HCC cell lines to the inhibitors. For example, the levels were higher in JHH-1, HepG2, and JHH-4 compared with that of HLF during Rot treatment (Fig. 4 A), which mirrored their viability under these conditions (Fig. 1 A). During AMA treatment, JHH-1 and JHH-2, both of which were remarkably resistant to the treatment, exhibited higher activation levels compared with the others (Fig. 4 B). In JHH-1, robust and sustained AMPK activation was consistently observed in response to AMA, whereas AMPK activation was moderate in HuH-7 and JHH-4 cells, even when AMA was treated with higher doses for longer periods (Fig. 4 C-D). These correlations between the activation of AMPK and cell viability suggest that AMPK underlies cell survival during ETC inhibition. To substantiate the role of AMPK in cell survival directly, we inhibited AMPK by an inhibitor and siRNA or activated AMPK with activators, and observed their effects on cell fates. The treatment with compound C, a well-known AMPK inhibitor, reversed the resistance phenotype of the mt-Normal JHH-1 and HepG2 cells during AMA treatment (Fig. 5 A). Similarly, cell survival was reduced by knockdown of AMPK with siRNA (Fig. 5 B). Conversely, the AMPK activator A-7, which potently activated AMPK among all the activators tested in the study, significantly promoted the resistance phenotype of the cells (Fig. 5 C). These results highlight the important role of AMPK activation in cell survival during ETC inhibition. Collectively, it was most likely that glycolysis upregulation and AMPK activation were the determinants of sensitivity to ETC inhibition. Effects of siRNA targeting the subunits of respiratory complex I and III on cell survival and proliferation Clinically, the selective and high sensitivity or vulnerability of mt-Low cells to ETC inhibition may represent a therapeutic target for mt-Low cancers. As substitutes for small molecular inhibitors, including Rot, AMA, and others, for which clinical trials were discontinued, we evaluated the efficacy of siRNAs targeting core subunits of respiratory complex I and III to inhibit cell survival and/or the proliferation of mt-Low HCC cells. We knocked down NDUFV1 (NADH: ubiquinone oxidoreductase core subunit V1) and NDUFS 1 (NADH: ubiquinone oxidoreductase core subunit S1) of complex I, and URCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1) of complex III. NDUFV1 and NDUFS1 constitute the electron input module, which accepts electrons from NADH through flavin mononucleotide and sequentially transfers them along a chain of iron-sulfur (FeS) clusters in complex I. UQCRFS1 is a subunit that receives an electron from ubiquinol and transfers it to cytochrome c 1 , thereby playing an essential enzymatic role in complex III. All three siRNAs downregulated the expression of the target gene (Fig. S3 ) and showed cytostatic effects on mt-Low JHH-4 and HLF cells as evidenced by decreased cell numbers (Fig. 6 A). Moreover, siRNAs for NDUFV1 and NDUFS1 exhibited cytotoxic effects and decreased cell viability. These results suggest that siRNAs targeting the core subunits of complex I and III, in particular those of complex I, are promising therapeutics for the mt-Low-type cancers. Discussion For the successful development of mitochondrial-targeted therapeutics, it is necessary to characterize cancerous mitochondria in detail, identify distinctive and druggable features or vulnerabilities, and target them specifically. Based on previous studies, cancerous mitochondria exhibit heterogeneous mtDNA/RNA content and OXPHOS function [ 20 , 21 , 34 , 35 ]. Toward the goal of developing mitochondrial precision medicine, we focused on the mt-Low-type of cancer, which occurs in approximately half of all cancer types [ 21 , 22 ]. We found that the mt-Low-type of cancer cells are distinctively vulnerable to the ETC inhibition. Previously, the preferential effect of OXPHOS inhibition on mitochondria was a concern with respect to normal OXPHOS activity in healthy organs, particularly those with a high reliance on OXPHOS, rather than tumor mitochondrial flux [ 20 ]. Our results indicate that it is possible to preferentially compromise tumor mitochondria flux while sparing healthy cells by targeting ETC functions in the case of mt-Low-type cancers. Mechanistically, mt-Low cancer cells are unable to upregulate glycolysis efficiently during ETC inhibition, resulting in cell death. The mt-Low cells may be forced to leverage glycolytic flux at nearly maximum levels because of their intrinsically low respiratory activity; therefore, it is likely that they cannot afford to upregulate glycolysis in response to ETC inhibition. By supplementing the culture medium with glucose, glycolysis was upregulated in mt-Low cells, and cell death was mitigated (Fig. 3 D). This highlights the importance of glycolysis upregulation for cell survival under ETC-limited conditions. Supplementation with pyruvate also ameliorated cell death induced by the ETC inhibition. Uridine was also effective in rescuing Rot-treated cells (Fig. 3 E). In AMA-treated cells, only pyruvate was effective (Fig. 3 F). The difference in effectiveness of uridine in relieving cell death induced by Rot or AMA may be attributed to the difference in TCA metabolite abundance, which was affected by Rot and AMA treatment as previously reported [ 10 ]. According to a recent study [ 36 ], OXPHOS dysfunction resulted in higher lactate/lower pyruvate abundance, lower levels of TCA intermediates and aspartic acid, and increased glycerol-3-phosphate levels. Interestingly, these metabolite imbalances were normalized by combined supplementation of pyruvate and uridine. Taken together, these beneficial effects of nutrient supplementation on cell viability during ETC inhibition suggest the compensatory network between ETC functions, glycolysis, and pyruvate/uridine metabolism. Therefore, a functional linkage has emerged between glycolysis, pyruvate/uridine metabolic pathways, and respiratory capacities, which is important as a safeguard for cell survival during ETC dysfunction. AMPK coordinates metabolic pathways and maintains energy homeostasis through the phosphorylation of various substrates [ 33 ]. It also promotes mitochondrial biogenesis and dynamics [ 37 ]. By performing these roles, AMPK contributes to cell survival under low-energy conditions and mitochondrial dysfunction. In this study, the mt-Normal cells activated the kinase in response to ETC inhibition as previously reported (Fig. 4 A, B) [ 33 , 37 – 39 ]. Conversely, the mt-Low HCC cells were unable to activate AMPK. A series of experiments using the inhibitor, the activators, and siRNA support the role for AMPK in cell survival during ETC inhibition. Although AMP is the most common activator of AMPK, a non-canonical mechanism independent of AMP [ 33 ] is suggested to be predominantly involved in activating AMPK in the ETC-inhibited HCC cells, because the ATP levels were unchanged (Fig. S2 A) and among the activators tested, A-7 was the most prominent activator in the HCC cells. Unlike AICAR and metformin, A-7 activates AMPK by a different mechanism than AMP [ 40 , 41 ]. Recently, glycolysis was found to be a key regulator of AMPK activation [ 42 ]. In this study, a correlation was observed between the ability of cells to activate AMPK and upregulate glycolysis, suggesting that the glycolytic pathway regulates its activation. However, in another report, AMPK activation was suggested to upregulate glycolysis [ 43 , 44 ]. Thus, the relationship between glycolysis and AMPK activation is context-dependent and requires further study. Oligonucleotide therapeutics represent a next-generation modality in medicine. In this study, we evaluated potential and efficacy of siRNAs targeting respiratory complex I and III for anticancer therapeutics. Complex I was targeted by siRNAs against the core subunits, NDUFV1 and NDUFS1 . Treatment with these siRNAs inhibits NADH oxidation and electron transfer along a chain of FeS clusters, which is the initial step in electron transfer for complex I [ 45 ]. Rot inhibits ubiquinone reduction following electron transfer [ 46 ]. The two siRNAs decreased cell viability, similar to Rot (Fig. 6 A). siRNA targeting UQCRFS1 , the Rieske FeS protein in complex III, was also effective in inhibiting cell proliferation; however, it could not induce cell death. AMA has both cytostatic and cytotoxic effects. Complex III ( bc 1 complex; ubiquinol cytochrome c oxidoreductase) contains three redox subunits along with the Rieske FeS protein. It catalyzes electron transfer from ubiquinol to cytochrome c . AMA inhibits electron transfer between the redox subunits, cytochrome b and c [ 47 ], whereas UQCRFS1 knockdown inhibits electron transfer between ubiquinol and cytochrome c 1 [ 48 ]. These results suggest that electron transfer between cytochrome b and c is critical for cell survival rather than that between ubiquinol and cytochrome c 1 . In conclusion, this study encourages continuous efforts in pursuing an ETC inhibition approach as an effective anticancer strategy. It is worth re-attempting the clinical trials with the previous inhibitors by restricting the study to a patient cohort/subset with the mt-Low-type cancer. Along with small molecule inhibitors, siRNAs targeting complex I and III exerted cytostatic and/or cytotoxic effects on HCC cells, particularly on mt-Low cells. After massive but unfruitful efforts at developing ETC-targeted medicine, pioneering approaches from a different perspective are expected in this field. Abbreviations A-7, A769622; AMA, antimycin A; AMPK, AMP-activated protein kinase; C.C, compound C; ETC, electron transport chain; FeS, iron-sulfur; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; GSH, glutathione; GSSG, glutathione disulfide; HCC, hepatocellular carcinoma; OligA, oligomycin A; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; Rot, rotenone; siRNA, small interfering RNA; TCA, tricarboxylic acid cycle; 2-DG, 2-deoxy-D-glucose. Declarations Acknowledgements: We thank Yui Shikama, Yuki Tsuchiya, Kohei Naka, Saki Fujiwara, Ken Hirabayashi, Yudai Suto, Misato Narisawa, Maho Betsuyaku, Eiji Funayama, Yoshihiro Watanabe, Takane Watari, Saki Kikuchi, and Ayaka Saito for their contribution to this work as part of their bachelor’s degrees. We would also like to thank Enago (www.enago.jp) for the English language review. Authorship contributions: Uchida M: Investigation, Data curation, Writing–original draft. 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Chem Biol Drug Des 83(1):71–80 Fernandez-Vizarra E, Zeviani M (2018) Mitochondrial complex III Rieske Fe-S protein processing and assembly. Cell Cycle 17(6):681–687 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.eps Supplementarylegends.docx FigS1.eps FigS2.eps FigS3.eps TableS1Antibody.eps TableS2Primer.eps Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 21 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviews received at journal 06 Apr, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviews received at journal 11 Mar, 2026 Reviewers agreed at journal 03 Mar, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviewers invited by journal 08 Jan, 2026 Editor assigned by journal 22 Dec, 2025 Submission checks completed at journal 01 Nov, 2025 First submitted to journal 01 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8003013","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":571563341,"identity":"8b314fac-81cf-4bc0-b21c-dabc58e27c6f","order_by":0,"name":"Momoko Uchida","email":"","orcid":"","institution":"Showa Medical University Graduate School of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Momoko","middleName":"","lastName":"Uchida","suffix":""},{"id":571563343,"identity":"c58e3a47-ff30-41a5-933a-42bbf1605289","order_by":1,"name":"Masato Higugrashi","email":"","orcid":"","institution":"Showa Medical University Graduate 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07:48:18","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133167,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/1f130d9f42d827d4a4cf5065.html"},{"id":100363207,"identity":"47595306-6667-4bb2-b4c2-a6173ddb68e4","added_by":"auto","created_at":"2026-01-16 07:49:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1025121,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivities to the inhibition of electron transport chain complexes and oxidative phosphorylation in mt-Normal and mt-Low HCC cells. \u003cstrong\u003eA, B \u003c/strong\u003eMeasurement of cell viability. In \u003cstrong\u003eA\u003c/strong\u003e, cells were treated with antimycin A and rotenone, and in \u003cstrong\u003eB\u003c/strong\u003e, with staurosporine, FCCP, and oligomycin A at the indicated concentrations for 24 h. Straight lines; mt-Normal cells, dotted lines; mt-Low cells. Plots represent the means ± standard deviation from two independent experiments with triplicate measurements for each experiment.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/ca8197e46e486cac6ccaae7d.png"},{"id":100362232,"identity":"3397857e-95e1-4cc3-8f5a-3495374337e7","added_by":"auto","created_at":"2026-01-16 07:46:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":705786,"visible":true,"origin":"","legend":"\u003cp\u003eATP synthetic capability and glycolysis availability of mt-Normal and mt-Low cells.\u003cstrong\u003e A, B\u003c/strong\u003e Cells were treated with 1.25 mM oligomycin A (OligA) or 22.5 mM 2-deoxy-D-glucose (2-DG) for 5 h. The cell lysates and culture supernatants were used for measuring ATP (\u003cstrong\u003eA)\u003c/strong\u003e and lactate (\u003cstrong\u003eB)\u003c/strong\u003e levels. The levels are shown as ratios to the vehicle-treated control (-). Cells marked with a downward arrow represent mt-Low cells. Bars represent the means ± standard deviation from three independent experiments with triplicate measurements for each experiment. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs. (-).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/b000b6f87e1010d483c32bac.png"},{"id":100028043,"identity":"9b7e9cfd-df6b-4903-8dbf-f62d2588f3b6","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1467249,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of glycolysis upregulation and nutrient supplementation on cell survival during ETC inhibition. \u003cstrong\u003eA\u003c/strong\u003eLactate levels in the culture supernatants were measured. Cells were treated with 0.2 mM antimycin A (AMA) with or without 22.5 mM 2-deoxy-D-glucose (2-DG) for 4 h. The values are expressed relative to the vehicle-treated control (-). Bars represent the means ± standard deviation from at least three independent experiments with triplicate measurements for each experiment. \u003cstrong\u003eB\u003c/strong\u003e Glucose consumption was measured in cells treated with 0.2 mM AMA for 12 h. Cells marked with a downward arrow represent mt-Low cells. Bars represent the means ± standard deviation from two or three independent experiments with triplicate measurements for each experiment. \u003cstrong\u003eC\u003c/strong\u003e In lactate assay, JHH-4 cells were treated with or without 10 mM D-glucose (Glc) for 24 h, followed by treatment with 0.2 mM AMA for 9 h. Lactate release into the culture medium was measured as described above. Values are expressed relative to the vehicle-treated control (-). \u003cstrong\u003eC, D\u003c/strong\u003eCell viability (%) was determined in JHH-4 cells treated with 0.2 mM AMA for 48 h in culture medium supplemented with 10 mM Glc (\u003cstrong\u003eC\u003c/strong\u003e) and in HLF cells treated with 0.2 mM AMA for 17 h in culture medium supplemented with 10 mM Glc or 2-DG (\u003cstrong\u003eD\u003c/strong\u003e). Bars represent means ± standard deviation from three independent experiments with triplicate measurements for each experiment. \u003cstrong\u003eE, F\u003c/strong\u003e HLF (\u003cstrong\u003eE\u003c/strong\u003e) and JHH-4 (\u003cstrong\u003eF\u003c/strong\u003e) cells were treated with 0.2 mM Rot (\u003cstrong\u003eE\u003c/strong\u003e) and 0.2 mM AMA (\u003cstrong\u003eF\u003c/strong\u003e) or vehicle (dimethyl sulfoxide: DMSO) for 48 h in medium supplemented with 10 mM Glc, 5 mM pyruvate (Pyr), 0.2 mM uridine (Uri), and 20 mM aspartate (Asp). LDH release assay was conducted to measure the LDH levels in the culture medium and cell lysate. The ratios (%) of LDH released into the medium to the total amount are shown. Bars represent the means ± standard deviation from at least three independent experiments with triplicate measurements for each experiment. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, and *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs (-).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/01bb26aeaa883fda4f847235.png"},{"id":100028045,"identity":"6b614314-60ce-4bdf-a313-02eba4164a8a","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1432813,"visible":true,"origin":"","legend":"\u003cp\u003eAMPK activation in response to ETC inhibition. \u003cstrong\u003eA, B\u003c/strong\u003e Cells were treated with 0.2 mM rotenone (Rot) for 20 h (\u003cstrong\u003eA\u003c/strong\u003e) and 0.2 mM antimycin A (AMA) (\u003cstrong\u003eB\u003c/strong\u003e) for 8 h and analyzed by western blotting using the indicated antibodies. \u003cstrong\u003eC, D\u003c/strong\u003e Cells were treated with 0.2 mM AMA for the indicated periods (\u003cstrong\u003eC\u003c/strong\u003e) and with AMA at the indicated concentrations for 8 h (\u003cstrong\u003eD\u003c/strong\u003e). Band intensities for AMPK phosphorylated at threonine 172 (pAMPK) measured using ImageJ software are shown relative to the control [(-) or 0 h] after normalization with those for total AMPK. b-actin and glyceraldehyde-3-phosphate dehydrogenase (GD) were used as the loading controls. Cells marked with bold and plain letters indicate the resistant and sensitive phenotypes, respectively, to cell death following treatment with Rot or AMA (Fig. 1A). Representative results from three independent experiments are shown.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/61292df7dad0dac1f88d0edc.png"},{"id":100363248,"identity":"3960c3d7-da24-48d7-9846-df173d52d1c4","added_by":"auto","created_at":"2026-01-16 07:49:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1442449,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of activation and inhibition of AMPK on cell survival under ETC inhibition. \u003cstrong\u003eA\u003c/strong\u003e JHH-1 (left) and HepG2 (right) cells were pretreated with 5 mM compound C for 1 h, followed by treatment with antimycin A (AMA) at the indicated concentrations for 24 h. LDH release assay was performed, and the ratios (%) of LDH released into the medium to the total amount are shown. \u003cstrong\u003eB\u003c/strong\u003eJHH-1 cells were incubated with 50 nM siRNA for AMPKa1 for 3 days, followed by treatment with 5 mM AMA for 20 h, and cell viability (%) was measured. \u003cstrong\u003eC\u003c/strong\u003e HepG2 cells were pretreated with 0.5 mM AICAR, 50 mM A-7, and 1 mM metformin for 1 h, followed by treatment with 0.2 mM AMA. After 6.5 h (left), western blot analysis was performed using the indicated antibodies. After 40 h (right), LDH release assay was performed. b-actin was used as the loading control. Representative blots from two independent experiments are shown. Bars represent the means ± standard deviation from two or three independent experiments with triplicate measurements for each experiment. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, and *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs (-).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/bc23f69d212dd90d6c56a8a3.png"},{"id":100028047,"identity":"176886f5-854f-4d8b-a7a4-7547f9697490","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1083915,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of siRNA for the core subunits of complex I and III on cell proliferation and survival. \u003cstrong\u003eA, B\u003c/strong\u003e Cells marked with a downward arrow represent mt-Low cells. The cells were incubated with 50 nM siRNAs for the core subunits of respiratory complex I (\u003cem\u003eNDUFV1\u003c/em\u003e and \u003cem\u003eNDUFS1\u003c/em\u003e) and III (\u003cem\u003eURCRFS1\u003c/em\u003e)\u003cem\u003e \u003c/em\u003eor\u003cem\u003e \u003c/em\u003enegative control siRNA (Ctr) for 3 days, and cell numbers (\u003cstrong\u003eA\u003c/strong\u003e) and cell viability (%) (\u003cstrong\u003eB\u003c/strong\u003e) were determined. Bars represent the means ± standard deviation from three independent experiments with triplicate measurements for each experiment. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs siCtr.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/fc56200ad1430d0974b7ea32.png"},{"id":100381359,"identity":"6afd0427-0672-4097-b670-a20fb6e7ea9a","added_by":"auto","created_at":"2026-01-16 10:38:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7943910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/9bc83637-ac4f-471f-a729-18d53d886eee.pdf"},{"id":100362432,"identity":"66078235-03a1-4a39-9e64-408f7fbbd80a","added_by":"auto","created_at":"2026-01-16 07:46:44","extension":"eps","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3778690,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.eps","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/35b81fe38d7b33dad1d77426.eps"},{"id":100028040,"identity":"8a472c47-3d3a-4776-a098-cab52055d852","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16836,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarylegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/9ce0be4fca6e994a2b74afd6.docx"},{"id":100363168,"identity":"ebdffcbf-5129-428c-9383-89093c59db75","added_by":"auto","created_at":"2026-01-16 07:49:01","extension":"eps","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1269822,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.eps","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/e527e197f40f797f42fa298c.eps"},{"id":100363116,"identity":"0009966e-fc12-4c2b-a114-7028751d660d","added_by":"auto","created_at":"2026-01-16 07:48:55","extension":"eps","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2181146,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.eps","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/6a09f79328388eb9be6bda3a.eps"},{"id":100028057,"identity":"47771dba-ec67-4bd0-9092-22840911da78","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"eps","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":968318,"visible":true,"origin":"","legend":"","description":"","filename":"FigS3.eps","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/8ccdaa672dd3d2cefaa955e0.eps"},{"id":100028056,"identity":"53192773-f0e5-4c23-a89e-fefbff4cab50","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"eps","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1140902,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1Antibody.eps","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/6d2fd313b8aaef7323c4d70a.eps"},{"id":100028052,"identity":"a59ebf5a-6cc2-4ec6-9499-74f1dc33b30a","added_by":"auto","created_at":"2026-01-12 08:59:59","extension":"eps","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1009218,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2Primer.eps","url":"https://assets-eu.researchsquare.com/files/rs-8003013/v1/1376046c2bd3b8cea3728c04.eps"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional linkage between mitochondrial electron transport, glycolysis, and AMP-activated protein kinase signaling underlying cancer cell survival","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStudies on cancer metabolism, which have been inspired by Otto Warburg\u0026rsquo;s discovery of aerobic glycolysis, have substantially progressed over the several decades to further elucidate the metabolic phenotype of cancer cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The molecular mechanisms explaining how cancer cells reprogram metabolism to cope with the increased anabolic demands for incessant and durable cell proliferation are more understandable [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Accordingly, our view on mitochondrial metabolism in cancer cells has also evolved from the original concept that misinterpreted aerobic glycolysis as a consequence of irreversible mitochondrial respiratory defects in cancer cells. Mitochondria in most tumors are intact and actively performing oxidative phosphorylation (OXPHOS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, mitochondrial respiration is indispensable for cell proliferation. Thus, cell proliferation is impeded by pharmacological or genetic inhibition of electron transport chain (ETC), specifically, respiratory complex I and III [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a conventional perspectives, ETC activity likely affects cell proliferation through ATP generation and/or reactive oxygen species (ROS) release; however, because of the diverse roles of ETC beyond ATP generation, including metabolite production through TCA cycle flux and pyrimidine synthesis pathway for macromolecule biosynthesis, alternatives are also possible. Recent studies have highlighted the importance of ETC in the production of electron acceptors, which fulfill a specific metabolic requirement to support cell proliferation. For example, de novo synthesis of aspartate, a key biosynthetic precursor of proteins, and purine and pyrimidine nucleotides, is critically dependent on NAD\u003csup\u003e+\u003c/sup\u003e regeneration by ETC complex I [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recently, we found that NAD\u003csup\u003e+\u003c/sup\u003e regenerated by complex I regulates p21\u003csup\u003eCip1\u003c/sup\u003e expression and cancer cell proliferation through SIRT activity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Ubiquinol oxidation to ubiquinone by ETC complex III is also required for tumor growth [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvidence supporting the role for ETC in cell proliferation has led to attempts that target ETC for cancer therapy; however, the translation of various ETC inhibitors into clinical practice has been challenging. Thus far, clinical trials have failed because of setbacks, such as dose-limiting toxicity, lack of potency and specificity, even when preclinical results were promising [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These failures have emphasized the importance of discriminating between noncancerous and cancerous mitochondria, or identifying and focusing on specific phenotypes of cancerous mitochondria and targeting their distinct vulnerabilities for the development of safe and effective mitochondrial-based therapies.\u003c/p\u003e \u003cp\u003eAccording to a recent mitochondrial phenotyping study, the expression/function of the OXPHOS complex is consistently lower in cancer tissues compared with matched normal tissues across several cancer types [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Genome-wide analyses of mitochondrial DNA (mtDNA) and mitochondrial RNA (mtRNA) showed a significant decrease in mtDNA copy number on average in 7 of 15 cancer types and a concomitant decrease in mtRNA levels [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These results suggest that the sizeable proportion of cancerous mitochondria exhibit a functionally lower mtDNA/RNA/OXPHOS phenotype compared with their normal counterparts in numerous types of cancers. This difference can be potentially exploited to develop mitochondrial medicines that can differentiate between cancerous and normal mitochondria as well as specifically target cancer cells with lower mitochondrial activity.\u003c/p\u003e \u003cp\u003eOur recent study of hepatocellular carcinoma (HCC) cell lines revealed that over half (4 of 7) of them exhibited a mt-Low phenotype with lower mtDNA/RNA and membrane potential compared with non-malignant hepatocytes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which agreed with the previous studies. Similarly, 61% (17 of 28) of in vivo HCC tissues were mt-Low cases [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, we characterized the metabolic traits of these HCC cell lines, especially focusing on mt-Low cells. Interestingly, mt-Low HCC cells are highly and selectively sensitive to ETC inhibition. Mechanistically, the sensitivity of mt-Low cancer cells to ETC inhibition results from the loss of a sufficient functional linkage between ETC, glycolysis, and AMP-activated protein kinase (AMPK) signaling.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAntimycin A (AMA) and rotenone (Rot) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Sigma-Aldrich, Inc. (St Louis, MO, USA), respectively. Staurosporine and 2-Deoxy-D-glucose were purchased from Wako Pure Chemical Corporation (Osaka, Japan). Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin A (OligoA) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Compound C and metformin were purchased from Merck KGaA (Darmstadt, Germany) and Enzo Life Sciences, Inc. (Farmingdale, NY, USA), respectively. AICAR and A-769622 (A-7) were purchased from FUJIFILM and Adooq Bioscience LLC (Irvine, CA, USA), respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) or the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan), and maintained in their respective media for subculture. For the experiments, the cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (Shimadzu Diagnostics Corp., Tokyo, Japan) supplemented with 10% fetal bovine serum to standardize the nutrient supply.\u003c/p\u003e\n\u003ch3\u003eCell proliferation and viability assay\u003c/h3\u003e\n\u003cp\u003eThe cells were seeded at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e or 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in 12-well plate. Total and viable cell numbers were counted with a Countess II Automated Cell Counter (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell viability was assessed by performing trypan blue staining.\u003c/p\u003e\n\u003ch3\u003eMeasurements of lactate and ATP levels\u003c/h3\u003e\n\u003cp\u003eCells (1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e or 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were seeded into a single well of a 12-well plate. Lactate released into the culture medium was measured using the Lactate Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s instructions. The culture supernatant was collected and diluted 10-fold with water and analyzed. The working solution was incubated with the supernatant for 30 min at 37\u0026deg;C, and the absorbance was measured at 450 nm using a Varioskan Lux (Thermo Fisher Scientific Inc.) microplate reader. The remaining cells were lysed, and intracellular ATP levels were measured using an ATP determination kit (Thermo Fisher Scientific, Inc.) as described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMeasurement of glucose consumption\u003c/h3\u003e\n\u003cp\u003eCells (0.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e or 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were plated in a single well of a 12-well plate. The following day, the cells were treated with or without reagents after aliquots of the supernatant were collected (sup 0 h). After 12 h, the supernatants (i.e., medium; sup 12 h) were collected from each well, and the cells were counted. Glucose concentrations in the sup (0 h) and (12 h) were measured using the Glucose Assay Kit-WST (Dojindo Laboratories) based on the manufacturer\u0026rsquo;s instructions, and glucose consumption (\u0026micro;mol/h) was calculated from the difference in glucose levels between sup (0 h) and sup (12 h), normalized with 10⁶ cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLDH release assay\u003c/h2\u003e \u003cp\u003eLactate dehydrogenase (LDH) activity retained in cells and released into the culture media was determined using the LDH-Cytotoxic Test wako (Wako Pure Chemical Industries, Ltd.) according to manufacturer\u0026rsquo;s instructions. Briefly, 0.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e or 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were seeded into a single well of a 12-well plate and treated with the reagent the following day. After 24\u0026ndash;72 h, the medium was centrifuged, and the resulting supernatant and pellets were collected. The remaining cells in each well were solubilized with phosphate buffer solution (PBS) containing 0.2% Tween-20 together with the cells pelleted from the medium. Equivalent aliquots of the supernatant and cell lysate were incubated with the coloring solution after clearing by centrifugation, and the absorbance at 570 nm was measured using a Varioskan Lux (Thermo Fisher Scientific, Inc.). Fresh medium and PBS/0.2% Tween were used as a control for the supernatants and cell lysates, respectively. LDH release was calculated as the ratio of LDH activity in the medium to the total LDH activity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eWestern blotting was performed as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The primary antibodies used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The specific bands were quantified using ImageJ software (version 1.53 k, National Institutes of Health, Bethesda, MD, USA) to compare densities.\u003c/p\u003e\n\u003ch3\u003eRNA interference\u003c/h3\u003e\n\u003cp\u003eSmall interfering RNA (siRNA) (FlexiTube siRNA) was purchased from Qiagen (Venlo, the Netherlands). Transfection of the siRNA was achieved using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Inc.) as described previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation with *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Comparisons between two groups were analyzed using a two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. For multiple comparisons, the data were analyzed using one-way ANOVA with Bonferroni\u0026rsquo;s post hoc correction to identify datasets that differed from the control data. A p-value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSelective and high sensitivity of mt-Low HCC cells to ETC inhibition\u003c/h2\u003e \u003cp\u003eWe previously categorized seven HCC cell lines into an mt-Normal or mt-Low phenotype based on normal or low mtDNA/RNA content and membrane potential compared with primary hepatocytes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Oxygen consumption rates confirmed that the respiratory activities were higher and lower in mt-Normal cells and mt-Low cells, respectively (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We further characterized the metabolic properties of the two groups (mt-Normal cells; HepG2, JHH-1, and JHH-6, mt-Low cells; HLF, HuH-7, JHH-2, and JHH-4). Specifically, we disrupted respiratory activity using the established ETC inhibitors, AMA and Rot, which inhibit complexes III and I, respectively, and observed the response of the mt-Normal and mt-Low cells. In the experiments, all cell lines were cultured in DMEM throughout the study to standardize the nutrient supply.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mt-Normal cells were largely tolerable to inhibitor treatment within the concentration range used in the experiments (IC50 of AMA and Rot are 16 nM and 56 nM, respectively [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]). However, the inhibitors, especially AMA, were remarkably cytotoxic to the mt-Low cells, except JHH-2, at lower doses, where the majority of the mt-Normal cells were alive (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Therefore, the sensitivity to the ETC inhibition was markedly different between the two groups. However, the sensitivities to FCCP and OligA, which interfere with ATP synthesis at the final step of OXPHOS, were indistinguishable between the two groups and independent of respiratory levels of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, they were comparably tolerable to FCCP and OligA treatment over the concentration range used in this study (IC50 of FCCP and OligA; 4.8\u0026ndash;8.5 \u0026micro;M and 0.21\u0026ndash;3.8 \u0026micro;M, respectively [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]). Sensitivity to staurosporine (IC50; 0.3 \u0026micro;M [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]), a representative toxic substance, was also independent of the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Collectively, the results indicated the selective and high sensitivity of mt-Low HCC cells to ETC inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe sensitivity to ETC inhibition is dependent upon a variety of cellular processes that sustain energy homeostasis. Because a major role of ETC is to support ATP synthesis through OXPHOS, the ATP synthetic potential of cells is the most likely determinant of sensitivity to ETC inhibition. However, this was unlikely because ATP levels were sustained for several hours following AMA treatment in mt-Normal and mt-Low cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), possibly because of metabolic plasticity as described below. Moreover, in contrast to the intolerance toward ETC inhibition, the mt-Low and mt-Normal cells were tolerant to direct ATP synthesis inhibition by FCCP and OligA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting that they are equally adaptable for the inhibition of OXPHOS-driven ATP synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCellular antioxidant capacity is likely an alternative determinant of sensitivity to ETC inhibition. The inhibition of ETC by Rot and AMA accompanies ROS production [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Accordingly, cells require a sufficient antioxidant defense during ETC inhibition. Therefore, we quantified total glutathione (GSH) levels as a major antioxidant with the ratio of GSH/glutathione disulfide. Both parameters varied among the HCC cell lines at steady-state levels and during AMA treatment, but appeared unrelated to the sensitivity to ETC inhibition or mt-Normal/Low status (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003eAmong cellular signaling, PI3K/AKT pathway plays an important role in cell survival and is frequently implicated in resistance to toxic substances [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This may be relevant to atypical resistant phenotypes of JHH-2 to these inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD, AKT was distinctly activated in JHH-2. In conclusion, the sensitivity of the mt-Low HCC cells to ETC inhibition was independent of ATP synthetic potential, antioxidant defense capacity, and PI3K/AKT survival signaling, except JHH-2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDifferences in glycolysis availability between mt-Normal and mt-Low HCC cells\u003c/h2\u003e \u003cp\u003eTo gain insight into determinants of sensitivity to ETC inhibition in the mt-Low cells, we compared fundamental metabolic characteristics, glycolytic/mitochondrial ATP synthetic capacity and glycolysis availability, between mt-Normal and mt-Low HCC cells. We first evaluated glycolytic/mitochondrial ATP synthetic capacity by disrupting the two synthetic pathways using 2-deoxy-D-glucose (2-DG) or OligA, which inhibit OXPHOS and glycolytic ATP synthesis, respectively. Consistent with the above observation of sustained ATP levels under ETC inhibition (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), the results indicated that the cancer cells in both groups are highly metabolically plastic and flexibly generate ATP through glycolysis or OXPHOS in response to the availability of these two pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Alternatively, a surplus quantity of ATP may be normally reserved in cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConversely, glycolysis availabilities, which are evaluated by measuring lactate release from cells in the presence of OligA, were different between the two groups. During OligA treatment, in which OXPHOS is inhibited, cells generally enhance glycolysis by mobilizing their reserve capacity for glycolysis to compensate for OXPHOS, releasing more lactate into the medium. Accordingly, the amount of released lactate in the presence of OligA reflects a maximum level of glycolysis availability of cells, and the difference from the control conditions without OligA treatment corresponds to the reserve capacity for glycolysis. In this study, lactate release was significantly increased during OligA treatment in mt-Normal HepG2 and JHH-1cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB); however, the release was only marginally increased in mt-Low cells, such as HLF and JHH-4, under these conditions, suggesting that mt-Low cancer cells lack a sufficient reserve capacity for glycolysis or are mechanistically defective in upregulating glycolysis. In summary, the mt-Normal and mt-Low cells were similarly able to synthesize ATP by glycolytic and mitochondrial pathways, but differed in the upregulation of glycolysis during ETC inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eUpregulation of glycolysis is important for cell survival during limited ETC activity\u003c/h2\u003e \u003cp\u003eBased on the above results, the difference between mt-Normal and mt-Low cells with regard to sensitivity for ETC inhibition may be attributed to their difference in glycolysis availability. Consistent with this idea, the mt-Normal cells, which are resistant to ETC inhibition, exhibited enhanced glycolysis during ETC inhibition with AMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), concomitant with increased glucose consumption (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, glycolysis and glucose consumption were only slightly enhanced in mt-Low cells under these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results suggested that if glycolysis were sufficiently upregulated, mt-Low cells could resist ETC inhibition, similar to mt-Normal cells. In fact, the mt-Low cells became tolerant of AMA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; viability) when glycolysis was enhanced following glucose supplementation into the medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; lactate assay). Conversely, glycolysis inhibition with 2-DG markedly decreased cell survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results highlight the importance of upregulating glycolysis for cell survival during limited ETC activity.\u003c/p\u003e \u003cp\u003eIn addition to glucose, supplementation with other biosynthetic precursors or intermediates was also effective at mitigating cell death induced by ETC inhibition. Pyruvate and uridine significantly decreased LDH release and cell death at supra-physiological concentrations during ETC inhibition by Rot treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). When ETC was inhibited by AMA, cell death was partially relieved by pyruvate supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results suggest that metabolic pathways driven by glucose and other metabolites substitute for ETC function, which is important for cell survival.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eActivation of AMP-activated protein kinase underlies cell survival during limited ETC activity\u003c/h2\u003e \u003cp\u003eAMPK is an energy-sensing kinase, which is activated by various metabolic stresses that lower cellular energy levels, and has various roles in cellular energy homeostasis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the HCC cell lines, an active form of AMPK phosphorylated at threonine 172 was observed at various levels during ETC inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). Interestingly, the activation levels of AMPK correlated with the resistance of the HCC cell lines to the inhibitors. For example, the levels were higher in JHH-1, HepG2, and JHH-4 compared with that of HLF during Rot treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which mirrored their viability under these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). During AMA treatment, JHH-1 and JHH-2, both of which were remarkably resistant to the treatment, exhibited higher activation levels compared with the others (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In JHH-1, robust and sustained AMPK activation was consistently observed in response to AMA, whereas AMPK activation was moderate in HuH-7 and JHH-4 cells, even when AMA was treated with higher doses for longer periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). These correlations between the activation of AMPK and cell viability suggest that AMPK underlies cell survival during ETC inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo substantiate the role of AMPK in cell survival directly, we inhibited AMPK by an inhibitor and siRNA or activated AMPK with activators, and observed their effects on cell fates. The treatment with compound C, a well-known AMPK inhibitor, reversed the resistance phenotype of the mt-Normal JHH-1 and HepG2 cells during AMA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similarly, cell survival was reduced by knockdown of AMPK with siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Conversely, the AMPK activator A-7, which potently activated AMPK among all the activators tested in the study, significantly promoted the resistance phenotype of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results highlight the important role of AMPK activation in cell survival during ETC inhibition. Collectively, it was most likely that glycolysis upregulation and AMPK activation were the determinants of sensitivity to ETC inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of siRNA targeting the subunits of respiratory complex I and III on cell survival and proliferation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eClinically, the selective and high sensitivity or vulnerability of mt-Low cells to ETC inhibition may represent a therapeutic target for mt-Low cancers. As substitutes for small molecular inhibitors, including Rot, AMA, and others, for which clinical trials were discontinued, we evaluated the efficacy of siRNAs targeting core subunits of respiratory complex I and III to inhibit cell survival and/or the proliferation of mt-Low HCC cells. We knocked down \u003cem\u003eNDUFV1\u003c/em\u003e (NADH: ubiquinone oxidoreductase core subunit V1) and \u003cem\u003eNDUFS\u003c/em\u003e1 (NADH: ubiquinone oxidoreductase core subunit S1) of complex I, and \u003cem\u003eURCRFS1\u003c/em\u003e (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1) of complex III. NDUFV1 and NDUFS1 constitute the electron input module, which accepts electrons from NADH through flavin mononucleotide and sequentially transfers them along a chain of iron-sulfur (FeS) clusters in complex I. UQCRFS1 is a subunit that receives an electron from ubiquinol and transfers it to cytochrome \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e, thereby playing an essential enzymatic role in complex III.\u003c/p\u003e \u003cp\u003eAll three siRNAs downregulated the expression of the target gene (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) and showed cytostatic effects on mt-Low JHH-4 and HLF cells as evidenced by decreased cell numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Moreover, siRNAs for \u003cem\u003eNDUFV1\u003c/em\u003e and \u003cem\u003eNDUFS1\u003c/em\u003e exhibited cytotoxic effects and decreased cell viability. These results suggest that siRNAs targeting the core subunits of complex I and III, in particular those of complex I, are promising therapeutics for the mt-Low-type cancers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor the successful development of mitochondrial-targeted therapeutics, it is necessary to characterize cancerous mitochondria in detail, identify distinctive and druggable features or vulnerabilities, and target them specifically. Based on previous studies, cancerous mitochondria exhibit heterogeneous mtDNA/RNA content and OXPHOS function [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Toward the goal of developing mitochondrial precision medicine, we focused on the mt-Low-type of cancer, which occurs in approximately half of all cancer types [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We found that the mt-Low-type of cancer cells are distinctively vulnerable to the ETC inhibition. Previously, the preferential effect of OXPHOS inhibition on mitochondria was a concern with respect to normal OXPHOS activity in healthy organs, particularly those with a high reliance on OXPHOS, rather than tumor mitochondrial flux [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Our results indicate that it is possible to preferentially compromise tumor mitochondria flux while sparing healthy cells by targeting ETC functions in the case of mt-Low-type cancers.\u003c/p\u003e \u003cp\u003eMechanistically, mt-Low cancer cells are unable to upregulate glycolysis efficiently during ETC inhibition, resulting in cell death. The mt-Low cells may be forced to leverage glycolytic flux at nearly maximum levels because of their intrinsically low respiratory activity; therefore, it is likely that they cannot afford to upregulate glycolysis in response to ETC inhibition. By supplementing the culture medium with glucose, glycolysis was upregulated in mt-Low cells, and cell death was mitigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This highlights the importance of glycolysis upregulation for cell survival under ETC-limited conditions.\u003c/p\u003e \u003cp\u003eSupplementation with pyruvate also ameliorated cell death induced by the ETC inhibition. Uridine was also effective in rescuing Rot-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In AMA-treated cells, only pyruvate was effective (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The difference in effectiveness of uridine in relieving cell death induced by Rot or AMA may be attributed to the difference in TCA metabolite abundance, which was affected by Rot and AMA treatment as previously reported [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. According to a recent study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], OXPHOS dysfunction resulted in higher lactate/lower pyruvate abundance, lower levels of TCA intermediates and aspartic acid, and increased glycerol-3-phosphate levels. Interestingly, these metabolite imbalances were normalized by combined supplementation of pyruvate and uridine. Taken together, these beneficial effects of nutrient supplementation on cell viability during ETC inhibition suggest the compensatory network between ETC functions, glycolysis, and pyruvate/uridine metabolism. Therefore, a functional linkage has emerged between glycolysis, pyruvate/uridine metabolic pathways, and respiratory capacities, which is important as a safeguard for cell survival during ETC dysfunction.\u003c/p\u003e \u003cp\u003eAMPK coordinates metabolic pathways and maintains energy homeostasis through the phosphorylation of various substrates [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It also promotes mitochondrial biogenesis and dynamics [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. By performing these roles, AMPK contributes to cell survival under low-energy conditions and mitochondrial dysfunction. In this study, the mt-Normal cells activated the kinase in response to ETC inhibition as previously reported (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Conversely, the mt-Low HCC cells were unable to activate AMPK. A series of experiments using the inhibitor, the activators, and siRNA support the role for AMPK in cell survival during ETC inhibition.\u003c/p\u003e \u003cp\u003eAlthough AMP is the most common activator of AMPK, a non-canonical mechanism independent of AMP [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] is suggested to be predominantly involved in activating AMPK in the ETC-inhibited HCC cells, because the ATP levels were unchanged (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA) and among the activators tested, A-7 was the most prominent activator in the HCC cells. Unlike AICAR and metformin, A-7 activates AMPK by a different mechanism than AMP [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recently, glycolysis was found to be a key regulator of AMPK activation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, a correlation was observed between the ability of cells to activate AMPK and upregulate glycolysis, suggesting that the glycolytic pathway regulates its activation. However, in another report, AMPK activation was suggested to upregulate glycolysis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, the relationship between glycolysis and AMPK activation is context-dependent and requires further study.\u003c/p\u003e \u003cp\u003eOligonucleotide therapeutics represent a next-generation modality in medicine. In this study, we evaluated potential and efficacy of siRNAs targeting respiratory complex I and III for anticancer therapeutics. Complex I was targeted by siRNAs against the core subunits, \u003cem\u003eNDUFV1\u003c/em\u003e and \u003cem\u003eNDUFS1\u003c/em\u003e. Treatment with these siRNAs inhibits NADH oxidation and electron transfer along a chain of FeS clusters, which is the initial step in electron transfer for complex I [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Rot inhibits ubiquinone reduction following electron transfer [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The two siRNAs decreased cell viability, similar to Rot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). siRNA targeting \u003cem\u003eUQCRFS1\u003c/em\u003e, the Rieske FeS protein in complex III, was also effective in inhibiting cell proliferation; however, it could not induce cell death. AMA has both cytostatic and cytotoxic effects. Complex III (\u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex; ubiquinol cytochrome \u003cem\u003ec\u003c/em\u003e oxidoreductase) contains three redox subunits along with the Rieske FeS protein. It catalyzes electron transfer from ubiquinol to cytochrome \u003cem\u003ec\u003c/em\u003e. AMA inhibits electron transfer between the redox subunits, cytochrome \u003cem\u003eb\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], whereas \u003cem\u003eUQCRFS1\u003c/em\u003e knockdown inhibits electron transfer between ubiquinol and cytochrome \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These results suggest that electron transfer between cytochrome \u003cem\u003eb\u003c/em\u003e and \u003cem\u003ec\u003c/em\u003e is critical for cell survival rather than that between ubiquinol and cytochrome \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, this study encourages continuous efforts in pursuing an ETC inhibition approach as an effective anticancer strategy. It is worth re-attempting the clinical trials with the previous inhibitors by restricting the study to a patient cohort/subset with the mt-Low-type cancer. Along with small molecule inhibitors, siRNAs targeting complex I and III exerted cytostatic and/or cytotoxic effects on HCC cells, particularly on mt-Low cells. After massive but unfruitful efforts at developing ETC-targeted medicine, pioneering approaches from a different perspective are expected in this field.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eA-7, A769622; AMA, antimycin A; AMPK, AMP-activated protein kinase; C.C, compound C; ETC, electron transport chain; FeS, iron-sulfur; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; GSH, glutathione; GSSG, glutathione disulfide; HCC, hepatocellular carcinoma; OligA, oligomycin A; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; Rot, rotenone; siRNA, small interfering RNA; TCA, tricarboxylic acid cycle; 2-DG, 2-deoxy-D-glucose.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe thank Yui Shikama, Yuki Tsuchiya, Kohei Naka, Saki Fujiwara, Ken Hirabayashi, Yudai Suto, Misato Narisawa, Maho Betsuyaku, Eiji Funayama, Yoshihiro Watanabe, Takane Watari, Saki Kikuchi, and Ayaka Saito for their contribution to this work as part of their bachelor\u0026rsquo;s degrees.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would also like to thank Enago (www.enago.jp) for the English language review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions:\u003c/strong\u003e Uchida M: Investigation, Data curation, Writing\u0026ndash;original draft. Higugrashi M: Investigation, Data curation, Writing\u0026ndash;original draft. Nakagawa H: Writing\u0026ndash;review and editing. Ishikawa F: Writing\u0026ndash;review and editing. Mori K: Supervision, Writing\u0026ndash;review and editing. Shibanuma M: Conceptualization, Project administration, Funding acquisition, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e JSPS KAKENHI Grant Number 21K06809.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e No datasets were generated or analyzed during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKoppenol WH, Bounds PL, Dang CV (2011) Otto Warburg's contributions to current concepts of cancer metabolism. 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Cell Cycle 17(6):681\u0026ndash;687\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"respiratory chain complex, mitochondrial electron transport, glycolysis, AMP-activated protein kinase","lastPublishedDoi":"10.21203/rs.3.rs-8003013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8003013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMitochondrial metabolism has emerged as a potential target for cancer therapy because of its essential role in cell proliferation and survival beyond ATP production. However, translation into clinical practice is challenging owing to several limitations, including the difficulty in distinguishing cancerous mitochondria from noncancerous mitochondria. The heterogeneity of cancerous mitochondria further complicates this effort. Herein, we focused on cancerous mitochondria exhibiting low DNA/RNA levels and respiratory function compared with those exhibited by normal cells and determined the effect of low mitochondrial respiratory activity on cancer cells. Interestingly, mitochondria low-type (mt-Low) cancer cells derived from hepatocellular carcinoma were selectively and highly sensitive to electron transport chain (ETC) inhibition. Specifically, cells died under the treatment with the ETC inhibitors, rotenone and antimycin A, at lower doses compared with cells exhibiting normal respiratory activity (mt-Normal), although ATP levels were sustained in both types of cells under these conditions. In mt-Normal cells, glycolysis increased and AMP-activated protein kinase was activated upon ETC inhibition, which critically contributed toward cell survival. However, mt-Low cells could not induce these responses, which resulted in cell death. Based on these results, therapeutics targeting respiratory function have emerged as promising precision medicines for mt-Low cancers. Similar to conventional ETC inhibitors, small interfering RNAs targeting core subunits of respiratory complex I or III were effective in inhibiting cell proliferation (complex I and III) and survival (complex I) of mt-Low cancer cells, encouraging pionerring anticancer approaches using the next-generation modality.\u003c/p\u003e","manuscriptTitle":"Functional linkage between mitochondrial electron transport, glycolysis, and AMP-activated protein kinase signaling underlying cancer cell survival","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 08:59:54","doi":"10.21203/rs.3.rs-8003013/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-21T12:52:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"221826914455290299240076242424363723695","date":"2026-04-07T14:35:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T19:58:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203794753580338948426333164800815876035","date":"2026-03-24T03:43:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T02:39:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271547577177110391809122727022323794515","date":"2026-03-03T14:04:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204730839356179912757627822713392908096","date":"2026-01-14T19:53:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-08T14:37:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-22T14:19:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-01T15:14:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2025-11-01T05:20:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"42c1bd25-e51b-40bc-aa09-811bbdc389c2","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T12:56:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 08:59:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8003013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8003013","identity":"rs-8003013","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}