Inhibition Of Mitochondrial Respiration Research Articles

Inhibition of the mitochondrial respiratory chain reduces catecholamine‑stimulated lipolysis via increasing lactate production in 3T3‑L1 adipocytes.

Adipose tissue serves a significant role in the regulation of energy metabolism in the body. The re‑esterification of the fatty acids generated during lipolysis is critical for efficient lipolysis. However, the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis is not yet fully understood. The present study aimed to assess the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis. 3T3‑L1 adipocytes were incubated with mitochondrial respiratory chain inhibitors, oligomycin A or rotenone, during isoproterenol stimulation; and glycerol, glucose and lactate concentrations in the medium were measured. Western blot analysis was performed to examine the phosphorylation levels of cAMP‑dependent protein kinase A (PKA). The results showed that inhibition of mitochondrial ATP synthesis decreased catecholamine‑stimulated lipolysis without affecting PKA signaling. The inhibition of mitochondrial respiration increased glucose uptake and lactate production, indicating that a large amount of glucose taken up into the cell was preferentially used for ATP production rather than for re‑esterification. In conclusion, the results of the present study suggested that the energy state during lipolysis may influence lipolytic activity by suppressing fatty acid re‑esterification.

Torpid 13-lined ground squirrel liver mitochondria resist anoxia-reoxygenation despite high levels of protein damage.

Hibernation confers resistance to ischemia-reperfusion injury in tissue, but the underlying mechanisms remain unclear. Suppression of mitochondrial respiration during torpor may contribute to this tolerance. To explore this concept, we subjected isolated liver mitochondria from torpid, interbout euthermic (IBE) and summer 13-lined ground squirrels (Ictidomys tridecemlineatus) to 5 min of anoxia, followed by reoxygenation (A/R). We also included rat liver mitochondria as a non-hibernating comparison group. Maximum respiration rates of mitochondria from torpid ground squirrels were not affected by A/R, but in IBE and summer, these rates decreased by 50% following A/R and in rats they decreased by 80%. Comparing net ROS production rates among groups, revealed seasonal differences; mitochondria from IBE and torpor produced 75% less ROS than summer ground squirrels and rats. Measurements of oxidative damage to these mitochondria, both freshly isolated, as well as pre- and post-A/R, demonstrated elevated damage to protein, but not lipids, in all groups. Hibernation likely generates oxidative stress, as freshly isolated mitochondria had greater protein damage in torpor and IBE than in summer and rats. When comparing markers of damage pre- and post-A/R, we found that when RET was active, rat macromolecules were more damaged than when RET is inhibited, but in TLGS markers of damage were similar. This result suggests that suppression of RET during hibernation, both in torpor and IBE, lessens oxidative stress produced during arousal. Taken together our study suggests that ischemia-reperfusion tolerance at the mitochondrial level is associated with metabolically suppressed oxidative phosphorylation during hibernation.

Amino acid transporter SLC38A5 is a tumor promoter and a novel therapeutic target for pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) cells have a great demand for nutrients in the form of sugars, amino acids, and lipids. Particularly, amino acids are critical for cancer growth and, as intermediates, connect glucose, lipid and nucleotide metabolism. PDAC cells meet these requirements by upregulating selective amino acid transporters. Here we show that SLC38A5 (SN2/SNAT5), a neutral amino acid transporter is highly upregulated and functional in PDAC cells. Using CRISPR/Cas9-mediated knockout of SLC38A5, we show its tumor promoting role in an in vitro cell line model as well as in a subcutaneous xenograft mouse model. Using metabolomics and RNA sequencing, we show significant reduction in many amino acid substrates of SLC38A5 as well as OXPHOS inactivation in response to SLC38A5 deletion. Experimental validation demonstrates inhibition of mTORC1, glycolysis and mitochondrial respiration in KO cells, suggesting a serious metabolic crisis associated with SLC38A5 deletion. Since many SLC38A5 substrates are activators of mTORC1 as well as TCA cycle intermediates/precursors, we speculate amino acid insufficiency as a possible link between SLC38A5 deletion and inactivation of mTORC1, glycolysis and mitochondrial respiration, and the underlying mechanism for PDAC attenuation. Overall, we show that SLC38A5 promotes PDAC, thereby identifying a novel, hitherto unknown, therapeutic target for PDAC.

Tissue-specific mitochondrial toxicity of cigarette smoke concentrate: consequence to oxidative phosphorylation.

Cigarette smoke exposure is a well-known risk factor for developing numerous chronic health conditions, including pulmonary disease and cardiometabolic disorders. However, the cellular mechanisms mediating the toxicity of cigarette smoke in extrapulmonary tissues are still poorly understood. Therefore, the purpose of this study was to characterize the acute dose-dependent toxicity of cigarette smoke on mitochondrial metabolism by determining the susceptibility and sensitivity of mitochondrial respiration from murine skeletal (gastrocnemius and soleus) and cardiac muscles, as well as the aorta to cigarette smoke concentrate (CSC). In all tissues, exposure to CSC inhibited tissue-specific respiration capacity, measured by high-resolution respirometry, according to a biphasic pattern. With a break point of 451 ± 235 μg/mL, the aorta was the least susceptible to CSC-induced mitochondrial respiration inhibition compared with the gastrocnemius (151 ± 109 μg/mL; P = 0.008, d = 2.3), soleus (211 ± 107 μg/mL; P = 0.112; d = 1.7), and heart (94 ± 51 μg/mL; P < 0.001; d = 2.6) suggesting an intrinsic resistance of the vascular smooth muscle mitochondria to cigarette smoke toxicity. In contrast, the cardiac muscle was the most susceptible and sensitive to the effects of CSC, demonstrating the greatest decline in tissue-specific respiration with increasing CSC concentration (P < 0.001, except the soleus). However, when normalized to citrate synthase activity to account for differences in mitochondrial content, cardiac fibers' sensitivity to cigarette smoke inhibition was no longer significantly different from both fast-twitch gastrocnemius and slow-twitch soleus muscle fibers, thus suggesting similar mitochondrial phenotypes. Collectively, these findings established the acute dose-dependent toxicity of cigarette smoke on oxidative phosphorylation in permeabilized tissues involved in the development of smoke-related cardiometabolic diseases.NEW & NOTEWORTHY Despite numerous investigations into the mechanisms underlying cigarette smoke-induced mitochondrial dysfunction, no studies have investigated the tissue-specific mitochondrial toxicity to cigarette smoke. We demonstrate that, while aorta is least sensitive and susceptible to cigarette smoke-induced toxicity, the degree of cigarette smoke-induced toxicity in striated muscle depends on the tissue-specific mitochondrial content. We conclude that while the mitochondrial content influences cigarette smoke-induced toxicity in striated muscles, aorta is intrinsically protected against cigarette smoke-induced mitochondrial toxicity.

Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

Costunolide ameliorates angiotensin II-induced atrial inflammation and fibrosis by regulating mitochondrial function and oxidative stress in mice: A possible therapeutic approach for atrial fibrillation

Atrial fibrillation (AF) is a cardiac disease characterized by disordered atrial electrical activity. Atrial inflammation and fibrosis are involved in AF progression. Costunolide (COS) is a sesquiterpene lactone containing anti-inflammatory and anti-fibrotic activities. This study aims to explore the underlying mechanisms by which COS protects against AF. Male C57BL/6 mice (8- to 10-week-old) were infused with angiotensin (Ang) II for 3 weeks. Meanwhile, different doses of COS (COS-L: 10 mg/kg, COS-H: 20 mg/kg) were administered to mice by intragastric treatment. The results showed irregular and rapid heart rates in Ang II-treated mice. Moreover, the levels of inflammatory cytokines and fibrotic factors were elevated in mice. COS triggered a reduction of Ang II-induced inflammation and fibrosis, which conferred a protective effect. Mechanistically, mitochondrial dysfunction with mitochondrial respiration inhibition and aberrant ATP levels were observed after Ang II treatment. Moreover, Ang-II-induced excessive reactive oxygen species caused oxidative stress, which was further aggravated by inhibiting Nrf2 nuclear translocation. Importantly, COS diminished these Ang-II-mediated effects in mice. In conclusion, COS attenuated inflammation and fibrosis in Ang-II-treated mice by alleviating mitochondrial dysfunction and oxidative stress. Our findings represent a potential therapeutic option for AF treatment.

Mitochondrial vulnerability to oxidation in human brain organoids modelling Alzheimer's disease

Reactive Oxygen Species (ROS) and mitochondrial dysfunction are implicated in the pathogenesis of Alzheimer's disease (AD), a common neurodegenerative disorder characterized by abnormal metabolism of the amyloid precursor protein (APP) in brain tissue. However, the exact mechanism by which abnormal APP leads to oxidative distress remains unclear. Damage to mitochondrial membrane and inhibition of mitochondrial respiration are thought to contribute to the progression of the disease. However, the lack of suitable human models that replicate pathological features, together with impaired cellular pathways, constitutes a major challenge in AD studies. In this work, we induced pluripotency in patient-derived skin fibroblasts carrying the Swedish mutation in App (APPswe), to generate human brain organoids that model AD, and studied redox regulation and mitochondrial homeostasis. We found time-dependent increases in AD-related pathological hallmarks in APPswe brain organoids, including elevated Aβ levels, increased extracellular amyloid deposits, and enhanced tau phosphorylation. Interestingly, using live-imaging spinning-disk confocal microscopy, we found an increase in mitochondrial fragmentation and a significant loss of mitochondrial membrane potential in APPswe brain organoids when subjected to oxidative conditions. Moreover, ratiometric dyes in a live imaging setting revealed a selective increase in mitochondrial superoxide anion and hydrogen peroxide levels in APPswe brain organoids that were coupled to impairments in cytosolic and mitochondrial redoxin protein expression. Our results suggest a selective increase in mitochondrial vulnerability to oxidative conditions in APPswe organoids, indicating that the abnormal metabolism of APP leads to specific changes in mitochondrial homeostasis that enhance the vulnerability to oxidation in AD.

Ultra-stable MOF@MOF nanoplatform for photodynamic therapy sensitized by relieved hypoxia due to mitochondrial respiration inhibition

Metal-organic frameworks (MOFs) with periodically arranged porphyrinic linkers avoiding the self-quenching issue of porphyrins in photodynamic therapy (PDT) have been widely applied. However, the porphyrinic MOFs still face challenges of poor stability under physiological conditions and limited photodynamic efficiency by the hypoxia condition of tumors. Herein, we fabricate the MOF@MOF structure with a protective MOF shell to improve the stability and relieve the hypoxia condition of tumors for sensitized PDT. Under protection of the MOF shell, the MOF@MOF structure can keep intact for 96 h under physiological conditions. Consequently, the tumoral accumulation efficiency is two folds of the MOF core. Furthermore, the MOF shell decomposes under acidic environment, and the loaded inhibitor of mitochondria pyruvate carrier (7-amino carboxycoumarins-2, 7ACC2) will be released. 7ACC2 inhibits the mitochondrial pyruvate influx and simultaneously blocks glucose and lactate from fueling the mitochondrial respiration, thereupon relieving the hypoxia condition of tumors. Under a 5-min laser irradiation, the 7ACC2 carrying MOF@MOF nanoplatforms induced doubled cellular apoptosis and reduced 70% of the tumor growth compared with the cargo-free MOF@MOF. In summary, the design of this stable and hypoxia self-relievable MOF@MOF nanoplatform will enlighten the future development of MOF-based nanomedicines and PDT. Statement of significanceThough widely used for photodynamic therapy (PDT) in previous studies, porphyrinic metal-organic frameworks (MOFs) still face challenges in poor stability under physiological conditions and limited photodynamic efficiency due to the hypoxia condition of tumors. In order to solve these problems, (1) we develop the MOF@MOF strategy to improve the physiological stability; (2) an inhibitor of mitochondria pyruvate carrier, 7-amino carboxycoumarins-2 (7ACC2), is loaded to inhibit the mitochondrial pyruvate influx and simultaneously block glucose and lactate from fueling the mitochondrial respiration, thereupon relieving the hypoxia condition of tumors. In comparison with previous studies, our strategy simultaneously improves stability and overcomes the limited PDT efficiency in the hypoxia tumor tissue, which will enlighten the future development of MOF-based nanomedicines and PDT.

Isocitrate dehydrogenase 1 sustains a hybrid cytoplasmic-mitochondrial tricarboxylic acid cycle that can be targeted for therapeutic purposes in prostate cancer.

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

Hyaluronic acid-based supramolecular nanomedicine with optimized ratio of oxaliplatin/chlorin e6 for combined chemotherapy and O2-economized photodynamic therapy.

Dual- or multi-modality combination therapy has become one of the most effective strategies to overcome drug resistance in cancer therapy, and the optimized ratio of the therapeutic agents working on the tumor greatly affects the therapeutic outcomes. However, the absence of a facile method to optimize the ratio of therapeutic agents in nanomedicine has, at least in part, impaired the clinical potential of combination therapy. Herein, a new cucurbit[7]uril (CB[7])-conjugated hyaluronic acid (HA) based nanomedicine was developed, in which both chlorin e6 (Ce6) and oxaliplatin (OX) were co-loaded non-covalently at an optimized ratio via facile host-guest complexation, for optimal, combined photodynamic therapy (PDT)/chemotherapy. To maximize the therapeutic efficacy, a mitochondrial respiration inhibitor, atovaquone (Ato), was also loaded into the nanomedicine to limit consumption of oxygen by the solid tumor, sparing oxygen for more efficient PDT. Additionally, HA on the surface of nanomedicine allowed targeted delivery to cancer cells with over-expressed CD44 receptors (such as CT26 cell lines). Thus, this supramolecular nanomedicine platform with an optimal ratio of photosensitizer and chemotherapeutic agent not only provides an important new tool for enhanced PDT/chemotherapy of solid tumors, but also offers a CB[7]-based host-guest complexation strategy to facilely optimize the ratio of therapeutic agents for multi-modality nanomedicine. STATEMENT OF SIGNIFICANCE: Chemotherapy remains the most common modality for cancer treatment in clinical practice. Combination therapy by co-delivery of two or more therapeutic agents has been recognized as one of the most effective strategies to improve therapeutic outcome of cancer treatment. However, the ratio of loaded drugs could not be facilely optimized, which may greatly affect the combination efficiency and overall therapeutic outcome. Herein, we developed a hyaluronic acid based supramolecular nanomedicine with facile method to optimize the ratio of two therapeutic agents for improved therapeutic outcome. This supramolecular nanomedicine not only provides an important new tool for enhanced photodynamic therapy/chemotherapy of solid tumors, but also offers insights in using macrocyclic molecule-based host-guest complexation to facilely optimize the ratio of therapeutic agents in multi-modality nanomedicine.

Smart Nanosystem-Mediated Inhibition of Mitochondrial Respiration for Enhanced Phototherapy-Induced Antitumor Immunity.

Here, based on oxygen-dependent photodynamic therapy (PDT) and oxygen-consumed oxidative phosphorylation of cancer tissues, we designed and developed a nanosystem (named CyI&Met-Liposome, LCM) to co-encapsulate the photosensitizer CyI and mitochondrial respiration inhibitor metformin (Met) as a PDT enhancer. We synthesized nanoliposomes encapsulating Met and CyI with excellent photodynamic/photothermal and anti-tumor immune properties using a thin film dispersion method. Confocal microscopy and flow cytometry were used to assess the cellular uptake, PDT, photothermal therapy (PTT) and immunogenicity of nanosystem in vitro. Finally, two tumor models in mice were constructed to investigate the tumor suppression and immunity in vivo. The resulting nanosystem relieved hypoxia in tumor tissues, enhanced PDT efficiency, and amplified antitumor immunity induced by phototherapy. As a photosensitizer, CyI effectively killed the tumor by generating toxic singlet reactive oxygen species (ROS), while the addition of Met reduced oxygen consumption in tumor tissues, thereby evoking an immune response via oxygen-boosted PDT. Both in vitro and in vivo results illustrated that LCM effectively restricted the respiration of tumor cells to reduce tumor hypoxia, thus providing continuous oxygen for enhanced CyI-mediated PDT. Furthermore, T cells were recruited and activated at high levels, providing a promising platform to eliminate the primary tumors and synchronously realize effective inhibition of distant tumors.

Bioengineered nanogenerator with sustainable reactive oxygen species storm for self-reinforcing sono-chemodynamic oncotherapy

Oxidative stress-based antitumor modalities derived from reactive oxygen species (ROS) storms have attracted increasing attention. Nevertheless, low delivery efficiency, poor selectivity, hypoxia and overexpressed glutathione (GSH) have severely restricted the sustainable generation of the ROS storm in tumor cells. Herein, we design a bioengineered nanogenerator by coordination-driven co-assembly of sonosensitizer indocyanine green (ICG), Fenton-like agent copper ion (CuⅡ) and mitochondrial respiratory inhibitor metformin (MET), which is then camouflaged by a cancer cytomembrane to induce a sustainable intracellular ROS storm for on-demand self-reinforcing sono-chemodynamic oncotherapy. Such a nanogenerator with a core–shell structure, suitable diameter and outstanding stability can efficiently accumulate in tumor regions and then internalize into tumor cells through the camouflaging and homologous targeting strategy of the cancer cytomembrane. The nanogenerator shows an exceptional instability under the triple stimulations of acidic lysosomes, overexpressed GSH and ultrasound (US) radiation, thereby resulting in the rapid disassembly and burst drug release. Interestingly, the released MET significantly enhances the sonodynamic therapy (SDT) efficacy of the released ICG by inhibiting mitochondrial respiration and meanwhile the released CuⅡ obviously reduces ROS elimination by downregulating overexpressed GSH for self-amplifying and self-protecting the intracellular ROS storm. Moreover, such a nanogenerator almost completely achieves the tumor ablation in vivo in a single therapy cycle. Taken together, our bioengineered nanogenerator with a sustainable ROS storm can provide a promising strategy for ROS storm-based oncotherapy.

Nutrient-Dependent Mitochondrial Fission Enhances Osteoblast Function

Background: The bone synthesizing function of osteoblasts (OBs) is a highly demanding energy process that requires nutrients. However, how nutrient availability affects OBs behavior and bone mineralization remain to be fully understood. Methods: MC3T3-E1 cell line and primary OBs (OBs) cultures were treated with physiological levels of glucose (G; 5.5 mM) alone or with the addition of palmitic acid (G+PA) at different concentrations. Mitochondria morphology and activity were evaluated by fluorescence microscopy, qPCR, and oxygen consumption rate (OCR) measurement, and OBs function was assessed by mineralization assay. Results: The addition of non-lipotoxic levels of 25 μM PA to G increased mineralization in OBs. G+25 μM PA exposure reduced mitochondria size in OBs, which was associated with increased activation of dynamin-related protein 1, a mitochondrial fission protein, enhanced mitochondria OCR and ATP production, and increased expression of oxidative phosphorylation genes. Treatment with Mdivi-1, a putative inhibitor of mitochondrial fission, reduced osteogenesis and mitochondrial respiration in OBs. Conclusions: Our results revealed that OBs function was enhanced in the presence of glucose and PA at 25 μM. This was associated with increased OBs mitochondrial respiration and dynamics. These results suggest a role for nutrient availability in bone physiology and pathophysiology.

Respiratory Syncytial Virus Matrix Protein Is Sufficient and Necessary to Remodel Host Mitochondria in Infection.

Although respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants, immunosuppressed adults and the elderly worldwide, there is no licensed RSV vaccine or widely applicable antiviral therapeutics We previously reported a staged redistribution of mitochondria with compromised respiratory activities and increased reactive oxygen species (ROS) generation during RSV infection. Here, we show for the first time that the RSV matrix protein (M) is sufficient and necessary to induce these effects. Ectopically expressed M, but not other RSV proteins, was able to induce mitochondrial perinuclear clustering, inhibition of mitochondrial respiration, loss of mitochondrial membrane potential (Δψm), and enhanced generation of mitochondrial ROS (mtROS) in infection. Truncation and mutagenic analysis revealed that the central nucleic acid-binding domain of M is essential for the effects on host mitochondria, with arginine/lysine residues 170/172 being critically important. Recombinant RSV carrying the arginine/lysine mutations in M was unable to elicit effects on host mitochondria. Further, wild-type but not mutant RSV was found to inhibit the mRNA expression of genes encoding mitochondrial proteins, including Complex I subunits. Importantly, the RSV mutant was impaired in virus production, underlining the importance of M-dependent effects on mitochondria to RSV infection. Together, our results highlight M's unique ability to remodel host cell mitochondria and its critical role in RSV infection, representing a novel, potential target for future anti-RSV strategies.

UDP-glucose Regulates Dendritic Cell Mitochondrial Respiration via a Nitric Oxide-dependent Mechanism

Abstract Dendritic cells (DCs) are key innate immune cells specialized in fine tuning an immune response. Activation of DCs through Toll-Like Receptors (TLRs) is accompanied by a metabolic switch to increased use of glycolytic metabolism fueled by carbohydrates glucose and glycogen. Our lab has shown that DCs require glucose and glycogen to perform their immune effector functions including: production of cytokines and inflammatory mediators, antigen presentation to T cells, and initiation of the T cell-mediated immune response. Recent studies in inflammatory macrophages showed a secondary metabolite in the glycogen synthetic pathway, uridine diphosphate glucose (UDP-glucose), is upregulated through TLR4 stimulation. UDP-glucose exits the cell and signals in an autocrine manner through its specific purinergic receptor P2Y 14. Additionally, P2Y 14R signaling modulates the expression of proinflammatory pathways including upregulation of inducible nitric oxide synthase (iNOS). Activated bone marrow-derived DCs (BMDCs) upregulate iNOS and produce nitric oxide (NO), which has pleiotropic effects, including inhibiting mitochondrial respiration. Therefore, we hypothesized that the UDP-glucose/P2Y 14R/iNOS axis regulates BMDC metabolism, which has further ramifications on DC cell survival and proinflammatory phenotypes. To investigate the impact of UDP-glucose as an autocrine signaling molecule, we characterized proinflammatory gene transcript and protein levels, as well as the functional outcomes of increased iNOS protein in the cell. These studies showed that DCs use the UDP-glucose/P2Y 14R/iNOS axis to bolster inflammatory phenotypes including inhibition of mitochondrial respiration leading to exacerbated cell death. Supported by grants from NIH (R01 AI158710-01, 1R21AI135385-01A1) and UVM (COBRE VCIID Funding Support and Pilot Project)

Targeting tumor energy metabolism via simultaneous inhibition of mitochondrial respiration and glycolysis using biodegradable hydroxyapatite nanorods

Tumor cells obtain energy supply from the unique metabolic pathways of mitochondrial respiration and glycolysis, which can be used interchangeably to produce adenosine triphosphate (ATP) for survival. To simultaneously block the two metabolic pathways and sharply cut off ATP supply, a multifunctional “nanoenabled energy interrupter” (called as HNHA-GC) was prepared by attaching glucose oxidase (GOx), hyaluronic acid (HA), and 10-hydroxycamptothecin (CPT) on the surface of degradable hydroxyapatite (NHA) nanorods. After targeted delivery of HNHA-GC to the tumor site by HA, the tumor-selective acid degradation of HNHA-GC as well as the subsequent deliveries of Ca2+, drug CPT, and GOx take place. The released Ca2+ and CPT induce mitochondrial dysfunction by Ca2+ overload and chemotherapy respectively, while the GOx-triggered glucose oxidation inhibits glycolysis by starvation therapy (exogenous effect). The generated H2O2 and released CPT increase the intracellular reactive oxygen (ROS) level. Moreover, the generated H+ and enhanced ROS promote Ca2+ overload by accelerating the degradation of HNHA-GC and preventing intracellular Ca2+ efflux, respectively (endogenous effect). As a result, the HNHA-GC displays a promising therapeutic modality for simultaneously cutting off mitochondrial and glycolytic ATP production through a combination of Ca2+ overload, chemotherapy, and starvation therapy.

Improved Tumor Control Following Radiosensitization with Ultrasound-Sensitive Oxygen Microbubbles and Tumor Mitochondrial Respiration Inhibitors in a Preclinical Model of Head and Neck Cancer.

Tumor hypoxia (oxygen deficiency) is a major contributor to radiotherapy resistance. Ultrasound-sensitive microbubbles containing oxygen have been explored as a mechanism for overcoming tumor hypoxia locally prior to radiotherapy. Previously, our group demonstrated the ability to encapsulate and deliver a pharmacological inhibitor of tumor mitochondrial respiration (lonidamine (LND)), which resulted in ultrasound-sensitive microbubbles loaded with O2 and LND providing prolonged oxygenation relative to oxygenated microbubbles alone. This follow-up study aimed to evaluate the therapeutic response to radiation following the administration of oxygen microbubbles combined with tumor mitochondrial respiration inhibitors in a head and neck squamous cell carcinoma (HNSCC) tumor model. The influences of different radiation dose rates and treatment combinations were also explored. The results demonstrated that the co-delivery of O2 and LND successfully sensitized HNSCC tumors to radiation, and this was also enhanced with oral metformin, significantly slowing tumor growth relative to unsensitized controls (p < 0.01). Microbubble sensitization was also shown to improve overall animal survival. Importantly, effects were found to be radiation dose-rate-dependent, reflecting the transient nature of tumor oxygenation.

Temporal inhibition of the electron transport chain attenuates stress-induced cellular senescence by prolonged disturbance of proteostasis in human fibroblasts.

We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC-5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short-term co-treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. By co-treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt ) were remarkably suppressed. Furthermore, AA co-treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.

Abstract 1387: Novel thienopyrimidine-hydrazinyl analog, TPH104m inhibits mitochondrial respiration and induces caspase-independent cell death in triple-negative breast cancer cells

Abstract A triple negative breast cancer (TNBC) is an aggressive and difficult-to-treat breast cancer subtype. Neoadjuvant chemotherapy using apoptosis-inducing taxanes and/or anthracyclines has been shown to produce early therapeutic responses. Unfortunately, drug resistance often results from mutations in apoptotic pathways, which can lead to metastasis and poor prognosis. There are emerging studies suggesting that TNBC tumors display altered mitochondrial dynamics and have overexpressed dynamin related protein 1 (Drp1), which drives mitochondrial fission. Drp1 deregulation also influences metabolic changes and mitochondrial energetics, which support TNBC tumor growth. Therefore, bypassing apoptosis signaling cascade, suppressing Drp1 and reversing aberrant mitochondrial dynamics may be an effective therapy for TNBC. We report here for the first time the identification of novel thienopyrimidine-hydrazinyl analog of parent TPH104, namely, TPH104m, which inhibits Drp1 phosphorylation and induces a non-apoptotic TNBC cell death. TPH104m significantly inhibited TNBC proliferation at nanomolar concentrations, and was more selective for TNBC than normal human mammary epithelial cells. TPH104m induced a unique type of non-apoptotic cell death, where cells swelled without shrinkage, nuclear fragmentation or apoptotic bleb formation typical of apoptosis. Moreover, TPH104m did not induce the cleavage of initator (caspase-8, -9) and executioner caspases (caspase-3, -7), and zVAD.fmk, a pan-caspase inhibitor, did not rescue TNBC cell death when incubated with TPH104m. Furthermore, TPH104m did not significantly increase reactive oxygen species (ROS) production in BT-20 cells compared to Paclitaxel treated cells. Although mitochondrial membrane potential (MMP) was significantly reduced, cytochrome C release was not increased, but instead decreased, explaining why MMP loss did not lead to apoptosis. TPH104m remarkably downregulated the phosphorylated and total forms of Drp1 protein in TNBC cells. A docking-based reverse screening approach and Surface Plasmon Resonance (SPR) data indicated that TPH104m binds to Drp1 more strongly than Mdivi-1, a weak non-specific Drp1 inhibitor. A complete and partial Drp1 KO cell line was created in SUM-159 TNBC cells to investigate the role of Drp1 in TPH104m-induced non-apoptotic cell death. Genetically deleting Drp1 from TNBC cells reduced TPH104m's cytotoxic potential suggesting Drp1's role in TPH104m's anticancer mechanism. Moreover, mito-stress assay showed that TPH104m severely impaired oxygen consumption rate, resulting in decreased ATP production and inhibition of mitochondrial respiration. Development of alternative treatments for TNBC will be facilitated by a deeper understanding of mitochondrial role in non-apoptotic cell death. Citation Format: Saloni Malla, David Terrero, Sushma Karki, Mariam Sami Abou-Dahech, Shikha Kumari, Amit K. Tiwari. Novel thienopyrimidine-hydrazinyl analog, TPH104m inhibits mitochondrial respiration and induces caspase-independent cell death in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1387.

Abstract 2554: Phosphorylationdependent control of protein synthesis in response to inhibitors of mRNA translation and mitochondrial respiration in HeLa cancer cell line

Abstract Control of protein turnover via regulation of the rates of protein synthesis and degradation is one of the principal means by which cell signaling networks can be modulated. This regulation can also be achieved through alterations in post-translational modifications such as phosphorylation. The human genome encodes ~21,300 proteins, and over 95% of these are subjected to reversible phosphorylation at nearly a million sites by at least 568 protein kinases (PK) and about 156 protein phosphatases (PP). The aim of current study was to use differential treatments of cultured cells with diverse agents to uncover connectivities between protein kinases and their substrates to elucidate the architecture of signaling networks. Western blot analyses showed that the protein synthesis inhibitor anisomycin (Alomone labs, Israel) and the mitochondrial respiratory chain inhibitor arsenate (Sigma, Saint Louis, MO) both increased ribosomal proteins S6 (RPS6) S235+S236+S240 phosphorylation. There were similar patterns of activatory phosphorylations observed for upstream kinases of RPS6, including p70S6K at T255 and RSK1 at S380. Anisomycin and arsenate also enhanced activatory phosphorylation of the kinases p38α at T180+Y182 and CK2α at Y255. We explored whether or not the effect of anisomycin or arsenate on these protein kinases were dependent on mTOR signaling with its inhibitor rapamycin (Calbiochem, EMD Chemicals Inc.), which by itself slightly increased the RPS6 S235+S236+S240 phosphorylation. However, rapamycin completely blocked both anisomycin and arsenate induced RPS6 phosphorylation. Rapamycin did not block phosphorylation of p38α MAPK T180 and Y182 in response to anisomycin or arsenate. The results of CK2α Y255 phosphorylation were particularly interesting, because rapamycin treatment alone increased the phosphorylation at this site as did anisomycin alone, but the combination of these inhibitors blocked this phosphorylation. However, CK2α Y255 phosphorylation in response to arsenate alone and in combination with rapamycin showed that the arsenate induction of phosphorylation was not dependent on mTOR signaling. We hypothesized that this mTOR-independent signaling pathway involves the activation of protein-tyrosine kinases that may target both CK2a and p38 MAPK. Therefore, we designed an in vitro experiment and we found that some protein-tyrosine kinases like Blk, Bmx, Btk, EGFR, Fer, Fes, Frk, Fyn, LynA, Pyk2, Src, Syk, Tec, and TrkB can significantly phosphorylate p38 MAPK at Y182 and CK2α at Y255, whereas some other protein-tyrosine kinases such as Fgr, InsR, Jak2, Met, and Txk primarily targeted CK2α at Y255. We also demonstrated that p38 MAPK is able to directly phosphorylate CK2α near its C-terminus at T360 and S362, and this correlated with activation of its casein phosphotransferase activity. Citation Format: Hamidreza Galavi, Steven Pelech. Phosphorylationdependent control of protein synthesis in response to inhibitors of mRNA translation and mitochondrial respiration in HeLa cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2554.