Inhibition Of Mitochondrial Function Research Articles

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-29-01.

Cone photoreceptor phosphodiesterase PDE6H inhibition regulates cancer cell growth and metabolism, replicating the dark retina response

BackgroundPDE6H encodes PDE6γ′, the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth.MethodsFrom an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model.ResultsPDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ′-deficient tumours.ConclusionsOur results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ′ depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

Novel ability of diflubenzuron as an inhibitor of mitochondrial function

Compounds classified as benzoylphenylurea (BPU), such as diflubenzuron (DFB), are used as insecticides. Although BPU disrupts molting by inhibiting chitin biosynthesis and exhibits insecticidal activity, their exact mode of action remains unknown. Since epidermal cells proliferate and morphologically change from squamous to columnar cells during the early stages of insect molting, we speculate that a transition similar to that from epithelium to mesenchyme occurs and that BPU may inhibit this transition. Here, we addressed this possibility. We found that DFB decreases actin expression in insect cells (the tissue cultures of insect integument). Detailed analysis in Schneider S2 cells reveals that DFB inhibits the expression of actin isoforms (Act5C and Act42A) and the Drosophila ortholog of myocardin-related transcription factor (Mrtf), leading to cell growth suppression. Proteomics identified the Drosophila ortholog of prohibitin (Phb1D and Phb2E) as one of the DFB-binding proteins. DFB inhibits the interaction between Phb1D and Phb2E and induces mitochondrial dysfunction. The knock-down of Phb2E suppresses the expression of Act5C, Act42A, and Mrtf, leading to cell growth inhibition. Thus, the disruption of Phb function is a possible novel target of DFB.

The clarithromycin-binding proteins NIPSNAP1 and 2 regulate cytokine production through mitochondrial quality control

The mechanism underlying the anti-inflammatory effect of macrolide antibiotics, such as clarithromycin (CAM), remains to be clarified. The CAM-binding proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25 (SNAP25)-like protein homolog (NIPSNAP) 1 and 2 are involved in the immune response and mitochondrial homeostasis. However, the axis between CAM-NIPSNAP-mitochondria and Toll-like receptor (TLR) and their molecular mechanisms remain unknown. In this study, we sought to elucidate the relationship between mitochondrial homeostasis mediated by NIPSNAP1 and 2 and the immunomodulatory effect of CAM. NIPSNAP1 or 2 knockdown (KD) by RNA interference impaired TLR4-mediated interleukin-8 (IL-8) production. Similar impairment was observed upon treatment with mitochondrial function inhibitors. However, IL-8 secretion was not impaired in NIPSNAP1 and 2 individual knockout (KO) and double KO (DKO) cells. Moreover, the oxygen consumption rate (OCR) in mitochondria measured using a flex analyzer was significantly reduced in NIPSNAP1 or 2 KD cells, but not in DKO cells. CAM also dose-dependently reduced the OCR. These results indicate that CAM suppresses the IL-8 production via the mitochondrial quality control regulated by temporary functional inhibition of NIPSNAP1 and 2. Our findings provide new insight into the mechanisms underlying cytokine production, including the TLR-mitochondria axis, and the immunomodulatory effects of macrolides.

Inhibition of mitochondrial function by approved drugs overcomes nasopharyngeal carcinoma chemoresistance.

The development of chemo-resistance in nasopharyngeal carcinoma (NPC) presents a significant therapeutic challenge, and its underlying mechanisms remain poorly understood. In our previous studies, we highlighted the association between isoprenylcysteine carboxylmethyltransferase (ICMT) and chemoresistance in NPC. In this current research, we revealed that both 5-FU and cisplatin-resistant NPC cells exhibited elevated mitochondrial function and increased expression of mitochondrial genes, independent of ICMT. Our investigations further showed that classic mitochondrial inhibitors, such as oligomycin, antimycin, and rotenone, were notably more effective in reducing viability in chemo-resistant NPC cells compared to parental cells. Moreover, we identified two antimicrobial drugs, tigecycline and atovaquone, recognized as mitochondrial inhibitors, as potent agents for decreasing chemo-resistant NPC cells by targeting mitochondrial respiration. Remarkably, tigecycline and atovaquone, administered at tolerable doses, inhibited chemo-resistant NPC growth in mouse models and extended overall survival rates. This work unveils the efficacy of mitochondrial inhibition as a promising strategy to overcome chemo-resistance in NPC. Additionally, our findings highlight the potential repurposing of clinically available drugs like tigecycline and atovaquone for treating NPC patients who develop chemoresistance.

Plumbagin Suppresses Growth, Induces Apoptosis, and Inhibits Migration in Cholangiocarcinoma via Reactive Oxygen Species Generation and Mitochondrial Function

Objectives: Plumbagin has been displayed on growth inhibition in several cancer cells; however, the anticancer effects of plumbagin on cholangiocarcinoma (CCA) cell are still less information. This article will explore the potential anticancer effects and underlying mechanisms of plumbagin on CCA cells. Materials and Methods: Two types of CCA cell, including KKU-100 with slow growth and migration and KKU-M452 with rapid growth and migration, were treated with plumbagin and measured the growth inhibition and migratory suppression by sulforhodamine B, colony formation, acridine orange/ethidium bromide staining, wound healing, and Matrigel migration. Apoptosis induction, reactive oxygen species (ROS) formation, and mitochondrial function were analyzed by the flow cytometric method. Results: To compare the plumbagin effects on two types of CCA cells, the data indicated that plumbagin strongly reduced cell proliferation in a concentration- and time-dependent manner. At 72 h, IC50 values were 1.11 ± 0.12 µM for KKU-100 and 2.52 ± 0.40 µM for KKU-M452 cells, with inhibition of the cell cycle in the G0/G1 phase for KKU-100 and the G2/M phase for KKU-M452, respectively. Moreover, plumbagin decreased cell migration in both CCA cells in a dose-dependent manner. Furthermore, late apoptosis was significantly induced after treatment with plumbagin. Mechanistically, plumbagin increased ROS along with decreased mitochondrial function in CCA cells. Finally, the position of 5-hydroxy on the naphthoquinone ring of plumbagin was in a good position to form an H-bond with the amino group for all caspase 3, cytochrome C, and p21 binding sites, and these data indicated that plumbagin suppressed CCA cells via modulation of these proteins. Conclusion: Plumbagin strongly suppresses cell viability and stimulates apoptosis in CCA cells via induction of ROS and inhibition of mitochondrial function.

Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures.

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

The Antimelanoma Biological Assessment of Triterpenic Acid Functionalized Gold Nanoparticles.

One of several promising strategies for increasing the bioavailability and therapeutic potential of high-lipophilic biologically active compounds is gold nanoparticle formulation. The current study describes the synthesis and biological antimelanoma evaluation of three triterpen-functionalized gold nanoparticles, obtained using our previously reported antimelanoma benzotriazole-triterpenic acid esters. Functionalized gold nanoparticle (GNP) formation was validated through UV-VIS and FTIR spectroscopy. The conjugate's cytotoxic effects were investigated using HaCaT healthy keratinocytes and A375 human melanoma cells. On A375 cells, all three conjugates demonstrated dose-dependent cytotoxic activity, but no significant cytotoxic effects were observed on normal HaCaT keratinocytes. GNP-conjugates were found to be more cytotoxic than their parent compounds. After treatment with all three GNP-conjugates, 4,6'-diamidino-2-phenylindole (DAPI) staining revealed morphological changes consistent with apoptosis in A375 melanoma cells. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis revealed that the triterpene-GNP conjugate treated A375 melanoma cells had a fold change increase in Bcl-2-associated X protein (BAX) expression and a fold change decrease in B-cell lymphoma 2 (Bcl-2) expression. In A735 melanoma cells, high-resolution respirometry studies revealed that all three GNP-conjugates act as selective inhibitors of mitochondrial function. Furthermore, by examining the effect on each mitochondrial respiratory rate, the results indicate that all three conjugates are capable of increasing the production of reactive oxygen species (ROS), an apoptosis trigger in cancer cells.

Inhibition of mitochondrial function: An alternative explanation for the antipyretic and hypothermic actions of acetaminophen

AimsAcetaminophen is the medication of choice when treating fever because of its limited anti-inflammatory effects. However at overdose it can cause mitochondrial dysfunction and damage, often associated with metabolism to N-acetyl-p-benzoquinone imine (NAPQI). What has never been investigated is whether the inhibition of mitochondrial function, particularly fatty acid uptake and oxidation could be the key to its antipyretic and hypothermic properties. MethodsMitochondrial function and fatty acid oxidation (FAO) was determined by measuring oxygen consumption rate (OCR) in isolated mitochondria and in 3T3-L1 adipocytes using the XFp Analyser. Basal fatty acids and adrenergic stimulated OCR of mitochondria and 3T3-L1 adipocytes were assessed with acetaminophen and compared to NAPQI, etomoxir, and various mitochondrial stress compounds. Key findingsUsing the XFp Analyser, acetaminophen (10 mM) decreased FAO by 31 % and 29 % in basal and palmitate stimulated adipocytes. NAPQI (50 μM) caused a 63 % decrease in both basal and palmitate stimulated FAO. Acetaminophen (10 mM) caused a 34 % reduction in basal and adrenergic stimulated OCR. In addition acetaminophen also inhibited complex I and II activity at 5 mM. NAPQI was far more potent at reducing mitochondrial respiratory capacity, maximum respiratory rates and ATP production than acetaminophen. SignificanceThese studies demonstrate the direct inhibition of mitochondrial function by acetaminophen at concentrations which have been shown to reduce fever and hypothermia in mammals. Understanding how antipyretics directly affect mitochondrial function and heat generation could lead to the development of new antipyretics which are not compromised by the anti-inflammatory and toxicity of the current medications.

Involvement of Mitochondrial Dysfunction in the Inflammatory Response in Human Mesothelial Cells from Peritoneal Dialysis Effluent.

Recent studies have related mitochondrial impairment with peritoneal membrane damage during peritoneal dialysis (PD) therapy. Here, we assessed the involvement of mitochondrial dysfunction in the inflammatory response in human mesothelial cells, a hallmark in the pathogenesis of PD-related peritoneal membrane damage. Our ex vivo studies showed that IL-1β causes a drop in the mitochondrial membrane potential in cells from peritoneal effluent. Moreover, when mitochondrial damage was induced by inhibitors of mitochondrial function, a low-grade inflammatory response was generated. Interestingly, mitochondrial damage sensitized mesothelial cells, causing a significant increase in the inflammatory response induced by cytokines, in which ROS generation and NF-κB activation appear to be involved, since inflammation was counteracted by both mitoTEMPO (mitochondrial ROS scavenger) and BAY-117085 (NF-κB inhibitor). Furthermore, the natural anti-inflammatory antioxidant resveratrol significantly attenuated the inflammatory response, by reversing the decline in mitochondrial membrane potential and decreasing the expression of IL-8, COX-2 and PGE2 caused by IL-1β. These findings suggest that IL-1β regulates mitochondrial function in mesothelial cells and that mitochondrial dysfunction could induce an inflammatory scenario that sensitizes these cells, causing significant amplification of the inflammatory response induced by cytokines. Resveratrol may represent a promising strategy in controlling the mesothelial inflammatory response to PD.

HMGA2 mediates Cr (VI)-induced metabolic reprogramming through binding to mitochondrial D-Loop region.

Hexavalent chromium [Cr (VI)] exists environmentally and occupationally. It has been shown to pose a carcinogenic hazard in certain occupations. This study was to investigate the role of high mobility group A2 (HMGA2) in Cr (VI)-induced metabolism reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis in A549 and HELF cells. First, knockdown of HMGA2 by siHMGA2 significantly attenuated Cr (VI)-reduced expression of OXPHOS-related proteins (COX IV and ND1) and mitochondrial mass, indicating that HMGA2 was involved in Cr (VI)-reduced OXPHOS. Overexpression of HMGA2 by transfection of HMGA2-DNA plasmids reduced the expression of COX IV, ND1 and mitochondrial mass, suggesting the negative role of HMGA2 in OXPHOS. Secondly, both CCCP, the inhibitor of mitochondrial function, and the ER stress inhibitor, 4-phenylbutyric acid (4-PBA), decreased the level of HMGA2, indicating that the interaction of mitochondrial dysfunction and ER stress resulted in Cr (VI)-induced HMGA2 expression. Further study demonstrated that ER stress/HMGA2 axis mediated the metabolism rewiring from OXPHOS to aerobic glycolysis. Notably, Cr (VI) induced the accumulation of HMGA2 proteins in mitochondria and ChIP assay demonstrated that HMGA2 proteins could bind to D-loop region of mitochondrial DNA (mtDNA), which provided the proof for HMGA2-modulating OXPHOS. Taken together, our results suggested that the interaction of mitochondria and ER stress-enhanced HMGA2 played an important role in Cr (VI)-induced metabolic reprogramming from OXPHOS to glycolysis by binding directly to D-loop region of mtDNA. This work informs on the potential mode of action for Cr (VI)-induced tumors and builds on growing evidence regarding the contribution of cellular metabolic disruption contributing to carcinogenicity.

Proteasome granule formation is regulated through mitochondrial respiration and kinase signaling.

In the yeast Saccharomyces cerevisiae, proteasomes are enriched in cell nuclei, in which they execute important cellular functions. Nutrient stress can change this localization, indicating that proteasomes respond to the metabolic state of the cell. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules. Surprisingly, we observed strongly reduced levels of proteasome granules when cells had active cellular respiration prior to starvation. This suggests that the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function, we observed that proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a mitogen-activated protein kinase (MAPK) cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, but it is dispensable for granule formation following carbon starvation. We propose a model in which mitochondrial activity promotes nuclear localization of the proteasome. This article has an associated First Person interview with the first author of the paper.

Baicalin promotes antibacterial defenses by modulating mitochondrial function

Natural flavonoids, such as baicalin, have been extensively studied for their role in bacterial infection. However, the underlying mechanisms remain poorly understood. We demonstrated that baicalin coordinates mitochondrial function and dynamics to promote antibacterial response. Baicalin protected against Staphylococcus aureus infections and alleviates inflammatory responses in vivo and in vitro. An increase in mitochondrial mass and elevated expression of factors regulating mitochondrial fission and fusion were observed in baicalin-treated macrophages. Baicalin induced Drp1-dependent biogenesis, which contributes to the generation of additional mitochondria. Baicalin improved the mitochondrial membrane potential, ATP levels, and mitochondrial reactive oxygen species (mtROS) production. Importantly, the inhibition of mitochondrial function by rotenone or MitoTEMPO suppressed the antimicrobial activity of baicalin in macrophages. We conclude that baicalin can regulate immune responses during S. aureus infection by improving mitochondrial function and dynamics, implying that it is a promising therapeutic agent for controlling infection and inflammatory diseases.

Anisomycin inhibits angiogenesis, growth, and survival of triple-negative breast cancer through mitochondrial dysfunction, AMPK activation, and mTOR inhibition.

Aberrant upregulation of mitochondrial biogenesis is observed in breast cancer and holds potential therapeutic option. In our work, we showed that inhibition of mitochondrial function by anisomycin is effective against triple-negative breast cancer (TNBC). Anisomycin inhibits growth and induces caspase-dependent apoptosis in a panel of TNBC cell lines. Of note, anisomycin at a tolerable dose remarkably suppresses growth of TNBC in mice. In addition, anisomycin effectively targets breast cancer angiogenesis through inhibiting capillary network formation, migration, proliferation, and survival. Mechanistic studies show that although anisomycin activates p38 and JNK, their activations are not required for anisomycin's action. In contrast, anisomycin inhibits mitochondrial respiration, and decreases mitochondrial membrane potential and adenosine triphosphate (ATP) level. The inhibitory effect of anisomycin is significantly reversed in mitochondria respiration-deficient ρ0 cells. As a consequence, anisomycin activates AMPK and inhibits mammalian target-of-rapamycin signaling pathways. Our work demonstrated that anisomycin is a useful addition to the treatment armamentarium for TNBC.

Abstract 5808: Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions

Abstract Esophageal cancer is one of the most aggressive forms of human malignancy with a 5-yearsurvival rate of less than 20%. Mitochondria are dynamic organelles that play essential roles in various cellular processes, including energy metabolism, redox homeostasis, and apoptotic cell death. Alterations in mitochondrial biology are associated with esophageal carcinogenesis and esophageal cancer cell response to therapy. As the anti-inflammatory drug diclofenac (DCF)induces mitochondrial dysfunction, we hypothesized that DCF may inhibit esophageal carcinogenesis by affecting mitochondrial processes. In the human esophageal squamous cell carcinoma (ESCC) cell line TE11, we performed MTT assay and Annexin-V/Propidium Iodide (PI) to evaluate the impact of DCF on proliferation and cell death, respectively. RNA sequencing and Ingenuity Pathway Analysis (IPA) identified differentially expressed genes (DEGs) and altered canonical pathways in DCF-treated TE11 cells. To assess metabolic function, we measured the levels of pyruvate, lactate, and ATP. Mitochondrial membrane potential and ROS production were assessed by flow cytometry for MitoTracker red/green and MitoSox red, respectively. Mice bearing subcutaneous syngeneic ESCC tumors were treated with DCF.DCF inhibited proliferation of TE11 in dose dependent manner with an IC50 concentration of 76.7µM. By contrast, the IC50 concentration of DCF in normal esophageal keratinocytes was 876.2µM, supporting selectivity of DCF for esophageal cancer cells. In TE11, 200 µM DCF induced apoptosis in 60% of cells. RNA-sequencing identified that DCF significantly altered expression of3287 genes (FC ≥ 1.50) in TE11, including the top cancer-associated genes: PLK1, MCM2, MCM3,MCM7, MCM10 and SKP2. IPA analysis revealed that DCF activated p53 signaling while inhibited Gluconeogenesis I, Glycolysis I and Oxidative Phosphorylation. DCF significantly inhibited glycolysis as demonstrated by reduction in concentrations of pyruvate (1.76-fold) and lactate (7.30-fold) and downregulation of the glycolysis- associated genes PKM2, PFKM, LDHA, and PKM1. A decreased production of ATP (5-fold) was also noted in DCF-treated TE11 cells. In addition, DCF depleted mitochondrial membrane potential (2.5-fold) and increased mitochondrial ROS production (5-fold). A reduction in ROS by mitochondrial antioxidant MitoTempo increased viability of the DCF-treated TE11 cells, indicating that ROS contributes to the anticancer activity of DCF. Consistent with antitumor activity of DCF in TE11, the drug significantly decreased the tumor volume in syngeneic ESCC tumors in vivo. Our preclinical findings indicate that DCF may limit esophageal cancer cell growth through the inhibition of mitochondrial functions, identifying a novel experimental therapeutic for ESCC. Future studies will define the precise molecular mechanisms through which DCF promotes cell death in vitro and in vivo. Citation Format: Mohammad Faujul Kabir, Adam Karami, Anbin Mu, Don-Gerard Conde, Kelly A. Whelan. Diclofenac inhibits esophageal cancer cell growth by depleting mitochondrial functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5808.

Lipoic acid blocks autophagic flux and impairs cellular bioenergetics in breast cancer and reduces stemness

Autophagy inhibition is currently considered a novel therapeutic strategy for cancer treatment. Lipoic acid (LA), a naturally occurring compound found in all prokaryotic and eukaryotic cells, inhibits breast cancer cell growth; however, the effect of LA on autophagy-mediated breast cancer cell death remains unknown. Our study identified that LA blocks autophagic flux by inhibiting autophagosome-lysosome fusion and lysosome activity which increases the accumulation of autophagosomes in MCF-7 and MDA-MB231 cells, leading to cell death of breast cancer cells. Interestingly, autophagic flux blockade limits the recycling of cellular fuels, resulting in insufficient substrates for cellular bioenergetics. Therefore, LA impairs cellular bioenergetics by the inhibition of mitochondrial function and glycolysis. We show that LA-induced ROS generation is responsible for the blockade of autophagic flux and cellular bioenergetics in breast cancer cells. Moreover, LA-mediated blockade of autophagic flux and ROS generation may interfere with the regulation of the BCSCs/progenitor phenotype. Here, we demonstrate that LA inhibits mammosphere formation and subpopulation of BCSCs. Together, these results implicate that LA acts as a prooxidant, potent autophagic flux inhibitor, and causes energetic impairment, which may lead to cell death in breast cancer cells/BCSCs.

MO712: Involvement of Mitochondrial Dysfunction in the Inflammatory Response in Human Mesothelial Cells from Peritoneal Dialysis Effluent

Abstract BACKGROUND AND AIMS Recent studies have related mitochondrial impairment with peritoneal membrane damage in peritoneal dialysis (PD) therapy. Here we assessed the involvement of mitochondrial dysfunction in the inflammatory response in human mesothelial cells, a hallmark in the pathogenesis of PD-related peritoneal membrane damage. METHOD Ex vivo studies of effluent-derived human mesothelial cells were carried out. Mitochondria function was evaluated by analysing the mitochondrial membrane potential with flow cytometry using the fluorescent dye tetramethylrhodamine. Mitochondrial dysfunction was induced with the inhibitors oligomycin and paraquat. The effects of mitochondrial dysfunction on inflammatory mediators were studied. Mitochondrial ROS scavenger (MitoTEMPO) and a NF-κB inhibitor (BAY-117 085) were used to investigate the pathways involved. The natural anti-inflammatory antioxidant resveratrol was also tested. RESULTS Our ex vivo studies showed that IL-1β causes a drop in the mitochondrial membrane potential in cells from peritoneal effluent. Besides, when mitochondrial damage was induced by inhibitors of mitochondrial function a low-grade inflammatory response was generated. Interestingly, mitochondrial damage sensitizes mesothelial cells causing a significant increase in the inflammatory response induced by cytokines, in which ROS generation and NF-κB activation are involved since inflammation was counteracted by both MitoTEMPO and BAY-117 085. Furthermore, pretreatment with resveratrol significantly reduced the inflammatory response by reversing the decline in mitochondrial membrane potential and decreasing the expression of IL-8, COX-2 and PGE2 caused by IL-1β. CONCLUSION These findings suggest that IL-1β regulates mitochondrial function in mesothelial cells and that mitochondrial dysfunction could induce an inflammatory scenario and sensitize these cells, causing a significant amplification of the inflammatory response induced by cytokines. Resveratrol may represent a promising strategy in controlling the mesothelial inflammatory response.

Proteasome Localization is Regulated Through Mitochondrial Respiration and Kinase Signaling

In most organisms, proteasomes are enriched in cell nuclei under optimal physiological conditions, indicating an important need for protein degradation in this organelle. However, stress conditions can result in proteasome nuclear export, possibly reflecting changes in demand for protein degradation. To understand this adaptive response, it is crucial to know when and how proteasome localization and activity are altered upon changing cellular needs. In yeast, carbon starvation triggers a reversible re‐localization of proteasomes to cytosolic granules known as proteasome storage granules (PSGs). These PSGs behave like liquid‐liquid phase separated structures and highlight a link between cellular metabolism and proteasome localization. We hypothesized that mitochondrial respiration is important in this process. To test this, we manipulated the glycolytic and respiratory activity in yeast using different carbon sources as well as chemical inhibitors of mitochondrial function and monitored proteasome localization by fluorescent microscopy. Growing cells in carbon sources that necessitate respiration prior to starvation, caused a strong reduction in proteasome storage granule formation upon carbon starvation. This suggests that the mitochondrial activity of cells is a determining factor in proteasome localization. Consistent with this, upon chemical inhibition of mitochondrial function, we observed proteasomes re‐localize to cytosolic granules independent of starvation stress. Interestingly, the stress response kinase Snf1, which is not required for proteasome re‐localization upon carbon starvation, was required for proteasome re‐localization following mitochondrial inhibition. In addition, MAP kinases of the cell wall integrity cascade were required for proteasome granule formation, specifically following respiratory but not glycolytic growth, upon carbon starvation or mitochondrial inhibition. In all, our data point to a model where mitochondria regulate proteasome localization via kinases that sense the cells metabolic state.

Exogenous Melatonin Activating Nuclear Factor E2-Related Factor 2 (Nrf2) Pathway via Melatonin Receptor to Reduce Oxidative Stress and Apoptosis in Antler Mesenchymal Stem Cells.

Antler growth depends on the proliferation and differentiation of mesenchymal stem cells (MSCs), and this process may be adversely affected by oxidative stress. Melatonin (MLT) has antioxidant functions, but its role in Cervidae remains largely unknown. In this article, flow cytometry, reactive oxygen species (ROS) identification, qPCR, and other methods were used to investigate the protective mechanism of MLT in H2O2-induced oxidative stress of antler MSCs. The results showed that MLT significantly increases cell viability by relieving the oxidative stress of antler MSCs. MLT inhibits cell apoptosis by protecting mitochondrial function. We blocked the melatonin receptor with luzindole (Luz) and found that the receptor blockade significantly increases H2O2-induced hyperoxide levels and causes significant inhibition of mitochondrial function. MLT treatment activates the nuclear factor E2-related factor 2 (Nrf2) antioxidant signaling pathway, up-regulates the expression of NAD(P)H quinone oxidoreductase 1 (NQO1) and other genes and it could inhibit apoptosis. In contrast, the melatonin receptor blockade down-regulates the expression of Nrf2 pathway-related genes, but significantly up-regulates the expression of apoptotic genes. It was indicated that MLT activates the Nrf2 pathway through the melatonin receptor and alleviates H2O2-induced oxidative stress and apoptosis in antler MSCs. This study provides a theoretical basis for further studying the oxidative stress and antioxidant process of antler MSCs and, thereby, increasing antler yields.

Milrinone effects on cardiac mitochondria, hemodynamics, and death in catecholamine-infused rats.

BackgroundCatecholamine-storm is considered the major cause of enterovirus 71-associated cardiopulmonary death. To elucidate the effect of milrinone on cardiac mitochondria and death, a rat model of catecholamine-induced heart failure was investigated.MethodsYoung male Spray-Dawley rats received a continuous intravenous infusion of norepinephrine then followed by co-treatment with and without milrinone or esmolol. Vital signs were monitored and echocardiography was performed at indicated time points. At the end of experiments, hearts were extracted to study mitochondrial function, biogenesis, and DNA copy numbers.ResultsHypernorepinephrinemia induced persistent tachycardia, hypertension, and high mortality and significantly impaired the activities of the electron transport chain and suppressed mitochondrial DNA copy number, mitochondrial transcription factor A and peroxisome proliferator-activated receptor-gamma coactivator 1-α. Norepinephrine-induced hypertension could be significantly suppressed by milrinone and esmolol. Milrinone improved but esmolol deteriorated the survival rate. The left ventricle was significantly enlarged shortly after norepinephrine infusion but later gradually reduced in size by milrinone. The impairment and suppression of mitochondrial function could be significantly reversed by milrinone but not by esmolol.ConclusionsMilrinone may protect the heart via maintaining mitochondrial function from hypernorepinephrinemia. This study warrants the importance of milrinone and the preservation of mitochondrial function in the treatment of catecholamine-induced death.Impact Milrinone may protect the heart from hypernorepinephrinemia-induced death via maintaining myocardial mitochondrial activity, function, and copy number.Maintenance of cardiac mitochondrial function may be a potential therapeutic strategy in such catecholamine-induced heart failure.