Cellular ATP Levels Research Articles

Local glycolysis fuels actomyosin contraction during axonal retraction.

In response to repulsive cues, axonal growth cones can quickly retract. This requires the prompt activity of contractile actomyosin, which is formed by the non-muscle myosin II (NMII) bound to actin filaments. NMII is a molecular motor that provides the necessary mechanical force at the expense of ATP. Here, we report that this process is energetically coupled to glycolysis and is independent of cellular ATP levels. Induction of axonal retraction requires simultaneous generation of ATP by glycolysis, as shown by chemical inhibition and genetic knock-down of GAPDH. Co-immunoprecipitation and proximal-ligation assay showed that actomyosin associates with ATP-generating glycolytic enzymes and that this association is strongly enhanced during retraction. Using microfluidics, we confirmed that the energetic coupling between glycolysis and actomyosin necessary for axonal retraction is localized to the growth cone and near axonal shaft. These results indicate a tight coupling between on-demand energy production by glycolysis and energy consumption by actomyosin contraction suggesting a function of glycolysis in axonal guidance.

Deacetylation of ACO2 Is Essential for Inhibiting Bombyx mori Nucleopolyhedrovirus Propagation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a specific pathogen of Bombyx mori that can significantly impede agricultural development. Accumulating evidence indicates that the viral proliferation in the host requires an ample supply of energy. However, the correlative reports of baculovirus are deficient, especially on the acetylation modification of tricarboxylic acid cycle (TCA cycle) metabolic enzymes. Our recent quantitative analysis of protein acetylome revealed that mitochondrial aconitase (ACO2) could be modified by (de)acetylation at lysine 56 (K56) during the BmNPV infection; however, the underlying mechanism is yet unknown. In order to understand this regulatory mechanism, the modification site K56 was mutated to arginine (Lys56Arg; K56R) to mimic deacetylated lysine. The results showed that mimic deacetylated mitochondrial ACO2 restricted enzymatic activity. Although the ATP production was enhanced after viral infection, K56 deacetylation of ACO2 suppressed BmN cellular ATP levels and mitochondrial membrane potential by affecting citrate synthase and isocitrate dehydrogenase activities compared with wild-type ACO2. Furthermore, the deacetylation of exogenous ACO2 lowered BmNPV replication and generation of progeny viruses. In summary, our study on ACO2 revealed the potential mechanism underlying WT ACO2 promotes the proliferation of BmNPV and K56 deacetylation of ACO2 eliminates this promotional effect, which might provide novel insights for developing antiviral strategies.

Hypoxia and HIF-1α Regulate the Activity and Expression of Na,K-ATPase Subunits in H9c2 Cardiomyoblasts.

The optimal function of the Na,K-ATPase (NKA) pump is essential for the heart. In ischemic heart disease, NKA activity decreases due to the decreased expression of the pump subunits. Here, we tested whether the hypoxia-inducible transcription factor (HIF-1α), the key signaling molecule regulating the adaptation of cells to hypoxia, is involved in controlling the expression and cellular dynamics of α1- and β1-NKA isoforms and of NKA activity in in-vitro hypoxic H9c2 cardiomyoblasts. HIF-1α was silenced through adenoviral infection, and cells were kept in normoxia (19% O2) or hypoxia (1% O2) for 24 h. We investigated the mRNA and protein expression of α1-, β1-NKA using RT-qPCR and Western blot in whole-cell lysates, cell membranes, and cytoplasmic fractions after labeling the cell surface with NHS-SS-biotin and immunoprecipitation. NKA activity and intracellular ATP levels were also measured. We found that in hypoxia, silencing HIF-1α prevented the decreased mRNA expression of α1-NKA but not of β1-NKA. Hypoxia decreased the plasma membrane expression of α1-NKA and β1- NKA compared to normoxic cells. In hypoxic cells, HIF-1α silencing prevented this effect by inhibiting the internalization of α1-NKA. Total protein expression was not affected. The decreased activity of NKA in hypoxic cells was fully prevented by silencing HIF-1α independent of cellular ATP levels. This study is the first to show that in hypoxic H9c2 cardiomyoblasts, HIF-1α controls the internalization and membrane insertion of α1-NKA subunit and of NKA activity. The mechanism behind this regulation needs further investigation.

Host cell sensing and restoration of mitochondrial function and metabolism within Helicobacter pylori VacA intoxicated cells.

Persistent human gastric infection with Helicobacter pylori is the single most important risk factor for development of gastric malignancy, which is one of the leading causes of cancer-related deaths worldwide. An important virulence factor for Hp colonization and severity of gastric disease is the protein exotoxin VacA, which is secreted by the bacterium and modulates functional properties of gastric cells. VacA acts by damaging mitochondria, which impairs host cell metabolism through impairment of energy production. Here, we demonstrate that intoxicated cells have the capacity to detect VacA-mediated damage, and orchestrate the repair of mitochondrial function, thereby restoring cellular health and vitality. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies.

Unraveling the mechanistic insights of sophorolipid-capped gold nanoparticle-induced cell death in Vibrio cholerae.

Cholera is one of the leading causes of mortality and morbidity worldwide. The treatment of cholera includes rehydration therapy, often combined with the administration of antibiotics (in severe cases). With the emergence and rapid spread of antimicrobial resistance, such microbial pathogens have become a compelling medical problem and a global threat. To combat the resistance to antibiotics, there is an urgent need for the discovery of novel and potent antimicrobial agents. The discovery of antimicrobial nanoparticles as an alternative to the existing antimicrobial regime has increased the hope for its use as one of the most promising tools against an array of microbial pathogens, including multidrug-resistant bacteria. Our recent report demonstrated that sophorolipid-capped gold nanoparticles (AuNPs-SL) exhibit potent antibacterial activity against Vibrio cholerae; however, the killing mechanism remained obscure. Metal nanoparticle-mediated killing of microbes occurs through non-specific mechanisms. To develop antimicrobial nanoparticles with better efficacy, knowledge of their specific mechanism of action is essential. In this work, we deduced the mechanistic insights of AuNPs-SL mediated cell death of V. cholerae. We observed that AuNPs-SL treatment evokes reactive oxygen species (ROS) formation. The surge in the ROS level triggers the overexpression of ROS-responsive genes, depolarization of the membrane, change in membrane permeability and leakage of intracellular proteins and DNA, depletion of ATP levels in the cell, DNA damage, and subsequent cell death. This study shows the possibility of use of AuNPs-SL as an alternative potential antimicrobial agent. IMPORTANCE Vibrio cholerae, a Gram-negative bacterium, is the causative agent of a fatal disease, "cholera." Prevention of cholera outbreak is possible by eliminating the bacteria from the environment. However, antimicrobial resistance developed in microorganisms has posed a threat and challenges to its treatment. Application of nanoparticles is a useful and effective option for the elimination of such microorganisms. Metal-based nanopaticles exhibit microbial toxicity through non-specific mechanisms. To prevent resistance development and increase antibacterial efficiency, rational designing of nanoparticles is required. Thus, knowledge on the exact mechanism of action of nanoparticles is highly essential. In this study, we explore the possible mechanisms of antibacterial activity of AuNPs-SL against V. cholerae. We show that the interaction of AuNPs-SL with V. cholerae enhances ROS production and membrane depolarization, change in permeability, and leakage of intracellular content. This action leads to the depletion of cellular ATP level, DNA damage, and subsequent cell death.

AXL transcriptionally up-regulates TMEM14A expression to mediate cell proliferation in non-small-cell lung cancer cells

In non-small cell lung cancer (NSCLC), the receptor tyrosine kinase AXL has been identified as a potent activator of tumor progression and resistance to therapies. However, the molecular mechanisms behind AXL-mediated oncogenesis remain elusive. Current study thus aimed to uncover potential downstream genes regulated by AXL in NSCLC. Through transcriptomic RNA sequencing of AXL-silenced NSCLC cells, TMEM14A was identified as a significantly up-regulated gene. Clinical evaluations using GEPIA2 revealed that TMEM14A mRNA expression was notably higher in lung adenocarcinoma (LUAD) tumor tissues compared to normal tissues. Further, significantly increased TMEM14A levels were associated with poorer overall survival in LUAD patients. Experimentally, silencing TMEM14A in NSCLC cells led to reduced cellular proliferation and ATP levels, highlighting a key role of TMEM14A in NSCLC progression. Moreover, our promoter analysis demonstrated that AXL-mediated regulation of TMEM14A transcription could involve binding of transcription factors STAT and NF-κB to 5′-promoter of TMEM14A. Collectively, current study unveils TMEM14A as a novel downstream target of AXL, suggesting its potential as a therapeutic target to counteract resistance in future NSCLC patients undergoing AXL-targeted therapies.

SARS-CoV-2 viral genes Nsp6, Nsp8, and M compromise cellular ATP levels to impair survival and function of human pluripotent stem cell-derived cardiomyocytes

BackgroundCardiovascular complications significantly augment the overall COVID-19 mortality, largely due to the susceptibility of human cardiomyocytes (CMs) to SARS-CoV-2 virus. SARS-CoV-2 virus encodes 27 genes, whose specific impacts on CM health are not fully understood. This study elucidates the deleterious effects of SARS-CoV-2 genes Nsp6, M, and Nsp8 on human CMs.MethodsCMs were derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, using 2D and 3D differentiation methods. We overexpressed Nsp6, M, or Nsp8 in hPSCs and then applied whole mRNA-seq and mass spectrometry for multi-omics analysis. Co-immunoprecipitation mass spectrometry was utilized to map the protein interaction networks of Nsp6, M, and Nsp8 within host hiPSC-CMs.ResultsNsp6, Nsp8, and M globally perturb the transcriptome and proteome of hPSC-CMs. SARS-CoV-2 infection and the overexpression of Nsp6, Nsp8, or M coherently upregulated genes associated with apoptosis and immune/inflammation pathways, whereas downregulated genes linked to heart contraction and functions. Global interactome analysis revealed interactions between Nsp6, Nsp8, and M with ATPase subunits. Overexpression of Nsp6, Nsp8, or M significantly reduced cellular ATP levels, markedly increased apoptosis, and compromised Ca2+ handling in hPSC-CMs. Importantly, administration of FDA-approved drugs, ivermectin and meclizine, could restore ATP levels, thereby mitigating apoptosis and dysfunction in hPSC-CMs overexpressing Nsp6, Nsp8, or M.ConclusionOverall, our findings uncover the extensive damaging effects of Nsp6, Nsp8, and M on hPSC-CMs, underlining the crucial role of ATP homeostasis in CM death and functional abnormalities induced by these SARS-CoV-2 genes, and reveal the potential therapeutic strategies to alleviate these detrimental effects with FDA-approved drugs.

CS-NO suppresses inhibits glycolysis and gastric cancer progression through regulating YAP/TAZ signaling pathway.

Gastric cancer (GC) is a significant contributor to global mortality and is recognized for its elevated prevalence and fatality rates. Nitric Oxide (NO) plays a role in multiple aspects of cancer metastasis and progression. CS-NO is a polysaccharide-based biomaterial with NO-releasing properties that shows promising therapeutic potential. Nonetheless, the action mechanism of CS-NO in GC is still largely unclear. The present study employed various experimental techniques, including CCK-8 assay, colony formation assay, EdU staining, and transwell assays, to evaluate the proliferation, migration, and invasion of GC cells. Additionally, ELISA was utilized to measure glucose uptake, lactate production, and cellular ATP levels in GC cells. In vivo investigations on nude mice were conducted to validate the in vitro results. The present study aimed to examine the potential anti-tumor properties of CS-NO on GC through in vitro and in vivo investigations, while also exploring the underlying mechanisms involved. Our data suggested that CS-NO might prevent GC cell invasion and migration. Decreased expressions of GLUT1, HK2, and LDHA further demonstrated that CS-NO significantly suppressed aerobic glycolysis in GC cells. The administration of CS-NO resulted in a significant reduction of YAP and TAZ levels in GC cells. Our data further show that CS-NO treatment could inhibit GC cancer growth in mice, consistent with the significant decrease in Ki67, GLUT1 and YAP expression levels. These findings could reveal the good effects of CS-NO therapy on inhibiting GC.

Abstract P2105: Store-Operated Channels Mediate Metabolic Adaptations In Activated Fibroblast During Cardiac Fibrosis

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are associated with developing fibrotic disorders in multiple organs, including the heart. Many ER resident chaperones require calcium (Ca2+) as a co-factor to mediate protein folding. Store-operated Ca2+ entry (SOCE) mediated by the Orai1 channels primarily maintains the ER- Ca2+ homeostasis. However, the role of the Orai1 channels in the modulation of ER-stress/UPR and downstream cellular processes that lead to cardiac fibrosis is not entirely understood. To investigate the role of Orai1 channels on the ER-stress/UPR signaling we utilized a multilayered approach that includes both genetic and pharmacological inhibition of Orai and a combination of long-term real-time cell imaging combined with genetically encoded sensors for several metabolites signaling. Our results showed that Orai1 expression was upregulated in the fibrotic mouse heart. Transcriptome analysis of fibrotic tissue from a fibroblast-specific Orai1 knock-out mouse model revealed the dysregulation of many genes associated with cell metabolism and collagen turnover. Pharmacological inhibition of Orai1 channel activity in cultured cardiac fibroblast decreased ER-Ca2+ and mitochondrial Ca2+ levels and was sufficient to trigger ER-stress/UPR and promote mitochondrial dysfunction. Like the in vivo fibrotic model, comparative transcriptome and pathway analysis between cells treated with Orai1 blocker (YM-58483, 48 hours) and controls identified dysregulated metabolic pathways and ER-stress/UPR signaling. Bioenergetic analysis showed that Orai1 inhibition induced an overall decrease in oxygen consumption rate (OCR), and mitochondrial membrane potential and reduced cellular ATP levels. Accordingly, we found that Orai1 loss-of-function increased glycolysis and reduced fatty acid oxidation (FAO), substantially reducing fibroblast fuel flexibility. This is a remarkable novel function of Orai1 channels because perturbations in fibroblast metabolic dynamic are the initial steps in the injury response. Our results suggest that Orai1 could be a novel target for developing treatments for fibrosis.

HSP90/CDC37 inactivation promotes degradation of LKB1 protein to suppress AMPK signaling in bronchial epithelial cells exposed to sulfur mustard analog, 2-chloroethyl ethyl sulfide

To investigate the role of the liver kinase (LK) B1 protein, an activator of AMP-activated protein kinase (AMPK), in AMPK signaling suppression when exposed to vesicant, a kind of chemical warfare agent. Cultured human bronchial epithelial cells were inflicted with sulfur mustard (SM) analog, 2-chloroethyl ethyl sulfide (CEES) of 0.2–1.0 mM concentration, and cell proliferation, apoptosis, autophagy, and cellular ATP level were analyzed up to 24 h after the exposure. Focusing on LKB1, heat shock protein (HSP) 90, and cell division cycle (CDC) 37 proteins, the protein expression, phosphorylation, and interaction were examined with western blot, immunofluorescence staining, and/or immunoprecipitation. AMPK signaling was found to be inhibited 24 h after being exposed to either sub-cytotoxic (0.5 mM) or cytotoxic (1.0 mM) concentration of CEES based on MTS assay. Consistently, the degradation of the LKB1 protein and its less interaction with the HSP90/CDC37 complex was confirmed. It was found that 1.0, not 0.5 mM CEES also decreased the CDC37 protein, proteasome activity, and cellular ATP content that modulates HSP90 protein conformation. Inhibiting proteasome activity could alternatively activate autophagy. Finally, either 0.5 or 1.0 mM CEES activated HSP70 and autophagy, and the application of an HSP70 inhibitor blocked autophagy and autophagic degradation of the LKB1 protein. In conclusion, we reported here that AMPK signaling inactivation by CEES was a result of LKB1 protein loss via less protein complex formation and enhanced degradation.

Mutant SPART causes defects in mitochondrial protein import and bioenergetics reversed by Coenzyme Q

Pathogenic variants in SPART cause Troyer syndrome, characterized by lower extremity spasticity and weakness, short stature and cognitive impairment, and a severe mitochondrial impairment. Herein, we report the identification of a role of Spartin in nuclear-encoded mitochondrial proteins. SPART biallelic missense variants were detected in a 5-year-old boy with short stature, developmental delay and muscle weakness with impaired walking distance. Patient-derived fibroblasts showed an altered mitochondrial network, decreased mitochondrial respiration, increased mitochondrial reactive oxygen species and altered Ca 2+ versus control cells. We investigated the mitochondrial import of nuclear-encoded proteins in these fibroblasts and in another cell model carrying a SPART loss-of-function mutation. In both cell models the mitochondrial import was impaired, leading to a significant decrease in different proteins, including two key enzymes involved in CoQ10 (CoQ) synthesis, COQ7 and COQ9, with a severe reduction in CoQ content, versus control cells. CoQ supplementation restored cellular ATP levels to the same extent shown by the re-expression of wild-type SPART, suggesting CoQ treatment as a promising therapeutic approach for patients carrying mutations in SPART .

Differentiated Neurons Are More Vulnerable to Organophosphate and Carbamate Neurotoxicity than Undifferentiated Neurons Due to the Induction of Redox Stress and Accumulate Oxidatively-Damaged Proteins.

Organophosphate (OP) and carbamate pesticides are toxic to pests through targeted inhibition of acetylcholinesterase (AChE). However, OPs and carbamates may be harmful to non-target species including humans and could induce developmental neurotoxicity if differentiated or differentiating neurons are particularly vulnerable to neurotoxicant exposures. Hence, this study compared the neurotoxicity of OPs, chlorpyrifos-oxon (CPO), and azamethiphos (AZO) and the carbamate pesticide, aldicarb, to undifferentiated versus differentiated SH-SY5Y neuroblastoma cells. OP and carbamate concentration-response curves for cell viability were undertaken using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and cellular bioenergetic capacity assessed via quantitation of cellular ATP levels. Concentration-response curves for inhibition of cellular AChE activity were also generated and the production of reactive oxygen species (ROS) was monitored using a 2',7'-dichlorofluorescein diacetate (DCFDA) assay. The OPs and aldicarb reduced cell viability, cellular ATP levels, and neurite outgrowth in a concentration-dependent fashion, from a threshold concentration of ≥10 µM. Neurotoxic potency was in the order AZO > CPO > aldicarb for undifferentiated cells but CPO > AZO > aldicarb for differentiated cells and this toxic potency of CPO reflected its more extensive induction of reactive oxygen species (ROS) and generation of carbonylated proteins that were characterized by western blotting. Hence, the relative neurotoxicity of the OPs and aldicarb in part reflects non-cholinergic mechanisms that are likely to contribute to developmental neurotoxicity.

An Evaluation of the Cytotoxic and Genotoxic Effects of the Marine Toxin C17-SAMT in Human TK6 and HepaRG Cell Lines.

This study investigates the genotoxicity and cytotoxicity of C17-sphinganine analog mycotoxin (C17-SAMT) using in vitro assays. C17-SAMT was previously identified as the cause of unusual toxicity in cultured mussels from the Bizerte Lagoon in northern Tunisia. While a previous in vivo genotoxicity study was inconclusive, in vitro results demonstrated that C17-SAMT induced an increase in micronucleus formation in human lymphoblastoid TK6 cells at concentrations of 0.87 µM and 1.74 µM. In addition, multiparametric cytotoxicity assays were performed in the human hepatoma HepaRG cell line, which showed that C17-SAMT induced mitochondrial dysfunction, decreased cellular ATP levels, and altered the expression of various proteins, including superoxide dismutase SOD2, heme oxygenase HO-1, and NF-κB. These results suggest that C17-SAMT is mutagenic in vitro and can induce mitochondrial dysfunction in HepaRG cells. However, the exact mode of action of this toxin requires further investigation. Overall, this study highlights the potential toxicity of C17-SAMT and the need for further research to better understand its effects.

D-xylose accelerated death of pentose metabolizing Saccharomyces cerevisiae

Rapid and effective consumption of d-xylose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. Hence, heterologous d-xylose metabolic pathways have been introduced into S. cerevisiae. An effective solution is based on a xylose isomerase in combination with the overexpression of the xylulose kinase (Xks1) and all genes of the non-oxidative branch of the pentose phosphate pathway. Although this strain is capable of consuming d-xylose, growth inhibition occurs at higher d-xylose concentrations, even abolishing growth completely at 8% d-xylose. The decreased growth rates are accompanied by significantly decreased ATP levels. A key ATP-utilizing step in d-xylose metabolism is the phosphorylation of d-xylulose by Xks1. Replacement of the constitutive promoter of XKS1 by the galactose tunable promoter Pgal10 allowed the controlled expression of this gene over a broad range. By decreasing the expression levels of XKS1, growth at high d-xylose concentrations could be restored concomitantly with increased ATP levels and high rates of xylose metabolism. These data show that in fermentations with high d-xylose concentrations, too high levels of Xks1 cause a major drain on the cellular ATP levels thereby reducing the growth rate, ultimately causing substrate accelerated death. Hence, expression levels of XKS1 in S. cerevisiae needs to be tailored for the specific growth conditions and robust d-xylose metabolism.

Acai Berry (Euterpe sp.) Extracts Are Neuroprotective against L-Glutamate-Induced Toxicity by Limiting Mitochondrial Dysfunction and Cellular Redox Stress.

Aberrant accumulation of the neurotransmitter L-glutamate (L-Glu) has been implicated as a mechanism of neurodegeneration, and the release of L-Glu after stroke onset leads to a toxicity cascade that results in neuronal death. The acai berry (Euterpe oleracea) is a potential dietary nutraceutical. The aim of this research was to investigate the neuroprotective effects of acai berry aqueous and ethanolic extracts to reduce the neurotoxicity to neuronal cells triggered by L-Glu application. L-Glu and acai berry effects on cell viability were quantified using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and effects on cellular bioenergetics were assessed via quantitation of the levels of cellular ATP, mitochondrial membrane potential (MMP), and production of reactive oxygen species (ROS) in neuroblastoma cells. Cell viability was also evaluated in human cortical neuronal progenitor cell culture after L-Glu or/and acai berry application. In isolated cells, activated currents using patch-clamping were employed to determine whether L-Glu neurotoxicity was mediated by ionotropic L-Glu-receptors (iGluRs). L-Glu caused a significant reduction in cell viability, ATP, and MMP levels and increased ROS production. The co-application of both acai berry extracts with L-Glu provided neuroprotection against L-Glu with sustained cell viability, decreased LDH production, restored ATP and MMP levels, and reduced ROS levels. Whole-cell patch-clamp recordings showed that L-Glu toxicity is not mediated by the activation of iGluRs in neuroblastoma cells. Fractionation and analysis of acai berry extracts with liquid chromatography-mass spectrometry identified several phytochemical antioxidants that may have provided neuroprotective effects. In summary, the acai berry contains nutraceuticals with antioxidant activity that may be a beneficial dietary component to limit pathological deficits triggered by excessive L-Glu accumulations.

Defective Mitochondrial Dynamics and Protein Degradation Pathways Underlie Cadmium-Induced Neurotoxicity and Cell Death in Huntington's Disease Striatal Cells.

Exposure to heavy metals, including cadmium (Cd), can induce neurotoxicity and cell death. Cd is abundant in the environment and accumulates in the striatum, the primary brain region selectively affected by Huntington's disease (HD). We have previously reported that mutant huntingtin protein (mHTT) combined with chronic Cd exposure induces oxidative stress and promotes metal dyshomeostasis, resulting in cell death in a striatal cell model of HD. To understand the effect of acute Cd exposure on mitochondrial health and protein degradation pathways, we hypothesized that expression of mHTT coupled with acute Cd exposure would cooperatively alter mitochondrial bioenergetics and protein degradation mechanisms in striatal STHdh cells to reveal novel pathways that augment Cd cytotoxicity and HD pathogenicity. We report that mHTT cells are significantly more susceptible to acute Cd-induced cell death as early as 6 h after 40 µM CdCl2 exposure compared with wild-type (WT). Confocal microscopy, biochemical assays, and immunoblotting analysis revealed that mHTT and acute Cd exposure synergistically impair mitochondrial bioenergetics by reducing mitochondrial potential and cellular ATP levels and down-regulating the essential pro-fusion proteins MFN1 and MFN2. These pathogenic effects triggered cell death. Furthermore, Cd exposure increases the expression of autophagic markers, such as p62, LC3, and ATG5, and reduces the activity of the ubiquitin-proteasome system to promote neurodegeneration in HD striatal cells. Overall, these results reveal a novel mechanism to further establish Cd as a pathogenic neuromodulator in striatal HD cells via Cd-triggered neurotoxicity and cell death mediated by an impairment in mitochondrial bioenergetics and autophagy with subsequent alteration in protein degradation pathways.

HIGD2A silencing impairs hepatocellular carcinoma growth via inhibiting mitochondrial function and the MAPK/ERK pathway

BackgroundThe Hypoxia inducible gene domain family member 2A (HIGD2A) protein is indispensable for the assembly of the mitochondrial respiratory supercomplex, which has been implicated in cell proliferation and cell survival under hypoxic conditions. Because the liver has a naturally low oxygen microenvironment, the role of HIGD2A in the development of hepatocellular carcinoma (HCC) remains largely unknown.MethodsGene expression data and clinical information were obtained from multiple public databases. A lentivirus-mediated gene knockdown approach was conducted to explore the function and mechanism of HIGD2A activity in HCC cells. In vivo and in vitro assays were performed to investigate the biological roles of HIGD2A.ResultsHIGD2A was overexpressed in HCC tissues and cell lines and was associated with a worse prognosis. Silencing HIGD2A expression significantly attenuated cell proliferation and migration, caused S-phase cell cycle arrest, and decreased tumor formation in nude mice. Mechanistically, HIGD2A depletion greatly decreased cellular ATP levels by disrupting mitochondrial ATP production. Moreover, HIGD2A knockdown cells displayed impaired mitochondrial function, such as mitochondrial fusion, increased expression of the mitochondrial stress response protein, and decreased oxygen consumption. Furthermore, knockdown of HIGD2A markedly attenuated the activation of the MAPK/ERK pathway.ConclusionsHIGD2A promoted liver cancer cell growth by fueling mitochondrial ATP synthesis and activating the MAPK/ERK pathway, suggested that targeting HIGD2A may represent a new strategy for HCC therapy.

Regulation of Autophagy via Carbohydrate and Lipid Metabolism in Cancer.

Metabolic changes are an important component of tumor cell progression. Tumor cells adapt to environmental stresses via changes to carbohydrate and lipid metabolism. Autophagy, a physiological process in mammalian cells that digests damaged organelles and misfolded proteins via lysosomal degradation, is closely associated with metabolism in mammalian cells, acting as a meter of cellular ATP levels. In this review, we discuss the changes in glycolytic and lipid biosynthetic pathways in mammalian cells and their impact on carcinogenesis via the autophagy pathway. In addition, we discuss the impact of these metabolic pathways on autophagy in lung cancer.

Abstract 4246: Synergistic inhibition of prostate tumor growth and progression by the combination of curcumin and ursolic acid in HiMyc mice

Abstract Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. According to the World Health Organization, approximately one-third of all cancer deaths could be prevented through an increased consumption of natural compounds. Through the use of a two-tiered screening method in PCa cells (depletion of cellular ATP level followed by glutamine uptake) of a phytochemical library, curcumin and ursolic acid were selected for further investigation. Twenty-four hour treatment of LNCaP, 22Rv1 and HMVP2 cells with the combination resulted in significant decreases in expression of members of the miR-17-92 and miR-106b-25 clusters, which are upregulated in PCa and have been shown to be involved in tumorigenesis, cell survival, and metastasis. In the HiMyc mouse model, where mice spontaneously develop primary PCa, supplementing diets with the combination of curcumin (1%) and ursolic acid (0.2%) resulted in significant inhibition of tumor growth and progression compared to diets containing either compound alone. There were no differences in body weight or food consumption in any of the diet groups during the tumor experiment. The current data suggests that the combination of curcumin and ursolic acid synergistically inhibits PCa growth and progression in HiMyc mice, which may be in part due to their effects on microRNA expression. Ongoing studies are further examining the role of specific miRNAs as well as oncogenic signaling pathways potentially involved in the mechanism of synergistic chemopreventive action of this combination. The current data suggest that this novel combination could be a valuable therapeutic option for prevention and/or treatment of PCa. Funding: This work was supported by NIH Grant CA228404. Citation Format: Chelsea A. Friedman, Achinto Saha, John DiGiovanni. Synergistic inhibition of prostate tumor growth and progression by the combination of curcumin and ursolic acid in HiMyc mice. [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 4246.

Leaching components and initial biocompatibility of novel bioactive restorative materials.

Bioactive restorative materials were developed on the premise that direct restorations should not only serve the purpose of reconstructing dental hard tissue defects but also exhibit biological features that prevent secondary caries development, without having adverse effects on the host cells. This study focuses on assessing the in vitro biocompatibility of two novel bioactive restorative materials. Specimens of the bioactive restorative materials, Cention Forte (CF) and ACTIVA BioACTIVE RESTORATIVE (AB), a glass ionomer cement/glass hybrid (EQUIA Forte HT, EF) and an established nanohybrid composite (Venus Diamond, VD) were produced and finished. The specimens were eluted in water and methanol and the resulting eluates were analyzed via gas chromatography-mass spectrometry. hGF-1 cells were exposed to eluates prepared in cell culture medium. Cellular ATP levels, oxidized glutathione concentration, caspase-3/7 activity and the inflammatory response (IL-6 and PGE2 levels) were determined. Microscopic images were taken to examine the cell morphology. Methyl methacrylate and 2-Hydroxyethyl methacrylate were the main monomers detected in CF and AB eluates. All materials inhibited cell proliferation and led to significantly reduced ATP-levels. The cells exhibited a healthy morphology in the presence of CF and AB. Cells exposed to VD showed increased oxidized glutathione levels. Only EF led to enhanced caspase-3/7 activity. CF and AB caused IL-6 levels to increase, while EF and AB led to enhanced PGE2 levels. CF and AB are promising materials from a biological point of view and seem to have improved bioactive properties compared to glass ionomer cements.