Real-time ATP Research Articles

Transient inhibition of mitochondrial function by chrysin and apigenin prolong longevity via mitohormesis in C. elegans.

Mild inhibition of mitochondrial function leads to longevity. Genetic disruption of mitochondrial respiratory components either by mutation or RNAi greatly extends the lifespan in yeast, worms, and drosophila. This has given rise to the idea that pharmacologically inhibiting mitochondrial function would be a workable strategy for postponing aging. Toward this end, we used a transgenic worm strain that expresses the firefly luciferase enzyme widely to evaluate compounds by tracking real-time ATP levels. We identified chrysin and apigenin, which reduced ATP production and increased the lifespan of worms. Mechanistically, we discovered that chrysin and apigenin transiently inhibit mitochondrial respiration and induce an early ROS, and the lifespan-extending effect is dependent on transient ROS formation. We also show that AAK-2/AMPK, DAF-16/FOXO, and SKN-1/NRF-2 are required for chrysin or apigenin-mediated lifespan extension. Temporary increases in ROS levels trigger an adaptive response in a mitohormetic way, thereby increasing oxidative stress capacity and cellular metabolic adaptation, finally leading to longevity. Thus, chrysin and apigenin represent a class of compounds isolated from natural products that delay senescence and improve age-related diseases by inhibiting mitochondrial function and shed new light on the function of additional plant-derived polyphenols in enhancing health and delaying aging. Collectively, this work provides an avenue for pharmacological inhibition of mitochondrial function and the mechanism underlining their lifespan-extending properties.

IGF-1 Stimulates Glycolytic ATP Production in MCF-7L Cells.

The Insulin-like Growth Factor (IGF) system in breast cancer progression has been a matter of interest for decades, but targeting this system did not result in a successful clinical strategy. The system's complexity and homology of its two receptors-insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R)-are possible causes. The IGF system maintains cell proliferation and also regulates metabolism, making it a pathway to explore. To understand the metabolic phenotype of breast cancer cells, we quantified their real-time ATP production rate upon acute stimulation with ligands-insulin-like growth factor 1 (1GF-1) and insulin. MCF-7L cells express both IGF-1R and IR, while tamoxifen-resistant MCF-7L (MCF-7L TamR) cells have downregulated IGF-1R with unchanged IR levels. Treating MCF-7L cells with 5 nM IGF-1 increased the glycolytic ATP production rate, while 10 nM insulin did not affect metabolism when compared with the control. Neither treatment altered ATP production in MCF-7L TamR cells. This study provides evidence of the relationship between metabolic dysfunction, cancer, and the IGF axis. In these cells, IGF-1R, and not IR, regulates ATP production.

Abstract 3969: Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay

Abstract Cancer cells are metabolically reprogrammed through tumor initiation and progression, and there are increasing developments in cancer therapies exploiting the potential metabolic vulnerability. A better understanding of cancer cell metabolic regulation can also contribute to modulating the harsh tumor microenvironment, which strongly affects the efficacy of emerging anti-cancer immunotherapies. As one of the critical contributors to cancer therapy design, there is increasing demand for time-efficient and reliable analytic tools when searching for drugs and genes modulating cancer cell metabolism. The XF Real-Time ATP Rate Assay measures ATP production from glycolysis and mitochondrial respiration simultaneously and provides a quantitative overview of the bioenergetic phenotype of the cells. By using ATP production rate as a universal unit of bioenergetic metabolism, this assay quickly detects metabolic modulations induced by chemical stimuli or genetic modifications. It allows the identification of potential targets for therapy design. In this study, we used the Seahorse XF Pro Analyzer combined with the XF ATP Rate assay to screen 80 kinase inhibitors for the suppressive effects on energy metabolism and/or inducing a metabolic phenotype shift in THP-1 cells, a monocytic leukemia cell line, using PBMC as the control. We identified four mitochondrial and two glycolytic suppressors using the screening and dose-response views available in Seahorse Analytics, a cloud-based data analysis tool for Seahorse XF assays. These compounds showed moderate or no effects on the net ATP production rates but induced a significant shift in the metabolic phenotype of THP-1 cells. Among the selected kinase inhibitors, AG-879 (ErbB2 inhibitor), SU-1498 (VEGFR inhibitor), and U-0126 (MEK1/2 inhibitor) showed the most potent inhibitory effect on mitochondrial energy production in THP-1 cells. Dose-response studies indicated that SU-1498 and U-0126 have higher potency in THP-1 than PBMC, and thus they can be considered good cancer-cell targets for mitochondrial suppressors. In contrast, AG-879 showed a similar or higher inhibitory effect on PBMC even though it is the most potent mitochondrial suppressor among the selected compounds. These results demonstrate that the XF Real-Time ATP Rate Assay can be used as a primary assay for screening and validating drug candidate(s) targeting the bioenergetic metabolism of cancer cells. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay. [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 3969.

Spike Protein Impairs Mitochondrial Function in Human Cardiomyocytes: Mechanisms Underlying Cardiac Injury in COVID-19.

COVID-19 has a major impact on cardiovascular diseases and may lead to myocarditis or cardiac failure. The clove-like spike (S) protein of SARS-CoV-2 facilitates its transmission and pathogenesis. Cardiac mitochondria produce energy for key heart functions. We hypothesized that S1 would directly impair the functions of cardiomyocyte mitochondria, thus causing cardiac dysfunction. Through the Seahorse Mito Stress Test and real-time ATP rate assays, we explored the mitochondrial bioenergetics in human cardiomyocytes (AC16). The cells were treated without (control) or with S1 (1 nM) for 24, 48, and 72 h and we observed the mitochondrial morphology using transmission electron microscopy and confocal fluorescence microscopy. Western blotting, XRhod-1, and MitoSOX Red staining were performed to evaluate the expression of proteins related to energetic metabolism and relevant signaling cascades, mitochondrial Ca2+ levels, and ROS production. The 24 h S1 treatment increased ATP production and mitochondrial respiration by increasing the expression of fatty-acid-transporting regulators and inducing more negative mitochondrial membrane potential (Δψm). The 72 h S1 treatment decreased mitochondrial respiration rates and Δψm, but increased levels of reactive oxygen species (ROS), mCa2+, and intracellular Ca2+. Electron microscopy revealed increased mitochondrial fragmentation/fission in AC16 cells treated for 72 h. The effects of S1 on ATP production were completely blocked by neutralizing ACE2 but not CD147 antibodies, and were partly attenuated by Mitotempo (1 µM). S1 might impair mitochondrial function in human cardiomyocytes by altering Δψm, mCa2+ overload, ROS accumulation, and mitochondrial dynamics via ACE2.

RIP140-Mediated NF-κB Inflammatory Pathway Promotes Metabolic Dysregulation in Retinal Pigment Epithelium Cells.

Metabolic dysregulation of the retinal pigment epithelium (RPE) has been implicated in age-related macular degeneration (AMD). However, the molecular regulation of RPE metabolism remains unclear. RIP140 is known to affect oxidative metabolism and mitochondrial biogenesis by negatively controlling mitochondrial pathways regulated by PPAR-γ co-activator-1 α(PGC-1α). This study aims to disclose the effect of RIP140 on the RPE metabolic program in vitro and in vivo. RIP140 protein levels were assayed by Western blotting. Gene expression was tested using quantitative real-time PCR (qRT-PCR), ATP production, and glycogen concentration assays, and the release of inflammatory factors was analyzed by commercial kits. Mice photoreceptor function was measured by electroretinography (ERG). In ARPE-19 cells, RIP140 overexpression changed the expression of the key metabolic genes and lipid processing genes, inhibited mitochondrial ATP production, and enhanced glycogenesis. Moreover, RIP140 overexpression promoted the translocation of NF-κB and increased the expression and production of IL-1β, IL-6, and TNF-α in ARPE-19 cells. Importantly, we also observed the overexpression of RIP140 through adenovirus delivery in rat retinal cells, which significantly decreased the amplitude of the a-wave and b-wave measured by ERG assay. Therapeutic strategies that modulate the activity of RIP140 could have clinical utility for the treatment of AMD in terms of preventing RPE degeneration.

Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway

BackgroundSalidroside (Sal), an active component from Rhodiola crenulata, has been confirmed to exert neuroprotective effects against hypoxia. However, its molecular mechanisms of intensifying mitochondrial function still largely unknown. In the present study, we aimed to explore the mechanisms by which Sal heightened mitochondrial function in CoCl2-induced HT22 hypoxic injury. MethodsThe hypoxic condition of HT22 cells was performed by CoCl2 stimulus. We then investigated the effects of Sal on the viability of hypoxic HT22 cells by cell counting kit-8. The contents of lactate dehydrogenase (LDH) release in cultured supernatant were detected by using commercial biochemical kit. Superoxide free radical scavenging activity, total antioxidant capacity assay kit with ferric reducing ability of plasma and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) methods were employed to detect the free radical scavenging ability and antioxidant capacity of Sal. Meanwhile, intracellular reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (MMP) were determined by corresponding specific labeled probes. Mitochondrial morphology was tested by Mito-tracker green with confocal microscopy. Hoechst 33342 and Annexin V-FITC/propidium iodide staining were also employed to evaluate the effect of Sal on cell apoptosis. Oxygen consumption rate (OCR), real-time ATP production and proton efflux rate were measured using a Seahorse analyzer. Additionally, the potential interactions of Sal with PI3K-AKT signaling pathway-related proteins were predicted and tested by molecular docking, molecular dynamics simulation (MDS) and localized surface plasmon resonance (LSPR) techniques, respectively. Furthermore, the protein levels of p-PI3K, PI3K, p-AKT, AKT, p-JNK, JNK, p-p38 and p38 were estimated by western blot analysis. ResultsSal alleviated CoCl2-induced hypoxic injury in HT22 cells as evidenced by increased cell viability and decreased LDH release. In vitro antioxidant test confirmed that Sal had marvelous antioxidant abilities. The protected mitochondrial function by Sal treatment was illustrated by the decrease of ROS, Ca2+, mitochondrial fragment and the increase of MMP. In addition, Sal ameliorated the apoptosis of HT22 cells by decreasing Hoechst 33342 positive cells and the rate of apoptotic cells. Enhancement of energy metabolism in HT22 by Sal was demonstrated by increased OCR, real-time ATP generation and proton efflux rate. The molecular docking confirmed the potential binding of Sal to PI3K, AKT and CaMK II proteins with calculated binding energy of -1.32, -4.21 and -4.38 kcal/mol, respectively. The MDS test revealed the average hydrogen bond of complex Sal-PI3K and Sal-AKT were 0.79 and 4.46, respectively. The results of LSPR verified the potential binding of Sal to proteins PI3K, AKT and HIF-1α with affinity values of 5.20 × 10 − 3, 2.83 × 10 − 3 and 3.97 × 10 − 3 KD, respectively. Western blot analysis further argued that Sal consolidated the levels of p-PI3K and p-AKT. Meanwhile, Sal could downregulate the proteins expression of p-JNK and p-p38. ConclusionCollectively, our findings suggested that Sal can intensify mitochondrial function of CoCl2-simulated hypoxia injury in HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway. Sal is a potential agent for mitochondrial protection against hypoxia with the underlying molecular mechanisms of energy metabolism being further elucidated.

Transcriptomic-Based in silico Drug Screening Reveals Dual PI3K/mTOR Inhibitors Overcome Cabozantinib-Resistance of FLT3-ITD AML Cells

Background: Elucidating the mechanisms involved in tyrosine kinase inhibitor (TKI) resistance is critical for developing effective therapies in acute myeloid leukemia (AML). Cabozantinib (CBZ), an oral multi-target TKI, is well tolerated in AML patients and effectively inhibits the FLT3-mutated AML. We previously established CBZ-resistant MV4-11 cells (MV4-11-XR). However, the precise mechanisms of CBZ resistance are still not fully studied. Aims: To elucidate the molecular mechanism in MV4-11-XR and explore the potential therapeutic approaches to overcome CBZ-resistance. Materials and Methods: MV4-11-P and MV4-11-XR myeloid leukemia cell lines were used in this study. Cabozantinib (BMS-907351), omipalisib (GSK2126458), and gedatolisib (PF-05212384) were used for experiments. Cell proliferation was measured by Cell Titer 96 AQueous One Solution Cell Proliferation Assay. The protein expression was analyzed by immunoblotting. RNA-seq was performed using an Illumina NextSeq 500 platform. Sanger's sequencing and pyrosequencer were used to characterize the SNVs (single nucleotide variants). FusionCatcher was also used to explore potential fusion events related to resistance. Differentially expressed genes (DEGs) between MV4-11-P and MV4-11-XR were identified by EBseq. A threshold of p ≤ 0.05 was used to select the DEGs. Connectivity map was used to predict small molecules which had potentials to reverse enriched metabolic pathways in MV4-11-XR. The quantitative reverse transcription PCR (qRT-PCR) was performed by QuantStudio 3 Real-Time PCR Systems. The cellular ATP production rate was analyzed by the XFe 24 extracellular flux analyzer. Flow cytometry was used for cell cycle and apoptosis analysis. Results: We demonstrated that the anti-proliferative effect of CBZ on MV4-11-XR and parental MV4-11 (MV4-11-P). MV4-11-XR was significantly resistant to CBZ with IC50 of 1500 nM compared with MV4-11-P with IC50 of 9.5 nM. Subsequently, we performed RNA-seq to evaluate SNVs, indels, and enriched pathways in MV4-11-XR compared to MV4-11-P. We found 376 SNVs in MV4-11-XR. Of them, we noticed the emergence of FLT3 c.2503G>T (p.D835Y) mutation in MV4-11-XR. Pyrosequencing analysis confirmed that the frequency of mutant allele increased from 5 % in MV4-11-P to 99 % in MV4-11-XR. Immunoblotting confirmed the hyperactivation of FLT3 downstream signaling pathways in MV4-11-XR compared to MV4-11-P. FusionCatcher analysis indicated no novel fusion transcript related to CBZ resistance. Transcriptomic analysis revealed that a total of 1113 (366 upregulated and 725 downregulated) DEGs were detected in MV4-11-XR compared to MV4-11-P. Metascape analysis revealed that those up-regulated DEGs were enriched in "HALLMARK MTORC1 SIGNALING” and "HALLMARK HYPOXIA", indicating increased glycolysis in MV4-11-XR. qRT-PCR validated the overexpression of glycolytic genes including HIF1A, GLUT1, HK2, PKM2, LDHA, and PDK1 in MV4-11-XR. Real-time ATP rates analysis showed that MV4-11-XR had significantly increased rates of glycolytic ATP production. To explore small molecules that have the potential to reverse the metabolic phenotypes and overcome CBZ resistance, we performed a connectivity map analysis and predicted a PI3K inhibitor (wortmannin) and an mTOR inhibitor (sirolimus) as candidates. Therefore, we selected and evaluated the effects of gedatolisib and omipalisib, which have dual PI3K/mTOR inhibitory capacity, on MV4-11-XR. qRT-PCR showed that omipalisib significantly inhibited the expression of glycolytic genes in MV4-11-XR. Further experiments showed that gedatolisib and omipalisib significantly inhibited the proliferation of MV4-11-XR with IC50 of 28.33 nM and 37.56 nM, respectively. More importantly, gedatolisib and omipalisib significantly suppressed PI3K-AKT-mTOR signaling, promoted cell apoptosis, and induced cell cycle arrest in MV4-11-XR. Summary: In this study, we identified the emergence of FLT3 D835Y mutation and the upregulation of glycolysis in MV4-11-XR. As suggested by connectivity map analysis, we identified PI3K/mTOR dual inhibitor gedatolisib and omipalisib had potent anti-leukemic effects and could reverse the metabolic signature of CBZ-resistant cells. These findings highlight the potentials of PI3K/mTOR dual inhibitors as therapeutic molecules in AML to overcome TKI resistance.

Abstract B009: Multi-omics analysis of glutathione S-transferase P1 (GSTP1) knockdown model suggests role in pancreatic ductal adenocarcinoma (PDAC) cell metabolism

Abstract GSTP1 belongs to a large family of detoxification enzymes essential for cellular homeostasis in healthy cells. GSTP1 is overexpressed in many cancers and is associated with poor survival in PDAC patients. Studies in glioblastoma and breast cancer have suggested crucial roles of GST family members in metabolism through increased lactate dehydrogenase A and increased GAPDH activity, respectively. The mechanisms underlying the action of GSTP1 in PDAC metabolism and cell proliferation are not well studied. To begin characterizing the diverse functions of GSTP1 in cancer cells, we knocked down GSTP1 expression in three different PDAC cell lines. We revealed unique changes in the global transcriptomic, proteomic, and metabolomic signatures associated with GSTP1 knockdown compared to control cells through multi-omics techniques, real-time ATP production assays, and comprehensive hydrophilic metabolite and phospholipid panels. Through preliminary transcriptomic and proteomic analyses, we found 461 genes and 57 proteins differentially expressed in our GSTP1 knockdown MIA PaCa-2 cells. Among these, 41 were similarly upregulated or downregulated at the mRNA and protein levels. Pathway analysis of these changes revealed profound effects on energy production and lipid metabolism in GSTP1 knockdown cells. Hydrophilic metabolite and phospholipid panels and real-time ATP production assays supported our findings. Our results indicate that GSTP1 knockdown may directly influence mitochondrial dysfunction and lipid peroxidation pathways through associated reductions in ALDH7A1, GLUT3, and SLC7A11 levels. Continued research to further elucidate precise mechanisms and understand GSTP1’s role in driving cancer cell metabolism will aid in establishing GSTP1 targeting as a therapeutic opportunity for PDAC treatment. Citation Format: Jenna Duttenhefner, Katie M. Reindl. Multi-omics analysis of glutathione S-transferase P1 (GSTP1) knockdown model suggests role in pancreatic ductal adenocarcinoma (PDAC) cell metabolism [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B009.

DDDR-20. TARGETING THE SELECTIVE VULNERABILITY OF IDH-MUTANT GLIOMA WITH ZOTIRACICLIB

Abstract BACKGROUND IDH mutations are common in diffuse gliomas and confer an increased dependency on oxidative mitochondrial function. Zotiraciclib, a multi-kinase inhibitor, not only suppresses CDK9-mediated gene transcription process but also causes mitochondrial dysfunction in preclinical models. We investigated the selective activity of zotiraciclib in IDH-mutant gliomas. METHODS Cytotoxicity of zotiraciclib was determined in patient-derived glioma cells with/without IDH mutations. Immunoblotting and flow cytometry were used to evaluate the cell death pathways. Real-time ATP production was measured by Seahorse assay. RESULTS The EC50 of zotiraciclib in IDH-wt cells was twice as high as in IDH-mutant cells. In low-dose zotiraciclib (15nM)-treated IDH-mutant cells, RNA Pol II phosphorylation and expression of antiapoptotic proteins, including XIAP, MCL-1 and survivin were decreased significantly. Cleaved caspase 3/7, 8, 9 and LC3B were also detected in treated IDH-mutant cells but not in IDH-wt cells, suggesting activation of intrinsic and extrinsic cell apoptosis pathways, and subsequent autophagy of IDH-mutant cells. Low-dose treated IDH-mutant, but not IDH-wt cells, induced decreased expression of a panel of mitochondrial respiration genes, and mitochondrial respiration complexes, cellular ATP depletion and increased ROS production, suggesting a greater disruption of mitochondrial function in zotiraciclib-treated IDH-mutant cells. Zotiraciclib treatment prolonged the survival in mice bearing intracranial IDH-mutant gliomas (30 days in DMSO-treated group vs 33 days in zotiraciclib-treated group, p = 0.01), but not in mice with glioma established with the isogenic IDH-wt glioma cells (18 days in DMSO-treated vs 20 days in zotiraciclib-treated group, p = 0.10). CONCLUSIONS There is selective activity of zotiraciclib in IDH-mutated glioma. Low-dose exposure of zotiraciclib induces cell death and suppresses transcriptional process and mitochondrial biogenesis in IDH-mutant glioma, not in IDH-wt tumors. These findings support a clinical trial testing zotiraciclib in IDH-mutated glioma. The treatment efficacy seen even at low concentrations further supports testing it as a single agent, thereby reducing toxicity concerns.

Impaired mitochondria of Tregs decreases OXPHOS-derived ATP in primary immune thrombocytopenia with positive plasma pathogens detected by metagenomic sequencing

BackgroundPrimary immune thrombocytopenia (ITP) is an autoimmune disease. Some ITP patients are associated with pathogen infection undetected with conventional technologies. Investigating the changes of T cells and potential metabolic mechanism are important for better understanding of ITP.MethodsThe study enrolled 75 newly diagnosed ITP patients. The pathogens of patients were detected by metagenomic next-generation sequencing (mNGS). Plasma lipids were measured by liquid chromatography-mass spectrometry (LC–MS). CD4 T cell and CD8 T cell were analyzed using flow cytometry. Mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential were measured by flow cytometry. Seahorse XF real-time ATP rate assay was used to investigate the change of cellular metabolism.ResultsPositive plasma pathogens were detected in seven ITP patients. Of them, 5 (71.4%) positive pathogen-ITP patients were no response (NR) after first-line treatment with corticosteroids. Regulatory T cells (Tregs) increased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and healthy controls (HC). Mitochondrial membrane potential of Th17 and Tregs were decreased in positive pathogen-ITP and negative pathogen-ITP patients, compared to HC (all p < 0.05). The overall metabolism flux of positive pathogen-ITP patients was decreased, as compared to HC (p = 0.004), of them a higher proportion of glycolysis-derived ATP and a smaller proportion of oxidative phosphorylation (OXPHOS)-derived ATP were found in Tregs. The ATP rate index of Tregs was decreased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and HC (p < 0.05).ConclusionsImpaired mitochondria function of Tregs in positive pathogen-ITP patients caused a decrease of OXPHOS-derived ATP and overall metabolism flux that might be the cause of steroid resistance in ITP patients.

Phosphofructokinase 1 platelet isoform induces PD-L1 expression to promote glioblastoma immune evasion.

Overexpression of PD-L1 is observed in many types of human cancer, including glioblastoma (GBM) and contributes to tumor immune evasion. In addition, GBM shows highly-activated aerobic glycolysis due to overexpression of phosphofructokinase 1 platelet isoform (PFKP), which the key enzyme in the glycolysis. However, it remains unclear whether the metabolic enzyme PFKP plays a role in the regulation of PD-L1 expression and GBM immune evasion. We aimed to investigate the non-metabolic role of PFKP in PD-L1 expression-induced GBM immune evasion. The mechanisms of PFKP-induced PD-L1 expression were studied by several experiments, including real-time PCR, immunoblot analysis, and ATP production. The coculture experiments using GBM cell and T cells were performed to evaluate the effect of PFKP on T cell activation. The clinical relationship between PFKP and PD-L1 was analyzed in The Cancer Genome Atlas (TCGA) database and in human GBM specimens. We showed that PFKP promotes EGFR activation-induced PD-L1 expression in human GBM cells. Importantly, we demonstrated that EGFR-phosphorylated PFKP Y64 plays an important role in AKT-mediated β-catenin transactivation and subsequent PD-L1 transcriptional expression, thereby enhancing the GBM immune evasion. In addition, based on our findings, the levels of PFKP Y64 phosphorylation are positively correlated with PD-L1 expression in human GBM specimens, highlighting the clinical significance of PFKP Y64 phosphorylation in the GBM immune evasion. These findings provide new mechanistic insight into the regulation of PD-L1 expression by a non-metabolic function of PFKP on tumor cells.

Abstract 2323: A bioenergetic functional screening assay workflow to identify, validate, and characterize anti-cancer compounds

Abstract As bioenergetic metabolism is recognized as a critical activity supporting cell proliferation, differentiation, and even death, many proposed targets for cancer therapy are functionally linked to cellular bioenergetic metabolism. Here, we introduce an assay workflow for screening and analyzing potential anticancer drugs affecting cellular metabolism using extracellular flux analysis. The Agilent Seahorse XF Real-Time ATP rate assay can be used as a starting point to identify therapeutic compounds that induce a perturbation in metabolic phenotype. It provides a quantitative comparison of basal mitochondrial and glycolytic activities using the universal unit of ATP production rate. Through this initial comparative screening, the effect of different compounds on basal energetic state and pathway-specific metabolic perturbations can be identified providing information that cannot be assessed by intracellular ATP level measurements or cytotoxicity assays. A more comprehensive functional analysis can then be applied to validate the target mode of action through pathway-specific analysis tools such as the XF Cell Mito Stress Test and the XF Glycolytic Rate Assay. In this study, we tested the proposed workflow in three different NSCLC cell lines with different genetic backgrounds using a mock panel of known metabolic modulators including thiosemicarbazones, EGFR inhibitors, KRAS inhibitors, a glutaminase inhibitor, a Glut1 inhibitor, a chemotherapy drug, and a radiotherapy sensitizer candidate. The ATP production rate data from lung cancer cell lines exposed for 1 hour or 24 hours to the compounds, successfully identified the effects of metabolic pathway-specific modulation as well as metabolic phenotypic switching. In addition, dose-response studies using cell lines with different genetic backgrounds demonstrated differential susceptibility to the tested compounds. The workflow was then applied using a broader panel of 72 potential mitochondrial toxicants compounds. The assay could identify potent mitochondrial and glycolytic suppressor compounds effective on A549, a lung cancer cell line. A more comprehensive XF analysis then validated the mode of action for the identified metabolic suppressors. These results demonstrate the benefit of using the XF Real-Time ATP rate assay as an initial assay to uncover novel metabolic targets in early drug discovery research, guiding further research toward functional validation with the appropriate comprehensive XF assays. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. A bioenergetic functional screening assay workflow to identify, validate, and characterize anti-cancer compounds [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 2323.

Genetically Encoded ATP Biosensors for Direct Monitoring of Cellular ATP Dynamics.

Adenosine 5′-triphosphate, or ATP, is the primary molecule for storing and transferring energy in cells. ATP is mainly produced via oxidative phosphorylation in mitochondria, and to a lesser extent, via glycolysis in the cytosol. In general, cytosolic glycolysis is the primary ATP producer in proliferative cells or cells subjected to hypoxia. On the other hand, mitochondria produce over 90% of cellular ATP in differentiated cells under normoxic conditions. Under pathological conditions, ATP demand rises to meet the needs of biosynthesis for cellular repair, signaling transduction for stress responses, and biochemical processes. These changes affect how mitochondria and cytosolic glycolysis function and communicate. Mitochondria undergo remodeling to adapt to the imbalanced demand and supply of ATP. Otherwise, a severe ATP deficit will impair cellular function and eventually cause cell death. It is suggested that ATP from different cellular compartments can dynamically communicate and coordinate to adapt to the needs in each cellular compartment. Thus, a better understanding of ATP dynamics is crucial to revealing the differences in cellular metabolic processes across various cell types and conditions. This requires innovative methodologies to record real-time spatiotemporal ATP changes in subcellular regions of living cells. Over the recent decades, numerous methods have been developed and utilized to accomplish this task. However, this is not an easy feat. This review evaluates innovative genetically encoded biosensors available for visualizing ATP in living cells, their potential use in the setting of human disease, and identifies where we could improve and expand our abilities.

Linoleic Acid Reduces Oxidative Phosphorylation and Impairs Early Differentiation of MC3T3-E1 Osteoblast Precursor Cells

ObjectivesUntargeted metabolomics analyses by our group have shown that plasma linoleic acid (LA) was inversely associated with bone mineral density Z-score and that bone formation indices were associated with energy-generating metabolic pathways, including fatty acid b-oxidation, in adult cohorts. Here, we examined the effect of increasing LA concentrations on osteoblast precursor cell bioenergetics and osteoblast differentiation to determine whether high LA is detrimental to bone formation. MethodsWe treated MC3T3-E1 pre-osteoblastic cells with 0 μM (control), 1 μM, and 50 μM LA cultured in osteogenic differentiation media supplemented with 50 μM L-ascorbic acid and 2 mM β-glycerophosphate. To assess the effect of LA on early commitment/differentiation, cells were stained for alkaline phosphatase activity and late differentiation using Alizarin Red S staining for mineral deposition, at 7 and 18 days, respectively. To assess cellular bioenergetics, real-time ATP production rates in LA treated (1 or 50 μM) and control MC3T3-E1 cells were measured using an extracellular flux analyzer after 24 hours (normalized for total protein content). Differences in bioenergetic values were determined using one-way ANOVA or Kruskal-Wallis test with Tukey’s HSD or Dunn’s post hoc tests. ResultsWhile LA had no effect on late differentiation/mineralizing activity of MC3T3 cells, LA dose-dependently decreased commitment/early differentiation. LA also significantly altered the bioenergetic profile of MC3T3-E1 cells by decreasing basal oxygen consumption rate (P < 0.001), as well as mitochondrial and total ATP production rate (P < 0.05 and P < 0.001, respectively). There were no significant changes in glycolytic ATP production rate. ConclusionsOsteoblast differentiation is a highly bioenergetic process, and this study suggests excess LA may impair ATP production from oxidative phosphorylation. This, in turn, may impede commitment and early differentiation of osteoblasts. Our study supports further clinical and translational investigation into the role of LA and energy metabolism in osteoblast function, as well as bone formation. Funding SourcesNational Institutes of Health.

Single-Fluorescence ATP Sensor Based on Fluorescence Resonance Energy Transfer Reveals Role of Antibiotic-Induced ATP Perturbation in Mycobacterial Killing.

ABSTRACTThe rapid emergence of multidrug-resistant/extensively drug-resistant tuberculosis (TB) is responsible for treatment failure in patients with TB and significantly endangers global public health. Recently, bioenergetics has become a new paradigm for anti-TB drug discovery and is based on the link between bacterial ATP levels and drug efficacy. A better understanding of the role of ATP fluctuations during antibiotic treatment may provide insight into antibiotic-mediated killing of mycobacteria. Here, we employed an advanced single-fluorescence FRET (fluorescence resonance energy transfer)-based ATP biosensor, ATPser, for the stable and convenient detection of intracellular ATP fluctuations in mycobacteria. This strategy correlated closely with the results obtained from conventional luminescence ATP assays, indicating the reliability of the system for bioenergetics analysis in mycobacteria. Moreover, the reporter strains expressing ATPser displayed obvious ATP changes when subjected to different stresses, such as starvation and ATP depletion. Interestingly, we observed that different antibiotics induced fluctuations in cellular ATP levels in individual cells of various magnitudes, revealing a strong connection between ATP fluctuations and drug efficacy. Furthermore, drug combinations accelerated ATP perturbation, resulting in increased cell death. We concluded that ATPser enabled real-time measurement of ATP at the single-cell level in mycobacteria, and monitoring ATP dynamics in drug-treated bacteria may shed light on novel treatment strategies.IMPORTANCE Bioenergetics has emerged as a new paradigm for antituberculosis (anti-TB) drug discovery, and the cellular ATP level is the core indicator reflecting bacterial metabolic homeostasis. Although several bulk assays have been designed for the measurement of cellular ATP content, a more convenient strategy is required for real-time ATP measurement of single viable cells. In this study, by combining the ε-subunit of Bacillus subtilis FoF1-ATP synthase with a circularly permuted green fluorescent protein [(cp)GFP], we constructed a FRET-based single-fluorescence ATP sensor, ATPser, for real-time single-cell ATP detection among a mycobacterial population. Using the ATPser, we designed different drug combinations containing components that have similar/opposite effects on ATP alternation. Our results demonstrated that increased cellular ATP fluctuations were associated with depletion of mycobacterial viability, while counteracting ATP fluctuations weakened the killing effect of the drug regime. Thus, potentially efficient drug combinations can be considered based on their similar effects on mycobacterial ATP levels, and ATPser may be a useful tool to study mycobacterial bioenergetics and to guide drug regime design.

Increased Glycolysis and ATP Production Are Not Prerequisites for Cardiomyocytes Cell Cycle Induction

BackgroundIt has been reported that metabolic switch from fatty acid oxidation to glycolysis is associated with cardiomyocyte cell cycle induction. However, it is unknown if increased glycolysis and ATP production are indispensable for cardiomyocyte cell cycle induction. Here, we aim to address this question.MethodsCell cycle was induced by overexpression of CCND2 gene or treatment of FGF1 (100 ng/mL) and CHIR99021 (5 µM) on human induced pluripotent stem cells‐derived cardiomyocytes (hiPSC‐CMs). Contraction inhibition was induced by treatment of verapamil (0.5 μM). Cell cycle was evaluated by immunostaining using antibodies against cell cycle markers. Real‐time ATP production and mitochondrial oxidative phosphorylation were determined by Seahorse assay. Metabolic profile was assessed by liquid chromatography–mass spectrometry.ResultsTreatment of FGF1 and CHIR99021 induces cell cycle of hiPSC‐CMs, reduced carbohydrate metabolism and real‐time ATP production from both mitochondria and cytoplasm. Data from RNA Sequencing showed this treatment increased expression of CCND2 and CDK4 and decreased expression of sarcomeric contractile related genes. Overexpression of CCND2 in hiPSC‐CMs also induces cell cycle, increased aerobic glycolysis (Warburg effect) and no change of real‐time ATP production. Inhibition of contraction via treatment of verapamil resulted in reduced overall metabolism and real‐time ATP production.ConclusionOur data suggest that cardiomyocyte cell cycle may be induced without increased glycolysis or real‐time ATP production.

Adenosine receptor signaling regulates effector T cell activation through the limitation of aerobic glucose metabolism.

Abstract Background In a mouse model of colitis, we previously reported the protective role of the adenosine axis on effector T (Teff) cell responses and disease progression. Inflammatory reactions are paired with cell metabolic reprogramming from oxidative phosphorylation (OXPHOS) to aerobic glycolysis which is also a hallmark of T cell activation. Thus, we sought to evaluate the role of the adenosine 2A receptor (A2AR) signaling in the regulation of T cell metabolism. Methods Teff cells were reactivated in the presence or absence of A2AR agonists. Gene expression of mTOR, HIF-1α and cMyc (transcriptional factors of glycolysis-related genes) were quantified by qPCR. T cell metabolic phenotype was evaluated by transcriptomic profiling (Nanostring nCounter® Metabolic Pathway Panel), as well as real-time extracellular flux analysis, glucose uptake, and ATP production assays. Results mTOR, HIF-1α and cMyc gene expression increased as soon as 2hr post stimulation. At this time point, transcriptomic analysis revealed an A2AR agonist-induced downregulation of pathways related to T cell activation (i.e. mTOR, MAPK, PI3K, NFκB and cMyc pathways) and energy metabolism (i.e glycolysis, autophagy, fatty acid synthesis) whereas oxidative metabolism pathways (OXPHOS, fatty acid oxidation, glutamine metabolism) were upregulated (Pathway Score q-value &amp;lt;0.05). Functional assays showed a decreased de novo glucose uptake and glycolysis rate after A2AR agonist treatment while OXPHOS capacity and ATP production were not modulated. Conclusion A2AR signaling provokes a rewiring of the activated T cell metabolism towards an oxidative state making adenosine a potential T cell metabolic regulator that can be exploited in the treatment of IBD. Supported by NIH R01AI079145

Potential Mechanisms of NUR77‐Mediated Signaling in the Aging Retinal Pigment Epithelium: Implications for Age‐Related Macular Degeneration

PurposeRetinal pigmented epithelium (RPE) dysfunction is associated with age‐related macular degeneration (AMD), the leading cause of vision loss in the elderly in the western world. A better understanding of RPE‐related signaling pathways is the key to developing therapies and potential drug treatments for the early stages of AMD. Nuclear receptors are master regulators of cellular processes including lipid metabolic dysregulation, apoptosis, and inflammation, pathogenic pathways associated with AMD development and progression. Previously we found that orphan nuclear receptor member 1(NR4A1) or NUR77 is expressed by human RPE. The role of NUR77 in the eye or RPE‐mediated signaling pathways, however is not known. Given NUR77’s purported role in regulating inflammation, angiogenesis, and apoptosis in a number of body tissues, herein we investigated the role of NUR77 in RPE as a function of age.MethodsTotal RNA was extracted from RPE/choroid fractions of young and old mouse eyes, RPE/choroid from human donors, and human primary RPE cultures harvested from young and old eye tissue (ages 18‐93). Expression of NUR77/Nurr77 and its target genes were measured by qRT‐PCR. Cellular bioenergetics of young versus old RPE and NUR77 overexpressing RPE were measured using the Seahorse XF Real‐Time ATP Rate Assay. RPE cells overexpressing NUR77 were analyzed for expression of senescence‐related and epithelial‐mesenchymal transition (EMT) markers. To investigate the function of Nur77, in vivo, ocular imaging and retinal function assessments of 4‐8 month old Nur77‐null mice was done. Ex vivo, ocular morphology was examined. Statistical methods for data analysis included two‐tailed Student’s t‐test and two‐way ANOVA. Values were considered statistically significant at p &lt; 0.05.ResultsNur77/NUR77 expression in mouse and human RPE decreases with age. Overexpression of NUR77 (induction of the ‘young phenotype’) in RPE cells isolated from old donors results in a decrease in expression of the senescence associated genes p21 and p16, while low levels of NUR77 were associated with elevated senescence. Expression of the metabolic markers COX III, pyruvate kinase M1 and 2were differentially regulated in the RPE/choroid fractions from old versus young mice. Seahorse assays revealed that overexpression of NUR77 in RPE cells from young but not old donors resulted in a metabolic shift from glycolysis towards mitochondrial respiration. In the absence of Nur77, enhanced expression of markers of inflammation (CCL2, TNFa), oxidative stress (HMOX1, SOD1), metabolism (COXIII, PKM2) and EMT (N‐cadherin, Snail 2, ZEB1, FN1, Vimentin) was observed in the mouse RPE/choroid. Initial assessments of ocular imaging revealed the presence of hypopigmented regions within the posterior pole of Nur77‐null mice at 4 months of age.ConclusionsOur preliminary results indicate an age‐dependent role of NUR77 in balancing a metabolic switch towards mitochondrial respiration in the RPE. Studies are ongoing to further dissect the NUR77‐mediated signaling pathways in aging and ocular disease, and in determining if NUR77 overexpression may serve as potential therapy in diseases wherein RPE is compromised.

The enhanced mitochondrial dysfunction by cantleyoside confines inflammatory response and promotes apoptosis of human HFLS-RA cell line via AMPK/Sirt 1/NF-κB pathway activation

ObjectiveCantleyoside (CA) is a kind of iridoid glycosides in Pterocephalus hookeri (C. B. Clarke) Höeck. The purpose of this study was to investigate the effects of CA on human rheumatoid arthritis fibroblast synovial cells (HFLS-RA). MethodsCell proliferation of HFLS-RA was assessed by CCK-8. ELISA was used to detect cytokines NO, TNF-α, IL-1β/6, MCP-1, MMP-1/3/9 and metabolism-related ATPase activities and ATP levels. JC-1, DCFH-DA, Fluo-3 AM and Calcein AM probes were used to detect mitochondrial membrane potential (MMP), reactive oxygen species (ROS), Ca2+ and mitochondrial permeability conversion pore (MPTP), respectively. Isolated mitochondria assay was used to detect mitochondrial swelling. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and real-time ATP production were measured using a Seahorse analyzer. Apoptosis was detected by TUNEL and Hoechst staining. Western blot was used to detect the expressions of AMPK/p-AMPK, Sirt 1, IκBα, NF-κB p65/p-NF-κB p65, Bcl-2 and Bax. Cytoplasmic nuclear isolation was also performed to detect the translocation of NF-κB. ResultsCA significantly suppressed cell proliferation and the levels of NO, TNF-α, IL-1β/6, MCP-1 and MMP-1/3/9 in HFLS-RA. In addition, CA promoted the apoptosis of HFLS-RA by increasing TUNEL and Hoechst positive cells and the ratio of Bax/Bcl-2. Inhibition of energy metabolism in HFLS-RA by CA reduced OCR, ECAR and real-time ATP generation rate. Importantly, CA promoted p-AMPK and Sirt 1 expression, inhibited IκBα degradation to reduce p-NF-κB and translocation. ConclusionThe results suggest that CA activates the AMPK/Sirt 1/NF-κB pathway by promoting mitochondrial dysfunction, thereby exerting anti-inflammatory and pro-apoptotic effects.

The Use of Seahorse XF Assays to Interrogate Real-Time Energy Metabolism in Cancer Cell Lines.

The propensity of cancer cells to preferentially undergo anaerobic metabolism despite oxygen being abundant is referred to as the Warburg effect. Measuring cellular metabolism is therefore central to understanding the cellular physiology of cancer cells. The Seahorse XFe Analyzer series allows real-time measurement of cellular metabolism. In the basic assay, two parameters, the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), are used to determine real-time changes in the energy needs of live cells: OCR provides a measure of aerobic mitochondrial respiration; ECAR gives a measure of anaerobic glycolysis. Through the use of various respiration inhibitors, the Seahorse assay allows baseline respiration rate and total aerobic and anaerobic ATP production to be determined under a variety of experimental conditions. Here we describe the protocol for completing the Seahorse Real-Time ATP Rate Assay for adherent and suspension cancer cell lines. Depending on individual experimental results, more refined subsequent assays can then be performed to specifically determine, for example, the ability to utilize different substrates by the cell lines in the presence and absence of pharmacological and/or genetic interventions.