Biology subjects

Greenwood, B.

Publications and source records attributed to Greenwood, B..

6 recordsLinked to original sources

bioRxiv2024

Amino Acid and Glucose Fermentation Maintain ATP Content in Mouse and Human Malignant Glioma Cells

Energy is necessary for tumor cell viability and growth. Aerobic glucose-driven lactic acid fermentation is a common metabolic phenotype seen in most cancers including malignant gliomas. This metabolic phenotype is linked to abnormalities in mitochondrial structure and function. A luciferin-luciferase bioluminescence ATP assay was used to measure the influence of amino acids, glucose, and oxygen on ATP content and viability in mouse (VM-M3 and CT-2A) and human (U-87MG) glioma cells that differed in cell biology, genetic background, and species origin. Oxygen consumption was measured using the Resipher system. Extracellular lactate and succinate were measured as end products of the glycolysis and glutaminolysis pathways, respectively. The results showed that: 1) glutamine was a source of ATP content irrespective of oxygen. No other amino acid could replace glutamine in sustaining ATP content and viability; 2) ATP content persisted in the absence of glucose and under hypoxia, ruling out substantial contribution through either glycolysis or oxidative phosphorylation (OxPhos) under these conditions; 3) Mitochondrial complex IV inhibition showed that oxygen consumption was not an accurate measure for ATP production through OxPhos. The glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine (DON), reduced ATP content and succinate export in cells grown in glutamine. The data suggests that mitochondrial substrate level phosphorylation in the glutamine-driven glutaminolysis pathway contributes to ATP content in these glioma cells. A new model is presented highlighting the synergistic interaction between the high-throughput glycolysis and glutaminolysis pathways that drive malignant glioma growth and maintain ATP content through the aerobic fermentation of both glucose and glutamine. Summary statementMalignant gliomas, regardless of cell of origin or species, rely on fermentation mechanisms for ATP production due to OxPhos insufficiency. Glucose and glutamine together are necessary and sufficient for dysregulated tumor cell growth, whereas OxPhos is neither necessary nor sufficient.

cancer biology↗

bioRxiv2023

Ketogenic diet as a metabolic vehicle for enhancing the therapeutic efficacy of mebendazole and devimistat in preclinical pediatric glioma

Invasion of high-grade glioma (HGG) cells through the brain and spinal cord is a leading cause of cancer death in children. Despite advances in treatment, survivors often suffer from lifelong adverse effects of the current toxic therapies used for management. This study investigated the influence of nutritional ketosis on the therapeutic action of mebendazole (MBZ) and devimistat (CPI-613) against the highly invasive VM-M3 and non-invasive CT-2A glioblastoma cells grown orthotopically in juvenile syngeneic mice. Additionally, both drugs were tested in the human pediatric GBM cell line SF-188. DON (6-Diazo-5-oxo-L-norleucine) was used as a positive drug control for glutamine targeting. Cerebral implantation of the VM-M3 cells, which are mesenchymal origin, invaded throughout the brain and the spinal column similar to that seen in children with HGG. Neither the CT-2A nor the VM-NM1 glioblastoma stem cell tumors showed distal invasion in syngeneic juvenile mouse brains. The maximum therapeutic benefit of MBZ and CPI-613 on tumor invasion, growth, and mouse survival occurred only when the drugs were administered together with a ketogenic diet (KD). MBZ treatment inhibited both the glutaminolysis and the glycolysis pathways in VM-M3 cells grown either in vivo or in vitro. Both MBZ and CPI-613 significantly reduced the in vitro growth and viability of the SF-188 cells. Moreover, drug administration together with the KD allowed for lower dosing thus minimizing toxicity while improving overall survival of the mice. This preclinical study in two different HGGs, grown in syngeneic juvenile mice, highlights the potential importance of diet/drug therapeutic strategies for managing childhood brain cancer.

cancer biology↗

bioRxiv2022

Residual Complex I activity supports glutamate catabolism and mtSLP via canonical Krebs cycle activity during acute anoxia without OXPHOS

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in organello in real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U-13C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system inferred that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U-13C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.

biochemistry↗

bioRxiv2022

Serum metabolomic analysis of men on a low-carbohydrate diet for biochemically recurrent prostate cancer reveal the potential role of ketogenesis to slow tumor growth: A secondary analysis of the CAPS2 diet trial

BackgroundSystemic treatments for prostate cancer (PC) have significant side effects. Thus, newer alternatives with fewer side effects are urgently needed. Animal and human studies suggest the therapeutic potential of low carbohydrate diet (LCD) for PC. To test this possibility, Carbohydrate and Prostate Study 2 (CAPS2) trial was conducted in PC patients with biochemical recurrence (BCR) after local treatment to determine the effect of a 6-month LCD intervention vs. usual care control on PC growth as measured by PSA doubling time (PSADT). We previously reported the LCD intervention led to significant weight loss, higher HDL, and lower triglycerides and HbA1c with a suggested longer PSADT. However, the metabolic basis of these effects are unknown. MethodsTo identify the potential metabolic basis of effects of LCD on PSADT, serum metabolomic analysis was performed using baseline, month 3, and month 6 banked sera to identify the metabolites significantly altered by LCD and that correlated with varying PSADT. ResultsLCD increased the serum levels of ketone bodies, glycine and hydroxyisocaproic acid. Reciprocally, LCD reduced the serum levels of alanine, cytidine, asymmetric dimethylarginine (ADMA) and 2-oxobutanoate. As high ADMA level is shown to inhibit nitric oxide (NO) signaling and contribute to various cardiovascular diseases, the ADMA repression under LCD may contribute to the LCD-associated health benefit. Regression analysis of the PSADT revealed a correlation between longer PSADT with higher level of 2-hydroxybutyric acids, ketone bodies, citrate and malate. Longer PSADT was also associated with LCD reduced nicotinamide, fructose-1, 6-biphosphate (FBP) and 2-oxobutanoate. ConclusionThese results suggest a potential association of ketogenesis and TCA metabolites with slower PC growth and conversely glycolysis with faster PC growth. The link of high ketone bodies with longer PSADT supports future studies of ketogenic diets to slow PC growth.

cancer biology↗

bioRxiv2020

The effects of low-carbohydrate diets on the metabolic response to androgen-deprivation therapy in prostate cancer

Prostate cancer (PC) is the second most lethal cancer for men and metastatic PC is treated by androgen deprivation therapy (ADT). While effective, ADT has many metabolic side effects. Previously, serum metabolome analysis showed that ADT reduced androsterone sulfate, 3-hydroxybutyric acid, acyl-carnitines while increased serum glucose. Since ADT reduced ketogenesis, we speculate that low-carbohydrate diets (LCD) may reverse many ADT-induced metabolic abnormalities in animals and humans. To test this possibility, we conducted a multi-center trial of PC patients initiating ADT randomized to no diet change (control) or LCD. We previously showed LCD led to significant weight loss, reduced fat mass, improved insulin resistance and lipid profiles. To determine whether and how LCD affects ADT-induced metabolic effects, we analyzed serum metabolites after 3-, and 6-months of ADT on LCD vs. control. We found androsterone sulfate was most consistently reduced by ADT, and was slightly further reduced by LCD. Contrastingly, LCD increased 3-hydroxybutyric acid and various acyl-carnitines, counteracting their reduction during ADT. LCD also reversed the ADT-reduced lactic acid, alanine and S-adenosyl Methionine (SAM), elevating glycolysis metabolites, amino acids and sulfur-containing metabolites. While the degree of ADT-reduced androsterone was strongly correlated with glucose and indole-3-carboxaldehyde, LCD disrupted such correlation. However, many LCD-induced changes were seen at 3-but not 6-month, suggesting metabolic adaption. Together, LCD significantly reversed many ADT-induced metabolic changes while slightly enhancing androgen reduction. Future research is needed to confirm these findings and determine whether LCD can mitigate ADT-linked comorbidities and possibly delaying disease progression by further lowering androgens. Statement of translational relevanceProstate cancer (PC) is the most common non-skin cancer and second leading cause of cancer-related death in men. While androgen deprivation therapy (ADT) is the main treatment for metastatic PC, it has many metabolic side effects. Previous serum metabolome analysis of PC patients receiving ADT identified reduced ketogenesis. Therefore, low-carbohydrate diets (LCD), ketogenic in nature, may reverse many ADT-induced metabolic abnormalities. We conducted a 6-month multi-center trial of no diet change (control) vs. LCD in men initiating ADT. We found that LCD reversed many ADT-induced metabolic abnormalities while slightly further reducing androgen levels. Also, LCD disrupted the diabetogenic effects of ADT, but some effects seen at 3-month were lost at 6-month, suggesting metabolic adaptation to LCD. These data suggest the potential metabolic benefits of LCD with potential to enhance ADT efficacy. Larger studies testing whether LCDs mitigate metabolic side effects and slow disease progression are warranted given acceptable safety profiles, metabolic benefits, and possibly lowered androgens.

cancer biology↗

bioRxiv2020

KCNQ gene family members act as both tumor suppressors and oncogenes in gastrointestinal cancers

Voltage sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large scale analysis of 1594 patients with GI cancers coupled with in vitro models we find KCNQ family genes are mutated in ~30% of patients, and play therapeutically targetable roles in GI cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation, and its resultant effects on cadherins junctions. This also highlights novel roles for KCNQ3 in non-excitable tissues. We additionally discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors, and that inhibition of KCNQ activity reduces proliferation of GI cancer. These findings reveal a novel and exploitable role for potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GI cancers may exist through KCNQ inhibitors. SIGNIFICANCEKCNQ channels modulate the WNT pathway and MYC signalling, and drive growth of gastrointestinal cancers. Available drugs modulate these pathways and offer therapeutic potential in gastrointestinal cancer.

cancer biology↗