Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress.

Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.

Nitric oxide has diverse effects on head and neck cancer cell proliferation and glycolysis.

Glycolysis is a key energy-providing process and one of the hallmarks of cancer. Nitric oxide (NO), a free radical molecule, regulates glycolysis in various cancers. NO can alter the cell cycle and apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. However, the effect of NO on glycolysis in HNSCC cells remains unresolved. The present study investigated the effects of NO on cell proliferation, glucose transporter (GLUT) gene expression and glycolytic indicators in HNSCC cell lines. Two pairs of isogenic HNSCC cell lines, HN18/HN17 and HN30/HN31, were treated with a NO donor, diethylamine NONOate (DEA-NONOate), for 24, 48 and 72 h. Cell proliferation was assessed using MTT assay and NO concentration was measured using the Griess Reagent System. GLUT1, GLUT2, GLUT3, and GLUT4 gene expression was analyzed using reverse transcription-quantitative PCR. Furthermore, hexokinase (HK) activity and lactate production were measured in NO-treated cells using colorimetric assay. NO exhibited concentration-dependent pro- and anti-proliferative effects on the HNSCC cell lines. Lower NO concentrations (5-200 µM) had pro-proliferative effects, whereas NO >200 µM had an anti-proliferative effect on HNSCC cells. NO (5 µM) promoted proliferation and glycolysis in HN18 cells by upregulating GLUT1 and GLUT2 gene expression and increasing HK activity and lactate levels. At 5-20 µM, NO-induced HN17 and HN30 cells demonstrated enhanced proliferation and GLUT2, GLUT3 and GLUT4 gene expression, whereas the glycolytic pathway was not affected. In conclusion, the present study demonstrated distinct proliferative effects of NO on HNSCC cells. NO may promote cell proliferation by stimulating glucose consumption and the glycolytic rate in HN18 cells. The effects of NO in other cell lines may be mediated by a non-glycolysis mechanism and require further investigation.

Ovarian Cancer Cell-Conditioning Medium Induces Cancer-Associated Fibroblast Phenoconversion through Glucose-Dependent Inhibition of Autophagy.

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor-stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and response to therapy, making them attractive targets for anti-cancer treatment. Unraveling the mechanisms involved in CAFs activation and maintenance is therefore crucial for the improvement of therapy efficacy. Here, we report that CAFs phenoconversion relies on the glucose-dependent inhibition of autophagy. We show that ovarian cancer cell-conditioning medium induces a metabolic reprogramming towards the CAF-phenotype that requires the autophagy-dependent glycolytic shift. In fact, 2-deoxy-D-glucose (2DG) strongly hampers such phenoconversion and, most importantly, induces the phenoreversion of CAFs into quiescent fibroblasts. Moreover, pharmacological inhibition (by proline) or autophagy gene knockdown (by siBECN1 or siATG7) promotes, while autophagy induction (by either 2DG or rapamycin) counteracts, the metabolic rewiring induced by the ovarian cancer cell secretome. Notably, the nutraceutical resveratrol (RV), known to inhibit glucose metabolism and to induce autophagy, promotes the phenoreversion of CAFs into normal fibroblasts even in the presence of ovarian cancer cell-conditioning medium. Overall, our data support the view of testing autophagy inducers for targeting the tumor-promoting stroma as an adjuvant strategy to improve therapy success rates, especially for tumors with a highly desmoplastic stroma, like ovarian cancer.

Tethering of hexokinase 2 to mitochondria promotes resistance of liver cancer cells to natural killer cell cytotoxicity.

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.

Glycometabolism and lipid metabolism related genes predict the prognosis of endometrial carcinoma and their effects on tumor cells.

BACKGROUND: Glycometabolism and lipid metabolism are critical in cancer metabolic reprogramming. The primary aim of this study was to develop a prognostic model incorporating glycometabolism and lipid metabolism-related genes (GLRGs) for accurate prognosis assessment in patients with endometrial carcinoma (EC). METHODS: Data on gene expression and clinical details were obtained from publicly accessible databases. GLRGs were obtained from the Genecards database. Through nonnegative matrix factorization (NMF) clustering, molecular groupings with various GLRG expression patterns were identified. LASSO Cox regression analysis was employed to create a prognostic model. Use rich algorithms such as GSEA, GSVA, xCELL ssGSEA, EPIC,CIBERSORT, MCPcounter, ESTIMATE, TIMER, TIDE, and Oncoppredict to analyze functional pathway characteristics of the forecast signal, immune status, anti-tumor therapy, etc. The expression was assessed using Western blot and quantitative real-time PCR techniques. A total of 113 algorithm combinations were combined to screen out the most significant GLRGs in the signature for in vitro experimental verification, such as colony formation, EdU cell proliferation, wound healing, apoptosis, and Transwell assays. RESULTS: A total of 714 GLRGs were found, and 227 of them were identified as prognostic-related genes. And ten GLRGs (AUP1, ESR1, ERLIN2, ASS1, OGDH, BCKDHB, SLC16A1, HK2, LPCAT1 and PGR-AS1) were identified to construct the prognostic model of patients with EC. Based on GLRGs, the risk model's prognosis and independent prognostic value were established. The signature of GLRGs exhibited a robust correlation with the infiltration of immune cells and the sensitivity to drugs. In cytological experiments, we selected HK2 as candidate gene to verify its value in the occurrence and development of EC. Western blot and qRT-PCR revealed that HK2 was substantially expressed in EC cells. According to in vitro experiments, HK2 knockdown can increase EC cell apoptosis while suppressing EC cell migration, invasion, and proliferation. CONCLUSION: The GLRGs signature constructed in this study demonstrated significant prognostic value for patients with endometrial carcinoma, thereby providing valuable guidance for treatment decisions.

Virtually identical does not mean exactly identical: Discrepancy in energy metabolism between glucose and fructose fermentation influences the reproductive potential of yeast cells.

The physiological efficiency of cells largely depends on the possibility of metabolic adaptations to changing conditions, especially on the availability of nutrients. Central carbon metabolism has an essential role in cellular function. In most cells is based on glucose, which is the primary energy source, provides the carbon skeleton for the biosynthesis of important cell macromolecules, and acts as a signaling molecule. The metabolic flux between pathways of carbon metabolism such as glycolysis, pentose phosphate pathway, and mitochondrial oxidative phosphorylation is dynamically adjusted by specific cellular economics responding to extracellular conditions and intracellular demands. Using Saccharomyces cerevisiae yeast cells and potentially similar fermentable carbon sources i.e. glucose and fructose we analyzed the parameters concerning the metabolic status of the cells and connected with them alteration in cell reproductive potential. Those parameters were related to the specific metabolic network: the hexose uptake - glycolysis and activity of the cAMP/PKA pathway - pentose phosphate pathway and biosynthetic capacities - the oxidative respiration and energy generation. The results showed that yeast cells growing in a fructose medium slightly increased metabolism redirection toward respiratory activity, which decreased pentose phosphate pathway activity and cellular biosynthetic capabilities. These differences between the fermentative metabolism of glucose and fructose, lead to long-term effects, manifested by changes in the maximum reproductive potential of cells.

Icariin alleviates oxygen-induced retinopathy by targeting microglia hexokinase 2.

Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.

Complex genetic interaction between glucose sensor HXK1 and E3 SUMO ligase SIZ1 in regulating plant morphogenesis.

Sugar signaling forms the basis of metabolic activities crucial for an organism to perform essential life activities. In plants, sugars like glucose, mediate a wide range of physiological responses ranging from seed germination to cell senescence. This has led to the elucidation of cell signaling pathways involving glucose and its counterparts and the mechanism of how these sugars take control over major hormonal pathways such as auxin, ethylene, abscisic acid and cytokinin in Arabidopsis. Plants use HXK1(Hexokinase) as a glucose sensor to modulate changes in photosynthetic gene expression in response to high glucose levels. Other proteins such as SIZ1, a major SUMO E3 ligase have recently been implicated in controlling sugar responses via transcriptional and translational regulation of a wide array of sugar metabolic genes. Here, we show that these two genes work antagonistically and are epistatic in controlling responsiveness toward high glucose conditions.

Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.

A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Breakthroughs in Alzheimer's Research: A Path to a More Promising Future?

Background: Alzheimer's disease (AD) is a widespread neurodegenerative disorder with a significant global impact, affecting approximately 50 million individuals, and projections estimate that up to 152 million people will be affected by 2050. AD is characterized by beta-amyloid plaques and tau tangles in the brain, leading to cognitive decline. Summary: Recent research on AD has made significant strides, including the development of an "amyloid clock" biomarker that tracks AD progression through positron emission tomography (PET) scans. Surf4 and other genes have been discovered to play a role in regulating beta-amyloid toxicity, while inhibiting the enzyme hexokinase-2 has shown positive results in preclinical studies. New brain mapping techniques have identified early brain-based causes of cognitive changes in AD, and biomarkers such as neuronal pentraxin protein Nptx2 and astrocytic 7-subunit of the nicotinic acetylcholine receptors (7nAChRs) show potential for early detection. Other approaches, such as replenishing the enzyme Tip60, selectively degrading the modified protein p-p38 with PRZ-18002, and targeting the protein voltage-dependent anion channel-1 (VDAC1), have shown promise in enhancing cognitive function and preventing pathophysiological alterations linked to AD. Baseline blood samples and other biomarkers such as urine formic acid, p-tau 198, microRNAs, and glial fibrillary acidic protein (GFAP) have also been discovered for early detection and intervention of AD. Additionally, recent FDA approvals for medications such as aducanumab and lecanemab provide options for reducing AD symptoms and improving function, while clinical trials for dementia vaccines show promise for the nasal and beta-amyloid 40 vaccines as well as vaccinations targeting tau. Key Messages: These advancements in AD research, including biomarker discovery and the development of disease-modifying treatments, are crucial steps towards improving the lives of those affected by AD and finding a cure for this debilitating disease.

Abatement of the binding of human hexokinase II enzyme monomers by in-silico method with the design of inhibitory peptides.

The hexokinase II enzyme is bound to the (VDAC1) channel in the form of a dimer and prevents the release of cell death factors from mitochondria to the cytoplasm. Studies have shown that blocking the binding of hexokinase II enzyme to (VDAC1) led to the initiation of apoptosis in cancer cells. No peptide has been designed so far to inhibit hexokinase II. The aim of this study was to inhibit the dimerization of enzyme subunits in order to inhibition the formation of (VDAC1) and the hexokinase II complex. In this study, the molecular dynamics simulation of the enzyme in monomer and dimer states was investigated in terms of RMSF, RMSD and radius of gyration. The following process involves extracting and designing variable-length peptides from the interacting segments of enzyme monomers. Using molecular dynamics simulation, the stability of the peptide was determined in terms of RMSD. Molecular docking was used to investigate the interaction between the designed peptides. Finally, the inhibitory effect of peptides on subunit association was measured using dynamic light scattering (DLS) technique. Our results showed that the designed peptides, which mimic common amino acids in dimerization, interrupt the bona fide form of the enzyme subunits. The result of this study provides a new way to disrupt the assembly process and thereby decreased the function of the hexokinase II. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00201-8.

Sugar sensing in C4 source leaves: a gap that needs to be filled.

Plant growth depends on sugar production and export by photosynthesizing source leaves and sugar allocation and import by sink tissues (grains, roots, stems, and young leaves). Photosynthesis and sink demand are tightly coordinated through metabolic (substrate, allosteric) feedback and signalling (sugar, hormones) mechanisms. Sugar signalling integrates sugar production with plant development and environmental cues. In C3 plants (e.g. wheat and rice), it is well documented that sugar accumulation in source leaves, due to source-sink imbalance, negatively feeds back on photosynthesis and plant productivity. However, we have a limited understanding about the molecular mechanisms underlying those feedback regulations, especially in C4 plants (e.g. maize, sorghum, and sugarcane). Recent work with the C4 model plant Setaria viridis suggested that C4 leaves have different sugar sensing thresholds and behaviours relative to C3 counterparts. Addressing this research priority is critical because improving crop yield requires a better understanding of how plants coordinate source activity with sink demand. Here we review the literature, present a model of action for sugar sensing in C4 source leaves, and suggest ways forward.

The Hexokinase 1 5'-UTR Mutation in Charcot-Marie-Tooth 4G Disease Alters Hexokinase 1 Binding to Voltage-Dependent Anion Channel-1 and Leads to Dysfunctional Mitochondrial Calcium Buffering.

Demyelinating Charcot-Marie-Tooth 4G (CMT4G) results from a recessive mutation in the 5'UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial calcium homeostasis by binding to the Voltage-Dependent Anion Channel (VDAC), through its N-terminal porin-binding domain. Our hypothesis is that CMT4G mutation results in a broken interaction between mutant HK1 and VDAC, disturbing mitochondrial calcium homeostasis. We studied a cohort of 25 CMT4G patients recruited in the French gypsy population. The disease was characterized by a childhood onset, an intermediate demyelinating pattern, and a significant phenotype leading to becoming wheelchair-bound by the fifth decade of life. Co-IP and PLA studies indicated a strong decreased interaction between VDAC and HK1 in the patients' PBMCs and sural nerve. We observed that either wild-type HK1 expression or a peptide comprising the 15 aa of the N-terminal wild-type HK1 administration decreased mitochondrial calcium release in HEK293 cells. However, mutated CMT4G HK1 or the 15 aa of the mutated HK1 was unable to block mitochondrial calcium release. Taken together, these data show that the CMT4G-induced modification of the HK1 N-terminus disrupts HK1-VDAC interaction. This alters mitochondrial calcium buffering that has been shown to be critical for myelin sheath maintenance.

Discovery of Strong 3-Nitro-2-Phenyl-2H-Chromene Analogues as Antitrypanosomal Agents and Inhibitors of Trypanosoma cruzi Glucokinase.

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

HKDC1 enhances the proliferation, migration and glycolysis of pancreatic adenocarcinoma and is linked to immune infiltration.

Background: Understanding the molecular mechanisms of pancreatic adenocarcinoma (PAAD) development is vital for treating this disease, as the current prognosis and treatment options are highly discouraging. Objective: This study aimed to examine the involvement of Hexokinase Domain Containing 1 (HKDC1) in the progression of PAAD. Methods: The study utilized bioinformatics techniques to evaluate the relationship between the expression of HKDC1 and clinical characteristics. In vitro experiments were conducted to investigate the molecular mechanisms and biological functions of HKDC1 in PAAD. Results: The findings of this research indicate that the expression of HKDC1 was increased in various types of human cancers, and a significant correlation was observed between elevated HKDC1 expression in PAAD and unfavorable prognosis. According to the findings from univariate and multivariate Cox regression analyses, HKDC1 could potentially serve as a standalone prognostic indicator for individuals diagnosed with PAAD. After performing calculations, we determined that the HKDC1 high-expression group exhibited lower immunologic score and higher ESTIMATE score, indicating a difference in immune infiltration score. In order to validate the expression of HKDC1 in PAAD cell lines, we analyzed the PAAD cell lines through qPCR and protein blotting. The expression of HKDC1 in human PAAD tissues was also detected by western blotting. Additionally, we explored the involvement of HKDC1 in PAAD by conducting experiments such as colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, and wound healing assays. In our study, we discovered that disruption of HKDC1 expression in PAAD cell types resulted in a decrease in cell growth rate and inhibited cell movement and invasion. Conclusion: To conclude, our findings indicate that HKDC1 has a significant impact on the tumor microenvironment (TME) of PAAD and could potentially be a promising target for PAAD treatment, offering fresh perspectives on the management of PAAD.

Targeting hexokinase 2 for oral cancer therapy: structure-based design and validation of lead compounds.

The pursuit of small molecule inhibitors targeting hexokinase 2 (HK2) has significantly captivated the field of cancer drug discovery. Nevertheless, the creation of selective inhibitors aimed at specific isoforms of hexokinase (HK) remains a formidable challenge. Here, we present a multiple-pharmacophore modeling approach for designing ligands against HK2 with a marked anti-proliferative effect on FaDu and Cal27 oral cancer cell lines. Molecular dynamics (MD) simulations showed that the prototype ligand exhibited a higher affinity towards HK2. Complementing this, we put forth a sustainable synthetic pathway: an environmentally conscious, single-step process facilitated through a direct amidation of the ester with an amine under transition-metal-free conditions with an excellent yield in ambient temperature, followed by a column chromatography avoided separation technique of the identified lead bioactive compound (H2) that exhibited cell cycle arrest and apoptosis. We observed that the inhibition of HK2 led to the loss of mitochondrial membrane potential and increased mitophagy as a potential mechanism of anticancer action. The lead H2 also reduced the growth of spheroids. Collectively, these results indicated the proof-of-concept for the prototypical lead towards HK2 inhibition with anti-cancer potential.

LC/MS/MS and GC/MS/MS metabolic profiling of Leontodon hispidulus, in vitro and in silico anticancer activity evaluation targeting hexokinase 2 enzyme.

Leontodon hispidulus Boiss is a wild annual plant growing in Egypt. The present study aims for the first time, to evaluate the phytochemical profile of the main secondary metabolites of the optimized ethanolic extract of the plant using Quadrupole Time-of-Flight Liquid chromatography-mass spectrometry and Gas chromatography-mass spectrometry. It also aims to assess the anticancer activity of its different fractions against the prostate carcinoma cell line. Moreover, an in-silico docking study was performed using the Hexokinase-two enzyme. LC-qToF-MS analysis revealed the tentative identification of 36 phenolic compounds including the glycosides of (luteolin, quercetin, kaempferol, apigenin, isorhamnetin, and daidzein), coumarines (esculin, esculetin, and daphnetin), and phenolic acids (chlorogenic, caffeic, quinic, P-coumaric, and rosmarinic). GC-MS/MS analysis revealed the presence of 18 compounds where palmitic acid, myristic acid, alpha-amyrin, and beta-amyrin were the major ones. The cytotoxic activity results revealed that methylene chloride and ethyl acetate fractions showed the highest cytotoxic activity against the PC3 cell line, with IC50 values of 19, and 19.6 µg/ml, respectively. Interestingly, the docking study demonstrated that apigenin-7-O-glucoside, luteolin-7-O-glucoside, kaempferol-3-O-glucuronide, quercetin-4'-O-glucoside, esculin, rosmarinic acid, chlorogenic acid, and α-amyrin exhibited high affinity to the selected target, HEK-2 enzyme.

Poly(ADP-ribose) mediates bioenergetic defects and redox imbalance in neurons following oxygen and glucose deprivation.

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.

Genetic association of novel SNPs in HK-1 (rs201626997) and HK-3 (rs143604141) with type 2 diabetes mellitus in Bangladeshi population.

BACKGROUND: Hexokinase, a key enzyme in glycolysis, has isoforms like HK-1, HK-2, HK-3, and Glucokinase. Unpublished exome sequencing data showed that two novel polymorphisms in HK-1 rs201626997 (G/T) and HK-3 rs143604141 (G/A) exist in the Bangladeshi population. We investigated the possible relationship of these SNPs with T2DM. MATERIALS AND METHODS: Peripheral blood samples from the study participants were used to isolate their genomic DNA. An allele-specific PCR was standardized that can discriminate between the wild-type and mutant-type alleles of HK-1 (rs201626997) and HK-3 (rs143604141) polymorphisms. The data was analyzed by SPSS for statistics. RESULTS: We performed allele-specific PCR for 249 diabetic patients and 195 control samples. For HK-1 (rs201626997), 24 (5.4%) have a mutant allele, and for HK-3 (rs143604141), 25 (5.6%) are mutant. There is no significant relationship between the individuals' disease condition and the HK-1 polymorphism (P value 0.537). But the GA genotype of the HK-3 rs143604141 pertains to an increased risk of diabetes (P value 0.039). HK-3 rs143604141 polymorphism has a moderate correlation (P value 0.078, OR, 3.11, 95% CI, 0.88-10.94) with a family diabetic history. Both polymorphisms showed no significant correlation with gender or BMI. However, hexokinase-1 polymorphism significantly related with diastolic blood pressure (P value 0.048). CONCLUSION: This study will help us to easily detect the polymorphisms of HK-1 (rs201626997) and HK-3 (rs143604141) in different populations of the world. Further studies with a greater number of participants and more physiological information are required to better understand the underlying genetic causes of T2DM susceptibility in Bangladesh.

The Molecular Mechanisms behind Advanced Breast Cancer Metabolism: Warburg Effect, OXPHOS, and Calcium.

Altered metabolism represents a fundamental difference between cancer cells and normal cells. Cancer cells have a unique ability to reprogram their metabolism by deviating their reliance from primarily oxidative phosphorylation (OXPHOS) to glycolysis, in order to support their survival. This metabolic phenotype is referred to as the "Warburg effect" and is associated with an increase in glucose uptake, and a diversion of glycolytic intermediates to alternative pathways that support anabolic processes. These processes include synthesis of nucleic acids, lipids, and proteins, necessary for the rapidly dividing cancer cells, sustaining their growth, proliferation, and capacity for successful metastasis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with the poorest patient outcome due to its high rate of metastasis. TNBC is characterized by elevated glycolysis and in certain instances, low OXPHOS. This metabolic dysregulation is linked to chemotherapeutic resistance in TNBC research models and patient samples. There is more than a single mechanism by which this metabolic switch occurs and here, we review the current knowledge of relevant molecular mechanisms involved in advanced breast cancer metabolism, focusing on TNBC. These mechanisms include the Warburg effect, glycolytic adaptations, microRNA regulation, mitochondrial involvement, mitochondrial calcium signaling, and a more recent player in metabolic regulation, JAK/STAT signaling. In addition, we explore some of the drugs and compounds targeting cancer metabolic reprogramming. Research on these mechanisms is highly promising and could ultimately offer new opportunities for the development of innovative therapies to treat advanced breast cancer characterized by dysregulated metabolism.