Hexokinase II Research Articles

E. coli Biomolecules Increase Glycolysis and Invasive Potential in Lung Adenocarcinoma

Introduction: Recent studies have discovered that lung cancer subtypes possess distinct microbiome profiles within their tumor microenvironment. Additionally, the tumor-associated microbiome exhibits altered bacterial pathways, suggesting that certain bacterial families are more capable of facilitating tumor progression than others. We hypothesize that there exists a crosstalk between lung adenocarcinoma (LUAD) cells and bacterial cells. Methods and Materials: RNA sequencing (RNA-seq) was performed on LUAD cell lines to explore the paracrine signaling effects of bacterial biomolecules. Based on our RNA-seq data, we investigated glycolysis by measuring glucose uptake and lactate production, invasive potential through invasion assays, and epithelial-to-mesenchymal transition (EMT) markers. Since lipopolysaccharides (LPS), abundant on the cell walls of Gram-negative bacteria, can activate toll-like receptor 4 (TLR4), we inhibited TLR4 with C34 to assess its relationship with the observed phenotypic changes. To identify the bacterial biomolecules responsible for these changes, we treated the media with RNAse enzyme, charcoal or dialyzed away molecules larger than 3 kDa. Results and Discussion: RNA-seq revealed 948 genes upregulated in the presence of E. coli biomolecules. Among these, we observed increased expression of Hexokinase II (HKII), JUN proto-oncogene, and Snail Family Transcriptional Repressor 1. We verified the elevation of glycolytic enzymes through Western blot and saw elevation of 2-deoxyglucose uptake and lactate production in LUAD cell lines incubated in E. coli biomolecules. In addition to E. coli elevating glycolysis in LUAD cell lines, E. coli exposure enhanced invasive potential as demonstrated by Boyden chamber assays. Notably, inhibition of TLR4 did not reduce the impact of E. coli biomolecules on glycolysis or the invasive potential of LUAD. Modulating the E. coli-supplemented media with RNAse enzyme or dextran-coated charcoal or using a spin column to remove biomolecules smaller than 3 kDa resulted in changes in HKII and Claudin protein expression. These findings suggest a direct relationship between E. coli and LUAD, wherein several cancer hallmarks are upregulated. Future studies should further investigate these bacterial biomolecules and their role in the tumor microenvironment to fully understand the impact of microbial shifts on cancer progression.

Understanding the Molecular Mechanisms of Incomptine A in Treating Non-Hodgkin Lymphoma Associated with U-937 Cells: Bioinformatics Approaches, Part I.

Background: Incomptine A (IA) has been reported to have cytotoxic activity in non-Hodgkin lymphoma cancer cell lines and have effects on U-937 cells, including the induction of apoptosis, the production of reactive oxygen species, and the inhibition of glycolytic enzymes. Also, IA has cytotoxic activity in the triple-negative subtypes, HER2+, and luminal A of breast cancer cells, with its properties being associated with an effect on the antiapoptotic function of Hexokinase II (HKII). Objectives: In this research, we reviewed the altered levels of proteins present in the lymph nodes of male Balb/c mice inoculated with U-937 cells and treated with IA or methotrexate, as well as mice only inoculated with cancer cells. Methods: Five approaches, including Tandem Mass Tag (TMT), Gene ontology (GO), Reactome, KEGG pathway analysis, and molecular docking, were used. Results: TMT showed that 74 proteins were differentially expressed, out of which 12 presented overexpression (FC ≥ 1.5) and 62 were under expressed (FC ≤ 0.67). In general, the TMT approach showed that IA had a better effect on proteins than methotrexate. Gene ontology, Reactome, and KEGG pathway analysis showed that proteins with altered levels may be implicated in several processes, including gene silencing by RNA, oxidative phosphorylation, glycolysis/gluconeogenesis, cytoskeleton organization, and ATP metabolic and energetic processes. The molecular docking analysis, which used 23 altered proteins as targets, revealed that IA interacted with all the proteins used. Conclusions: The results obtained using the five bioinformatic approaches provide information and show that IA could be used to treat non-Hodgkin lymphoma induced with the U-937 cell line. Also, it could provide a basis for future research and the development of clinical trials.

Antioxidant Potential of Xanthohumol in Disease Prevention: Evidence from Human and Animal Studies.

Xanthohumol (XN) is a phenolic compound found in the largest amount in the flowers of the hop plant, but also in the leaves and possibly in the stalks, which is successfully added to dietary supplements and cosmetics. XN is known as a potent antioxidant compound, which, according to current research, has the potential to prevent and inhibit the development of diseases, i.e., cancer and neurodegenerative diseases. The review aims to examine the antioxidant role of XN in disease prevention, with an emphasis on the benefits and risks associated with its supplementation. The regulation by XN of the Nrf2/NF-kB/mTOR/AKT (Nuclear factor erythroid 2-related factor 2/Nuclear factor kappa-light-chain-enhancer of activated B cells/Mammalian target of rapamycin/Protein Kinase B) pathways induce a strong antioxidant and anti-inflammatory effect, among others the acceleration of autophagy through increased synthesis of Bcl-2 (B-cell lymphoma 2) proteins, inhibition of the synthesis of VEGF (Vascular-endothelial growth factor) responsible for angiogenesis and phosphorylation of HKII (Hexokinase II). It is the key function of XN to ameliorate inflammation and to promote the healing process in organs. However, existing data also indicate that XN may have adverse effects in certain diseases, such as advanced prostate cancer, where it activates the AMPK (activated protein kinase) pathway responsible for restoring cellular energy balance. This potential risk may explain why XN has not been classified as a therapeutic drug so far and proves that further research is needed to determine the effectiveness of XN against selected disease entities at a given stage of the disease.

Renal Inflammation, Oxidative Stress, and Metabolic Abnormalities During the Initial Stages of Hypertension in Spontaneously Hypertensive Rats.

Background: Hypertension is a major cause of mortality worldwide. The kidneys play a crucial role in regulating blood pressure and fluid volume. The relationship between the kidneys and hypertension is complex, involving factors such as the renin-angiotensin system, oxidative stress, and inflammation. This study aims to assess the levels of inflammatory markers, oxidative stress, and metabolic factors in the kidneys, focusing on their potential role in early renal damage and their association with the development of hypertension. Methods: This study was designed to compare the levels of selected inflammatory markers, e.g., interleukins, tumor necrosis factor-α (TNF-α), transforming growth factor, and serine/threonine-protein (mTOR); oxidative stress markers such as malondialdehyde, sulfhydryl group, and glucose (GLC); and metabolic markers among other enzymes, such as alanine transaminase (ALT), aspartate transaminase (AST), hexokinase II (HK-II), and hypoxia-inducible factor-1α (HIF-1α), as well as creatinine in the kidneys of spontaneously hypertensive rats (SHR/NCrl, n = 12) and Wistar Kyoto rats (WKY/NCrl, n = 12). Both juvenile (5 weeks old) and maturing (10 weeks old) specimens were examined using spectrophotometric methods, e.g., ELISA. Results: Juvenile SHRs exhibited reduced renal levels of all studied cytokines and chemokines, with lower oxidative stress and deficits in the mTOR and HK-II levels compared to the age-matched WKYs. Maturing SHRs showed increased renal levels of interleukin-1β (IL-1β), IL-6, IL-18, and TNF-α, alongside elevated carbonyl stress and increased HIF-1α as opposed to their control peers. The levels of all other studied markers were normalized in these animals, except for ALT (increased), ALP, and GLC (both reduced). Conclusions: This study underscores the significant impact of inflammatory, oxidative stress, and metabolic marker changes on renal function. Juvenile SHRs display lower marker levels, indicating an immature immune response and potential subclinical kidney damage that may contribute to hypertension development. In contrast, mature SHRs exhibit chronic inflammation, oxidative dysregulation, and metabolic disturbances, suggesting cellular damage. These changes create a feedback loop that worsens kidney function and accelerates hypertension progression, highlighting the kidneys' crucial role in both initiating and exacerbating this condition.

Acetazolamide suppresses the progression of hepatocellular carcinoma induced by diethylnitrosamine in Wistar albino rats.

Hepatocellular carcinoma (HCC) continues to be the most prevalent type of liver cancer worldwide. Diethylnitrosamine (DEN)-induced HCC is an extensively used hepatic cancer model in experimental animals. Acetazolamide (AZA) is a carbonic anhydrase enzyme inhibitor. This study aimed to assess the therapeutic mechanism of AZA against DEN-induced HCC. Thirty male Wistar albino rats were divided equally into three groups. Group I (C): control group, Group II (HCC): DEN-induced HCC, and Group III (HCC/AZA): AZA-treated HCC. Verification of the HCC induced by DEN was confirmed by elevated liver enzymes' activities, and increased α-fetoprotein (AFP) levels, as well as distinct liver architecture changes. On the other hand, the AZA-treated HCC group experienced decreases in the activities of serum liver enzymes and AFP levels, as well as, regulated liver architecture. Additionally, it downregulated p-p38 MAPK/p-JNK1/JNK2/p-C-Jun/p-NF-κB p65 protein expressions. Moreover, it ameliorated autophagy by controlling the expression of the p-AMPK/p-mTOR1/LC3 I/II proteins. Furthermore, it downregulated the relative gene expressions of carbonic anhydrase-IX (CAIX) and hexokinase-II (HKII). Histopathological examination of AZA-treated HCC liver tissues supported these findings. Conclusion: AZA provides a new dimension in ameliorating experimentally induced HCC through regulation of hepatic biomarkers, antioxidant status, inflammatory markers, and autophagy, mediated by amelioration of CAIX and HKII gene expressions.

Expression of the TGF-β Target Gene Mss51 Is Induced in Muscle From Tumor-Bearing Mice and Attenuated by Treatment With the AMPK Activator AICAR

Cachexia is a severe complication of cancer characterized by skeletal muscle atrophy and loss of function and is associated with a poor prognosis. AMPK is a cellular energy sensor that is upregulated in cancer cachexia. AMPK activation is well-established in promoting catabolic cell signaling in skeletal muscle, partly through the induction of muscle-specific ubiquitin ligases MAFBx and MuRF-1 expression. Despite AMPK’s acute catabolic effects, chronic treatment with the AMPK-activating drug 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) has been reported to preserve muscle function and metabolism in various muscle-wasting conditions, including cancer cachexia. Elevated TGF-β during cancer cachexia is known to disrupt mitochondrial homeostasis and lead to mitochondrial dysfunction. Mss51 is a skeletal muscle-specific mitochondrial gene that is upregulated through TGF-β signaling. Although the role of Mss51 in skeletal muscle cancer cachexia has not been studied as of yet, its expression is associated with mitochondrial and metabolic impairment. Therefore, we hypothesized that Mss-51 expression would be elevated in cachexic skeletal muscle and attenuated by chronic AMPK activation. To test this, we inoculated mice (n=4) with either Lewis Lung Carcinoma (LLC) cells or PBS. Mice were injected daily with 350 mg/kg AICAR or an equivalent volume of saline beginning seven days after inoculation. Twenty-four days after inoculation, the mice were euthanized, and tissues were harvested and weighed. RNA was isolated from the gastrocnemius-plantaris-soleus complex and analyzed by RT-PCR for Mss51, MuRF-1, and MAFBx expression. Protein expression for hexokinase II (HKII) was measured by western blotting. AICAR treatment did not attenuate muscle atrophy in tumor-bearing mice. Compared to the control, Mss-51 was elevated in LLC mice treated with saline and decreased in LLC mice treated with AICAR, consistent with the known effects of AMPK in blocking TGF-β signaling. Similarly, MuRF-1 and MAFBx tended to be elevated in LLC-saline. Still, this increase was attenuated in LLC-AICAR mice, though we were likely underpowered to detect significance in this measure. In conclusion, based on previous work, the rise in Mss51 in tumor-bearing mice may contribute to mitochondrial and metabolic dysfunction in cancer cachexia. The prevention of this increase by AICAR suggests that AMPK may benefit dysfunctional metabolism in the cachexic muscle through inhibition of Mss51. Funding for these experiments was from a BYU Widtsoe Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Advances in the Properties of Incomptine A: Cytotoxic Activity and Downregulation of Hexokinase II in Breast Cancer Cell Lines.

Breast cancer treatments are limited by the cancer subtype and its selectivity towards tumor cells, hence the importance of finding compounds that increase the survival of healthy cells and target any subtype. Incomptine A (IA) is a sesquiterpene lactone with demonstrated cytotoxic activity. In this study, through in vitro assays, it was observed that IA has similar cytotoxic activity between the subtypes triple negative, HER2+, and luminal A of the breast cancer cell lines. IA cytotoxic activity is higher in cancer than in nontumorigenic cells, and its selectivity index for cancer cells is more than that of the drug doxorubicin. Molecular docking and its in silico comparison with the 2-Deoxyglucose inhibitor suggest that IA could bind to Hexokinase II (HKII), decreasing its expression. Since we did not find changes in the expression of the glycolytic pathway, we suppose that IA could affect the antiapoptotic function of HKII in cancer cells. The IA-HKII union would activate the voltage-gated anion channel 1 (VDAC1), resuming apoptosis. Therefore, we suggest that IA could be used against almost any subtype and that its cytotoxic effect could be due to the reactivation of apoptosis in breast cancer cells.

588-P: Probing the Role of Muscle Glycogen Resynthesis on Postexercise AMPK Activation and Metabolic Protein Abundance Using a Novel Genetic Model

Acute exercise can alter the abundance of key metabolic proteins in skeletal muscle. These effects are influenced by postexercise (PEX) refeeding. Muscle glycogen resynthesis is a putative mediator of these refeeding effects. However, it is challenging to test this idea, because glycogen resynthesis is only one of many outcomes with refeeding. Therefore, we devised a novel genetic model to modify muscle expression of glycogen synthase 1 (GS1) using injection of rat skeletal muscle with adeno-associated viral (AAV) vectors for short hairpin RNA targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Rats were sedentary (SED) or exercised (120min swim-exercise). Muscles were collected immediately postexercise (IPEX) and 9-hours PEX (9hPEX), along with time-matched SED controls. The 9hPEX and their SED-control rats were either refed (RF) or not-refed (NRF). Muscles were analyzed for glycogen and phosphorylation and abundance of key metabolic proteins. Muscles injected with shRNA-GS1 vs shRNA-Scr had lower GS1 abundance in all groups. Regardless of genotype, in IPEX vs SED rats: glycogen fell to similarly low values and phosphorylation of AMPK Thr172 (pAMPK) and its substrate ACC Ser79 (pACC) were comparably increased. Muscle glycogen was increased in all groups of 9h-RF rats, but it was lower for shRNA-GS1 vs shRNA-Scr from RF-9hPEX rats. In the NRF-9hPEX rats, pAMPK and pACC were greater for shRNA-GS1 vs shRNA-Scr muscles. Muscle pyruvate dehydrogenase kinase-4 abundance from 9hPEX rats (both NRF and RF) was greater for shRNA-GS1 vs shRNA-Scr. Hexokinase II (HKII) abundance was greater for 9hPEX vs SED in NRF rats regardless of genotype. In RF-9hPEX rats, HKII abundance was greater for shRNA-GS1 vs shRNA-Scr. These results support the idea that some PEX effects on metabolic protein abundance are influenced by muscle GS1 abundance and the extent of glycogen resynthesis Disclosure S.Kwak: None. A.Zheng: None. E.B.Arias: None. H.Wang: None. X.Pan: None. D.Duan: None. Y.Yue: None. G.D.Cartee: None. Funding National Institutes of Health (R01DK071771); University of Michigan

The PI3K-Akt-mTOR pathway mediates renal pericyte-myofibroblast transition by enhancing glycolysis through HKII

BackgroundPericyte-myofibroblast transition (PMT) has been confirmed to contribute to renal fibrosis in several kidney diseases, and transforming growth factor-β1 (TGF-β1) is a well-known cytokine that drives PMT. However, the underlying mechanism has not been fully established, and little is known about the associated metabolic changes. MethodsBioinformatics analysis was used to identify transcriptomic changes during PMT. PDGFRβ + pericytes were isolated using MACS, and an in vitro model of PMT was induced by 5 ng/ml TGF-β1. Metabolites were analyzed by ultraperformance liquid chromatography (UPLC) and tandem mass spectrometry (MS). 2-Deoxyglucose (2-DG) was used to inhibit glycolysis via its actions on hexokinase (HK). The hexokinase II (HKII) plasmid was transfected into pericytes for HKII overexpression. LY294002 or rapamycin was used to inhibit the PI3K-Akt-mTOR pathway for mechanistic exploration.ResultsAn increase in carbon metabolism during PMT was detected through bioinformatics and metabolomics analysis. We first detected increased levels of glycolysis and HKII expression in pericytes after stimulation with TGF-β1 for 48 h, accompanied by increased expression of α-SMA, vimentin and desmin. Transdifferentiation was blunted when pericytes were pretreated with 2-DG, an inhibitor of glycolysis. The phosphorylation levels of PI3K, Akt and mTOR were elevated during PMT, and after inhibition of the PI3K-Akt-mTOR pathway with LY294002 or rapamycin, glycolysis in the TGF-β1-treated pericytes was decreased. Moreover, PMT and HKII transcription and activity were blunted, but the plasmid-mediated overexpression of HKII rescued PMT inhibition.ConclusionsThe expression and activity of HKII as well as the level of glycolysis were increased during PMT. Moreover, the PI3K-Akt-mTOR pathway regulates PMT by increasing glycolysis through HKII regulation.

Cannabidiol alters mitochondrial bioenergetics via VDAC1 and triggers cell death in hormone-refractory prostate cancer

In spite of the huge advancements in both diagnosis and interventions, hormone refractory prostate cancer (HRPC) remains a major hurdle in prostate cancer (PCa). Metabolic reprogramming plays a key role in PCa oncogenesis and resistance. However, the dynamics between metabolism and oncogenesis are not fully understood. Here, we demonstrate that two multi-target natural products, cannabidiol (CBD) and cannabigerol (CBG), suppress HRPC development in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model by reprogramming metabolic and oncogenic signaling. Mechanistically, CBD increases glycolytic capacity and inhibits oxidative phosphorylation in enzalutamide-resistant HRPC cells. This action of CBD originates from its effect on metabolic plasticity via modulation of VDAC1 and hexokinase II (HKII) coupling on the outer mitochondrial membrane, which leads to strong shifts of mitochondrial functions and oncogenic signaling pathways. The effect of CBG on enzalutamide-resistant HRPC cells was less pronounced than CBD and only partially attributable to its action on mitochondria. However, when optimally combined, these two cannabinoids exhibited strong anti-tumor effects in TRAMP mice, even when these had become refractory to enzalutamide, thus pointing to their therapeutical potential against PCa.

Expression of fructose-1,6-bisphosphatase 1 is associated with [18F]FDG uptake and prognosis in patients with mesial temporal lobe epilepsy.

To determine whether fructose-1,6-bisphosphatase 1 (FBP1) expression is associated with [18F]FDG PET uptake and postsurgical outcomes in patients with mesial temporal lobe epilepsy (mTLE) and to investigate whether the molecular mechanism involving gamma-aminobutyric acid type A receptor (GABAAR), glucose transporter-3 (GLUT-3), and hexokinase-II (HK-II). Forty-three patients with mTLE underwent [18F]FDG PET/CT. Patients were divided into Ia (Engel class Ia) and non-Ia (Engel class Ib-IV) groups according to more than 1year of follow-up after surgery. The maximum standard uptake value (SUVmax) and asymmetry index (AI) of hippocampus were measured. The relationship among the SUVmax, AI, prognosis, and FBP1 expression was analyzed. A lithium-pilocarpine acute mTLE rat model was subjected to [18F]FDG micro-PET/CT. Hippocampal SUVmax and FBP1, GABAAR, GLUT-3, and HK-II expression were analyzed. SUVmax was higher in the Ia group than in the non-Ia group (7.31 ± 0.97 vs. 6.56 ± 0.96, p < 0.05) and FBP1 expression was lower in the Ia group (0.24 ± 0.03 vs. 0.27 ± 0.03, p < 0.01). FBP1 expression was negatively associated with SUVmax and AI (p < 0.01). In mTLE rats, the hippocampal FBP1 increased (0.26 ± 0.00 vs. 0.17 ± 0.00, p < 0.0001), and SUVmax, GLUT-3 and GABAAR levels decreased significantly (0.73 ± 0.12 vs. 1.46 ± 0.23, 0.20 ± 0.01 vs. 0.32 ± 0.05, 0.26 ± 0.02 vs. 0.35 ± 0.02, p < 0.05); no significant difference in HK-II levels was observed. In mTLE patients and rats, FBP1 negatively correlated with SUVmax and GLUT-3 and GABAAR levels (p < 0.05). FBP1 expression was inversely associated with SUVmax in mTLE, which might inhibit [18F]FDG uptake by regulating GLUT-3 expression. High FBP1 expression was indicative of low GABAAR expression and poor prognosis. • It is of paramount importance to explore the deep pathophysiological mechanisms underlying the pathogenesis of mesial temporal lobe epilepsy and find potential therapeutic targets. • [18F]FDG PET has demonstrated low metabolism in epileptic regions during the interictal period, and hypometabolism may be associated with prognosis, but the pathomechanism of this association remains uncertain. • Our results support the possibility that FBP1 might be simultaneously involved in the regulation of glucose metabolism levels and the excitability of neurons and suggest that targeting FBP1 may be a viable strategy in the diagnosis and treatment of mesial temporal lobe epilepsy.

Enhanced Glycolysis Confers Resistance Against Photon but Not Carbon Ion Irradiation in Human Glioma Cell Lines.

Metabolic reprogramming is a key hallmark in various malignancies and poses a challenge in achieving success with various therapies. Enhanced glycolysis is known to confer resistance against photon irradiation while the tumor response to carbon ion irradiation (CII) has not been investigated. This study aimed to investigate the effects of enhanced glycolysis on the response of human glioma cell lines to CII compared to the response to X-rays. Glycolysis was stimulated using Dinitrophenol (DNP), a mild OXPHOS inhibitor, in three human glioma cell lines (U251, U87, and LN229) and assessed by monitoring glucose uptake and utilization as well as expression of regulators of glycolysis (glucose transporter protein type 1(Glut1), hexokinase-II (HKII), and Pyruvate Kinase-2 (PKM2). Radiation (X-rays and CII) induced loss of clonogenic survival growth inhibition and perturbations in cell cycle progression (G2+M block), cytogenetic damage (micronuclei formation), apoptosis, necrosis (reflecting interphase death), and cell migration (Scratch assay) were investigated as parameters of radiation response. DNP (1 mM) enhanced the expression levels of GLUT1, HKII, and PKM2 by 30-60% and glucose uptake as well as usage by nearly 3 folds in U251 cells suggesting the stimulation of glycolysis. Enhanced glycolysis attenuated the loss of clonogenic survival with D10 doses increasing by 20% to 65% in these cell lines, while no significant changes were noted following CII. Concomitantly, dose-dependent growth inhibition, and cytogenetic damage as well as apoptosis and necrosis induced by X-rays were also reduced by elevated glycolysis in U251 and LN229 cells by 20-50%. However, stimulation of glycolysis enhanced the X-ray-induced cell migration, while it had negligible effect on migration following CII. Our results suggest that enhanced glycolysis confers resistance against X-ray-induced cell death and migration, while it may not significantly alter the cellular responses to carbon ion irradiation.

Retraction for Huang et al., PEA15-HKII, stabilized by U2AF2, form a molecular switch governing cellular fate after spinal cord injury via MAPK pathway.

Spinal cord injury (SCI) is a devastating disease with poor prognosis. In this study, we sought to investigate the effects of proliferation and apoptosis adaptor protein 15 (PEA15) and hexokinase-Ⅱ (HKⅡ) on neuron cells after SCI. Firstly, we established the in vitro SCI model and cultured its neuron cells. Western blot detected the levels of autophagy- and apoptosis-related proteins in SCI neuron cells, and found high levels of LC3-Ⅱ, p62 and CASP12 in SCI neuron cells. Quantitative real time RT-PCR (RT-qPCR) showed the high levels of PEA15 and HKⅡ in SCI neuron cells. Functional experiments verified the positive regulation of PEA15 and HKⅡ on cell apoptosis. Furthermore, PEA15 and HKⅡ promote SCI neuron cell apoptosis by activating mitogen-activated protein kinase (MAPK) pathway. Co-immunoprecipitation (CoIP) and GST pull down assays showed that HKⅡ could interact with phosphorylated PEA15 in SCI neuron cells. Then the interaction of HKⅡ and PEA15-pSer116 was demonstrated to restrain cell apoptosis after SCI. Simultaneously, U2 small nuclear RNA auxiliary factor 2 (U2AF2) could stabilize the mRNA stability of PEA15 and HKⅡ. In summary, PEA15-HKII form a molecular switch to regulate the apoptosis of SCI neuron cells by regulating MAPK pathway, providing a new direction for SCI study.

Pepsin enhances glycolysis to promote malignant transformation of vocal fold leukoplakia epithelial cells with dysplasia

PurposeThe mechanism underlying malignant transformation of vocal fold leukoplakia (VFL) and the precise role of the expression of pepsin in VFL remain unclear. This study aimed to investigate the effects of acidified pepsin on VFL epithelial cell growth and migration, and also identify pertinent molecular mechanisms.MethodsImmunochemistry and Western blotting were performed to measure glucose transporter type 1 (GLUT1), monocarboxylate transporters 4 (MCT4), and Hexokinase-II (HK-II) expressions. Cell viability, cell cycle, apoptosis, and migration were investigated by CCK-8 assay, flow cytometry and Transwell chamber assay, respectively. Glycolysis-related contents were determined using the corresponding kits. Mitochondrial HK-II was photographed under a confocal microscope using Mito-Tracker Red.ResultsIt was found: the expression of pepsin and proportion of pepsin+ cells in VFL increased with the increased dysplasia grade; acidified pepsin enhanced cell growth and migration capabilities of VFL epithelial cells, reduced mitochondrial respiratory chain complex I activity and oxidative phosphorylation, and enhanced aerobic glycolysis and GLUT1 expression in VFL epithelial cells; along with the transfection of GLUT1 overexpression plasmid, 18FFDG uptake, lactate secretion and growth and migration capabilities of VFL epithelial cell were increased; this effect was partially blocked by the glycolysis inhibitor 2-deoxy-glucose; acidified pepsin increased the expression of HK-II and enhanced its distribution in mitochondria of VFL epithelial cells.ConclusionIt was concluded that acidified pepsin enhances VFL epithelial cell growth and migration abilities by reducing mitochondrial respiratory complex I activity and promoting metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis.

Virtual Screening and Biological Activity Evaluation of New Potent Inhibitors Targeting Hexokinase-II.

Hexokinase-II (HK-II), the rate-limiting step enzyme in the glycolysis pathway, expresses high levels of cancer cells compared with normal cells. Due to its pivotal role in the different aspects of cancer physiology including cellular proliferation, metastasis, and apoptosis, HK-II provides a new therapeutic target for cancer therapy. The structure-based virtual screening targeting HK-II was used to hit identifications from small molecule databases, and the select compounds were further evaluated in biological assays. Forty-seven compounds with the lowest binding energies were identified as potential HK-II inhibitors. Among them, nine compounds displayed the highest cytotoxicity to three different cancer cells. Based on the mechanism study, compounds 4244-3659 and K611-0094 showed an obvious inhibitory effect on the HK-II enzyme. This study identified two potential inhibitors of HK-II and can be helpful for developing potential drugs targeting HK-II in tumor therapy.

Aspirin blocks AMPK/SIRT3-mediated glycolysis to inhibit NSCLC cell proliferation

Non-small cell lung cancer (NSCLC) has the highest incidence and mortality in the world. Aspirin has been reported to promote apoptosis, inhibit proliferation, stemness, angiogenesis, cancer-associated inflammation and migration in NSCLC. But the effect of aspirin on aerobic glycolysis in NSCLC is less reported. In the present study, we investigated whether aspirin blocked aerobic glycolysis of NSCLC cells to inhibit proliferation. Our results showed that aspirin inhibited viability, PCNA expression, ability of colony formation, dimished extracellular acidification rate (ECAR), oxygen consumption rate (OCR) and production of pyruvic acid and lactic acid, accompanied with reduced mitochondrial membrane potential (MMP), PGC-1α expression and ROS production, indicating mitochondrial dysfunction in NSCLC cells. AMPK and mitochondrial-localized deacetylase sirtuin 3 (SIRT3) were identified as the relevant molecular targets in glycolysis, but mechanism and relationship between AMPK and SIRT3 for aspirin induced glycolysis inhibition remain unknown in cancer cells. The investigation of underlying mechanism indicated that aspirin activated AMPK pathway to inhibit aerobic glycolysis and proliferation by upregulating SIRT3 after application of compound C (CC), an inhibitor of AMPK activity or SIRT3 siRNA. Upon activation of SIRT3, aspirin promoted the release of hexokinase-II (HK-II) from mitochondrial outer membrane to cytosol by deacetylating cyclophilin D (CypD). Consistently, aspirin significantly inhibited the growth of NSCLC xenografts and exhibited antitumor activity probably through AMPK/SIRT3/HK-II pathway in vivo. Collectively, AMPK/SIRT3/HK-II pathway plays a critical role in anticancer effects of aspirin, and our findings might serve as potential target for clinical practice and chemoprevention of aspirin in NSCLC.

Hypothermia Prevents Cardiac Dysfunction during Acute Ischemia Reperfusion by Maintaining Mitochondrial Bioenergetics and by Promoting Hexokinase II Binding to Mitochondria.

Background Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨm), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.

Phosphodiesterase 5 inhibitor sildenafil potentiates the antitumor activity of cisplatin by ROS-mediated apoptosis: a role of deregulated glucose metabolism.

Cyclic nucleotide phosphodiesterase 5 (PDE5) has been recently identified to play a crucial role in the progression of many cancers. PDE5 promotes tumorigenesis by dysregulating various cellular processes such as proliferation, apoptosis, angiogenesis, and invasion and migration. Interestingly, multiple studies have reported the promising chemosensitizing potential of PDE5 inhibitor sildenafil in breast, colon, prostate, glioma, and lung cancers. However, to date, the chemosensitizing action of sildenafil is not evaluated in T cell lymphoma, a rare and challenging neoplastic disorder. Hence, the present investigation was undertaken to examine the chemosensitizing potential of sildenafil against T cell lymphoma along with elucidation of possible involvement of altered apoptosis and glucose metabolism. The experimental findings of this study showed that sildenafil enhances the cytotoxic ability of cisplatin by apoptosis induction through altering the levels of apoptosis regulatory molecules: Bcl-2, Bax, cytochrome c (Cyt c), cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP). These molecular alterations were possibly driven by sildenafil through reactive oxygen species (ROS). Sildenafil deregulates glucose metabolism by markedly lowering the expression of glycolysis regulatory molecules, namely glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), hexokinase II (HKII), pyruvate kinase M2 (PKM2), and pyruvate dehydrogenase kinase 1 (PDK1) via suppressing hypoxia-inducible factor 1-alpha (HIF-1α) expression. Hence, sildenafil potentiates the tumor cell killing ability of cisplatin by augmenting ROS production through switching the glucose metabolism from glycolysis to oxidative phosphorylation (OXPHOS). Overall, our study demonstrates that sildenafil might be a promising adjunct therapeutic candidate in designing novel combinatorial chemotherapeutic regimens against T cell lymphoma.

Abstract 2179: Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma

Abstract Background: BCAT1 (Branched Chain Amino Transferase 1) has been shown to be upregulated in human IDH (isocitrate dehydrogenase) wild-type glioblastoma (GBM) and BCAT1 knockdown leads to reduced cell proliferation in vitro and in vivo1. Here we investigated the role of BCAT1 in human patient-derived glioblastoma cells and the corresponding orthotopically implanted xenografts to understand how upregulation of the enzyme leads to a more aggressive phenotype. Methods: The expression of BCAT1 was investigated in a panel of patient derived IDH wild-type glioblastoma cells and BCAT1 expression modulation was achieved using lentiviral expression of a doxycycline-inducible shRNA, to knockdown BCAT1 and expression of a BCAT1 cDNA to overexpress BCAT1. Effects of BCAT1 knockdown and overexpression were investigated in vitro in cell culture and in vivo, following intracranial implantation of mice with the relevant cells. Results: Variable levels of BCAT1 expression were identified in different patient-derived cell lines. RNA sequencing analysis of BCAT1 knockdown in BCAT1 expressing glioblastoma cells revealed that knockdown led to significant downregulation of HIF 1α (Hypoxia Inducible Factor 1α) and its targets, which was confirmed by Western Blot analysis of Carbonic Anhydrase IX (CAIX) and Hexokinase II (HKII) in cell and tumor lysates. The opposite was observed following BCAT1 overexpression. BCAT1 knockdown also led to higher DNA demethylation levels. Treatment with membrane permeant dimethyl-alpha ketoglutarate mimicked the effect of BCAT1 knockdown. These data suggest that decreased cell proliferation following BCAT1 knockdown in BCAT1 expressing cells is mediated by accumulation of alpha-ketoglutarate, a substrate of BCAT1. Cells that had no BCAT1 expression showed a similar growth profile and overexpression had no effect on proliferation, suggesting that these cells are not dependent on BCAT1 expression. Conclusion: We conclude that in a subtype of GBM characterized by upregulated BCAT1, BCAT1 expression results in lowered levels of alpha-ketoglutarate, leading to reduced prolyl hydroxylase activity and stabilization of HIF 1α and therefore a pseudo-hypoxic state, resulting in a transcriptome that favors a high proliferation rate. This makes BCAT1 a potential therapeutic target, for tumors that show BCAT1 upregulation.1. Tönjes M, Barbus S, Park YJ, et al. BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1. Nat Med. 2013;19(7):901-908. doi:10.1038/nm.3217 Citation Format: Maria Fala, Susana Ros, Ashley Sawle, Kevin M. Brindle. Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma [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 2179.

Potential of Novel Methyl Jasmonate Analogs as Anticancer Agents to Metabolically Target HK-2 Activity in Glioblastoma Cells.

Change in the energy metabolism of cancer cells, which display significant differences compared to normal cells, is a rising phenomenon in developing new therapeutic approaches against cancers. One of the metabolic enzymes, hexokinase-II (HK-II) is involved in glycolysis, and inhibiting the HK-II activity may be a potential metabolic target for cancer therapy as most of the drugs in clinical use act on DNA damage. Methyl jasmonate (MJ) is one of the compounds blocking HK-II activity in cancer cells. In a previous study, we showed that the novel MJ analogs inhibit HK-II activity through VDAC detachment from the mitochondria. In this study, to evaluate the potential of targeting HK-2 activity, through patient cohort analysis, we first determined HK-2 expression levels and prognostic significance in highly lethal glioblastoma (GBM) brain tumor. We then examined the in vitro therapeutic effects of the novel analogs in the GBM cells. Here, we report that, among all, compound-10 (C-10) showed significant in vitro therapeutic efficacy as compared to MJ which is in use for preclinical and clinical studies. Afterward, we analyzed cell death triggered by C-10 in two different GBM cell lines. We found that C-10 treatment increased the apoptotic/necrotic cells and autophagy in GBM cells. The newly developed analog, C-10, was found to be lethal against GBM by the activation of cell death authorities, mostly in a necrotic and autophagic fashion at the early stages of the treatment. Considering that possibly decreased intracellular ATP levels by C-10 mediated inhibition of HK-2 activity and disabled VDAC interaction, a more detailed analysis of HK-2 inhibition–mediated cell death can provide a deep understanding of the mechanism of action on the oncosis/necroptosis axis. These findings provide an option to design clinically relevant and effective novel HK-II inhibitors and suggest novel MJ analogs to further study them as potential anticancer agents against GBM.