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.

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.

Long non-coding RNA HANR modulates the glucose metabolism of triple negative breast cancer via stabilizing hexokinase 2.

Increasing evidence has demonstrated the oncogenic roles of long non-coding RNA (lncRNA) hepatocellular carcinoma (HCC)-associated long non-coding RNA (HANR) in the development of HCC and lung cancer; however, the involvement of HANR in triple-negative breast cancer (TNBC) remains largely unknown. Our results demonstrated the significant overexpression of HANR in TNBC tissues and cells. Higher HANR levels significantly correlated with the poorer phenotypes in patients with TNBC. HANR down-regulation inhibited the proliferation and cell cycle progression and increased the apoptosis of TNBC cells. Mechanistically, immunoprecipitation-mass spectrometry revealed hexokinase II (HK2) as a direct binding target of HANR. HANR binds to and stabilizes HK2 through the proteasomal pathway. Consistent with the important role of HK2 in cancer cells, HANR depletion represses the glucose absorbance and lactate secretion, thus reprogramming the metabolism of TNBC cells. An in vivo xenograft model also demonstrated that HANR promoted tumor growth and aerobic glycolysis. This study reveals the role of HANR in modulating the glycolysis in TNBC cells by regulating HK2 stability, suggesting that HANR is a potential drug target for TNBC.

SMAD4 inhibits glycolysis in ovarian cancer through PI3K/AKT/HK2 signaling pathway by activating ARHGAP10.

BACKGROUND: ARHGAP10 is a tumor-suppressor gene related to ovarian cancer (OC) progression; however, its specific mechanism is unclear. AIMS: To investigate the effect of ARHGAP10 on OC cell migration, invasion, and glycolysis. METHODS AND RESULTS: Quantitative real-time PCR (qRT-PCR) quantified mRNA and protein expressions of AKT, p-AKT, HK2, and SMAD4 were tested by Western blot. EdU, Wound healing, and Transwell assay were utilized to evaluate OC cell proliferation, migration, and invasion. We used a Seahorse XF24 Extracellular Flux Analyzer to monitor cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Chromatin immunoprecipitation (ChIP) was used to analyze the transcriptional regulation of ARHGAP10 by SMAD4. ARHGAP10 expression in OC tissues was detected by immunohistochemistry. Our results showed that ARHGAP10 expression was negatively related to lactate levels in human OC tissues. ARHGAP10 overexpression can inhibit the migration, proliferation, and invasion of OC cells, and this function can be blocked by 2-Deoxy-D-glucose. Moreover, we found that ARHGAP10 expression can be rescued with the AKT inhibitor LY294002. CONCLUSIONS: This study revealed that the antitumor effects of ARHGAP10 in vivo and in vitro possibly suppress oncogenic glycolysis through the PI3K/AKT/HK2-regulated glycolysis metabolism pathway.

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.

ATF4 knockdown in macrophage impairs glycolysis and mediates immune tolerance by targeting HK2 and HIF-1α ubiquitination in sepsis.

Strengthened glycolysis is crucial for the macrophage pro-inflammatory response during sepsis. Activating transcription factor 4 (ATF4) plays an important role in regulating glucose and lipid metabolic homeostasis in hepatocytes and adipocytes. However, its immunometabolic role in macrophage during sepsis remains largely unknown. In the present study, we found that the expression of ATF4 in peripheral blood mononuclear cells (PBMCs) was increased and associated with glucose metabolism in septic patients. Atf4 knockdown specifically decreased LPS-induced spleen macrophages and serum pro-inflammatory cytokines levels in mice. Moreover, Atf4 knockdown partially blocked LPS-induced pro-inflammatory cytokines, lactate accumulation and glycolytic capacity in RAW264.7. Mechanically, ATF4 binds to the promoter region of hexokinase II (HK2), and interacts with hypoxia inducible factor-1α (HIF-1α) and stabilizes HIF-1α through ubiquitination modification in response to LPS. Furthermore, ATF4-HIF-1α-HK2-glycolysis axis launches pro-inflammatory response in macrophage depending on the activation of mammalian target of rapamycin (mTOR). Importantly, Atf4 overexpression improves the decreased level of pro-inflammatory cytokines and lactate secretion and HK2 expression in LPS-induced tolerant macrophages. In conclusion, we propose a novel function of ATF4 as a crucial glycolytic activator contributing to pro-inflammatory response and improving immune tolerant in macrophage involved in sepsis. So, ATF4 could be a potential new target for immunotherapy of sepsis.

High­risk pathological subtype associated FAM83A­AS1 promotes malignancy and glycolysis of lung adenocarcinoma via miR­202­3p/HK2 axis.

According to the diverse cellular morphology, lung adenocarcinoma (LUAD) was classified into five pathological subtypes, referred to as follows: High­risk group (micropapillary and solid), intermediate­risk group (acinar and papillary) and low­risk group (epidic). Nevertheless, little is known about the biological function of long non­coding RNA (lncRNA) in the molecular determination of LUAD histologic patterns. Screening the transcriptional expression data from TCGA­LUAD, the differentially expressed lncRNA across the divergent pathological subtypes were explored. Pan­cancer analysis revealed the characteristic of FAM83A­AS1, which was also confirmed in the LUAD tissues. The function of FAM83A­AS1 was uncovered through the in vitro assays. RNA immunoprecipitation and dual­luciferase reporter assays were performed to explore the molecular mechanisms of FAM83A­AS1. In the present study, it was identified that the expression of FAM83A­AS1 was increased from the low­risk group to the high, which was associated with a poorer prognosis and higher risk of recurrence. Pan­cancer analysis revealed that FAM83A­AS1 was positively correlated with high tumor mutational burden. Additionally, FAM83A­AS1 promoted cell migration, invasion and growth of LUAD cancer cells. Mechanistically, FAM83A­AS1 sponged miR­202­3p to regulate the expression of hexokinase II (HK2) in post­transcription, which facilitated the malignancy and glycolysis. The present study uncovered the biological roles of FAM83A­AS1/miR­202­3p/HK2 axis in regulating malignancy and glycolysis of LUAD, which provided novel avenues to addressing the determination of histologic patterns.

microRNA-143 interferes the EGFR-stimulated glucose metabolism to re-sensitize 5-FU resistant colon cancer cells via targeting hexokinase 2.

5-Fluorouracil (5-FU) is one of the frequently used chemotherapeutic agents against colorectal cancer (CRC). However, 5-FU treatment remains clinical challenges since a large fraction of patients with CRC developed resistance to 5-FU-based chemotherapies. Hexokinase 2 (HK II), coding for a rate-limiting enzyme of glutamine metabolism, is responsible for the dysregulated glycolysis of cancers. In this study, we report epidermal growth factor receptor (EGFR) and HK II were overexpressed in colon cancers and positively correlated with 5-FU resistance of CRC. In addition, expression of miR-143 was remarkedly suppressed in 5-FU resistant CRC patients and colon cancer cells. Moreover, miR-143 expression was effectively downregulated by EGFR and inversely associated with HK II expression in CRC cells. We identified HK II as a direct target of miR-143 in colon cancer cells. Overexpression of miR-143 inhibited glycolysis rate through direct targeting HK II, leading to re-sensitization of 5-FU resistant colon cancer cells to 5-FU treatment. Rescue experiments validated that recovering HK II in miR-143-overexpressing cells restored 5-FU resistance of CRC cells. In general, our study reveals critical roles of miR-143, which is a downstream effector of EGFR in 5-FU resistant CRC cells through direct targeting HK II, indicating miR-143 is an effectively therapeutic target for the treatment of patients with chemoresistant CRC.

The Promoting Role of HK II in Tumor Development and the Research Progress of Its Inhibitors.

Increased glycolysis is a key characteristic of malignant cells that contributes to their high proliferation rates and ability to develop drug resistance. The glycolysis rate-limiting enzyme hexokinase II (HK II) is overexpressed in most tumor cells and significantly affects tumor development. This paper examines the structure of HK II and the specific biological factors that influence its role in tumor development, as well as the potential of HK II inhibitors in antitumor therapy. Furthermore, we identify and discuss the inhibitors of HK II that have been reported in the literature.

3-Bromopyruvate inhibits pancreatic tumor growth by stalling glycolysis, and dismantling mitochondria in a syngeneic mouse model.

Pancreatic cancer (PC) is the fourth-most-deadly cancer in the United States with a 5-year survival rate of only 8%. The majority of patients with locally advanced pancreatic cancer undergo chemotherapy and/or radiation therapy (RT). However, current treatments are inadequate and novel strategies are desperately required. 3-Bromopyruvate (3-BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme (HK2) of the glycolytic pathway in cancer cells while not affecting the normal cells. 3-BP killed 95% of Panc-2 cells at 15 µM concentration and severely inhibited ATP production by disrupting the interaction between HK2 and mitochondrial Voltage Dependent Anion Channel-1 (VDAC1) protein. Electron microscopy data revealed that 3-BP severely damaged mitochondrial membrane in cancer cells. We further examined therapeutic effect of 3-BP in syngeneic mouse pancreatic cancer model by treating animals with 10, 15 and 20 mg/kg dose. 3-BP at 15 & 20 mg/kg dose level significantly reduced tumor growth by approximately 75-80% in C57BL/6 female mice. Immunohistochemistry data showed complete inhibition of hexokinase II (HK2) and TGFß, in animals treated with 3-BP drug. We also observed enhanced expression of active caspase-3 in tumor tissues exhibited apoptotic death. Flow Cytometry analysis showed significant inhibition in MDSC (CD11b) population in treated tumor which may have allowed infiltration of CD8+ T cells and inhibited tumor growth. Notably, metabolomic data also revealed severe inhibition in glycolysis, NADP, ATP and lactic acid production in cancer cells treated with 40 µM 3-BP. Importantly, we also observed inhibition in lactic acid production responsible for tumor aggression. These results provide new evidence that 3-BP severely inhibit glucose metabolism in cancer cells by blocking hexokinase II, and disrupting mitochondria by suppressing BCL2L1 in pancreatic cancer.

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.

Detachment of Hexokinase II From Mitochondria Promotes Collateral Sensitivity in Multidrug Resistant Chronic Myeloid Leukemia Cells.

Chronic Myeloid Leukemia is a neoplastic disease characterized by the abnormal expansion of hematopoietic cells with compromised functions. Leukemic cells often display a multidrug resistance phenotype, enabling them to evade a number of structurally unrelated cytotoxic compounds. One of those mechanisms relies on the high expression of efflux transporters, such as the ABC proteins, whose activity depends on the hydrolysis of ATP to reduce intracellular drug accumulation. In the present work, we employed a well-known erythroleukemia cell line, K562, and a multidrug resistant derivative cell, FEPS, to evaluate how hexokinase II, a key regulator for the rate-limiting step glycolysis, contributes to the establishment of the multidrug resistance phenotype. We found that multidrug resistant cells primarily resort to glycolysis to generate ATP. Clotrimazole reduced the expression of mitochondrial hexokinase II, which destabilized bioenergetic parameters such as reactive oxygen species production, ATP, and glutathione levels on multidrug resistant cells. This impaired the activity of ABCC1, leading to increased drug accumulation and cell death. In summary, we propose that decoupling of hexokinase II from the mitochondria emerges as a promising strategy to generate collateral sensitivity and aid in the management of chronic myeloid leukemia in chemotherapy-refractory patients.

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.

Targeted Inhibition of HK-II Reversed the Warburg Effect to Improve the Radiosensitivity of Laryngeal Carcinoma.

PURPOSE: Hexokinase-II (HK-II) is the key enzyme in the first rate-limiting step of glycolysis that catalyzes the conversion of glucose to glucose-6-phosphate. Here, we examined the association between HK-II expression and radioresistance in laryngeal carcinoma and whether the inhibition of HK-II expression can enhance the radiosensitivity of these tumors. METHODS: The effects of HK-II small interfering RNA (siRNA) on the radiosensitivity of Tu212 cells were examined in vitro and in vivo in a mouse model. Cells were irradiated using a 6-MV linear accelerator. The cell viability, cell survival, proliferation, apoptosis, and cell cycle of Tu212 cells were evaluated using trypan blue staining, colony formation assays, CCK-8 assays, and flow cytometry, respectively. Oxygen consumption, lactic acid production, glucose consumption, and the ATP level of Tu212 cells were also examined. The expression of glycolytic and regulatory enzymes involved in the tricarboxylic acid cycle was assessed using Western blotting. RESULTS: The HK-II siRNA and X-ray combination treatment led to a significantly greater reduction of cell viability, inhibition of cell survival and proliferation, increased apoptosis, and increased G2 phase arrest compared to either treatment alone (all, P<0.01). HK-II siRNA increased the oxygen consumption rate of cells, significantly inhibited lactic acid production and glucose consumption, and significantly suppressed the upregulation of HK-II, pyruvate kinase M2 (PKM2), pyruvate dehydrogenase (PDH), phosphofructokinase platelet (PFKP), lactate dehydrogenase (LD), and citrate synthase (CS) (all, P<0.01). CONCLUSION: The inhibition of HK-II by siRNA enhances the radiosensitivity of laryngeal carcinoma Tu212 cells by inhibiting glycolysis and partially inhibiting oxidative phosphorylation.

Molecular docking and simulation studies of phytocompounds derived from Centella asiatica and Andrographis paniculata against hexokinase II as mitocan agents.

Hexokinase II (HK2), a glycolytic enzyme is commonly overexpressed in most cancer types. The overexpression of HK2 is reported to promote the survival of cancer cells by facilitating the constant ATP generation and protecting the cancer cell against apoptotic cell death. Hence, HK2 is considered as potential target of many mitochondria targeting anticancerous agents (referred to as mitocans). Most of the existing mitocans are synthetic and hence such compounds are observed to exhibit adverse effects, witnessed through many experimental outcomes. These limitations necessitates hunting for an alternative source of mitocans with minimum/no side effects. The need for an alternative therapy points towards the ethnomedicinal herbs, known for their minimal side effects and effectiveness. Henceforth recent studies have put forth the effort to utilize anticancer herbs in formulating naturally derived mitocans as an add-on to improve cancer therapeutics. So, our study aims to explore the HK2 targeting potential of phytocompounds from the selected anticancerous herbs Andrographis paniculata (AP) and Centella asiatica (CA). 60 phytocompounds collectively from CA and AP were docked against HK2 and drug-likeness prediction of the selected phytocompounds was performed to screen the best possible ligand for HK2. Furthermore, the docked complexes were subjected to molecular dynamics simulations (MDS) to analyse the molecular mechanism of protein-ligand interactions. The results of the study suggest that the natural compounds asiatic acid and bayogenin (from CA) and andrographolide (from AP) can bepotential natural mitocans by targeting HK2. Further experimental studies (in-vitro and in-vivo) are required to validate the results.

Nuclear HKII-P-p53 (Ser15) Interaction is a Prognostic Biomarker for Chemoresponsiveness and Glycolytic Regulation in Epithelial Ovarian Cancer.

In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

Preconditioning or Postconditioning with 8-Br-cAMP-AM Protects the Heart against Regional Ischemia and Reperfusion: A Role for Mitochondrial Permeability Transition.

The cAMP analogue 8-Br-cAMP-AM (8-Br) confers marked protection against global ischaemia/reperfusion of isolated perfused heart. We tested the hypothesis that 8-Br is also protective under clinically relevant conditions (regional ischaemia) when applied either before ischemia or at the beginning of reperfusion, and this effect is associated with the mitochondrial permeability transition pore (MPTP). 8-Br (10 µM) was administered to Langendorff-perfused rat hearts for 5 min either before or at the end of 30 min regional ischaemia. Ca2+-induced mitochondria swelling (a measure of MPTP opening) and binding of hexokinase II (HKII) to mitochondria were assessed following the drug treatment at preischaemia. Haemodynamic function and ventricular arrhythmias were monitored during ischaemia and 2 h reperfusion. Infarct size was evaluated at the end of reperfusion. 8-Br administered before ischaemia attenuated ventricular arrhythmias, improved haemodynamic function, and reduced infarct size during ischaemia/reperfusion. Application of 8-Br at the end of ischaemia protected the heart during reperfusion. 8-Br promoted binding of HKII to the mitochondria and reduced Ca2+-induced mitochondria swelling. Thus, 8-Br protects the heart when administered before regional ischaemia or at the beginning of reperfusion. This effect is associated with inhibition of MPTP via binding of HKII to mitochondria, which may underlie the protective mechanism.

Neuronal Pentraxin 1 Promotes Hypoxic-Ischemic Neuronal Injury by Impairing Mitochondrial Biogenesis via Interactions With Active Bax[6A7] and Mitochondrial Hexokinase II.

Mitochondrial dysfunction is a key mechanism of cell death in hypoxic-ischemic brain injury. Neuronal pentraxin 1 (NP1) has been shown to play crucial roles in mitochondria-mediated neuronal death. However, the underlying mechanism(s) of NP1-induced mitochondrial dysfunction in hypoxia-ischemia (HI) remains obscure. Here, we report that NP1 induction following HI and its subsequent localization to mitochondria, leads to disruption of key regulatory proteins for mitochondrial biogenesis. Brain mitochondrial DNA (mtDNA) content and mtDNA-encoded subunit I of complex IV (mtCOX-1) expression was increased post-HI, but not the nuclear DNA-encoded subunit of complex II (nSDH-A). Up-regulation of mitochondrial proteins COXIV and HSP60 further supported enhanced mtDNA function. NP1 interaction with active Bax (Bax6A7) was increased in the brain after HI and in oxygen-glucose deprivation (OGD)-induced neuronal cultures. Importantly, NP1 colocalized with mitochondrial hexokinase II (mtHKII) following OGD leading to HKII dissociation from mitochondria. Knockdown of NP1 or SB216763, a GSK-3 inhibitor, prevented OGD-induced mtHKII dissociation and cellular ATP decrease. NP1 also modulated the expression of mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), regulators of mitochondrial biogenesis, following HI. Together, we reveal crucial roles of NP1 in mitochondrial biogenesis involving interactions with Bax[6A7] and mtHKII in HI brain injury.

Mesenchymal Stromal Cells Inhibit Aerobic Glycolysis in Activated T Cells by Negatively Regulating Hexokinase II Activity Through PD-1/PD-L1 Interaction.

Mesenchymal stromal cells (MSCs) have been shown to inhibit aerobic glycolysis in activated T cells, leading to increased autophagy. Although tryptophan depletion induced by indoleamine 2,3-dioxygenase (IDO) generated by MSCs has been suggested as a potential mechanism, we found that this inhibition was completely abolished when T cells were physically separated from MSCs using the Transwell system. Instead, in the current study, we demonstrate that programmed cell death 1 receptor (PD-1) and its ligand PD-L1, the expression of which is induced on activated T cells and MSCs, respectively, in response to IFN-γ are involved in this inhibition. Blockade of PD-1/PD-L1 interaction by blocking antibodies significantly restored glucose uptake, glycolytic activity, and cluster formation of activated T cells, whereas a specific inhibitor of IDO, 1-methyl-DL-tryptophan, had no effect. Neither surface nor cytoplasmic glucose transporter-1 expression on T cells was changed by MSCs. In addition, glycolytic gene expression in activated T cells was not inhibited despite the presence of MSCs. However, we found that hexokinase II (HK2) protein expression was markedly decreased in activated T cells that had been cocultured with MSCs. PD-1 blocking antibody restored HK2 expression. Taken together, our findings indicate that the PD-1/PD-L1 axis is involved in the MSC-mediated suppression of T cell glycolysis by negatively regulating HK2 activity at the protein level, but not at the mRNA level.

Knockout of VDAC1 in H9c2 Cells Promotes Oxidative Stress-Induced Cell Apoptosis through Decreased Mitochondrial Hexokinase II Binding and Enhanced Glycolytic Stress.

BACKGROUND/AIMS: The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1-/-) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1-/- in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells. METHODS: We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1-/- H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroperoxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer. RESULTS: We found that under control conditions, VDAC1-/- did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1-/- H9c2 cells. VDAC1-/- H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1-/- H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1-/- and WT H9c2 cells. CONCLUSION: Our results suggest that VDAC1-/- in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.