Acesulfame potassium upregulates PD-L1 in HCC cells by attenuating autophagic degradation.

Artificial sweeteners, which contain no or few calories, have been widely used in various foods and beverages, and are regarded as safe alternatives to sugar by the Food and Drug Administration. While several studies suggest that artificial sweeteners are not related to cancer development, some research has reported their potential association with the risk of cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether acesulfame potassium (Ace K), a commonly used artificial sweetener, induces immune evasion of HCC cells by upregulating programmed death ligand-1 (PD-L1). Ace K elevated the protein levels of PD-L1 in HCC cells without increasing its mRNA levels. The upregulation of PD-L1 protein levels in HCC cells by Ace K was induced by attenuated autophagic degradation of PD-L1, which was mediated by the Ace K-stimulated ERK1/2-mTORC1 signaling pathway. Ace K-induced upregulation of PD-L1 attenuated T cell-mediated death of HCC cells, thereby promoting immune evasion of HCC cells. In summary, the present study suggests that Ace K promotes HCC progression by upregulating the PD-L1 protein level.

Glial cell proteome using targeted quantitative methods for potential multi-diagnostic biomarkers.

Glioblastoma is one of the most malignant primary brain cancer. Despite surgical resection with modern technology followed by chemo-radiation therapy with temozolomide, resistance to the treatment and recurrence is common due to its aggressive and infiltrating nature of the tumor with high proliferation index. The median survival time of the patients with glioblastomas is less than 15 months. Till now there has been no report of molecular target specific for glioblastomas. Early diagnosis and development of molecular target specific for glioblastomas are essential for longer survival of the patients with glioblastomas. Development of biomarkers specific for glioblastomas is most important for early diagnosis, estimation of the prognosis, and molecular target therapy of glioblastomas. To that end, in this study, we have conducted a comprehensive proteome study using primary cells and tissues from patients with glioblastoma. In the discovery stage, we have identified 7429 glioblastoma-specific proteins, where 476 proteins were quantitated using Tandem Mass Tag (TMT) method; 228 and 248 proteins showed up and down-regulated pattern, respectively. In the validation stage (20 selected target proteins), we developed quantitative targeted method (MRM: Multiple reaction monitoring) using stable isotope standards (SIS) peptide. In this study, five proteins (CCT3, PCMT1, TKT, TOMM34, UBA1) showed the significantly different protein levels (t-test: p value ≤ 0.05, AUC ≥ 0.7) between control and cancer groups and the result of multiplex assay using logistic regression showed the 5-marker panel showed better sensitivity (0.80 and 0.90), specificity (0.92 and 1.00), error rate (10 and 2%), and AUC value (0.94 and 0.98) than the best single marker (TOMM34) in primary cells and tissues, respectively. Although we acknowledge that the model requires further validation in a large sample size, the 5 protein marker panel can be used as baseline data for the discovery of novel biomarkers of the glioblastoma.

SGLT2 inhibitors prevent LPS-induced M1 macrophage polarization and alleviate inflammatory bowel disease by downregulating NHE1 expression.

BACKGROUND: Classically activated M1 macrophages, characterized by aberrant glycolysis and secretion of inflammatory cytokines, play pivotal roles in inflammatory diseases, including inflammatory bowel disease (IBD). Recently, sodium-glucose co-transporter 2 (SGLT2) inhibitors were shown to suppress Na+/H+ exchanger 1 (NHE1) and Na+/Ca2+ exchanger 1 (NCX1) activity, regulating downstream intracellular Ca2+ concentrations in cardiomyocytes. However, whether SGLT2 inhibitors regulate M1 macrophage polarization by downregulating NHE1 and NCX1 remains unknown. METHODS: We analyzed cellular responses to SGLT2 inhibitors using mouse bone marrow-derived macrophages and peritoneal macrophages treated with lipopolysaccharide (LPS). To induce IBD, we used a dextran sulfate sodium salt-induced colitis mouse model. RESULTS: We observed that NHE1 and NCX1 were overexpressed in LPS-treated macrophages, leading to M1 macrophage polarization. Mechanistically, NHE1 and NCX1-mediated Ca2+ accumulation in the macrophage resulted in enhanced glycolysis by promoting PI3K/AKT/mTORC1 signaling. SGLT2 inhibitors suppressed both the expression levels and activities of NHE1 and NCX1, and consequently downregulated PI3K/AKT/mTORC1 signaling and glycolysis in LPS-treated macrophages. We observed inhibition of LPS-stimulated M1 polarization and cytokine production by SGLT2 inhibitors in vitro, ex vivo, and in an IBD mouse model. CONCLUSIONS: NHE1 promotes M1 macrophage polarization and SGLT2 inhibitors are a novel strategy to treat M1 macrophage-mediated inflammatory diseases, including IBD.

Cassiaside C Inhibits M1 Polarization of Macrophages by Downregulating Glycolysis.

Classically activated M1 macrophages reprogram their metabolism towards enhanced glycolysis to obtain energy and produce pro-inflammatory cytokines after activation by mammalian target of rapamycin complex 1 (mTORC1) and hypoxia-inducible factor (HIF)-1α. Thus, a strategy that constrains M1 polarization of macrophages via downregulation of glycolysis is essential for treating chronic inflammatory diseases. Cassiae semen has pharmacological activity against various inflammatory diseases. However, it is unclear whether specific compounds within Cassia seeds affect M1 polarization of macrophages. Here, we investigated whether Cassiaside C napthopyrone from Cassiae semen inhibits M1 polarization by downregulating glycolysis. We found that Cassiaside C reduced expression of inducible nitric oxide synthase and cyclooxygenase-2 and the phosphorylation of nuclear factor kappa B, all of which are upregulated in lipopolysaccharide (LPS)/interferon (IFN)-γ-treated Raw264.7 cells and peritoneal macrophages. Moreover, Cassiaside C-treated macrophages showed marked suppression of LPS/IFN-γ-induced HIF-1α, pyruvate dehydrogenase kinase 1, and lactate dehydrogenase A expression, along with downregulation of the phosphoinositide 3-kinases (PI3K)/AKT/mTORC1 signaling pathway. Consequently, Cassiaside C attenuated enhanced glycolysis and lactate production, but rescued diminished oxidative phosphorylation, in M1 polarized macrophages. Thus, Cassiaside C dampens M1 polarization of macrophages by downregulating glycolysis, which could be exploited as a therapeutic strategy for chronic inflammatory conditions.

V-9302 inhibits proliferation and migration of VSMCs, and reduces neointima formation in mice after carotid artery ligation.

Rapidly proliferating cells such as vascular smooth muscle cells (VSMCs) require metabolic programs to support increased energy and biomass production. Thus, targeting glutamine metabolism by inhibiting glutamine transport could be a promising strategy for vascular disorders such as atherosclerosis, stenosis, and restenosis. V-9302, a competitive antagonist targeting the glutamine transporter, has been investigated in the context of cancer; however, its role in VSMCs is unclear. Here, we examined the effects of blocking glutamine transport in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using V-9302. We found that V-9302 inhibited mTORC1 activity and mitochondrial respiration, thereby suppressing FBS- or PDGF-stimulated proliferation and migration of VSMCs. Moreover, V-9302 attenuated carotid artery ligation-induced neointima in mice. Collectively, the data suggest that targeting glutamine transport using V-9302 is a promising therapeutic strategy to ameliorate occlusive vascular disease.

Inhibitory Effect of a Glutamine Antagonist on Proliferation and Migration of VSMCs via Simultaneous Attenuation of Glycolysis and Oxidative Phosphorylation.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.

Integrated approach using multistep enzyme digestion, TiO2 enrichment, and database search for in-depth phosphoproteomic profiling.

Protein phosphorylation is a major PTM that regulates important cell signaling mechanisms. In-depth phosphoproteomic analysis provides a method of examining this complex interplay, yielding a mechanistic understanding of the cellular processes and pathogenesis of various diseases. However, the analysis of protein phosphorylation is challenging, due to the low concentration of phosphoproteins in highly complex mixtures and the high variability of phosphorylation sites. Thus, typical phosphoproteome studies that are based on MS require large amounts of starting material and extensive fractionation steps to reduce the sample complexity. To this end, we present a simple strategy (integrated multistep enzyme digestion, enrichment, database search-iMEED) to improve coverage of the phosphoproteome from lower sample amounts which is faster than other commonly used approaches. It is inexpensive and adaptable to low sample amounts and saves time and effort with regard to sample preparation and mass spectrometric analysis, allowing samples to be prepared without prefractionation or specific instruments, such as HPLC. All MS data have been deposited in the ProteomeXchange with identifier PXD001033 (http://proteomecentral.proteomexchange.org/dataset/PXD001033).

Proteomic analysis of mouse astrocytes and their secretome by a combination of FASP and StageTip-based, high pH, reversed-phase fractionation.

Astrocytes are the most abundant cells in the CNS, but their function remains largely unknown. Characterization of the whole-cell proteome and secretome in astrocytes would facilitate the study of their functions in various neurodegenerative diseases and astrocyte-neuron communication. To build a reference proteome, we established a C8-D1A astrocyte proteome to a depth of 7265 unique protein groups using a novel strategy that combined two-step digestion, filter-aided sample preparation, StageTip-based high pH fractionation, and high-resolution MS. Nearly, 6000 unique protein groups were identified from conditioned media of astrocyte cultures, constituting the largest astrocyte secretome that has been reported. High-confidence whole-cell proteomes and secretomes are valuable resources in studying astrocyte function by label-free quantitation and bioinformatics analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD000501 (http://proteomecentral.proteomexchange.org/dataset/PXD000501).

In-depth correlation analysis between tear glucose and blood glucose using a wireless smart contact lens.

Tears have emerged as a promising alternative to blood for diagnosing diabetes. Despite increasing attempts to measure tear glucose using smart contact lenses, the controversy surrounding the correlation between tear glucose and blood glucose still limits the clinical usage of tears. Herein, we present an in-depth investigation of the correlation between tear glucose and blood glucose using a wireless and soft smart contact lens for continuous monitoring of tear glucose. This smart contact lens is capable of quantitatively monitoring the tear glucose levels in basal tears excluding the effect of reflex tears which might weaken the relationship with blood glucose. Furthermore, this smart contact lens can provide an unprecedented level of continuous tear glucose data acquisition at sub-minute intervals. These advantages allow the precise estimation of lag time, enabling the establishment of the concept called 'personalized lag time'. This demonstration considers individual differences and is successfully applied to both non-diabetic and diabetic humans, as well as in animal models, resulting in a high correlation.

Targeting YAP Activity and Glutamine Metabolism Cooperatively Suppresses Tumor Progression by Preventing Extracellular Matrix Accumulation.

Cancer cells use multiple mechanisms to evade the effects of glutamine metabolism inhibitors. The pathways that govern responses to alterations in glutamine availability within the tumor may represent therapeutic targets for combinatorial strategies with these inhibitors. Here, we showed that targeting glutamine utilization stimulated Yes-associated protein (YAP) signaling in cancer cells by reducing cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent phosphorylation of large tumor suppressor (LATS). Elevated YAP activation induced extracellular matrix (ECM) deposition by increasing secretion of connective tissue growth factor (CTGF) that promoted production of fibronectin and collagen by surrounding fibroblasts. Consequently, inhibiting YAP synergized with inhibition of glutamine utilization to effectively suppress tumor growth in vivo, along with a concurrent decrease in ECM deposition. Blocking ECM remodeling also augmented the tumor suppressive effects of the glutamine utilization inhibitor. Collectively, these data reveal mechanisms by which targeting glutamine utilization increases ECM accumulation and identify potential strategies to reduce ECM levels and increase the efficacy of glutamine metabolism inhibitors.

Glutamine-derived aspartate is required for eIF5A hypusination-mediated translation of HIF-1α to induce the polarization of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are vital contributors to the growth, metastasis, and therapeutic resistance of various cancers, including hepatocellular carcinoma (HCC). However, the exact phenotype of TAMs and the mechanisms underlying their modulation for therapeutic purposes have not been determined. Here, we present compelling evidence that glutamine-derived aspartate in TAMs stimulates spermidine production through the polyamine synthesis pathway, thereby increasing the translation efficiency of HIF-1α via eIF5A hypusination. Consequently, augmented translation of HIF-1α drives TAMs to undergo an increase glycolysis and acquire a metabolic phenotype distinct from that of M2 macrophages. Finally, eIF5A levels in tumor stromal lesions were greater than those in nontumor stromal lesions. Additionally, a higher degree of tumor stromal eIF5A hypusination was significantly associated with a more advanced tumor stage. Taken together, these data highlight the potential of inhibiting hypusinated eIF5A by targeting glutamine metabolism in TAMs, thereby opening a promising avenue for the development of novel therapeutic approaches for HCC.

In-depth proteomic analysis of mouse microglia using a combination of FASP and StageTip-based, high pH, reversed-phase fractionation.

Microglia are major immune cells in the central nervous system. A characterization of microglia proteome would facilitate on the study of microglial functions in association with various neurodegenerative diseases. To build a reference proteome, we established a BV-2 microglial proteome to a depth of 5494 unique protein groups using a novel strategy that combined FASP, StageTip-based high pH fractionation, and high-resolution MS quickly and cost efficiently. By bioinformatics analysis, the BV-2 proteome is a valuable resource for studies of microglial function, such as in the immune response, inflammatory response, and phagocytosis. All MS data have been deposited in the ProteomeXchange with identifier PXD000168.

Verification of multimarkers for detection of early stage diabetic retinopathy using multiple reaction monitoring.

Diabetic retinopathy (DR) is a complication of diabetes and 80% of diabetes mellitus (DM) patients whose DM duration is over 10 years can be expected to suffer with DR. The diagnosis of DR depends on an ophthalmological examination, and no molecular methods of screening DR status exist. Nonproliferative diabetic retinopathy (NPDR) is the early DR which is hard to be noticed in early NPDR, showing significant cause of adult blindness in type 2 diabetes patients. Protein biomarkers have been valuable in the diagnosis of disease and the use of multiple biomarkers has been suggested to overcome the low specificity of single ones. For biomarker development, multiple reaction monitoring (MRM) has been spotlighted as an alternative method to quantify target proteins with no need for immunoassay. In this study, 54 candidate DR marker proteins from a previous study were verified by MRM in plasma samples from NPDR patients in 3 stages (mild, moderate and severe; 15 cases each) and diabetic patients without retinopathy (15 cases) as a control. Notably, 27 candidate markers distinguished moderate NPDR from type 2 diabetic patients with no diabetic retinopathy, generating AUC values (>0.7). Specifically, 28 candidate proteins underwent changes in expression as type 2 diabetic patients with no diabetic retinopathy progressed to mild and moderate NPDR. Further, a combination of 4 markers from these 28 candidates had the improved specificity in distinguishing moderate NPDR from type 2 diabetic patients with no diabetic retinopathy, yielding a merged AUC value of nearly 1.0. We concluded that MRM is a fast, robust approach of multimarker panel determination and an assay platform that provides improved specificity compared with single biomarker assay systems.

Targeting glutamine metabolism as a therapeutic strategy for cancer.

Proliferating cancer cells rely largely on glutamine for survival and proliferation. Glutamine serves as a carbon source for the synthesis of lipids and metabolites via the TCA cycle, as well as a source of nitrogen for amino acid and nucleotide synthesis. To date, many studies have explored the role of glutamine metabolism in cancer, thereby providing a scientific rationale for targeting glutamine metabolism for cancer treatment. In this review, we summarize the mechanism(s) involved at each step of glutamine metabolism, from glutamine transporters to redox homeostasis, and highlight areas that can be exploited for clinical cancer treatment. Furthermore, we discuss the mechanisms underlying cancer cell resistance to agents that target glutamine metabolism, as well as strategies for overcoming these mechanisms. Finally, we discuss the effects of glutamine blockade on the tumor microenvironment and explore strategies to maximize the utility of glutamine blockers as a cancer treatment.

Untargeted Metabolomics Analysis Reveals Toxicity Based on the Sex and Sexual Maturity of Single Low-Dose DEHP Exposure.

Di-(2-Ethylhexyl) phthalate (DEHP) is a prevalent environmental endocrine disruptor that affects homeostasis, reproduction, and developmental processes. The effects of DEHP have been shown to differ based on sex and sexual maturity. This study examines the metabolic profiles of mature adult rats from both sexes, aged 10 weeks, and adolescent female rats, aged 6 weeks, following a single 5 mg/kg of body weight DEHP oral administration. An untargeted metabolomic analysis was conducted on urine samples collected at multiple times to discern potential sex- and maturity-specific DEHP toxicities. Various multivariate statistical analyses were employed to identify the relevant metabolites. The findings revealed disruptions to the steroid hormone and primary bile acid biosynthesis. Notably, DEHP exposure increased hyocholic, muricholic, and ketodeoxycholic acids in male rats. Moreover, DEHP exposure was linked to heart, liver, and kidney damage, as indicated by increased plasma GOT1 levels when compared to the levels before DEHP exposure. This study provides detailed insights into the unique mechanisms triggered by DEHP exposure concerning sex and sexual maturity, emphasizing significant distinctions in lipid metabolic profiles across the different groups. This study results deepens our understanding of the health risks linked to DEHP, informing future risk assessments and policy decisions.

Novel Asian-Specific Visceral Adiposity Indices Are Associated with Chronic Kidney Disease in Korean Adults.

BACKGROUND: The Chinese visceral adiposity index (CVAI) and new visceral adiposity index (NVAI) are novel indices of visceral adiposity used to predict metabolic and cardiovascular diseases in Asian populations. However, the relationships of CVAI and NVAI with chronic kidney disease (CKD) have not been investigated. We aimed to characterize the relationships of CVAI and NVAI with the prevalence of CKD in Korean adults. METHODS: A total of 14,068 participants in the 7th Korea National Health and Nutrition Examination Survey (6,182 men and 7,886 women) were included. Receiver operating characteristic (ROC) analyses were employed to compare the associations between indices of adiposity and CKD, and a logistic regression model was used to characterize the relationships of CVAI and NVAI with CKD prevalence. RESULTS: The areas under the ROC curves for CVAI and NVAI were significantly larger than for the other indices, including the visceral adiposity index and lipid accumulation product, in both men and women (all P<0.001). In addition, high CVAI or NVAI was significantly associated with a high CKD prevalence in both men (odds ratio [OR], 2.14; 95% confidence interval [CI], 1.31 to 3.48 in CVAI and OR, 6.47; 95% CI, 2.91 to 14.38 in NVAI, P<0.05) and women (OR, 4.87; 95% CI, 1.85 to 12.79 in CVAI and OR, 3.03; 95% CI, 1.35 to 6.82 in NVAI, P<0.05); this association remained significant after adjustment for multiple confounding factors in men and women. CONCLUSION: CVAI and NVAI are positively associated with CKD prevalence in a Korean population. CVAI and NVAI may be useful for the identification of CKD in Asian populations, including in Korea.

N-methyl-D-aspartate receptors induce M1 polarization of macrophages: Feasibility of targeted imaging in inflammatory response in vivo.

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are considered to be involved in several physiological and pathophysiological processes in addition to the progression of neurological disorders. However, how NMDARs are involved in the glycolytic phenotype of M1 macrophage polarization and the possibility of using them as a bio-imaging probe for macrophage-mediated inflammation remain unclear. METHODS: We analyzed cellular responses to NMDAR antagonism and small interfering RNAs using mouse bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS). An NMDAR targeting imaging probe, N-TIP, was produced via the introduction of NMDAR antibody and the infrared fluorescent dye FSD Fluor™ 647. N-TIP binding efficiency was tested in intact and LPS-stimulated BMDMs. N-TIP was intravenously administered to mice with carrageenan (CG)- and LPS-induced paw edema, and in vivo fluorescence imaging was conducted. The anti-inflammatory effects of dexamethasone were evaluated using the N-TIP-mediated macrophage imaging technique. RESULTS: NMDARs were overexpressed in LPS-treated macrophages, subsequently inducing M1 macrophage polarization. Mechanistically, NMDAR-mediated Ca2+ accumulation resulted in LPS-stimulated glycolysis via upregulation of PI3K/AKT/mTORC1 signaling. In vivo fluorescence imaging with N-TIP showed LPS- and CG-induced inflamed lesions at 5 h post-inflammation, and the inflamed lesions could be detected until 24 h. Furthermore, our N-TIP-mediated macrophage imaging technique helped successfully visualize the anti-inflammatory effects of dexamethasone in mice with inflammation. CONCLUSION: This study demonstrates that NMDAR-mediated glycolysis plays a critical role in M1 macrophage-related inflammation. Moreover, our results suggest that NMDAR targeting imaging probe may be useful in research on inflammatory response in vivo.

Retinal proteome analysis in a mouse model of oxygen-induced retinopathy.

To identify proteins that are involved in the molecular mechanisms of oxygen-induced retinopathy (OIR), a well-established model of blinding ischemic retinopathy, we quantitatively analyzed the retinal proteome in a mouse model of OIR. OIR was induced by exposing C57BL/6 mice on postnatal day 7 (P7) to 75% hyperoxia for 5 days, followed by 5 days in room air. Retinas from mice on P12 and P17, the hyperoxic and hypoxic phases, respectively, and control groups were examined using isobaric tags for relative and absolute quantitation (iTRAQ) and nano-LC-ESI-MS/MS. In total, 1422 retinal proteins were identified: 699 from the iTRAQ experiment and 1074 by nano-LC-ESI-MS/MS. Compared with control retinas in the iTRAQ study, OIR retinas upregulated and downregulated 21 and 17 proteins, respectively, in P17 retinas and 25 and 14 proteins, respectively, in P12 retinas. Of the differentially expressed proteins, the retinal expression of crystallin proteins, Müller cell-associated proteins, neurodegeneration-associated proteins, and angiogenesis-associated proteins, such as 150-kDa oxygen-regulated protein (ORP150), were analyzed. ORP150 colocalized to the neovascular tufts, and knockdown of ORP150 by siRNA decreased the levels of secreted VEGF in cultured retinal pigment epithelial cells. Moreover, intravitreal administration of siRNA targeting ORP150 significantly reduced the retinal neovascularization in OIR. In conclusion, our proteomic discovery method, coupled with targeted approaches, revealed many proteins that were differentially regulated in the mouse model of OIR. These proteins, including ORP150, are potential novel therapeutic targets for the treatment of proliferative ischemic retinopathy.

Melatonin inhibits glycolysis in hepatocellular carcinoma cells by downregulating mitochondrial respiration and mTORC1 activity.

Various mechanisms have been suggested to explain the chemopreventive and tumor-inhibitory effects of melatonin. Despite the growing evidence supporting melatonin-induced mitochondrial dysfunction, it remains largely unknown how this phenomenon modulates metabolic reprogramming in cancer cells. The aim of our study was to identify the mechanism underlying the anti-proliferative and apoptotic effects of melatonin, which is known to inhibit glycolysis. We analyzed the time-dependent effects of melatonin on mitochondrial respiration and glycolysis in liver cancer cells. The results showed that from a cell bioenergetic point of view, melatonin caused an acute reduction in mitochondrial respiration, however, increased reactive oxygen species production, thereby inhibiting mTORC1 activity from an early stage post-treatment without affecting glycolysis. Nevertheless, administration of melatonin for a longer time reduced expression of c-Myc protein, thereby suppressing glycolysis via downregulation of HK2 and LDHA. The data presented herein suggest that melatonin suppresses mitochondrial respiration and glycolysis simultaneously in HCC cells, leading to anti-cancer effects. Thus, melatonin can be used as an adjuvant agent for therapy of liver cancer. [BMB Reports 2022; 55(9): 459-464].