The Globodera rostochiensis Gr29D09 Effector with a Role in Defense Suppression Targets the Potato Hexokinase 1 Protein.

The potato cyst nematode (Globodera rostochiensis) is an obligate root pathogen of potatoes. G. rostochiensis encodes several highly expanded effector gene families, including the Gr4D06 family; however, little is known about the function of this effector family. We cloned four 29D09 genes from G. rostochiensis (named Gr29D09v1/v2/v3/v4) that share high sequence similarity and are homologous to the Hg29D09 and Hg4D06 effector genes from the soybean cyst nematode (Heterodera glycines). Phylogenetic analysis revealed that Gr29D09 genes belong to a subgroup of the Gr4D06 family. We showed that Gr29D09 genes are expressed exclusively within the nematode's dorsal gland cell and are dramatically upregulated in parasitic stages, indicating involvement of Gr29D09 effectors in nematode parasitism. Transgenic potato lines overexpressing Gr29D09 variants showed increased susceptibility to G. rostochiensis. Transient expression assays in Nicotiana benthamiana demonstrated that Gr29D09v3 could suppress reactive oxygen species (ROS) production and defense gene expression induced by flg22 and cell death mediated by immune receptors. These results suggest a critical role of Gr29D09 effectors in defense suppression. The use of affinity purification coupled with nanoliquid chromatography-tandem mass spectrometry identified potato hexokinase 1 (StHXK1) as a candidate target of Gr29D09. The Gr29D09-StHXK1 interaction was further confirmed using in planta protein-protein interaction assays. Plant HXKs have been implicated in defense regulation against pathogen infection. Interestingly, we found that StHXK1 could enhance flg22-induced ROS production, consistent with a positive role of plant HXKs in defense. Altogether, our results suggest that targeting StHXK1 by Gr29D09 effectors may impair the positive function of StHXK1 in plant immunity, thereby aiding nematode parasitism. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Disrupting Circadian Rhythm via the PER1-HK2 Axis Reverses Trastuzumab Resistance in Gastric Cancer.

Trastuzumab is the only approved targeted drug for first-line treatment of HER2-positive advanced gastric cancer, but the high rate of primary resistance and rapid emergence of secondary resistance limit its clinical benefits. We found that trastuzumab-resistant (TR) gastric cancer cells exhibited high glycolytic activity, which was controlled by hexokinase 2 (HK2)-dependent glycolysis with a circadian pattern [higher at zeitgeber time (ZT) 6, lower at ZT18]. Mechanistically, HK2 circadian oscillation was regulated by a transcriptional complex composed of PPARγ and the core clock gene PER1. In vivo and in vitro experiments demonstrated that silencing PER1 disrupted the circadian rhythm of PER1-HK2 and reversed trastuzumab resistance. Moreover, metformin, which inhibits glycolysis and PER1, combined with trastuzumab at ZT6, significantly improved trastuzumab efficacy in gastric cancer. Collectively, these data introduce the circadian clock into trastuzumab therapy and propose a potentially effective chronotherapy strategy to reverse trastuzumab resistance in gastric cancer. SIGNIFICANCE: In trastuzumab-resistant HER2-positive gastric cancer, glycolysis fluctuates with a circadian oscillation regulated by the BMAL1-CLOCK-PER1-HK2 axis, which can be disrupted with a metformin-based chronotherapy to overcome trastuzumab resistance.

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.

Effect of Caralluma tuberculata on regulation of carbohydrate metabolizing genes in alloxan-induced rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma tuberculata (C. tuberculata) has traditionally been used in Pakistan and other parts of the world as a folk treatment for diabetes mellitus. A few studies indicated its antihyperglycemic effect, however, the mystery remained unfolded as how did it modify the pathophysiological condition. AIM OF STUDY: Hence, this study aimed to explore underlying mechanism(s) for its hypoglycemic activity at biochemical and molecular levels. MATERIALS AND METHODS: Methanol extract (ME) of C. tuberculata as well as its hexane (HF) and aqueous (AF) fractions were explored for their effect on total glycogen in liver and skeletal muscle of alloxan-induced rats by spectroscopy. Moreover, the expression of genes related to hepatic carbohydrate metabolizing enzymes was quantified. At molecular level, mRNA expression of glucose transporter 2 (GLUT-2), glycogen synthase (GS), glucokinase (GK), hexokinase 1 (HK-1), pyruvate kinase (PK), glucose 6 phosphate dehydrogenase (G-6-PDH), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G-6-Pase) was determined by using quantitative real time polymerase chain reaction (qRT-PCR) after administration of ME (350 mg), HF(3 mg), AF (10 mg) and metformin (500 mg). The doses were administered twice daily according to per kg of body weight. RESULTS: A significant reduction in hepatic and skeletal muscle glycogen content was exhibited. The data of qRT-PCR revealed that gene's expression of GLUT-2 was significantly decreased after treatment with ME and HF, whilst it was unaltered by AF, however, a significant decrease was observed in genes corresponding to GS, GK and HK-1 after treatment with ME. Similarly, there was a significant decrease in expression of genes corresponding to GS, GK and HK-1 following treatment with HF. Surprisingly, post-treatment with AF didn't modify the gene's expression of GS and GK, whilst it caused a profound decrease in expression of HK-1 gene. Contrarily, the expression of gene related to PK was significantly up-regulated post-administration with ME, HF and AF. The expression levels of G-6-PDH, however, remained unaltered after treatment with the experimental extract and fractions of the plant. In addition, HF and AF did not cause any modification in PEPCK, whereas ME caused a significant down-regulation of the gene. Treatment with all the extract and fractions of the plant caused a substantial decrease in the gene's expression of PC, while there was a significant increase in the expression of gene related to G-6-Pase. CONCLUSION: The three experimental extract and fractions caused a substantial decrease in glycogen content in liver and skeletal muscle tissues. The analysis by qRT-PCR showed that glucose transport via GLUT-2 was profoundly declined by ME and HF. The expression of genes related to various metabolic pathways involved in metabolism of carbohydrate in hepatocytes revealed explicitly that the ME, HF and AF decreased the phenomena of glycogenesis and gluconeogenesis. Contrarily, all the extract and fractions of the plant activated glycogenolysis and glycolysis but did not modify the pentose phosphate shunt pathway.

Bromide impairs the circadian clock and glycolytic homeostasis via disruption of autophagy in rat H9C2 cardiomyocytes.

BACKGROUND: Trace elements function as essential cofactors that are involved in various biochemical processes in mammals. Autophagy is vital for nutrient supplement, which is an important Zeitegber for the circadian homeostasis in heart. Here, we considered the possibility that autophagy, as well as the cardiomyocyte clock and glycolysis are interlinked. Detrimental effects were observed when cardiac system is exposed to bromine containing drugs. This study investigated the effects and mechanisms of bromide on the circadian clock and glycolytic metabolism of H9C2 cardiomyocytes. RESULTS: In the present study, bromide does not affect cell viability and apoptosis of H9C2 cardiomyocytes. Bromide dampens the clock and glycolytic (Hk2 and Pkm2) gene expression rhythmicity in a dose-dependent manner. Additionally, bromide inhibits autophagic process in H9C2 cardiomyocytes. In contrast, rapamycin (an autophagy inducer) dramatically restores the inhibitory effect of NaBr on the mRNA expression levels of clock genes (Bmal1, Cry1 and Rorα) and glycolytic genes (Hk2 and Pkm2). CONCLUSIONS: Our results reveal that bromide represses the clock and glycolytic gene expression patterns, partially through inhibition of autophagy.

FoxA2 inhibits the proliferation of hepatic progenitor cells by reducing PI3K/Akt/HK2-mediated glycolysis.

FoxA2 is an essential transcription factor for liver organogenesis and homeostasis. Although reduced expression of FoxA2 has been associated with chronic liver diseases, hepatic progenitor cells (HPCs) that are activated in these circumstances express FoxA2. However, the functional effects and underlying mechanism of FoxA2 in HPCs are still unknown. As revealed by immunostaining, HPCs expressed FoxA2 in human cirrhotic livers and in the livers of choline-deficient diet supplemented with ethionine (CDE) rats. Knocking down FoxA2 in HPCs isolated from CDE rats significantly increased cell proliferation and aerobic glycolysis. Moreover, gene transcription, protein expression, and the enzyme activities of hexokinase 2 (HK2) were upregulated, and blocking HK2 activities via 2-deoxyglucose markedly reduced cell proliferation and aerobic glycolysis. Kyoto Encyclopedia of Genes and Genomes analysis revealed that FoxA2 knockdown enhanced the transcription of genes involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway and triggered downstream Akt phosphorylation. Blocking the PI3K/Akt pathway by Ly294002 inhibited HK2 activities, aerobic glycolysis, and cell proliferation in FoxA2-knockdown cells. Therefore, FoxA2 plays an important role in the proliferation and inhibition of HPCs by suppressing PI3K/Akt/HK2-regulated aerobic glycolysis.

The Black Truffle, Tuber melanosporum (Ascomycetes), Ameliorates Hyperglycemia and Regulates Insulin Signaling Pathway in STZ-Induced Diabetic Rats.

Black truffle mushroom, Tuber melanosporum, is effective in treating various symptoms associated with diabetes mellitus such as hyperglycemia, oxidative stress, and hyperlipidemia and is used as traditional medicine. The aim of our study is to elucidate the antidiabetic potential of T. melanosporum. Male albino Wistar rats were administered a single dose of STZ (40 mg/kg b.w.) to induce mild diabetes mellitus (DM). After the confirmation of hyperglycemia, rats were treated with three different doses of truffle extract (TE) (200, 400, and 600 mg/kg b.w.) for the duration of 45 days. The various tissues were collected at the end of the study. The levels of glucose, oral glucose tolerance test, insulin, hexokinase, glucose 6 phosphatase, and fructose-1,6-bisphosphatase, and regulation of insulin signaling genes were quantified. The results showed that STZ- induced rats have a higher blood glucose level and a lower insulin level compared with the control groups and TE treated groups. Results also reveal that STZ suppressed the expressions of insulin signaling genes in diabetic rats and TE treatment resulted in a positive regulation of the insulin signaling pathway. The results of TE are similar to the results attained in glibenclamide (GB) group rats. Overall, the study provides scientific evidence for the medicinal properties of black truffle; future clinical studies can warrant a potential antidiabetic drug in the form of diet.

Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility.

There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

Hexokinase II inhibition by 3-bromopyruvate sensitizes myeloid leukemic cells K-562 to anti-leukemic drug, daunorubicin.

An increased metabolic flux towards Warburg phenotype promotes survival, proliferation and causes therapeutic resistance, in leukemic cells. Hexokinase-II (HK-II) is expressed predominantly in cancer cells, which promotes Warburg metabolic phenotype and protects the cancer cells from drug-induced apoptosis. The HK-II inhibitor 3- Bromopyruvate (3-BP) dissociates HK-II from mitochondrial complex, which leads to enhanced sensitization of leukemic cells to anti-leukemic drugs. In the present study, we analyzed the Warburg characteristics viz. HK-II expression, glucose uptake, endogenous reactive oxygen species (ROS) level of leukemic cell lines K-562 and THP-1 and then investigated if 3-BP can sensitize the leukemic cells K-562 to anti-leukemic drug Daunorubicin (DNR). We found that both K-562 and THP-1 cells have multi-fold high levels of HK-II, glucose uptake and endogenous ROS with respect to normal PBMCs. The combined treatment (CT) of 3-BP and DNR showed synergistic effect on the growth inhibition (GI) of K-562 and THP-1 cells. This growth inhibitory effect was attributed to 3-BP induced S-phase block and DNR induced G2/M block, resulted in reduced proliferation due to CT. Further, CT resulted in low HK-II level in mitochondrial fraction, high intracellular calcium and elevated apoptosis as compared with individual treatment of DNR and 3-BP. Moreover, CT caused enhanced DNA damage and hyperpolarized mitochondria, leading to cell death. Taken together, these results suggest that 3-BP synergises the anticancer effects of DNR in the chronic myeloid leukemic cell K-562, and may act as an effective adjuvant to anti-leukemic chemotherapy.

BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.

OsAGO2 controls ROS production and the initiation of tapetal PCD by epigenetically regulating OsHXK1 expression in rice anthers.

Proteins of the ARGONAUTE (AGO) family function in the epigenetic regulation of gene expression. Although the rice (Oryza sativa) genome encodes 19 predicted AGO proteins, few of their functions have thus far been characterized. Here, we show that the AGO protein OsAGO2 regulates anther development in rice. OsAGO2 was highly expressed in anthers. Knockdown of OsAGO2 led to the overaccumulation of reactive oxygen species (ROS) and abnormal anther development, causing premature initiation of tapetal programmed cell death (PCD) and pollen abortion. The expression level of Hexokinase 1 (OsHXK1) increased significantly, and the methylation levels of its promoter decreased, in plants with knocked-down OsAGO2 expression. Overexpression of OsHXK1 also resulted in the overaccumulation of ROS, premature initiation of PCD, and pollen abortion. Moreover, knockdown of OsHXK1 restored pollen fertility in OsAGO2 knockdown plants. Chromatin immunoprecipitation assays demonstrated that OsAGO2 binds directly to the OsHXK1 promoter region, suggesting that OsHXK1 is a target gene of OsAGO2. These results indicate that OsHXK1 controls the appropriate production of ROS and the proper timing of tapetal PCD and is directly regulated by OsAGO2 through epigenetic regulation.

Metastatic cancer cells compensate for low energy supplies in hostile microenvironments with bioenergetic adaptation and metabolic reprogramming.

Metastasis accounts for the majority of cancer-related mortalities, and the complex processes of metastasis remain the least understood aspect of cancer biology. Metabolic reprogramming is associated with cancer cell survival and metastasis in a hostile envi-ronment with a limited nutrient supply, such as solid tumors. Little is known regarding the differences of bioenergetic adaptation between primary tumor cells and metastatic tumor cells in unfavorable microenvironments; to clarify these differences, the present study aimed to compare metabolic reprogramming of primary tumor cells and metastatic tumor cells. SW620 metastatic tumor cells exhibited stronger bioenergetic adaptation in unfavorable conditions compared with SW480 primary tumor-derived cells, as determined by the sustained elevation of glycolysis and regulation of the cell cycle. This remarkable glycolytic ability of SW620 cells was associated with high expression levels of hexokinase (HK)1, HK2, glucose transporter type 1 and hypoxia-inducible factor 1α. Compared with SW480 cells, the expression of cell cycle regulatory proteins was effectively inhibited in SW620 cells to sustain cell survival when there was a lack of energy. Furthermore, SW620 cells exhibited a stronger mesenchymal phenotype and stem cell characteristics compared with SW480 cells; CD133 and CD166 were highly expressed in SW620 cells, whereas expression was not detected in SW480 cells. These data may explain why metastatic cancer cells exhibit greater microenvironmental adaptability and survivability; specifically, this may be achieved by upregulating glycolysis, optimizing the cell cycle and reprogramming cell metabolism. The present study may provide a target metabolic pathway for cancer metastasis therapy.

Euxanthone inhibits glycolysis and triggers mitochondria-mediated apoptosis by targeting hexokinase 2 in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the most prevalent gynaecological cancers. Euxanthone, an active ingredient of the medicinal plant Polygala caudata, exhibits a selective cytotoxic effect in tumour cells. The present study was aimed to determine whether euxanthone could suppress ovarian tumour growth, and to study the relevant mechanism. Two EOC cell lines, SKOV3 and A2780, were used as the in vitro model and treated with euxanthone. Cell viability and apoptosis were assayed using Cell Counting Kit-8 (CCK-8) and Annexin-V FITC/PI staining, respectively. Commercially available kits were used to measure the glucose consumption, lactate production, and intracellular ATP levels. Western blots assay was conducted to examine the level of apoptotic markers. To examine the roles of HK2 and STAT3 in the anti-tumour effect of euxanthone, cells were transfected with vectors overexpressing HK2 or STAT3, and assayed as above. Finally, SKOV3 cells were injected to mice models to appreciate the anti-neoplastic effect of euxanthone in vivo. We found that euxanthone impaired the cell viability and induced apoptosis via the intrinsic pathway in a concentration-dependent fashion in both SKOV3 and A2780 cells. Euxanthone also caused inhibition of glycolysis. Apoptosis and glycolysis inhibition was mediated by the downregulation of HK2, which in turn was a result of STAT3 inactivation. In vivo experiments also supported that euxanthone could exert anti-cancer activities without general toxicity. In conclusion, euxanthone triggered mitochondrial apoptosis and inhibited glycolysis in EOC cells. SIGNIFICANCE OF THE STUDY: Euxanthone triggered mitochondrial apoptosis and inhibited glycolysis in EOC cells. Our findings provide preliminary experimental data that support further studies on the potential therapeutic role of euxanthone in ovarian cancer.

Alleviation Mechanisms of Selenium on Cadmium-Spiked in Chicken Ovarian Tissue: Perspectives from Autophagy and Energy Metabolism.

Cadmium (Cd) is a kind of toxic heavy metal and it can cause damage to organs and tissues. Selenium (Se) can antagonize some metal element toxicity including Cd. The present study was designed to investigate Cd-induced damage to chicken ovary by autophagy and the protective mechanism of Se on Cd-induced damage. Administration of Cd for 12 weeks led to energy metabolism disorder of the chicken ovarian tissues, which resulted in autophagy. In addition, the mRNA expression of glucose-related genes including hexokinase II (HK2), pyruvate kinase (PK), pyruvate dehydrogenase complex (PDHX), and succinate dehydrogenase (SDH) and the activities of ATPase, including Na+-K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, were all downregulated remarkably compared with the control. However, combined with oral administration of Se at 2 mg/kg, the mRNA expression of glucose-related genes and the activities of ATPase increased. The mRNA expression of the autophagy-related genes by Cd treatment, including microtubule-associated protein light chain 3 (LC3), dynein, autophagy-related gene 5 (Atg5), and Beclin 1, was remarkably enhanced, whereas mammalian target of rapamycin (mTOR) was downregulated. However, besides mTOR, their levels displayed a downregulated trend beyond simultaneous Se treatment. The protein expression of autophagy genes was similar to those of mRNA. In conclusion, Cd toxicity affect energy metabolism and induce autophagy, which causes damage to chicken ovary, whereas Se could protect effectively this injury induced by Cd.

Jolkinolide B inhibits glycolysis by downregulating hexokinase 2 expression through inactivating the Akt/mTOR pathway in non-small cell lung cancer cells.

Jolkinolide B (JB), a bioactive compound isolated from herbal medicine, has been found to inhibit tumor growth by altering glycolysis. However, whether glycolysis is influenced by JB in non-small cell lung cancer (NSCLC) cells and the mechanism remain unknown. The aim of the present study was to evaluate the effect of JB on the glycolysis in NSCLC cells and the underlying molecular mechanism. The results showed that JB treatment inhibited cell viability of A549 and H1299 cells in a concentration-dependent manner. JB reduced the glucose consumption, lactate production, and HK2 expression. The expressions of p-Akt and p-mTOR were also decreased by JB treatment. Knockdown of HK2 reduced glucose consumption and lactate production. Inhibition of the Akt/mTOR pathway decreased HK2 expression and inhibited glycolysis. In conclusion, the results indicated that JB inhibits glycolysis by down-regulating HK2 expression through inactivating the Akt/mTOR pathway in NSCLC cells, suggesting that JB might be a potential therapeutic agent for the treatment of NSCLC.

Licochalcone A suppresses hexokinase 2-mediated tumor glycolysis in gastric cancer via downregulation of the Akt signaling pathway.

Licochalcone A (LicA) is a chalcone extracted from liquorice which has been used as a traditional Chinese medicine for many generations. Increased glucose consumption and glycolytic activity are important hallmarks of cancer cells, and hexokinase 2 (HK2) upregulation is a major contributor to the elevation of glycolysis. Recently, the antitumor activities of LicA have been reported in various cancers; however, its effect on tumor glycolysis in gastric cancer and the underlying mechanisms are completely unknown. In vitro, cell proliferation and clonogenic survival were substantially inhibited after LicA treatment. LicA reduced HK2 expression, and both glucose consumption and lactate production in gastric cancer cells were significantly suppressed. Mechanistic investigations revealed that multiple signaling pathways including Akt, ERK and NF­κB were suppressed by LicA. Further studies demonstrated that the inhibition of glycolysis by LicA was mainly attributed to the blockade of the Akt signaling pathway, and the suppression of glycolysis was substantially attenuated when Akt was exogenously overexpressed. In addition to the role in the inhibition of glycolysis, reduction in HK2 was confirmed to be involved in the induction of cell apoptosis. The apoptosis induced by LicA was substantially impaired after HK2 overexpression in gastric cancer cells. The in vivo experiment showed that MKN45 xenograft growth was markedly delayed after LicA treatment and HK2 expression in LicA­treated tissues was markedly decreased. All of these data demonstrated that blockade of the Akt/HK2 pathway was the underlying mechanism required for LicA to exert its biological activities in glycolysis inhibition and apoptosis induction.

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis.

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of ß-fructofuranosidase (sacA), fructokinase (scrK), UDP-glucose 4-epimerase (GALE), Mannose-1-phosphate guanylyltransferase (GMPP), and UDP-glucose 6-dehydrogenase (UGDH), but negatively correlated with the expression of Hexokinase (HK). Through qRT-PCR validation and comprehensive analysis, we determined that sacA, HK, and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions.

Astragalin Reduces Hexokinase 2 through Increasing miR-125b to Inhibit the Proliferation of Hepatocellular Carcinoma Cells in Vitro and in Vivo.

Astragalin (ASG) can be found in a variety of food components. ASG exhibits cytotoxic effects on several different types of malignant cells. However, its effects on hepatocellular carcinoma (HCC) cells and the underlying molecular mechanisms have remained to be fully elucidated. Here, we revealed that ASG remarkably suppressed the proliferation of HCC cells. In HCC cells, ASG inhibited glucose glycolysis and promoted oxidative phosphorylation, resulting in a surge of reactive oxygen species (ROS). Mechanistically, ASG suppressed the expression of hexokinase 2 (HK2). This event was indispensible for ASG-mediated metabolic reprogramming, ROS accumulation, and subsequent growth arrest. Our further investigations unveiled that ASG repressed HK2 expression via increasing miR-125b. In vivo experiments showed that gavage of ASG decreased the proliferation of Huh-7 HCC xenografts in nude mice and inhibited the growth of transplanted H22 HCC cells in Kunming mice. Declined HCC tumor growth in vivo was associated with boosted miR-125b and reduced expression of HK2 in tumor tissues. Collectively, our results demonstrated that ASG is able to suppress the proliferation of HCC cells both in vitro and in vivo. Inhibition of HK2 through upregulating miR-125b and subsequent metabolic reprogramming is implicated in the antiproliferative effects of ASG on HCC cells.

ITD mutation in FLT3 tyrosine kinase promotes Warburg effect and renders therapeutic sensitivity to glycolytic inhibition.

Internal tandem duplication (ITD) mutation in Fms-like tyrosine kinase 3 gene (FLT3/ITD) represents an unfavorable genetic change in acute myeloid leukemia (AML) and is associated with poor prognosis. Metabolic alterations have been involved in tumor progression and attracted interest as a target for therapeutic intervention. However, few studies analyzed the adaptations of cellular metabolism in the context of FLT3/ITD mutation. Here, we report that FLT3/ITD causes a significant increase in aerobic glycolysis through AKT-mediated upregulation of mitochondrial hexokinase (HK2), and renders the leukemia cells highly dependent on glycolysis and sensitive to pharmacological inhibition of glycolytic activity. Inhibition of glycolysis preferentially causes severe ATP depletion and massive cell death in FLT3/ITD leukemia cells. Glycolytic inhibitors significantly enhances the cytotoxicity induced by FLT3 tyrosine kinase inhibitor sorafenib. Importantly, such combination provides substantial therapeutic benefit in a murine model bearing FLT3/ITD leukemia. Our study suggests that FLT3/ITD mutation promotes Warburg effect, and such metabolic alteration can be exploited to develop effective therapeutic strategy for treatment of AML with FLT3/ITD mutation via metabolic intervention.