Pyruvate Kinase Isozyme Type M2 Research Articles

Glycolysis related lncRNA SNHG3 / miR-139-5p / PKM2 axis promotes castration-resistant prostate cancer (CRPC) development and enzalutamide resistance

Although androgen deprivation therapy (ADT) by the anti-androgen drug enzalutamide (Enz) may improve the survival level of patients with castration-resistant prostate cancer (CRPC), most patients may eventually fail due to the acquired resistance. The reprogramming of glucose metabolism is one type of the paramount hallmarks of cancers. PKM2 (Pyruvate kinase isozyme typeM2) is a speed-limiting enzyme in the glycolytic mechanism, and has high expression in a variety of cancers. Emerging evidence has unveiled that microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have impact on tumor development and therapeutic efficacy by regulating PKM2 expression. Herein, we found that lncRNA SNHG3, a highly expressed lncRNA in CRPC via bioinformatics analysis, promoted the invasive ability and the Enz resistance of the PCa cells. KEGG pathway enrichment analysis indicated that glucose metabolic process was tightly correlated with lncRNA SNHG3 level, suggesting lncRNA SNHG3 may affect glucose metabolism. Indeed, glucose uptake and lactate content determinations confirmed that lncRNA SNHG3 promoted the process of glycolysis. Mechanistic dissection demonstrated that lncRNA SNHG3 facilitated the advance of CRPC by adjusting the expression of PKM2. Further explorations unraveled the role of lncRNA SNHG3 as a ‘sponge’ of miR-139-5p and released its binding with PKM2 mRNA, leading to PKM2 up-regulation. Together, Our studies suggest that lncRNA SNHG3 / miR-139-5p / PKM2 pathway promotes the development of CRPC via regulating glycolysis process and provides valuable insight into a novel therapeutic approach for the disordered disease.

TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC), the predominant malignancy of the oral cavity, is characterized by high incidence and low survival rates. Emerging evidence suggests a link between circadian rhythm disruptions and cancer development. The circadian gene TIMELESS, known for its specific expression in various tumors, has not been extensively studied in the context of OSCC. This study aims to explore the influence of TIMELESS on OSCC, focusing on cell growth and metabolic alterations.MethodsWe analyzed TIMELESS expression in OSCC using western blot, immunohistochemistry, qRT-PCR, and data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). The role of TIMELESS in OSCC was examined through clone formation, MTS, cell cycle, and EdU assays, alongside subcutaneous tumor growth experiments in nude mice. We also assessed the metabolic impact of TIMELESS by measuring glucose uptake, lactate production, oxygen consumption, and medium pH, and investigated its effect on key metabolic proteins including silent information regulator 1 (SIRT1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), recombinant lactate dehydrogenase A (LDHA) and glucose transporter-1 (GLUT1).ResultsElevated TIMELESS expression in OSCC tissues and cell lines was observed, correlating with reduced patient survival. TIMELESS overexpression enhanced OSCC cell proliferation, increased glycolytic activity (glucose uptake and lactate production), and suppressed oxidative phosphorylation (evidenced by reduced oxygen consumption and altered pH levels). Conversely, TIMELESS knockdown inhibited these cellular and metabolic processes, an effect mirrored by manipulating SIRT1 levels. Additionally, SIRT1 was positively associated with TIMELESS expression. The expression of SIRT1, HK2, PKM2, LDHA and GLUT1 increased with the overexpression of TIMELESS levels and decreased with the knockdown of TIMELESS.ConclusionTIMELESS exacerbates OSCC progression by modulating cellular proliferation and metabolic pathways, specifically by enhancing glycolysis and reducing oxidative phosphorylation, largely mediated through the SIRT1 pathway. This highlights TIMELESS as a potential target for OSCC therapeutic strategies.

Effect and mechanism of Yueju Pills on breast cancer induced by DMBA in rats

This paper aims to explore the inhibitory effect of Yueju Pills on breast cancer and decipher the underlying mechanism. A total of 92 SPF-grade SD female rats were involved in this study, and 14 of them were randomly selected into control group. The remaining 78 rats were administrated with 7,12-dimethylbenzanthracene(DMBA) by gavage to establish the breast cancer model. The modeled rats were randomized into model, tamoxifen(1.9 mg·kg~(-1)·d~(-1)), and low-and high-dose(17, 34 g·kg~(-1)·d~(-1)) Yueju Pills groups. The mental state, food intake, and activities of the rats were observed daily, and the body weight was measured on alternate days. After 12 weeks of administration, the rats were sacrificed and the tumor weight was measured. The serum estrogen and progeste-rone levels were determined by enzyme-linked immunosorbent assay. The histopathological changes of the breast and tumor were observed by hematoxylin-eosin staining. Western blot was employed to measure the protein levels of glucose transporter 1(GLUT1), lactate dehydrogenase A(LDHA), phosphofructokinase muscle(PFKM), pyruvate kinase isozyme type M2(PKM2), hexokinase 2(HK2), nuclear factor-kappaB(NF-κB), and phosphorylated NF-κB. The intestinal microbiome was examined by 16S rRNA high-throughput sequencing. The results showed that compared with the model group, high and low-dose Yueju Pills showed the tumor inhibition rate of 15.8% and 64.5%, respectively, and the low dose group had stronger inhibitory effect. Compared with the control group, the model group presented elevated the levels of estrogen and progesterone in serum. The administration of Yueju Pills lowered such ele-vation, and the low-dose group showed stronger lowering effect(P<0.05). Compared with the model group, Yueju Pills reduced the glands with increased breast tissue, the degree of breast duct expansion, the number and area of acinar cavity, the secretions, and the layers of mammary epithelial cells. Furthermore, Yueju Pills down-regulated the expression of GLUT1, LDHA, PFKM, PKM2, HK2, and NF-κB(P<0.05) and altered the diversity, composition, structure, and abundance of intestinal flora. The results showed that Yueju Pills could inhibit breast cancer by regulating the secretion of estrogen and progesterone, glycolysis, inflammatory cytokines, and intestinal flora.

Adrenomedullin induces cisplatin chemoresistance in ovarian cancer through reprogramming of glucose metabolism.

The metabolic network of cancer cells has been reprogrammed - relying more on aerobic glycolysis to gain energy, which is an important reason for drug resistance. Expression of adrenomedullin (ADM) in ovarian cancer tissues is related to resistance to platinum-based drugs. In view of this, we intended to investigate the correlation between ADM and glucose metabolism reprogramming of tumor cells to clarify the possible mechanism of ADM-induced ovarian cancer cisplatin resistance through glucose metabolism reprogramming. Epithelial ovarian cancer (EOC) cell viability and apoptosis were determined. Different gene expression and protein levels were detected by real-time revere transcription polymerase chain reaction and western blotting. Oxygen consumption rate (OCR) and extracellular acidification rates (ECARs) were measured. ADM expression was upregulated in cisplatin-resistant EOC cells. ADM attenuated cisplatin-inhibited cell survival and cisplatin-induced apoptosis in sensitive EOC cells; knockdown of ADM enhanced cisplatin chemosensitivity of cisplatin-resistant EOC cells. ADM enhanced glycolysis in cisplatin-sensitive EOC cells; knockdown of ADM significantly inhibited glycolysis in cisplatin-resistant EOC cells. ADM significantly upregulated pyruvate kinase isozyme type M2 (PKM2) protein level, the key enzyme during glycolysis; PKM2 inhibitor significantly abolished the ADM-improved cell survival and ADM-inhibited apoptosis. ADM promoted proliferation and inhibited apoptosis of ovarian cancer cells through reprogramming of glucose metabolism, so as to promote cisplatin resistance. The study is expected to identify multidrug resistance markers of ovarian cancer and provide a target for the prevention and treatment of ovarian cancer, which is important for clinical translational research.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes.

Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Advances in research on the relationship between thymoquinone and pancreatic cancer.

Pancreatic cancer has one of the worst prognoses among the most common cancers in the world. Its characteristics include a high rate of metastasis and chemotherapeutic resistance, which present major challenges to the medical community. The potential anticancer effects of thymoquinone (TQ), which is the main bioactive compound of the black seeds of the Nigella sativa plant, have recently received widespread attention for their potential use in treating pancreatic cancer. TQ can inhibit cell proliferation, promote cancer cell apoptosis, inhibit cell invasion and metastasis, enhance chemotherapeutic sensitivity, inhibit angiogenesis, and exert anti-inflammatory effects. These anticancer effects predominantly involve the nuclear factor (NF)-κB, phosphoinositide 3 kinase (PI3K)/Akt, Notch, transforming growth factor (TGF)-β, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways as well as the regulation of the cell cycle, matrix metallopeptidase (MMP)-9 expression, and pyruvate kinase isozyme type M2 (PKM2) activity. TQ regulates the occurrence and development of pancreatic cancer at multiple levels and through multiple targets that communicate with each other. In this review, we summarize and discuss the analogs and carriers of TQ that have been developed in recent years. Given its multilevel anticancer effects, TQ may become a new therapeutic drug for treating pancreatic cancer in the future. This review presents a brief introduction to the research that has been conducted on TQ in relation to pancreatic cancer to provide a theoretical basis for future studies on the topic.

Osthole increases the radiosensitivity of hepatoma cells by inhibiting GSK-3β/AMPK/mTOR pathway-controlled glycolysis.

Osthole is a natural coumarin substance that has an inhibitory effect on hepatic cancer, but its radiosensitization effect on hepatoma cells has not been reported. This study aimed to investigate the effect of osthole. Human HCC-LM3 and SK-Hep-1 hepatoma cells were used and treated with or without osthole, irradiation, or their combination; the cell survival, migration, colony formation, DNA damage repair, intracellular lactic acid content, and glycolysis-related glycogen synthase kinase-3β (GSK-3β), p-GSK-3β, AMP-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), p-mTOR, glucose transporter-1 (GLUT-1), GLUT-3, and pyruvate kinase isozyme type M2 (PKM2) protein expressions were determined. Compared with the irradiation group, the osthole plus irradiation group could further decrease the survival rate, migration, colony formation, and DNA damage repair of both hepatoma cells, indicating a synergistic effect of the combination treatment. Moreover, the combination of osthole and irradiation could decrease the content of intracellular lactic acid, ratios of intracellular p-GSK-3β/GSK-3β and p-mTOR/mTOR proteins, and expressions of intracellular GLUT-1/3 and PKM2 proteins, and increase the ratio of intracellular p-AMPK/AMPK proteins. Osthole can increase the radiosensitivity of hepatoma cells, and its radiosensitization mechanisms may be related to glycolytic inhibition by attenuating the GSK-3β/AMPK/mTOR pathway.

Revisited Metabolic Control and Reprogramming Cancers by Means of the Warburg Effect in Tumor Cells.

Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a “metabolically abnormal system”. Carbohydrates are metabolically reprogrammed by its metabolizing and catabolizing enzymes in such abnormal cancer cells. Normal cells acquire their energy from oxidative phosphorylation, while cancer cells acquire their energy from oxidative glycolysis, known as the “Warburg effect”. Energy–metabolic differences are easily found in the growth, invasion, immune escape and anti-tumor drug resistance of cancer cells. The glycolysis pathway is carried out in multiple enzymatic steps and yields two pyruvate molecules from one glucose (Glc) molecule by orchestral reaction of enzymes. Uncontrolled glycolysis or abnormally activated glycolysis is easily observed in the metabolism of cancer cells with enhanced levels of glycolytic proteins and enzymatic activities. In the “Warburg effect”, tumor cells utilize energy supplied from lactic acid-based fermentative glycolysis operated by glycolysis-specific enzymes of hexokinase (HK), keto-HK-A, Glc-6-phosphate isomerase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, phosphofructokinase (PFK), phosphor-Glc isomerase (PGI), fructose-bisphosphate aldolase, phosphoglycerate (PG) kinase (PGK)1, triose phosphate isomerase, PG mutase (PGAM), glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase isozyme type M2 (PKM2), pyruvate dehydrogenase (PDH), PDH kinase and lactate dehydrogenase. They are related to glycolytic flux. The key enzymes involved in glycolysis are directly linked to oncogenesis and drug resistance. Among the metabolic enzymes, PKM2, PGK1, HK, keto-HK-A and nucleoside diphosphate kinase also have protein kinase activities. Because glycolysis-generated energy is not enough, the cancer cell-favored glycolysis to produce low ATP level seems to be non-efficient for cancer growth and self-protection. Thus, the Warburg effect is still an attractive phenomenon to understand the metabolic glycolysis favored in cancer. If the basic properties of the Warburg effect, including genetic mutations and signaling shifts are considered, anti-cancer therapeutic targets can be raised. Specific therapeutics targeting metabolic enzymes in aerobic glycolysis and hypoxic microenvironments have been developed to kill tumor cells. The present review deals with the tumor-specific Warburg effect with the revisited viewpoint of recent progress.

Natural shikonin and acetyl-shikonin improve intestinal microbial and protein composition to alleviate colitis-associated colorectal cancer

BackgroundColorectal cancer (CRC) and inflammatory bowel disease (IBD) are the most common diseases of human digestive system. Nowadays, the influence of the inflammatory microenvironment on tumorigenesis has become a new direction, and the exploration of relative molecular mechanism will facilitate the discovery and identification of novel potential anti-cancer molecules. MethodsNatural shikonin (SK) and acetyl-shikonin (acetyl-SK) was administered to azoxymethane (AOM)/dextran sodium sulphate (DSS)-induced colitis-associated colorectal cancer (CAC) mice model by gavage to investigate their therapeutic effects. Moreover, fresh feces and colon tissues were collected for determining the function of SK and acetyl-SK on the gut microbes and protein expression, respectively. ResultsBoth SK and acetyl-SK decreased AOM/DSS-induced CAC, and regulated the intestinal flora structure in CAC mouse model. They, especially SK, improved species richness, evenness and diversity of intestinal flora, recovered the upregulated ratio of Firmicutes to Bacteroidota (F/B ratio) which symbolizes gut microbiota dysbiosis. SK and its derivative increased the beneficial bacteria g__norank_f__Muribaculaceae, Lactobacillus, Lachnospiraceae_NK4A136_Group, and reduced those harmful ones including Ileibacterium and Coriobacteriaceae UCG–002. Notably, AOM/DSS caused significant increase in the abundance of Ileibaterium valens and g__norank_f__norank_o__Clostridia_UCG-014, which were not previously reported in studies of colonic inflammation or cancer, and the disorder was reversed by 20 mg/kg of SK. In our current study, the action of SK and acetyl-SK is dose-dependent, and 20 mg/kg SK exhibited the most effective functions, even better than the positive drug mesalazine.Moreover, differential proteomics and ELISA results showed that SK could recover the increase of pro-inflammatory cytokines (including IL-1β, IL-6 and TNF-α), the upregulation of pyruvate kinase isozyme type M2 (PKM2) and some other proteins (mainly concentrated in transcriptional mis-regulation in cancer and IL-17 signaling pathways), and the downregulation of Aldh1b1-Acc3-Maoa and Μgt2b34-Aldh1a1-Aldh1a7 involved in Wnt/β-catenin signaling pathway. ConclusionOur study identified SK and acetyl-SK, especially SK, as potential preventive agents for CAC through regulating both gut microbes and pathways involved in inflammation and cancer such as Wnt/β-catenin signaling pathway.

Chemoproteomics reveals berberine directly binds to PKM2 to inhibit the progression of colorectal cancer

SummaryColorectal cancer is one of the most serious tumors and berberine can inhibit the recurrence and transformation of colorectal adenoma into colorectal cancer. However, the direct binding target proteins of berberine in inhibiting colorectal cancer remain unclear. In this study, the chemical proteomics method was used and demonstrated that berberine is directly bound to pyruvate kinase isozyme type M2 (PKM2) in colorectal cancer cells. The triangular N-O-O triangular structure of berberine contributed to hydrophobic interaction with I119 amino acid residues and π-π interaction with F244 amino acid residues of PKM2 protein. Moreover, berberine was shown to inhibit the reprogramming of glucose metabolism and the phosphorylation of STAT3, down regulate the expression of Bcl-2 and Cyclin D1 genes, ultimately inhibiting the progression of colorectal cancer. This study uncovered the direct binding target protein and mechanism of berberine to improve metabolic reprogramming in colorectal cancer, which is helpful to guide the optimization of berberine.

Apigenin sensitizes radiotherapy of mouse subcutaneous glioma through attenuations of cell stemness and DNA damage repair by inhibiting NF-κB/HIF-1α-mediated glycolysis

We used radioresistant SU3-5R stem cells-inoculated subcutaneous glioma model to investigate the radiosensitization effect of apigenin. After treatment of glioma with apigenin 20 mg/kg for 12 days, irradiation 8 Gray twice or their combination, the tumor volume and weight were decreased, especially in the combination group. Apigenin inhibited the activities of glycolytic enzymes and expressions of nuclear factor kappa B (NF-κB) p65, hypoxia inducible factor-lα (HIF-1α), glucose transporter (GLUT)-1/3 and pyruvate kinase isozyme type M2 (PKM2) proteins in tumor tissues. After treatment of SU3-5R cells with apigenin 7.5 µM, the fluorescence intensity of CD133 positive cells was decreased, the percentage of cells with comet tails was increased, and the expressions of lipopolysaccharide-induced NF-κB p65, HIF-1α, GLUT-3 and PKM2 proteins were reduced. These results demonstrate that apigenin can sensitize the radiotherapy of glioma via the attenuations of cell stemness and DNA damage repair by inhibiting NF-κB/HIF-1α-mediated glycolytic enzymes and protein expressions.

PKM2 promotes angiotensin-II-induced cardiac remodelling by activating TGF-β/Smad2/3 and Jak2/Stat3 pathways through oxidative stress.

Hypertensive cardiac remodelling is a common cause of heart failure. However, the molecular mechanisms regulating cardiac remodelling remain unclear. Pyruvate kinase isozyme type M2 (PKM2) is a key regulator of the processes of glycolysis and oxidative phosphorylation, but the roles in cardiac remodelling remain unknown. In the present study, we found that PKM2 was enhanced in angiotensin II (Ang II)‐treated cardiac fibroblasts and hypertensive mouse hearts. Suppression of PKM2 by shikonin alleviated cardiomyocyte hypertrophy and fibrosis in Ang‐II‐induced cardiac remodelling in vivo. Furthermore, inhibition of PKM2 markedly attenuated the function of cardiac fibroblasts including proliferation, migration and collagen synthesis in vitro. Mechanistically, suppression of PKM2 inhibited cardiac remodelling by suppressing TGF‐β/Smad2/3, Jak2/Stat3 signalling pathways and oxidative stress. Together, this study suggests that PKM2 is an aggravator in Ang‐II‐mediated cardiac remodelling. The negative modulation of PKM2 may provide a promising therapeutic approach for hypertensive cardiac remodelling.

Apigenin increases radiosensitivity of glioma stem cells by attenuating HIF-1α-mediated glycolysis.

Apigenin, a natural flavonoid compound present in a variety of edible plants and health foods, has an anti-tumor effect and inhibits hypoxia inducible factor-lα (HIF-1α) expression in hypertrophic cardiac tissues. However, whether or not apigenin has a radiosensitization effect on glioma stem cells (GSCs) is unknown. Our present study aimed to investigate the effect of apigenin and its possible mechanisms. The human GSCs SU3 and its radioresistance line SU3-5R were treated with apigenin, radiation, or their combination, and the cell proliferation, migration, colony formation, and intracellular lactic acid and glycolytic related protein expressions were determined. Additionally, a cell model with hypoxia-induced HIF-1α expression was used and treated with apigenin. The current results displayed that the combination of apigenin and radiation could synergically reduce the viability, colony formation, and migration of the both GSCs. Moreover, this combination could also decrease the radiation-induced increments of glycolytic production lactic acid in the both GSCs and related protein expressions, including HIF-1α, glucose transporter (GLUT)-1/3, nuclear factor kappa B (NF-κB) p65, and pyruvate kinase isozyme typeM2 (PKM2). Further study confirmed that after treatment of hypoxia-cultured SU3 or SU3-5R cells with apigenin, the expression levels of HIF-1α, GLUT-1/3, NF-κB p65, and PKM2 proteins were reduced. These results demonstrated that apigenin could increase the radiosensitivity of GSCs and its radiosensitization mechanisms were attributable to the attenuation of glycolysis, which might result from the inhibition of HIF-1α expression and subsequent reductions of GLUT-1/3, NF-κB, and PKM2 expressions.

Fructose-1,6-bisphosphatase 2 represses cervical cancer progression via inhibiting aerobic glycolysis through promoting pyruvate kinase isozyme type M2 ubiquitination.

Growing evidence has shown that aerobic glycolysis, as a hallmark of cancer cells, plays a crucial role in cervical cancer. The aim of the study is to uncover whether fructose-1,6-bisphosphatase 2 (FBP2) is involved in cervical cancer progression via the aerobic glycolysis pathway. FBP2 levels were determined by quantitative PCR (qPCR) and western blotting. Cell growth viability and apoptosis were tested by cell counting kit-8 (CCK-8) and flow cytometry assays. Immunoprecipitation assay was applied for the detection of the FBP2 effect on pyruvate kinase isozyme type M2 (PKM2) ubiquitination. FBP2 level was decreased in cervical cancer, which is closely linked to shorter overall survival. FBP2 decreased cell growth and aerobic glycolysis and increased cell apoptosis, as well as decreased PKM2 expression and increased its ubiquitination level. The above-mentioned roles of FBP2 were weakened followed by PKM2 overexpression. FBP2 inhibited cervical cancer cell growth via inhibiting aerobic glycolysis by inducing PKM2 ubiquitination.

Rhubarb granule promotes diethylnitrosamine-induced liver tumorigenesis by activating the oxidative branch of pentose phosphate pathway via G6PD in rats

Ethnopharmacological relevanceRhubarb is a natural herbal medicine widely used clinically with numerous pharmacological activities including anti-cancer. Specifically, several studies reported that free anthraquinones from Rhubarb suppressed the proliferation of hepatoma cells. Nonetheless, recent studies revealed that Rhubarb caused hepatotoxicity in vivo, confirming its “two-way” effect on the liver. Therefore, the efficacy and safety of Rhubarb in the in vivo treatment of liver cancer should be further elucidated.Aim of the study: This study investigated the presence of hepatoprotection or hepatotoxicity of Rhubarb in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Material and methodsA total of 112 male Sprague-Dawley rats weighing 190–250 g were enrolled. The rats were induced hepatocarcinogenesis using diethylnitrosamine (0.002 g/rat) until 17 weeks. Starting at week 11, Rhubarb granules (4 g/kg and 8 g/kg) were intragastrically administered daily for 7 weeks. All rats were euthanized at week 20 and the livers were analyzed via non-targeted metabolomics analysis. We established hepatic glucose 6 phosphate (6PG) levels and glucose 6 phosphate dehydrogenase (G6PD) activities to assess the pentose phosphate pathway (PPP). And the liver injuries of rats were analyzed via histological changes, hepatic function, as well as hepatic protein levels of alpha-fetoprotein (AFP), pyruvate kinase isozyme type M2 (PKM2), and proliferating cell nuclear antigen (PCNA). Furthermore, polydatin (0.1 g/kg/d) as a specific inhibitor of G6PD was used to treat rats. Notably, their histological changes, hepatic function, hepatic 6PG levels, hepatic G6PD activities, PCNA levels, and PKM2 levels were recorded. ResultsNon-targeted metabolomics revealed that Rhubarb regulated the PPP in the liver of Rhubarb-DEN-treated rats. Besides, Rhubarb activated the oxidative branch of the PPP by activating G6PD (a rate-limiting enzyme in the oxidative PPP) in the liver of Rhubarb-DEN-treated rats. Meanwhile, Rhubarb promoted DEN-induced hepatocarcinogenesis. Moreover, polydatin attenuated the promoting effect of Rhubarb on DEN-induced hepatocarcinogenesis. ConclusionsRhubarb promoted DEN-induced hepatocarcinogenesis by activating the PPP, indicating that the efficacy and safety of Rhubarb in the treatment of liver cancer deserve to be deliberated.

Iridin Prevented Against Lipopolysaccharide-Induced Inflammatory Responses of Macrophages via Inactivation of PKM2-Mediated Glycolytic Pathways.

PurposeAbnormal glycolysis of immune cells contributed to the development of inflammatory response. Inhibition of this Warburg phenotype could be a promising strategy for preventing various inflammatory diseases. Iridin (IRD) is a natural isoflavone, and exerts anticancer, antioxidant, and anti-inflammatory effects. However, the underlying mechanism of IRD on acute inflammation remains unknown. In this study, the protective effects of IRD against lipopolysaccharide (LPS)-induced inflammation were investigated in murine macrophage RAW264.7 cells and in mice.MethodsThe inhibition of IRD on NO production in culture medium was detected by Griess assay while the levels of TNF-α, IL-1β, and MCP-1 were detected by ELISA assay. The effects of IRD on OCR and ECAR levels in LPS-treated macrophages were monitored by using Seahorse Analyzer. The apoptosis rate as well as the release of ROS and NO of RAW264.7 cells were analyzed by flow cytometric assay. The protective effects of IRD were investigated on LPS-induced inflammation in mice. The expressions of PKM2 and its downstream (p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2) in cells and in lung tissues were detected by Western blotting analysis.ResultsIRD treatment at the concentrations of 12.5–50 μM significantly inhibited the productions of TNF-α, IL-1β, MCP-1, and ROS, and suppressed the levels of glucose uptake and lactic acid in LPS-treated RAW264.7 cells. Oral administration with IRD (20–80 mg/kg) inhibited LPS-induced acute lung injury as well as inflammatory cytokine production in mice. Moreover, IRD targeted pyruvate kinase isozyme type M2 (PKM2) and suppressed its downstream p-JAK1, p-STAT1, p-STAT3, p-p65, iNOS, and COX2, which could be abolished by PKM2 agonist DASA-58 and antioxidant N-acetyl-L-cysteine, but partly be reversed by NF-κB activator CUT129 and JAK1 activator RO8191.ConclusionIRD alleviated LPS-induced inflammation through suppressing PKM2-mediated pathways, and could be a potential candidate for the prevention of inflammatory diseases.

Iminostilbene, a novel small-molecule modulator of PKM2, suppresses macrophage inflammation in myocardial ischemia–reperfusion injury

IntroductionInflammation is a key factor in myocardial ischemia/reperfusion (MI/R) injury. Targeting leucocyte-mediated inflammation is an important strategy for MI/R therapy. Iminostilbene (ISB), a simple dibenzoazepine small molecule compound, has a strong anti-neurodegenerative effect. However, no study has shown the cardioprotective effect of ISB. ObjectivesThis study aimed to investigate the role of ISB against MI/R injury and identify its molecular target. MethodsTo verify the cardiac protection of ISB in vivo and in vitro, we performed rat MI/R surgery and subjected inflammatory modeling of macrophages. In terms of molecular mechanisms, we designed and synthesized a small molecular probe of ISB and employed it on the click chemistry-activity-based protein profiling technique to fish for ISB targets in macrophages. To identify the target, we applied the competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. ResultsIn vivo, ISB showed robust anti-myocardial injury activity by improving cardiac function, reducing myocardial infarction, and inhibiting macrophage-mediated inflammation. In vitro, ISB strongly inhibited the transcription and the expression levels of inflammatory cytokines in macrophages. The pyruvate kinase isozyme type M2 (PKM2) was identified as the potential target of ISB through proteomic analysis and the competitive assay was performed for specific binding verification. Further thermodynamic and kinetic experiments showed that ISB was bound to PKM2 in a dose-dependent manner. Moreover, in terms of the biological function of ISB on PKM2, ISB reduced the expression of PKM2, thereby reducing the expression of HIF1α and the phosphorylation of STAT3. ConclusionThis study for the first time demonstrated that ISB targeted PKM2 to reduce macrophage inflammation thereby significantly alleviating MI/R injury.

Positive feedback in Cav-1-ROS signalling in PSCs mediates metabolic coupling between PSCs and tumour cells.

Caveolin‐1 (Cav‐1) is the principal structural component of caveolae, and its dysregulation occurs in cancer. However, the role of Cav‐1 in pancreatic cancer (PDAC) tumorigenesis and metabolism is largely unknown. In this study, we aimed to investigate the effect of pancreatic stellate cell (PSC) Cav‐1 on PDAC metabolism and aggression. We found that Cav‐1 is expressed at low levels in PDAC stroma and that the loss of stromal Cav‐1 is associated with poor survival. In PSCs, knockdown of Cav‐1 promoted the production of reactive oxygen species (ROS), while ROS production further reduced the expression of Cav‐1. Positive feedback occurs in Cav‐1‐ROS signalling in PSCs, which promotes PDAC growth and induces stroma‐tumour metabolic coupling in PDAC. In PSCs, positive feedback in Cav‐1‐ROS signalling induced a shift in energy metabolism to glycolysis, with up‐regulated expression of glycolytic enzymes (hexokinase 2 (HK‐2), 6‐phosphofructokinase (PFKP) and pyruvate kinase isozyme type M2 (PKM2)) and transporter (Glut1) expression and down‐regulated expression of oxidative phosphorylation (OXPHOS) enzymes (translocase of outer mitochondrial membrane 20 (TOMM20) and NAD(P)H dehydrogenase [quinone] 1 (NQO1)). These events resulted in high levels of glycolysis products such as lactate, which was secreted by up‐regulated monocarboxylate transporter 4 (MCT4) in PSCs. Simultaneously, PDAC cells took up these glycolysis products (lactate) through up‐regulated MCT1 to undergo OXPHOS, with down‐regulated expression of glycolytic enzymes (HK‐2, PFKP and PKM2) and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1). Interrupting the metabolic coupling between the stroma and tumour cells may be an effective method for tumour therapy.

TRAF4 acts as a fate checkpoint to regulate the adipogenic differentiation of MSCs by activating PKM2.

BackgroundMesenchymal stem cells (MSCs) selectively differentiate into adipocytes or osteoblasts, and several molecules control the fate determination of MSCs. Understanding these key checkpoints greatly contributes to the ability to induce specific MSC differentiation for clinical applications. In this study, we aimed to explore whether TNF receptor-associated factor 4 (TRAF4) affects MSC adipogenic differentiation, which we previously reported that could positively regulated the osteogenic differentiation.MethodsWestern blotting and Real-time Polymerase Chain Reaction were used to detected the expression pattern of TRAF4 during adipogenic differentiation. Lentivirus was constructed to regulate TRAF4 expression, and oil red O staining and Western blotting were used to assess its role in adipogenesis, which was confirmed in vivo by implanting an MSC-matrigel mixture into nude mice. Western blotting was used to detect the activated signaling pathways, and a specific inhibitor and agonist were used to clear the roles of the key signaling pathways. Additionaly, Co-Immunoprecipitation was conducted to find that Pyruvate kinase isozyme type M2 (PKM2) interacts with TRAF4, and to further explore their binding and functional domains. Finally, an RNA-binding protein immunoprecipitation assay and Western blotting were used to detect whether N6-methyladenosine mediates the decreased TRAF4 expression during adipogenic differentiation.FindingsThe results demonstrated that TRAF4 negatively regulates MSC adipogenesis in vitro and in vivo. Mechanistically, we revealed that TRAF4 binds to PKM2 to activate the kinase activity of PKM2, which subsequently activates β-catenin signaling and then inhibits adipogenesis. Furthermore, TRAF4 downregulation during adipogenesis is regulated by ALKBH5-mediated N6-methyladenosine RNA demethylation.InterpretationTRAF4 negatively regulates the adipogenesis of MSCs by activating PKM2 kinase activity, which may act as a checkpoint to fine-tune the balance of adipo-osteogenic differentiation, and suggests that TRAF4 may be a novel target of MSCs in clinical use and may also illuminate the underlying mechanisms of bone metabolic diseases.FundingThis study was supported by the National Natural Science Foundation of China (81871750 and 81971518) and the Science and Technology Project of Guangdong Province (2019B02023600 and 2017A020215070).

High glucose up-regulates MMP-1 expression in endothelial cells via mTORC2-PKM2 pathway.

To investigate the role of pyruvate kinase isozyme type M2 (PKM2) in regulating the expression of matrix metalloproteinase-1 (MMP-1) in human umbilical vein endothelial cells (HUVECs) under high glucose exposure and the underlying mechanisms. HUVECs were exposed to concentration gradient (5.5, 15.0, 30.0 mmol/L) of D-glucose for 72 h. Western blotting was used to detect the expression of PKM2, p-PKM2 Y105 as well as its upstream and downstream proteins. The formation of PKM2 tetramer/dimer was examined. Immunofluorescence was used to detect the translocation of PKM2. The specific siRNAs, TEPP-46, and rapamycin were used to analyze the regulatory relations among PKM2, mammalian target of rapamycin (mTOR) complex, and MMP-1. Co-immunoprecipitation was used to detect the interaction of mTOR complex and PKM2. High glucose exposure upregulated the level of p-PKM2 Y105, and promoted PKM2 dimer formation and nuclear translocation as well as the expression of MMP-1 and Rictor in HUVECs. Down-regulation of PKM2 expression or the treatment of TEPP-46 reversed the high glucose induced-upregulation of MMP-1. Inhibition of mTOR singnal pathway by rapacymin reversed the phosphorylation of PKM2 on tyrosine 105 and the expression of MMP-1 in high glucose-treated cells. Knockdown the expression of Rictor decreased the phosphorylation of PKM2 on tyrosine 105, while knockdown the expression of Raptor upregulated the phosphorylation of PKM2 on tyrosine 105 under high glucose condition. Co-immunoprecipitation indicated the direct interaction between Rictor and PKM2. The phosphorylation of PKM2 promotes high glucose-induced upregulation of MMP-1, and mTORC2 participates in the upregulation of PKM2 phosphorylation under high glucose condition.