Pyruvate Kinase Type M2 Research Articles

Point-of-care isoenzyme type M2 pyruvate kinase quick stool test for the detection of gastric cancer in Rwanda: A pilot study.

e18530 Background: Isoenzyme type M2 pyruvate kinase (M2-PK) can only be detected in gastrointestinal (GI) tumors. Detection of M2-PK on a small, portable platform with minimal training required, could offer a streamlined approach for the diagnosis of GI cancers especially in low-income countries (LIC's). We evaluated the feasibility of M2-PK stool test to detect gastric cancer in Rwanda. Methods: Participants with GI symptoms were screened and scheduled for upper endoscopy during the 5th Annual Rwanda Endoscopy Week. Participants who underwent gastric biopsy and provided a stool sample were enrolled. Three Rwandan medical centers participated. ScheBo M2-PK Quick test (Biotech AG, Giessen, Germany) was used to test fecal tumor M2-PK. Study approval was obtained from Dartmouth Health, UConn Health, and University of Rwanda IRB's. Descriptive statistics were estimated. Study is currently enrolling patients to reach the desired sample size (N = 110). Results: A total of 35 patients were consented; 26 patients did not have a biopsy, 1 did not provide a stool sample, 1 was lost to follow-up, and 7 were analyzable. 57.1% were male (n = 4) and 42.9% females (n = 3) with ages ranging from 50 to 71 years old. Males accounted for 66.6% of the cases of gastric cancer (n = 2). The most predominant symptoms reported were dyspepsia (66.7%), and weight loss (33.3%). Among those with hemoglobin levels available the mean was 8.63 g/dL (SD ± 0.71). Only one patient completed staging imaging with CT scans. Two patients with positive M2-PK stool tests had a proven diagnosis of gastric cancer (100%). One of them had a diagnosis of GIST. There was only 1 confirmed gastric adenocarcinoma in 5 patients with a negative M2-PK stool test, yet two patients were told to repeat the endoscopy procedure. Conclusions: A point-of-care (POC) quick stool test to screen gastric cancer is feasible in Rwanda. Study participation interest was high among Rwandan patients. Due to a lack of flexible endoscopes in LIC's, screening is restricted to patients living near regional or urban endoscopy centers, which contributes to the growing prevalence of gastric cancer and health disparities. Cancer control programs in LIC's can be implemented and maintained iteratively with realistic expectations and interventions tailored to the local population, its cultural values, and economic infrastructure. Thus, an emerging POC M2-PK stool test can triage patients to early medical attention when suspected gastric cancer.

Stool DNA testing for early detection of colorectal cancer: systematic review using the HTA Core Model® for Rapid Relative Effectiveness Assessment.

Stool DNA testing for early detection of colorectal cancer (CRC) is a non-invasive technology with the potential to supplement established CRC screening tests. The aim of this health technology assessment was to evaluate effectiveness and safety of currently CE-marked stool DNA tests, compared to other CRC tests in CRC screening strategies in an asymptomatic screening population. The assessment was carried out following the guidelines of the European Network for Health Technology Assessment (EUnetHTA). This included a systematic literature search in MED-LINE, Cochrane and EMBASE in 2018. Manufacturers were asked to provide additional data. Five patient interviews helped assessing potential ethical or social aspects and patients' experiences and preferences. We assessed the risk of bias using QUADAS-2, and the quality of the body of evidence using GRADE. We identified three test accuracy studies, two of which investigated a multitarget stool DNA test (Cologuard®, compared fecal immunochemical test (FIT)) and one a combined DNA stool assay (ColoAlert®, compared to guaiac-based fecal occult blood test (gFOBT), Pyruvate Kinase Isoenzyme Type M2 (M2-PK) and combined gFOBT/M2-PK). We found five published surveys on patient satisfaction. No primary study investigating screening effects on CRC incidence or on overall mortality was found. Both stool DNA tests showed in direct comparison higher sensitivity for the detection of CRC and (advanced) adenoma compared to FIT, or gFOBT, respectively, but had lower specificity. However, these comparative results may depend on the exact type of FIT used. The reported test failure rates were higher for stool DNA testing than for FIT. The certainty of evidence was moderate to high for Cologuard® studies, and low to very low for the ColoAlert® study which refers to a former version of the product and yielded no direct evidence on the test accuracy for ad-vanced versus non-advanced adenoma. ColoAlert® is the only stool DNA test currently sold in Europe and is available at a lower price than Cologuard®, but reliable evidence is lacking. A screening study including the current product version of ColoAlert® and suitable comparators would, therefore, help evaluate the effectiveness of this screening option in a European context.

Myeloid-Specific Pyruvate-Kinase-Type-M2-Deficient Mice Are Resistant to Acute Lung Injury.

Infiltration of polymorphonuclear neutrophils (PMNs) plays a central role in acute lung injury (ALI). The mechanisms governing PMN inflammatory responses, however, remain incompletely understood. Based on our recent study showing a non-metabolic role of pyruvate kinase type M2 (PKM2) in controlling PMN degranulation of secondary and tertiary granules and consequent chemotaxis, here we tested a hypothesis that Pkm2-deficient mice may resist ALI due to impaired PMN inflammatory responses. We found that PMN aerobic glycolysis controlled the degranulation of secondary and tertiary granules induced by fMLP and PMA. Compared to WT PMNs, Pkm2-deficient (Pkm2-/-) PMNs displayed significantly less capacity for fMLP- or PMA-induced degranulation of secondary and tertiary granules, ROS production, and transfilter migration. In line with this, myeloid-specific Pkm2-/- mice exhibited impaired zymosan-induced PMN infiltration in the peritoneal cavity. Employing an LPS-induced ALI mouse model, LPS-treated Pkm2-/- mice displayed significantly less infiltration of inflammatory PMNs in the alveolar space and a strong resistance to LPS-induced ALI. Our results thus reveal that PKM2 is required for PMN inflammatory responses and deletion of PKM2 in PMN leads to an impaired PMN function but protection against LPS-induced ALI.

Research progress of pyruvate kinase type M2 in hepatocellular carcinoma

Primary hepatocellular carcinoma is one of the most common high-grade malignant tumors in the world. Its incidence ranks fifth among malignant tumors in China, and various therapeutic measures have poor curative effect. Pyruvate kinase type M2 is a key enzyme in the glycolytic pathway, and its abnormal expression in liver cancer is closely related to the proliferation, metastasis, diagnosis, treatment, prognosis, as well as drug and radiation resistance. Therefore, multi-pathway targeted regulation of pyruvate kinase type M2 use is expected to become a new direction for the treatment of primary liver cancer.

Posttranslational modification of pyruvate kinase type M2 (PKM2): novel regulation of its biological roles to be further discovered.

PKM2, pyruvate kinase type M2, has been shown to play a key role in aerobic glycolysis and to regulate the malignant behaviors of cancer cells. Recently, PKM2 has been revealed to hold dual metabolic and nonmetabolic roles. Working as both a pyruvate kinase with catalytic activity and a protein kinase that phosphorylates its substrates, PKM2 stands at the crossroads of glycolysis and tumor growth. Recently, it was revealed that the catalytic activity of PKM2 can be regulated by its posttranslational modification (PTM). Several PTM types, including phosphorylation, methylation, acetylation, oxidation, hydroxylation, succinylation, and glycylation, have been gradually identified on different amino acid residues of the PKM2 coding sequence. In this review, we highlight the recent advancements in understanding PKM2 PTMs and the regulatory roles conferred by PTMs during anaerobic glycolysis in tumors.

Pyruvate Kinase M2 Coordinates Metabolism Switch between Glycolysis and Glutaminolysis in Cancer Cells.

SummaryCancer cells alter their nutrition metabolism to cope the stressful environment. One important metabolism adjustment is that cancer cells activate glutaminolysis in response to the reduced carbon from glucose entering into the TCA cycle due to inactivation of several enzymes in glycolysis. An important question is how the cancer cells coordinate the changes of glycolysis and glutaminolysis. In this report, we demonstrate that the pyruvate kinase inactive dimer PKM2 facilitates activation of glutaminolysis. Our experiments show that growth stimulations promote PKM2 dimer. The dimer PKM2 plays a role in regulation of glutaminolysis by upregulation of mitochondrial glutaminase I (GLS-1). PKM2 dimer regulates the GLS-1 expression by controlling internal ribosome entry site (IRES)-dependent c-myc translation. Growth stimulations promote PKM2 interacting with c-myc IRES-RNA, thus facilitating c-myc IRES-dependent translation. Our study reveals an important linker that coordinates the metabolism adjustment in cancer cells.

Enhanced patterns on intraoperative contrast-enhanced ultrasonography predict outcomes after curative liver resection in patients with hepatocellular carcinoma.

This study aimed to clarify what hepatocellular carcinoma (HCC) phenotype, as categorized by intraoperative contrast-enhanced ultrasonography (CEUS), showed a high risk of recurrence after hepatic resection. Patients who underwent initial curative hepatectomy with intraoperative CEUS for a single HCC nodule were retrospectively assigned to three patterns of fine (FI), vascular (VA), and irregular (IR) according to the maximum intensity projection pattern based on intraoperative CEUS. Staining was performed for Ki-67, pyruvate kinase type M2 (PKM2), and vascular endothelial growth factor (VEGF) to assess the tumor proliferative activity, tumor glucose metabolism, and angiogenesis, respectively. Of 116 patients, 18, 50, and 48 were assigned to the FI, VA and IR patterns, respectively. IR patients demonstrated a significantly worse prognosis for both the recurrence-free survival (RFS) and overall survival (OS) (P = 0.0002, 0.0262, respectively) than did patients with other patterns. A multivariate analysis revealed an IR pattern in intraoperative CEUS to be an independent predictive factor for a poor RFS, and major hepatectomy and an IR pattern were independent predictive factors for a poor OS. An IR pattern was closely related to the tumor size (≥ 3.3cm) and poor histological differentiation and showed a high Ki-67 index, low VEGF expression, and high PKM2 expression. IR-pattern HCCs as classified by intraoperative CEUS may be associated with a higher risk of recurrence and worse outcomes in HCC patients after hepatic resection than other patterns.

Experimental Study of Hepatocellular Carcinoma Treatment by Shikonin Through Regulating PKM2.

ObjectiveShikonin is a natural product with many activities, including anti-cancer effects. Pyruvate kinase type M2 (PKM2) plays a crucial role in the growth of tumor cells. However, the effect of shikonin on PKM2 in hepatocellular carcinoma (HCC) is unclear.MethodsCell viability, apoptosis level, glucose uptake, and lactate production were detected in HCC cells. Lentivirus-overexpressed and –shRNA of PKM2 were used to verify the key target of shikonin. A xenograft mouse model was used to detect the efficacy of shikonin and its combination with sorafenib in vivo.ResultsShikonin inhibited proliferation and glycolysis and induced apoptosis in HCC cells. Either PKM2-overexpressed or PKM2-shRNA alleviated or enhanced this effect. The results of CCK-8 showed that shikonin significantly inhibited cell viability of HCC cells. The levels of glucose uptake and lactate production were dramatically decreased by shikonin-treated. Results of flow cytometry and Western blot showed that the levels of apoptosis of HCC cells were significantly increased in a dose-dependent manner after shikonin treatment. In addition, shikonin enhanced the anti-cancer effect of sorafenib in vitro and in vivo. Our results showed that SK combined with sorafenib markedly inhibits tumor growth in HCC-transplanted nude mice compared to SK or sorafenib alone.ConclusionBy inhibiting PKM2, shikonin inhibited proliferation and glycolysis and induced cell apoptosis in HCC cells. The effect of shikonin on tumor cell proliferation, apoptosis and glycolsis will make it promising drug for HCC patients.

P16 and its putative interplay with metabolic factors in prostate cancer: An analysis based on public TCGA data

p16 is one of the most common tumour suppressor genes, mainly due to its genetic inactivation. However, the clinical significance of p16 in prostate cancer is not yet fully understood, and although p16 acts as a tumour suppressor gene, stress or oncogenic factors or alternative molecular events may overcome the role of p16 as a negative cell cycle regulator. p16 seems to be involved in the metabolic switch to glycolysis during tumorigenesis, possibly interacting with NADPH oxidase 4 (NOX4) and pyruvate kinase type M2 (PKM2), involved in energy metabolism, with differences depending on cell type. The aim of this study was to assess the putative crosstalk between p16, NOX4 and PKM2, with an involvement of miRNA‑mediated regulation, in prostate cancer. Transcriptome data from a cohort of 243 patients were extracted from The Cancer Genome Atlas (TCGA) database. An elevated p16 expression level was significantly associated a high Gleason score, decreasing with the score (P<0.0001). NOX4 and PKM2 expression exhibited a similar trend as p16, with higher values in the samples with Gleason scores of 9‑10 samples (P<0.0001 and P=0.02, respectively). Moreover, bioinformatics analysis by TargetScan revealed that miR‑625‑5p could bind to the 3'UTR of p16. A consequential pairing of the NOX4 and PKM2 target region with miR‑23a‑3p and miR‑122‑5p, respectively was also found. Of note, the miR‑625‑5p levels inversely correlated with p16 expression, miR‑23a‑3p and miR‑122 with NOX4 and PKM2, respectively (data not shown). Taken together, these data suggest an interplay between p16 and metabolic factors, such as NOX4 and PKM2, and a miRNA regulation, with a potential clinical impact for the development of novel therapeutic strategies in prostate tumours.

Suppressed "Warburg Effect" in Nasopharyngeal Carcinoma Via the Inhibition of Pyruvate Kinase Type M2-Mediated Energy Generation Pathway.

Warburg effect describes the abnormal energy metabolism in cancer cells and pyruvate kinase type M2 is involved in the regulation of this effect. In the current study, the role of pyruvate kinase type M2 in the initiation of Warburg effect in nasopharyngeal carcinoma cells was explored. The expression status of pyruvate kinase type M2 was detected in nasopharyngeal carcinoma samples and analyzed by different clinicopathological characteristics. Then the level of pyruvate kinase type M2 was suppressed in 2 nasopharyngeal carcinoma cell lines. The effects of pyruvate kinase type M2 inhibition on cell viability, apoptosis, invasion, glucose uptake, ATP generation, and glycolysis metabolism were determined. The data showed that the high expression of pyruvate kinase type M2 in nasopharyngeal carcinoma tissues was associated with the larger tumor size and advanced metastasis in the patients. The inhibition of pyruvate kinase type M2 resulted in the repressed proliferation and invasion in nasopharyngeal carcinoma cells, along with the increased apoptotic rate. The lack of pyruvate kinase type M2 function inhibited glucose uptake, while increased ATP generation in nasopharyngeal carcinoma cells. Moreover, the production of glycolysis metabolites, including pyruvic acid, lactate, citrate, and malate, was also suppressed by pyruvate kinase type M2 inhibition. At molecular level, the expressions of glucose transporter and hexokinase 2 were downregulated by pyruvate kinase type M2 inhibition, confirming the changes in glucose metabolism. Collectively, the current study demonstrated that the function of pyruvate kinase type M2 was important to maintain the proliferation and invasion of nasopharyngeal carcinoma cells, and the inhibition of the factor would antagonize nasopharyngeal carcinoma by blocking Warburg effect.

Biomarkers of inflammatory bowel disease activity.

The review presents data on calprotectin, lactoferrin, leukocytes labeled with isotope indium 111In, calgranulin C and pyruvate kinase type M2 - highly sensitive biomarkers to assess the severity of intestinal inflammation. Their importance in diagnostics, determination of treatment efficiency, including as predictors of recurrence of ulcerative colitis and Crohn's disease is shown.

Glucose and Lactate Transport in Pancreatic Cancer: Glycolytic Metabolism Revisited.

Membrane transporters fulfill essential roles in maintaining normal cellular function in health. In cancer, transporters likewise facilitate the aberrant characteristics typical of proliferating tumor cells. Pancreatic ductal adenocarcinoma is remarkable in its aggressiveness, and its metabolism is supported by a variety of membrane transporters. Glucose transporter 1 is upregulated in pancreatic cancer, enables rapid cellular uptake of glucose, and contributes to the invasiveness and metastatic ability of the disease. Likewise, the machinery of glycolysis, enzymes such as pyruvate kinase type M2 and hexokinase 2, is particularly active and ultimately leads to both lactate and tumor formation. Lactic acid channels and transporters include monocarboxylate transporters 1 and 4, connexin43, and CD147. In conjunction with glucose transporters and glycolytic metabolism, lactic acid transport helps perpetuate tumor cell metabolism and contributes to the formation of the unique tumor microenvironment in pancreatic cancer. These transporters may serve as potential therapeutic targets.

Overexpression of Pyruvate Kinase Type M2 (PKM2) Promotes Ovarian Cancer Cell Growth and Survival Via Regulation of Cell Cycle Progression Related with Upregulated CCND1 and Downregulated CDKN1A Expression.

BackgroundMany findings have shown that pyruvate kinase type M2 (PKM2) plays crucial roles in regulating the occurrence and development of various human cancers; however, its roles in ovarian cancer oncogenesis remain to be determined.Material/MethodsThe expression intensity of PKM2 in ovarian cancer tissues was examined by immunohistochemistry (IHC), and was then correlated to patient clinicopathologic characteristics. The roles of PKM2 in ovarian cancer cell proliferation, growth, and survival were examined by CCK-8, colony forming, and flow cytometry assays. The potentially involved molecular were then investigated by Western blot analysis.ResultsIHC results showed that PKM2 was overexpressed in 100 of 114 (87.7%) serous ovarian cancer tissues as compared with 50 cases of non-cancerous ovarian tissues, and was associated with tumor size ≥7.5 cm and <7.5 cm (p<0.05). Overexpression of PKM2 in SKOV3 and HEY ovarian cancer cells by transfection with PKM2 lentivirus vector led to increased cell proliferation, growth, and survival, which may be related with PKM2 being able to increase cell cycle progress: G1 stage decreased, whereas S stage significantly increased. In contrast, all functions of SKOV3 and HEY cells described above were reversed by knocked down PKM2 expression using siRNA. Further data showed that overexpressed PKM2 led to increased CCND1 and decreased CDKN1A expression, whereas underexpressed PKM2 led to decreased CCND1 and increased CDKN1A expression in ovarian cancer cells.ConclusionsPKM2 may play important roles in ovarian cancer development and may be a treatment target for this cancer.

The Diagnostic Value of Pyruvate Kinase Isoenzyme Type M2 for Biliary Tract Carcinoma. A Systematic Review and Meta-Analysis.

Growing evidence has shown that M2-PK is involved in cancer diagnosis and prognosis. The overall diagnostic accuracy of the pyruvate kinase isoenzyme type M2 (M2-PK) in biliary tract carcinoma (BTC) remains controversial. We performed a meta-analysis to evaluate the diagnostic value of M2-PK for BTC. The online PubMed, Cochrane, Web of Science, and Embase databases were searched for eligible studies published until August 8th, 2017. The Quality Assessment for Diagnostic Accuracy Studies 2 (QUADAS-2) was used to evaluate study quality. All statistical analyses were conducted with Stata 12.0. We included 7 studies from 5 articles with 410 patients with BTC and 438 controls. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and AUC for M2-PK in the diagnosis of BTC were 0.79 (95%CI 0.70-0.86), 0.81 (95%CI 0.71-0.88), 4.1 (95%CI 2.5-6.8), 0.26 (95%CI 0.16-0.41), 17.159 (95%CI 5.468-54.071), and 0.87 (95%CI 0.83-0.89), respectively. The same indicators assessed for CA19-9 were as follows: 0.70 (95%CI 0.62-0.77), 0.71 (95%CI 0.45-0.87), 2.38 (95%CI 1.2-4.73), 0.43 (95%CI 0.34-0.53), 6.28 (95%CI 2.4-16.44) and 0.73 (95%CI 0.69-0.77), respectively. Additionally, the diagnostic value of M2-PK varied based on characteristics of golden methods and different cut-off values. This meta-analysis showed that M2-PK had a better diagnostic accuracy for BTC compared with CA19-9, with moderate diagnostic performance. However, prospective studies are required to confirm its diagnostic value.

Pyruvate kinase type M2 contributes to the development of pancreatic ductal adenocarcinoma by regulating the production of metabolites and reactive oxygen species.

The majority of cancer cells maintain a high glycolytic activity and an increased lactate production, even in a well oxygenated environment. This phenomenon is known as the Warburg effect. Previous studies have revealed that various types of cancer selectively express the pyruvate kinaseM2 isoform(PKM2), and that PKM2 plays a pivotal role in the Warburg effect. Although elevated PKM2 levels have been observed in pancreatic cancer and other types of cancer, little is known about the biological function of PKM2. In this study, in order to examine the expression and role of PKM2 in pancreatic ductal adenocarcinoma(PDAC), we knocked down PKM2 in PDAC cells by introducing small interfering and short hairpin RNAs, and examined the gene expression profiles in the cells by microarray analysis. We analyzed the energy-producing pathways in the cells by XFe Extracellular Flux Analyzers, and detected intracellular metabolites by capillary electrophoresis time-of-flight mass spectrometry. We found that the RNAi-mediated knockdown of PKM2 diminished the proliferative, migratory and tumorigenic ability of the PDAC cell-lines. PKM2 knockdown also resulted in lower glycolytic activities and decreased levels of some intracellular metabolites, such as pyruvate and polyamine; however, it led to elevated levels of reactive oxygen species. Microarray analysis revealed the functional association between PKM2 and the expression of genes that drive the cell cycle. On the whole, the findings of this study demonstrate that PKM2 plays an important role in metabolic activities, as well as in the malignancy of PDAC cells.

Lapatinib Inhibits Breast Cancer Cell Proliferation by Influencing PKM2 Expression.

Pyruvate kinase type M2, which is expressed in multiple tumor cell types and plays a key role in aerobic glycolysis, also has nonglycolytic functions and can regulate transcription and cell proliferation. The results of this study show that epidermal growth factor receptor activation induces pyruvate kinase type M2 nuclear translocation. To further determine the relationship between pyruvate kinase type M2 and epidermal growth factor receptor, we analyzed pathological data from mammary glands and performed epidermal growth factor receptor/human epidermal growth factor receptor 2 knockdown to reveal that pyruvate kinase type M2 is associated with epidermal growth factor receptor and human epidermal growth factor receptor 2. Lapatinib is a small molecule epidermal growth factor receptor tyrosine kinase inhibitor that can inhibit epidermal growth factor receptor and human epidermal growth factor receptor 2, though its effect on pyruvate kinase type M2 remains elusive. Accordingly, we performed Western blotting and reverse transcription polymerase chain reaction and analyzed pathological data from mammary glands, with results suggesting that lapatinib inhibits pyruvate kinase type M2 expression. We further found that the antitumor drug lapatinib inhibits breast cancer cell proliferation by influencing pyruvate kinase type M2 expression, as based on Cell Counting Kit-8 analyses and pyruvate kinase type M2 overexpression experiments. Signal transducer and activator of transcription 3, which is a transcription factor-associated cell proliferation and the only transcription factor that interacts with pyruvate kinase type M2, we performed pyruvate kinase type M2 knockdown experiments in Human breast cancer cells MDA-MB-231 and Human breast cancer cells SK-BR-3 cell lines and examined the effect on levels of Signal transducer and activator of transcription 3 and phosphorylated Signal transducer and activator of transcription 3. The results indicate that pyruvate kinase type M2 regulates Signal transducer and activator of transcription 3 and phospho-Stat3 (Tyr705) expression. Together with previous reports, our findings show that lapatinib inhibits breast cancer cell proliferation by influencing pyruvate kinase type M2 expression, which results in a reduction in both Signal transducer and activator of transcription 3 and phosphorylated Signal transducer and activator of transcription 3.

Role of pyruvate kinase type M2 in the migration and invasion of renal cell carcinoma and possible mechanisms

Objective To analyze the alterations of glucose metabolism upon the knockdown of pyruvate kinase type M2 (PKM2) in renal cell carcinoma cell line ACHN, and explore the potential role of PKM2 in migration and invasion of renal cell carcinoma and the possible mechnisms. Methods The pLV lentivirus vector with PKM2-short hairpin RNA (shRNA) was constructed and the stably expressed cell line was generated. The effects of PKM2 knockdown on proliferation of ACHN cells were detected by cell counting kit-8 (CCK-8) assay, migration and invasion ability detected by Transwell assay, and alteration of Warburg effect detected by measuring glucose uptake and lactate production. Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were performed to examine the expression of E-cadherin and Vimentin after the knockdown of PKM2. Results CCK-8 assay showed that cell viability of shPKM2 group was significantly lower than that in control group. The glucose uptake and lactate production in control group were (3.493±0.179) and (3.553±0.185) nmol/106 cells/min repectively, while those in shPKM2 group were (1.457±0.081) and (5.820±0.352) nmol/106 cells/min (P=0.001, P=0.005). The number of migrating cells in control and shPKM2 groups was 62±6 and 39±5 (P=0.001), and that of invasive cells in control and shPKM2 groups was 40±7 and 26±6 (P=0.009), respectively. The relative expressions of E-cadherin protein and mRNA in shPKM2 group (1.560±0.110 and 2.534±0.342) were higher than that in control group (0.833±0.070 and 1.312±0.284) by Western blotting and RT-qPCR (P=0.001, P=0.009). The relative expressions of Vimentin protein and mRNA in shPKM2 group (0.340±0.110 and 1.631±0.123) were lower than that in control group (0.653±0.100 and 3.530±0.511, P=0.022, P=0.003). Conclusion Knockdown of PKM2 can impair the glucose metabolism and suppress cell proliferation, migration and invasion in vitro and in vivo. Knockdown of PKM2 decreases the expression of Vimentin, and increase the expression of E-cadherin. Key words: Clear cell renal cell carcinoma; Pyruvate kinase type M2; Migration; Invasion; Epithelial-mesenchymal transition

Knockdown of pyruvate kinase type M2 suppresses tumor survival and invasion in osteosarcoma cells both in vitro and in vivo

Osteosarcoma (OS) is the mostly diagnosed primary bone malignancy. Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. In the present study, the effect of PKM2 knockdown on the proliferation and migration of OS cells were assessed both in vitro and in vivo. Small hairpin RNA (shRNA) technology were employed to suppress the expression of PKM2 in MG-63 and Saos-2 cell lines. In vitro, shRNA-mediated knockdown of PKM2 efficiently inhibited cell proliferation, and induced G1 cell cycle arrest and apoptosis in both cell lines, which was associated with decreased expressions of cyclin D1 and Bcl-2 as well as increased expressions of Bax, cleaved-caspase-3, and cleaved-PARP. The invasion and migration potential of OS cell lines were also inhibited by PKM2 knockdown through the regulating effect of PKM2 on MMP-2 and VEGF signaling. In vivo, knockdown of PKM2 decelerated tumor growth rate and induced structure deterioration in tumor tissues. The current study for the first time showed that the activity of PKM2 was indispensable for the development and metastasis of OS, thereby providing the basic information for the future development of PKM2-based anti-OS therapies.

Effects of shRNA-Mediated Silencing of PKM2 Gene on Aerobic Glycolysis, Cell Migration, Cell Invasion, and Apoptosis in Colorectal Cancer Cells.

This study aims to explore the effects of shRNA-mediated silencing on Pyruvate kinase type M2 (PKM2) gene during aerobic glycolysis in colorectal cancer (CRC) cells. CRC tissues and adjacent normal tissues were obtained from 136 patients diagnosed with qRT-PCR, Western blotting, and immunohistochemistry (IHC) were performed to detect mRNA and protein expressions of PKM2. CRC cells were divided into a blank, vector, and PKM2-shRNA groups. Hexokinase (HK) and PKM2 activity were both determined by glucose-6-phosphate dehydrogenase (G-6-PD) coupled colorimetric assay and enzyme coupling rate method. The extracellular lactate concentration was measured by ultraviolet spectrophotometer and caspase activity was measured using spectrophotometry. The proliferation, cell cycle, apoptosis, invasion, and migration of CRC cells were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, transwell assay, and scratch test. Three groups of nude mice were injected with 0.2 mL single-cell suspension from the blank, vector, and PKM2-shRNA groups, respectively. PKM2 protein content in CRC tissues was higher than that in adjacent normal tissues. Results showed that the PKM2-shRNA group exhibited significantly lower mRNA and protein expressions of PKM2, decreased PKM2 activity, reduced lactate metabolism level, increased cell apoptosis rate, elevated caspase-3 and caspase-9 activity, weakened proliferation, and a reduction in cell invasion and migration ability compared to the vector and blank groups. The optical density (OD) value was lower in the PKM2-shRNA group than in the blank and vector groups. These findings indicate that shRNA-mediated silencing of PKM2 gene promotes apoptosis and inhibits aerobic glycolysis, proliferation, migration, and invasion in CRC cells. J. Cell. Biochem. 118: 4792-4803, 2017. © 2017 Wiley Periodicals, Inc.

Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis.

Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy.