Overexpression Of Pyruvate Kinase M2 Research Articles

PKM2 interacts with and phosphorylates PHB2 to sustain mitochondrial quality control against septic cerebral-cardiac injury.

Sepsis induces profound disruptions in cellular homeostasis, particularly impacting mitochondrial function in cardiovascular and cerebrovascular systems. This study elucidates the regulatory role of the Pyruvate Kinase M2 (PKM2)- Prohibitin 2 (PHB2) axis in mitochondrial quality control during septic challenges and its protective effects against myocardial and cerebral injuries. Employing LPS-induced mouse models, we demonstrate a significant downregulation of PKM2 and PHB2 in both heart and brain tissues post-sepsis, with corresponding impairments in mitochondrial dynamics, including fission, fusion, and mitophagy. Overexpression of PKM2 and PHB2 not only restores mitochondrial function, as evidenced by normalized ATP production and membrane potential but also confers resistance to oxidative stress by mitigating reactive oxygen species generation. These cellular mechanisms translate into substantial in vivo benefits, with transgenic mice overexpressing PKM2 or PHB2 displaying remarkable resistance to sepsis-induced cardiomyocyte and neuronal apoptosis, and organ dysfunction. Our findings highlight the PKM2-PHB2 interaction as a novel therapeutic target for sepsis, providing a foundation for future research into mitochondrial-based interventions to treat this condition. The study's insights into the molecular underpinnings of sepsis-induced organ failure pave the way for potential clinical applications in the management of sepsis and related pathologies.

2′-Hydroxycinnamaldehyde mitigates acetaminophen-induced acute liver injury by inhibiting the Warburg effect-mediated pyroptosis via the PKM2-NLRP3 axis

Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver injury. However, the underlying mechanisms appear to be multifaceted and there are few effective treatments. In this study, we used the 2′-Hydroxycinnamaldehyde (HCA), an active ingredient extracted from cinnamon stem bark to evaluate the therapeutic effect against APAP-induced ALI. The results indicated that pyruvate kinase M2 (PKM2)-mediated aerobic glycolysis may activate NLRP3 inflammasome in APAP-induced ALI, However, this process was prevented by HCA. Additionally, overexpression of PKM2 by adeno-associated virus (AAV9-PKM2) in vivo and plasmid (PKM2-OE) in vitro reversed the inhibition of NLRP3 inflammasome by HCA. Intriguingly, the HCA-triggered repressive pyroptosis was mediated through dephosphorylation and nuclear translocation of PKM2. Furthermore, the therapeutic effect of HCA on APAP-induced ALI was achieved by partially stimulating the Akt-mTOR signaling pathway, thus inhibiting the PKM2-NLRP3 axis. These findings elucidate that a potential drug to the clinical treatment of APAP overdose by HCA.

Effect of PKM2 on Osteogenic and Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Myeloma Bone Disease

To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs. BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs. Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2. The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Cynaropicrin Induces Cell Cycle Arrest and Apoptosis by Inhibiting PKM2 to Cause DNA Damage and Mitochondrial Fission in A549 Cells.

Metabolic reprogramming is critical for tumorigenesis. Pyruvate kinase M2 (PKM2) is overexpressed in lung carcinoma cells and plays a critical role in the Warburg effect, making the enzyme a research hotspot for anticancer drug development. Cynaropicrin (CYN), a natural sesquiterpene lactone compound from artichoke, has received increasing consideration due to its consumable esteem and pharmacological properties. Our data reveal that CYN not only inhibited the purified PKM2 activity but also decreased the cellular PKM2 expression in A549 cells. The inhibition of PKM2 leads to the upregulation of p53 and the downregulation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), and subsequently causes the cell cycle arrest. Additionally, CYN inhibits the interaction of PKM2 and Nrf2, resulting in the impairment of cellular antioxidant capacity, induction of oxidative stress, and mitochondrial damages. Overexpression of PKM2 attenuates the CYN-induced DNA damage, mitochondrial fission, and cell viability. Thus, targeting PKM2 provides an original mechanism for understanding the pharmacological impact of CYN and assists in the further development of CYN as an anticancer agent.

LncRNA OIP5-AS1 reduces renal epithelial cell apoptosis in cisplatin-induced AKI by regulating the miR-144-5p/PKM2 axis

BackgroundThe abnormal expression of long non-coding RNA (lncRNA) Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) has been observed in many human cancers and the underlying mechanisms have been well studied. However, the function of OIP5-AS1 in acute kidney injury (AKI) remains unclear. MethodsTo explore the role of OIP5-AS1 in the progression of AKI, the cisplatin-induced AKI mouse and cell model were established. To confirm the potential protective effect of OIP5-AS1 during cisplatin-induced AKI, rescue experiments were performed. Targetscan was used to predict the potential targets of miR-144-5p. To further determine whether the effect of miR-144-5p during cisplatin-induced AKI was mediated by PMK2, the recuse experiments using PMK2 overexpressing vector was applied. ResultsOIP5-AS1 was significantly downregulated both in cisplatin-induced AKI mice and human renal tubular cell line HK-2 cells. Moreover, overexpression of OIP5-AS1 efficiently promoted cell growth and reduced cisplatin-induced apoptosis of HK-2 cells. Furthermore, OIP5-AS1 was identified as a sponge of miR-144-5p, and upregulation of miR-144-5p could significantly reverse overexpression of OIP5-AS1-induced protective effect on the damage of cisplatin to HK-2 cells. In addition, pyruvate kinase M2 (PKM2) was found to be a direct target of miR-144-5p, and overexpression of PKM2 efficiently reversed the effect of miR-144-5p mimics on the damage in cisplatin-stimulated HK-2 cells. ConclusionsOIP5-AS1 reduced the apoptosis of cisplatin-stimulated renal epithelial cells by targeting the miR-144-5p/PKM2 axis, which extended the regulatory network of lncRNAs in cisplatin-induced AKI and also provided a novel therapeutic target for AKI treatment.

Succinate Dehydrogenase-Deficient Renal Cancer Featuring Fructose-1,6-Biphosphatase Loss, Pyruvate Kinase M2 Overexpression, and SWI/SNF Chromatin Remodeling Complex Aberrations: A Rare Case Report

Succinate dehydrogenase (SDH)‐deficient renal cancer is a rare renal cancer subtype recently accepted by the World Health Organization as a unique subtype of renal cell carcinoma (RCC). Here we report a case of 17‐year‐old man. The detailed evaluation indicated occurrence of the SDHB‐deficient RCC. The genetic testing revealed no germline mutation in SDH genes. Immunohistochemistry showed SDHB deficiency, overexpression of pyruvate kinase M2 and dramatic downregulation of fructose‐1,6‐bisphosphatase metabolic enzymes, and unaltered levels of phosphorylated AMP‐activated protein kinase and mammalian target of rapamycin. Strong upregulation of INI1 and BRG1 and overexpression of BAF180, subunits of SWI/SNF ATP‐dependent chromatin remodeling complex, were also found. The identified tumor pathologically did not resemble clear cell renal cell carcinoma (ccRCC), but some metabolic alterations are common for both cancer types. Thus, we postulate that the phenotypical differences between ccRCC and SDHB‐deficient RCC may be related to distinct molecular and metabolic alterations.Implications for PracticeSuccinate dehydrogenase (SDH)‐deficient renal cell carcinoma (RCC) is a rare renal tumor occurring even in young patients. Until now, in all described and genetically tested cases, mutations and deletions in SDH genes have been found. This article describes SDHB‐deficient RCC without any germline mutations in SDH genes. Therefore, genetic analysis for germline mutations in SDH genes in SDH‐deficient RCC, especially in young individuals, should be strongly recommended, although as of now it is not obligatory. This knowledge will allow improvement of patient monitoring including both disease recurrence and new cancer appearance.

Pyruvate Kinase M2 Contributes to TLR-Mediated Inflammation and Autoimmunity by Promoting Pyk2 Activation.

Toll-like receptors (TLRs) play critical roles in regulating the abnormal activation of the immune cells resulting in the pathogenesis of inflammation and autoimmune diseases. Pyruvate kinase M2 (PKM2), which governs the last step of glycolysis, is involved in multiple cellular processes and pathological conditions. However, little is known about the involvement of PKM2 in regulating TLR-mediated inflammation and autoimmunity. Herein, we investigated the role of PKM2 in the activation of the TLR pathways and the pathogenesis of inflammation and autoimmune diseases. The activation of TLR4, TLR7 and TLR9 pathways was found to induce the up-regulation of PKM2 expression in macrophages, dendritic cells (DCs) and B cells. The over-expression of PKM2 promotes the activation of TLR4, TLR7 and TLR9 pathways while interference with the PKM2 expression or the addition of the PKM2 inhibitor (PKM-IN) markedly inhibited the activation of TLR4, TLR7 and TLR9 pathways. Mechanistically, PKM2 augmented the activation of TLR4, TLR7 and TLR9 pathways by promoting the activation of the proline-rich tyrosine kinase 2 (Pyk2). Intriguingly, the PKM2 inhibitor PKM2-IN significantly protected the mice from the endotoxic shock mediated by the TLR4-agonist LPS. Additionally, it alleviated the progression in the TLR7-agonist imiquimod-mediated lupus mice and spontaneous lupus MRL/lpr mice. Moreover, PKM2 expression was highly elevated in the monocytes, DCs and B cells from systemic lupus erythematous (SLE) patients compared with those from the healthy donors. Besides, the PKM2 expression level was positively correlated with the degree of activation of these immune cells. In summary, PKM2 contributed to TLR-mediated inflammation and autoimmunity and can be a valuable target to control inflammation and autoimmunity.

Isovitexin potentiated the antitumor activity of cisplatin by inhibiting the glucose metabolism of lung cancer cells and reduced cisplatin-induced immunotoxicity in mice

The increased resistance and toxicity have become the main causes of chemotherapy failure for treating lung cancer. The combination of chemotherapeutic drugs with other agents has been recognized as a promising strategy to overcome these difficulties. Isovitexin (IVT) is a well-known flavone C-glycoside found in many plants and has attracted wide attention due to its obvious antitumor and antioxidant effects. In this study, we investigated the synergistic effects of IVX and cisplatin (DDP) in non-small cell lung cancer (NSCLC) A549 and H1975 cells. The results showed that the combined treatment with IVT and DDP markedly inhibited proliferation and induced apoptosis of the two NSCLC cells. Using a mouse model of A549 xenograft, IVT potentiated the inhibition of DDP on tumor growth, but reduced DDP-induced hepatotoxicity and nephrotoxicity in mice. Remarkedly, IVT promoted lipopolysaccharide (LPS)- and lectin- stimulated splenocyte proliferation, and enhance cytotoxic T lymphocyte (CTL) and natural killer (NK) cell activities as well as the production of IL-2 and TNF-α. Furthermore, IVT significantly reduced glucose uptake, lactate production, and ATP production, and downregulated the protein expressions of pyruvate kinase M2 (PKM2)-mediated pathway in both A549 and H1975 cells. After the over-expression of PKM2 in the NSCLC cells, the synergistic antitumor effect of IVT and DDP was markedly weakened. Therefore, IVT not only inhibited cell proliferation and glucose metabolism via downregulating the expression of PKM2 to enhance the antitumor activity of DDP against lung cancer cells, and improved DDP-induced immunotoxicity in mice. It also presented a novel strategy to enhance the anti-tumor effect of platinum-based chemotherapy against NSCLC.

Pyruvate Kinase M2 Mediates Glycolysis in the Lymphatic Endothelial Cells and Promotes the Progression of Lymphatic Malformations

Metabolism plays a pivotal role in the formation of the lymphatic vasculature. Pyruvate kinase M2 (PKM2) is typically a metabolic marker of proliferating cells and maintains the growth of vascular endothelial cells. In this study, the potential status of PKM2 in lymphatic endothelial cells and the pathogenesis of lymphatic malformations (LMs) was investigated. The glycolysis index, including glucose uptake, ATP, and lactate production, stayed at a relatively high level in human dermal lymphatic endothelial cells (HDLECs) compared with human umbilical vein endothelial cells, whereas the inhibition of PKM2 by shikonin or PKM2 knockdown significantly suppressed glycolysis, migration, tubular formation, and invasion of HDLECs. Moreover, compared with lymphatic vessels in healthy skin, lymphatic vessels of LMs expressed PKM2 highly, and this expression correlated with infection of LMs. Meanwhile, the overexpression of PKM2 in HDLECs strengthened the proliferation, migration, tubular formation, and invasion of HDLECs. The findings from further experiments in a rat LM model support that targeting PKM2 by shikonin significantly impedes the progression of LMs, even in an infected LM rat model. Taken together, these results indicate that PKM2 plays a pivotal role in the activation of LECs and promotes the progression of LMs, whereas the inhibition of PKM2 can effectively suppress the pathogenesis of LM lesions in the rat model.

PKM2 Drives Hepatocellular Carcinoma Progression by Inducing Immunosuppressive Microenvironment.

Background and AimsPyruvate kinase M2 (PKM2) is an essential regulator of the Warburg effect, but its biological function promoting immune escape of hepatocellular carcinoma (HCC) is unclear.MethodsGEPIA web tool and immunohistochemistry (IHC) analysis were employed to evaluate the clinical relevance of PKM2 in HCC patients. Both in vitro CCK-8, colony formation, and transwell assays, and in vivo xenografts were performed to evaluate the malignancy of HCC cells. PKM2 and PD-L1 levels were examined by Western blot, qRT-PCR, and IHC. The role of PKM2 on in vivo immune response was also investigated.ResultsPKM2 was significantly upregulated in HCC and associated with a poor prognosis of HCC patients. Knockdown of PKM2 inhibited in vitro proliferation, migration, and invasion of HCC cells, as well as in vivo tumor growth. Strikingly, PKM2 showed a strong correlation with the expression of immune inhibitory cytokines and lymphocyte infiltration in HCC. The overexpression of PKM2 sensitized HCC to immune checkpoint blockade, which enhanced IFN-γ positive CD8 T cells in HCC mice models.ConclusionPKM2 might be a predictor and a potential therapeutic target for immune checkpoint inhibitors in HCC.

Antitumor Effect of miR-1294/Pyruvate Kinase M2 Signaling Cascade in Osteosarcoma Cells.

BackgroundMicroRNAs (miRNAs) can act as negative regulators of gene expression, and play a crucial role in cancer progression. The aim of this study was to investigate the role of miR-1294/pyruvate kinase M2 (PKM2) axis in osteosarcoma cells in vitro and in vivo.MethodsThe function of miR-1294 and its association with PKM2 in osteosarcoma cells were studied by real-time PCR, CCK-8, Western blot, scratch test, transwell assay, flow cytometry, and dual-luciferase reporter assays. The effect of miR-1294 on tumor growth in vivo was evaluated in a subcutaneous xenograft model of osteosarcoma.ResultsmiR-1294 was downregulated in osteosarcoma cells. Forced overexpression of miR-1294 inhibited cell proliferation, migration, and invasion, and induced G0/G1 arrest and apoptosis. Consistently, protein expression levels of proliferating cell nuclear antigen, c-Myc, cyclin D1, active matrix metalloproteinase 2, and active matrix metalloproteinase 9 were decreased, and cleaved caspase 3 and cleaved PARP were increased following miR-1294 overexpression. Moreover, we demonstrated that PKM2 was a target of miR-1294 in osteosarcoma cells, and the effects caused by miR-1294 mimic were reversed by the overexpression of PKM2. Furthermore, we found that upregulation of miR-1294 inhibited tumorigenesis of osteosarcoma cells in vivo, which was accompanied by downregulation of PKM2.ConclusionOur results revealed that miR-1294/PKM2 signaling cascade exerts important roles in the regulation of tumor progression, implying that this pathway may serve as a potential therapeutic target in osteosarcoma.

Патогенетические механизмы развития гиперплазии эндометрия в репродуктивном возрасте

Objective. To analyze pathogenetic mechanisms underlying the development of endometrial hyperplasia in women of reproductive age. Patients and methods. We have examined 143 women of reproductive age with endometrial hyperplasia (EH). Study participants were divided into three groups: Group I included EH patients without atypia; Group II included patients with atypical hyperplasia of the endometrium; Group III (control group) comprised 56 women with abnormal uterine bleeding, in whom we excluded adenomyosis, uterine fibroids, endometrial hyperplasia, endometrial cancer, and iatrogenic causes of uterine bleeding. Genomic DNA was isolated using phenol-chloroform extraction. Real-time polymerase chain reaction (RT-PCR) was used to detect microRNA-210, -18a, -221, and -222. The detection of tumor pyruvate kinase M2 was performed using the ScheBo® Tumor M2-PK kit designed for quantitative assessment of this metabolic cancer marker in plasma and endometrial tissue samples. Results. Significant risk factors triggering the pathogenetic mechanism of EH development in reproductive age include extragenital disorders (obesity, thyroid diseases, diseases of the urinary system, hypertension) and gynecological diseases (pelvic inflammatory diseases, adenomyosis, benign breast dysplasia, uterine fibroids). Alterations affecting estrogen receptors lead to changes in microRNA messengers, which, in turn, affect target genes and cause changes in the adaptive abilities of the cell. Expression of pyruvate kinase M2 in this chain confirms proapoptotic changes in the cell and the risk of its atypia. Conclusion. The pathogenesis of EH is based on the following factors: polymorphism of the ERS1 and PRG genes, increased expression of miRNA-210, -18a, and -222, decreased expression of miRNA-221, and overexpression of pyruvate kinase M2. Key words: endometrial hyperplasia, miRNA, pyruvate kinase M2, progesterone receptors, estrogen receptors

Knockdown of Pyruvate Kinase M2 Inhibits Cell Proliferation, Metabolism, and Migration in Renal Cell Carcinoma.

Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. However, the molecular mechanism underlying the function of PKM2 in renal cancer is undetermined. Here, we reveal the overexpression of PKM2 in the proximal tubule of renal tumor tissues from 70 cases of patients with renal carcinoma. The functional role of PKM2 in human renal cancer cells following small-interfering RNA-mediated PKM2 knockdown, which retarded 786-O cell growth was examined. Targeting PKM2 affected the protein kinase B (AKT)/mechanistic target of the rapamycin 1 (mTOR) pathway, and downregulated the expression of glycolytic enzymes, including lactate dehydrogenase A and glucose transporter-1, and other downstream signaling key proteins. PKM2 knockdown changed glycolytic metabolism, mitochondrial function, adenosine triphosphate (ATP) level, and intracellular metabolite formation and significantly reduced 786-O cell migration and invasion. Acridine orange and monodansylcadaverine staining, immunocytochemistry, and immunoblotting analyses revealed the induction of autophagy in renal cancer cells following PKM2 knockdown. This is the first study to indicate PKM2/AKT/mTOR as an important regulatory axis mediating the changes in the metabolism of renal cancer cells.

Dihydroartemisinin represses esophageal cancer glycolysis by down-regulating pyruvate kinase M2

Esophageal cancer, especially esophageal squamous cell carcinoma (ESCC) threatens so many lives in China every year. Traditional treatment of ESCC has usually been disappointing. The development of novel therapy is worth investigation. We have previously demonstrated that dihydroartemisinin (DHA) has anticancer effect on esophageal cancer. However, the mechanism has not been completely known. In this present study, we explored the effect of DHA on cancer cell glycolysis, also known as Warburg effect. Pyruvate kinase M2 (PKM2) is a key regulatory factor of glycolysis, and our results showed that it is significantly overexpressed in patients with ESCC and ESCC cell lines. In DHA treatment cells, PKM2 was down-regulated and lactate product and glucose uptake were inhibited. Overexpression of PKM2 by lentiviral transfection abrogated the inhibition effect of DHA. These results suggested that DHA might repress esophageal cancer glycolysis partly by down-regulating PKM2 expression. We believe that DHA might be a prospective agent against esophageal cancer.

Prognostic implications of markers of the metabolic phenotype in human cutaneous melanoma.

Reprogramming of energy metabolism to enhanced aerobic glycolysis has been defined as a hallmark of cancer. To investigate the role of the mitochondrial proteins, β-subunit of the H+ -ATP synthase (β-F1-ATPase), and heat-shock protein 60 (HSP60), and the glycolytic markers, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as the bioenergetic cellular (BEC) index, in melanoma progression. The expression of energy metabolism proteins was assessed on a set of different melanoma cells representing the natural biological history of the disease: primary cultures of melanocytes, radial (WM35) and vertical (WM278) growth phases, and poorly (C81-61-PA) and highly (C8161-HA) aggressive melanoma cells. Cohorts of 63 melanocytic naevi, 55 primary melanomas and 35 metastases were used; and 113 primary melanoma and 33 metastases were used for validation. The BEC index was significantly reduced in melanoma cells and correlated with their aggressive characteristics. Overexpression of HSP60, GAPDH and PKM2 was detected in melanoma human samples compared with naevi, showing a gradient of increased expression from radial growth phase to metastatic melanoma. The BEC index was also significantly reduced in melanoma samples and correlated with worse overall and disease-free survival; the multivariate Cox analysis showed that the BEC index (hazard ratio 0·64; 95% confidence interval 0·4-1·2) is an independent predictor for overall survival. A profound alteration in the mitochondrial and glycolytic proteins and in the BEC index occurs in the progression of melanoma, which correlates with worse outcome, supporting that the alteration of the metabolic phenotype is crucial in melanoma transformation.

Overexpression of Pyruvate Kinase M2 in Tumor Tissues Is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, with a high degree of malignancy and a poor prognosis. The aim of this study was to investigate the relationship between expression of pyruvate kinase M2 (PKM2) and prognosis in patients with HCC. The expression levels of PKM2 and PKM1 in 86 cases of HCC were detected by immunohistochemistry. An H score was used to evaluate the expression of PKM, and all patients were further divided into PKM high-expression and PKM low-expression groups. The relationship between PKM2 expression and the clinicopathological parameters and prognosis of patients were subsequently analyzed. Our data suggested that the expression level of PKM2 was significantly higher in HCC tissues than in adjacent tissues and the negatively expression of PKM1 in HCC tissues. Kaplan-Meier analysis revealed that PKM2 expression was strongly associated with survival in HCC patients (P = 0.001). The patients in the PKM2 high-expression group had significantly shorter survival times than the patients in the PKM2 low-expression group (hazard ratio for death, 2.358; 95% confidence interval [1.156, 4.812]; P = 0.018). In conclusion, these data indicate that PKM2 expression in HCC tissue samples can be used as a prognostic factor for patients with HCC and that high PKM2 expression is correlated with a poor prognosis in HCC patients.

From the Editor’s desk....: November 2018

From the Editor’s desk....: November 2018

Effect of pyruvate kinase M2-regulating aerobic glycolysis on chemotherapy resistance of estrogen receptor-positive breast cancer.

This study aims to explore the effect and mechanism of pyruvate kinase M2 (PKM2) on chemotherapy resistance of estrogen receptor-positive breast cancer (ER BC) by regulating aerobic glycolysis. The expression of PKM2 in ER BC MCF-7 cells, T47D cells and MCF-7/ADR cells (which are subject to adriamycin/ADR induction) were determined by quantitative real-time PCR and western blot. MCF-7/ADR (M/A) cells were grouped into blank group (M/A), negative group (M/A+NC), low expression of PKM2 group (M/A+si-PKM2 group), overexpression of PKM2 group (M/A+PKM2 group) and glycolysis inhibition group (M/A+PKM2+2-DG group). Quantitative real-time PCR and western blot were applied to measure the expressions of PKM2, multidrug resistance, and glutathione-S-transferase π. Glucose and lactic acid kit was used to detect the amount of glucose uptake and lactic production. Cell variability, clone formation ability, and cell apoptosis were respectively measured by MTT, clone formation assay, and flow cytometry. Transwell assay and scratch assay were applied for cell invasion and migration ability. By overexpressing PKM2 in MCF-7 and T47D cells and using 2-DG, the effect on sensitivity of adriamycin amycin was explored. MCF-7/ADR cells have both elevated mRNA and protein expressions of PKM2 when compared with MCF-7 cells (both P<0.05). The cell activity of the M/A+si-PKM2+ADR group was notably lower than that in the M/A+ADR group and M/A+NC+ADR group (both P<0.05). In the M/A+si-PKM2 group, expressions of PKM2, multidrug resistance, and glutathione-S-transferase π, along with the amount of glucose uptake and lactic production, as well as cell variability, clone formation ability, and cell invasion and migration ability were inhibited, whereas cell apoptosis was increased in comparison with the M/A group and M/A+NC group (all P<0.05). On comparing with both the M/A group and the M/A+NC group, the M/A+si-PKM2 group displayed contrary tendency with the M/A+PKM2 group. The M/A+PKM2+2-DG group had elevated PKM2 expression compared with the M/A group and the M/A+NC group (all P<0.05). In MCF-7 and T47D cells with overexpression of PKM2, the sensitivity to adriamycin amycin, and cell apoptosis were suppressed, whereas the clone formation, invasion, and migration ability were enhanced. After 2-DG, the sensitivity on MCF-7 and T47D cells was enhanced while clone formation, invasion, migration and cell apoptosis rate were decreased (all P<0.05). PKM2 enhances chemotherapy resistance on ER BC by promoting aerobic glycolysis.

PKM2 under hypoxic environment causes resistance to mTOR inhibitor in human castration resistant prostate cancer.

The aim of this study was to explore the efficacy of mTOR inhibitor for castration-resistant prostate cancer (CRPC) under hypoxia. Although under normoxia C4-2AT6, it is a CRPC cell line, expressed elevated pAkt, pS6 and Pyruvate kinase M2 (PKM2) accompanied by elevated HIF-1a expression, 5% hypoxic condition further induced expression of these proteins. These results indicate hypoxic environment elevated PI3K/Akt/mTOR pathway in aggressive prostate cancer. However, C4-2AT6 cells treated with mTOR inhibitor under hypoxia less decreased compared to cells treated with the same dose drugs under normoxia. Western blot analysis showed mTOR inhibitor: RAD001 not only inhibited pS6, but also increased the expression of PKM2 in a dose and time dependent manner. Pyruvate kinase acts on glycolysis. PKM2, which is frequently express in tumor cells, is one isoform of pyruvate kinase. PKM2 is reported to act as a transcription factor. In the present study overexpression of PKM2 in C4-2AT6 induced resistance to RAD001 under normoxia. To evaluate the therapeutic effect of targeting PKM2, we inhibited PKM2 in C4-2AT6 under hypoxia using si-PKM2. The number of C4-2AT6 under chronic hypoxia exposed to siPKM2 significantly decreased compared to intact C4-2AT6 under chronic hypoxia. Furthermore, si-PKM2 improved resistance to mTOR inhibitor in C4-2AT6. When examined using clinical samples, high PKM2 expression was correlated with a high Gleason score and poor PSA free survival. These results suggested that up-regulation of PKM2 is one possibility of resistance to mTOR inhibitor in CRPC. And it is possible that PKM2 is a useful therapeutic target of CRPC.

Efficacy of Shikonin against Esophageal Cancer Cells and its possible mechanisms in vitro and in vivo.

Increasing evidences indicate that shikonin can suppress the tumor growth. However, the mechanisms remain elusive. In the present study, we investigated the effects and mechanisms of shikonin against esophageal cancer. The expression of hypoxia inducible factor 1α (HIF1α) and pyruvate kinase M2 (PKM2) in esophageal cancer tissues and cells was detected by immunohistochemistry and Western blot. CCK-8 was used to examine the esophageal cancer cell viability. Apoptosis and cell cycle were analyzed by flow cytometry. The expression of EGFR, PI3K, Akt, p-AKT, mTOR, HIF1α and PKM2 was detected by Western blot. EC109/pkm2 was established by lentivirus transducer. Ec109 tumor model was founded to observe the antitumor effect of shikonin in vivo.We found that HIF1α and PKM2 protein expression levels were higher in esophageal cancer tissues and cells than normal esophageal tissues and cells. Shikonin reduced esophageal cancer cells viability and induced cell cycle arrest and apoptosis. Shikonin decreased EGFR, PI3K, p-AKT, HIF1α and PKM2 expression. Overexpression of PKM2 could enhance resistance of esophageal cancer cells to shikonin. In vivo we found that shikonin reduced tumor burden, inducing cell arrest and apoptosis. Taken together, shikonin has a significant antitumor effect in the esophageal cancer by regulating HIF1α/PKM2 signal pathway.