PKM2 Regulates HSP90-Mediated Stability of the IGF-1R Precursor Protein and Promotes Cancer Cell Survival during Hypoxia.

Insulin-like growth factor-1 receptor (IGF-1R), an important factor in promoting cancer cell growth and survival, is commonly upregulated in cancer cells. However, amplification of the IGF1R gene is extremely rare in tumors. Here, we have provided insights into the mechanisms underlying the regulation of IGF-1R protein expression. We found that PKM2 serves as a non-metabolic protein that binds to and increases IGF-1R protein expression by promoting the interaction between IGF-1R and heat-shock protein 90 (HSP90). PKM2 depletion decreases HSP90 binding to IGF-1R precursor, thereby reducing IGF-1R precursor stability and the basal level of mature IGF-1R. Consequently, PKM2 knockdown inhibits the activation of AKT, the key downstream effector of IGF-1R signaling, and increases apoptotic cancer cell death during hypoxia. Notably, we clinically verified the PKM2-regulated expression of IGF-1R through immunohistochemical staining in a tissue microarray of 112 lung cancer patients, demonstrating a significant positive correlation (r = 0.5208, p < 0.0001) between PKM2 and IGF-1R expression. Together, the results of a previous report demonstrated that AKT mediates PKM2 phosphorylation at serine-202; these results suggest that IGF-1R signaling and PKM2 mutually regulate each other to facilitate cell growth and survival, particularly under hypoxic conditions, in solid tumors with dysregulated IGF-1R expression.

Evaluation of Surgical Pleth Index and Analgesia Nociception Index as surrogate pain measures in conscious postoperative patients: an observational study.

We evaluated the performance of the Surgical Plethysmographic Index (SPI) and the Analgesia Nociception Index (ANI) as surrogate pain measures and determined their respective cut-off values for detecting pain in conscious postoperative patients. In total, 192 patients after elective surgery were enrolled. Baseline SPI and ANI data were acquired for 10 min in the operating room prior to surgery when the patients rated their pain as 0 on the numerical rating scale (NRS). Upon arrival in the post-anaesthesia care unit (PACU) after surgery, SPI and ANI data were recorded for 10 min. The means of the recorded data at OR and PACU were defined as the values representing baseline and postoperative pain, respectively. SPI and ANI data obtained from 189 patients were analysed, who were anesthetized with propofol (n = 149) or sevoflurane (n = 40). Remifentanil was continuously infused intraoperatively in all patients. The values of SPI and ANI were significantly different in conscious patients without (NRS = 0) and with pain (NRS > 0). The areas under the receiver operating curves for SPI and ANI were 0.73 (P < 0.0001) and 0.67 (P < 0.0001), respectively. The cut-off values for SPI and ANI in predicting postoperative pain were 44 (sensitivity: 84%, specificity: 53%) and 63 (sensitivity: 52%, specificity: 82%), respectively, which are different from those suggested by their respective manufacturers for use in intraoperative state under general anaesthesia. The cut-off values of SPI and ANI for detecting pain were similar regardless of the type of anesthesia.

Characteristics of 419 patients with acquired middle ear cholesteatoma / Características de 419 pacientes com colesteatoma adquirido de orelha média

Abstract Introduction: Cholesteatoma is a destructive lesion that can result in life-threatening complications. Typically, it presents with hypoacusis and continuous otorrhea as symptoms. Because it is a rare disease, there are few studies in Brazil describing the characteristics of patients with the disease. Objective: This study aimed to determine the prevalence of cholesteatoma in patients with chronic otitis media and describe clinical, audiological and surgical characteristics of patients with acquired middle ear cholesteatoma treated at a referral hospital in the public health system. Methods: Cross-sectional and prospective cohort study, including 1710 patients with chronic otitis media, treated between August 2000 and June 2015, without prior surgery. Detailed clinical history, videotoscopy, and audiometry were performed, in addition to review of medical records to search for surgical data. Cholesteatomas were classified according to their route of formation. Results: Of the patients with chronic otitis media, 419 (24.5%) had cholesteatoma; mean age of 34.49 years; 53.5% female and 63.8% adults. Bilateral cholesteatoma was observed in 17.1%. Anterior epitympanic cholesteatoma corresponded to 1.9%; posterior epitympanic, 32.9%; posterior mesotympanic, 33.7%; two routes, 14.8%; and indeterminate, 16.7%. The mean air-bone gap was 29.84 dB and did not differ between routes of formation. There were no correlations between gap size and patient age or duration of symptoms. Of the surgical cases, 16.8% underwent closed tympanomastoidectomy and 75.2% open tympanomastoidectomy. Conclusion: The prevalence of cholesteatoma in patients with chronic otitis media was 24.5% and it was more common in adults than in children. Posterior mesotympanic cholesteatoma was more frequent, with no difference in mean air-bone gap between the different routes of formation. In patients undergoing surgery, open tympanomastoidectomy was the procedure most frequently chosen.

Resumo Introdução: Colesteatoma é uma lesão destrutiva que pode levar a complicações potencialmente letais. Tipicamente, apresenta hipoacusia e otorreia contínua como sintomas. Por ser uma doença rara, existem poucos estudos no Brasil que descrevam as características destes pacientes. Objetivo: O presente estudo teve como objetivos determinar a prevalência de colesteatoma entre os pacientes com otite média crônica (OMC) e descrever as características clínicas, audiológicas e cirúrgicas dos pacientes com colesteatoma adquirido de orelha média atendidos em um hospital de referência do sistema público de saúde. Método: Estudo transversal e de coorte prospectivo, incluindo 1.710 pacientes com OMC, atendidos entre agosto de 2000 e junho de 2015, sem tratamento cirúrgico prévio. Foram feitas anamnese detalhada, videotoscopia e audiometria, além de revisão de prontuários para busca de dados cirúrgicos. Os colesteatomas foram classificados conforme sua via de formação. Resultados: Dos pacientes com otite média crônica, 419 (24,5%) apresentavam colesteatoma. Média de 34,49 anos; 53,5% do sexo feminino e 63,8% adultos. Colesteatoma foi observado bilateralmente em 17,1%. Os epitimpânicos anteriores corresponderam a 1,9%; os epitimpânicos posteriores a 32,9%; os mesotimpânicos posteriores a 33,7%; duas vias a 14,8% e indeterminados a 16,7%. A média tritonal dos gaps aeroósseos foi de 29,84 dB e não diferiu entre os grupos segundo as vias de formação. Não foram observadas correlações entre tamanho do gap e idade do paciente ou duração dos sintomas. Dos pacientes operados, 16,8% foram submetidos a timpanomastoidectomia fechada e 75,2% a timpanomastoidectomia aberta. Conclusão: A prevalência de colesteatoma em pacientes com otite média crônica foi de 24,5% e foi mais frequente em adultos do que em crianças. Os mesotimpânicos posteriores foram mais frequentes, não foi observada diferença na média dos gaps aeroósseos entre diferentes vias de formação. Nos pacientes submetidos a cirurgia, a timpanomastoidectomia aberta foi o procedimento escolhido.

Characteristics of 419 patients with acquired middle ear cholesteatoma.

INTRODUCTION: Cholesteatoma is a destructive lesion that can result in life-threatening complications. Typically, it presents with hypoacusis and continuous otorrhea as symptoms. Because it is a rare disease, there are few studies in Brazil describing the characteristics of patients with the disease. OBJECTIVE: This study aimed to determine the prevalence of cholesteatoma in patients with chronic otitis media and describe clinical, audiological and surgical characteristics of patients with acquired middle ear cholesteatoma treated at a referral hospital in the public health system. METHODS: Cross-sectional and prospective cohort study, including 1710 patients with chronic otitis media, treated between August 2000 and June 2015, without prior surgery. Detailed clinical history, videotoscopy, and audiometry were performed, in addition to review of medical records to search for surgical data. Cholesteatomas were classified according to their route of formation. RESULTS: Of the patients with chronic otitis media, 419 (24.5%) had cholesteatoma; mean age of 34.49 years; 53.5% female and 63.8% adults. Bilateral cholesteatoma was observed in 17.1%. Anterior epitympanic cholesteatoma corresponded to 1.9%; posterior epitympanic, 32.9%; posterior mesotympanic, 33.7%; two routes, 14.8%; and indeterminate, 16.7%. The mean air-bone gap was 29.84dB and did not differ between routes of formation. There were no correlations between gap size and patient age or duration of symptoms. Of the surgical cases, 16.8% underwent closed tympanomastoidectomy and 75.2% open tympanomastoidectomy. CONCLUSION: The prevalence of cholesteatoma in patients with chronic otitis media was 24.5% and it was more common in adults than in children. Posterior mesotympanic cholesteatoma was more frequent, with no difference in mean air-bone gap between the different routes of formation. In patients undergoing surgery, open tympanomastoidectomy was the procedure most frequently chosen.

AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth.

Pyruvate kinase muscle type 2 (PKM2) exhibits post-translational modifications in response to various signals from the tumor microenvironment. Insulin-like growth factor 1 (IGF-1) is a crucial signal in the tumor microenvironment that promotes cell growth and survival in many human cancers. Herein, we report that AKT directly interacts with PKM2 and phosphorylates it at Ser-202, which is essential for the nuclear translocation of PKM2 protein under stimulation of IGF-1. In the nucleus, PKM2 binds to STAT5A and induces IGF-1-stimulated cyclin D1 expression, suggesting that PKM2 acts as an important factor inducing STAT5A activation under IGF-1 signaling. Concordantly, overexpression of STAT5A in cells deficient in PKM2 expression failed to restore IGF-induced growth, whereas reconstitution of PKM2 in PKM2 knockdown cells restored the IGF-induced growth capacity. Our findings suggest a novel role of PKM2 in promoting the growth of cancers with dysregulated IGF/phosphoinositide 3-kinase/AKT signaling.

Pyruvate kinase isoenzyme M2 is a therapeutic target of gemcitabine-resistant pancreatic cancer cells.

Despite its wide use as a first-line therapeutic agent, gemcitabine has shown limited efficacy in advanced pancreatic cancer due to chemoresistance by as yet unidentified mechanisms. Our goal here was to identify molecular features involved in gemcitabine chemoresistance. Pyruvate kinase M2 (PKM2), a key enzyme of aerobic glycolysis, has recently emerged as an important therapeutic target for cancer treatment. It is involved in the metabolic reprogramming of cancer cells and has previously unexpected non-metabolic functions that are heavily involved in tumor growth and survival. Herein, we report that the chemoresistance of pancreatic cancer to gemcitabine was dependent on PKM2 expression and its non-metabolic function. Knocking-down of PKM2 significantly enhanced gemcitabine-induced cell apoptosis through the activation of caspase 3/7 and PARP cleavage, and this inhibitory activity was associated with p38-mediated activation of p53 phosphorylation at serine 46. Our findings support the potential of PKM2 as a novel target for gemcitabine chemoresistance and suggest the feasibility of combining gemcitabine and PKM2 inhibition for the improved chemotherapy of pancreatic cancer.

A novel pyruvate kinase M2 activator compound that suppresses lung cancer cell viability under hypoxia.

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 µM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

The inhibitory effect of a synthetic compound, (Z)-5-(2,4-dihydroxybenzylidene) thiazolidine-2,4-dione (MHY498), on nitric oxide-induced melanogenesis.

Nitric oxide (NO) and the NO/PKG signaling pathway play crucial roles in ultraviolet (UV)-induced melanogenesis, which is known to be related to the induction of tyrosinase. In an attempt to find a novel anti-melanogenic agent, we synthesized (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498). The purpose of this study was to investigate the effect of MHY498 on NO levels and on the NO-mediated signaling pathway using an in vitro model of melanogenesis. MHY498 inhibited 200 µM sodium nitroprusside (SNP, a NO donor)-induced NO generation, dose-dependently and suppressed tyrosinase activity and melanin synthesis induced by SNP in B16F10 melanoma cells. To investigate the effect of MHY498 on NO-mediated signaling pathway, guanosine cyclic 3',5'-monophosphate (cGMP) activities were measured using a cGMP EIA Kit and western blotting was performed to determine the effects of MHY498 on the gene expressions of tyrosinase and microphthalmia-associated transcription factor (MITF). The increased activity of cGMP by SNP was reduced dose-dependently by pretreatment with MHY498. Furthermore, MHY498 suppressed the expressions of tyrosinase and MITF stimulated by SNP. This study shows that enhancement of tyrosinase gene expression via the cGMP pathway is a probable primary mechanism of NO-induced melanogenesis and that the NO-mediated signaling pathway with the expression of MITF enhances melanogenesis. In addition, MHY498 was found to scavenge NO and to suppress the activity of the NO-mediated signaling pathway, and thus, to subsequently down-regulate tyrosinase expression and melanogenesis. This study suggests that MHY498 is a promising anti-melanogenic agent that targets the NO-induced cGMP signaling pathway.

Metabolic signature genes associated with susceptibility to pyruvate kinase, muscle type 2 gene ablation in cancer cells.

Pyruvate kinase, muscle type 2 (PKM2), is a key factor in the aerobic glycolysis of cancer cells. In our experiments, liver cancer cell lines exhibited a range of sensitivity to PKM2 knockdown-mediated growth inhibition. We speculated that this differential sensitivity is attributable to the variable dependency on glycolysis for the growth of different cell lines. Transcriptome data revealed overexpression of a glucose transporter (GLUT3) and a lactate transporter (MCT4) genes in PKM2 knockdown-sensitive cells. PKM2 knockdown-resistant cells expressed high levels of the lactate dehydrogenase B (LDHB) and glycine decarboxylase (GLDC) genes. Concordant with the gene expression results, PKM2 knockdown-sensitive cells generated high levels of lactate. In addition, ATP production was significantly reduced in the PKM2 knockdown-sensitive cells treated with a glucose analog, indicative of dependency of their cellular energetics on lactate-producing glycolysis. The PKM2 knockdown-resistant cells were further subdivided into less glycolytic and more (glycolysis branch pathway-dependent) glycolytic groups. Our findings collectively support the utility of PKM2 as a therapeutic target for high lactate-producing glycolytic hepatocellular carcinoma (HCC).

In vitro and in vivo genotoxic effects of somatic cell nuclear transfer cloned cattle meat.

Although the nutritional composition and health status after consumption of the meat and milk derived from both conventionally bred (normal) and somatic cell nuclear transferred (cloned) animals and their progeny are not different, little is known about their food safeties like genetic toxicity. This study is performed to examine both in vitro (bacterial mutation and chromosome aberration) and in vivo (micronucleus) genotoxicity studies of cloned cattle meat. The concentrations of both normal and cloned cattle meat extracts (0-10×) were tested to five strains of bacteria (Salmonella typhimurium: TA98, TA100, TA1535, and TA1537; Escherichia coli: WP2uvrA) for bacterial mutation and to Chinese hamster lung (CHL/IU) cells for chromosome aberration, respectively. For micronucleus test, ICR mice were divided into five dietary groups: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) normal cattle meat, and pellets containing 5% (C-5) and 10% (C-10) cloned cattle meat. No test substance-related genotoxicity was noted in the five bacterial strains, CHL/IU cells, or mouse bone marrow cells, suggesting that the cloned cattle meat potentially may be safe in terms of mutagenic hazards. Thus, it can be postulated that the cloned cattle meat do not induce any harmful genotoxic effects in vitro and in vivo.

Inhibitory Effect of 1-O-Hexyl-2,3,5-Trimethylhydroquinone on Dimethylnitrosamine-induced Liver Fibrosis in Male SD Rats.

Hepatic fibrosis represents the main complication of most chronic liver disorders and, regardless of its etiology, is characterized by excessive deposition of extracellular matrix components. In this study, we examined that 1-O-Hexyl-2,3,5-Trimethylhydroquinone (HTHQ) , a potent anti-oxidative agent, could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in male SD rats. Except for vehicle control group, other groups were induced hepatic fibrosis by intraperitoneal injection with DMN (10 mg/ml/kg) on 3 consecutive days weekly for 4 weeks. During the same 4 weeks, control and DMN groups were given vehicle and HTHQ 50, 100 and 200 groups were orally administered HTHQ (50, 100, 200 mg/kg respectively) . In HTHQ 100 and 200 groups, relative liver weight and serum chemistry level improved significantly. HTHQ reduced hydroxyproline (p < 0.05) and malondialdehyde (p < 0.05) level in the liver. Histopathological examination of H&E, Masson's trichrome stain showed the reduced fibrotic septa in HTHQ 100 and 200 groups. HTHQ administration showed reduced mRNA level of PDGF (Plateletderived growth factor) , α-SMA (α-smooth muscle actin) and TGF-ß (transforming growth factor-ß) than DMN-induced hepetic fibrosis animals in the liver tissue. In this study, we showed that HTHQ improves against DMN-induced liver fibrosis in male SD rats.