Hypoxia promotes acquisition of aggressive phenotypes in human malignant mesothelioma.

BACKGROUND: Hypoxia is a hallmark of the solid tumor microenvironment and is associated with poor outcomes in cancer patients. The present study was performed to investigate mechanisms underlying the hypoxia-induced phenotypic changes using human malignant mesothelioma (HMM) cells. METHODS: Hypoxic conditions were achieved by incubating HMM cells in the air chamber. The effect of hypoxia on phenotype changes in HMM cells was investigated by performing in vitro clonogenicity, drug resistance, migration, and invasion assays. Signaling pathways and molecules involved in the more aggressive behaviors of HMM cells under hypoxia were investigated. A two-tailed unpaired Student's t-test or one-way ANOVA with Bonferroni post-test correction was used in this study. RESULTS: Hypoxic conditions upregulated hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α in parallel with the upregulation of its target, Glut-1, in HMM cells. In vitro clonogenicity of HMM cells was significantly increased in hypoxic conditions, but the proliferation of cells at a high density in hypoxia was lower than that in normoxic conditions. The expression levels of HIF-2α and Oct4 were increased in hypoxic HMM cells. The percentage of cells with high CD44 expression was significantly higher in HMM cells cultured in hypoxia than those cultured in normoxia. Hypoxia significantly enhanced the resistance of HMM cells to cisplatin, which occurred through cytoprotection against cisplatin-induced apoptosis. While cisplatin treatment decreased the ratio of Bcl-2 to Bax in normoxic condition, hypoxia conversely increased the ratio in HMM cells treated with cisplatin. Hypoxia increased the mobility and invasiveness of HMM cells. Epithelial to mesenchymal transition was promoted, which was indicated by the repression of E-cadherin and the concomitant increase of vimentin in HMM cells. CONCLUSIONS: The data illustrated that hypoxic conditions augmented the aggressive phenotypes of HMM cells at the biological and molecular levels. The present study provides valuable background information beginning to understand aggressiveness of HMM in tumor microenvironments, suggesting that a control measure for tumor hypoxia may be an effective therapeutic strategy to reduce the aggressiveness of cancer cells in HMM patients.

Metastatic intestinal adenocarcinoma with osseous metaplasia in two Domestic Korean Shorthair cats.

Two Domestic Korean Shorthair cats presented with dyschezia and vomiting. Computed tomography revealed a colonic mass with calcification and lymph node metastasis in case 1, and a small intestinal mass with disseminated mesenteric metastasis and calcification in case 2. Histopathology revealed intestinal adenocarcinoma with osseous metaplasia. Case 1 died two months after surgery from distant metastasis; and case 2 showed no metastasis for five months but presented with anorexia, euthanized seven months after diagnosis. Metastatic intestinal adenocarcinoma with bone formation should be considered as differential diagnosis for calcification on imaging, and lymph node metastasis at diagnosis may indicate poor prognosis.

Temozolomide abrogates the aggressiveness of urothelial carcinoma cells by enhancing senescence and depleting the side population.

Patients with advanced urothelial carcinoma (UC) generally have poor prognoses due to therapeutic resistance. Furthermore, there are limited treatment options for advanced UC. Therefore, novel or effective chemotherapeutic agents are needed to improve patient survival. The present study was conducted to investigate the effect of temozolomide (TMZ) on UC cells so as to identify a potential method to overcome therapeutic resistance. TMZ is an alkylating agent with a target different from that of other anticancer drugs used to treat UC, such as cisplatin. TMZ enhanced the autophagic response and senescence, which was mediated via the p53 and p21 pathways. Inhibiting the autophagic response using chloroquine synergistically augmented the cytotoxic effect of TMZ on UC cells. TMZ significantly reduced the invasiveness of UC cells. Notably, the abundance of side population fraction was also significantly reduced following TMZ treatment. Considering that side population fraction is known to confer therapeutic resistance, it is noteworthy that the TMZ treatment markedly decreased side population fraction. Altogether, TMZ may have the potential to be applied as a part of an alternative treatment strategy to reduce the malignancy of UC cells.

Reduction in mitochondrial oxidative stress mediates hypoxia-induced resistance to cisplatin in human transitional cell carcinoma cells.

Tumor hypoxia is known to promote the acquisition of more aggressive phenotypes in human transitional cell carcinoma (TCC), including drug resistance. Accumulating evidence suggests that mitochondria play a central role in the chemoresistance of TCC. However, the role of mitochondria in the hypoxia-induced drug resistance in TCC remains elusive. The present study investigated the function of mitochondria in the drug resistance using a TCC cell line under hypoxic conditions. In vitro hypoxia (0.1% O2, 48 h) was achieved by incubating TCC cells in air chamber. Mitochondrial events involving hypoxia-induced drug resistance were assessed. Hypoxia significantly reduced the cisplatin-induced apoptosis of TCC cells. Additionally, hypoxia substantially decreased the level of mitochondrial reactive oxygen species (ROS) generated by cisplatin treatment. Analogously, elimination of mitochondrial ROS significantly rescued cells from cisplatin-induced apoptosis. Hypoxia enhanced mitochondrial hyperpolarization, which was not related to ATP production or the reversal of ATP synthase activity. The mitochondrial DNA (mtDNA) amplification efficiency data illustrated that hypoxia significantly prevented oxidative damage to the mitogenome. Moreover, transmission electron microscopy revealed that cisplatin-induced disruption of the mitochondrial ultrastructure was abated under hypoxic conditions. Notably, depletion of mtDNA by ethidium bromide abrogated hypoxia-induced resistance to cisplatin. Taken together, the present study demonstrated that TCC cells exposed to hypoxic conditions rendered mitochondria less sensitive to oxidative stress induced by cisplatin treatment, leading to enhanced drug resistance.

Evaluation of a human glycated hemoglobin test in canine diabetes mellitus.

Glycated hemoglobin A1c (HbA1c) is widely used for monitoring and diagnosing human diabetes mellitus, but is rarely used in veterinary clinics. The goal of our study was to validate the commercial HbA1c testing system SD A1cCare analyzer (Bionote, Gyeoggi-do, South Korea) for use in dogs. Dogs were recruited with owner's consent. Diabetic status was determined based on clinical signs, fasting hyperglycemia, and glycosuria. Intra-assay precision and linearity were evaluated with EDTA, heparin, or citrate as anticoagulants, and had excellent precision with mean coefficients of variation (CVs) of 2.47%, 2.26%, and 1.92%, respectively. Diluted anticoagulated blood samples showed excellent linear relationships with R2 of 0.991, 0.996, and 0.994, respectively. Inter-assay precision revealed that the mean CV of the normal control was 2.18% and that of the high control was 2.01% (30 repeats). Observed total error of a normal control was 7.81%, and 6.12% for the high control. HbA1c level measured before and after removal of plasma and replacement by saline showed minimal interference by lipid contents ( p = 0.929). The HbA1c concentrations of diabetic dogs were significantly higher than those of non-diabetic dogs ( p < 0.001). HbA1c value >6.2% indicated canine diabetes through a classification and regression tree model. In most cases, fructosamine and HbA1c were highly correlated ( r = 0.674, p < 0.001). The HbA1c testing system could be a valuable testing system to evaluate canine diabetes mellitus, providing an alternative in-house option for use by veterinary clinicians.

Integrated analysis of microRNA and mRNA expressions in peripheral blood leukocytes of Warmblood horses before and after exercise.

Exercise capacity is a valuable trait in horses, and it has been used as a horse selection criterion. Although exercise affects molecular homeostasis and adaptation in horses, the mechanisms underlying these effects are not fully described. This study was carried out to identify changes in the blood profiles of microRNAs (miRNAs) and mRNAs induced by exercise in horse leukocytes. Total RNAs isolated from the peripheral blood leukocytes of four Warmblood horses before and after exercise were subjected to next-generation sequencing (NGS) and microarray analyses to determine the miRNA and mRNA expression profiles, respectively. The expressions of 6 miRNAs, including 4 known and 2 novel miRNAs, were altered by exercise. The predicted target genes of the differentially expressed miRNAs identified by NGS were matched to the exercise-induced mRNAs determined by microarray analysis. Five genes (LOC100050849, LOC100054517, KHDRBS3, LOC100053996, and LOC100062720) from the microarray analysis were matched to the predicted target genes of the 6 miRNAs. The subset of mRNAs and miRNAs affected by exercise in peripheral blood leukocytes may be useful in elucidating the molecular mechanisms of exercise-associated physiology in horses.

Anthropogenic food provisioning and immune phenotype: Association among supplemental food, body condition, and immunological parameters in urban environments.

Direct or indirect supplemental feeding of free-ranging animals occurs worldwide, resulting in significant impacts on population density or altered demographic processes. Another potential impact of increased energy intake from supplemental feeding is altered immunocompetence. As immune system maintenance is energetically costly, there may be trade-offs between immune responses and other energy-demanding physiological processes in individual animals. Although increased availability of food sources through supplemental feeding is expected to increase the overall immunocompetence of animals, empirical data verifying the association between supplemental feeding and different immune parameters are lacking. Understanding the potential influence of supplemental feeding on immune phenotypes is critical, as it may also impact host-pathogen dynamics in free-ranging animals. Using urban stray cats as a study model, we tested for associations between the intensity of supplemental feeding due to cat caretaker activity (CCA); body condition; and immune phenotype (bacterial killing assay (BKA), immunoglobulin G (IgG) concentration, and leukocyte counts). Significantly higher bacterial killing ability was observed in cats from high CCA districts, whereas higher IgG concentration and eosinophil counts were observed in cats from low CCA districts. Other leukocyte counts and body condition indices showed no significant association with CCA. We observed varying patterns of different immune components in relation to supplemental feeding. Out data suggest that supplemental feeding influences immune phenotype, not only by means of energy provisioning, but also by potentially reducing exposure rates to parasite infections through stray cat behavioral changes.

A subset of microRNAs defining the side population of a human malignant mesothelioma cell line.

This study was performed to investigate the global expression profile of microRNAs in distinct subpopulations of a human malignant mesothelioma cell line. Total RNAs were isolated from the sorted side population and non-side population of MS1. The RNAs were subjected to analysis using Affymetrix GeneChip microRNA Arrays. After data extraction and normalization, a subset of microRNAs defining cell subpopulations was identified using bioinformatics softwares. Based on the criteria of 2-fold difference and the p-value of < 0.05, a total of 95 microRNAs were differentially expressed in the side population compared to the non-side population. Functional ontology revealed that target genes of the miRNAs were categorized into various gene ontology terms, such as stem cell maintenance, cell proliferation, programmed cell death, cell migration, and cellular response to stress. The Kyoto Encyclopedia of Genes and Genomes analysis showed that ErbB-2 receptor tyrosine kinases signaling pathway was the most represented. Integrated analysis of MiRTarBase and RNA-seq identified 12 target genes of microRNAs defining side population, including DDIT4 and ROCK2. The present study indicates that a distinct set of microRNAs may be critically involved in the generation and maintenance of heterogeneous subpopulations of cancer cells. They could be a plausible target for the eradication of more aggressive cancer cell subpopulations.

An Integrated Analysis of the Genome-Wide Profiles of DNA Methylation and mRNA Expression Defining the Side Population of a Human Malignant Mesothelioma Cell Line.

Intratumoral heterogeneity is a hallmark of all cancers and functions as the major barrier against effective cancer therapy. In contrast to genetic mutations, the role of epigenetic modifications in the generation and maintenance of heterogeneous cancer cells remains largely undetermined. This study was performed to evaluate the epigenetic mechanisms involved in the tumor cell heterogeneity using side population (SP) and non-SP cells isolated from a human malignant mesothelioma (HMM) cell line. The subpopulations of cancer cells were analyzed by methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) and RNA-seq methodology. The RNA-seq data were analyzed with the MeDIP-seq data in an integrated way to identify the epigenetically modified genes that defined the SP. Concomitant changes in mRNA expression and DNA methylation were found in 122 genes, including 118 down-regulated genes with hypermethylation and 4 up-regulated genes with hypomethylation. Gene ontology revealed that a large portion of the genes belonged to the groups of biological processes such as stem cell maintenance, stem cell development, stem cell differentiation, and the negative regulation of the developmental process. Among these genes, BNC1, RPS6KA3, TWSG1 and DUSP15 contained aberrant methylation in the CpG islands of the promoter region, indicating that the genes regulated by DNA methylation characterized a distinct subpopulation of HMM cells. The present study provided valuable information to shed light on the epigenetic contributions to the generation and maintenance of tumor cell heterogeneity.