Frequent Epigenetic Suppression of Tumor Suppressor Gene Glutathione Peroxidase 3 by Promoter Hypermethylation and Its Clinical Implication in Clear Cell Renal Cell Carcinoma

The goal of this study is to identify novel tumor suppressor genes silenced by promoter methylation in clear cell renal cell carcinoma (ccRCC) and discover new epigenetic biomarkers for early cancer detection. Reactive oxygen species (ROS) is a major cause of DNA damage that correlates with cancer initiation and progression. Glutathione peroxidase 3 (GPX3), the only known extracellular glycosylated enzyme of GPXs, is a major scavenger of ROS. GPX3 has been identified as a tumor suppressor in many cancers. However, the role of GPX3 in ccRCC remains unclear. This study aimed to investigate its epigenetic alteration in ccRCC and possible clinicopathological association. In our study, GPX3 methylation and down-regulation were detected in 5 out of 6 ccRCC cell lines and the GPX3 mRNA and protein expression level in ccRCC tumors was significantly lower than in adjacent non-malignant renal tissues (p < 0.0001). Treatment with 5-Aza-2''-deoxycytidine restored GPX3 expression in ccRCC cells. Aberrant methylation was further detected in 77.1% (162/210) of RCC primary tumors, but only 14.6% (7/48) in adjacent non-malignant renal tissues. GPX3 methylation status was significantly associated with higher tumor nuclear grade (p = 0.014). Thus, our results showing frequent GPX3 inactivation by promoter hypermethylation in ccRCC may reveal the failure in the cellular antioxidant system in ccRCC and may be associated with renal tumorigenesis. GPX3 tumor specific methylation may serve as a biomarker for early detection and prognosis prediction of ccRCC.     

Lentiviral-Mediated Short Hairpin RNA Knockdown of MTDH Inhibits Cell Growth and Induces Apoptosis by Regulating the PTEN/AKT Pathway in Hepatocellular Carcinoma

The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). Metaherin (MTDH), also called astrocyte elevated gene-1 (AEG-1), is frequently amplified in a variety of cancers, but the roles of MTDH with regard to growth and apoptosis in HCC have not yet been studied. In the present study, we first analyzed the expression of MTDH in HCC samples. We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher in HCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. Knockdown MTDH promoted PTEN and p53 expression in HCC cells and inhibited AKT phosphorylation. Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN. Therefore, MTDH might be an effective targeted therapy gene for HCC.     

Liver Lipids of Patients with Hepatitis B and C and Associated Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.     

Age-Related DNA Methylation in Normal Kidney Tissue Identifies Epigenetic Cancer Risk Susceptibility Loci in the ANKRD34B and ZIC1 Genes

Both age-dependent and age-independent alteration of DNA methylation in human tissues are functionally associated with the development of many malignant and non-malignant human diseases. TCGA-KIRC data were biometrically analyzed to identify new loci with age-dependent DNA methylation that may contribute to tumor risk in normal kidney tissue. ANKRD34B and ZIC1 were evaluated as candidate genes by pyrosequencing of 539 tissues, including 239 normal autopsy, 157 histopathologically tumor-adjacent normal, and 143 paired tumor kidney samples. All candidate CpG loci demonstrated a strong correlation between relative methylation levels and age (R = 0.70–0.88, p < 2 × 10⁻¹⁶) and seven out of 10 loci were capable of predicting chronological age in normal kidney tissues, explaining 84% of the variance (R = 0.92). Moreover, significantly increased age-independent methylation was found for 9 out of 10 CpG loci in tumor-adjacent tissues, compared to normal autopsy tissues (p = 0.001–0.028). Comparing tumor and paired tumor-adjacent tissues revealed two patient clusters showing hypermethylation, one cluster without significant changes in methylation, and a smaller cluster demonstrating hypomethylation in the tumors (p < 1 × 10⁻¹⁰). Taken together, our results show the presence of additional methylation risk factors besides age for renal cancer in normal kidney tissue. Concurrent tumor-specific hypermethylation suggests a subset of these loci are candidates for epigenetic renal cancer susceptibility.     

A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin,Tested Over a Wide Range of Concentrations

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10⁻¹¹–10⁻² M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10⁻⁹–10⁻⁵ M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high—but not low—melatonin concentrations, although the mechanism of action still remains to be elucidated.     

Differential DNA Methylation in Relation to Age and Health Risks of Obesity

The aim of this study was to evaluate whether genome-wide levels of DNA methylation are associated with age and the health risks of obesity (HRO); defined according to BMI categories as “Low HRO” (overweight and class 1 obesity) versus “High HRO” (class 2 and class 3 obesity). Anthropometric measurements were assessed in a subsample of 48 volunteers from the Metabolic Syndrome Reduction in Navarra (RESMENA) study and 24 women from another independent study, Effects of Lipoic Acid and Eicosapentaenoic Acid in Human Obesity (OBEPALIP study). In the pooled population; the methylation levels of 55 CpG sites were significantly associated with age after Benjamini-Hochberg correction. In addition, DNA methylation of three CpG sites located in ELOVL2; HOXC4 and PI4KB were further negatively associated with their mRNA levels. Although no differentially methylated CpG sites were identified in relation to HRO after multiple testing correction; several nominally significant CpG sites were identified in genes related to insulin signaling; energy and lipid metabolism. Moreover, statistically significant associations between BMI or mRNA levels and two HRO-related CpG sites located in GPR133 and ITGB5 are reported. As a conclusion, these findings from two Spanish cohorts add knowledge about the important role of DNA methylation in the age-related regulation of gene expression. In addition; a relevant influence of age on DNA methylation in white blood cells was found, as well as, on a trend level, novel associations between DNA methylation and obesity.     

A Pleiotropic Role of the Hepatitis B Virus Core Protein in Hepatocarcinogenesis

Chronic hepatitis B virus (HBV) infection is one of the most common factors associated with hepatocellular carcinoma (HCC), which is the sixth most prevalent cancer among all cancers worldwide. However, the pathogenesis of HBV-mediated hepatocarcinogenesis is unclear. Evidence currently available suggests that the HBV core protein (HBc) plays a potential role in the development of HCC, such as the HBV X protein. The core protein, which is the structural component of the viral nucleocapsid, contributes to almost every stage of the HBV life cycle and occupies diverse roles in HBV replication and pathogenesis. Recent studies have shown that HBc was able to disrupt various pathways involved in liver carcinogenesis: the signaling pathways implicated in migration and proliferation of hepatoma cells, apoptosis pathways, and cell metabolic pathways inducing the development of HCC; and the immune system, through the expression and production of proinflammatory cytokines. In addition, HBc can modulate normal functions of hepatocytes through disrupting human host gene expression by binding to promoter regions. This HBV protein also promotes HCC metastasis through epigenetic alterations, such as micro-RNA. This review focuses on the molecular pathogenesis of the HBc protein in HBV-induced HCC.     

Analysis of Different Ploidy and Parent–Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus

In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid) and hybrid F₁ generation obverse cross (4 × 2) and inverse cross (2 × 4) by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism) reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F₁ generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F₁ generation is significantly higher than the orthogonal F₁ generation (p < 0.01). After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively.     

Blood-Based Biomarkers in Hepatitis B Virus-Related Hepatocellular Carcinoma,Including the Viral Genome and Glycosylated Proteins

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) development and is a global public health issue. High performance biomarkers can aid the early detection of HCC development in HBV-infected individuals. In addition, advances in the understanding of the pathogenesis of HBV infection and in clinical laboratory techniques have enabled the establishment of disease-specific tests, prediction of the progression of liver diseases, including HCC, and auxiliary diagnosis of HCC, using blood-based methods instead of biopsies of liver or HCC tissues. Viral factors such as the HBV genotype, HBV genetic mutations, HBV DNA, and HBV-related antigens, as well as host factors, such as tumor-associated proteins and post-translational modifications, especially glycosylated proteins, can be blood-based, disease-specific biomarkers for HCC development in HBV-infected patients. In this review, we describe the clinical applications of viral biomarkers, including the HBV genome and glycosylated proteins, for patients at a risk of HBV-related HCC, based on their molecular mechanisms. In addition, we introduce promising biomarker candidates for practical use, including colony stimulating factor 1 receptor (CSF1R), extracellular vesicles, and cell-free, circulating tumor DNA. The clinical use of such surrogate markers may lead to a better understanding of the risk of disease progression and early detection of HCC in HBV-infected patients, thereby improving their prognosis.     

LIM Mineralization Protein-1 Inhibits the Malignant Phenotypes of Human Osteosarcoma Cells

Osteosarcoma (OS), also known as osteogenic sarcoma, is the most common primary malignancy of bone tumor in children and adolescents. However, its underlying molecular pathogenesis is still only vaguely understood. Recently, LIM mineralization protein-1 (LMP-1) was reported to be an essential positive regulator of osteoblast differentiation. In the present study, we found that the expression of LMP-1 is downregulated in OS tissues compared with adjacent normal tissues. Moreover, we restored the expression of LMP-1 through a recombinant adenovirus. Overexpression of LMP-1 inhibited cell proliferation and invasion, arrested cell cycle progression, and induced apoptosis in vitro. Finally, ectopic LMP-1 expression suppressed the expression of Runx2 and BMP-2 in OS cells. These data demonstrate that LMP-1 is an essential tumor suppressor in the OS pathological process, which will provide a new opportunity for discovering and identifying novel effective treatment strategies.     

Physcomitrella patens Activates Defense Responses against the Pathogen Colletotrichum gloeosporioides

The moss Physcomitrella patens is a suitable model plant to analyze the activation of defense mechanisms after pathogen assault. In this study, we show that Colletotrichum gloeosporioides isolated from symptomatic citrus fruit infects P. patens and cause disease symptoms evidenced by browning and maceration of tissues. After C. gloeosporioides infection, P. patens reinforces the cell wall by the incorporation of phenolic compounds and induces the expression of a Dirigent-protein-like encoding gene that could lead to the formation of lignin-like polymers. C. gloeosporioides-inoculated protonemal cells show cytoplasmic collapse, browning of chloroplasts and modifications of the cell wall. Chloroplasts relocate in cells of infected tissues toward the initially infected C. gloeosporioides cells. P. patens also induces the expression of the defense genes PAL and CHS after fungal colonization. P. patens reporter lines harboring the auxin-inducible promoter from soybean (GmGH3) fused to β-glucuronidase revealed an auxin response in protonemal tissues, cauloids and leaves of C. gloeosporioides-infected moss tissues, indicating the activation of auxin signaling. Thus, P. patens is an interesting plant to gain insight into defense mechanisms that have evolved in primitive land plants to cope with microbial pathogens.