Reduced mRNA expression levels of MBD2 and MBD3 in gastric carcinogenesis.

Aberrant methylation has been reported in several neoplasias, including gastric cancer. The methyl-CpG-binding domain (MBD) family proteins have been implicated in the chromatin remodeling process, leading to the modulation of gene expression. To evaluate the role of MBD2 and MBD3 in gastric carcinogenesis and the possible association with clinicopathological characteristics, we assessed the mRNA levels and promoter methylation patterns in gastric tissues. In this study, MBD2 and MBD3 mRNA levels were determined by RT-qPCR in 28 neoplastic and adjacent nonneoplastic and 27 gastritis and non-gastritis samples. The promoter methylation status was determined by bisulfite sequencing, and we found reduced MBD2 and MBD3 levels in the neoplastic samples compared with the other groups. Moreover, a strong correlation between the MBD2 and MBD3 expression levels was observed in each set of paired samples. Our data also showed that the neoplastic tissues exhibited higher MBD2 promoter methylation than the other groups. Interestingly, the non-gastritis group was the only one with positive methylation in the MBD3 promoter region. Furthermore, a weak correlation between gene expression and methylation was observed. Therefore, our data suggest that DNA methylation plays a minor role in the regulation of MBD2 and MBD3 expression, and the presence of methylation at CpGs that interact with transcription factor complexes might also be involved in the modulation of these genes. Moreover, reduced mRNA expression of MBD2 and MBD3 is implicated in gastric carcinogenesis, and thus, further investigations about these genes should be conducted for a better understanding of the role of abnormal methylation involved in this neoplasia.

Association study of SNPs of genes IFNGR1 (rs137854905), GSTT1 (rs71748309), and GSTP1 (rs1695) in gastric cancer development in samples of patient in the northern and northeastern Brazil.

Cancer is a multifactorial disease with a high mortality rate in Brazil and worldwide. Gastric cancer (GC) is considered the fourth type of malignancy more frequent in the population worldwide and the second leading cause of death. This work aimed to evaluate single nucleotide polymorphisms (SNPs) of IFNGR1, GSTT1, and GSTP1 genes samples in gastric cancer. We analyzed 60 samples of gastric cancer, 26 diffuse and 34 intestinal types, totaling 120 alleles for each SNP. The results were obtained by PCR and allele-specific PCR. Statistical analyzes performed using BioEstat 5.0 software, applying the Fisher's exact test and chi-square. Only the SNP gene GSTP1 (rs1695) were significantly associated with gastric cancer in the samples analyzed (χ(2) = 8.73, P < 0.05). Our results suggest that the GSTP1 gene SNP (rs1695) can be considered a risk factor associated with gastric carcinogenesis.

hTERT, MYC and TP53 deregulation in gastric preneoplastic lesions.

BACKGROUND: Gastric cancer is a serious public health problem in Northern Brazil and in the world due to its high incidence and mortality. Despite the severity of the disease, more research is needed to better understand the molecular events involved in this intestinal-type gastric carcinogenesis process. Since precancerous lesions precede intestinal-type gastric cancer, here, we evaluated the hTERT, MYC, and TP53 mRNA and protein expression, as well as TP33 copy number, in gastric preneoplastic lesions. METHODS: We evaluated 19 superficial gastritis, 18 atrophic gastritis, and 18 intestinal metaplasia from cancer-free individuals of Northern Brazil. Quantitative reverse transcription PCR was used to analyze the mRNA expression and immunohistochemical methods were used to assess protein immunoreactivity in tissue samples. The number of TP53 gene copies was investigated in gastric diseases by quantitative PCR. RESULTS: We observed hTERT, MYC, and p53 immunoreactivity only in intestinal metaplasia samples. The immunoreactivity of these proteins was strongly associated with each other. A significantly higher MYC mRNA expression was observed in intestinal metaplasia compared to gastritis samples. Loss of TP53 was also only detected in intestinal metaplasia specimens. CONCLUSIONS: We demonstrated that hTERT, MYC, and TP53 are deregulated in intestinal metaplasia of individuals from Northern Brazil and these alterations may facilitate tumor initiation.

Lymphocyte proliferation stimulated by activated Cebus apella macrophages treated with a complex homeopathic immune response modifiers.

INTRODUCTION: Canova is a complex homeopathic medicine that enhances a specific immunologic responses against several exogenous and endogenous conditions. Canova activates macrophages both in vivo and in vitro. AIM AND METHOD: We evaluated the effects of macrophages activated by Canova in vivo and ex vitro in the proliferation of lymphocytes. Canova was used to activate Cebus apella macrophages in vivo or ex vitro with Canova. Lymphocytes were cultured with the macrophage culture medium. The analysis of Canova effects in cultured lymphocytes was performed according to the cell cycle phase using flow cytometry. The Interferon gamma and Interleukin-5 cytokines quantification in these lymphocyte culture media was performed by Enzyme-linked immunosorbent assay (ELISA). RESULTS: We observed that Canova actives macrophages in vivo and ex vitro. The lymphocytes cultured in a supplemented medium with macrophages activated by Canova treatment presented a higher number of proliferation cells than lymphocytes not exposed to macrophages activated by Canova. The Interferon gamma and Interleukin-5 cytokines were only observed in the medium of lymphocytes exposed to macrophages activated by Canova. Thus, Canova has potential as a new adjuvant therapy.

MYC, TP53, and chromosome 17 copy-number alterations in multiple gastric cancer cell lines and in their parental primary tumors.

We evaluated whether MYC, TP53, and chromosome 17 copy-number alterations occur in ACP02, ACP03, and AGP01 gastric cancer cell lines and in their tumor counterpart. Fluorescence in situ hybridization for MYC and TP53 genes and for chromosome 17 was applied in the 6th, 12th, 60th, and 85th passages of the cell lines and in their parental primary tumors. We observed that three and four MYC signals were the most common alterations in gastric cell lines and tumors. ACP02 presented cells with two copies of chr17 and loss of one copy of TP53 more frequently than ACP03 and AGP01. Only ACP03 and AGP01 presented clonal chr17 trisomy with three or two TP53 copies. The frequency of MYC gain, TP53 loss, and chromosome 17 trisomy seems to increase in gastric cell lines compared to their parental tumors. Our findings reveal that these cell lines retain, in vitro, the genetic alterations presented in their parental primary tumors.

Dysregulated Expression of Apoptosis-Associated Genes and MicroRNAs and Their Involvement in Gastric Carcinogenesis.

PURPOSE: Analyze the expression of caspase-9, Smac/DIABLO, XIAP, let-7a, and let-7b in patients with normal gastric tissue, chronic gastritis, and gastric adenocarcinoma. METHODS: The expression of caspase-9, Smac/DIABLO, XIAP, let-7a, and let-7b by qRT-PCR was analyzed in 158 samples from 53 patients with normal gastric mucosa, 86 with chronic gastritis, and 19 with gastric cancer. RESULTS: The comparison between the gastric cancer and the control group revealed a decreased expression of caspase-9 in gastric cancer tissues; considering the Helicobacter pylor presence, comparable results were revealed. Smac/DIABLO was increased in gastric cancer cells, while XIAP demonstrated no significant difference in the gene expression. The microRNA analysis revealed a decreased expression of let-7a and let-7b in samples positive to H. pylori infection and in gastric cancer group, regardless of the presence of the bacterium. CONCLUSION: Our study provided some evidence of low activity of the intrinsic apoptosis pathway, as well as the influence of H. pylori on let-7a and let-7b expression.

Chromosome X aneuploidy in Brazilian schizophrenic patients.

The identification of cytogenetic abnormalities in schizophrenic patients may provide clues to the genes involved in this disease. For this reason, a chromosomal analysis of samples from 62 schizophrenics and 70 controls was performed with trypsin-Giemsa banding and fluorescence in situ hybridization of the X chromosome. A clonal pericentric inversion on chromosome 9 was detected in one male patient, and we also discovered mosaicism associated with X chromosome aneuploidy in female patients, primarily detected in schizophrenic and normal female controls over 40 years old. When compared with age-matched female controls, the frequency of X chromosome loss was not significantly different between schizophrenics and controls, except for the 40- to 49-year-old age group. Our findings suggest that the X chromosome loss seen in schizophrenic patients is inherent to the normal cellular aging process. However, our data also suggest that X chromosome gain may be correlated with schizophrenia in this Brazilian population.

The impact of DNA demethylation on the upregulation of the NRN1 and TNFAIP3 genes associated with advanced gastric cancer.

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Very few therapeutic options are currently available in this neoplasia. The use of 5-Aza-2'-deoxycytidine (5-AZAdC) was approved for the treatment of myelodysplastic syndromes, and this drug can treat solid tumours at low doses. Epigenetic manipulation of GC cell lines is a useful tool to better understand gene expression regulatory mechanisms for clinical applications. Therefore, we compared the gene expression profile of 5-AZAdC-treated and untreated GC cell lines by a microarray assay. Among the genes identified in this analysis, we selected NRN1 and TNFAIP3 to be evaluated for gene expression by RT-qPCR and DNA methylation by bisulfite DNA next-generation sequencing in 43 and 52 pairs of GC and adjacent non-neoplastic tissue samples, respectively. We identified 83 candidate genes modulated by DNA methylation in GC cell lines. Increased expression of NRN1 and TNFAIP3 was associated with advanced tumours (P < 0.05). We showed that increased NRN1 and TNFAIP3 expression seems to be regulated by DNA demethylation in GC samples: inverse correlations between the mRNA and DNA methylation levels in the promoter of NRN1 (P < 0.05) and the intron of TNFAIP3 (P < 0.05) were detected. Reduced NRN1 promoter methylation was associated with III/IV TNM stage tumours (P = 0.03) and the presence of Helicobacter pylori infection (P = 0.02). The identification of demethylated activated genes in GC may be useful in clinical practice, stratifying patients who are less likely to benefit from 5-AZAdC-based therapies. KEY MESSAGES: Higher expression of NRN1 and TNFAIP3 is associated with advanced gastric cancer (GC). NRN1 promoter hypomethylation contributes to gene upregulation in advanced GC. TNFAIP3 intronic-specific CpG site demethylation contributes to gene upregulation in GC. These findings may be useful to stratify GC patients who are less likely to benefit from DNA demethylating-based therapies.

Interrelationship between MYC gene numerical aberrations and protein expression in individuals from northern Brazil with early gastric adenocarcinoma.

Gastric cancer is the second leading cause of death by cancer in Brazil. Early gastric cancer represents approximately 10% of gastric cancer cases in some services of Brazil, which underscores the need for early gastric cancer diagnosis that could lead to better prognosis. There are few published studies of cytogenetic alterations in early gastric cancer. To evaluate MYC copy number and its protein expression, we performed fluorescence in situ hybridization and immunohistochemical analyses in five early gastric adenocarcinomas in individuals from northern Brazil. Three signals of MYC and MYC immunoreactivity were observed in all five samples, regardless of histologic type, tumor extension, or lymph nodal status. These novel findings concerning MYC copy number alteration in early gastric cancer suggest that MYC alteration is observed in the beginning of gastric carcinogenesis and could be used as a therapeutic target.

Decreased MicroRNA miR-181c Expression Associated with Gastric Cancer.

PURPOSE: This study investigated miRNA-181c expression in control patients (healthy gastric mucosa), patients with gastritis, and patients with gastric cancer. The presence of Helicobacter pylori was determined, and the associations between H. pylori infection, levels of miRNA-181c expression, and gastric disease were also analyzed. METHODS: A total of 158 subjects were included in the study, and the three groups were respectively composed of 53 controls, 86 patients with gastritis, and 19 patients with gastric cancer. miRNA-181c expression and H. pylori infection were determined by quantitative real-time PCR and PCR, respectively. The subsequent target gene analysis was performed using the bioinformatics approach to understand the possible mechanisms of gastric cancer. RESULTS: We determined significantly lower miRNA-181c expression in the gastric cancer group when compared to the control and gastritis groups, regardless of the presence of H. pylori. There was no difference in miRNA-181c expression between the control group and gastritis group, whether the presence of H. pylori was considered or not. The bioinformatics approach identified several genes as possible targets for miRNA-181c, including the X-linked inhibitor of apoptosis (XIAP) gene (which encodes a protein that belongs to a family of apoptotic suppressor proteins), the caspase 9 gene, and the caspase 3 gene. All target genes identified may be involved in gastric cancer and apoptosis pathways. CONCLUSION: The results suggest that the presence of H. pylori has no influence on microRNA expression and that the downregulation of miR-181c may play an important role in gastric cancer progression by controlling important genes associated with apoptosis. Therefore, miRNA-181c may be a potential marker of gastric cancer.

Anticancer potential of benzothiazolic derivative (E)-2-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-4-nitrophenol against melanoma cells.

Malignant melanoma is an important type of cancer worldwide due to its aggressiveness and poor survival rate. Significant efforts to understand the biology of melanoma and approaches to treat the advanced disease are focused on targeted gene inhibitors. Frequently mutated genes, such as NRAS, B-RAF and TP53, significantly exceed the frequency of mutations of other genes, emphasizing their importance for future targeted therapies. Considering the antitumor activity of benzothiazolic derivatives, this study aimed to demonstrate the action of benzothiazolic (E)-2-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-4-nitrophenol (AFN01) against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of its genetic alterations and mutations, such as the TP53, NRAS and B-RAF genes. The results presented here indicate that AFN01, as a significant cytostatic and cytotoxic drug due to its induction of DNA fragmentation, causes single and double DNA strand breaks, consequently inhibiting cell proliferation, migration and invasion by promoting apoptosis. Our data suggest that AFN01 might be considered as a future therapeutic option for managing melanoma.

Interrelationships among chromosome aneuploidy, promoter hypermethylation, and protein expression of the CDKN2A gene in individuals from northern Brazil with gastric adenocarcinoma.

Numerical alterations of chromosome 9, the status of promoter methylation and protein expression of the CDKN2A gene (aliases include p16 and p16(INK4a)), the possible association with gain of chromosome X, and the interrelation of these findings with clinic and pathological characteristics were investigated in gastric adenocarcinomas. Fluorescence in situ hybridization analysis with centromeric DNA probes, immunohistochemical staining, and methylation-specific polymerase chain reaction assays were performed in 15 gastric adenocarcinomas samples from individuals from northern Brazil. Aneuploidies of chromosomes X and 9 were found in all samples, both intestinal and diffuse type. Monosomy of chromosome 9 and gain of a copy of chromosome X (in both sexes) were observed in 100% of cases. Hypermethylation frequency and protein expression of CDKN2A were also found in all cases analyzed. No association of genetic and epigenetic alterations with histological type, tumor aggressiveness, and invasion was found (P > 0.05), which may be attributable to small sample size. There was a high level of association between absence of p16 protein expression levels, CDKN2A gene promote hypermethylation, and chromosome 9 aneuploidy (100% of cases). Thus, in the present samples, the apparent mechanisms behind p16 silencing include loss of chromosome 9 and promoter region hypermethylation.

Interleukin-6 polymorphisms, Helicobacter pylori infection in adult Brazilian patients with chronic gastritis and gastric adenocarcinoma.

BACKGROUND: The discovery of Helicobacter pylori offered the etiologic agent of the initiating event of the inflammatory cascade. It has been confirmed that the development of gastric cancer spans over several decades sequentially starting with the acquisition of H. pylori infection and the development of chronic gastritis. The IL-6 gene (Il-6), inflammatory cytokine and the single-nucleotide polymorphisms (SNPs) at the 5' flanking region of the Il-6 gene promoter (G or C at -174 base and at -572 or -597 C or A) have been identified with increased Il-6 levels. METHODS: Biopsies were collected from 168 patients. SNPs in Il-6-174 were analyzed by PCR-RFLP. RESULTS: Promoter SNP of Il-6 at -174 base were within Hardy-Weinberg equilibrium. We did not find any association between the frequencies of -174 polymorphism with specific histological type of gastric adenocarcinoma, but the G (guanine) allele at -174 was significantly higher in gastric adenocarcinoma than in patients with chronic gastritis. CONCLUSIONS: We observed an association between GG allele on -174 base with gastric cancer.

Apolipoprotein A-V gene polymorphism -1131T>C and Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly and is also considered a progeroid genetic syndrome. The etiology of AD is complex and the mechanisms underlying its pathophysiology remain to be clarified. It has been suggested that a high serum cholesterol level is a risk factor for (AD), and that some polymorphisms of genes encoding proteins regulating cholesterol metabolism are associated with AD development. APOA5 is a recently discovered apolipoprotein involved primarily with triglyceride (TG) metabolism disorder. This study investigates the association of AD with the APOA5 gene -1131T>C polymorphisms in samples of 106 patients with Alzheimer's disease (AD), 76 elderly healthy controls and 93 young healthy controls. DNA samples were isolated from blood cells, amplified by PCR and digested with Tru1l. We observed that the genotype distributions of APOA5 variants were within Hardy-Weinberg equilibrium in all subject samples. Furthermore, chi-square test comparison for genotype distributions and allele frequencies did not reveal any significant difference among the three groups of subjects P>0.05). These results support the idea that these variants are not involved as a risk factor for developing AD.

C-MYC locus amplification as metastasis predictor in intestinal-type gastric adenocarcinomas: CGH study in Brazil.

BACKGROUND: The genetic events involved in gastric cancer, the third most frequent cancer in the world with a high incidence in Pard State, Brazil, remain largely unknown. MATERIALS AND METHODS: Twenty-one primary gastric adenocarcinomas were investigated by comparative genomic hybridization (CGH) and the relationships between genomic abnormalities and histopathological features were evaluated. RESULTS: Eighty-one percent of cases presented DNA copy-number changes. Chromosomal gains were the most frequent finding, losses occurring only in the diffuse type. The main copy-number gains were on chromosome 8, principally on 8q24.1 (8/21 cases), 8p21 (3/21) and 8p23.2-8p12 (2/21). Gain of region 8q24. 1, where C-MYC is located, was the main finding, exclusively in the intestinal type with metastasis. CONCLUSION: C-MYC locus amplification may be predictor of aggressiveness in intestinal-type gastric cancer, playing an important role in its development and progression. Moreover, other genes on 8q24 should be investigated. Gastric adenocarcinomas of differing histopathological features were associated with distinct genetic alterations, supporting the hypothesis that they evolve through distinct genetic pathways.

Interrelationship between chromosome 8 aneuploidy, C-MYC amplification and increased expression in individuals from northern Brazil with gastric adenocarcinoma.

AIM: To investigate chromosome 8 numerical aberrations, C-MYC oncogene alterations and its expression in gastric cancer and to correlate these findings with histopathological characteristics of gastric tumors. METHODS: Specimens were collected surgically from seven patients with gastric adenocarcinomas. Immunostaining for C-MYC and dual-color fluorescence in situ hybridization (FISH) for C-MYC gene and chromosome 8 centromere were performed. RESULTS: All the cases showed chromosome 8 aneuploidy and C-MYC amplification, in both the diffuse and intestinal histopathological types of Lauren. No significant difference (P < 0.05) was observed between the level of chromosome 8 ploidy and the site, stage or histological type of the adenocarcinomas. C-MYC high amplification, like homogeneously stained regions (HSRs) and double minutes (DMs), was observed only in the intestinal-type. Structural rearrangement of C-MYC, like translocation, was observed only in the diffuse type. Regarding C-MYC gene, a significant difference (P < 0.05) was observed between the two histological types. The C-MYC protein was expressed in all the studied cases. In the intestinal-type the C-MYC immunoreactivity was localized only in the nucleus and in the diffuse type in the nucleus and cytoplasm. CONCLUSION: Distinct patterns of alterations between intestinal and diffuse types of gastric tumors support the hypothesis that these types follow different genetic pathways.

Biflorin induces cytotoxicity by DNA interaction in genetically different human melanoma cell lines.

Cancer is a public health problem and the second leading cause of death worldwide. The incidence of cutaneous melanoma has been notably increasing, resulting in high aggressiveness and poor survival rates. Taking into account the antitumor activity of biflorin, a substance isolated from Capraria biflora L. roots that is cytotoxic in vitro and in vivo, this study aimed to demonstrate the action of biflorin against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of genetic alterations and mutations, such as the TP53, NRAS and BRAF genes. The results presented here indicate that biflorin reduces the viability of melanoma cell lines by DNA interactions. Biflorin causes single and double DNA strand breaks, consequently inhibiting cell cycle progression, replication and DNA repair and promoting apoptosis. Our data suggest that biflorin could be considered as a future therapeutic option for managing melanoma.

The Emerging Role of miRNAs and Their Clinical Implication in Biliary Tract Cancer.

Biliary tract cancers are aggressive malignancies that include gallbladder cancer and tumors of intra- and extrahepatic ducts and have a poor prognosis. Surgical resection remains the main curative therapy. Nevertheless, numerous patients experience recurrence even after radical surgery. This scenario drives the research to identify biliary tract cancer biomarkers despite the limited progress that has been made. Recently, a large number of studies have demonstrated that deregulated expression of microRNAs is closely associated with cancer development and progression. In this review, we highlight the role and importance of microRNAs in biliary tract cancers with an emphasis on utilizing circulating microRNAs as potential biomarkers. Additionally, we report several single-nucleotide polymorphisms in microRNA genes that are associated with the susceptibility of biliary tract tumors.

Cancer type-specific epigenetic changes: gastric cancer.

Gastric cancer (GC) remains a major cause of mortality despite declining rate in the world. Epigenetic alterations contribute significantly to the development and progression of gastric tumors. Epigenetic refers to the number of modifications of the chromatin structure that affect gene expression without altering the primary sequence of DNA, and these changes lead to transcriptional activation or silencing of the gene. Over the years, the study of epigenetic processes has increased, and novel therapeutic approaches have emerged. This chapter summarizes the main epigenomic mechanisms described recently involved in gastric carcinogenesis, focusing on the roles that aberrant DNA methylation, histone modifications (histone acetylation and methylation), and miRNAs (oncogenic and tumor suppressor function of miRNA) play in the onset and progression of gastric tumors. Clinical implications of these epigenetic alterations in GC are also discussed.