Tiago Donizetti Silva

DNA methylation as an epigenetic biomarker in colorectal cancer.

Sporadic colorectal cancer (CRC) is a consequence of the accumulation of genetic and epigenetic alterations that result in the transformation of normal colonic epithelial cells to adenocarcinomas. Studies have indicated that a common event in the tumorigenesis of CRC is the association of global hypomethylation with discrete hypermethylation at the promoter regions of specific genes that are involved in cell cycle regulation, DNA repair, apoptosis, angiogenesis, adhesion and invasion. The present study aimed to investigate the epigenetic changes (DNA methylation) in 24 candidate genes in CRC. A total of 10 candidate hypermethylated (HM) and unmethylated (UM) genes were identified that may be useful epigenetic markers for non-invasive CRC screening. The five genes that had the highest average UM percentages in the control group were MLH1 (71.7%), DKK2 (69.6%), CDKN2A (68.4%), APC (67.5%) and hsa-mir-342 (67.4%). RUNX3 (58.9%), PCDH10 (55.5%), SFRP5 (52.1%), IGF2 (50.4%) and Hnf1b (50.0%) were the five genes with the highest average HM percentages in the test group. In summary, the present preliminary study identified the methylation profiles of normal and cancerous colonic epithelial tissues, and provided the groundwork for future large-scale methylation studies.

What are the most effective methods for assessment of nutritional status in outpatients with gastric and colorectal cancer

OBJECTIVE To evaluate methods for the identification of nutrition risk and nutritional status in outpatients with colorectal (CRC) and gastric cancer (GC), and to compare the results to those obtained for patients already treated for these cancers. METHODS A cross-sectional study was conducted on 137 patients: group 1 (n = 75) consisting of patients with GC or CRC, and group 2 (n = 62) consisting of patients after treatment of GC or CRC under follow up, who were tumor free for a period longer than 3 months. Nutritional status was assessed in these patients using objective methods [body mass index (BMI), phase angle, serum albumin]; nutritional screening tools [Malnutrition Universal Screening Tool (MUST), Malnutrition Screening Tool (MST), Nutritional Risk Index (NRI)], and subjective assessment [Patient-Generated Subjective Global Assessment (PGSGA)]. The sensitivity and specificity of each method was calculated in relation to the PG-SGA used as gold standard. RESULTS One hundred thirty seven patients participated in the study. Stage IV cancer patients were more common in group 1. There was no difference in BMI between groups (p = 0.67). Analysis of the association between methods of assessing nutritional status and PG-SGA showed that the nutritional screening tools provided more significant results (p < 0.05) than the objective methods in the two groups. PG-SGA detected the highest proportion of undernourished patients in group 1. The nutritional screening tools MUST, NRI and MST were more sensitive than the objective methods. Phase angle measurement was the most sensitive objective method in group 1. CONCLUSION The nutritional screening tools showed the best association with PG-SGA and were also more sensitive than the objective methods. The results suggest the combination of MUST and PG-SGA for patients with cancer before and after treatment.

N-Acetyltransferase 2 genetic polymorphisms and risk of colorectal cancer

AIM To investigate the possible association between meat intake, cigarette smoking and N-acetyltransferase 2 (NAT2) genetic polymorphisms on colorectal cancer (CRC) risk. METHODS Patients with CRC were matched for gender and age to healthy controls. Meat intake and cigarette smoking were assessed using a specific frequency questionnaire. DNA was extracted from peripheral blood and the genotypes of the polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism. Five NAT2 alleles were studied (WT, M1, M2, M3 and M4) using specific digestion enzymes. RESULTS A total of 147 patients with colorectal cancer (76 women and 90 men with colon cancer) and 212 controls were studied. The mean age of the two groups was 62 years. More than half the subjects (59.8% in the case group and 51.9% in the control group) were NAT2 slow acetylators. The odds ratio for colorectal cancer was 1.38 (95% CI: 0.90-2.12) in slow acetylators. Although the number of women was small (n = 76 in the case group), the cancer risk was found to be lower in intermediate (W/Mx) acetylators [odds ratio (OR): 0.55, 95% confidence interval (95% CI): 0.29-1.02]. This difference was not observed in men (OR: 0.56, 95% CI: 0.16-2.00). Among NAT2 fast acetylators (W/W or W/Mx), meat consumption more than 3 times a week increased the risk of colorectal cancer (OR: 2.05, 95% CI: 1.01-4.16). In contrast, cigarette smoking increased the risk of CRC among slow acetylators (OR: 1.97, 95% CI: 1.02-3.79). CONCLUSION The risk of CRC was higher among fast acetylators who reported a higher meat intake. Slow NAT2 acetylation was associated with an increased risk of CRC.

Oral Concentrated Grape Juice Suppresses Expression of NF-kappa B, TNF-α and iNOS in Experimentally Induced Colorectal Carcinogenesis in Wistar Rats

UNLABELLED The aim of this study was to evaluate the effects of grape juice on colon carcinogenesis induced by azoxymethane (AOM) and expression of NF-kB, iNOS and TNF- α. METHODS Forty male Wistar rats were divided into 7 groups: G1, control; G2, 15 mg/kg AOM; G3, 1% grape juice 2 weeks before AOM; G4, 2% grape juice 2 weeks before AOM; G5, 1% grape juice 4 weeks after AOM; G6, 2% grape juice 4 weeks after AOM; G7, 2% grape juice without AOM. Histological changes and aberrant crypt foci (ACF) were studied, while RNA expression of NF- kB, TNF- and iNOS was evaluated by qPCR. RESULTS The number of ACF was higher in G2, and G4 presented a smaller number of crypts per focus than G5 (p=0.009) and G6. Small ACF (1-3) were more frequent in G4 compared to G2, G5 and G6 (p=0.009, p=0.009 and p=0.041, respectively). RNA expression of NF-kB was lower in G3 and G4 compared to G2 (p=0.004 and p=0.002, respectively). A positive correlation was observed between TNF- α and NF-kB gene expression (p=0.002). In conclusion, the administration of 2% grape juice before AOM reduced the crypt multiplicity, attenuating carcinogenesis. Lower expression of NF-kB was observed in animals exposed to grape juice for a longer period of time, regardless of concentration.

RNA interference: a promising therapy for gastric cancer.

Gastric cancer (GC) remains a virtually incurable disease when metastatic and requires early screening tools for detection of early tumor stages. Therefore, finding effective strategies for prevention or recurrence of GC has become a major overall initiative. RNA-interference (RNAi) is an innovative technique that can significantly regulate the expression of oncogenes involved in gastric carcinogenesis, thus constituting a promising epigenetic approach to GC therapy. This review presents recent advances concerning the promising biomolecular mechanism of RNAi for GC treatment.

Genetic polymorphisms of vitamin D receptor (VDR), CYP27B1 and CYP24A1 genes and the risk of colorectal cancer.

Introduction Vitamin D receptor (VDR) and proteins encoded by the genes CYP27B2 and CYP24A1 involved in the production and inactivation of vitamin D can influence vitamin D and the susceptibility to colorectal cancer (CRC). The objective of this study was to investigate the relationship between the risk of CRC and polymorphisms in VDR, CYP27B1 and CYP24A1, lifestyle and dietary habits. Methods The study included 152 patients with CRC and 321 controls. All participants answered a questionnaire on their dietary habits, alcohol consumption and smoking habits. DNA was extracted from peripheral blood. Polymorphisms of BsmI and ApaI were identified by performing PCR-RFLP. Identification of CYP24A1 (rs6013897, rs158552 and rs17217119) and CYP27B1 (rs10877012) polymorphisms was performed by gene sequencing. Results Smoking, alcohol use, and low or no consumption of fruit, cereals and dairy products were associated with an increased risk of CRC. A heterozygous genotype Aa or an association genotype aa + Aa of the VDR ApaI polymorphism increased the risk of CRC. The VDR BsmI polymorphism was not significantly associated with the risk of CRC. Multivariate analysis showed that heterozygous and association genotype AT + AA of the rs6013897 polymorphism, genotype CT of the rs158552 polymorphism, association genotype CT + CC and genotypes AA and GG of the rs17217119 polymorphism of CYP24A1, and heterozygous genotype GT and association genotype GT + TT of the rs10877012 polymorphism in CYP27B1 were associated with a higher risk of CRC. Conclusions Dietary habits, lifestyle, and polymorphisms in VDR (ApaI), CYP24A1 (rs6013897, rs158552, rs17217119) and CYP27B1 (rs10877012) were associated with a higher risk of CRC.

Establishment and Partial Characterization of an Epirubicin- Resistant Gastric Cancer Cell Line with Upregulated ABCB1

Multidrug resistance (MDR) is a major impediment to successful chemotherapy of gastric cancer. Our aim was to establish an epirubicin-resistant cell subline (AGS/EPI) and to elucidate the mechanisms involved in acquired EPI resistance. The AGS/EPI cell subline developed by exposing parental AGS cells to stepwise increasing concentrations of EPI demonstrated 2.52-fold resistance relative to the AGS cell line, and mRNA expression of the ATP-dependent drug-efflux pump P-glycoprotein (Pgp), more recently known as ABCB1 protein, was similarly upregulated. An AGS/EPI cell subline could thus be effectively established, and MDR mechanism of these cells was shown to be related to the overexpression of mRNA of the ABCB1 gene.

Genetic polymorphisms of vitamin D metabolism genes and serum level of vitamin D in colorectal cancer

Background The metabolism of vitamin D is complex, its receptor (VDR) and proteins encoded by the genes CYP27B2 and CYP24A1 can influence vitamin D serum levels. The aim of this study was to investigate the relationship of the polymorphisms of VDR (ApaI and BsmI), CYP27B1 and CYP24A1 with serum vitamin D levels in both forms, 25(OH)D3 (circulating form) and 1,25(OH)2D3 (active form), in colorectal cancer (CRC) patients. Methods One hundred fifty-two CRC patients and 321 controls were included. DNA was extracted from peripheral blood. Polymorphisms of BsmI and ApaI were identified by PCR-RFLP. Those of CYP24A1 (rs6013897, rs158552 and rs17217119) and CYP27B1 (rs10877012) were determined by gene sequencing. Results The median serum levels of circulating vitamin D were not different between CRC patients and controls; however, the percentage of those with deficient vitamin D was higher in patients with cancer. The active form of the vitamin D was higher in CRC patients. VDR, CYP27B1 and CYP24A1 polymorphic genotypes had no influence on serum levels of circulating vitamin D. The correlation between circulating and active vitamin D forms was lower among patients with CRC, regardless of the presence or absence of any genetic polymorphism. The mean serum levels of active vitamin D were higher among patients with polymorphic genotype variants of Apa1 or Bsm1. Conclusions CRC patients had a higher frequence of insufficient vitamin D and a higher concentration of active vitamin D. These concentration were higher between patients with polymorphic genotypes variants of ApaI and BsmI, CYP24A1 and CYP27B1. Polymorphic genotypes cause a lower correlation between the forms of vitamin D.

HUMAN DNA QUANTIFICATION IN THE STOOLS OF PATIENTS WITH COLORECTAL CANCER

BACKGROUND Colorectal cancer is one of the main cause of cancer in the world. Colonoscopy is the best screen method, however the compliance is less than 50%. Quantification of human DNA (hDNA) in the feces may be a possible screen non-invasive method that is a consequence of the high proliferation and exfoliation of cancer cells. OBJECTIVE To quantify the human DNA in the stools of patients with colorectal cancer or polyps. METHODS Fifty patients with CRC, 26 polyps and 53 with normal colonoscopy were included. Total and human DNA were analyzed from the frozen stools. RESULTS An increased concentration of hDNA in the stools was observed in colorectal cancer patients compared to controls and polyps. Tumors localized in the left side of the colon had higher concentrations of hDNA. There were no difference between polyps and controls. A cut off of 0.87 ng/mL of human DNA was determined for colorectal cancer patients by the ROC curve, with a sensitivity of 66% and a specificity of 86.8%. For polyps the cut off was 0.41, the sensitivity was 41% and the specificity 77.4%. CONCLUSION A higher concentration of hDNA had been found in colorectal cancer patients The quantification of hDNA from the stools can be a trial method for the diagnosis of colorectal cancer.

The influence of nutritional status and disease on adiponectin and TNF-α levels in colorectal cancer patients

BACKGROUND The aim of this study was to evaluate the association between adiponectin and tumor necrosis factor-α;(TNF-α;) serum levels in colorectal cancer (CRC) patients and compare these levels to clinical stage and nutritional status. METHODS A total of 79 patients were enrolled in the study (39 with CRC and 40 in the control). Nutritional status was assessed by Patient-Generated Subjective Global Assessment (PG-SGA), body mass index (BMI), and phase angle (PhA). Adiponectin and TNF-α;serum concentrations were determined using an enzyme-linked immunosorbent assay. RESULTS Serum adiponectin levels were higher among CRC patients (p = 0.001). TNF-α;serum levels were not significantly different between the groups, but patients with stage III or IV CRC had higher levels of TNF-α;than those with lower stage disease (p = 0.037). The three tools used for the assessment of nutritional status (BMI, PhA, and PG-SGA) demonstrated that patients with a more severe nutritional deficit had higher adipocytokine levels, although these differences were significant only to TNF- , when distributed PhA in tertiles. CONCLUSIONS Adiponectin levels were higher among CRC patients. Although TNF-α;serum levels from CRC patients did not differ significantly to the control group, CRC patients with stage III or IV had higher levels compared to those with stage I and II tumors. Nutritional status, as determined by BMI, PhA, and PG-SGA, demonstrated that patients with a greatest nutritional deficit, had higher levels of adipocytokines; however, these differences were significant only for TNF-, when distributed PhA in tertiles.