Marcelo Soares da Mota e Silva

Efeito do álcool perílico na expressão gênica de células de adenocarcinoma de pulmão humano

OBJECTIVE: To study the effect of perillyl alcohol on the gene expression of human pulmonary adenocarcinoma cells. METHODS: Pulmonary adenocarcinoma cells were incubated with perillyl alcohol in dilutions ranging from 0.03% to 0.0003% for 48 hours. Alterations were observed in the cell morphology, and cell viability was quantified using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Protein synthesis of samples previously targeted with S35 was analyzed using electrophoresis on a polyacrylamide gel. Expression of the proteins p53 and p44/42 was determined using the Western blot method. RESULTS: After 48 hours of incubation, greater nsumbers of morphological alterations were observed in cells treated with the 0.03% perillyl alcohol dilution than in those treated with perillyl alcohol diluted to 0.003% or further. Treatment with perillyl alcohol dilutions of 0.03%, 0.003% and 0.0003% inhibited cellular viability by 60.17% (p < 0.001), 15.62% (p < 0.001) and 11.53% (p < 0.05), respectively. The results show that 28-kDa, 42-kDa and 110-kDa proteins were induced. No statistically significant effect on p53 expression was observed. In comparison with the expression of a-tubulin, the 0.003% perillyl alcohol dilution induced an increase in p42 phosphorylation and a marked decrease in p44 phosphorylation. CONCLUSION: The results suggest that there are other, previously undescribed, metabolic pathways for perillyl alcohol effects in human pulmonary adenocarcinoma cells.

Detoxification enzymes: cellular metabolism and susceptibility to various diseases

Detoxification enzymes act in the cellular metabolism of substances that are strange to the organism (xenobiotic) and endogenous compounds that could cause cellular and tissue damage, such as compounds derived from the oxidative stress. Cellular metabolism occurs in three successive phases: in phase 1, potentially toxic compounds are activated, especially through the action of P-oxidases cytochrome (CYP) enzymes.1 In phase 2, the compounds that were activated in the previous phase are conjugated to substances that will make them more water soluble and easily excreted. In this phase, glutathione S-transferase (GSTs), UDP-glucuronosyltransferases (UGT), Sulfotransferases (SULT) and Nacetyl-transferase (NAT) enzymes are the main players.2 In phase 3, conjugates compounds are eliminated extracellularly through membrane transporters, which are proteins with ion and molecule transport function through the cellular membrane.3 Figure 1 shows a scheme of the cellular metabolism by the action of detoxification enzymes. FIGURE 1 Scheme of cellular metabolism by the action of detoxification enzymes. Author: Marcelo S. M. Silva

DNA detection of JC and BK virus in archival urine cytospin slides

To identify decoy cells, cytological examination was performed in urine cytospin slides. Decoy cells are related to Polyomaviruses (JC virus [JCV] and BK virus [BKV]), which are recognized worldwide due to potential infection and morbidity in kidney transplant recipients. Cytologically, it is difficult to evaluate the cytopathic effect of JCV and BKV in urine of patients with urothelial neoplasia. For this reason, there is a need for molecular approaches. To evaluate the incidence of BKV and JCV DNA in archival slides of urine cytospin material with benign and malignant characteristics. A total of 176 urine specimens were used for cytological examination of neoplastic or decoy cells. The samples were analyzed for the presence of JCV and BKV, by polymerase chain reaction (PCR) in DNA Isolated from archival slides of urine cytospin material. A typical samples (n = 48) were compared with the remaining 128 samples without atypia/neoplasia for the presence of JCV or BKV DNA. A statistically nonsignificant result was observed correlating the presence of JCV or BKV. The results show that DNA Isolated from archival slides of urine cytospin material can be used for detection of BKV and JCV.

Abstract 389: Epigenetic and proteomic analysis of gastric tumor and its histologically free proximal and distal margins

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Gastric cancer (GC) corresponds to the fourth most common malignancy among men and sixth among women in Brazil. GC is a multifactorial disease that results from individual genetic predisposition and exposure to ambient factors such as diet, alcohol consumption, smoking, chronic Helicobacter pylori infection or Epstein-Barr virus (EBV). H. pylori and EBV are known to up-regulate DNA methyltransferases. Aim: Compare the promoter methylation profiles of E-cadherin, p16, DAPK, and Rb genes of histologically tumor free proximal and distal margins from fresh tissues and perform a quantitative proteomic comparison of the tumor, distal and proximal resection margins profiles from the same patient. Methods: 18 samples consisting of six gastric carcinomas, their corresponding proximal margins (PM), and distal margins (DM) were obtained from six patients subjected to gastric resection at the Federal University of Rio de Janeiro, Brazil. DNA was extracted from the fresh tissues by using proteinase K digestion and Phenol-chloroform isoamilic alcohol followed by ethanol precipitation. Methylation-specific PCR analysis was used to determine the methylation status of E-cadherin, p16, DAPK, and Rb genes promoter by bisulfite modification. The presence of EBV was investigated by PCR and a shotgun proteomic analysis of all tissues from the H pilory and EBV negative patient was performed using an Orbitrap Velos. Results: 3 of 6 patients were positive for EBV viz: 3 tumors plus 6 margins. The total methylation for the 4 genes in all 9 samples (p16, E-cadherin, DAPK, and Rb genes) were: 5/36 in PM (esophagus); 5/36 in tumor, and 9/36 for DM (intestinal); in the negative EBV samples: 6/36 in PM; 4/36 in tumor and 8/36 in the DM. The proteomic analysis disclosed 786 proteins identified in the tumor fragment (58 proteins uniquely identified in this tissue), 777 to histologically normal proximal margin (87 unique proteins) and 750 to histologically normal distal margin (132 unique proteins). In all three fragments analyzed, unique proteins related to tumor progression or metastasis were identified; examples are: hepatoma- derived growth (HDGF) in the tumor, Annexin 1A in the PM and Mucin 1 in the DM. Conclusion: Our results show that histologically free tumor margins are molecularly compromised by methylation and by up regulation of proteins correlated to tumor progression and metastasis even in samples not infected with EBV or H.pylori Financial support CNPq Citation Format: Paulo Costa Carvalho, Carlos Eduardo Carvalho, Guilherme Pinto Bravo Neto, Juliana Fischer Carvalho, Thais Mac Cormick, Priscila F. Aquino, Marcelo Mota Silva, Maria da Gloria da Costa Carvalho. Epigenetic and proteomic analysis of gastric tumor and its histologically free proximal and distal margins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 389. doi:10.1158/1538-7445.AM2014-389