Navegando por Autor "Camargo, Elaine Aparecida de"
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Item Cell cycle kinetics, apoptosis rates, DNA damage and TP53 geneexpression in bladder cancer cells treated with allyl isothiocyanate(mustard essential oil).(2014) Sávio, André Luiz Ventura; Silva, Glenda Nicioli da; Camargo, Elaine Aparecida de; Salvadori, Daisy Maria FáveroAllyl isothiocyanate (AITC) is present in plants of the cruciferous family and is abundant in mustard seed.Due to its high bioavailability in urine after ingestion, AITC has been considered a promising antineoplasticagent against bladder cancer. Because TP53 mutations are the most common alterations in bladder cancercells and are frequently detected in in situ carcinomas, in this study, we investigated whether the AITCeffects in bladder cancer cells are dependent on the TP53 status. Two bladder transitional carcinoma celllines were used: RT4, with wild-type TP53; and T24, mutated TP53 gene. AITC was tested at concentrationsof 0.005, 0.0625, 0.0725, 0.0825, 0.0925, 0.125 and 0.25 _M in cytotoxicity, cell and clonogenic survivalassays, comet and micronucleus assays and for its effects on cell cycle and apoptosis by flow cytometry andon TP53 gene expression. The data showed increased primary DNA damage in both cell lines; however,lower concentrations of AITC were able to induce genotoxicity in the mutant cells for the TP53 gene.Furthermore, the results demonstrated increased apoptosis and necrosis rates in the wild-type cells, butnot in mutated TP53 cells, and cell cycle arrest in the G2 phase for mutated cells after AITC treatment.No significant differences were detected in TP53 gene expression in the two cell lines. In conclusion,AITC caused cell cycle arrest, increased apoptosis rates and varying genotoxicity dependent on the TP53status. However, we cannot rule out the possibility that those differences could reflect other intrinsicgenetic alterations in the examined cell lines, which may also carry mutations in genes other than TP53.Therefore, further studies using other molecular targets need to be performed to better understand themechanisms by which AITC may exert its antineoplastic properties against tumor cells.Item MRE11A and SKP2 genes are associated with the increased cytotoxicity induced by the synergistic effects of cisplatin and gemcitabine in bladder cancer cells.(2014) Silva, Glenda Nicioli da; Camargo, Elaine Aparecida de; Sávio, André Luiz Ventura; Salvadori, Daisy Maria FáveroThe combination of gemcitabine and cisplatin has been shown previously to elicit a synergistic therapeutic effect on bladder cancer cell lines and result in reduced cell survival. However, the precise mechanism by which cells die has not been elucidated. Cell cycle-related genes are the predominant targets of chemotherapeutic protocols. Therefore, molecular biomarkers that are predictive of therapeutic outcomes associated with tumor sensitivity might be important for optimal treatment protocol selection. The aim of this study was to investigate the changes in gene expression in cell cycle-related genes that were induced by cisplatin, gemcitabine or a combined treatment using both agents in a low-grade urinary bladder transitional carcinoma cell line (RT4). The following three treatment protocols were used: 1.0 lM cisplatin, 1.56 lM gemcitabine and a combination of 1.0 lM cisplatin and 1.56 lM gemcitabine. Cytometry and morphology analysis (by phase-contrast photomicrography) were performed in addition to pathway-specific gene expression analysis using quantitative RT-PCR gene arrays. The following results were observed after 1.0 lM cisplatin treatment: (1) a decrease in cell number, (2) an increased percentage of scattered cells and (3) downregulated expression of genes related to cell cycle arrest, G1/S-to-mitotic cell cycle transition, DNA repair, apoptosis, transcription and mitosis. Treatment with 1.56 lM gemcitabine, or with both drugs simultaneously, induced the following effects: (1) a decrease in cell number, (2) an increased percentage of scattered and elongated cells, (3) the modulation of genes that are predominantly involved in DNA repair and (4) a significant upregulation of genes related to cell cycle arrest. Reduced cell density was observed after the combined treatment compared to the two other single-agent protocols. The downregulation of MRE11A and SKP2 was observed only in cells subjected to the combined treatment. In conclusion, cisplatin, gemcitabine and the combination of both drugs elicited distinct toxicogenomic effects in the RT4 bladder transitional carcinoma cell line, although disruptions in the expression of cell cycle control-related genes and other pathways responsible for cell survival were observed for all of the protocols. MRE11A and SKP2 downregulation appeared to be responsible for the synergistic therapeutic effects elicited by cisplatin and gemcitabine.Item No relationship between the amount of DNA damage and the level of hMLH1 and RASSF1A gene expression in bladder cancer cells treated with cisplatin and gemcitabine.(2013) Camargo, Elaine Aparecida de; Silva, Glenda Nicioli da; Gobette, Camila Pereira; Marcondes, João Paulo de Castro; Salvadori, Daisy Maria FáveroTumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.