AMANDA IKEGAMI
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Artigo IPEN-doc 24344 Knockdown of NF-κB1 by shRNA inhibits the growth of renal cell carcinoma in vitro and in vivo2018 - IKEGAMI, AMANDA; TEIXEIRA, LUIZ F.S.; BRAGA, MARINA S.; DIAS, MATHEUS H. dos S.; LOPES, EDUARDO C.; BELLINI, MARIA H.Renal cell carcinoma (RCC) accounts for approximately 2-3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-кB transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-kB in RCC, and many implicated NF- κB1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-κB1 into mouse renal cell carcinoma (Renca) cells. It was determined that the knockdown of the NF-κB1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G2/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-кB1 cells have significantly diminished tumorigenicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNANF- кB1 tumors. Thus, this study indicates that downregulation of NF-кB1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-кB1 may be a potential therapeutic target for RCC.Resumo IPEN-doc 23797 shRNA Knockdown of NFKB1 expression inhibits proliferation and promotes apoptosis of renal cell carcinoma2016 - IKEGAMI, A.; TEIXEIRA, L.F.; BRAGA, M.S.; SILVA, E.C. da; BELLINI, M.H.Renal cell carcinoma (RCC) represents approximately 2‐3 % of human malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. The transcription factor KB (NFKB) comprises a family of transcription factors which has been associated with apoptosis resistance and progression of RCC. In this study, shRNA plasmid vector against NFKB1 gene was stably transduced into the Renca murine RCC cell line. Knockdown of NFKB1 was confirmed by quantitative real time PCR, Western blot and immunofluorescence analysis. The biological effects of decreased NFKB1 protein levels were evaluated, in vitro, by cell cycle and doubling time analysis and, in vivo, by tumor growth, cell proliferation (PCNA staining) and apoptosis (Caspase‐3 staining) and necrosis (morphometry). The results revealed that NFKB1 knockdown efficiently inhibited the growth of RencashRNA cells in culture, induced cell cycle arrest at the G2/M phase and led to a significant decrease of the doubling‐time. Moreover, NFKB1 shRNA vector suppressed tumor growth, enhanced apoptosis and necrosis compared with a wild type and mock control groups. In conclusion, our results suggest that specific silencing of NFKB is a potential therapeutic strategy for the treatment of RCC. This research was supported by FAPESP (2014/19265‐6)