AMANDA IKEGAMI
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Resumo IPEN-doc 24634 Downregulation of NF-ΚB1 enhances the radiosensitivity of renal cell carcinoma2017 - IKEGAMI, AMANDA; SILVA, LUIZ F.T. da; BELLINI, MARIA H.Introduction: Clear cell renal cell carcinoma (ccRCC) accounts for ∼80% of all renal cell carcinomas (RCC) and has the Von Hippel–Lindau (VHL) tumor suppressor gene mutated. The lack of VHL protein leads to a constitutionally active Hypoxia Inducible Factor (HIF) pathway that confers both chemoresistance and radioresistance for renal tumor. HIF pathway is known to interact with the transcription factor nuclear factor kappa B (NF-kB). Increased NF-κB activity is associated with the development and progression of RCC (IKEGAMI A, TEIXEIRA LF. BRAGA MS et al. The American Society for Cell Biology 2016; 26: 3948-3955). Objective: Evaluate the synergistic effect of NF-kB1 knockdown and ionizing radiation in murine renal adenocarcinoma cell line. Methods: The murine renal adenocarcinoma cell line (Renca cells) (ATCC, USA) was cultured in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin. Lentiviral shRNA vector was used to knockdown of NF-KB1 gene in Renca cells, as described previously (1). In the clonogenic cell survival assay, the cells were irradiated by 60Co source in the range from 0 to 10 Gy, using the GammaCell 220 – Irradiation Unit of Canadian-Atomic Energy Commision Ltd. (CTR-IPEN). After 10-14 days of culture, cell colonies were fixed and stained with formaldehyde 4% and rhodamine B 2% and counted. To assess cell viability, tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-MTS] was performed within 24 hours after irradiation at a dose of 10Gy. The survival variables α e β were fitted according to the linear quadratic equation (SF=exp[-αD-βD2]); SF=survival fraction, D=dose of irradiation and P value was determined by F test. Multiple comparisons were assessed by One-way ANOVA followed by Bonferroni´s tests with GraphPad Prism version 6.0 software. P< 0.05 was considered statistically significant. Data are shown as the mean ± SD. Results: The Renca-shRNA-NF-kB1 cells were found to be significantly more radiosensitive than controls - Renca-WT and Renca-Mock, (P<0.001 vs Renca-Mock). The ratio α/β was increased in Renca-shRNA-NF-kB1: -0.177±0.677 compared with 7.368±1.833 and 11.960±5.240 of the Renca- WT and Renca-Mock, respectively. There was no significant difference in the survival fraction between Renca-WT and Renca-Mock groups. The lethal dose 50% (LD50) of Renca-WT was 3.33 Gy and Renca-Mock was 3.288 Gy whereas for the Renca-shRNA-NF-kB1 group it was 2.08 Gy. Corroborating these data, the Renca-shRNA-NF-kB1 showed reduction of 16.75±0.06% in the viability when compared to the Renca-Mock (P<0.001). Conclusion: The knockdown of NF-kB1 gene mediated by shRNA on Renca cells led to a decrease in the radioresistance. Therefore, this gene can be a therapeutic target for CCR treatment.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)Resumo IPEN-doc 22980 A regulação negativa da expressão do gene NF-κβ1 induz a diminuição da proliferação em linhagem celular de carcinoma renal2016 - IKEGAMI, A.; TEIXEIRA, L.F.S.; BELLINI, M.H.Resumo IPEN-doc 22987 A regulação negativa da expressão do gene NF-κβ1 induz o retardamento da migração e invasão e redução da clonogenicidade em linhagem celular de carcinoma renal2016 - TEIXEIRA, L.F.S.; IKEGAMI, A.; BELLINI, M.H.