Análise da expressão de Sirtuína-1 e da microdensidade vascular no melanoma cutâneo humano e em modelo experimental in vitro de melanoma murino

Abstract

The current study evaluated the proliferation index and immunohistochemical expression of SIRT1 on cutaneous melanocytic nevi (CMN) and cutaneous malignant melanoma (CMM). Formalin-fixed paraffin-embedded tissue samples from 43 CMN, 22 CMM and 17 normal skin were obtained and clinical data were abstracted from the electronic medical record. SIRT1 and Ki67 proteins expressions were evaluated regarding to clinic pathological behavior in CMM. The level of significance was set at α = 5% (p < 0.05). Our findings showed that SIRT1 positivity was significantly higher in benign melanocytic nevi than that in cutaneous malignant melanoma (p = 0.035). As expected, the proliferation index was significantly higher in samples of cutaneous malignant melanoma as compared to the normal skin and melanocytic nevi (p < 0.001). However, the expression of Ki67 protein was not also significantly related to the expression of SIRT1 (p > 0.05). In conclusion, low expression of SIRT1 and high proliferation index may play an important role in progression of cutaneous melanoma. The second objective aimed to evaluate the effect of resveratrol and sirtinol, activator and inhibitor of sirtuins, respectively, on cell viability and SIRT1 mRNA expression in murine metastatic skin melanoma (B16F10) cell line. B16F10 and non-tumoral murine melanocytes (Melan-A) cell lines were treated with resveratrol and sirtinol and evaluated for cell viability, DNA fragmentation and SIRT1 gene expression. All experiments were performed in triplicate and submitted to specific statistical tests with significance level at α = 5% (p < 0.05). Treatments with resveratrol and sirtinol significantly affected the B16F10 cell viability (p < 0.05) and promoted DNA fragmentation (p < 0.05). Significant reductions in the viability and DNA fragmentation after exposure to resveratrol and sirtinol were also observed in Melan-A cells. However, both treatments did not change the SIRT1 expression on the same studied conditions. Results of the present study revealed that use of these drugs may be a promising chemotherapeutic strategy against melanoma cancer, even in the absence of changes in the transcription of the SIRT1 gene. Moreover, it was evaluated expression of PECAM-1 and endoglin for a correlation with clinicopathological behavior in CMM. Control (n = 12), CMN (n = 48), and CMM (n = 44) samples were submitted for immunohistochemistry. PECAM-1 and endoglin expression were counted in the stroma (hot spots) of all samples in order to calculate the MVD. Data analyses were performed using univariate statistical tests, with significance set at p<0.05. Our findings showed that CMM exhibited higher MVD estimates for both PECAM-1 and endoglin compared to control and CMN samples (p < 0.001, for all associations). Moreover, CMN samples exhibited higher MVD compared to control samples (p<0.001 for all associations). CMM from subjects with metastatic disease showed higher MVD by PECAM-1 (p=0.036) and endoglin (p=0.015) compared to non-metastatic CMM. In conclusion, increasing MVD from normal skin to benign and malignant melanocytic tumors suggest the importance of a rich vascular network in the peritumoral stroma to support greater metabolic and energetic demands, which favors the dissemination of melanocytic tumor cells.