RESUMO
Resveratrol (RES), a polyphenol compound with antiproliferative properties, has been previously evaluated for its beneficial effects against a variety of tumour cells. The current study elucidated the means by which RES enhances the antiproliferative effects of cisplatin (CIS) on MCF7 cells, focusing on the inhibitory effects on DNA repair of doublestrand breaks (DSBs). Chemoresistant MCF7 cells (MCF7R) were generated by continuous exposure to low concentrations of CIS (10 µM CISIC40) during 5 passages, with the IC50 value increasing ~3fold. Using an MTT assay, we estimated the changes in IC50 for CIS in MCF7, T47D, MDAMB231 and MCF7R cells in the presence of RES. The relative transcript level of Nbs1, Mre11 and Rad50 genes was assessed using RTqPCR analysis. Rad51 and H2AX [pSer139] protein expression was determined by western blot analysis. RES at 50 and 100 µM significantly enhanced the antiproliferative effects of CIS in both MCF7 and MCF7R cells, decreasing the IC50 values for CIS to onetenth and onesixth, respectively. A total of 100 µM RES decreased the relative transcript levels of homologous recombination (HR) initiation complex components and the Rad51 protein level in MCF7 and MCF7R cells. After 48 h of CIS DNA damage, the levels of Rad51 protein increased, but this effect was inhibited by 100 µM RES. RES also maintained serine 139 phosphorylation of histone H2AX, suggesting that RES prevents the repair of DSBs. It was observed that RES exerts an antagonistic effect over CIS on the activation of Rad51 and sustained phosphorylation of H2AX. The results suggest that RES in combination with DNA damagebased therapy has potential as a strategy to overcome resistance and provide much safer and more effective treatment for breast cancer.
Assuntos
Neoplasias da Mama/genética , Cisplatino/farmacologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Rad51 Recombinase/genética , Estilbenos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Células MCF-7 , Fosforilação/efeitos dos fármacos , Rad51 Recombinase/metabolismo , ResveratrolRESUMO
The transcriptional terminator tI generates the 3'end of the integrase (int) gene transcript that is read from the lambda PI promoter in lambda phage. We have studied the factors that affect transcription termination in vitro and in vivo at the lambda tI terminator. In vitro transcriptional studies showed that tI is about 80% efficient in the presence of purified NusA protein, whereas it is only about 50% efficient in its absence. In vivo studies, where the readthrough transcript of lambda tI was measured by quantitative dot blot analysis, gave about 80% efficiency in wild-type strains, but only 60% in the nusA1 mutant strain at non-permissive temperatures. These results support the idea that termination at lambda tI in vivo involves interaction with the NusA factor.