Investigação do papel de APE1 em modelos de câncer de mama: em busca de novas funções moleculares

Abstract

Breast cancer is the second cancer that kills more women in developed countries and the first cause of death in developing countries. The basic excision (BER) repair pathway is responsible for repairing the free radical damage to DNA; however, the malfunction contributes to the formation and progression of tumors. APE1 endonuclease, the main BER enzyme, is responsible for preparing the abasic sites for repair continuity and increased expression has been related to aggressiveness and a poor prognosis. Thus, in the present study, the function of APE1 endonuclease in proliferation, aggressiveness, and gene regulation of cellular models of breast cancer was evaluated. WST-1 assay was performed to evaluate the viability of cell lines after APE1 inhibition with CRT0044876. Annexin V/7-AAD assay was performed to investigate the mechanism of death activated by CRT0044876. Immunofluorescence assay with Phalloidin was then performed to evaluate the actin cytoskeleton. In addition, microarray assay was performed to investigate possible gene expression changes caused by APE1 inhibition and real-time PCR validation of gene expression related to cytoskeleton pathway regulation. Wound-healing and cellular invasion cell migration trials were performed in Transwell® with matrigel to investigate the metastatic potential of cell models. Finally, TCGA analysis was used to investigate possible correlations of gene signature of APE1 activity and expression of altered microarray pathway genes. Cell viability assay showed that inhibition of APE1 endonuclease function is important for viability of MDA-MB-231 and MCF-7 cell lines, reducing viability in MDA-MB-231 cell line by approximately 50 %, and viability in MCF-7 cell line by 25 %7 incubated with CRT0044876 at concentrations greater than 1000 µM after 6 h of treatment. Apoptosis/necrosis assay showed that both mechanisms are triggered simultaneously in MDA-MB-231 cells, where apoptosis and necrosis rates were approximately 57% and 59%, respectively. Immunofluorescence in MDA-MB-231 cell line showed formation of lamellipodia and stress fibers. Microarray assay showed alteration of 2399 positively regulated genes and 496 negatively regulated genes in MDA-MB-231 cells, with the main altered pathway being related to cytoskeleton regulation. In addition, cell migration assay showed significant variation, approximately 10%, after treatment with CRT0044876 in MDA-MB-231 cells. Finally, through PCR, it was possible to observe an increase of approximately 11 times in MMP1 transcripts, increased 8.25 times in microarray, in MDA-MB-231 cells. In MCF-7 cells there was a reduction of approximately 4 times in PAK2 transcripts, which appears in the increased microarray 2.92 times. GA analyses indicated that there is a mean correlation between APE1 activity and MMP1 expression. In the basal-like subtype there is more expression of ENAH, NRAS, NCKAP1, DIAPH3, and MMP1. Thus, the results indicate that inhibition of APE1 endonuclease function is important for viability in MDA-MB-231 and MCF-7 cell cultures, but only induces morphological alteration in MDA-MB-231 cells. Such inhibition also suggests a gene regulation of APE1, mainly in relation to the cytoskeleton and MDA-MB-231 cell migration process