Análise de diferentes programas de morte e sobrevivência celular, relacionadas ao mecanismo de quimiorresistência na linhagem celular de câncer de ovário SKOV-3

Abstract

Ovarian cancer presents the highest rate of mortality among gynecological tumors. Chemotherapy based on platin and taxane compounds is the first line of treatment available for patients, therefore the initial response, about 60-80% will present relapse of the disease-associated to chemoresistance. TNF-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R) have become a target for cancer therapy since the discovery that this protein can induce death in tumoral cells and spare normal cell. Draw the mechanisms by which TRAIL acts in different tumoral types can orientate its right use in clinical practice. Thus, this work aims to evaluate the participation of targets related to the TRAIL/TRAIL-R system and chemoresistance in the ovarian cancer cell line SKOV-3. Characterization of the SKOV-3 cell line regarding TRAIL-R2 and TRAIL-R3 receptors was accomplished by flow cytometry and immunofluorescence. The results showed that these receptors were not present in the surface of the cell membrane, cytoplasm, or nucleus. At the transcriptional level, both mRNAs of TRAIL-R2 and TRAIL-R3 were detected, and by that, it can be assumed that post-transcriptional regulation of these receptors is happening in this cell line. Viability assays were performed by MTT and the concentrations found to cisplatin were 0.015mg/mL, 100ng/mL for rhTRAIL, and 0.025mg/mL;100ng/mL for cisplatin and rhTRAIL combinations to treat cells and perform the RNA extraction. Evaluation of gene expression profile of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RIPK1, MLKL, NFKB1, RELA, TP53, CASP8 and CFLAR was performed by RT-qPCR. Three differential responses in the profile of gene expression were observed. Cisplatin's treatment up-regulated the expression of TP53. rhTRAIL's treatment up-regulated NFKB1 and RELA gene expression, suggesting that the NF-kB pathway may have initiated. Already in the combination of both drugs, RIPK1 and MLKL were up-regulated, indicating that death by necroptosis may have activated. These results demonstrate that chemotherapy treatment not always reaches apoptosis but also transcriptionally regulates survival pathways and inflammatory death.