RESUMO
Prostate cancer (PCa) is the second most common type of male malignancy worldwide. The transcription factor zinc finger Ebox binding homeobox 1 (ZEB1) is associated with epithelialmesenchymal transition and is also involved in regulation of androgen receptor (AR) expression, the main ligands of which are testosterone and dihydrotestosterone (DHT). These androgens are synthesized through the steroidogenic pathway within the prostate, and their synthesis is altered in PCa. The present study aimed to determine the ZEB1induced alterations in androgen synthesis and AR expression in the DU145 PCa cell line. Reverse transcriptionquantitative polymerase chain reaction, western blotting and immunocytochemistry were used to determine the mRNA and protein expression levels, and cellular localization of steroidogenic pathway enzymes in the DU145 cell line in response to ZEB1 silencing. Furthermore, the concentrations of testosterone and DHT were detected in cell culture medium using ELISA. ZEB1silenced cells exhibited an increase in testosterone and DHT production, an increase in AR expression and an alteration in the steroidogenic pathway. In particular, steroidogenic acute regulatory protein and 5αreductase 2 expression levels were decreased, whereas cytochrome P450 family 17 subfamily A member 1, 5αreductase 1, aldoketo reductase family 1 member D1 and aldoketo reductase family 1 member C2 expression levels were increased. In conclusion, the present study provided novel information regarding the regulation of intratumoral androgen production in PCa, which is relevant for the progression of the disease to a castrationresistant form.
Assuntos
Di-Hidrotestosterona/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Testosterona/biossíntese , Homeobox 1 de Ligação a E-box em Dedo de Zinco/fisiologia , Linhagem Celular Tumoral , Di-Hidrotestosterona/análise , Inativação Gênica , Humanos , Masculino , Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/química , Receptores Androgênicos/metabolismo , Testosterona/análise , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
In the early stages, prostate cancer is androgen dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.