Efeitos do citrato de clomifeno e do letrozol no comportamento da linhagem celular de melanoma humano A375

Abstract

Melanoma is a cancer type originated from the malignant transformation of melanocytes, melanin-producing cells that can be found in the skin, as well as in other anatomic sites. Despite its low incidence, melanoma has high lethality rates due to its notably aggressive cell profile. Its occurrence depends on the interaction of multiple factors, and the association between the use of fertility drugs and the development of melanoma, amongst other cancer types, has already been suggested in the literature. Clomiphene citrate (CC) and letrozole (LET) are widely used for the treatment of infertility in anovulatory patients, and for preserving cancer patients’ fertility, for instance. These drugs are used for controlled ovarian stimulation (COS), which aims to induce ovulation. The correlation between CC use for COS and the development of melanoma is indicated by several studies, but their observational character limits the interpretation of their results. Regarding LET, the literature has already mentioned its protective role against estrogen-receptor positive breast cancer, when LET was used in association with tamoxifen. However, up to date there are no studies that investigated its effects in the development nor treatment of other cancer types. Thus, the present work aimed to investigate the effects of CC and LET in the A375 human melanoma cell line, by evaluating aspects such as cell viability, cell cycle, migratory profile, and mRNA expression of genes associated with epithelial-mesenchymal transition (NCAD and ECAD), mitochondrial oxidative stress (SOD2) and cell death (BAX). The cells were treated with CC (200 to 2000 ng/mL) and LET (400 to 4000 ng/mL) for 24 and 48 hours. An 80% reduction in cell viability was observed 48 hours after treatment with 2000 ng/mL CC; with induction of cell cycle arrest in G1 phase 24h after treatment, and in sub-G1 phase 48h following treatment. LET induced only mild changes in cell viability and cell cycle. After 48 hours of CC treatment (2000 ng/mL), A375 cells’ migration ability in the wound healing assay decreased. Additionally, gene expression results indicated statistically significant differences in the expression of BAX and SOD2 after cell treatment with 2000 ng/mL of CC, as well as 400 and 4000 ng/mL of LET. After 24h and 48h of incubation with 2000 ng/mL of CC, there was a reduction in the expression of BAX (0.044 and 0.485 fold change, respectively), with an increase in the expression of SOD2 48h after the treatment (1.399 fold change). However, 48h after treatment with LET there was an increase in the expression of BAX and SOD2 at 400 ng/mL (1.836 and 2.321 fold change, respectively), and at 4000 ng/mL of the drug (5.193 and 8.587 fold change, respectively). Furthermore, LET down-regulated NCAD expression after 24 and 48 h of incubation at concentrations of 400 ng/mL (0.261 and 0.319 fold change, respectively) and 4000 ng/mL (0.272 and 0.462 fold change, respectively). In conclusion, the results of the present study indicate that CC and LET reduce A375 cells’ viability, induce cell cycle arrest in the G1 phase, reduce cells’ migration ability, and increase oxidative stress; indicated by the up-regulation of SOD2 and BAX. Therefore, these ovulation-inducing drugs did not favor the aggressiveness of A375 melanoma cell line.