Efeitos do citrato de clomifeno e letrozol na transição-epitélio-mesenquimal e na carga viral do HPV das células HeLa (adenocarcinoma cervical humano)

Abstract

Cervical cancer is the third most frequent type of neoplasm among Brazilian women, accounting for 7.5% of new cancer cases in 2020. It is known that high-risk Human Papillomaviruses (HPV) are the main cause of the development of this disease. However, there are multiple factors associated with the prevalence of the virus, such as smoking, long-term use of oral contraceptives, multiparity, co-infection with other sexually transmitted agents and hormonal imbalance. In this last context, assisted human reproduction techniques use ovulation-inducing drugs to collect ovarian follicles, such as Clomiphene Citrate (CC) and Letrozole (LET). As they act on the hypothalamic-pituitary-ovarian axis, these drugs lead to a hormonal imbalance and, therefore, are associated with the promotion and progression of various neoplasms, such as cervical cancer. The aim of the present study was to evaluate the effects of CC and LET on epithelial-mesenchymal transition (TEM) and on HPV18 viral load in HeLa cells (cervical adenocarcinoma). A 24- and 48-hours treatment of HeLa cells was performed with 2000 ng/mL of CC and 4000 ng/mL of LE and, then, the mRNA expression of ECAD, NCAD, ZEB1, ZEB2 and SNAIL was quantified, as well as HPV18 DNA, by real-time PCR. The results showed that, after 24 hours of treatment with CC and LET, there was an overexpression of transcription factors ZEB1, ZEB2 and SNAIL, which was reduced significantly after 48 hours. NCAD, on the other hand, showed aberrant expression after 48 hours of treatment with both drugs. Regarding the HPV viral load, it was observed that HeLa cells treated with CC and LET for 24 hours have a lower number of virus copies than the control condition. However, after 48 hours, there was an increase in viral load in both treatments. Therefore, based on these results, it is suggested that the use of CC and LET stimulate viral replication over time, and induce a more aggressive behavior of HeLa cervical cancer cells, since it stimulates EMT by activating transcription in the first 24 hours of treatment and thus increase NCAD expression after 48 hours. This profile is compatible with a mesenchymal phenotype, which has a high invasive, migratory and, consequently, metastatic capacity, associated with a worse prognosis.