Regulação da expressão de genes de controle do ciclo celular em linhagem de leucemia mielóide aguda (kg-1): caracterização de alvos de um novo fármaco

Abstract

Alterations in the cell cycle control mechanism led to uncontrolled proliferation, invasion, and metastasis, which are key features in cancer development. Additionally, abnormal differentiation during the division cycles of hematopoietic stem/progenitor cells is a hallmark of oncohematologies such as leukemias. Chemotherapeutic agents that act on the "negative" control of the cell cycle, called "cyclo-specific drugs", have been shown to be effective tumor agents in cells that are constantly dividing. The natural isoflavonoids present important biological activities such as antifungal, antibacterial, insecticidal and antitumoral. The present study sought to evaluate the cytotoxic profile (in vitro) of an isoflavonoid against a panel of leukemic cell lines and to determine its ability to modulate the expression of genes involved in the cell cycle. The antiproliferative profile of the compound was evaluated using the MTT method. The blocking profile in the cell cycle phase was evaluated by flow cytometry and the gene expression profile was done by the real-time PCR technique. The compound showed antiproliferative potential in all leukemic cell lines tested, where the IC50 ranged from 0.41 (EMP ± 0.13) to 7.51 (EMP ± 0.29) µM. The HL-60 (CI50 = 0.41µM - EMP ± 0.13) and KG-1 (CI50 = 1.0µM – EMP ± 0.09) strains showed higher sensitivity to the compound, respectively. Treatment with the isoflavonoid led to blockade of cell division in the G2/M phase at all tested times (12, 24 and 48h, p<0.0001) when compared to the drug-free negative control. Furthermore, during all times of treatment with the compound, several changes in the morphology of KG-1 cells were observed, such as an increase in the number of vacuoles, reduction of nuclear content and formation of plasma membrane "blebs". The results of CEP55, AURKB, MAD2, CDC20 and ATM gene expression after 12 and 24 hours of incubation showed that the compound significantly reduced AURKB gene expression (p<0.02) only at 24 hours, when compared to the untreated negative control. The results presented confirmed that the isoflavonoide evaluated had an antiproliferative and cytotoxic profile, showing blockade in G2/M, corroborating other studies that had already demonstrated its arrest profile during this phase of the cell cycle. The isoflavonoids tested seem to act in the inhibition of the AURKB gene, with no effects on the other genes evaluated. The auroras kinases have a critical role in cell division, acting in the mechanisms of chromosome alignment, mitotic spindle formation, and cytokinesis. The modulation of this gene represents an important target in anticancer therapy considering the widely investigated group of non-tubulin antimitotic drugs are among the first line chemotherapies for a wide spectrum of cancers.