Avaliação do mecanismo de ação citotóxico de análogos sintéticos de alcaloides da classe dos 3-alquilpiridínicos em linhagens celulares humanas de Leucemia Promielocítica Aguda.

Abstract

Leukemia is a malignant disease of clonal disorder that originates in the bone marrow, where blood cells are produced. Acute Promyelocytic Leukemia (APL) in particular, without treatment, has an unfavorable evolution because it causes an intense hemorrhagic syndrome. Chemotherapy treatment, therefore, is one of the alternatives to improve patient survival. In this context, the search for new compounds with potential antitumor action is justified and relevant. The present work evaluated the cytotoxic activity of 16 synthetic analog molecules of 3-alkylpyridine alkaloids (3-APA). Cytotoxic profiles were determined by in vitro assay of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium (MTT) against acute promyelocytic leukemia with PML-RARα(NB4); acute promyelocytic leukemia with PML-RARα and ATRA-resistant (NB4-R2) and acute promyelocytic leukemia (HL-60) and PBMC (peripheral blood mononuclear cell). Assessment of apoptosis, as well as analysis of cell cycle arrest, was performed by flow cytometry; investigation of the mechanism of action, by analyzing the cell cycle; gene expression alterations of apoptosis genes (TP53, p21, BAK, Bcl-2, AKT) and oxidative stress (NOX1, NOX2, NOX4, P47phox) were performed by RT-qPCR. In order to enhance the investigation on substance interaction, a trivial chemical similarity analysis was performed by ChEMBL to predict probable targets of the substances. Substances 6,7 were the most active and selective for NB4 (IC50 of 38.81μM and 37.40μM respectively), substances 1 and 10 were more active for NB4-R2 (IC50 of 42.40μM and 41.05μM ) and substances 1,2,3, and 4 were more cytotoxic for HL60 (IC50 of 18.02 μM; 9.90 μM; 25.40μM; and 31.42 μM, respectively). All these molecules induced apoptosis in the cell lineages tested. With regard to the investigation of the mechanism of action of the compounds, it was observed that there is a predominance of decreased expression of anti-apoptosis genes (p21, Bcl-2, and AKT) inducing the cell death process. An attempt at cell survival by oxidative stress genes was noted by increasing the expression of NOX2 and p47phox in the NB4 lineage. The increased expression of NOX1 and NOX4 in the NB4-R2 lineage could be related to cell damage. Trivial ChEMBL chemical similarities search returned ROS1 and PARP1 as possible targets for molecules studied in this work. The data presented in this work indicate that the 3-alkylpyridine alkaloid analogues are a potential class of compounds with cytotoxic action (in vitro)