Estudo in vitro dos mecanismos de ação antitumoral do composto LQB-461 em células de leucemia Jurkat

Abstract

Leukemias stand out as the main type of childhood cancer in the world and current treatments have strong side effects, with the possibility of developing therapy resistance. Several researches seek to develop new hybrid molecules that present better therapeutic efficacy. Previous studies related to the in vitro antileukemic structure-activity of LQB-278 analogues, highlighted the LQB-461 (benzaldehyde and cinnamoyl hybrid with insertion of a nitro group) due to its potentiated effect and increase of p21, with inhibition of proliferation without inducing death (2 and 3 μM), or accompanied by death by apoptosis (5 μM). In this context, this work evaluated the mechanisms of action of LQB 461 involved in its cytostatic and apoptosis-inducing effects in Jurkat cells. To analyze the genes and pathways involved in the mechanisms of action of LQB-461, a transcriptome assay was performed (Microarray). The expression of the CDKN1A gene (which encodes p21) was increased, confirmed by PCRq (p<0.01) with 5 μM. An increase (p<0.05) of apoptosis-inducing biological processes (3 and 5 μM) was also observed, highlighting the intrinsic apoptosis pathway. Through flow cytometry (FACS), a significant increase in the Bax/Bcl-2 ratio was demonstrated at tested concentrations, with changes in Bax (p<0.05) and Bcl -2 (p<0.001) also observed by RT-PCR at some concentrations. Treatment with LQB-461 increased (p<0.001) the expression of Fas receptor and active caspase-3 (p<0.001) with 5 μM, by FACS. Through the transcriptome, we observed inhibition (p<0.05) of biological processes related to protein synthesis (3 μM), and negative regulation for mediators of the mTOR pathway, the main regulator of cell proliferation and protein translation. Preliminary Western Blotting (WB) assays suggest a decrease in the mTOR-P/mTOR (3 μM) ratio. By the same technique, LQB-461 seems to increase (with 2 and 3 μM) the expression of the S6KP70 protein, which induces protein synthesis, and decrease it at 5 μM. In the transcriptome, we found reduced expression of the RPS6KB1 gene, encoding the S6K1 isoform, activated by mTOR, at LQB-461 3μM and 5μM. Also by WB, the RB1 protein, which inhibits the Akt/mTOR pathway, seems to have its expression increased at 2 and 3 μM and decreased at 5 μM. Modulation of autophagy was also evaluated, as it can promote apoptosis, resistance and proliferation. The transcriptome showed an increase (p<0.05) of autophagic biological processes (3 μM) and inhibition (5 μM). We also observed, by WB, increased (p<0.05) expression of the autophagic marker LC-3 at 2 and 3 μM, and decreased one at 5 μM (p<0.001). In conclusion, LQB-461 promotes a cytostatic effect in Jurkat cells (3 μM), inhibiting proliferation, by decreasing protein synthesis. Apoptosis is not observed at this concentration, suggesting action of cellular resistance mechanisms, including autophagy. At the highest concentration (5 μM) LQB-461 seems to inhibit autophagic mechanisms, activating the intrinsic and extrinsic apoptosis pathways in a caspase-dependent manner. LQB-461 seems to act in multiple mechanisms of action, standing out as a promising alternative in the treatment of acute lymphocytic leukemia.