Investigation of Phenothiazine-Induced Cell Death Mechanisms In Tumor Cells: Changes in Gene Expression, Role of Bcl-2 Family Proteins, and ER Stress

Abstract

Literature data have shown that psycotropic drugs derived from phenothiazines present antitumor activity. Working with isolated rat liver mitochondria, we previously showed that phenothiazine derivatives induced the mitochondrial permeability transition associated with cytochrome c release. After, we established the cytotoxic potential of different phenothiazine derivatives and determined the structural requirements for the induction of cell death by these drugs. Additionally, we proposed that mitochondrial permeabilization process plays a central role in thioridazine-induced cell death. In leukemia cells, the cell death induced by phenothiazines is a complex process with involvement of apoptosis and autophagy pathways, and our preliminary results show its modulation by Bcl-2 family proteins. Thus, the aim of this project is to investigate the molecular mechanisms of thioridazine-induced cell death in leukemic cells, particularly evaluating the role of Bcl-2 family proteins, the changes in gene expression of cell death-related pathways and the involvement of endoplasmic reticulum stress. Also, the antitumor effect of thioridazine in vivo will be evaluated. A quantitative PCR array with selected genes from apoptosis, autophagy and necrosis pathways will be performed to screen the possible targets of thioridazine. Also, it will be investigated the alterations in the amount of Bcl-2 expressed proteins, including the pro and anti-apoptotic members (BCL-2, BCL-XL, MCL-1, BAX, BAK, BID, BAD, PUMA and NOXA) and other proteins that interact with this family (Beclin), using specific monoclonal antibodies by western blotting or flow cytometry. Also, disruption of calcium homeostasis and endoplasmic reticulum stress response induced by thioridazine, as well as their modulation by Bcl-2 proteins, will be also studied. It was expected that these results will contribute not only for a better comprehension of the mechanisms of cell death in tumor cells, but also to the discovery of new therapeutic targets and drugs to be used in the antitumor chemotherapy. (AU)