Study of the mechanisms of thioridazine-induced cell death in tumor cells: focus on signaling pathways and expression of BCL-2 family proteins

Abstract

Cancer is a worldwide occurrence disease, which is considered a public health problem since it is the leading cause of population death. In 2007, it was registered 7.9 millions of deaths caused by this disease and the expectations for the next years are an increase in this number. Data indicates that will occur at least 12 millions of deaths until the year of 2030. The costs with cancer therapy are estimated in approximately 40 million dollars per year in the world and that will increase more than 173 billion by the year of 2020. Cancer chemotherapy is medicine field in constant innovation and the attention given to this issue is rising. Consequently, it was observed a significant progress in this area during the last years leading to successful treatments of a variety of tumors and an increase in patient's survival rates. Nevertheless, despite the availability of many therapeutic options, drug resistance still is the major problem observed during the evolution of the disease. Therefore, increase in the efficacy and selectivity of antitumor agents associated to the reduction of drug resistance are the main goals in the development of new therapies nowadays. Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem-cells characterized by a genetic abnormality called the Philadelphia chromosome, which originates the oncogene protein BCR-ABL responsible for the pathogenicity of this disease. The expression of this protein in leukemia cells makes them able to overcome cell death mechanisms resulting in resistance to chemotherapeutic agents. Since it is not a solid tumor, there are not many therapeutic options available for CML treatment. Thus, the search for new chemotherapeutic agents is extremely important. Literature data have shown that psychotropic drugs derived from phenothiazines presented antitumor activity. Unplublished data from our group have evidenced the effects of phenothiazines and its analogues on mitochondrial and lysossomal morphology and function. These effects have been associated to the ability of inducing cell death. Among the studied phenothiazines, the most potent was thioridazine, which was chosen for the present work. The aim of this work is to underlie the molecular mechanisms of thioridazine-induced cell death in leukemic cells, evaluating the role of BCL-2 family proteins, as well as the changes in the signaling pathways associated to cell death. Since our previous results demonstrated that phenothiazines were able to promote mitochondrial dysfunction associated to cell death, alterations in the quantity and localization of BCL-2 family 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 analysis or flow cytometry. Additionally, specific signaling pathways related to apoptotic/authophagic cell death such as PI3K/Akt/mTOR, Ras/MAPK and JAK/STAT will be carefully investigated by quantitative techniques. Also, the effects of several inhibitors and modulators in thioridazine-induced cytotoxicity will be investigated. As proposed, the study of the modulation of cell death-related signaling pathways and the evaluation of the alterations of BCL-2 family proteins induced by thioridazine will help not only in the comprehension of cell death induction mechanisms, but also in the discovery of new therapeutic targets for a possible association with conventional drugs.