Evaluation of cannabinoid-mediated cytoprotection via autophagic modulation in induced Pluripotent Stem Cells (iPS) and C2C12 myoblasts: establishment of an in vitro model for studies of neoplastic cachexia

Abstract

Human-induced pluripotent stem cells (iPS) are an unlimited source of cells with pluripotent potential similar to embryonic stem cells, but generated from somatic cells. In vitro studies of disease modeling with these cells have stood out in drug screening, as they represent human diseases much better than the animal model. Thus, using myogenic progenitors and myotubes obtained from iPS cells, we will study the role of autophagy and its modulation by cannabinoids in protecting these cells after cytotoxic insults, aiming to identify the mechanisms involved in the loss of muscle mass, which is triggered by several chronic diseases, including advanced stage cancer (cancerous cachexia or neoplastic cachexia). Our goal is more precisely to identify specific molecular / cellular targets that can control the progression of proteolysis and/or cell death events. For this, we will use the iPS myogenic progenitors (PLZ cell line) as a study model, kindly provided for the accomplishment of this project by Prof. Dr. Perlingeiro (University of Minneosta, MN, USA). These progenitors were obtained after reprogramming human fibroblasts during the development of my activities at the University of Minnesota, MN, USA (BPE-FAPESP 18/07633-2). These iPS cells, as well as the murine C2C12 myoblasts, will be subjected to treatment with pharmacological autophagy modulators (rapamycin and chloroquine), and then challenged with chemotherapeutic agents that induce cachexia in individuals with cancer, such as cisplatin. Cardiotoxin (CTX), conditioned medium of tumor cells (RXT393) and palmitate will also be evaluated in the induction of cytotoxicity in these cells, as they represent models of induction of muscle mass loss in different pathophysiological conditions. From these tests, where we hope to understand the role of autophagy in the entire process of inducing cytotoxicity by the compounds described above, we will move on to studies on the cytoprotective effects of cannabinoids CDB (cannabidiol), CBDV (Cannabidivarin) and THCV (tetrahydrocannabidivarin) and possible participation of autophagy in these events. To meet all of these objectives, culture, proliferation and cell death assays will be used by flow cytometry and the TUNEL technique. Immunofluorescence tests will be applied in the morphological evaluation and quantification of myotubes obtained in different treatments. The proteins involved in the autophagy pathway and contractile muscle processes will be studied using the Western blotting technique. The qRT-PCR assay will also be applied to understand the modulation of genes involved in the autophagic and contractile processes. As a result, we hope to obtain solid data on the molecular mechanisms involved in the modulation of autophagy by cannabinoids with possible control in the loss of muscle mass. The establishment of a culture of human iPS cells in the screening of drugs capable of controlling the events of neoplastic cachexia is also the focus of this project. We emphasize that few studies are verified in the literature on the studies proposed here, which makes this project innovative in all aspects. Several orientations can be performed in this project due to its comprehensive proposal, involving different models of proteolysis/cell death induction and its possible pharmacological protection by phyto-cannabinoids and other compounds. The possibility of exchange between students from the University of Minnesota and UNIFESP, is also a differential of this proposal. (AU)