Role of pioglitazone on gene/protein expression profile, bioenergetics and TGFβ/SMAD signaling pathway in NSCLC

Abstract

Background: Pioglitazone is an antidiabetic drug of the Thiazolidinediones (TZDs) class that acts as ligand for PPAR-γ, a member of peroxisome proliferator activated receptors (PPARs), that regulates lipid and glucose cell metabolism. Prior studies in in vitro and in vivo models of non-small cell lung cancer (NSCLC) showed that PPAR-γ modulation affects cancer cells proliferation and differentiation but few reported studies have investigated molecular pathways involved in the potential role of PPAR-γ agonists as anti-cancer agents. Methods: NSCLC cell lines H460 and H1299, were treated with different doses of Pioglitazone. Anti-proliferative effect was determined by MTT, colony-forming assay and flow-cytometry. Protein expression was detected by Western Blot analysis while functional mitochondrial measurements were performed with SeaHorse © stress test. Finally, cell lines samples were analyzed with a cDNA microarray assay. Results: Pioglitazone significantly reduces cell proliferation and invasion with an IC50 of 1-5 μM. Analysis of apoptosis confirmed the data. Western blot analysis demonstrated a dose-related reduction of Survivin and phosphorylated proteins of MAP kinase pathway and cDNA microarray expression profiling showed a down-regulation of MAPK, Myc and RAS genes. Oxygen Consumption Rate (OCR) and proportional Glut-1 protein expression reduction of treated cells demonstrated cell bioenergetics modulation. Interestingly cDNA microarray analysis showed that also TGFβ pathway is regulated by Pioglitazione through pTGFβR1 and pSMAD3 down-regulation and consecutive up-regulation of total TGFβR1. Conclusions: Pioglitazone regulates NSCLC cell lines proliferation and bioenergetics. Moreover, affecting TGFβ/SMAD signaling pathway, it could have a role in epitelialto- mesenchymal transition (EMT) and cancer invasive phenotype development. These results encourage the study of PPAR-γ agonists as anti-cancer agents and promote research to explore the mechanisms beyond their activity in NSCLC models.