Abstract 5044: P144, an inhibitory peptide of the TGF-beta signaling pathway, inhibits growth and increases apoptosis in brain tumor cell lines

Abstract

Transforming growth factor beta (TGF-beta), a cytokine with potent promalignant properties, is overexpressed in different tumors. Therefore, the development of TGF-beta inhibitory molecules is of paramount importance to improve the efficacy of antitumor therapies. Several strategies using neutralizing antibodies, TGF-beta soluble binding proteins, antisense compounds, or small molecule inhibitors targeting TGF-beta signaling, have shown that TGF-beta inhibition helps in antitumor therapy. Some of these compounds have shown their value by directly inhibiting tumor growth and metastasis, whereas others have been able to enhance antitumor immune responses. The aim of our study was to investigate the effect of two peptide inhibitors of TGF-beta, namely P144 (1) and P17 (2), in 6 human tumor cell lines derived from medulloblastoma (DAOY), rhabdomyosarcoma (TE671), neuroblastoma (SK-NSH and SH-SY5Y), and glioblastoma (A172 and U87-MG). P144 and P17 are derived from human TGF-beta type III receptor sequence (1) and phage display library technology (2) respectively. Both peptides have high affinity for soluble TGF-beta and potent inhibitory effects on TGF-beta binding to its receptors. MTT assay and an apoptosis test (Cell Death Detection ELISA PLUS, Roche) were performed before and after treatment with the peptides, which were added to the media at a final concentration of 100 ug/ml. P17 did not produce any effect on the cell lines, while P144 induced growth inhibition and apoptosis in three brain tumor cell lines (DAOY, A172, and U87-MG). The level of phosphorylated Smad2 (that initiates the activation of the TGF-beta intracelular signaling pathway) was decreased in presence of P144 in all cases, as determined by Western blot, confirming that P144 inhibits TGF-beta signaling. qRT-PCR was performed in order to analyze different genes related with the regulation of the TGF-beta signaling, such as Smad7, Ski, SnoN, FoxG1, which are inhibitors of the pathway, and p21CIP. Our preliminary data show that P144 affects the transcription of Smad dependent genes, e.g. Smad7, SnoN and Ski, although confirmatory experiments are still required. Our data suggest that inhibition of the TGF-beta pathway by P144 inhibits cell growth and increases apoptosis in brain tumor cell lines, possibly in a Smad-dependent manner. Therefore, P144 should be further tested as a therapeutic agent against brain tumors. 1. Ezquerro IJ, et al. A synthetic peptide from transforming growth factor beta type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury. Cytokine 22:12-20, 2003. 2. Dotor J, et al. Identification of peptide inhibitors of transforming growth factor beta 1 using a phage-displayed peptide library. Cytokine. 39:106-115, 2007. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5044. doi:10.1158/1538-7445.AM2011-5044