ABSTRACT
Title
Antiangiogenic and antitumour activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38
Authors
A. Fioravanti1, C. La Motta2, P. Orlandi1, B. Canu1, T. Di Desidero1, L. Mugnaini2, S. Cosconati2, S. Sartini2, R. Frati1, A. Antonelli3, P. Berti4, P. Miccoli4, F. Da Settimo2, R. Danesi1, G. Bocci1
1Div. of Pharmacology, Dept. of Internal Medicine, University of Pisa, Pisa, Italy; 2Dept. of Pharmaceutical Sciences, University of Pisa, Pisa, Italy; 3Metabolism Unit, Dept. of Internal Medicine, University of Pisa, Pisa, Italy; 4Dept. of Surgery, University of Pisa, Pisa, Italy
1Div. of Pharmacology, Dept. of Internal Medicine, University of Pisa, Pisa, Italy; 2Dept. of Pharmaceutical Sciences, University of Pisa, Pisa, Italy; 3Metabolism Unit, Dept. of Internal Medicine, University of Pisa, Pisa, Italy; 4Dept. of Surgery, University of Pisa, Pisa, Italy
Abstract
Tyrosine kinase inhibitors (TKIs) have significantly changed the perspectives of current cancer therapy. Understanding the mechanisms of normal and aberrant tyrosine kinase signalling and strategies to inhibit them in angiogenesis and cancer, further promote the development of novel agents (Gotink et al., 2010). Tyrosine kinase inhibitors (TKIs) have significantly changed the perspectives of current cancer therapy. Understanding the mechanisms of normal and aberrant tyrosine kinase signalling and strategies to inhibit them in angiogenesis and cancer, further promote the development of novel agents (Gotink et al., 2010). Vascular endothelial growth factors (VEGFs) and their respective family of receptor tyrosine kinases (VEGFRs) are key proteins modulating normal and pathological angiogenesis.The EGFR is required for normalcellular proliferation, survival, adhesion, migration, and differentiation buta dysregulation of this pathway can lead to oncogenesis.EGFR signaling plays a key role in promoting the growth and survival of various types of solid tumors, including non-small cell lung cancer (NSCLC) and breast, gastric, prostate, thyroid and colorectal cancer (Yoshida et al., 2010). The role of the RET oncogene in the development of medullary thyroid cancer (MTC) has been well descibed. The activation of RET germline and somatic mutations has been identified as the primary cause of hereditary and sporadic MTC cases (Ball 2007). Based on these considerations, tyrosine kinases of growth factor receptors, such as EGFR and VEGFR-2, are highly attractive targets for the development of new specific anticancer agents. The aim of the present study is to demonstrate the antiproliferative and pro-apoptotic activity of the novel pyrazolopyrimidine derivative multiple tyrosine kinase inhibitor CLM3, alone and in combination with SN-38 (the active metabolite of irinotecan), on endothelial and tumour cells and to show its mechanism of action. Proliferation and apoptotic assays were performed on microvascular endothelial (HMVEC-d) and lung (A549) and thyroid cancer (8305C, TT) cell lines exposed to CLM3 and to the simultaneous combination with SN38 for 72h. Cell-based phospho-VEGFR-2, phospho-EGFR and phospho-RETinhibition assays were performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 gene expression was performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA.A strong effect on antiproliferative and pro-apoptotic activity was found with the CLM3 on endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2, phospho-EGFR and phospho-RET levels significantly decreased after CLM3 treatments in activated endothelial and cancer cells; ERK1/2 and Akt phosphorylation were significantly inhibited by lower concentrations of the pyrazolopyrimidinedrug in endothelial cells if compared to cancer cells. Moreover, CLM3 treatment greatly inhibited the expression of the cyclin D1 gene in endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration.The pyrazolopyrimidine derivative CLM3 demonstrated a highly significant and promising antiproliferative and proapoptotic activity, alone and in combination with SN-38, for activated endothelial and cancer cells. These effects are mainly due to its inhibition of phosphorylation of VEGFR-2, EGFR and RET tyrosine kinases and their related signalling pathways.
1) Gotink KJ et al. (2010). Angiogenesis. 13,1-14.
2) Yoshida T et al. (2010). Biochem Pharmacol. 80,613-23.
3) Ball DW. (2007). Endocrinol Metab Clin North Am. 36,823-37.
1) Gotink KJ et al. (2010). Angiogenesis. 13,1-14.
2) Yoshida T et al. (2010). Biochem Pharmacol. 80,613-23.
3) Ball DW. (2007). Endocrinol Metab Clin North Am. 36,823-37.