title: Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells.
abstract: OBJECTIVE
  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 tumor cells and to show its mechanism of action.
  
  
  METHODS
  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-RET inhibition 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.
  
  
  RESULTS
  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 pyrazolopyrimidine drug 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.
  
  
  CONCLUSIONS
  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 signaling pathways.
authors: Division of Pharmacology, Department of Internal Medicine, University of Pisa, Via Roma, Pisa, Italy.
authors: Guido
authors: G
authors: Bocci
authors: Anna
authors: A
authors: Fioravanti
authors: Concettina
authors: C
authors: La Motta
authors: Paola
authors: P
authors: Orlandi
authors: Bastianina
authors: B
authors: Canu
authors: Teresa
authors: T
authors: Di Desidero
authors: Laura
authors: L
authors: Mugnaini
authors: Stefania
authors: S
authors: Sartini
authors: Sandro
authors: S
authors: Cosconati
authors: Rita
authors: R
authors: Frati
authors: Alessandro
authors: A
authors: Antonelli
authors: Piero
authors: P
authors: Berti
authors: Paolo
authors: P
authors: Miccoli
authors: Federico
authors: F
authors: Da Settimo
authors: Romano
authors: R
authors: Danesi
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D002166:Camptothecin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000235:genetics; D004359:Drug Therapy, Combination; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000077146:Irinotecan; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D011720:Pyrazoles / Q000008:administration & dosage / Q000494:pharmacology*; D011743:Pyrimidines / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C556683:CLM3 compound; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D019938:Cyclin D1; D000077146:Irinotecan; D002166:Camptothecin