Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells
Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells
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DOI：
10.1016/j.bcp.2011.03.022
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发表时间：
2011-06-01
影响因子：
5.8
通讯作者：
Danesi, Romano
中科院分区：
文献类型：
--
作者：
Bocci, Guido;Fioravanti, Anna;Danesi, Romano
目的：证明新型吡唑并嘧啶衍生物多重酪氨酸激酶抑制剂CLM 3单独和与SN-38联合的抗增殖和促凋亡活性（伊立替康的活性代谢产物）对内皮细胞和肿瘤细胞的作用并显示其作用机制。对微血管内皮细胞（HMVEC-d）和肺（A549）和甲状腺癌进行增殖和凋亡测定（8305 C，TT）细胞系暴露于CLM 3并同时与SN 38组合72小时。进行基于细胞的磷酸-VEGFR-2、磷酸-EGFR和磷酸-RET抑制测定，并通过ELISA试剂盒定量ERK 1/2和Akt磷酸化。细胞周期蛋白D1基因的表达进行实时PCR和细胞周期蛋白D1细胞内浓度测定ELISA.Results：一个强大的抗增殖和促凋亡活性的影响被发现与CLM 3对内皮细胞和癌细胞，协同增强SN 38。在活化的内皮细胞和癌细胞中，经CLM 3处理后，磷酸-VEGFR-2、磷酸-EGFR和磷酸-RET水平显著降低;与癌细胞相比，内皮细胞中较低浓度的吡唑并嘧啶药物显著抑制ERK 1/2和Akt磷酸化。此外，CLM 3治疗极大地抑制了内皮细胞和癌细胞中的细胞周期蛋白D1基因的表达，降低了细胞周期蛋白D1蛋白的细胞内concentration.Conclusions：吡唑并嘧啶衍生物CLM 3表现出非常显着的和有前途的抗增殖和促凋亡活性，单独和SN-38相结合，激活内皮细胞和癌细胞。这些作用主要是由于其抑制VEGFR-2、EGFR和RET酪氨酸激酶的磷酸化及其相关信号通路。(C)2011 Elsevier Inc. All rights reserved.
Aims: 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 72 h. 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. (C) 2011 Elsevier Inc. All rights reserved.