Division of Endocrinology, Diabetes and Metabolism, The Ohio State University and The Arthur G. James Cancer Hospital, Columbus, Ohio 43210, USA.
Endocr Relat Cancer. 2012 Jan 9;19(1):29-38. doi: 10.1530/ERC-11-0155. Print 2012 Feb.
Clinical trials using kinase inhibitors have demonstrated transient partial responses and disease control in patients with progressive medullary thyroid cancer (MTC). The goal of this study was to identify potential combinatorial strategies to improve on these results using sorafenib, a multikinase inhibitor with activity in MTC, as a base compound to explore signaling that might predict synergystic interactions. Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to sorafenib, everolimus, and AZD6244 alone and in combination. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and poly (ADP-ribose) polymerase (PARP) cleavage assays were performed to measure cell survival and apoptosis. Western blots were performed to confirm activity of the compounds and to determine possible mechanisms of resistance and predictors of synergy. As a solitary agent, sorafenib was the most active compound on MTT assay. Western blots confirmed that sorafenib, everolimus, and AZD6244 inhibited their anticipated targets. At concentrations below its IC(50), sorafenib-treated TT and MZ-CRC-1 cells demonstrated transient inhibition and then re-activation of Erk over 6 h. In concordance, synergistic effects were only identified using sorafenib in combination with the Mek inhibitor AZD6244 (P<0.001 for each cell line). Cells treated with everolimus demonstrated activation of Akt and Ret via TORC2 complex-dependent and TORC2 complex-independent mechanisms respectively. Everolimus was neither additive nor syngergistic in combination with sorafenib or AZD6244. In conclusion, sorafenib combined with a Mek inhibitor demonstrated synergy in MTC cells in vitro. Mechanisms of resistance to everolimus in MTC cells likely involved TORC2-dependent and TORC2-independent pathways.
临床研究表明，激酶抑制剂在进展期甲状腺髓样癌（MTC）患者中具有短暂的部分缓解和疾病控制作用。本研究的目的是确定潜在的联合策略，以使用索拉非尼作为基础化合物来改善这些结果，索拉非尼是一种在 MTC 中具有活性的多激酶抑制剂，以探索可能预测协同相互作用的信号。两种人 MTC 细胞系 TT 和 MZ-CRC-1 分别携带内源性 C634W 或 M918T RET 突变，单独或联合暴露于索拉非尼、依维莫司和 AZD6244 中。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）和多聚（ADP-核糖）聚合酶（PARP）裂解测定用于测量细胞存活和凋亡。进行 Western blot 以确认化合物的活性，并确定可能的耐药机制和协同作用的预测因子。作为单一药物，索拉非尼在 MTT 测定中是最活跃的化合物。Western blot 证实索拉非尼、依维莫司和 AZD6244 抑制了它们预期的靶点。在低于其 IC50 的浓度下，索拉非尼处理的 TT 和 MZ-CRC-1 细胞在 6 小时内表现出 Erk 的短暂抑制和随后的再激活。一致地，仅在用索拉非尼与 Mek 抑制剂 AZD6244 联合使用时才鉴定出协同作用（每个细胞系的 P<0.001）。用依维莫司处理的细胞通过 TORC2 复合物依赖性和 TORC2 复合物非依赖性机制分别激活 Akt 和 Ret。依维莫司与索拉非尼或 AZD6244 联合使用既没有相加作用也没有协同作用。总之，索拉非尼与 Mek 抑制剂联合在 MTC 细胞系中表现出协同作用。MTC 细胞对依维莫司的耐药机制可能涉及 TORC2 依赖性和 TORC2 非依赖性途径。
