背景:含TBC1结构域的激酶(TBCK)蛋白在某些细胞类型中起生长抑制因子的作用,在其他细胞类型中起肿瘤启动子的作用。虽然TBCK敲低增加了癌细胞对抗癌药物的反应,TBCK敲除增加抗癌药物敏感性的详细机制尚不清楚.
目的:本研究分析了TBCK在人肾癌细胞中对肿瘤坏死因子相关凋亡诱导配体(TRAIL)和阿霉素的敏感性中的作用。
方法:采用流式细胞术评估细胞凋亡的程度。西方印迹,瞬时转染,采用慢病毒感染技术研究TBCK对细胞凋亡相关蛋白表达和丝裂原活化蛋白激酶(MAPK)的影响。
结果:肾癌细胞中TBCK敲低抑制ERK和Akt信号通路,并增加TRAIL和阿霉素敏感性。在TBCK敲低Caki-1细胞中,与对照细胞系相比,ERK和Akt磷酸化受到抑制,TRAIL和阿霉素敏感性在这些细胞中增加。此外,PDK1的磷酸化在TBCK抑制细胞中被抑制,表明TBCK可能参与PDK1和Akt信号通路。在TBCK抑制的细胞中引入显著活性的Akt恢复了它们对TRAIL的敏感性。此外,TBCK下调增强了不同肾癌细胞系的TRAIL敏感性。
结论:这些数据表明,TBCK可能通过调节人肾癌细胞中涉及Akt和PDK1的信号通路,在影响包括TRAIL在内的抗癌药物的作用方面具有关键功能。
BACKGROUND: TBC1 domain-containing kinase (
TBCK) protein functions as a growth suppressor in certain cell types and as a tumor promoter in others. Although
TBCK knockdown increases the responsiveness of cancer cells to anticancer drugs, the detailed mechanisms by which
TBCK knockdown increases susceptibility to anticancer drugs remain unknown.
OBJECTIVE: This study analyzed the role of TBCK in sensitivities to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and doxorubicin in human renal cancer cells.
METHODS: Flow cytometry was employed to evaluate the extent of apoptosis. Western blotting, transient transfection, and lentiviral infection techniques were conducted to investigate the impact of TBCK on apoptosis-related protein expression and mitogen-activated protein kinase (MAPK).
RESULTS: TBCK knockdown in renal cancer cells inhibits ERK and Akt signaling pathways and increases TRAIL and doxorubicin sensitivity. In TBCK-knockdown Caki-1 cells, ERK and Akt phosphorylation was suppressed compared to control cell lines, and TRAIL and doxorubicin sensitivities were increased in these cells. In addition, the phosphorylation of PDK1 was suppressed in
TBCK-suppressed cells, indicating that
TBCK may be involved in the PDK1 and Akt signaling pathways. The introduction of dominantly active Akt into TBCK-suppressed cells restored their sensitivity to TRAIL. In addition,
TBCK downregulation enhanced TRAIL sensitivity in different renal cancer cell lines.
CONCLUSIONS: These data suggest that TBCK could potentially have a crucial function in influencing the effects of anti-cancer drugs including TRAIL by modulating the signaling pathway involving Akt and PDK1 in human renal cancer cells.