目的:mTOR/S6K1信号轴,以细胞生长调节而闻名,在多种癌症中过度激活。在这项研究中,我们研究了核糖体蛋白p70-S6激酶1(S6K1)相关的转化人肝细胞(THH)生长调节的机制。
方法:THH用p70-S6K1抑制剂处理并分析细胞活力,细胞周期分布,蛋白质印迹特异性标记蛋白表达,和异种移植小鼠模型中的肿瘤抑制。我们通过使用特异性siRNA敲低p70-S6K1来验证我们的结果。
结果:p70-S6K1抑制剂治疗导致体外肝细胞生长受损,并在G1期阻止细胞周期进程。Further,p70-S6K1抑制剂治疗显示FAK和Erk活化降低,其次是整合素-β1表达改变,半胱天冬酶8和PARP裂解似乎是失巢凋亡样生长抑制。p70-S6K1抑制剂还解聚了肌动蛋白微丝,并减少了活性Rac1/Cdc42复合物的形成,从而失去了细胞附着。用其它转化的人肝细胞细胞系获得类似的结果。p70-S6K1抑制也导致磷酸EGFR减少,Slug和Twist;暗示抑制上皮-间质转化(EMT)状态。异种移植肿瘤模型,由裸鼠植入的THH产生,腹膜内注射S6K1抑制剂后,阻止了肿瘤的进一步生长。
结论:我们的结果表明p70-S6K1抑制改变了细胞周期进程的安排,诱导细胞脱离,并使肝细胞生长受损敏感。通过抑制剂靶向S6K1的p70同工型可能被证明是与其他治疗肝细胞癌(HCC)治疗的有希望的方法。
OBJECTIVE: The mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation.
METHODS: THH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA.
RESULTS: p70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-β1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth.
CONCLUSIONS: Our results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.