金复康通过p16/RB信号通路诱导肺癌循环肿瘤细胞衰老

Jinfukang induced senescence of circulating tumor cells in lung cancer by p16/RB signal pathway

目的:研究金复康诱导非小细胞肺癌患者循环肿瘤细胞(circulating tumor cells,CTCs)衰老的分子机制。方法:采用不同质量浓度的金复康(0、125、250、500和1 000μg/mL)处理人肺癌CTCs(CTC-TJH-01细胞),CCK-8法检测对CTC-TJH-01细胞增殖的影响,β-半乳糖苷酶染色法检测对细胞衰老的影响;FCM法和免疫荧光染色法检测CTC-TJH-01细胞内增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达水平,并用蛋白质印迹法检测增殖和衰老相关蛋白p16、p21、视网膜母细胞瘤蛋白(retinoblastoma protein,RB)以及磷酸化RB蛋白的表达水平。结果:金复康质量浓度为500和1 000μg/mL时可明显抑制CTC-TJH-01细胞的增殖(P值均<0.05);质量浓度为350和700μg/mL时即可诱导CTC-TJH-01细胞发生衰老(P值均<0.05),并促进CTC-TJH-01细胞核内PCNA蛋白的表达(P值均<0.05);蛋白质印迹法检测结果显示,金复康可上调p16和p21蛋白的表达水平,同时下调p-RB蛋白的表达水平(P值均<0.05)。结论:金复康通过调控p16/RB信号通路诱导CTCs发生衰老,这可能与临床上金复康防治肺癌复发和转移的机制相关。

Objective： To explore the molecular mechanism of Jinfukang inducing the senescence of circulating tumor cells （CTCs） in patients with non- small cell lung cancer. Methods： Human with different co of Jinfukang. Th by CCK-8 assay. lung cancer CTCs （CTC-TJH-01 ncentrations （0, 125, 250, 500 e proliferation of CTC-TJH-01 The state of cellular senescen cells） were treated and 1000μg/mL） cells was detected ce was detected by-galactosidase staining. The expression of proliferating cell nuclear antigen （PCNA） protein in CTC-TJH-01 cells was detected by flow cytometry and immunofluorescence staining. The expression levels of proliferation- and senescence-associated proteins including p16, p21, retinoblastoma protein （RB） and phospho-RB （p-RB） were detected by Western blotting. Results：Jinfukang inhibited the proliferation of CTC-TJH-01 cells （500 and 1 000 μg/mL： both P 〈 0.05）, induced the senescence of CTC-TJH-01 cells （350 and 700 μg/mL： both P 〈 0.05）, and promoted the expression of PCNA protein in CTC-TJH-01 cells （350 and 700 μg/mL： both P 〈 0.05）. The expression levels of p16 and p21 proteins were up-regulated, but the expression level of p-RB protein was down-regulated after treatment （all P 〈 0.05）. Conclusion： Jinfukang induces the senescence of CTCs by regulating p16/RB signal pathway which may be the mechanism of preventing the recurrence and metastasis of lung cancer.