Effects and Mechanism of Anlotinib on Radiosensitivity of Non-small Cell Lung Cancer Cell Lines A549
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摘要:
目的 研究安罗替尼对肺腺癌细胞放疗增敏作用及机制。 方法 采用安罗替尼处理人肺腺癌细胞株A549，使用CCK8法测定安罗替尼对细胞增殖的影响；采用克隆形成实验测定安罗替尼联合放疗对细胞的生长抑制作用；使用流式细胞术测定细胞周期和细胞凋亡的变化；采用免疫荧光测定安罗替尼联合放疗对细胞内DNA损伤的影响；采用Western blot检测细胞内DNA损伤标志因子DNA-PKcs的表达变化。 结果 安罗替尼对人肺腺癌细胞A549增殖具有抑制作用，第2、3、4、5天，（P < 0.05）。体外培养克隆形成实验显示，安罗替尼联合放疗组的准予剂量（Dq）、平均致死剂量（D0）及4 Gy照射时的存活分数（SF）均明显低于单纯放疗组（ P < 0.05）。安罗替尼能够使细胞阻滞于G1/G0期，与放疗联合作用后，进一步降低了G2和S期细胞比例。安罗替尼能够增加放疗诱导的细胞凋亡比例（31.94±2.25）%和（44.44±2.30）%，（ P < 0.001），增加γH2AX阳性细胞数量（25.67±2.52）%和(54.67±3.79）%，（ P < 0.001）。安罗替尼能够降低放疗诱导的DNA断裂损伤标志因子DNA-PKcs的表达强度（0.90±0.06）和（0.40±0.06），（ P < 0.001）。 结论 安罗替尼具有放疗增敏作用，其机制可能与减少G2/S期细胞、增加细胞凋亡和维持DNA持续损伤有关。 Abstract:Objective To investigate the effect and mechanism of anlotinib on radiosensitization of human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinom cell line A549 was treated with anlotinib and/or radiotherapy, then divided in to four groups, control group （Ctrl）, Anlotinib treatment group （A）, irradiation group （RT） and anlotinib combined with irradiation group （A + RT）. CCK8 method was used to determine cell proliferation; the clone formation experiment was used to determine the inhibitory effect on cell growth; flow cytometry was used to determine cell cycle and apoptosis; immunofluorescence of γ-H2AX was used to determine DNA damage; expression of DNA-PKcs were detected by Western blot. Results Anlotinib inhibited proliferation and clonogenic survival following irradiation. The dose （Dq）, the average lethal dose （D0） and the survival score （SF2） in the anlotinib combined radiotherapy group was significantly lower than those in the radiotherapy group. Anlotinib decreased G2/M phase arrest and promoted the cells apoptosis induced by in irradiation. The confocal microscopy results showed the average number of γ-H2AX foci in the A+RT group was more than that in RT group. The protein levels of DNA-PKcs were higher in A + RT group than that in RT group. Conclusion Anlotinib enhances the radiosensitivity of A549 cells, which may be attributed to the delay DNA damage repair. It provides a rationale strategy by Anlotinib combined with irradiation for NSCLC. -
Key words:
- Anlotinib /
- Non-small cell lung cancer /
- Radiosensitization /
- DNA damage repair
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图 1 安罗替尼对A549细胞增殖的抑制图
A：不同浓度安罗替尼对细胞的抑制率；B：对照组（Ctrl组）和安罗替尼组（A组）细胞生长曲线。与Ctrl相比，*P < 0.05。
Figure 1. Effect of anlotinib on the proliferation of A549 cell. （A） Inhibition rate of anlotinib in different concentrations
图 2 单击多靶模型拟合A549细胞生存曲线和放射增敏比
A：不同浓度安罗替尼联合放疗（RT+A组）降低A549细胞的存活率；B：不同浓度安罗替尼联合放疗（RT+A组）促进A549细胞放射增敏比。 与RT相比，*P < 0.05。
Figure 2. Survival curve and radiosensitization ratio of A549 cell by multitarget-single hitting model
图 3 安罗替尼单独或联合放疗对肺腺癌细胞凋亡的影响
A：通过Annexin V/PI双染检测4组细胞的凋亡情况；B：4组细胞的凋亡率。 与Ctrl组相比，*P < 0.05；与RT组相比，#P < 0.05。
Figure 3. Effects of anlotinib and/or combined with radiation on the apoptosis of A549 cells
图 4 安罗替尼单独或联合放疗对肺腺癌细胞周期的影响。
A：流式细胞术检测4组细胞的细胞周期情况；B：4组细胞的细胞周期比例比较。
Figure 4. Effects of anlotinib and/or combined with radiation on cell cycle redistribution of A549 cells
图 5 安罗替尼能够维持放疗诱导的DNA双链断裂。
A：安罗替尼对放疗肺腺癌A549细胞中DNA损伤标志物γ-H2AX灶形成的影响；B：γ-H2AX灶的量化。 与Ctrl组相比，*P < 0.05；与RT组相比，#P < 0.05。
Figure 5. Anlotinib leads to persistent DNA double-strand breaks. A，Fluorescence images of γ-H2AX foci in A549 cells treated with anlotinib and/or combined with radiation
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