摘要
[目的]探究甲磺酸阿帕替尼联合放疗对结直肠癌细胞体外抑制作用。[方法]体外培养结直肠癌细胞株HCT116、SW480和HT29,采用MTT法检测HCT116、SW480和HT29细胞株对甲磺酸阿帕替尼的敏感性,以药物敏感性最高的细胞进行后续联合治疗,分为对照组、药物组、放疗组和联合组,应用MTT检测不同处理组细胞的增殖抑制能力,筛选最强联合抑制效果时的药物浓度作为后续试验;AnnexinⅤ-FITC/PI联合流式细胞术分析不同处理组对HCT116细胞凋亡和细胞周期的影响;ELISA分析不同处理组上清中VEGF分泌水平;Western blot试验检测VEGFR2、VEGFA、ERK/p-ERK、STAT3/p-STAT3、BAX和BCL2蛋白的表达。[结果]甲磺酸阿帕替尼抑制结直肠癌细胞HCT116、SW480和HT29体外增殖且具有剂量依赖性,半数致死浓度(IC_(50))分别为14.90μmol/L、25.99μmol/L和24.19μmol/L,HCT116细胞药物敏感性显著高于SW480和HT29细胞(P均<0.05),以敏感性最高的HCT116细胞进行不同分组,结果显示甲磺酸阿帕替尼浓度为8μmol/L时,联合组对比药物组和放疗组显示更强的增殖抑制能力(65.97%vs25.83%vs 21.97%,P均<0.001);不同处理组细胞凋亡率有差异(F=237.930,P=0.000),对比药物组(8μmol/L)和放疗组,联合组细胞凋亡率显著增加[(6.55±0.58)%vs (12.69±0.71)%vs (29.11±2.08)%,P均<0.001];联合组未能增强阿帕替尼(8μmol/L)对细胞周期的阻滞(P=0.09);细胞上清中VEGF分泌水平在4组间无差异(P>0.05),联合组VEGFA蛋白的表达显著下调;但放疗未增加阿帕替尼(8μmol/L)对VEGFR2的抑制作用;对比药物组(8μmol/L)和放疗组,联合组增加了促凋亡基因BAX蛋白表达,并抑制抗凋亡基因BCL2蛋白表达,此外联合组处理后p-STAT3蛋白的磷酸化水平显著降低,STAT3和ERK总蛋白水平无改变。[结论]甲磺酸阿帕替尼联合放疗可能通过抑制STAT3通路诱导细胞凋亡,从而抑制HCT116细胞的增殖。
[Purpose] To investigate the inhibition effect of Apatinib combined with radiation on colorectal cancer cells in vitro. [Methods] The sensitivity of human colorectal cancer HCT116, SW480 and HT29 cells to Apatinib was tested by MTT assay. The cells with the highest sensitivity were used in the experiments and cells were divided into control group, Apatinib group, irradiation group and combination group. Annexin VFITC/PI and flow cytometry were used to detect cell apoptosis and cell cycle, and VEGF secretion was analyzed by ELISA. The expression of VEGFR2, VEGFA, ERK/p-ERK, STAT3/p-STAT3, BAX and BCL2 protein were detected by Western blot. [Results] HCT116 cells showed more sensitive to Apatinib than SW480and HT29 cells and Apatinib at concentration of 8 μmol/L showed stronger proliferation inhibition. 8 μmol/L Apatinib was used in the following experiments. HCT116 cells were treated with 8 μmol/L Apatinib alone or combination with irradiation. The apoptosis rate was significantly different among 4 groups(F =237.930, P =0.000). Compared with Apatinib group and radiation group, the apoptosis rate in combination group was significantly increased[(29.11±2.08)% vs(6.55±0.58)% vs(12.69±0.71)%, P<0.001)]. The cell cycle arrest was not enhanced in Apatinib group compared with the combination group(P=0.09). There was no significant difference in VEGF levels in supernatant among 4 groups(P >0.05), however, VEGFA protein was down-regulated in combination group, and radiation did not increase the inhibition of Apatinib on VEGFR2 protein. The expression of pro-apoptotic gene BAX was increased in the combined group and the expression of the anti-apoptotic gene BCL2 was inhibited. Meanwhile, the level of p-STAT3 decreased significantly, while the levels of STAT3and ERK had no difference in 4 groups. [Conclusion] Apatinib combined with radiation may inhibit the proliferation of HCT116 cells and induce apoptosis by STAT3 pathway.
作者
杨永净
赵玲
许德权
王雪
刘婷婷
柯天洋
刘士新
YANG Yong-jing;ZHAO Ling;XU De-quan;WANG Xue;LIU Ting-ting;KE Tian-yang;LIU Shi-xin(Jilin Cancer Hospital,Changchun 130012,China)
出处
《中国肿瘤》
CAS
CSCD
北大核心
2022年第10期839-845,共7页
China Cancer
基金
北京市希思科临床肿瘤学研究基金项目(20180902(HZ)0002)。
