甲磺酸阿帕替尼调控Ras/Raf/MEK/ERK和JAK2/STAT3信号通路影响食管癌细胞增殖迁移凋亡和荷瘤鼠移植瘤生长的机制  

The inhibition effects of apatinib on cell proliferation, migration and apoptosis in esophageal carcinoma via Ras/Raf/MEK/ERK and JAK2/STAT3 pathways

作　　者：封悦 周梦耘 孙菲[1] 孔泽 王坚 孙志强 胡莉钧 汪建林 华秋 于静萍[2] Feng Yue;Zhou Mengyun;Sun Fei;Kong Ze;Wang Jian;Sun Zhiqiang;Hu Lijun;Wang Jianlin;Hua Qiu;Yu Jingping(Department of Radiotherapy, Zhoushan Branch of Shanghai Ruijin Hospital, Zhoushan 316011, China;Department of Radiotherapy and Oncology, Changzhou No.2 People′s Hospital, the Affiliated Hospital of Nanjing Medical University, Changzhou 213003, China)

机构地区：[1]上海交通大学医学院附属瑞金医院舟山分院放疗科,316011 [2]南京医科大学附属常州市第二人民医院放疗科,213003

出　　处：《中华肿瘤杂志》2019年第4期263-275,共13页Chinese Journal of Oncology

基　　金：国家自然科学基金(11705095);常州市高层次卫生人才培养计划(2016C2BJ007);常州市科技支撑社会发展项目(CE20165024);常州市科技局应用基础研究项目(CJ20159050).

摘　　要：目的探讨阿帕替尼对食管癌细胞生物学功能、食管癌裸鼠移植瘤生长情况的影响及其机制。方法采用2.5、5、10、20、40μmol/L阿帕替尼处理食管癌细胞KYSE-150和ECA-109,采用四甲基偶氮唑蓝法检测食管癌细胞的增殖活性,采用Transwell小室法检测食管癌细胞的迁移能力,采用克隆形成法检测食管癌细胞的克隆形成率,采用流式细胞术检测阿帕替尼对细胞周期和细胞凋亡的影响,采用实时荧光定量PCR法检测食管癌细胞中血管内皮细胞生长因子(VEGF)mRNA和VEGF受体2(VEGFR-2)mRNA的表达,采用酶联免疫吸附法检测食管癌细胞上清液中VEGF浓度,采用Westernblot法检测正常培养条件下和VEGF刺激下食管癌细胞中MEK、ERK、磷酸化MEK(p-MEK)、磷酸化ERK(p-ERK)、JAK2、STAT3和磷酸化STAT3(p-STAT3)的表达。建立人食管鳞癌裸鼠移植瘤模型,采用随机数字表法随机分为健康对照组、250mg阿帕替尼组和500mg阿帕替尼组,计算肿瘤抑制率,采用免疫组化法检测移植瘤瘤组织中VEGF和VEGFR-2的表达。结果20、40μmol/L阿帕替尼处理细胞24h后,KYSE-150细胞的迁移数分别为(428.67±4.16)个和(286.67±1.53)个,ECA-109细胞的迁移数分别为(1123.67±70.00)个和(477.33±26.84)个,均低于空白对照组[分别为(874.67±22.75)个和(1749.67±65.77)个],差异均有统计学意义(均P<0.05)。10、20、40μmol/L阿帕替尼处理细胞7d后,KYSE-150细胞的克隆形成率分别为(65.12±25.48)%、(58.19±24.73)%和(29.10±22.40)%,ECA-109细胞的克隆形成率分别为(70.61±15.14)%、(61.12±17.21)%和(43.09±11.13)%,均低于空白对照组(100%),差异均有统计学意义(均P<0.05)。KYSE-150细胞中,20μmol/L阿帕替尼组的G2/M期细胞比例和凋亡率分别为(26.27±3.30)%和(15.65±1.54)%,空白对照组分别为(12.14±2.13)%和(3.49±0.74)%,差异均有统计学意义(均P<0.05)。ECA-109细胞中,20μmol/L阿帕替尼组细胞的G2/M期细胞比例和凋亡率分别为(22.64±2.36)%�Objective To investigate the in vitro and in vivo effects of apatinib in esophageal squamous cell carcinoma and the underlying mechanisms. Methods The esophageal cancer cells, KYSE-150 and ECA-109, were divided into control group and apatinib treatment group at the concentrations of 2.5, 5, 10, 20 and 40 μmol/L respectively. All of experiments were performed in triplicate. MTT and colony formation assays were used to measure cell proliferation. Transwell assay was used to determine the migration capacity. The effect of apatinib on cell cycle and apoptosis was analyzed by flow cytometry. The expression of VEGF and VEGFR-2 was measured by real-time quantitative PCR (qRT-PCR). The concentration of VEGF in the cell supernatant was assessed by enzyme-linked immunosorbent assay (ELISA). The expression levels of MEK, ERK, p-MEK, p-ERK, JAK2, STAT3 and p-STAT3 after VEGF stimulation were detected by Western blot. Furthermore, the nude mice xenograft model was established. The tumor-bearing mice were randomly divided into control group, apatinib low dose treatment group (250 mg) and apatinib high dose treatment group (500 mg), respectively. Tumor inhibition rates of different groups were calculated. And then the expressions of VEGF and VEGFR2 were detected in xenograft tissues by immunohistochemical staining. Results In the presence of 20 μmol/L and 40 μmol/L of apatinib for 24 hours, the migration cell numbers of KYSE-150 and ECA-109 were 428.67±4.16 and 286.67±1.53 as well as 1 123.67±70.00 and 477.33±26.84, respectively, that were significantly lower than control group (P<0.05 for all). In addition, after treatment with 10 μmol/L, 20 μmol/L and 40 μmol/L of apatinib for 7 days on KYSE-150 and ECA-109, the colony formation rates were (65.12±25.48)%,(58.19±24.73)% and (29.10±22.40)% as well as (70.61±15.14)%,(61.12±17.21)% and (43.09±11.13)%, respectively. The colony formation rates of 20 μmol/L and 40 μmol/L of apatinib treatment groups were significantly lower than control group (100.00±0.00, P<0.05)

关 键 词：食管肿瘤 阿帕替尼 凋亡 裸鼠 血管内皮生长因子 

分 类 号：R735.1[医药卫生—肿瘤]