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
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、40 μmol／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Western bl...
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| Published in | 中华肿瘤杂志 Vol. 41; no. 4; pp. 263 - 275 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
上海交通大学医学院附属瑞金医院舟山分院放疗科316011%南京医科大学附属常州市第二人民医院放疗科213003
01.04.2019
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-3766 |
| DOI | 10.3760/cma.j.issn.0253-3766.2019.04.005 |
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| Abstract | 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、40 μmol／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Western blot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、250 mg阿帕替尼组和500 mg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、40 μmol／L阿帕替尼处理细胞24 h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(1 123.67±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(1 749.67±65.77)个],差异均有统计学意义(均P＜0.05). 10、20、40 μmol／L阿帕替尼处理细胞7 d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,20 μmol／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). |
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| AbstractList | 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、40 μmol／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Western blot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、250 mg阿帕替尼组和500 mg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、40 μmol／L阿帕替尼处理细胞24 h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(1 123.67±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(1 749.67±65.77)个],差异均有统计学意义(均P＜0.05). 10、20、40 μmol／L阿帕替尼处理细胞7 d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,20 μmol／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). |
| Abstract_FL | 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). The cell cycle ratio of G2／M phase and apoptosis rate of control group and 20 μmol／L apatinib group in KYSE?150 cells were (12.14±2.13)% and (3.49±0.74)% as well as (26.27±3.30)% and (15.65± 1.54)%, respectively. The corresponding ratios in ECA?109 cells were (3.44±0.57)% and (6.31±1.43)%as well as (22.64±2.36)% and (49.26± 1.62)%, respectively. The results show that apatinib suppressed cell cycle progression at G2／M phase and induced cell apoptosis in both KYSE?150 and ECA?109 cells (P＜0.05 for all). In the presence of 20 μmol／L and 40 μmol／L of apatinib in KYSE?150 cells, the relative levels of VEGF mRNA were (42.57± 10.43)% and ( 25.69± 1.24)%, and those of VEGF?2 mRNA were (36.09±10.82)% and (13.99±6.54)%, which were all significantly decreased compared to control group (100.00±0.00, P＜0.05 for all). For ECA?109 cells, the relative expression of VEGF and VEGFR2 showed similar tendency (P＜0.05 for all). Moreover, after treatment with 20 μmol／L and 40 μmol／L of apatinib in KYSE?150 cells, the VEGF concentrations were ( 766.48± 114.27) pg／ml and ( 497.40± 102.18) pg／ml, which were significantly decreased compared to control group [(967.41± 57.75) pg／ml, P＜0.05)]. The results in ECA?109 were consistent (P＜0.05). Furthermore, after treatment with 40 μmol／L of apatinib in KYSE?150 and ECA?109, the relative expression of p?MEK and p?ERK were 0.49±0.05 and 0.28±0.03 as well as 0.63±0.03 and 1.22±0.15, which were significantly lower than control group (1.23±0.19 and 0.66± 0.07 as well as 1.03±0.20 and 1.76±0.20; P＜0.05). The relative expression of STAT3, p?STAT3 in control group and experimental group were 0.96 ± 0.15 and 0.85 ± 0.16 as well as 0.62 ± 0.09 and 0.36 ± 0.13, respectively. The results showed that the protein levels of STAT3 and p?STAT3 were significantly lower than the control group (P＜0.05 for all). The inhibition rates of apatinib in xenograft nude mice were 29.25% and 19.96% for 250 mg and 500 mg treatment groups. The concentration of VEGF were (25.11±4.12) pg／ml, (16.40 ± 2.81) pg／ml and ( 15.04 ± 4.88) pg／ml for control, 250 mg and 500 mg treatment groups, respectively. Conclusions Apatinib can inhibit cell proliferation, induce apoptosis and suppress migration of esophageal cancer cells in vitro and in vivo. This effect was mainly mediated via the alterations of Ras／Raf／MEK／ERK pathway and JAK2／STAT3 pathway. |
| Author | 周梦耘 王坚 孙菲 孔泽 汪建林 华秋 胡莉钧 于静萍 封悦 孙志强 |
| AuthorAffiliation | 上海交通大学医学院附属瑞金医院舟山分院放疗科316011%南京医科大学附属常州市第二人民医院放疗科213003 |
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| Author_FL | Wang Jianlin Wang Jian Kong Ze Yu Jingping Sun Zhiqiang Feng Yue Hu Lijun Zhou Mengyun Sun Fei Hua Qiu |
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| Keywords | Apatinib Nude mice [主题词] 食管肿瘤 阿帕替尼 裸鼠 [Subject words] Esophageal neoplasms 凋亡 血管内皮生长因子 Vascular endothelial growth factor Apoptosis |
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| Publisher | 上海交通大学医学院附属瑞金医院舟山分院放疗科316011%南京医科大学附属常州市第二人民医院放疗科213003 |
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| Title | 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 |
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