芥子碱硫氰酸盐对人皮肤鳞状细胞癌SCL-1细胞增殖、上皮间质转化、转移的影响及其机制研究
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篇名: 芥子碱硫氰酸盐对人皮肤鳞状细胞癌SCL-1细胞增殖、上皮间质转化、转移的影响及其机制研究
TITLE: Study on the Effects and Its Mechanism of Sinapine Thiocyanate on the Proliferation ,Epithelial Mesenchymal Transition and Metastasis of Human Cutaneous Squamous Cell Carcinoma SCL- 1 Cells
摘要: 目的:研究芥子碱硫氰酸盐(ST)对人皮肤鳞状细胞癌SCL-1细胞增殖、上皮间质转化(EMT)和转移的影响,并考察其可能的作用机制。方法:将人皮肤鳞状细胞癌SCL-1细胞分为空白对照组(0.1%二甲基亚砜)和ST不同浓度组(5、10、20μmol/L),分别通过CCK-8实验、5-乙炔基-2′脱氧尿嘧啶核苷染色实验、细胞划痕实验和Transwell小室侵袭实验测定各组细胞的增殖、迁移和侵袭能力,通过Westernblot实验和免疫荧光实验分别测定各组细胞中EMT相关指标N-钙黏着蛋白(N-cadherin)、E-钙黏着蛋白(E-cadherin)的表达水平。另将SCL-1细胞分为空白对照组(0.1%二甲基亚砜)、ST单用组(20μmol/L)、ST+NSC228155组[20μmol/LST+100μmol/LNSC228155(EGFR激动剂)]和ST+SC79组[20μmol/LST+20μmol/LSC79(PI3K/Akt激动剂)],分别通过CCK-8实验、细胞划痕实验和Transwell小室侵袭实验测定各组细胞的增殖、迁移和侵袭能力,并通过Westernblot实验测定空白对照组(0.1%二甲基亚砜)和ST不同浓度组(5、10、20μmol/L)组细胞中表皮生长因子受体(EGFR)、磷脂酰肌醇3激酶(PI3K)、磷酸化磷脂酰肌醇3激酶(p-PI3K)、蛋白激酶B(Akt)和磷酸化Akt(p-Akt)蛋白的表达水平,以验证ST的作用与EGFR/PI3K/Akt信号通路的关系。另将SCL-1细胞和人正常皮肤成纤维细胞WS1分别分为空白对照组(0.1%二甲基亚砜)、ST组(20μmol/L)、ZD1839组(阳性对照,20μmol/L,EGFR抑制剂)和LY294002组(阳性对照,20μmol/L,PI3K/Akt抑制剂),采用CCK-8实验测定各组细胞的增殖能力,以评价ST的细胞毒性。结果:与空白对照组比较,5、10、20μmol/LST组SCL-1细胞的增殖、迁移和侵袭能力均显著减弱(P<0.05);Westernblot和免疫荧光实验结果显示,5、10、20μmol/LST组SCL-1细胞中N-cadherin蛋白的表达均显著下调(P<0.05),E-cadherin蛋白的表达均显著上调(P<0.05),并且细胞中EGFR、p-PI3K、p-Akt蛋白的表达水平均显著降低(P<0.05)。与ST单用组比较,ST+NSC228155组和ST+SC79组SCL-1细胞的增殖、迁移和侵袭能力均显著增强(P<0.05)。与空白对照组比较,ST组WS1细胞的增殖能力差异无统计学意义(P>0.05),SCL-1细胞增殖能力显著减弱(P<0.05),ZD1839组和LY294002组两种细胞的增殖能力均显著减弱(P<0.05);与ST组比较,ZD1839组和LY294002组WS1细胞的增殖能力均显著减弱(P<0.05),但SCL-1细胞的增殖能力差异无统计学意义(P>0.05)。结论:ST可能通过抑制EGFR/PI3K/Akt信号通路的活化而抑制人皮肤鳞癌SCL-1细胞的增殖、EMT和转移,且其毒副作用较小。
ABSTRACT: OBJECTIVE:To stud y the effects of sinapine thiocyanate (ST) on the proliferation ,epithelial mesenchymal transformation(EMT)and metastasis of human cutaneous squamous cell carcinoma SCL- 1 cells,and to investigate its possible mechanism. METHODS :Human cutaneous squamous cell carcinoma SCL- 1 cells were divided into blank control group (0.1% DMSO) and ST different concentration groups (5,10,20 μmol/L). CCK- 8 assay,5-ethynyl-2′-deoxyuridine(EDU)test, scratch test and Transwell chamber invasion test were adopted to test the proliferation ,migration and invasion ability. The expression of N-cadherin and E-cadherin were detected by Western blot and immunofluorescence assay . Other SCL- 1 cells were collected and divided into blank control group (0.1% DMSO),ST group (20 μmol/L),ST+NSC228155 group [ 20 μmol/L ST+100 μmol/L NSC228155(EGFR agonist )] and ST+SC 79 group [ 20 μmol/L ST+20 μmol/L SC79(PI3K/Akt agonist )]. The proliferation ,migration and invasion ability of SCL- 1 cells in each group were detected by CCK- 8 assay,scratch test and Transwell chamber invasion assay. The expression of epidermal growth factor receptor (EGFR),phosphatidylinositol 3 kinase(PI3K),phosphorylated phosphatidylinositol 3 kinase(p-PI3k),protein kinase B (Akt)and phosphorylated protein Akt (p-Akt)protein of cells in blank control group and ST different concentration groups(5,10,20 μmol/L)were determined by Western blot assay so as to validate the relationship between ST effect and EGFR/ PI3K/Akt signaling pathway. SCL- 1 cells and human normal skin fibroblasts cell WS 1 were divided into blank control group (0.1% DMSO),ST group (20 μmol//L),ZD1839 group(positive control ,20 μmol//L,EGFR inhibitor )and LY 294002 group(positive control,20 μmol//L,PI3K/Akt inhibitor ). CCK- 8 assay was used to detect the cell proliferation in order to evaluate the cells cytotoxicity of ST. RESULTS :Compared with blank control group ,the proliferation ,migration and invasion ability of SCL- 1 cells were significantly decreased in 5,10,20 μmol/L ST groups(P<0.05). Western blot and immunofluorescence assay showed that the expression of N-cadherin in SCL- 1 cells were decreased significantly in 5,10,20 μmol/L ST groups(P<0.05),while the protein expression of E-cadherin was increased significantly (P<0.05);the protein expressions of EGFR ,p-PI3K and p-Akt were significantly decreased (P<0.05). Compared with ST group ,the proliferation ,migration and invasion ability of SCL- 1 cells were increased significantly in ST + NSC 228155 group and ST + SC 79 group (P<0.05). Compared with blank control group ,the proliferation ability of WS 1 cells had no significant change in ST group ,while the proliferation ability of SCL- 1 cells was decreased significantly (P<0.05);the proliferation ability of the two kinds of cells were decreased significantly in ZD 1839 group and LY 294002 group(P<0.05). Compared with ST group ,the proliferation ability of WS 1 cells was decreased significantly in ZD1839 group and LY 294002 group(P<0.05),but there was no significant difference in the proliferation ability of SCL- 1 cells (P>0.05). CONCLUSIONS :ST may inhibit the proliferation ,EMT and metastasis of SCL- 1 cells through inhibiting the activation of EGFR/PI 3K/Akt signaling pathway ,and its side effects are few.
期刊: 2021年第32卷第08期
作者: 苏羽屾,曾智锐,荣冬芸,王叶,李丹,唐姗姗,汪涛,龙雪梅,曹煜
AUTHORS: SU Yushen, ZENG Zhirui,RONG Dongyun,WANG Ye,LI Dan,TANG Shanshan,WANG Tao,LONG Xuemei,CAO Yu
关键字: 芥子碱硫氰酸盐;人皮肤鳞状细胞癌SCL-1细胞;增殖;上皮间质转化;转移;EGFR/PI3K/Akt信号通路
KEYWORDS: Sinapine thiocyanate ; Human cutaneous squamous cell carcinoma SCL-1 cells; Proliferation; Epithelial
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