[关键词]
[摘要]
目的 通过研究木鳖子中提取的对羟基桂皮醛(p-hydroxylcinnamaldehyde,CMSP)对食管癌细胞株Kyse30细胞增殖、迁移、细胞周期及肿瘤标志蛋白表达水平等方面的影响,探讨CMSP对食管癌细胞的诱导分化作用及其机制。方法 分别用0、10、20、40 μg/mL CMSP处理食管癌Kyse30、Eca109、Kyse180细胞24、48、72 h,MTS检测CMSP对Kyse30、Eca109、Kyse180细胞增殖的影响;光学显微镜和电镜下观察Kyse30细胞形态变化;流式细胞术检测CMSP对Kyse30细胞周期和凋亡的影响;ELISA检测CMSP对Kyse30细胞表达肿瘤分化相关抗原癌胚抗原(CEA)、鳞状细胞癌抗原(SCC)、和肿瘤标志物白细胞介素-6(IL-6)和巨噬细胞抑制因子1(MIC-1)的影响;Transwell、克隆形成和细胞划痕实验检测CMSP对Kyse30细胞迁移和侵袭能力的影响;免疫印迹检测CMSP对食管癌细胞C-myc、N-myc肿瘤标志蛋白及RhoA-MAPK信号通路中相关蛋白水平的影响。结果 CMSP可呈时间和剂量依赖性抑制食管癌细胞的增殖,并将Kyse30细胞周期阻滞于G0/G1期。CMSP能够抑制Kyse30细胞的迁移和侵袭能力,还可呈时间和剂量依赖性抑制肿瘤标志物的表达及RhoA-MAPK途径的活化。结论 CMSP通过调控RhoA-MAPK信号通路的活性抑制食管癌Kyse30细胞的增殖,诱导其分化,为其抗肿瘤临床应用提供参考依据。
[Key word]
[Abstract]
Objective To investigate the effects of p-hydroxylcinnamaldehyde (CMSP) on cell proliferation, migration, cell cycle and the expression level of malignant biomarkers, and to investigate the underlying mechanism of differentiation of esophageal carcinoma Kyse30 cells (ESCC cells).Methods The effect of different concentration of CMSP at 0, 10, 20, and 40 μg/mL on viabilities of ESCC cell lines (Kyse30, Eca109, and Kyse180) for 24, 48, and 72 h was determined by MTS assay. Optical microscope and scanning electronic microscopy (SEM) were used to observe the morphologic changes of Kyse30 cells. The effect of CMSP at different concentration on cell cycle distribution and apoptosis of Kyse30 cells was assessed by flow cytometry analysis. ELISA was used to detect the effect of CMSP on expression of tumor related antigens (CEA and SCC) and malignant biomarkers (IL-6 and MIC-1) in Kyse30 cells at protein secretion level. Influence of different concentration of CMSP on migration and invasiveness of Kyse30 cells were determined by colony-formation, wound healing and Transwell assays. Western blotting was used to evaluate the effect of CMSP on expression of protein biomarkers C-myc and N-myc of Kyse30 cells and the related proteins in RhoA-MAPK pathway.Results The proliferation of esophageal cancer cell lines (Kyse30, Eca109, and Kyse180) was significantly inhibited by CMSP in a dose-and time-dependent manner. The cell cycle Kyse30 was blocked in G0/G1 phase. After the treatment with CMSP, Kyse30 cells showed typical dendrite-like cellular protrusions, and the percentage of such elongated cells was significantly and progressively increased with the increase in CMSP concentration (P < 0.01). The results of flow cytometry revealed that the treatment with CMSP increased the number of Kyse30 cells in G0/G1 phase in a dose-and time-dependent manner (P < 0.01), while the number of cells in S phase decreased (P < 0.05); However, the apoptosis rate showed no obvious change (P > 0.05). CMSP could decrease the expression of CEA, SCC, IL-6, and MIC-1 both in protein secretion levels significantly in a dose-and time-dependent manner (P < 0.05, 0.01). Western blotting analysis showed that C-myc and N-myc proteins were all decreased significantly in Kyse30 cells after treatment with CMSP (P < 0.05). CMSP significantly inhibited the proliferation and migration ability of Kyse30 cells (P < 0.05) and induced cell differentiation; The protein levels of p-P38 was significantly increased (P < 0.01), while protein levels of ERK1/2, SAPK/JNK, and GTP-RhoA were obviously decreased in Kyse30 cells after treatment with CMSP (P < 0.01).Conclusion CMSP suppressed the proliferation and induced the differentiation of Kyse30 cells through regulating the RhoA-MAPK signal pathway, which might provide new potential strategies for ESCC treatment.
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[基金项目]
国家自然科学基金青年基金资助项目(81502032);河北省医学科学研究重点课题计划资助项目(20120120)