目的 探讨蟾毒灵对食管癌增殖的影响,并初步探讨其可能分子机制。方法 采用四甲基偶氮唑蓝 (MTT)法和乳酸脱氢酶细胞毒性检测法 (LDH Assay Kit)检测蟾毒灵对食管癌细胞株KYSE-70活力的影响。通过细胞克隆形成法和EdU (5-ethynyl-2-deoxyuridine)细胞增殖实验研究蟾毒灵对KYSE-70细胞增殖的抑制作用。利用DAPI染色、TUNEL、流式细胞术检测蟾毒灵对KYSE-70细胞凋亡的影响。采用实时荧光定量PCR技术 (quantitative real time-PCR, qPCR)、蛋白免疫印迹 (Western blotting)法检测细胞凋亡相关因子Bcl-2、Bax、NF-κBp65、NF-κB p65 mRNA和蛋白表达水平的变化。通过JC-1荧光探针试剂盒和ATP检测试剂盒研究蟾毒灵作用后对KYSE-70线粒体功能的影响。最后,利用裸鼠体外成瘤实验研究蟾毒灵在体内对食管癌生长的影响。结果 MTT和LDH实验结果表明,蟾毒灵对KYSE-70细胞活力具有明显的抑制作用。克隆形成实验和EdU结果表明,蟾毒灵可显著抑制KYSE-70细胞增殖。DAPI染色结果显示,蟾毒灵处理后KYSE-70细胞的核出现染色质不均匀、浓缩、聚集及碎裂等凋亡形态。TUNEL染色和流式细胞检测结果显示,蟾毒灵作用后可促进KYSE-70细胞凋亡。qPCR和Western blot结果显示,蟾毒灵处理后KYSE-70细胞内Bcl-2表达下调,Bax表达上调,Bax/Bcl-2表达比例升高。另外,蟾毒灵作用后,KYSE-70细胞的线粒体膜电位呈现明显的下降趋势 (P<0.01),ATP的生成量也显著减少。裸鼠体外成瘤实验表明,蟾毒灵能够明显抑制体内肿瘤的生长。结论 蟾毒灵能通过抑制食管癌的增殖,并通过线粒体途径促进食管癌细胞凋亡。
Abstract
OBJECTIVE To investigate the effect of bufalin on the proliferation of esophageal cancer and its possible molecular mechanism. METHODS The effects of bufalin on the activity of esophageal cancer cell line KYSE-70 were determined by MTT assay and LDH assay kit. Colony-forming and EdU (5-ethynyl-2-deoxyuridine) assay were performed to detect the inhibitory effect of bufalin on the proliferation of KYSE-70 cells. DAPI staining, TUNEL and flow cytometry were used to assess the effects of bufalin on the apoptosis of KYSE-70 cells. Quantitative real-time PCR (qPCR) and Western blotting were used to determine the relevant expression changes in mRNA and protein of apoptosis-related factors Bcl-2, Bax, NF-κBp65, NF-κBpp65. The mitochondrial function changes of KYSE-70 cells were studied by using JC-1 fluorescent probe kit and ATP detection kit after bufalin treatment. Finally, the effect of bufalin on esophageal cancer proliferation in vivo was studied by xenograft model in nude mice. RESULTS The results of MTT and LDH assay shown that bufalin inhibited the activity of KYSE-70 cells. Colony-forming and EdU assay showed that bufalin significantly suppressed KYSE-70 cells proliferation. DAPI staining showed that chromatin heterogeneity, nuclear concentration and fragmentation were observed after bufalin treatment. It was found that bufalin treatment significantly promoted KYSE-70 cells apoptosis by TUNEL staining and flow cytometry assay of qPCR and Western blot showed that Bcl-2 expression was down-regulated, Bax expression was up-regulated and Bax/ Bcl-2 expression ratio was increased after bufalin treatment. The mitochondrial membrane potential and the ATP production of KYSE-70 cells were significant reduced after bufalin treatment. In vivo, the growth of xenograft tumors was significantly inhibited in bufalin group. CONCLUSION Bufalin markedly inhibits KYSE-70 cells proliferation by promoting apoptosis, and the possible mechanism of apoptosis may be related to mitochondrial pathway. Our results indicate that bufalin may be a potential therapeutic agent for esophageal cancer.
关键词
蟾毒灵 /
食管癌细胞 /
细胞增殖 /
细胞凋亡 /
线粒体
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Key words
bufalin /
esophageal carcinoma cells /
cell proliferation /
apoptosis /
mitochondria
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参考文献
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脚注
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基金
河南省医学科技攻关计划省部共建项目资助(SB201902030);河南省重点研发与推广专项(科技攻关)资助(182102310120);中国博士后基金项目资助(2019M652542)
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