目的 探究维替泊芬是否具有诱导多种子宫内膜癌细胞发生内质网应激及自噬的作用。方法 维替泊芬处理子宫内膜癌细胞系KLE、EFE184、NOU-1和SKUT-2，并分别使用二甲基亚砜（DMSO）、蛋白酶体抑制剂MG132、硼替佐米和光敏剂原卟啉处理细胞作为对照组。维替泊芬作用浓度为0.1~10 μmol/L，作用时间为0~48 h。光学显微镜和活细胞成像系统观察细胞形态，磺酰罗丹明B法测定细胞增殖能力。Western blot法检测内质网应激过程中相关蛋白质水平变化，免疫荧光法检测自噬小体。结果 MG132和硼替佐米抑制子宫内膜癌细胞增殖、诱导细胞肿胀与死亡，维替泊芬诱导细胞出现肿胀并死亡，而原卟啉无此作用。维替泊芬促进细胞中肌醇蛋白-1a减少、钙敏感伴侣蛋白增加、C/EBP同源蛋白（CHOP）增加、蛋白质二硫键异构酶降低。同时CHOP蛋白质水平受维替泊芬浓度和作用时间的影响，呈现双向性：当降低维替泊芬浓度同时增加作用时间时，CHOP蛋白质水平增加;当作用浓度在0~5 μmol/L时，CHOP蛋白质水平随维替泊芬浓度增加而增加，但当作用浓度提高至10 μmol/L时，CHOP蛋白质水平降低。维替泊芬促进细胞膜关键蛋白Rab11水平下降，细胞核出现形态皱缩，溶酶体增加。结论 维替泊芬可能通过诱导子宫内膜癌细胞发生内质网应激，引起细胞自噬，最终导致细胞死亡。
Abstract
Objective To investigate whether verteporfin can induce endoplasmic reticulum (ER)stress and autophagy in a variety of endometrial cancer cells.Methods Endometrial cancer cell lines KLE, EFE184,NOU-1 and SKUT-2 were treated with verteporfin at different concentrations (0.1-10 μmol/L) and times (0-48 h).Dimethyl sulfoxide (DMSO),proteasome inhibitor MG132,bortezomib and photosensitizer protoporphyrin were served as controls. Cell morphology was observed using an optical microscope and a living cell imaging system.Cell proliferation ability was determined by the sulfonyl rhodamine B method. Western blot was used to detect the changes in protein levels during ER stress,and immunofluorescence was used to detect autophagy.Results MG132 and bortezomib inhibited cell proliferation and induced cell swelling and death,verteporfin also induced swelling and cell death of endometrial cancer cells,but protoporphyrin had no such effect.After verteporfin treatment in KLE and EFE184 cell lines,changes in ER stress-related proteins included:decreased inositol-requiring enzyme-1a (IRE-1a),increased binding immunoglobulin protein (BIP),increased CCAAT/enhancer-binding protein homologous protein (CHOP),decreased protein disulfide isomerase (PDI).When we decreased the concentration but increased the acting time of verteporfin,the level of CHOP increased.As the concentration went higher,the expression of CHOP was more obvious when verteporfin was in the range of 0-5 μmol/L.However,the CHOP level decreased instead when the concentration of verteporfin increased to 10 μmol/L.In addition,endometrial cancer cells showed a decreased expression of membrane key protein Rab11 after verteporfin treatment.At the same time,the shrinkage nuclear and increased lysosomes were observed.Conclusion Verteporfin may induce ER stress in endometrial cancer cells,causing cell autophagy and eventually leading to cell death.
关键词
子宫内膜癌 /
维替泊芬 /
内质网应激 /
自噬 /
机制
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Key words
endometrial cancer /
verteporfin /
endoplasmic reticulum stress /
autophagy /
mechanism
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中图分类号：
R737.33
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脚注
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基金
国家自然科学基金（81772777）；上海市科委医学引导计划（18411963700）；上海市卫生系统优秀青年培训计划（XYQ2011062）；上海市科委“浦江人才”课题（17PJ1401400）
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