Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells
Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells
复制标题
DOI：
10.1111/j.1745-7254.2007.00530.x
复制
发表时间：
2007-04
影响因子：
8.2
通讯作者：
Ci-hui Yan;Ya-ping Yang;Zheng-Hong Qin;Z. Gu;P. Reid;Zhong-Qin Liang
中科院分区：
文献类型：
--
作者：
Ci-hui Yan;Ya-ping Yang;Zheng-Hong Qin;Z. Gu;P. Reid;Zhong-Qin Liang
摘要目的：探讨crotoxin（CrTX）诱导的自噬在MCF-7细胞死亡中的作用，MCF-7细胞是一种caspase-3缺陷的人乳腺癌细胞系，方法：培养的MCF-7细胞用不同剂量的CrTX处理，CrTX是一种从南美响尾蛇Crotalus durissus terrificus毒液中分离的磷脂酶A2（PLA 2）。用MTT法和LDH法检测CrTX对caspase抑制剂的细胞毒性。透射电镜观察细胞自噬活性，单丹酰尸胺（MDC）标记法检测自噬活性。通过免疫印迹和免疫荧光检测溶酶体酶的上调、细胞色素c（cyto-c）的释放和凋亡诱导因子（AIF）的核转位。结果：CrTX以剂量和时间依赖性方式抑制MCF-7细胞的活力。CrTX激活的自噬通过点状MDC标记揭示，并且自噬体的形成以及凋亡增加，如核浓缩和碎裂所证明的。CrTX处理后，除了细胞色素c的释放和AIF重新定位到细胞核中之外，还观察到组织蛋白酶B、D和L的活化。自噬抑制剂3-甲基腺嘌呤（3-MA）、NH 4Cl和泛caspase抑制剂Z-Val-Ala-Asp-fluoromethylketone（Z-Vad-factorone）可减轻CrTX诱导的MCF-7细胞凋亡。
AbstractAim:To investigate the role of crotoxin (CrTX)-induced autophagy in the death of MCF-7 cells, a caspase-3-deficient, human breast cancer cell line.Methods:Cultured MCF-7 cells were treated with various doses of CrTX, a phospholipase A2 (PLA2) isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin (MDC) labeling. The upregulation of lysosomal enzymes, the release of cytochrome c (cyto-c), and the nuclear translocation of the apoptosis inducing factor (AIF) were examined by immunoblotting and immunofluorescence.Results:CrTX inhibited the viability of MCF-7 cells in a dose- and time-dependent manner. CrTX-activated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3-methyladenine (3-MA), NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-Vad-fmk), attenuated CrTX-induced cell death.Conclusion:An autophagic mechanism contributes to the apoptosis of MCF-7 cells induced by CrTX.