Ustundag Yucel, Bronk Steven F, Gores Gregory J
Mayo Clinic College of Medicine, 200 First Street SW, Rochester, Minnesota 55905, USA.
World J Gastroenterol. 2007 Feb 14;13(6):851-7. doi: 10.3748/wjg.v13.i6.851.
To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms.
Studies were performed in the human KMCH, KMBC, and Mz-ChA-1 cholangiocarcinoma, and normal rat cell lines. MG132, a peptide aldehyde, which inhibits the chymotrypsin-like activity of the proteasome was employed for this study. Apoptosis was assessed morphologically by 4'-6-Diamidino-2-phenylindole (DAPI) nuclear staining and fluorescence microscopy. Mitochondrial membrane potential was examined using a fluorescent unquenching assay. Ultrastructural changes during cell death were examined using transmission electron microscopy (TEM). Caspase 3/7 activity was assessed using an enzymatic-based fluorescent assay. Cytosolic-free calcium concentrations were measured using Fura-2 and digitized fluorescent microscopy.
MG132, a proteasome inhibitor, induced apoptosis in all the cholangiocarcinoma cell lines examined. In contrast, minimal cytotoxicity was observed in normal rat cholangiocytes. Apoptosis was time- and -concentration-dependent. There was no change in the mitochondrial membrane potential between treated and untreated cells. Ultrastructural examination by transmission electron microscopy displayed the classic features of apoptosis, but in addition, there was also dramatic vacuolization of the endoplasmic reticulum (ER). Unexpectedly, no increase in caspase 3/7 activity was observed in MG132 treated cells, nor did the pancaspase inhibitor, Q-VD-OPh prevent cell death. The protein synthesis inhibitor, cycloheximide, blocked apoptosis induced by proteasome inhibitor indicating that ER dysfunction was dependent upon the formation of new proteins.
Proteasome inhibition induces ER dysfunction and caspase-independent cell death selectively in human cholangiocarcinoma cells. Proteasome inhibitors warrant evaluation as anticancer agents for the treatment of human cholangiocarcinoma.
确定蛋白酶体抑制是否会诱导人胆管癌细胞凋亡，若会，则阐明其细胞机制。
在人KMCH、KMBC和Mz-ChA-1胆管癌细胞系以及正常大鼠细胞系中开展研究。本研究采用了一种肽醛MG132，它可抑制蛋白酶体的胰凝乳蛋白酶样活性。通过4′-6-二脒基-2-苯基吲哚（DAPI）核染色和荧光显微镜对凋亡进行形态学评估。使用荧光非淬灭测定法检测线粒体膜电位。利用透射电子显微镜（TEM）检查细胞死亡过程中的超微结构变化。使用基于酶的荧光测定法评估半胱天冬酶3/7活性。使用Fura-2和数字化荧光显微镜测量胞质游离钙浓度。
蛋白酶体抑制剂MG132在所有检测的胆管癌细胞系中均诱导了凋亡。相比之下，在正常大鼠胆管细胞中观察到的细胞毒性极小。凋亡呈时间和浓度依赖性。处理过的细胞与未处理的细胞之间线粒体膜电位没有变化。透射电子显微镜的超微结构检查显示出凋亡的典型特征，但此外，内质网（ER）也出现了显著的空泡化。出乎意料的是，在MG132处理的细胞中未观察到半胱天冬酶3/7活性增加，泛半胱天冬酶抑制剂Q-VD-OPh也未阻止细胞死亡。蛋白质合成抑制剂环己酰亚胺可阻断蛋白酶体抑制剂诱导的凋亡，这表明内质网功能障碍依赖于新蛋白质的形成。
蛋白酶体抑制在人胆管癌细胞中选择性地诱导内质网功能障碍和非半胱天冬酶依赖性细胞死亡。蛋白酶体抑制剂值得作为治疗人胆管癌的抗癌药物进行评估。
