8-氯腺苷对HL-60细胞中蛋白酶体活性的抑制作用

Inhibition of 8-Chloroadenosine on Proteasome Activity of HL-60 Cells

背景与目的:蛋白酶体是真核细胞中具有多相催化活性的蛋白酶复合体,国外报道,蛋白酶体可作为新靶点来筛选抗肿瘤药物。8-氯腺苷(8-chloroadenosine,8-CA)由本室合成,研究已证实8-CA对肉瘤细胞株S180、肝癌细胞株H22及人胃癌异种移植瘤生长有抑制作用。但8-CA抑制肿瘤生长与蛋白酶体的关系还不清楚。本研究拟探讨8-CA对人早幼粒细胞性白血病细胞HL-60中20S蛋白酶体的三种酶活性(包括糜蛋白酶样活性、胰蛋白酶样活性、多肽-谷氨酰-多肽水解酶活性)的影响。方法:不同浓度的8-CA作用于HL-60细胞24、48、72h后,提取细胞总蛋白,分别检测总蛋白提取液中20S蛋白酶体的三种酶活性,并采用免疫沉淀法测定纯化蛋白酶体的糜蛋白酶样活性,酶活性均以特异底物被蛋白酶体水解后产生的荧光吸光度表示。结果:0.1μmol/L8-CA作用于HL-60细胞48h,对总蛋白提取液中20S蛋白酶体的三种酶活性均有显著抑制作用,并且随着8-CA浓度的增加,其抑制作用逐渐增强。5μmol/L8-CA作用48h,对糜蛋白酶样活性、胰蛋白酶样活性、多肽-谷氨酰-多肽水解酶活性抑制率分别为44.96%、54.52%和48.36%;作用72h,对三种酶活性的抑制率分别为67.53%、70.48%和64.08%。1μmol/L和5μmol/L8-CA作用48h,对HL-60中蛋白酶体的糜蛋白酶样活性抑制率分别为68.

BACKGROUND &OBJECTIVE: The proteasome is a multicatalytic proteinase complex in eukaryotic cells, and it can be used as a new target for screening anti tumor drugs according to certain previous researches. The inhibition activity of 8 CA (8 chloroadenosine, a new anti tumor drug synthesized in our lab) in sarcoma 180, hepatocarcinoma 22, and human gastric carcinoma xenograft tumor has been confirmed. However,the relationship between the mechanism of the inhibition and proteasome is still unclear. The present study was designed to explore the effect of 8 CA on 3 kinds of enzyme activities (chymotrypsin like activity, trypsin like activity, and peptidyl glutamyl peptide hydrolyzing activity) of 20S proteasome in human progranulocyte leukemia HL 60 cells. METHODS: HL 60 cells were treated with 8 CA at different concentrations for 24, 48, and 72 hours. Then total protein of HL 60 cells was extracted, and the three enzyme activities of 20S proteasomes in the total protein was examined. And the chymotrypsin like activity of the purified proteasome from the total protein was also examined by immunoprecipitation. The enzyme activity was indicated by fluorescence absorbance (A) produced by degradation of specific substrates by proteasomes. RESULTS: In HL 60 cells, three enzyme activities of the proteasomes were significantly inhibited after exposure to 0.1 μmol/L 8 CA for 48 hours;and the inhibition effect was concentration dependent. When the concentration of 8 CA reached 5 μmol/L, the inhibition rates of three proteasome activities (chymotrypsin like activity,trypsin like activity,and peptidyl glutamyl peptide hydrolyzing activity) were 44.96%, 54.52%, 48.36%for 48 hours and 67.53%, 70.48%, 64.08%for 72 hours, respectively. The inhibition rate of 1 μmol/L and 5 μmol/L 8 CA on chymotrypsin like proteasome activity of HL 60 cells for 48 hours were 68.14%(P< 0.01) and 92.75%(P< 0.001), respectively. CONCLUSION: 8 CA inhibits proteasome activity of HL 60 cells with a time and concentration dependent manner.