CAJ | 학술논문

目的：评价以酵母细胞为模式菌高通量筛选Pin1抑制剂的方法。方法以“生型酵母菌W303-1a和温度敏感型突变株L94P为模式菌，筛选Pin1抑制剂；四甲基偶氮唑盐(MTT)比色法检验候选化合物对肿瘤细胞的杀伤作用；体外酶学实验验证候选化合物对Pin1的抑制作用；流式细胞仪检测候选化合物对肿瘤细胞周期和凋亡的阻滞和诱导作用；免疫印迹(Western Blotting)分析候选化合物对肿瘤细胞中Cyclin D1和Pin1表达量的影响。结果候选化合物显示出对温度敏感型突变株的特异性抑制作用；MTT实验证实化合物能抑制肿瘤细胞的增殖；酶学实验和Western Blotting共同阐释了化合物对重组纯化的人Pin1具抑制作用但并不影响Pin1蛋白的表达；流式结果表明化合物能诱导细胞发生凋亡和G0/G1期阻滞；化合物还能抑制肿瘤细胞中Cyclin D1的蛋白表达并呈剂量依赖性。结论首次发现Pin1为苯并呋喃衍生物抗肿瘤作用的靶点之一。
목적：평개이효모세포위모식균고통량사선Pin1억제제적방법。방법이“생형효모균W303-1a화온도민감형돌변주L94P위모식균，사선Pin1억제제；사갑기우담서염(MTT)비색법검험후선화합물대종류세포적살상작용；체외매학실험험증후선화합물대Pin1적억제작용；류식세포의검측후선화합물대종류세포주기화조망적조체화유도작용；면역인적(Western Blotting)분석후선화합물대종류세포중Cyclin D1화Pin1표체량적영향。결과후선화합물현시출대온도민감형돌변주적특이성억제작용；MTT실험증실화합물능억제종류세포적증식；매학실험화Western Blotting공동천석료화합물대중조순화적인Pin1구억제작용단병불영향Pin1단백적표체；류식결과표명화합물능유도세포발생조망화G0/G1기조체；화합물환능억제종류세포중Cyclin D1적단백표체병정제량의뢰성。결론수차발현Pin1위분병부남연생물항종류작용적파점지일。
Objective To evaluate potential small molecular inhibitors through high throughput screening (HTS) using budding yeast cells. Methods In vitro biochemical assay using purified recombinant human Pin1 was performed to test the inhibition of candidate determined by HTS using wild-type yeast cells and L94P temperature sensitive mutant. MTT proliferation assay was carried out to detect ZY0625’s cellular toxicity. Flow cytometric analysis was operated to assess ZY0625’s influence on cell cycle progression and apoptosis. Western Blotting was performed to determine the effects of candidate on Pin1 and Cyclin D1 expression. Results ZY0625 inhibited the growth of L94P temperature sensitive mu-tant more strongly than wild-type yeast cells, moreover ZY0625 inhibited purified recombinant human Pin1 in v itro without influencing the expression of Pin1 in tumor cells. Cell culture based tests of ZY0625 activity showed that ZY0625 potently inhibited the proliferation of human cancer cell lines. Flow cytometry confirmed that ZY0625 could induce apoptosis of tumer cells and modulate cell cycle distribution moderately with an elevated percentage of cells in G0/G1, as well. Like other Pin1 inhibitors, ZY0625 increased the protein expression of Cyclin D1 in a dose dependent fashion. Conclusion Pin1 is for the first time verified as one of the targets that contributes to the benzofuran deriva-tives' anticancer activity.