CAJ | 학술논문

目的：利用酵母模型进行保罗样激酶1(Polo-like kinase 1,PLK1)抑制剂的高通量筛选,并初步评估活性化合物的体外抗肿瘤效果。方法采用噻唑蓝比色法(MTT 法)检测初筛阳性化合物对不同细胞系增生的影响,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)分析化合物对体外纯化的 PLK1激酶的作用效果,流式细胞术(flow cytometry,FCM)检测细胞周期分布和凋亡率,免疫印迹(Western blotting)检测化合物对周期蛋白 B1(cyclin B1)和 PLK1的作用。结果利用酵母模型初筛得到3个阳性化合物,MTT 法检测显示1号和3号化合物能抑制多种肿瘤细胞的增生而对正常细胞的毒性很小,ELISA 结果显示2号和3号化合物对体外纯化的 PLK1几乎无抑制作用,FCM 检测显示1号化合物处理组 G2/ M 期细胞比例高于对照组, Western blotting 表明1号化合物不影响 PLK1的蛋白表达,能导致 cyclin B1的表达增加。结论1号化合物通过抑制 PLK1激酶的活性发挥抗肿瘤作用而不影响 PLK1的蛋白表达,有望成为靶向 PLK1的抗肿瘤先导化合物。
목적：이용효모모형진행보라양격매1(Polo-like kinase 1,PLK1)억제제적고통량사선,병초보평고활성화합물적체외항종류효과。방법채용새서람비색법(MTT 법)검측초사양성화합물대불동세포계증생적영향,매련면역흡부측정법(enzyme-linked immunosorbent assay,ELISA)분석화합물대체외순화적 PLK1격매적작용효과,류식세포술(flow cytometry,FCM)검측세포주기분포화조망솔,면역인적(Western blotting)검측화합물대주기단백 B1(cyclin B1)화 PLK1적작용。결과이용효모모형초사득도3개양성화합물,MTT 법검측현시1호화3호화합물능억제다충종류세포적증생이대정상세포적독성흔소,ELISA 결과현시2호화3호화합물대체외순화적 PLK1궤호무억제작용,FCM 검측현시1호화합물처리조 G2/ M 기세포비례고우대조조, Western blotting 표명1호화합물불영향 PLK1적단백표체,능도치 cyclin B1적표체증가。결론1호화합물통과억제 PLK1격매적활성발휘항종류작용이불영향 PLK1적단백표체,유망성위파향 PLK1적항종류선도화합물。
Objective To find potential small-molecular PLK1 inhibitors through high throughput screening using budding yeast assay and examine the in vitro anti-tumor effects of these compounds. Methods The influences of the positive compounds on cells' proliferation were measured by MTT assays. ELISA assay was employed to explore their inhibition activities on purified recombinant human PLK1. Flow cytometry(FCM) was conducted to determine their effects on cell cycle and apoptosis in Hela cells. The levels of Cyclin B1 and PLK1 were detected by Western blotting. Results We got 3 hits from preliminary screening by yeast assay. In vitro antitumor results revealed that compounds 1 and 3 showed the enhanced inhibition on the cell proliferation of human cancer cell lines with concentration increasing, while the activity of compound 2 was very weak. Compound 1 had lower nanomolar activity against the PLK1 enzyme without influencing its protein expression compared with compound 2 and 3. FCM showed that cells treated with compound 1 were arrested in G2 /M phase. Western blotting showed an increase in expression of Cyclin B1 in Hela cells after treated with compound 1. Conclusion The inhibition effect of compound 1 on PLK1 was associated with kinase activity without influencing the PLK1 protein expression. Compound 1 is expected to become one of the anti-cancer drug candidates targeted at PLK1.