吴茱萸碱联合CDK1抑制剂RO3306对鼠结肠癌CT26的协同杀伤作用  

Synergistic killing effect of evodiamine combined with RO3306, a CDK1 inhibitor, on murine colon cancer CT26 cells

作　　者：崔娟[1] 吴映雅[1] 谭宇蕙[1] 张广献[1] 杜标炎[2] 常金荣[3] 

机构地区：[1]广州中医药大学生化教研室,广东省广州市510006 [2]广州中医药大学病理教研室,广东省广州市510006 [3]广州中医药大学中医基础公共实验室,广东省广州市510006

出　　处：《世界华人消化杂志》2011年第12期1244-1250,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金面上资助项目;No.30973811~~

摘　　要：目的:探讨吴茱萸碱(EVO)联合CDK1的特异抑制剂RO3306对鼠结肠癌细胞CT26的生长抑制、诱导凋亡是否有协同增效作用.方法:采用MTT法求出EVO对CT26作用24h的IC50及诱导CT26细胞发生不可逆凋亡的时间点,比较EVO和RO3306同时用药与序贯用药(EVO先作用24h,再加入RO3306共同作用6h)对CT26细胞的抑制作用.采用金正均q值法检验其联合作用是否有协同性(q为实际药效与理论药效比值,q>1.15为协同性).同时用药实验与序贯用药实验的分组情况均为对照组、2mg/LEVO组,4mg/LEVO组,15mg/LRO3306组(加药时间同相应联合组),2mg/LEVO+15mg/LRO3306联合组,4mg/LEVO+15mg/LRO3306联合组.采用克隆集落形成法检测药物单独和联合作用下对CT26细胞的抑制率.采用流式细胞术检测药物作用对CT26凋亡率的影响.结果:MTT法结果显示EVO对结肠癌细胞CT26具有显著抑制作用,其抑制作用有明显的浓度依赖性,作用24h的IC50为10.8mg/L;EVO诱导CT26细胞进入不可逆凋亡的时间点在24h左右.MTT检测EVO和RO3306序贯用药的各组抑制率依次分别是22.0%±4.4%、30.4%±3.2%、12.3%±4.8%、48.0%±3.2%、62.2%±2.2%(序贯用药联合组q=1.52,1.60>1.15,同时加药联合组q=0.68,0.72).克隆集落形成法显示相应各组的抑制率依次分别是9.7%±5.8%、38.9%±3.8%,10.8%±3.7%,29.8%±10.7%,68.3%±12.7%(q>1.15).流式细胞术显示各组细胞凋亡率依次分别是5.5%±1.1%,18.3%±1.9%,25.6%±1.5%,9.2%±1.1%,39.1%±9.8%,54.6%±1.2%(q>1.15).结论:EVO能抑制鼠结肠癌细胞CT26的生长,其作用呈剂量依赖关系,EVO诱导CT26发生不可逆凋亡的时间点约在24h左右;EVO联合CDK1抑制剂RO3306并序贯用药对CT26的抑制作用具有协同增效效应,同时加药联合作用未显示协同性.AIM： To explore whether there is a synergisticeffect between evodiamine （EVO） and RO3306,a specific cyclin-dependent kinase 1 （CDK1）inhibitor, on the proliferation and apoptosis ofmurine colon cancer CT26 cells.METHODS： The inhibitory effect of EVO on the proliferation of CT26 cells was determined by MTT assay to calculate IC50 at 24 h and the time required for the induction of irreversible apoptosis. The inhibitory effect of combination treatment with EVO and RO3306 either in a simultaneous or sequential way （pretreatment withEVO for 24 h followed by addition of RO3306 for another 6 h） on cell proliferation was also detected. CT26 cells were divided into six groups：control group, 2 mg/L EVO group, 4 mg/L EVO group, 15 mg/L RO3306 group, 2 mg/L EVO ＋ 15 mg/L RO3306 group, and 4 mg/L EVO ＋ 15 mg/L RO3306 group. Colony-forming assay and flow cytometry （FCM） assay were used to detect the effect of these treatments on cell proliferang, tion and apoptosis. q-value analysis was used to estimate the synergistic effect of evodiamine and RO3306. A q value of ≥1.15 indicates synergism.RESULTS： Treatment with EVO alone for 24 h had a significant inhibitory effect on CT26 cell proliferation, and IC50 was around 10.8 mg/L. The time required for the induction of irreversible apoptosis was 24 h. Combination treatment with EVO and RO3306 in a sequential way resulted in the rates of reduced proliferation of 22.0 ± 4.4%, 30.4 ± 3.2%, 12.3 ± 4.8%, 48.0 ± 3.2%, and 62.2±2.2% in each treatment group. The q values of the two sequential treatment groups were 1.52 and 1.60, while those of simultaneoustreatment groups were 0.68 and 0.72, respective-ly. Colony-forming assay showed the reduced rates of colony formation were 9.7 ± 5.8%, 38.9 ± 3.8%, 10.8 ± 3.7%, 29.8 ± 10.7%, and 68.3 ± 12.7% in each treatment group. The q values of the two sequential treatment groups were 1.41 and 1.47. FCM assay showed that the apoptotic rates were 5.5±1.1%, 18.3 ± 1.9%, 25.6 ± 1.5%, 9.2 ± 1.1%, 39.1 ± 9.8%, an

关 键 词：吴茱萸碱 CDK1抑制剂 细胞凋亡 M期阻滞 M期滑移 

分 类 号：R735.35[医药卫生—肿瘤]