CAJ | 학술논문

目的探讨细胞外信号调节激酶(ERK)信号通路对蛋白酶体抑制剂MG262诱导卵巢上皮性癌(卵巢癌)细胞凋亡的调控作用.方法不同浓度(1、10、20、40、60、80 nmol/L)的MG262分别处理卵巢癌细胞株SKOV3细胞24、48 h后,四甲基偶氮唑蓝(MTT)比色法检测SKOV3细胞的存活率.MG262及ERK抑制剂PD98059分别及联合处理24 h后,流式细胞仪检测SKOV3细胞及胚肾上皮细胞株293T细胞的凋亡率;酶联免疫吸附试验(ELISA)及蛋白印迹法分别检测SKOV3细胞培养上清液中血管内皮生长因子(VEGF)的浓度及细胞内VEGF蛋白的表达.MG262处理不同时间(3、6,9、12 h)后,蛋白印迹法检测SKOV3细胞内磷酸化ERK(p-ERK)及野生型p53蛋白的表达.以上实验以未加药物者为对照.结果 MTT比色法检测发现,随着MG262浓度的增加,SKOV3细胞的存活率明显下降(P＜0.05).流式细胞仪检测发现,MG262、PD98059分别或联合处理SKOV3细胞后,其细胞凋亡率分别为(30.7±4.3)%、(26.8±8.6)%、(50.3±10.6)%,与对照细胞的(7.9±1.9)%比较,差异均有统计学意义(P＜0.05);两药单独处理者分别与联合处理者比较,差异也均有统计学意义(P＜0.01).而两药分别或联合处理293T细胞后,其细胞凋亡率分别为(14.5±5.3)%、(16.2±7.5)%、(10.8±7.3)%,与对照细胞的(12.2±6.3)%比较,差异均无统计学意义(P＞0.05).ELISA及蛋白印迹法检测显示,MG262处理后SKOV3细胞培养上清液中VEGF的浓度及细胞内VEGF蛋白的表达水平均明显下降(P＜0.05).MG262处理不同时间后,SKOV3细胞内p-ERK蛋白的表达水平明显下降(P＜0.05);但SKOV3细胞内野生型p53蛋白在MG262处理前、后均无表达.结论 MG262可通过ERK信号通路诱导卵巢癌细胞凋亡,抑制卵巢癌细胞的增殖及血管生成.
목적 탐토세포외신호조절격매(ERK)신호통로대단백매체억제제MG262유도란소상피성암(란소암)세포조망적조공작용.방법 불동농도(1、10、20、40、60、80 nmol/L)적MG262분별처리란소암세포주SKOV3세포24、48 h후,사갑기우담서람(MTT)비색법검측SKOV3세포적존활솔.MG262급ERK억제제PD98059분별급연합처리24 h후,류식세포의검측SKOV3세포급배신상피세포주293T세포적조망솔;매련면역흡부시험(ELISA)급단백인적법분별검측SKOV3세포배양상청액중혈관내피생장인자(VEGF)적농도급세포내VEGF단백적표체.MG262처리불동시간(3、6,9、12 h)후,단백인적법검측SKOV3세포내린산화ERK(p-ERK)급야생형p53단백적표체.이상실험이미가약물자위대조.결과 MTT비색법검측발현,수착MG262농도적증가,SKOV3세포적존활솔명현하강(P＜0.05).류식세포의검측발현,MG262、PD98059분별혹연합처리SKOV3세포후,기세포조망솔분별위(30.7±4.3)%、(26.8±8.6)%、(50.3±10.6)%,여대조세포적(7.9±1.9)%비교,차이균유통계학의의(P＜0.05);량약단독처리자분별여연합처리자비교,차이야균유통계학의의(P＜0.01).이량약분별혹연합처리293T세포후,기세포조망솔분별위(14.5±5.3)%、(16.2±7.5)%、(10.8±7.3)%,여대조세포적(12.2±6.3)%비교,차이균무통계학의의(P＞0.05).ELISA급단백인적법검측현시,MG262처리후SKOV3세포배양상청액중VEGF적농도급세포내VEGF단백적표체수평균명현하강(P＜0.05).MG262처리불동시간후,SKOV3세포내p-ERK단백적표체수평명현하강(P＜0.05);단SKOV3세포내야생형p53단백재MG262처리전、후균무표체.결론 MG262가통과ERK신호통로유도란소암세포조망,억제란소암세포적증식급혈관생성.
Objective To investigate whether the proteasomes inhibitor MG262 exerts its anticancer function by inducing apoptosis in human ovarian cancer cells,and whether the extracellular signal regulated kinase (ERK) signaling pathway is involved in the regulation of apoptosis induction.Method Human ovarian cancer cell line SKOV3 was incubated with different concentrations of MG262 for 24 and 48 hours.Cell viability was evaluated with 3-(4,5-dimethyhhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points of culturing.Flow cytometry was used to detect cell apoptosis rate.The expression of vascular endothelial growth factor (VEGF) was evaluated with western blot and enzyme-linked immtmosorbent assay (ELISA).Western blot was used to detect the expression of phosphorylated ERK(pERK) .Results The viability of SKOV3 cells was decreased by MG262 in a concentration-dependent fashion(P＜0.05).After 24 h incubation with MG262 at 1,10,20,40,60 and 80 nmol/L,the viability rates of SKOV3 were (94.6±3.1)%,(92.7±3.7)%,(89.5±7.7)%,(84.2±5.1)%,(82.0±7.4)%and(76.8±11.0) % respectively,and after 48 h incubation,those figures were further decreased to (91.3±10.1)%,(86.8±4.5)%,(74.6±4.2)%,(56.8±2.1)%,(49.3±4.5)% and (37.4±5.4) %,respectively(P＜0.05).Apoptosis rate of SKOV3 cells induced by MG262,PD98059 or their combination was (30.7±4.3)%,(26.8±8.6)% and (50.3±10.6)%,respectively,which were significantly different compared with controls (P＜0.05).In contrast to SKOV3 cells,apoptosis rate of 293T ceils induced by MG262,PD98059 or their combination was (14.5±5.3) %,(16.2±7.5) % and (10.8±7.3)%,respectively,which were not significantly different compared with controls (P＞0.05).pERK expression decreased gradually in a time-dependent manner. And wild-type p53 expression was not significantly different.There was no significant difference between experimental and control 293T cells(P＜0.05).In addition,MG262 down-regulated VEGF secretion and expression in SKOV3 ceils (P＜0.05).Conclusions Proteasome inhibitors can induce apoptosis and inhibit cell proliferation and angiogenesis through ERK signal pathway in SKOV3 cells.