Objective To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells. Methods Cisplatin-induced apoptosis were stained with DAPI and was ...Objective To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells. Methods Cisplatin-induced apoptosis were stained with DAPI and was assessed microscopically in human epithelial adenocarcinoma ovarian cell line SKOV3 cells. ERK activation was determined by Western blotting using an anti-phospho-ERK antibody to detect ERK activity. The effect of PD98059 on ERK activity induced by cisplatin was detected by MTT assay. Results Marked apoptosis of SKOV3 cells resulted from 48 hours treatment with 20 μg/mL cisplatin. Strong activation of ERK was led to by 15 μg/mL cisplatin. Dose response and time course of cisplatin induced apoptosis in SKOV3 cells. Cisplatin-induced ERK activation occurred at 12 hours and increased to highest induction at 24 hours by Western blotting. The effect of PD 98059 on ERK activity induced by cisplatin at the concentration of 100 μmol/L PD 98059. Statistically significant decreased in cell survival were observed with 100 μmol/L PD 98059 at 15 and 20 μg/mL cisplatin (P< 0.05). Conclusions Cisplatin activates the ERK signaling pathway in ovarian cancer cell line SKOV3. Inhibition of ERK acti-vity enhances sensitivity to cisplatin cytotoxity in ovarian cancer cell line SKOV3. Evaluation of ERK activity could be useful in predicting which ovarian cancer will response most favorably to cisplatin therapy.展开更多
目的:检测flavopiridol联合泰素对人卵巢癌细胞系SKOV3、裸鼠人卵巢癌皮下及腹腔移植瘤模型的治疗作用。方法:应用CCK-8法、流式细胞术和原位细胞凋亡检测法(TUNEL)检测flavopiridol和泰素作用后卵巢癌细胞系SKOV3的细胞存活率和凋亡率...目的:检测flavopiridol联合泰素对人卵巢癌细胞系SKOV3、裸鼠人卵巢癌皮下及腹腔移植瘤模型的治疗作用。方法:应用CCK-8法、流式细胞术和原位细胞凋亡检测法(TUNEL)检测flavopiridol和泰素作用后卵巢癌细胞系SKOV3的细胞存活率和凋亡率;应用逆转录-多聚酶链反应(RT-PCR)检测作用前后细胞中cyclinD1的表达:应用ELISA法检测细胞中活性caspase-3的表达。建立裸鼠人卵巢癌皮下及腹腔移植瘤模型,观测flavopiridol和泰素治疗前后裸鼠肿瘤体积的变化并计算抑瘤率,用TUNEL和免疫组化法分别检测作用前后肿瘤组织中的凋亡情况和微血管密度(MVD)值。结果:flavopiri-dol(300nmol/L)或泰素(1μmol/L)单独应用24h均能显著降低细胞存活率,增强caspase-3活性,诱导细胞凋亡,并下调细胞中cyclinD1的表达。Taxol先作用24h再用flavopiridol作用24h的联合用药对二者的上述作用产生显著的协同效应:联合用药后细胞存活率为(5.2±0.8)%,凋亡率为(51.10±2.52)%,caspase-3活性相对值为0.602±0.008,cy-clinD1相对表达水平为0.264±0.076。Flavopiridol和泰素单独应用均能显著抑制皮下移植瘤生长(抑瘤率分别为84.5%和85.7%),并降低肿瘤组织MVD值(分别为12.4±4.7vs 35.2±10.3和15.2±2.9 vs 35.2±10.3)。二者联合应用抑瘤作用稍差(抑瘤率68.9%),抑制MVD作用(23.0±5.1 vs 35.2±10.3)也不及单药治疗。结论:Flavopiridol和泰素均能通过致凋亡作用显著抑制卵巢癌细胞及移植瘤生长,二者联合应用对体外培养的卵巢癌细胞具有协同杀伤作用,但体内治疗中无协同抑瘤作用。展开更多
基金Supported by Heilongjiang Province Natural Science Funds (D03-55) and Heilongjiang Province Sanitary Bureau Science Funds (2003-003)
文摘Objective To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells. Methods Cisplatin-induced apoptosis were stained with DAPI and was assessed microscopically in human epithelial adenocarcinoma ovarian cell line SKOV3 cells. ERK activation was determined by Western blotting using an anti-phospho-ERK antibody to detect ERK activity. The effect of PD98059 on ERK activity induced by cisplatin was detected by MTT assay. Results Marked apoptosis of SKOV3 cells resulted from 48 hours treatment with 20 μg/mL cisplatin. Strong activation of ERK was led to by 15 μg/mL cisplatin. Dose response and time course of cisplatin induced apoptosis in SKOV3 cells. Cisplatin-induced ERK activation occurred at 12 hours and increased to highest induction at 24 hours by Western blotting. The effect of PD 98059 on ERK activity induced by cisplatin at the concentration of 100 μmol/L PD 98059. Statistically significant decreased in cell survival were observed with 100 μmol/L PD 98059 at 15 and 20 μg/mL cisplatin (P< 0.05). Conclusions Cisplatin activates the ERK signaling pathway in ovarian cancer cell line SKOV3. Inhibition of ERK acti-vity enhances sensitivity to cisplatin cytotoxity in ovarian cancer cell line SKOV3. Evaluation of ERK activity could be useful in predicting which ovarian cancer will response most favorably to cisplatin therapy.
基金This project was supported by Heilongjiang Province government natural science funds (No:D03-55) and Heilongjiang Province sanitary bureau scientific funds (No:2003-003)
文摘目的:检测flavopiridol联合泰素对人卵巢癌细胞系SKOV3、裸鼠人卵巢癌皮下及腹腔移植瘤模型的治疗作用。方法:应用CCK-8法、流式细胞术和原位细胞凋亡检测法(TUNEL)检测flavopiridol和泰素作用后卵巢癌细胞系SKOV3的细胞存活率和凋亡率;应用逆转录-多聚酶链反应(RT-PCR)检测作用前后细胞中cyclinD1的表达:应用ELISA法检测细胞中活性caspase-3的表达。建立裸鼠人卵巢癌皮下及腹腔移植瘤模型,观测flavopiridol和泰素治疗前后裸鼠肿瘤体积的变化并计算抑瘤率,用TUNEL和免疫组化法分别检测作用前后肿瘤组织中的凋亡情况和微血管密度(MVD)值。结果:flavopiri-dol(300nmol/L)或泰素(1μmol/L)单独应用24h均能显著降低细胞存活率,增强caspase-3活性,诱导细胞凋亡,并下调细胞中cyclinD1的表达。Taxol先作用24h再用flavopiridol作用24h的联合用药对二者的上述作用产生显著的协同效应:联合用药后细胞存活率为(5.2±0.8)%,凋亡率为(51.10±2.52)%,caspase-3活性相对值为0.602±0.008,cy-clinD1相对表达水平为0.264±0.076。Flavopiridol和泰素单独应用均能显著抑制皮下移植瘤生长(抑瘤率分别为84.5%和85.7%),并降低肿瘤组织MVD值(分别为12.4±4.7vs 35.2±10.3和15.2±2.9 vs 35.2±10.3)。二者联合应用抑瘤作用稍差(抑瘤率68.9%),抑制MVD作用(23.0±5.1 vs 35.2±10.3)也不及单药治疗。结论:Flavopiridol和泰素均能通过致凋亡作用显著抑制卵巢癌细胞及移植瘤生长,二者联合应用对体外培养的卵巢癌细胞具有协同杀伤作用,但体内治疗中无协同抑瘤作用。
