细胞周期蛋白依赖性蛋白激酶抑制剂flavopiridol对卵巢上皮性癌细胞及移植瘤的干预作用

Therapeutic effect of flavopiridol,a small molecular cyclin-dependent kinase inhibitor,in human ovarian carcinoma

目的检测细胞周期蛋白依赖性蛋白激酶抑制剂 flavopiridol 对卵巢上皮性癌(卵巢癌)细胞及移植瘤的干预作用。方法应用流式细胞仪和脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)检测 flavopiridol 作用后卵巢癌细胞系 AO 的细胞凋亡率和细胞周期,应用实时荧光定量 PCR 技术检测 flavopiridol 作用前、后 AO 细胞中细胞周期蛋白(cyclin)D 和活性半胱氨酸天冬氨酸蛋白酶(caspase)3的表达情况。建立卵巢癌裸鼠皮下及腹腔移植瘤模型,观测 flavopiridol 干预后裸鼠的生存情况及肿瘤体积的变化,TUNEL 和免疫组化方法分别检测肿瘤组织中的细胞凋亡情况和微血管密度(MVD)。结果 AO 细胞在150、300、500 nmol/L 浓度的 flavopiridol 作用下,凋亡率分别为4.1%、10.7%和7.6%;G_1期细胞比例显著增加,S 期比例显著降低(P<0.05)。flavopiridol作用后 AO 细胞 cyclin D 的表达量(0.25)显著下降,活性 caspase-3的表达量(2.55)轻度升高,高于flavopiridol 作用前的0.69(P<0.05)、2.49(P>0.05)。flavopiridol 干预后裸鼠的累积生存率显著提高(P<0.05);裸鼠的平均生存时间为(141±14)d,显著高于磷酸盐缓冲液(PBS)作用后的(106±11)d,两者比较,差异有统计学意义(P<0.05);在 flavopiridol 干预后53 d 时抑瘤率为40%。flavopiridol 干预后裸鼠的肿瘤组织中有细胞凋亡发生,MVD 为(12±5)个,显著高于 PBS 作用后的(35±10)个,两者比较,差异有统计学意义(P<0.05)。结论 flavopiridol 能显著抑制卵巢癌细胞及移植瘤的生长,延长荷瘤裸鼠的生存期。

Objective To investigate the antitumor effect of flavopiridol in ovarian cancer. Methods After the treatment with flavopiridol of AO cells, cell apoptotic rate and cell cycle distribution were detected by flow cytometer and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling （TUNEL）. Real time PCR was used to detect the expression of cyclin D and active caspase-3 in AO cells. Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/c nude mice were established. The mouse survival rates were measured for abdominally spread tumor models and the volume of tumor nodules was determined for subcutaneous tumor models following the treatments of flavopiridol. TUNEL was used to detect cell apoptosis, and immunohistochemistry was used to measure microvessel density （MVD） in tumor tissues. Results AO cells showed apoptotic rates of 4. 1%, 10. 7% and 7.6% following the treatments with flavopiridol at 150,300 and 500 nmol/L respectively, accompanied by an increase in G1 progression and a decrease in S phase progression. The level of active caspase-3 increased （2. 55 vs 2.49） and the level of cyclin D expression decreased significantly （0. 25 vs 0. 69, P 〈 0. 05 ） after treatments with flavopiridol. Flavopiridol prolonged mouse survival [ mean survival time of （ 141 ± 14 ） days ] and suppressed tumor growth significantly （tumor growth suppression rate of 40% ）, when compared with treatment using phosphate-buffered saline [ （106 ± 11 ） days, P 〈 0. 05 ]. Apoptosis was detected in tumor tissues treated with flavopiridol. MVD of tumor tissue was 12 ± 5 following flavopiridol treatment, significantly higher than that of 35 ± 10 treated with phosphate-buffered saline （ P 〈 0. 05 ）. Conclusion Flavopiridol results in significant suppression of ovarian carcinoma cell growth and prolongs survival of mice.