喜树碱药物NSC606985抗卵巢癌细胞活性及其机制的研究

The anticancer effect and mechanism of NSC606985,a camptothecin analog,against human ovarian cancer cells.

目的:体外观察喜树碱药物NSC606985对人卵巢癌细胞的生长抑制效应、凋亡诱导效应以及抗癌机制。方法:采用MTT法检测不同浓度(6.25,12.5,25,50,100nmol/L)NSC606985作用特定时间(24,48,72h)后对人卵巢癌COC1细胞的生长抑制效应,用AnnexinV-FITC/PI双染法FCM检测NSC606985对COC1细胞的凋亡诱导效应,逆转录聚合酶链反应(RT-PCR)法检测缺氧(2%O2)对HIF-1α,AMFR及RhoC mRNA表达水平的影响。用免疫印迹技术(Western blot)检测物理性缺氧及氯化钴(CoCl2)化学模拟缺氧下HIF-1α蛋白的表达水平以及NSC606985对HIF-1α的调控效应。结果:纳摩尔浓度的NSC606985可以显著抑制人卵巢癌COC1细胞生长,呈现剂量及时间依赖效应。不同浓度(50,100,200nmol/L)NSC606985作用24h凋亡细胞比率分别是68.44%,80.86%,91.98%,具有剂量依赖性。在人卵巢癌细胞系COC1和SKOV3中,缺氧(2%O2)14h对HIF-1α、AMFR及RhoC mRNA的表达无影响,却可以上调HO-8910PM细胞HIF-1αmRNA水平。物理性缺氧(2%O2 14h)及CoCl2化学模拟缺氧(50μmol/L或150μmol/L 24h)诱导COC1,HO-8910PM及SKOV3等卵巢癌细胞株HIF-1α蛋白高水平表达,NSC606985(20～400nmol/L 14h或24h)可呈剂量依赖性显著下调缺氧诱导的HIF-1α蛋白。结论:NSC606985抑制卵巢癌细胞增殖具有剂量和时间依赖性,可能与诱导细胞凋亡相关。缺氧下HIF-1α在卵巢癌细胞的恶性生物学行为中发挥重要作用,NSC606985可通过下调缺氧诱导的HIF-1α蛋白发挥独特的抗癌机制。

Objective：To investigate the cytotoxicity,apoptosis-inducing effect and anticancer mechanism of NSC606985,a camptothecin analog,against human ovarian cancer cells.Methods：The human ovarian cancer COC1 cells were cultured with different concentration（6.25,12.5,25,50,100nmol/L）of NSC606985 for indicated time（24h,48h,2h）,then MTT assay was used to measure the anticancer activity of NSC606985.NSC606985-induced apoptotic cell death were measured by Annexin V-fluorescein isothiocyanate/Propiduim Iodide（AnnexinV-FITC/PI）staining.The mRNA expression of HIF-1α,AMFR and RhoC was examined by RT-PCR.The regulation of HIF-1α protein under normoxia or hypoxia conditions in the presence or absence of NSC606985 was detected by Western Blot.Results：The cytotoxicity of NSC606985 with the concentration of nanomolar（nM）against human ovarian cancer COC1 cells was dose-and time-dependent.Treatment of COC1 cells with NSC606985 at the concentration of 50,100 or 200nmol/L for 24h produced a dose-dependent increase of apoptotic cells,the ratio of which increased from 68.44%,80.86% to 91.98%.The expression of HIF-1α,AMFR and RhoC mRNA were not altered under 2% O2 for 14h in COC1 and SKOV3 cells,but the HIF-1αmRNA expression increased significantly in HO-8910PM cells.The HIF-1α protein level was drastically dramestically induced in both 2% O2 and hypoxia mimetic agent cobalt chloride（CoCl2）-treated human ovarian cancer cells（COC1、HO-8910PM and SKOV3）.NSC606985 significantly inhibited the activation of HIF-1α protein under hypoxia conditions in dose-dependent fashion.Conclusion：NSC606985 can inhibit proliferation of human ovarian cancer cells in a dose-and time-dependent manner,which is partly correlated with apoptotic cell death.HIF-1α plays an important role in the biologic properties of ovarian cancer under hypoxia condition,but NSC606985 can exert its unique anticancer activity through down-regulating the high level of HIF-1α protein induced by hypoxia.