Hu Wei, Wu WeiGuo, Verschraegen Claire F, Chen Ling, Mao Li, Yeung Sai-Ching Jim, Kudelka Andrzej P, Freedman Ralph S, Kavanagh John J
Department of Gynecologic Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, USA.
Proteomics. 2003 Oct;3(10):1904-11. doi: 10.1002/pmic.200300547.
Farnesyl transferase inhibitors (FTIs) are novel antitumor drugs with clinical activity. FTIs inhibit cell growth not only by preventing direct Ras farnesylation but also through a Ras-independent pathway. Proteomics has been shown to be a powerful tool to monitor and analyze molecular networks and fluxes within the living cells and to identify the proteins that participate in these networks upon perturbation of the cellular environment. To observe early and dynamic protein changes in the cellular response to FTI in ovarian cancer cells, total proteins were extracted from 2774 cells treated or not with 10 microM manumycin, an FTI, for 3, 6 and 16 h. The proteins in the cells that were differentially expressed following treatment with manumycin for 3, 6 and 16 h were noted by two-dimensional electrophoresis and further identified by peptide mass fingerprinting as stress proteins. Both heat shock protein 70 (HSP70) and altered HSP70 were significantly up-regulated as early as 16 h in 2774 cells after exposure to manumycin. Since HSP70 plays an important role in protecting cells under stress, we treated the 2774 cells with the HSP inhibitor quercetin in combination with FTI. Quercetin dramatically enhanced the manumycin-mediated apoptosis in 2774 cells. Inducible HSP70 by manumycin in surviving ovarian cancer cells was also inhibited by quercetin as demonstrated by enzyme-linked immunosorbent assay. The inhibition of HSP70 by quercetin was correlated with enhancement of manumycin-induced mediated apoptosis in 2774 cells. The inhibition of HSP70 by 50 microM quercetin was also correlated with a decreased expression of procaspase-3 and enhancement of specific cleavage of poly (ADP-ribose) polymerase into apoptotic fragment in 2774 cells treated with manumycin. The interaction between the HSP70 inhibitor and FTI confirms the functional significance of the up-regulation of HSP70 as a protective mechanism against FTI-induced apoptosis and provides the framework for combination treatment of ovarian cancer.
法尼基转移酶抑制剂（FTIs）是具有临床活性的新型抗肿瘤药物。FTIs不仅通过阻止直接的Ras法尼基化来抑制细胞生长，还通过一条不依赖Ras的途径发挥作用。蛋白质组学已被证明是监测和分析活细胞内分子网络和通量，以及识别在细胞环境受到扰动时参与这些网络的蛋白质的有力工具。为了观察卵巢癌细胞对FTI的细胞反应中早期和动态的蛋白质变化，从2774个细胞中提取总蛋白，这些细胞分别用10微摩尔的FTI（法尼基转移酶抑制剂）曼诺霉素处理3小时、6小时和16小时，或不进行处理。用二维电泳记录在用曼诺霉素处理3小时、6小时和16小时后细胞中差异表达的蛋白质，并通过肽质量指纹图谱进一步鉴定为应激蛋白。在暴露于曼诺霉素后，热休克蛋白70（HSP70）和改变的HSP70在2774个细胞中早在16小时就显著上调。由于HSP70在保护应激状态下的细胞中起重要作用，我们用HSP抑制剂槲皮素与FTI联合处理2774个细胞。槲皮素显著增强了曼诺霉素介导的2774个细胞的凋亡。酶联免疫吸附测定表明，槲皮素也抑制了曼诺霉素在存活的卵巢癌细胞中诱导的HSP70。槲皮素对HSP70的抑制与曼诺霉素诱导的2774个细胞凋亡增强相关。50微摩尔槲皮素对HSP70的抑制也与在用曼诺霉素处理的2774个细胞中procaspase-3表达的降低以及聚（ADP-核糖）聚合酶特异性切割成凋亡片段的增强相关。HSP70抑制剂与FTI之间的相互作用证实了HSP70上调作为针对FTI诱导凋亡的保护机制的功能意义，并为卵巢癌的联合治疗提供了框架。
