Hu Wei, Wu Weiguo, Yeung Sai-Ching Jim, Freedman Ralph S, Kavanagh John J, Verschraegen Claire F
Department of Gynecologic and Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Anticancer Res. 2002 Mar-Apr;22(2A):665-72.
Since farnesylation by farnesyltransferase (FTase) is the first obligatory step in the signaling transduction pathway of ras p21, FTase has been the target of new anticancer treatment modalities. Famesyl transferase inhibitors (FTIs), a novel class of antitumor drugs that blocks the oncogenic activity of ras, were first developed as therapy for ras-mutated human tumors. FTIs are also capable of inhibiting tumors without ras mutations, while different mechanisms of cell growth inhibition by FTI have been proposed. In cell culture and in animal models, FTIs have been shown to be potent inhibitors of cancer cell growth. We showed that manumycin, a farnesyl pyrophosphate competitive inhibitor, inhibits ovarian and mesothelioma cancer cell growth. To examine the molecular changes after exposure to manumycin, total proteins from treated and untreated 2774 cell line were extracted and separated by two dimensional gel electrophoresis (2-DE) and detected by colloidal coomassie blue staining. The 2-DE was found reliable for comparison of protein profiles before and after manumycin treatment. Two protein spots (spot 1 and 2) appeared after manumycin treatment. Generated peptide peaks were matched by database query (prowl.rockefeller.edu/cgi-bin/ProFound) to a heat shock protein (HSP) and a modified HSP, both with MW 70 KD. The expression status of HSP 70 was confirmed by Western blot and HSP 70 ELISA using an antibody against HSP 70. The expression of HSP70 increased with the length of exposure to, and the dose of manumycin as demonstrated by ELISA. Increase in HSP70 expression was also observed by Western blot after a 48-hour treatment with manumycin in ovarian cancer cell line OVCAR3 and three other cell cultures derived from samples of patients diagnosed with ovarian cancer and mesothelioma. Our study is the first report demonstrating an up-regulation of HSP 70 in ovarian cancer cell lines and mesothelioma cell cultures after treatment with the FTI, manumycin. This up-regulation of HSP 70 may be a cellular mechanism of cell self-protection against the apoptotic process and cell death, and it may enhance resistance and account for the altered sensitivity of certain cancers to FTIs agents. HSPs may therefore be an important molecular target for drug intervention strategies.
由于法尼基转移酶（FTase）介导的法尼基化是ras p21信号转导途径中的首个必需步骤，因此FTase一直是新型抗癌治疗方法的靶点。法尼基转移酶抑制剂（FTIs）是一类新型抗肿瘤药物，可阻断ras的致癌活性，最初被开发用于治疗ras突变的人类肿瘤。FTIs也能够抑制无ras突变的肿瘤，同时人们提出了FTIs抑制细胞生长的不同机制。在细胞培养和动物模型中，FTIs已被证明是癌细胞生长的有效抑制剂。我们发现，法尼基焦磷酸竞争性抑制剂番红菌素可抑制卵巢癌细胞和间皮瘤癌细胞的生长。为了检测暴露于番红菌素后的分子变化，我们提取了经处理和未经处理的2774细胞系的总蛋白，通过二维凝胶电泳（2-DE）进行分离，并用考马斯亮蓝胶体染色进行检测。结果发现，2-DE可可靠地比较番红菌素处理前后的蛋白质谱。番红菌素处理后出现了两个蛋白点（点1和点2）。通过数据库查询（prowl.rockefeller.edu/cgi-bin/ProFound），将生成的肽峰与一种热休克蛋白（HSP）和一种修饰的HSP进行匹配，二者的分子量均为70 KD。使用抗HSP 70抗体通过蛋白质免疫印迹法和HSP 70 ELISA法证实了HSP 70的表达状态。ELISA结果表明，HSP70的表达随着暴露于番红菌素的时间长度和剂量的增加而增加。在用番红菌素处理48小时后，在卵巢癌细胞系OVCAR3以及另外三种源自被诊断患有卵巢癌和间皮瘤患者样本的细胞培养物中，通过蛋白质免疫印迹法也观察到了HSP70表达的增加。我们的研究是首份证明用FTIs番红菌素处理后，卵巢癌细胞系和间皮瘤细胞培养物中HSP 70上调的报告。HSP 70的这种上调可能是细胞针对凋亡过程和细胞死亡的一种自我保护机制，它可能增强耐药性，并解释某些癌症对FTIs药物敏感性改变的原因。因此，热休克蛋白可能是药物干预策略的一个重要分子靶点。
