Lee Keun-Ho, Yim Eun-Kyoung, Kim Chan-Joo, Namkoong Sung-Eun, Um Soo-Jong, Park Jong-Sup
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Kangnam St. Mary's Hospital, Seoul 137-040, Korea.
Gynecol Oncol. 2005 Jul;98(1):45-53. doi: 10.1016/j.ygyno.2005.04.010.
Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used in the treatment of ovarian, lung and breast cancer. At present, there is little information regarding the anti-cancer mechanism of paclitaxel against cervical carcinoma cells. We thus tried to show the anti-cancer effect of paclitaxel on cervical carcinoma cell line carrying HPV by using a proteomic analysis and to investigate the mechanism of actions.
We treated paclitaxel to cervical carcinoma cells and then carried out MTT assay to observe the anti-proliferate activity. Using proteomics analysis including two-dimensional (2-DE) gel electrophoresis and MALDI-TOF-MS, we tried to find the anti-proliferate activity-related proteins. Among them, paclitaxel treatment suppressed the expression of the mitotic checkpoint protein BUB3. Functional proteomic analysis by small interfering RNA (siRNA) targeting was tried to illuminate a role of mitotic checkpoint protein BUB3 in cell cycle progression.
The cytotoxicity effects of paclitaxel were determined in HPV-16 positive CaSki, HPV-18 positive HeLa and HPV-negative C33A cervical carcinoma cell lines. Using efficient proteomics methods including 2-DE/MALDI-TOF-MS, we identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells. Paclitaxel treatment elevated mainly apoptosis-related, immune response-related and cell cycle check point-related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Paclitaxel showed anti-proliferate activity through the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with a TRAIL-dependent fashion as well as the mitochondrial-mediated pathway involving down-regulation of bcl-2 by cytochrome c release. Furthermore, we found siRNA-induced BUB3 knock down on cell cycle progression blocked by cell cycle arrest after paclitaxel treatment.
The proteome profiling technique provided a broad-base and effective approach for the identification of protein changes induced by paclitaxel and showed anti-proliferate activity through the membrane death receptor-mediated apoptotic pathway, the mitochondrial-mediated pathway. This study shows the power of proteomic profiling with functional analysis using RNAi technology for the discovery of novel molecular targets and a better understanding of the actions of paclitaxel at the molecular level in cervical carcinoma cells.
紫杉醇（泰素）是从太平洋紫杉树皮中分离出的一种强效天然药物，广泛用于治疗卵巢癌、肺癌和乳腺癌。目前，关于紫杉醇对宫颈癌细胞的抗癌机制的信息很少。因此，我们试图通过蛋白质组学分析来显示紫杉醇对携带人乳头瘤病毒（HPV）的宫颈癌细胞系的抗癌作用，并研究其作用机制。
我们用紫杉醇处理宫颈癌细胞，然后进行MTT试验以观察其抗增殖活性。通过包括二维（2-DE）凝胶电泳和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）在内的蛋白质组学分析，我们试图找到与抗增殖活性相关的蛋白质。其中，紫杉醇处理抑制了有丝分裂检查点蛋白BUB3的表达。通过靶向小干扰RNA（siRNA）进行功能蛋白质组学分析，试图阐明有丝分裂检查点蛋白BUB3在细胞周期进程中的作用。
在HPV-16阳性的CaSki、HPV-18阳性的HeLa和HPV阴性的C33A宫颈癌细胞系中测定了紫杉醇的细胞毒性作用。使用包括2-DE/MALDI-TOF-MS在内的高效蛋白质组学方法，我们在HeLa细胞中鉴定了几种对紫杉醇处理有反应的细胞蛋白。紫杉醇处理主要上调了与凋亡相关、免疫反应相关和细胞周期检查点相关的蛋白。另一方面，紫杉醇处理减少了生长因子/癌基因相关蛋白和转录调控相关蛋白。紫杉醇通过膜死亡受体（DR）介导的凋亡途径显示出抗增殖活性，该途径以TRAIL依赖的方式激活半胱天冬酶-8，以及线粒体介导的途径，该途径通过细胞色素c释放下调bcl-2。此外，我们发现siRNA诱导的BUB3敲低会导致紫杉醇处理后细胞周期停滞，从而阻断细胞周期进程。
蛋白质组分析技术为鉴定紫杉醇诱导的蛋白质变化提供了一种广泛且有效的方法，并通过膜死亡受体介导的凋亡途径、线粒体介导的途径显示出抗增殖活性。这项研究展示了蛋白质组分析与使用RNAi技术进行功能分析相结合的强大力量，可用于发现新的分子靶点，并更好地理解紫杉醇在宫颈癌细胞分子水平上的作用。
