Sugimura Masaki, Sagae Satoru, Ishioka Shin-Ichi, Nishioka Yoshihiro, Tsukada Kuniko, Kudo Ryuichi
Department of Obstetrics and Gynecology, Sapporo Medical University, School of Medicine, Sapporo, Japan.
Oncology. 2004;66(1):53-61. doi: 10.1159/000076335.
To understand the complicated network of paclitaxel (PTX)-induced apoptosis pathways and to elucidate mechanisms of drug resistance in ovarian cancer, we looked at PTX-induced apoptosis by using cDNA microarray. We also quantitated the changes in apoptosis-related proteins in the process of apoptosis.
An ovarian cancer cell line KF, and its PTX-resistant clone KFTX, were treated with PTX or carboplatin (CBDCA). After exposure to PTX or CBDCA, the induction of apoptosis was examined by internucleosomal DNA fragmentation. Changes in mRNA expression after 12 h of exposure to PTX were studied using cDNA microarray and RT-PCR. Changes in P53 and Bcl-2 levels were also measured over 24 h by ELISA.
With increased doses of PTX or CBDCA, an increase in apoptosis was noted in both cell lines. cDNA microarray revealed that PTX treatment upregulated expression of caspase 1, 2, 3, 4, 6, 9, 10, their activator apaf-1, and stress reaction-related genes, gadd34, gadd153 in KF, although most of them were unchanged or downregulated in KFTX. bag-1 and hsc70 were markedly upregulated in KFTX. p53 and bcl-2 were not upregulated in either cell line. Results from protein studies also supported the cDNA microarray data.
p53-independent mitochondrial pathways and stress-reaction-induced pathways play critical roles in PTX-induced apoptosis in ovarian cancer cells. Suppression of those pathways and upregulation of bag-1 and hsp-70 played an important role in acquiring resistance to PTX.
为了解紫杉醇（PTX）诱导的凋亡途径的复杂网络并阐明卵巢癌的耐药机制，我们使用cDNA微阵列观察PTX诱导的凋亡。我们还定量了凋亡过程中凋亡相关蛋白的变化。
用PTX或卡铂（CBDCA）处理卵巢癌细胞系KF及其PTX耐药克隆KFTX。暴露于PTX或CBDCA后，通过核小体间DNA片段化检测凋亡诱导情况。使用cDNA微阵列和逆转录聚合酶链反应（RT-PCR）研究暴露于PTX 12小时后mRNA表达的变化。还通过酶联免疫吸附测定（ELISA）在24小时内测量P53和Bcl-2水平的变化。
随着PTX或CBDCA剂量的增加，两个细胞系中的凋亡均增加。cDNA微阵列显示，PTX处理上调了KF中半胱天冬酶1、2、3、4、6、9、10、其激活剂凋亡蛋白酶激活因子-1（apaf-1）以及应激反应相关基因gadd34、gadd153的表达，尽管其中大多数在KFTX中未发生变化或下调。bag-1和热休克蛋白70（hsc70）在KFTX中显著上调。p53和bcl-2在两个细胞系中均未上调。蛋白质研究结果也支持cDNA微阵列数据。
不依赖p53的线粒体途径和应激反应诱导的途径在PTX诱导的卵巢癌细胞凋亡中起关键作用。这些途径的抑制以及bag-1和热休克蛋白70（hsp-70）的上调在获得对PTX的耐药性中起重要作用。
