Fishel Melissa L, He Ying, Smith Martin L, Kelley Mark R
Department of Pediatrics (Section of Hematology/Oncology), Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana 46202, USA.
Clin Cancer Res. 2007 Jan 1;13(1):260-7. doi: 10.1158/1078-0432.CCR-06-1920.
To improve the treatment of women with ovarian cancer, we are investigating the modulation of a prominent DNA-damaging agent, temozolomide, by manipulating the DNA base excision repair (BER) pathway via BER inhibitor, methoxyamine, and overexpression of N-methylpurine DNA glycosylase (MPG).
Enhancement of temozolomide via methoxyamine and MPG overexpression was analyzed using in vitro assays, including 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay, apoptosis via Annexin staining, and Western blotting for H2AX phosphorylation to quantitate DNA damage.
Our data show that we can effectively modulate the activity of the chemotherapeutic agent, temozolomide, via modulator methoxyamine, in three ovarian cancer cell lines, SKOV-3x, Ovcar-3, and IGROV-1. This enhancement of temozolomide-induced cytotoxicity is not dependent on p53 status as we transfected an ovarian cancer cell line with a dominant-negative p53-expressing plasmid (IGROV-1mp53) and obtained similar results. Our results show that MPG-overexpressing IGROV-1 and IGROV-1mp53 cells are significantly more sensitive to the clinical chemotherapeutic temozolomide in combination with methoxyamine as assayed by cytotoxicity, apoptosis, and levels of DNA damage than either agent alone.
These studies show that although clinical trials in ovarian cancer to determine temozolomide single-agent efficacy are in development, through manipulation of the BER pathway, an increase in response to temozolomide is achieved. The combination of temozolomide plus methoxyamine has potential for second-line therapy for patients who have failed standard platinum plus paclitaxel chemotherapy.
为了改善卵巢癌女性患者的治疗效果，我们正在研究通过BER抑制剂甲氧基胺和N - 甲基嘌呤DNA糖基化酶（MPG）的过表达来调控一种主要的DNA损伤剂替莫唑胺。
使用体外试验分析通过甲氧基胺和MPG过表达增强替莫唑胺的效果，包括3 -（4 - 5 - 二甲基噻唑 - 2 - 基）- 5 -（3 - 羧基甲氧基苯基）- 2 -（4 - 磺基苯基）- 2H - 四氮唑盐（MTS）试验、通过膜联蛋白染色检测细胞凋亡以及通过蛋白质免疫印迹法检测H2AX磷酸化以定量DNA损伤。
我们的数据表明，在三种卵巢癌细胞系SKOV - 3x、Ovcar - 3和IGROV - 1中，我们可以通过调节剂甲氧基胺有效地调控化疗药物替莫唑胺的活性。替莫唑胺诱导的细胞毒性增强不依赖于p53状态，因为我们用表达显性负性p53的质粒转染了一种卵巢癌细胞系（IGROV - 1mp53）并获得了相似的结果。我们的结果表明，通过细胞毒性、细胞凋亡和DNA损伤水平检测，与单独使用任何一种药物相比，过表达MPG的IGROV - 1和IGROV - 1mp53细胞对临床化疗药物替莫唑胺联合甲氧基胺的敏感性显著更高。
这些研究表明，尽管确定替莫唑胺单药疗效的卵巢癌临床试验正在进行中，但通过操纵BER途径，可以提高对替莫唑胺的反应。替莫唑胺加甲氧基胺的联合用药对标准铂类加紫杉醇化疗失败的患者有二线治疗的潜力。
