Manipulation of base excision repair to sensitize ovarian cancer cells to alkylating agent temozolomide
Manipulation of base excision repair to sensitize ovarian cancer cells to alkylating agent temozolomide
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DOI：
10.1158/1078-0432.ccr-06-1920
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发表时间：
2007-01-01
影响因子：
11.5
通讯作者：
Kelley, Mark R.
中科院分区：
文献类型：
--
作者：
Fishel, Melissa L.;He, Ying;Kelley, Mark R.
目的：为了改善女性卵巢癌的治疗，我们正在研究一种著名的DNA损伤剂替莫唑胺，通过BER抑制剂甲氧胺和n -甲基嘌呤DNA糖基化酶（MPG）的过表达来调控DNA碱基切除修复（BER）途径。实验设计：通过体外实验分析甲氧胺增强替莫唑胺和MPG过表达，包括3-（4- 5-二甲基噻唑-2-基）-5-（3-羧基甲氧基苯基）-2-（4-磺苯基）- 2h -四氮唑盐（MTS）实验，Annexin染色细胞凋亡，以及H2AX磷酸化的Western blotting来定量DNA损伤。结果：我们的数据表明，我们可以通过调节剂甲氧基胺有效地调节化疗药物替莫唑胺在三种卵巢癌细胞系SKOV-3x、Ovcar-3和IGROV-1中的活性。替莫唑胺诱导的细胞毒性增强并不依赖于p53的状态，因为我们用p53表达质粒（IGROV-1mp53）转染了卵巢癌细胞系，并获得了类似的结果。我们的研究结果表明，mpg过表达的IGROV-1和IGROV-1mp53细胞对替莫唑胺联合甲氧基胺的临床化疗药物明显更敏感，通过细胞毒性、细胞凋亡和DNA损伤水平来检测。结论：这些研究表明，虽然在卵巢癌中确定替莫唑胺单药疗效的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。替莫唑胺联合甲氧基胺对于标准铂加紫杉醇化疗失败的患者具有二线治疗的潜力。
Purpose: 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).Experimental Design: 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.Results: 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.Conclusions: 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.