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Manipulation of Base Excision Repair to Sensitize Ovarian Cancer Cells to Alkylating Agent Temozolomide

Authors' Affiliations: ¹ Department of Pediatrics (Section of Hematology/Oncology), Herman B. Wells Center for Pediatric Research and Departments of ² Microbiology and Immunology, ³ Pharmacology and Toxicology, and ⁴ Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana

Requests for reprints: Mark R. Kelley, Department of Pediatrics, Herman B. Wells Center for Pediatric Research, 1044 West Walnut, R4-302C, Indianapolis, IN 46202. Phone: 317-274-2755; Fax: 317-278-9298; E-mail: mkelley{at}iupui.edu.

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.

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