Manipulation of base excision repair to sensitize ovarian cancer cells to alkylating agent temozolomide.
OBJECTIVE
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).


METHODS
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.
Fishel
Melissa L
ML
Department of Pediatrics (Section of Hematology/Oncology), Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana 46202, USA.
He
Ying
Y
Smith
Martin L
ML
Kelley
Mark R
MR
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism*; D000077204:Temozolomide
D018906:Antineoplastic Agents, Alkylating; D006898:Hydroxylamines; D003606:Dacarbazine; C005214:methoxyamine; D045647:DNA Glycosylases; D000077204:Temozolomide