The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model.
OBJECTIVE
Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients. The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum.


METHODS
The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days. Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate. The LD(50) for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan. Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD(50) was reassessed for each cell population by a second MTT assay. Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and beta-Actin.


RESULTS
The LD(50) to cisplatin was unchanged in A2780-CR cells treated by topotecan. Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay (P < 0.01). The second MTT assay demonstrated that the LD(50) for cisplatin in every cell line decreased significantly after a 4-week drug-free interval (P < 0.01). There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure. The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 (P < 0.01).


CONCLUSIONS
The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance. This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment. This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression. While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.
Horowitz
Neil S
NS
Department of Obstetrics and Gynecology, Washington University School of Medicine, St Louis, MO 63110, USA.
Hua
Jun
J
Gibb
Randall K
RK
Mutch
David G
DG
Herzog
Thomas J
TJ
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000008:administration & dosage; D004334:Drug Administration Schedule; D004347:Drug Interactions; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005260:Female; D019450:Genes, MDR / Q000235:genetics; D006801:Humans; D009364:Neoplasm Recurrence, Local / Q000188:drug therapy*; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D017239:Paclitaxel / Q000008:administration & dosage; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019772:Topotecan / Q000008:administration & dosage / Q000494:pharmacology*
D012333:RNA, Messenger; D019772:Topotecan; D017239:Paclitaxel; D002945:Cisplatin