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Overcoming S-phase checkpoint-mediated resistance: sequence-dependent synergy of gemcitabine and 7-ethyl-10-hydroxycamptothecin (SN-38) in human carcinoma cell lines.

ABSTRACT: Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine (2',2'-difluoro 2'-deoxycytidine) and 7-ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of irinotecan), two S-phaseselective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony-forming assays revealed that simultaneous treatment of Ovcar-5 ovarian cancer cells or BxPC-3 pancreatic cancer cells with gemcitabine and SN-38 resulted in antagonistic effects. In contrast, sequential treatment with these two agents in either order resulted in synergistic anti-proliferative effects, although the mechanism of synergy varied with the sequence. In particular, SN-38 arrested cells in S phase, enhanced the accumulation of gemcitabine metabolites, and diminished checkpoint kinase 1, thereby sensitizing cells in the SN-38 --> gemcitabine sequence. Gemcitabine treatment followed by removal allowed prolonged progression through S phase, contributing to synergy of the gemcitabine --> SN-38 sequence. These results collectively suggest that S-phase-selective agents might exhibit more cytotoxicity when administered sequentially rather than simultaneously.

SUBMITTER: Galvez-Peralta M

PROVIDER: S-EPMC2574763 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Overcoming S-phase checkpoint-mediated resistance: sequence-dependent synergy of gemcitabine and 7-ethyl-10-hydroxycamptothecin (SN-38) in human carcinoma cell lines.

Gálvez-Peralta Marina M Dai Nga T NT Loegering David A DA Flatten Karen S KS Safgren Stephanie L SL Wagner Jill M JM Ames Matthew M MM Karnitz Larry M LM Kaufmann Scott H SH

Molecular pharmacology 20080528 3

Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine (2',2'-difluoro 2'-deoxycytidine) and 7-ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of irinotecan), two S-phaseselective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony-forming assays r ...[more]

PMID: 18509065

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Project description:BackgroundDNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.MethodsThree SN-38 resistant (20-67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.ResultsTwo of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.ConclusionsThis study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.

Dataset Information

Overcoming S-phase checkpoint-mediated resistance: sequence-dependent synergy of gemcitabine and 7-ethyl-10-hydroxycamptothecin (SN-38) in human carcinoma cell lines.

Publications

Overcoming S-phase checkpoint-mediated resistance: sequence-dependent synergy of gemcitabine and 7-ethyl-10-hydroxycamptothecin (SN-38) in human carcinoma cell lines.

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