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Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo
Published on: February 6, 2015
Comparisons between sensitive and resistant human tumor cell lines regarding effects of polyamine depletion on
1Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Abstract:
We have reported that 2-difluoromethylornithine (DFMO)-induced polyamine (PA) depletion sensitized five chloroethylnitrosourea (CENU)-resistant, O6-alkylguanine repair-proficient (Mer+) human tumor cell lines to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), but failed to alter BCNU efficacy in a single CENU-sensitive, repair-deficient (Mer-) line. Further, alkaline elution assays of DNA interstrand cross-links (ISC) found no BCNU-induced ISC in either PA-depleted or control Mer+ cells, suggesting that targets other than ISC may be involved in the DFMO/BCNU drug interaction. To verify that DFMO-induced enhancement of BCNU action segregates with Mer phenotype, we tested three additional Mer- lines for effects of DFMO pretreatment on BCNU efficacy. We found no potentiation of BCNU by PA depletion in any of our human Mer- lines. We also used streptozotocin (STZ) to deplete the repair capacity of Mer+ cell lines, thus converting their BCNU sensitivity to near that of Mer- cells. Combined pretreatment with DFMO then STZ did enhance BCNU cell kill relative to STZ pretreatment alone. Exogenous putrescine restored BCNU sensitivity of (DFMO plus STZ)-pretreated cells to that of cells pretreated with STZ alone. Measurements of O6-alkylguanine DNA alkyltransferase activity verified that in at least one of the Mer+ lines (HT-29), STZ did deplete repair capacity to below detectable limits. These results suggest that in HT-29 cells, STZ and DFMO probably act via differing mechanisms to potentiate BCNU. Our observations also imply that targets for CENUs may differ between Mer+ and Mer- cells, with importance of ISC possibly limited to Mer- cells. Our data further suggest that PA depletion may potentiate CENUs only at targets critical in Mer+ cells. We also noted that 48-h treatments with DFMO markedly reduced clonogenicity of Mer- cells. Exogenous putrescine restored Mer- cell survival after DFMO to near that of controls. In contrast, Mer+ cells showed little, if any, effect of DFMO treatment on plating efficiency. These results suggest that PA depletion may be cytocidal to some Mer- cells.
Insights
2-difluoromethylornithine (DFMO) depletes polyamines, sensitizing cancer cells to BCNU, but only in repair-proficient (Mer+) cells. This suggests differing drug targets between Mer+ and Mer- cells and that DFMO may be toxic to Mer- cells.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Chloroethylnitrosoureas (CENUs) like BCNU are DNA cross-linking agents used in cancer therapy.
- Tumor cell resistance to CENUs can arise from proficient DNA repair mechanisms, particularly O6-alkylguanine repair (Mer+).
- Polyamines (PAs) play roles in cell growth and DNA repair, and their depletion via 2-difluoromethylornithine (DFMO) is being investigated as a chemosensitization strategy.
Purpose of the Study:
- To investigate the role of the O6-alkylguanine repair (Mer) phenotype in mediating the chemosensitizing effects of polyamine (PA) depletion by DFMO on BCNU efficacy.
- To explore potential differences in BCNU targets between Mer+ and Mer- cells.
- To assess the impact of PA depletion on the cytotoxicity of DFMO itself in different cell lines.
Main Methods:
- Utilized human tumor cell lines with varying O6-alkylguanine repair proficiency (Mer+ and Mer-).
- Pretreated cells with DFMO to induce polyamine depletion and assessed BCNU efficacy.
- Employed streptozotocin (STZ) to reduce repair capacity in Mer+ cells.
- Measured O6-alkylguanine DNA alkyltransferase activity.
- Assessed DNA interstrand cross-links (ISC) using alkaline elution assays.
- Evaluated cell viability and clonogenicity after DFMO treatment, with and without exogenous putrescine.
Main Results:
- DFMO-induced PA depletion sensitized Mer+ cells to BCNU but not Mer- cells, indicating a dependence on the Mer phenotype.
- No BCNU-induced DNA interstrand cross-links (ISC) were detected in PA-depleted Mer+ cells, suggesting non-ISC targets for DFMO/BCNU interaction in these cells.
- Combined DFMO and STZ pretreatment enhanced BCNU-induced cell kill in Mer+ cells more than STZ alone, and this effect was reversible with putrescine.
- STZ effectively reduced repair capacity in Mer+ cells (HT-29).
- DFMO treatment alone reduced clonogenicity in Mer- cells but had minimal effect on Mer+ cells, with putrescine restoring survival in Mer- cells.
Conclusions:
- The chemosensitizing effect of PA depletion by DFMO against BCNU is dependent on the Mer+ phenotype, suggesting distinct therapeutic targets in Mer+ versus Mer- cells.
- BCNU's mechanism may involve ISC in Mer- cells, while other targets are implicated in Mer+ cells.
- DFMO itself exhibits cytocidal effects on some Mer- cells, independent of BCNU potentiation.
- PA depletion may potentiate CENUs primarily through targets critical in Mer+ cells.
