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Novel cellular determinants for reversal of multidrug resistance in cells expressing P170-glycoprotein
1Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Abstract:
The newly synthesized calcium channel blocker, Ro44-5912, significantly potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Dox-resistant human ovarian cell line, A2780/DX5, overexpressing P170-glycoprotein (Pgp). Induction of DNA single- and double-strand breaks (ssbs and dsbs) was measured using alkaline elution and constant-field gel electrophoresis (CFGE) assays. The results indicate that potentiation of the cytotoxicity of Dox by Ro44-5912 was accompanied by significant increases in both, Dox-induced DNA ssbs and dsbs in the resistant cells. Pulsed-field gel electrophoresis (PFGE) analysis showed that Dox induced DNA fragments in the 50-800 kilobase (kb) and 0.8-5.7 megabase (Mb) ranges. The majority of the newly synthesized DNA fragments were in the 50-800 kb range. Ro44-5912 treatment resulted in significant potentiation of DNA fragmentation in the 50-800 kb range with a minor increase in 0.8-5.7 Mb DNA fragments, suggesting that the modulator functions by potentiating nascent DNA fragmentation in the resistant cells. Exposure to Dox with Ro44-5912 was associated with a prolonged blockage of cells in the S-phase. In contrast, exposure to Dox alone resulted in temporary blockage of cells in G2/M phase (approximately 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure. Incorporation of BrdUrd by flow cytometric analysis was inhibited by Dox in the presence of Ro44-5912, showing that there is a block of DNA replication. An increased damage in newly synthesized DNA could concur with a blocked DNA replication. Moreover, slowing progression through the S-phase in cells exposed to Dox in combination with Ro44-5912 is accompanied by increased sensitivity of Dox poisons, indicating a correlation of specific S-phase perturbation with the reversal of Dox resistance by Ro44-5912 in cells expressing Pgp. The results suggest that drug-induced augmentation of nascent DNA fragmentation and specific cell-cycle perturbation are potentially important molecular determinants for reversal of multidrug resistance in addition to restoration of intracellular drug retention.
Insights
A new drug, Ro44-5912, enhances doxorubicin
Area of Science:
- Molecular Pharmacology
- Cancer Cell Biology
- Drug Resistance Mechanisms
Background:
- Multidrug resistance (MDR) in cancer, particularly in ovarian cancer, limits the efficacy of chemotherapy.
- P-glycoprotein (Pgp) overexpression is a common mechanism of MDR, leading to reduced intracellular drug accumulation.
- Doxorubicin (Dox) resistance in A2780/DX5 cells is mediated by Pgp, hindering effective treatment.
Purpose of the Study:
- To investigate the potential of Ro44-5912, a novel calcium channel blocker, to overcome doxorubicin resistance.
- To elucidate the molecular mechanisms by which Ro44-5912 modulates doxorubicin cytotoxicity and DNA damage in resistant cancer cells.
Main Methods:
- Utilized alkaline elution and constant-field gel electrophoresis (CFGE) to measure DNA single- and double-strand breaks (ssbs and dsbs).
- Employed pulsed-field gel electrophoresis (PFGE) to analyze DNA fragmentation patterns.
- Assessed cell cycle progression and DNA replication using flow cytometry and BrdUrd incorporation.
Main Results:
- Ro44-5912 potentiated doxorubicin-induced cytotoxicity in doxorubicin-resistant A2780/DX5 cells at non-cytotoxic concentrations.
- Co-treatment with Ro44-5912 significantly increased doxorubicin-induced DNA ssbs and dsbs, particularly in newly synthesized DNA fragments.
- Ro44-5912 prolonged S-phase arrest and inhibited DNA replication, correlating with enhanced doxorubicin sensitivity and reversal of Pgp-mediated resistance.
Conclusions:
- Ro44-5912 effectively reverses doxorubicin resistance in Pgp-overexpressing ovarian cancer cells.
- The mechanism involves potentiation of nascent DNA fragmentation and specific cell-cycle perturbation, leading to increased sensitivity to doxorubicin.
- Ro44-5912 represents a promising therapeutic strategy for overcoming multidrug resistance in cancer treatment.