Novel cellular determinants for reversal of multidrug resistance in cells expressing P170-glycoprotein.
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.
Yin
M B
MB
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Guo
B
B
Voigt
W
W
Vanhoefer
U
U
Gibbs
J F
JF
Skenderis
B S
BS
Frank
C
C
Wrzosek
C
C
Rustum
Y M
YM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004249:DNA Damage; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000187:drug effects*; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000502:physiology*; D019008:Drug Resistance, Neoplasm; D005260:Female; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D010051:Ovarian Neoplasms; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000031:analogs & derivatives / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D002121:Calcium Channel Blockers; D004273:DNA, Neoplasm; C095183:Ro 44-5912; D004317:Doxorubicin; D014700:Verapamil