Inhibition of Pgp activity and cell cycle-dependent chemosensitivity to doxorubicin in the multidrug-resistant LoVo human colon cancer cell line.
To determine whether the cell cycle affects multidrug resistance (MDR) and its reversal, doxorubicin (DOX) cytotoxicity and the effect of inhibition of P-glycoprotein (Pgp) activity by verapamil (VER) were investigated in MDR LoVo cell lines (LoVo-R) in different phases of the cell cycle. Synchronised cells were obtained by exposing cells for 24 h to non-toxic concentrations (40 nmol/l) of methotrexate (MTX), which induced a reversible blockade in the S phase. DOX cytotoxicity was higher if cells were exposed to DOX shortly after the pretreatment with MTX, when most cells were in the S phase of the cell cycle. At that time, the DOX concentration inhibiting cell growth by 50% (IC10) was decreased by approximately 4-fold compared to non-synchronised cycling cells. DOX cytotoxicity remained high during the transition from the S to the G2M phase, but was reduced when the cells had shifted to the G2M phase. Inhibition of Pgp activity by VER (6 mumol/l) enhanced DOX uptake and resulted in an intracellular nuclear compartmentalisation of DOX in LoVo-R cells. These effects were not significantly different (P = NS) in the different phases of the cell cycle. However, similar increases in intracellular DOX uptake due to the inhibitory effect of VER on Pgp greatly potentiated DOX cytotoxicity in LoVo-R cells synchronised in the S or G2M phase compared with non-synchronised cycling cells. The ratio between DOX IC50 in the absence and presence of VER in LoVo-R cells synchronised in the S phase and in cycling cells was 11.1 and 4.1, respectively (P < 0.01). This greater potentiation could be explained by the increased chemosensitivity of the S- and G2M-phase cells to intracellular DOX concentration compared with the non-synchronised cells. Finally, the combination of synchronisation by MTX and of inhibition of Pgp activity by VER produced a considerable reduction in DOX IC50 (approximately 50-fold) in LoVo-R cells compared with the cells not treated with MTX and VER. In conclusion, this study demonstrates that, in LoVo-R cells, the effect of Pgp inhibition on DOX cytotoxicity is dependent on cell cycle phase. DOX cytotoxicity is maximal when inhibition of Pgp activity occurs during the S/G2M phases.
Toffoli
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Department of Experimental Oncology 1, Centro di Riferimento Oncologico, Aviano, Italy.
Corona
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G
Gigante
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Boiocchi
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002453:Cell Cycle / Q000502:physiology; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D016195:G2 Phase / Q000502:physiology; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016196:S Phase / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D002121:Calcium Channel Blockers; D004317:Doxorubicin; D014700:Verapamil; D008727:Methotrexate