In vitro studies on the mechanisms of oxaliplatin resistance.
OBJECTIVE
We have previously reported that elevation of glutathione mediated by gamma-glutamyl transpeptidase is one mechanism of oxaliplatin resistance. This study explored other potential oxaliplatin resistance mechanisms with two aims: (1) to identify the differences between cisplatin and oxaliplatin in terms of drug accumulation, DNA-Pt adduct formation and repair, and (2) to determine whether defects in drug accumulation and enhanced repair of the DNA-Pt adduct contribute to oxaliplatin resistance.


METHODS
The human ovarian carcinoma cell line A2780, an oxaliplatin-resistant variant A2780/C25 and a cisplatin-resistant variant A2780/CP along with an inherently cisplatin-resistant HT-29 colon carcinoma cell line were used in the study. The methods consisted of sulforhodamine-B assays, atomic absorption spectrophotometry and real-time quantitative RT-PCR.


RESULTS
Significantly higher drug accumulation and DNA-Pt adduct formation were observed after exposure to cisplatin compared to after oxaliplatin in the parent A2780 cells and the oxaliplatin-resistant A2780/C25 cells. The DNA-Pt adduct formed after treatment with either drug was repaired with equal efficiency by all cell lines except A2780/CP, which repaired the DNA-cisplatin adduct more efficiently than the DNA-oxaliplatin adduct. Relative to the parent line, oxaliplatin-resistant A2780/C25 cells showed reduced Pt accumulation and DNA-Pt adduct levels following exposure to oxaliplatin, but only reduced accumulation after exposure to cisplatin. The cisplatin-resistant A2780/CP cells showed reduced accumulation and DNA-Pt adduct levels after exposure to cisplatin, but only reduced DNA-Pt adduct after exposure to oxaliplatin. In comparison to A2780 cells, the inherently cisplatin-resistant HT-29 cells showed lower accumulation and DNA-Pt adduct levels after exposure to cisplatin, but displayed no difference after exposure to oxaliplatin. An enhanced repair of the DNA-cisplatin adduct was observed only in A2780/CP cells relative to A2780 cells in an 8-h period. The steady-state levels of ERCC-1 mRNA, but not of XPA, were moderately elevated in the resistant cells. Exposure to either one of the drugs resulted in an induction of XPA in all the cell lines and of ERCC-1 in cisplatin-resistant cells. There was no relationship between the level of expression of the repair genes and the DNA-Pt adduct levels or repair.


CONCLUSIONS
Relative to cisplatin a lower intracellular concentration and fewer DNA-Pt adducts are sufficient for oxaliplatin to exert its cytotoxicity. Resistance to oxaliplatin is mediated by similar mechanisms of reduced drug accumulation and DNA-Pt adduct formation as resistance to cisplatin. There is no clear evidence that enhanced repair is a mechanism of oxaliplatin resistance in the cell line (A2780/C25) studied here. The findings are suggestive of yet unidentified differences between the two drugs with respect to cellular uptake and/or efflux and repair of DNA-Pt adducts.
Hector
S
S
Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Bolanowska-Higdon
W
W
Zdanowicz
J
J
Hitt
S
S
Pendyala
L
L
D000970:Antineoplastic Agents / Q000494:pharmacology*; D018736:DNA Adducts / Q000032:analysis; D004260:DNA Repair; D004268:DNA-Binding Proteins; D019008:Drug Resistance, Neoplasm; D004720:Endonucleases; D019073:HT29 Cells; D006801:Humans; D009944:Organoplatinum Compounds / Q000493:pharmacokinetics / Q000494:pharmacology*; D000077150:Oxaliplatin; D010984:Platinum / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
D000970:Antineoplastic Agents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009944:Organoplatinum Compounds; D011506:Proteins; D012333:RNA, Messenger; D000077150:Oxaliplatin; D010984:Platinum; C071447:ERCC1 protein, human; D004720:Endonucleases