Mechanisms of synergism between cisplatin and gemcitabine in ovarian and non-small-cell lung cancer cell lines.
2',2'-Difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum (cisplatin, CDDP) are active agents against ovarian cancer and non-small-cell lung cancer (NSCLC). CDDP acts by formation of platinum (Pt)-DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index (CI; CI < 1 indicates synergism). With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the CIs after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all CIs were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt-DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines (1.6-, 1.4-, 2.9- and 1.6-fold respectively). This increase in Pt-DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt-DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt-DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt-DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA.
van Moorsel
C J
CJ
Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Pinedo
H M
HM
Veerman
G
G
Bergman
A M
AM
Kuiper
C M
CM
Vermorken
J B
JB
van der Vijgh
W J
WJ
Peters
G J
GJ
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D018827:Carcinoma, Lewis Lung; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D002455:Cell Division / Q000187:drug effects; D002945:Cisplatin / Q000008:administration & dosage* / Q000378:metabolism; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D005470:Fluorometry; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy*; D051379:Mice; D010051:Ovarian Neoplasms / Q000188:drug therapy*; D010984:Platinum / Q000378:metabolism; D012313:RNA / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D000093542:Gemcitabine
D018736:DNA Adducts; D003841:Deoxycytidine; D010984:Platinum; D012313:RNA; D004247:DNA; D002945:Cisplatin; D000093542:Gemcitabine