Mechanisms of synergism between cisplatin and gemcitabine in ovarian and non-small-cell lung cancer cell lines

Abstract

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 IC 25 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 IC 25 , the average CIs calculated at the IC 50 , IC 75 IC 90 and IC 95 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 IC 25 , the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the Cls after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all Cls 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. © 1999 Cancer Research Campaign

Human Cytosolic 5′-Nucleotidase I

“…Hypoxic tumors have poorer clinical prognosis, are often resistant to both radiation and chemotherapy (33), and are more likely to metastasize (34). The nucleoside analogs 5-FU and dFdC are used clinically in the treatment of solid tumors (35,36), and we have shown that elevated expression of cN-I increases the IC 50 for 5-FU 5.2-fold and the IC 50 for dFdC 21.6-fold in the HEK cells (Table V). Although the effect of hypoxia on drug resistance in the context of cN-I activity remains to be investigated, this study provides support for the hypothesis that increased ADP concentrations in ischemic tumors may facilitate dephosphorylation of several nucleoside-based drugs and lower their pharmacological efficacy.…”

Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines

“…After harvesting, nucleotides were extracted and quantitated with HPLC as previously described . Incorporation studies were performed using 96-well filter plates essentially as described previously (Van der Wilt et al, 1993;Van Moorsel et al, 1999a). A total of 1.0 Â 10 5 cells per well were plated in a volume of 100 ml, whereafter 100 ml of [ 3 H]gemcitabine containing medium (4 Ci mmol À1 ) was added resulting in final concentrations of 0.1 or 1.0 mM.…”

Gemcitabine, cisplatin and methylprednisolone (GEM-P) is an effective salvage regimen in patients with relapsed and refractory lymphoma

“…Phase I studies evaluating this combination in relapsed and refractory HL and NHL have also reported higher RRs (Aviles et al, 2004;Emmanouilides et al, 2004) compared to either agent given as monotherapy (Fossa et al, 1999;Santoro et al, 2000;Savage et al, 2000;Zinzani et al, 2000). The mechanisms underlying this synergy may be due to increased incorporation of gemcitabine into DNA and RNA, and increased cisplatin -DNA adduct formation by inhibition of exonuclease and DNA repair (van Moorsel et al, 1999). Given their synergism, absence of significant overlapping toxicities and non-cross-resistance with other regimens, gemcitabine in combination with cisplatin, presents an attractive treatment option.…”