Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

Abstract

The growth inhibitory effects of cisplatin and etoposide on neuroblastoma cell lines were investigated in several scheduled combinations. Results were analyzed using median effect and combination index analyses. In all schedules in which cisplatin was administered prior to etoposide a synergistic effect was observed. Conversely, an antagonistic effect was seen in all schedules where etoposide was administered before cisplatin. The design of the widely used OPEC protocol (oncovins, platinum agents, epipodophyllotoxins and cyclophosphamide) for management of the paediatric solid tumour neuroblastoma was based on clinical and in vitro evaluations of sequentially scheduled cisplatin and the epipodophyllotoxin, teniposide. These studies indicated that, for optimal anti-tumour effect, cisplatin should be administered prior to the epipodophyllotoxin (Hayes et al, 1981;Shafford et al, 1984;Pritchard et al, 1985).In several more recent clinical protocols for neuroblastoma, this apparently optimal order of drug administration has not been retained. The order of administration has been reversed in some regimens (Gordon et al, 1992;Tweddle et al, 2001) or the drugs are administered concurrently (reviewed in Pinkerton et al, 2000). If the OPEC rationale was correct, this altered scheduling pattern could result in sub-optimal response. Furthermore, in many of these schedules, cisplatin has been replaced by carboplatin. This will accentuate the change in order of administration because, despite the fact that carboplatin forms essentially the same final DNA adducts as cisplatin, the cytotoxic cross-linked structures develop more slowly than with cisplatin (Knox et al, 1986).The present study was aimed at determining, using contemporary methods of analysis, whether the currently used epipodophyllotoxin, etoposide, in combination with cisplatin, caused effects on neuroblastoma cell lines that were dependent upon the relative timings of the drug exposures. MATERIALS AND METHODS Cell culture and drug solutionsThe neuroblastoma cell lines SHSY5Y (Ciccarone et al, 1989) and NGP (Brodeur et al, 1977) were kindly provided by Drs P Lovat and D Tweddle (University of Newcastle upon Tyne). Cells were grown in RPMI 1640 (Dutch modification, supplemented with 10% v/v foetal bovine serum, and antibiotics (Gibco BRL)) at 378C/5% CO 2 . Frequent tests for mycoplasma infection were always negative. Etoposide was dissolved in methanol and stored at 7208C. Cisplatin was freshly dissolved in DMSO just prior to each experiment and immediately diluted into medium. During drug exposures, the concentrations of methanol and DMSO were kept below 1% and 0.001% respectively in treated and control wells. Sulphorhodamine B (SRB) assayFor each experiment, cells were inoculated into three 96-well tissue culture plates and incubated for 24 h prior to starting drug exposures (time zero). Plates were exposed to graded concentrations of either: the first drug in the schedule only followed by the diluent for the second drug; the diluent for the first drug in the s...

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