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NGP cells were slightly more sensitive to cisplatin than SHSY5Y cells ( Table 1). The responses of SHSY5Y and NGP cells to cisplatin, were independent of the time interval between seeding and drug exposure ( Table 1). Sensitivity of NGP cells to etoposide was not dependent on the time interval between seeding and drug exposure and was approximately 5–10-fold greater than the SHSY5Y cells ( Table 1). Results are shown for the mutually exclusive assumption of modes of activity of the drugs, however, applying the alternative assumption showed the same pattern of results. CI=1 indicates an additive effect 1, antagonism. Determination of synergy or antagonism was based on the multiple drug effect equation of Chou and Talalay (1977, 1983) and was quantified by the combination index (CI).
#Calcusyn combination index software#
This software calculates the median effect dose, Dm (analogous to the IC 50), of the drug combinations using the median effect equation. Median effect and combination index analysisĭrug interactions were analyzed using CalcuSyn ( Chou and Hayball, 1996). Procedures for fixing, staining and reading (OD570) were carried out as described by Skehan et al (1990). After the final wash plates were returned to the incubator for 5 days.
#Calcusyn combination index free#
The plates were washed twice in drug free medium in between each drug/diluent exposure. 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 schedule followed by the second drug in the schedule or both drugs in the schedule according to the schedule design. Sulphorhodamine B (SRB) assayįor each experiment, cells were inoculated into three 96-well tissue culture plates and incubated for 24 h prior to starting drug exposures (time zero). During drug exposures, the concentrations of methanol and DMSO were kept below 1% and 0.001% respectively in treated and control wells. Cisplatin was freshly dissolved in DMSO just prior to each experiment and immediately diluted into medium. Etoposide was dissolved in methanol and stored at −20☌. Frequent tests for mycoplasma infection were always negative. Cells were grown in RPMI 1640 (Dutch modification, supplemented with 10% v/v foetal bovine serum, and antibiotics (Gibco BRL)) at 37☌/5% CO 2. The 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).
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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. 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). Furthermore, in many of these schedules, cisplatin has been replaced by carboplatin. If the OPEC rationale was correct, this altered scheduling pattern could result in sub-optimal response.
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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). In several more recent clinical protocols for neuroblastoma, this apparently optimal order of drug administration has not been retained. 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). 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.