Abstract
BackgroundWe observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug) and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies.MethodsThree immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of < 0.05 considered significant.ResultsThe multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P < 0.05) and lowered dCK protein (24% to 56%, P < 0.05) without affecting CDA protein. However, paclitaxel increased both dCK (10% to 50%) and CDA (75% to 153%) activity (P < 0.05). Paclitaxel caused substantial declines in the accumulation of the deaminated and phosphorylated metabolites in H520 cells (P < 0.05); the metabolites were not measurable in the remaining two cell lines. The ratio of dCK to CDA mRNA levels corresponded to the combination index (CI) estimated for sequential paclitaxel-gemcitabine.ConclusionIn summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the selection of an appropriate anticancer drug regimen.
Highlights
We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells
The odd lanes were treated with vehicle-control and the even lanes were treated with paclitaxel at the observed IC50 value for 24 hours. (b) The mean (± standard deviation) relative protein levels of deoxycytidine kinase (dCK) to β-actin after exposure to paclitaxel at the observed IC-50 value for 24 hours compared to vehicle-control from three independent experiments. (c) A representative Western immunoblot of crude cellular extracts from H460, H520, and H838
The odd lanes were treated with vehicle-control and the even lanes were treated with paclitaxel at the observed IC50 values for 24 hours. (d) The mean (± standard deviation) relative protein levels of cytidine deaminase (CDA) to β-actin treated with paclitaxel at the observed IC-50 value for 24 hours compared to relative protein levels of CDA to β-actin treated with vehicle-control from three independent experiments
Summary
We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. We classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug) and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. Gemcitabine is a fluorinated pyrimidine analog that causes masked chain termination and inhibits ribonucleotide reductase (RNR) [5]. The diphosphate metabolite (dFdCDP) potentiates the incorporation of the dFdCTP into DNA by inhibiting RNR This reduces the intracellular accumulation of deoxycytidine triphosphate (dCTP) and promotes the incorporation of dFdCTP into DNA. The accumulation of the triphosphate and alterations in either dCK or RNR are associated with either sensitivity or resistance to gemcitabine in various cell lines and animal models [6,7,8,9,10]
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