Synergistic Cytotoxicity and Pharmacogenetics of Gemcitabine and Pemetrexed Combination in Pancreatic Cancer Cell Lines

Abstract

Purpose: Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA synthesis and is an effective agent in the treatment of pancreas cancer. The present study investigates whether the multitargeted antifolate pemetrexed would be synergistic with gemcitabine against MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cell lines.Experimental Design: Cells were treated with gemcitabine and pemetrexed, and the type of drug interaction was assessed using the combination index. Cytotoxicity of gemcitabine was examined with inhibitors of (a) deoxycytidine kinase (dCK), which activates gemcitabine by phosphorylation, and (b) 5-nucleotidase (drug dephosphorylation) and cytidine deaminase (drug deamination), the main inactivating enzymes. The effects of gemcitabine and pemetrexed on cell cycle were analyzed by flow cytometry, and apoptosis was examined by fluorescence microscopy. Finally, quantitative, real-time PCR was used to study the pharmacogenetics of the drug combination.Results: Synergistic cytotoxicity and enhancement of apoptosis was demonstrated, mostly with the sequence pemetrexed3gemcitabine. Pemetrexed increased cells in S phase, the most sensitive to gemcitabine, and a positive correlation was found between the expression ratio of dCK:RR and gemcitabine sensitivity. Indeed, pemetrexed significantly enhanced dCK gene expression (؉227.9, ؉86.0, and ؉135.5% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), and the crucial role of this enzyme was confirmed by impairment of gemcitabine cytotoxicity after dCK saturation with 2-deoxycytidine.Conclusions: These data demonstrate that the gemcitabine and pemetrexed combination displays scheduledependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells.

Phase I/II trial of doxorubicin and fixed dose-rate infusion gemcitabine in advanced soft tissue sarcomas: a GEIS study

“…The lower dFdCTP synthesis on day 1 would not be secondary to a decrease in gemcitabine membrane transport, due to the higher gemcitabine deamination observed on day 1 and that most of gemcitabine deamination occurs intracellularly, with plasma cytidine deaminase activity playing a limited role . It seems unlikely that the dose of gemcitabine given on day 1 would induce an increase in PBMC deoxycytidine kinase activity, as it has been detected in pancreatic cell lines in vitro (Giovannetti et al, 2004). The pharmacokinetics of DXR during the limited period studied was not affected by gemcitabine when compared with historical controls, similar to the results of other studies (Pérez-Manga et al, 2000;Fogli et al, 2002).…”

In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

“…Studies on colorectal cancer cell lines HCT-8, LoVo, WiDr, LRWZ and Calu-1 lung cancer cells showed maximal synergistic cytotoxicity when gemcitabine was followed by pemetrexed in cells (Tesei et al, 2002;Adjei, 2002;Giovannetti et al, 2005). On the contrary, other studies demonstrated that the schedule-dependent synergism was maximal when pemetrexed preceded gemcitabine in HT29 colon cancer cells (Tonkinson et al, 1999), MIA PaCa-2, PANC-1 and Capan-1 pancreatic cancer cells (Giovannetti et al, 2004) and A549 and Calu-6 lung cancer cells (Giovannetti et al, 2005). In vitro experimental data obtained in the present study indicate that in bladder cancer T24 cells the highest chemotherapeutic synergism was observed with the sequence pemetrexed-gemcitabine, while both sequences were almost equivalent in J82 cells.…”

“…These findings are in agreement with Table 2 Cell cycle modulation after drug treatments, followed by 24-h culture in drug-free medium. Values (%) are means from three independent experiments and the differences (D) are calculated with respect to controls previous data obtained in colon and pancreatic cells, which were synchronised after 24-h pemetrexed exposure, and showed a significant increase of apoptosis after treatment with gemcitabinepemetrexed combinations (Tonkinson et al, 1999;Giovannetti et al, 2004). One potential antiapoptotic signal transduction system that has been linked to chemoresistance of human cancer cells is the PI3K-Akt pathway.…”

Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level

“…Drug interaction was assessed by treating cells with increasing concentrations of both vinblastine and TBB, at a 1:10 concentration ratio (Vbl:TBB), using the combination index (CI) (Giovannetti et al, 2004), where CIo1, CI ¼ 1, and CI>1 indicate synergistic, additive and antagonistic effects, respectively.…”

“…Viability was plotted as function of Vbl concentrations (left panel), or TBB concentrations (right panel). Combination index (CI) was calculated as described by Giovannetti et al (2004), where a value o1 indicates synergism.…”