Synergistic cytotoxicity and pharmacogenetics of gemcitabine and pemetrexed combination in pancreatic cancer cell lines.
OBJECTIVE
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.


METHODS
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 pemetrexed-->gemcitabine. 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 schedule-dependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells.
Giovannetti
Elisa
E
Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine, Pisa, Italy.
Mey
Valentina
V
Danesi
Romano
R
Mosca
Irene
I
Del Tacca
Mario
M
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D003842:Deoxycytidine Kinase / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D000068437:Pemetrexed; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000235:genetics; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D005971:Glutamates; D000068437:Pemetrexed; D003841:Deoxycytidine; D006147:Guanine; D012264:Ribonucleotide Reductases; D003842:Deoxycytidine Kinase; D000093542:Gemcitabine