Interaction between gemcitabine and topotecan in human non-small-cell lung cancer cells: effects on cell survival, cell cycle and pharmacogenetic profile.
The pyrimidine analogue gemcitabine is an established effective agent in the treatment of non-small-cell lung cancer (NSCLC). The present study investigates whether gemcitabine would be synergistic with the topoisomerase I inhibitor topotecan against the NSCLC A549 and Calu-6 cells. Cells were treated with gemcitabine and topotecan for 1 h and the type of drug interaction was assessed using the combination index (CI). Cell cycle alterations were analysed by flow cytometry, while apoptosis was examined by the occurrence of DNA internucleosomal fragmentation, nuclear condensation and caspase-3 activation. Moreover, the possible involvement of the PI3K-Akt signalling pathway was investigated by the measurement of Akt phosphorylation. Finally, quantitative, real-time PCR (QRT-PCR) was used to study modulation of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) and the cellular target enzyme ribonucleotide reductase (RR). In results, it was found that simultaneous and sequential topotecan --> gemcitabine treatments were synergistic, while the reverse sequence was antagonistic in both cell lines. DNA fragmentation, nuclear condensation and enhanced caspase-3 activity demonstrated that the drug combination markedly increased apoptosis in comparison with either single agent, while cell cycle analysis showed that topotecan increased cells in S phase. Furthermore, topotecan treatment significantly decreased the amount of the activated form of Akt, and enhanced the expression of dCK (+155.0 and +115.3% in A549 and Calu-6 cells, respectively), potentially facilitating gemcitabine activity. In conclusion, these results indicate that the combination of gemcitabine and topotecan displays schedule-dependent activity in vitro against NSCLC cells. The gemcitabine --> topotecan sequence is antagonistic while drug synergism is obtained with the simultaneous and the sequential topotecan --> gemcitabine combinations, which are associated with induction of decreased Akt phosphorylation and increased dCK expression.
Giovannetti
E
E
Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, 55 Via Roma, 56126 Pisa, Italy. elisagio@tiscalinet.it
Mey
V
V
Danesi
R
R
Basolo
F
F
Barachini
S
S
Deri
M
M
Del Tacca
M
M
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D053938:DNA Fragmentation / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003842:Deoxycytidine Kinase / Q000187:drug effects / Q000378:metabolism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy*; D010766:Phosphorylation / Q000187:drug effects; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D059004:Topoisomerase I Inhibitors; D019772:Topotecan / Q000494:pharmacology*; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D059004:Topoisomerase I Inhibitors; D003841:Deoxycytidine; D019772:Topotecan; D003842:Deoxycytidine Kinase; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000093542:Gemcitabine