Interaction between gemcitabine and topotecan in human non-small-cell lung cancer cells: effects on cell survival, cell cycle and pharmacogenetic profile

Abstract

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 ( þ 155.0 and þ 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.

Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines

“…It has been already established in other tumor models that the combination of active drugs is not necessarily associated with greater anti-neoplastic efficacy. In lung cancer models, gemcitabine has been demonstrated to be either synergistic or antagonistic with topotecan depending on the type and timing of combination (Giovannetti et al, 2005). However, caution should be used to translate our results into clinically meaningful data.…”

Preclinical relevance of dosing time for the therapeutic index of gemcitabine–cisplatin

“…Gemcitabine is an S-phase specific agent (Tolis et al, 1999) and elicits apoptosis (Tolis et al, 1999;Giovannetti et al, 2005). This process can be prevented by BCL-2 expression and favoured by BAX expression (Hao et al, 2003).…”

Preparation and characterization of biodegradable paclitaxel loaded alginate microparticles for pulmonary delivery

“…Human non-small cell lung cancer, A549 [37][38][39] and Calu-6 [40] (American Type Culture Collection) were cultivated in essential medium (RPMI, Biosera) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 • C in humidified environment of 5% CO 2 . The medium was replenished every other day and the cells were subcultured after reaching confluence [39,40].…”

“…The medium was replenished every other day and the cells were subcultured after reaching confluence [39,40].…”