The efficiency of drug combinations is often sequence dependent. In our cell line system we observed additive to synergistic drug interaction for parallel drug combinations of 5-FU and FWGE. These data confirm the results of Szende et al, who observed no decrease in the antiproliferative activity of 5-FU, doxorubicin or navelbine by

the simultaneous exposure to nontoxic concentrations of FWGE [23]. In drug sequence experiments the additive to synergistic effect was abolished dependent on the sequence resulting in either additive effects or even a trend to antagonism (table 2). FWGE is known to interfere with ribonucleotide reductase which catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides [11]. Since these are the building blocks for DNA

replication, pretreatment of cells with FWGE decreases AZD2171 cost DNA-synthesis which might hamper the activity of the antimetabolite 5-FU. In line with this hypothesis, it was recently demonstrated in HT29 and HL-60 cells, that pretreatment of cells with FWGE significantly reduced the deoxyribonucleotide triphosphate pools and the incorporation of 14C-cytidine into DNA [3, 8]. In the event of impaired DNA-synthesis 5-FU might lose one of its targets which might at least in part explain the observed trend to antagonism in mTOR inhibitor our model system when FWGE treatment precedes 5-FU by 24 hours. Taken together, for further development of drug combinations with FWGE not just the combination partner but also the chosen drug schedule appeared to be crucial and should be considered. Based on its documented preclinical activity profile and mechanisms of drug action as well as on the available clinical data, FWGE appeared to be a good combination partner for drug regimens, in particular as modulator of drug activity and attenuator of drug toxicity. In conclusion, FWGE O-methylated flavonoid exerted significant antiproliferative activity in a broad spectrum of tumor cell lines. Simultaneous administration

of FWGE with 5-FU, oxaliplatin or irinotecan did not impair the cytotoxic activity of these cytostatic drugs in our colon cancer model. Our findings Autophagy inhibitor suggest that simultaneous application of 5-FU and FWGE, which resulted in additive to synergistic drug interactions, seems superior to sequential scheduling. The sequential administration of 5-FU followed by FWGE may be appropriate, while the reverse sequence should be avoided. Overall, based on its preclinical activity profile and clinical available data, further evaluation of combinations FWGE and conventional cytostatic drugs seems safe and warranted. Authors’ contribution TM carried out the cell line studies and contributed significantly to the design of the study. KJ performed the data analysis and preparation of figures. WV participated in the design of the study and data analysis. He prepared the manuscript and raised funding.