Gene-directed enzyme/prodrug therapy (GDEPT) mediated by mesenchymal stromal cells (MSC) was already approved for clinical study on a progressive disease refractory to standard therapy. In this work, we examined the effect of several GDEPT approaches on chemoresistant cells. First, we derived 5-fluorouracil (5-FU)-resistant variant of human colorectal adenocarcinoma cells HT-29 designated HT-29/EGFP/FUR. Our data show that the upregulation of thymidylate synthase (TS) and downregulation of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) contributed to the 5-FU resistance in cancer cells. Next, we combined the MSC expressing either yeast cytosine deaminase (CD-MSC) or fusion yeast CD::uracil phosphoribosyl transferase (CD::UPRT-MSC) and prodrug 5-fluorocytosine (5-FC) in a cell-mediated GDEPT approach. Bystander cytotoxic effect in the direct co-cultures of the tumor and therapeutic cells mixed in a 5:1 ratio resulted in 55% and 70% inhibition of proliferation, respectively. However, the acquired chemoresistance to 5-FU can be overcome by introducing the prodrug-converting transgene into the tumor cells. When the transgene CD::UPRT was expressed in the chemoresistant cells (CD::UPRT-FUR), substantial suicide effect and a 90% decrease in viability was observed using non-toxic concentration of 62.5 µg/ml 5-FC. In summary, we demonstrate here that the transgene introduction circumvented 5-FU resistance in the tumor cells.

Department of Analytical Chemistry Faculty of Natural Sciences Comenius University Mlynska dolina Ilkovicova 6 842 15 Bratislava Slovakia

Department of Analytical Chemistry Faculty of Natural Sciences Comenius University Mlynska dolina Ilkovicova 6 842 15 Bratislava Slovakia Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceske Budejovice South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Research Institute of Fish Culture and Hydrobiology Zatisi 728 2 389 25 Vodnany Czech Republic

Department of Molecular Oncology Cancer Research Institute Biomedical Research Center of Slovak Academy of Sciences Dubravska cesta 9 845 05 Bratislava Slovakia

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