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  • Previously we have demonstrated that AT MSCs express enzymes

    2018-11-06

    Previously we have demonstrated that AT-MSCs express enzymes relevant for the intracellular metabolism of 5FU such as thymidylate synthase (TS), thymidylate phosphorylase (TP), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) (Kucerova et al., 2008). DPD rapidly degrades 5FU and contributes to 5FU catabolism to produce inactive metabolite dihydrofluorouracil (Longley et al., 2003). Following entry into the nucleotide salvage pathway, 5-FU is further metabolized. CDy::UPRT recombinant fusion enzyme possesses both capability to convert 5FC to 5FU and further metabolizes 5FU to 5-fluorouridine-5′-monophospahte (FUMP). We anticipate that the ppar agonist abundant production of FUMP by exogenously delivered UPRT plays significant role in chemosensitization of CDy::UPRT-MSCs to 5FU by avoiding DPD mediated 5FU degradation. Exogenous UPRT combined with tissue specific expression significantly increased efficiency of 5FU-based chemotherapy in prostate tumor ppar agonist via suicide effect (Zhao et al., 2009). In our study we have demonstrated pronounced bystander cytotoxicity of CDy::UPRT/5FU treatment modality in breast cancer cells. This combined treatment of EGFP-MDA-MB-231 cells was much more efficient when compared to 5FU treatment alone or CDy::UPRT/5FC that has shown limited efficiency in vivo ((Kucerova et al., 2011; Kucerova et al., 2010) and unpublished data). As serum and intratumoral levels achievable by 5FC and 5FU administration are limited, we suggest that therapeutic combination of CDy::UPRT-MSCs with low dose of 5FU would be more effective in comparison to CDy::UPRT-MSCs with maximum tolerated dose of 5FC in vivo. Cyclophosphamide, cisplatin and some other treatments have been described to induce TP expression, which is beneficial for the anticancer effect (Toi et al., 2005; Walko and Lindley, 2005). In our work, we have demonstrated that combined 5FU treatment with either cyclophosphamide or cisplatin led to strong apoptosis induction as demonstrated by activation of Caspase 3/7 in CDy::UPRT-MSCs. Leucovorin combination treatment with 5FU has an apparently different mechanism of action although with similar outcome on decreased cell proliferation in CDy::UPRT-MSCs ((Thirion et al., 2004) and our data). Another study has reported cisplatin-mediated depression in enzyme activity of DPD leading to cytotoxic synergistic action of 5FU (Miyazaki et al., 2006). Responses to nucleotide analogs 5-azacytidine have not been affected in engineered MSCs, however cytosine arabinoside inhibited proliferation of CDy::UPRT-MSCs to a higher extent. We have found increased resistance of these MSCs to low concentrations of anthracycline antibiotic doxorubicin. We hypothesize that the enzymatic action of CDy::UPRT transgene could have contributed to cellular resistance to cytotoxic drug by interferring with the nucleotide salvage pathways and provided the transduced cells with a survival advantage. Moreover, these data add further indications to the preclinical testing of our therapeutic paradigm in order to increase its efficiency. Based on the presented data we suggest to augment the CDy::UPRT-MSC-mediated tumor targeted therapy with (low doses of) 5FU and/or in combination with doxorubicin. In order to achieve maximum tumor growth suppression the drugs which induce rapid apoptosis induction in therapeutic CDy::UPRT-MSCs should be avoided. In summary, our data add further knowledge to the use of engineered MSCs for the experimental applications. We have shown that unless specific transgene had been introduced into these cells to interfere directly with the metabolic pathway(s) of cytotoxic drug, no significant difference in behavior could have been determined between the naïve and transduced MSCs. Fluorescent and dominant selection marker expressing MSCs are useful tools to further examine the role of MSCs and the function in a stromal tumor compartment. These transgenes could be also used for the downstream analysis of homing, persistence, differentiation and other attributes of the MSCs\' progeny. Nevertheless, these studies should be also corroborated with those employing endogenous markers within the MSCs (Klopp et al., 2011) as it was shown, that EGFP expression declined rapidly to undetectable level post-transplantation in vivo and was reversed only upon re-establishment of EGFP transduced cells in cell cultures (Ronsyn et al., 2007). However, the future clinical application of genetically modified MSCs still requires further investigation and their translation into cancer treatment remains the matter of intense debate (Momin et al., 2010).