Anthracyclines effects on polyamine uptake and proliferation of T. cruzi epimastigotes

MD. RUIZ; L. FRACCAROLI; D. BALCAZAR; ML. SBARAGLINI; L. LAROCCA; C. CARRILLO

Buenos Aires

Congreso; Drug Discovery for Neglected Diseases International Congress 2018 and 4th Scientific Meeting of ResNet NPND; 2018

Background: Chagas disease is an endemic parasitosis originally from Latin America, caused by the protozoan Trypanosoma cruzi (T. cruzi). The current therapies are limited in efficacy and show multiple side effects. Thus, there is a need to identify new effective and specific trypanocidal strategies.Anthracyclines, used as anticancer agents, exert their citotoxicity by blocking DNA metabolism. In humans, these are transported into cells by the organic cation transporter hOCT1 [1]. Previous results have shown that these compounds inhibit DNA topoisomerases from trypanosomatids [2], however no specific transporters have been identifed. Aim: As hOCT1 functions as a low affinity polyamine permease, we were interested in analyzing the interaction and/or incorporation of anthracyclines in T. cruzi by the putrescine permease TcPAT12 [3], a member of the TcAAAP transporters family [4]. Methods & Materials: Anthracyclines tested were Daunorubicin (Dnr) and Doxorubicin (Dxr). We evaluated T. cruzi epimastigotes proliferation and viability by growth curves and MTT assay under different culture conditions. The strains assayed were Y-GFP (control) and Y-TcPAT12-GFP (overexpressing TcPAT12 permease). To evaluate the effects of Dnr y Dxr, transport assays were performed for putrescine (TcPAT12), arginine (TcAAAP411) and riboflavin (TcRibj) [5]. Results: Dnr and Dxr significantly decreased T. cruzi epimastigotes proliferation rate, in a dose dependent manner, assesed by MTT and cell counting using a Neubauer chamber. IC50 values ranged between 0.3 M for Dnr and 3 M for Dxr in control and Y-TcPAT12-GFP strains. These strains became more sensitive to Dnr and Dxr when epimastigotes were putrescine deprived during 15 days (IC50 values ranging 0.1 M for Dnr and 2 M for Dxr).Putrescine uptake was affected by Dnr and Dxr in both strains under study. Percentage of putrescine uptake respect to untreated epimastigotes: control + Dnr 50M: 46.4±8.8% vs TcPAT12 + Dnr 50M: 78.2±2.5%; control + Dxr 50M: 59.9±2.4% vs TcPAT12 + Dxr 50M: 91.4±8.6%. Arginine and riboflavin uptake was not affected under these conditions. Conclusion: The findings presented herein showed that Dnr and Dxr affect T. cruzi epimastigotes survival and proliferation. This effect could be related to the decrease of polyamine uptake and/or to anthracyclines incorporation by TcPAT12 transporter and their intracellular toxic effects. References:[1] Andreev E, et al. Sci Rep 2016, 6:20508[2] Das A, et al. Trends Parasitol 2004, 20(8):381-7[3] Carrillo C, et al. Biochem Biophys Res Commun 2006, 344(3):936-40[4] Canepa GE, et al. FEMS Microbiol Lett 2004, 236(1): 79-84[5] Balcazar DE, et al. Plos Negl Trop Dis 2017, 11(4):e0005513Acknowledgements: The authors thank CONICET and ANPCyT (PICT 2013-0664 and PICT 2015-0962) for the financial means to afford the study.