Pgp expression or not, is that the question in T. cruzi drug differential sensitivity?
Chagas disease is a parasitosis caused by Trypanosoma cruzi (T. cruzi). Current treatments, Nifurtimox (NFX) and Benznidazole (BZN), discovered 40 years ago, are limited in efficacy. Several compounds are being investigated to find new therapies; using very promising approaches such as drug repurposing and drug combination. Previous results showed that Ivermectin (IVM- an anthelmintic drug) is a trypanocidal compound and inhibits nuclear import mediated by importin α in T. cruzi. Moreover, IVM-NFX combination had additive or antagonistic effects on T. cruzi epimastigotes proliferation, depending on the drug concentration evaluated.
One mechanism of drug resistance in T. cruzi has been associated with the efflux and ATPase activity of P-glycoprotein (Pgp) (a member of the ABC transporter superfamily), and with the overexpression of the TcPGP1 and TcPGP2 genes. Besides, IVM is a substrate of Pgp and other efflux transporters, and it has been reported in different cell types that IVM may be able to regulate the expression level of Pgp through transcriptional or post-transcriptional mechanisms. Sensitivity or resistance to different drugs results from a complex combination between detox mechanisms and the drug target or its way of action. The aim of this work is to assess the relation between TcPgp and TcImportin expression and T. cruzi sensitivity to NFX and IVM.
Growth curves were performed for T. cruzi epimastigotes of CL Brener (DTU VI), Y (DTU II) and RA (DTU VI) strains in the presence of increasing concentrations of IVM and NFX. EC50 values were calculated at 72 h of culture. The three strains showed similar sensitivity to IVM according to its EC50 values: 9.2 (7.9 a 10.7) µM for CL Brener, 13.6 (11.3 a 16.5) µM for Y and 8.8 (7.3 a 10.8) µM for RA strain. However, for NFX treatment, CL Brener strain was significantly more sensitive than Y and RA strains as EC50 was 1.7 (1.3 a 2.1) µM for CL Brener and 8.6 (7.2 a 10.3) and 7 (5.7 a 8.5) µM for Y and RA strains respectively.
To evaluate possible mechanisms in the response to IVM and NFX, Pgp1, Pgp2, Importin α and β expression was evaluated by RT-PCR in epimastigotes of the three strains under study. Preliminary results showed that Y and RA strains have an increased expression of Pgp1 and 2 as well as of Importin α and β compared to CL Brener epimastigotes. An increased expression of Pgp1 and Pgp2 could be related to an improved detoxifying cellular mechanism that accounts for the greater resistance observed for Y and RA epimastigotes to NFX treatment. Instead, similarities in IVM effect on the strains could be related to different factors, among them the Importin α differential expression.
Drug sensitivity depends on many factors; not only on their molecular targets but also on the detoxifying mechanisms Our results indicate that increased sensitivity to NFX in CL Brener epimastigotes could be related to the decreased Pgp1 and Pgp2 expression. However, for other drugs, as IVM, sensitivity could depend on multiple factors, such as different aspects of the molecular target (importin α). Further studies are necessary to unravel the effect of these drugs on the modulation of their own molecular targets and the detoxifying mechanisms in T. cruzi.