Abstract
The control of the protozoan parasite Leishmania relies on few drugs with unknown cellular targets and unclear mode of action. Several antileishmanials, however, were shown to induce apoptosis in Leishmania and this death mechanism was further studied in drug-sensitive and drug-resistant Leishmania infantum. In sensitive parasites, antimonials (SbIII), miltefosine (MF) and amphotericin B (AMB), but not paromomycin (PARO), triggered apoptotic cell death associated with reactive oxygen species (ROS). In contrast, Leishmania mutants resistant to SbIII, MF or AMB not only failed to undergo apoptosis following exposure to their respective drugs, but also were more tolerant towards apoptosis induced by other antileishmanials, provided that these killed Leishmania via ROS production. Such tolerance favored the rapid acquisition of multidrug resistance. PARO killed Leishmania in a non-apoptotic manner and failed to produce ROS. PARO resistance neither protected against drug-induced apoptosis nor provided an increased rate of acquisition of resistance to other antileishmanials. However, the PARO-resistant mutant, but not SbIII-, MF- or AMB-resistant mutants, became rapidly cross-resistant to methotrexate, a model drug also not producing ROS. Our results therefore link the mode of killing of drugs to tolerance to cell death and to a facilitated emergence of multidrug resistance. These findings may have fundamental implications in the field of chemotherapeutic interventions.
Highlights
Apoptotic features have been reported in the eukaryotic parasite Leishmania following exposure to several drugs.[4,5,6,7,8] Leishmania are unicellular parasites responsible for a wide range of human diseases ranging from self-healing cutaneous lesions to fatal visceral infections
The L. infantum Sb2000.1, MF200.5, AMB1000.1 and PARO1200.1 cell lines were selected for resistance to SbIII, MF, amphotericin B (AMB) and PARO, respectively, in a stepwise manner until they reached at least a 10-fold increased resistance compared with the parental clonal strain (Table 1)
L. infantum-sensitive parasites subjected to increasing concentrations of SbIII, MF and AMB exhibited a dose-dependent increase in dichlorofluorescein diacetate (DCFDA) fluorescence signals (Figure 1), confirming increased reactive oxygen species (ROS) production upon exposure to these antileishmanials
Summary
Apoptotic features have been reported in the eukaryotic parasite Leishmania following exposure to several drugs.[4,5,6,7,8] Leishmania are unicellular parasites responsible for a wide range of human diseases ranging from self-healing cutaneous lesions to fatal visceral infections. Most antileishmanials were shown to induce apoptosis features in Leishmania, which include an increase in intracellular calcium levels, an accumulation of reactive oxygen species (ROS), a drop of the mitochondrial membrane potential (Dcm), genomic DNA degradation, exposure of phosphatidyl serine and induction of caspase-like activity.[7,16,17,26,27] Interestingly, the antimony-resistant phenotype of field isolates was reported to be accompanied by a decreased propensity to undergo apoptosis following exposure to either SbIII, MF or AMB.[7] it is possible that the common mechanisms involved in cell killing described for. We sought to determine the putative apoptotic features induced by SbIII, MF, AMB and PARO in Leishmania infantum drug-sensitive and drug-resistant parasites, and to assess whether an increased tolerance to apoptosis might accompany the selection of drug resistance. Our results are suggesting two types of drug-induced death in Leishmania, and are linking the mode of killing to tolerance to cell death and to a facilitated emergence of multidrug resistance
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