Sodium Antimony Gluconate Induces Generation of Reactive Oxygen Species and Nitric Oxide via Phosphoinositide 3-Kinase and Mitogen-Activated Protein Kinase Activation inLeishmania donovani-Infected Macrophages

Abstract

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.Visceral leishmaniasis, caused by Leishmania donovani, is fatal if left untreated. The pentavalent antimony (Sb V ) compound urea stibamine first emerged as an effective chemotherapeutic agent against Indian kala-azar (6). Although different forms of pentavalent antimony complexes (chelates, i.e., Sb V chelated to an organic backbone), namely, sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), are still the first choice for treatment of leishmaniases (21, 42), their mechanism of action is still largely unknown. Previous studies indicated that sodium antimony gluconate (SAG) failed to act in immunocompromised hosts, such as patients who are suffering from AIDS or receiving immunosuppressive agents (17, 38) and nude (36) and severe combined immunodeficient (SCID) mice (15). Several studies have shown that endogenous interleukin-2 (IL-2) (34), IL-4 (1, 43), and IL-12 (41) influence the effectiveness of chemotherapy with pentavalent antimony. These findings are inclined to indicate the requirement of a somewhat functional T-cell compartment for SAG action. Moreover, SAG has been found to inhibit selective protein tyrosine phosphatases (Src homology 2 domain-containing tyrosine phosphatase 1 [SHP1] and SHP2) in vitro and augment cytokine signaling and responses in hematopoietic cell lines (46), suggesting the role of phosphatases and possibly other signal transduction pathways in SAG-induced control of Leishmania infection. In addition, the dose of SAG that kills the axenic amastigotes in vitro is 50 times higher than the concentration of the drug required for killing the parasite w...

Antimonial treatment of visceral leishmaniasis: are current in vitro susceptibility assays adequate for prognosis of in vivo therapy outcome?

“…To understand it, the proposed dual action mode of SSG should be repeated: (i) SSG is converted to SbIII, which in turn has direct leishmanicidal activity [18,19], and (ii) SSG stimulates infected cells to produce microbicidal compounds such as ROI and nitric oxide which kill the intracellular parasites [20,21]. On the one hand, SbIII susceptibility was not analyzed in previous studies on L.(L.) donovani, but here this was done on a sub-sample of isolates.…”

“…On the other hand, considering the stimulating effect of SSG on the infected cells, a differential magnitude might be expected in the in vitro systems compared to in vivo. In vivo, the response of the infected cells to SSG leads to a more substantial involvement of the host immune system to attack the parasites [20,22,23], resulting in a synergistic activity between SSG and the specific T-cell response of the host [24e26]. In comparison, the in vitro system used for susceptibility assays does not include any immune components.…”

Leishmania antimony resistance: what we know what we can learn from the field

“…Microbicidal molecules, like nitric oxide (NO) or hydrogen peroxide (H 2 O 2 ) generated by the host during antimony treatment, also play a role in the in vivo leishmanicidal activity of antimony formulations (Carter et al 2005;Mookerjee-Basu et al 2006;Mandal et al 2007;Sarkar et al 2011). Therefore, the in vivo chemotherapeutic outcome of antimonial therapy depends on the susceptibility of the parasite not only to the antimonial compound but also to other host-derived microbicidal molecules.…”

Mitochondrial Proteomics of Antimony and Miltefosine Resistant Leishmania infantum

“…Mitochondria are also involved in the parasite response to pharmacological perturbation, being the main source of ROS and controlling cell death. It has been shown that ROS are produced when Leishmania are treated with either MIL or antimony [12,13,25,26]. Parasites belonging to the Kinetoplastida eukaryotic branch are unusual in that each cell contains one, enlarged mitochondrion, stretching the majority of the length of the cell.…”