Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani

Abstract

The unicellular organism Leishmania undergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3-O,28-O-disuccinyl betulin (DiSB), a potent topoisomerase type IB inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (⌬⌿ m ), caused a loss in ATP production in Leishmania parasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases. DiSB treatment translocated Leishmania donovani endonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown of L. donovani metacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G 1 phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in antileishmanial chemotherapy.

Green Synthesis of Silver and Titanium Dioxide Nanoparticles Using Euphorbia prostrata Extract Shows Shift from Apoptosis to G 0 /G 1 Arrest followed by Necrotic Cell Death in Leishmania donovani

“…However, other phospholipid classes which participate in apoptosis, like phosphatidylethanol sphingomyelin and phosphatidyl choline, can also enable annexin V staining, and therefore annexin V binding is used as an early indicator of apoptosis by many other investigators (35,50). Our results of annexin V-FITC and PI indicated necrosis as shift from the UR to the UL quadrant of synthesized-Ag NP-treated parasites.…”

“…3B, panel a). The antileishmanial activity of synthesized Ag NPs was also checked against the J774A.1 cell line to determine whether the doses used for IC 50 determination for intracellular amastigotes were toxic to the cell itself; experiments revealed that the 50% cytotoxic dose (CC 50 ) was far higher (115.5 g/ml) than the IC 50 (3.89 g/ml) for intracellular amastigotes (Fig. 3B, panel b).…”

“…IC 50 , the concentration of drug that is cytotoxic to 50% of the amastigotes, was obtained by plotting the graph of percentage of cell viability versus different concentrations of synthesized Ag NPs.…”

Cell death offers exceptional cellular and molecular windows for pharmacological interventions in protozoan parasites

“…The unicellular organism Leishmania donovani undergoes apoptosis-like cell death in response to external stress or exposure to anti-leishmanial agents, such as 3-O, 28-O-disuccinylbetulin (DiSB) resembled the PCD of higher eukaryotes. High level of metacaspase results in the translocation of endonuclease G (LdEndoG) from mitochondria to the nucleus for DNA degradation [67]. Surprisingly, the initiator proteins, for example, MCA-Arginine protease from both protozoan parasite species, such as Plasmodium falciparum and Leishmania major can elicit apoptotic cell death through a Z-VAD-FMK inhibitable effector protease [68].…”

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase

“…The sodium antimony gluconate (SAG)-sensitive (Sb s ) MHOM/IN/1983/AG83 (AG83) strain of L. donovani, the laboratory-grown SAG-resistant (Sb r ) strain GE1, laboratorygrown miltefosine-resistant (MIL r ) cells, and camptothecin-resistant (CPT r ) cells (raised in hamsters) were used (46,47). Amastigotes obtained from the spleens of infected hamsters were cultured at 22°C to obtain promastigotes and cultured in M199 containing 20% (vol/vol) heat-inactivated fetal bovine serum (FBS) supplemented with 100 IU/ml of penicillin and 100 mg/ml of streptomycin at 22°C to obtain promastigotes.…”