Mitochondria-Dependent Reactive Oxygen Species-Mediated Programmed Cell Death Induced by 3,3′-Diindolylmethane through Inhibition of F0F1-ATP Synthase in Unicellular Protozoan ParasiteLeishmania donovani

Abstract

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3Ј-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (⌬⌿ m ). Because ⌬⌿ m is the driving force for mitochondrial ATP synthesis, loss of ⌬⌿ m results in depletion of cellular ATP level. The loss of ⌬⌿ m causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of ⌬⌿ m leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.Apoptosis, a form of programmed cell death (PCD), is a genetically regulated active physiological process of cell suicide that causes cell deletion without inflammation, scarring, or release of cellular contents. Mitochondria of living cells play a pivotal role in controlling life and death (Green and Reed, 1998). Mitochondria are an important cellular source for the generation of reactive oxygen species (ROS) inside the cells (Halliwell and Gutteridge, 1990). Maintenance of proper mitochondrial transmembrane potential (⌬⌿ m ) is essential for the survival of the cell because it derives the synthesis of ATP and maintains oxidative phosphorylation (Gottlieb, 2001).3,3Ј-Diindolylmethane (DIM) is a major acid condensation product of indole-3-carbinol, a natural compound found in vegetables of the genus Brassica. It has an anticarcinogenic effect and inhibits the growth of human cancer cells. DIM-

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“…ROS overproduction in Leishmania is frequently associated with programmed cell death processes through mitochondrial dysfunction (37). The decrease in intracellular L. pifanoi parasites was also confirmed by electron microscopy.…”

Topoisomerase I Gene Mutations at F270 in the Large Subunit and N184 in the Small Subunit Contribute to the Resistance Mechanism of the Unicellular Parasite Leishmania donovani towards 3,3′-Diindolylmethane

“…In addition to its antitumor effects, DIM has also been recognized to specifically target L. donovani topoisomerase I (16). Moreover, we recently demonstrated that DIM induces programmed cell death through the inhibition of mitochondrial F o F 1 ATP synthase in the unicellular protozoan parasite L. donovani and reduces the parasitic loads in the livers and spleens of infected hamsters (17). Thus, DIM could be of interest in the treatment of human leishmaniasis.…”

“…The effect of the drug on the viability of the WT and LdDR50 cell lines was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (17). The cells were collected at the exponential phase and transferred to a 24-well plate (2 ϫ 10 6 cells per well).…”

“…Total RNA was prepared from WT and DIM-resistant promastigotes using the Total RNA isolation kit (Roche Biochemicals). cDNA was synthesized from 60 ng of total RNA using Superscript II RNaseH Ϫ reverse transcriptase (Invitrogen) and oligo(dT) [12][13][14][15][16][17][18] primers (Invitrogen) according to the manufacturer's instructions. Semiquantitative PCR was performed in a 25-l volume using 50 pmol each of sense and antisense primers corresponding to LdTOP1DRL, LdTOP1DRS, and L. donovani glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using the following profile: initial denaturation at 95°C for 5 min; 25 cycles with denaturation at 95°C for 1 min, annealing at 55°C for 40 s, and extension at 68°C for 40 s; and a final extension of 3 min.…”

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