Leishmania donovani: Intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells

Abstract

Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Δψ_m and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Δψ_m in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells.

Introduction

Apoptosis is a form of programmed cell death (PCD) that is initiated and completed in an orderly manner and where purpose is to eliminate the cells without releasing their content into the extracellular environment (Green and Reed, 1998). Mitochondria are central integrating organelle in apoptosis, with the capacity to directly activate the execution pathways in both multicellular (Bossy-Wetzel and Green, 1999) and unicellular organisms like Leishmania (Debrabant et al., 2003, Das et al., 2001, Arnoult et al., 2002, Zangger et al., 2002, Sen et al., 2004). Moreover, a fall of Δψ_m, induced by so-called permeability transition was observed as one of the first events in the apoptotic cascade, prior to fragmentation of nuclear DNA (Scheffler, 1999, Fontaine and Bernardi, 1999). Other common factors for PCD involving mitochondria include ROS and Ca⁺⁺ homeostasis and these factors were also shown to be associated with cell death (Ridgley et al., 1999).

Mitochondrial function depends on proteins that are encoded by both nuclear DNA and mitochondrial DNA (mtDNA). The mtDNA network of leishmanial cells contains two types of DNA molecules; maxicircles and minicircles (Simpson, 1986). Maxicircles encode ribosomal RNA (two ribosomal RNA genes, 9S and 12S) and several other proteins including six subunits of complex I, apocytochrome b of CoQ reductase, cytochrome c reductase (complex III), three subunits of cytochrome oxidase (complex IV), subunit A6 of ATP synthase (complex V), and a ribosomal protein (RPS 12), which are involved in the mitochondrial energy transduction. The genetic functions of the minicircles are known to encode small guide RNAs (gRNAs), which are 70 nt in size with a short oligo-U tail at the 3′ end and they govern the specificity of mRNA editing during post-transcriptional stages (Feagin, 2000).

Several flavonoid compounds have been shown to exert their action by interacting with DNA topoisomerases. The non-intercalating flavonoids like genistein and orobol and the DNA intercalating flavonoids like quercetin, myricetin and baicalein promote topoisomerase mediated site-specific DNA cleavage in mammalian cells in vitro and in vivo (Yamashita et al., 1990). From earlier studies it is known that, luteolin has both anti-inflammatory and anti-allergic properties and mediates its action by inhibiting production of nitric oxide inside cells (Kim et al., 1999). Moreover, a recent report establishes that, luteolin is a potent inhibitor of human mast cell activation and exerts its action through the inhibition of protein kinase C activation and Ca⁺⁺ influx. The growth and metabolism of human leukemic cell lines like CEM-C1 and CEM-C7 are also inhibited by luteolin (Post and Varma, 1992).

From our laboratory it was previously established that luteolin is similar to camptothecin, a class I inhibitor with respect to its ability to form the topoisomerase I mediated cleavable complex Das et al. (2004). But unlike CPT, luteolin interacts with both free enzyme and substrate DNA in vitro (Chowdhury et al., 2002) and it promotes topoisomerase II mediated linearization of kDNA minicircles in vivo (Mittra et al., 2000). It inhibits the growth of L. donovani promastigotes as well as amastigotes in infected macrophages. This cytotoxic lesion causes cell cycle arrest at the G1 phase and ultimately leads to apoptosis-like death (Mittra et al., 2000). But the molecular mechanisms connecting topoisomerase II inhibition inside leishmanial cells to the activation of apoptotic machinery are still unknown and these aspects remain to be investigated for apoptotic cell death in unicellular parasite like L. donovani during treatment with a flavone like luteolin.

Previously, we have found that a non-oxidant CPT induces apoptosis-like death through generation of ROS inside cells. Endogenous toxic ROS was responsible for collapse of mitochondrial Δψ_m in leishmanial cells (Sen et al., 2004a). Here in the case of treatment with luteolin, oxidative stress was not generated inside leishmanial cells. This may be due to the fact that luteolin has a strong scavenging ability for peroxide radicals. So the pathway of apoptosis-like death induced by luteolin is different from signaling pathway induced by another topoisomerase I poison CPT in leishmanial cells.

Here, we have demonstrated the mechanism of luteolin induced apoptosis-like death of leishmanial cells. In the present study, we show that luteolin induces the loss of mt-DNA and resulted in the formation of dyskinetoplastid cells. These cells undergo apoptosis-like death by enhancing caspase like proteases via cytochrome c dependent pathway. Moreover, our results also provide the evidence for the involvement of glycolytic ATP to enhance the apoptotic pathway in leishmanial cells. Such information has potentially useful value in determining the role of mitochondria in apoptosis-like death of leishmanial cells and help in designing of better drugs for leishmaniasis.