Abstract
Leishmania, a unicellular trypanosomatid protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. However, it undergoes a process of programmed cell death during treatment with the topoisomerase I poison camptothecin (CPT). The present study shows that CPT-induced formation of reactive oxygen species increases the level of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (DeltaPsim), which is followed by a significant decrease in intracellular pH levels. CPT-induced oxidative stress also causes impairment of the Na+ - K+ -ATPase pump and subsequently decreases the intracellular K+ level in leishmanial cells. A decrease in both intracellular pH and K+ levels propagates the apoptotic process through activation of caspase 3-like proteases by rapid formation of cytochrome c-mediated apoptotic complex. In addition to caspase-like protease activation, a lower level of intracellular K+ also enhances the activation of apoptotic nucleases at the late stage of apoptosis. This suggests that the physiological level of pH and K+ are inhibitory for apoptotic DNA fragmentation and caspase-like protease activation in leishmanial cells. Moreover, unlike mammalian cells, the intracellular ATP level gradually decreases with an increase in the number of apoptotic cells after the loss of DeltaPsim. Taken together, the elucidation of biochemical events, which tightly regulate the process of growth arrest and death of Leishmania donovani promastigotes, allows us to define a more comprehensive view of cell death during treatment with CPT.
Highlights
DNA topoisomerases are ubiquitous enzymes that catalyze the breakage and rejoining of DNA strands to permit topological changes in DNA [1]
We have shown by confocal microscopy and spectrofluorometric analysis that over the time course of drug treatment leishmanial cells showed a significant increase in intracellular calcium (Fig. 3)
Apoptosis or programmed cell death can be activated in variety of cells through diverse signaling pathways
Summary
Parasite Culture and Maintenance—Leishmania strain AG 83 promastigotes were grown at 22 °C in M199 liquid media supplemented with 10% fetal calf serum. Cells with differently treated groups were harvested and washed twice with wash buffer containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 5.5 mM glucose, 1 mM CaCl2, and 50 mM MOPS, pH 7.4. Cells (2 ϫ 107) after different treatments were harvested, suspended in cell extraction buffer (20 mM HEPES-KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 200 M PMSF, 10 g/ml leupeptin and pepstatin), and lysed by a process of freeze-thaw using nitrogen cavitation and a 37 °C water bath simultaneously. For the DNA fragmentation assay, 2 ϫ 107 nuclei were suspended in CPT-treated nuclear extract of leishmanial cells and incubated at 37 °C for 3 h in the presence of 2 mM MgCl2 and 1 mM CaCl2. Comparisons were made between different treatments using unpaired Student’s t test
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