Camptothecin-induced Imbalance in Intracellular Cation Homeostasis Regulates Programmed Cell Death in Unicellular Hemoflagellate Leishmania donovani

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 (ΔΨ_m), 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 ΔΨ_m. 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. DNA topoisomerases are ubiquitous enzymes that catalyze the breakage and rejoining of DNA strands to permit topological changes in DNA (1). They are classified into two types. Type I topoisomerase breaks and rejoins one strand of duplex DNA, whereas type II topoisomerase breaks and rejoins both strands of DNA by using ATP as cofactor (2). These enzymes play a pivotal role in the maintenance of genome integrity and are essential for many chromosomal functions including DNA replication, recombinations, transcription, and chromosome segregation (3). Other than the orderly synthesis of nucleic acids, these enzymes also have been identified as the molecular targets for numerous clinically important antibacterial and antitumor agents like fluoroquinolone, etoposide, and camptothecin (CPT), ¹ etc. (2, 3). CPT, an inhibitor of DNA topoisomerase I, has been widely used to induce apoptosis under experimental conditions and is in phase III clinical trials for colon cancer (4, 5). Poisoning of topoisomerase I by CPT causes protein-linked single strand breaks, but the breaks are by themselves not sufficient for cell death. The collision between the DNA replication fork with CPT-stabilized topoisomerase I-DNA covalent complex is thought to be responsible for cell killing (6). The double strand breaks resulting from the fork arrest are repaired very slowly and lead to prolonged S phase or G2 arrest of the cell cycle, followed by apoptosis (7). Apoptosis, a physiological mode of cell death, results from the action of a genetically encoded suicide program that leads to series of characteristic morphological and biochemical changes (8). These changes include activation of caspases, cell shrinkage, chromatin condensation, and nucleosomal degradation (9). But the most significant event in apoptosis is mitochondrial dysfunction, which was shown to be involved in an early phase of apoptosis in a variety of cells upon induction of a number of stimuli including tumor necrosis factor (10), glucocorticoids (11), ceramides (12), and oxidative stress (13, 14). As compared with necrosis, apoptosis is an energy-dependent process requiring functional mitochondria. Without the supply of ATP, cell cannot transmit apoptotic death signals from the cytoplasm to the nucleus (15). Moreover, changes in the intracellular concentration of cations are responsible for alterations in cell volume, which is one of the most striking morphological changes during the process of apoptosis (16). But very little is known about the effects of these ionic changes on the activity of underlying apoptotic machineries, including caspases and nucleases. Caspases are synthesized and maintained in the cytoplasm as proenzymes, which themselves must undergo a proteolytic activation, perhaps triggering apoptosis. The substrates cleaved by these enzymes are numerous, including structural proteins (17, 18) and degradative enzymes (19). In addition to caspase activation, nucleases are activated, which destroy the genome to produce DNA fragments, recognized as the DNA ladder. Leishmaniasis, one of the dreaded protozoal diseases threatening mankind, does not have enough combative measures. CPT has been shown to inhibit type I DNA topoisomerase of Leishmania donovani promastigotes and leads to apoptosis. To characterize the cellular events associated with apoptosis, we have found that CPT-induced oxidative stress causes depolarization of mitochondrial membrane potential. This is followed by the activation of caspase-like proteases inside leishmanial cells after the release of cytochrome c into the cytosol (20). But the molecular mechanisms connecting ion fluxes to the apoptotic machinery are still unknown, and these aspects remain to be investigated for the apoptotic cell death in unicellular parasites like L. donovani during treatment with CPT. Here we have dissected the mechanism of action of CPT by analyzing the nuclear, mitochondrial, and cytosolic changes associated with apoptosis of leishmanial cells. In the present study we show that changes in the level of both cytosolic cations (calcium and potassium) and alterations in the level of ATP and pH regulate the apoptotic process by controlling the mitochondrial membrane potential and activity of caspase-like proteases and endonucleases. Taken together, our results provide the first insight into the mechanistic pathway of apoptosis in leishmanial cells where a decrease in cytosolic K⁺ ion concentrations and alterations in pH homeostasis by the topoisomerase I poison CPT appears to be an essential event responsible for the propagation of apoptosis.