Arsenite resistance in Leishmania and possible drug targets.

Pregarded as the second-most dreaded parasitic disease after malaria (WHO). Visceral leishmaniasis or kala-azar, caused by Leishmania donovani, is the most fatal form of leishmaniasis afflicting millions of people worldwide. No vaccination is available against leishmaniasis and fast spreading drug resistance in these parasitic organisms is posing a major medical threat. All these emphasize the need for new drugs and molecular targets along with reappraisal of existing therapeutics. Identification and characterization of cellular targets and answering the problem of drug resistance in Leishmania has always been the main thrust of protozoal research worldwide. Model drug resistance phenotypes against drugs, viz. arsenite (an antimony related metal ion, the first line of treatment against leishmaniasis), have been widely used to address and understand mechanism of drug resistance. The present discussion is an attempt to understand the different factors associated with arsenite resistance in Leishmania.

Altered tubulin dynamics, localization and post-translational modifications in sodium arsenite resistant Leishmania donovani in response to paclitaxel, trifluralin and a combination of both and induction of apoptosis-like cell death.

In this study the anti-leishmanial activity and anti-microtubule effects of paclitaxel, trifluralin and a combination of paclitaxel and trifluralin have been tested in a wild type and sodium arsenite-resistant strain of Leishmania donovani. Both paclitaxel and trifluralin have been shown to be effective in limiting parasite growth. Specific alterations in morphology, tubulin polymerization dynamics, post-translational modifications and cellular distribution of the tubulins have been confirmed to be a part of the intracellular anti-microtubule-events that occur in arsenite-resistant L. donovani in response to these agents, ultimately leading to death of the parasite. DNA analyses of the drug-treated wild type and arsenite-resistant strains revealed an apoptosis-like death in response to paclitaxel and the combination but not to trifluralin. Data provide valuable information for further development of chemotherapeutic strategies based on anti-microtubule agents against drug resistant Leishmania parasites.

Novobiocin induces apoptosis-like cell death in topoisomerase II over-expressing arsenite resistant Leishmania donovani.

Leishmaniasis affects millions of people worldwide every year. Lack of effective vaccination, co-infection with other dreaded diseases like AIDS and generation of drug resistant strains demand immediate attention into this neglected area of research. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which shows a 13-fold increase in resistance compared with wild type. Here we report that the arsenite resistant strain of L. donovani promastigotes shows cross-resistance to novobiocin, a catalytic inhibitor of topoisomerase II, with IC50 value of 320 microg ml-1 as compared with 242 microg ml-1 for wild type L. donovani. Leishmanicidal action of novobiocin induces dose- and time-dependent increase in cell death. Treatment with IC50 of novobiocin caused morphological and biochemical changes which lead to induction of cell death exhibiting characteristic features of metazoan apoptosis. Phosphatidylserine externalization, cytochrome C release to cytoplasm, activation of caspases, oligonucleosomal DNA fragmentation and in situ labelling of condensed and fragmented nuclei in both wild type and arsenite resistant L. donovani promastigotes strongly suggest the apoptosis-like mode of cell death. Cross-resistance to novobiocin in arsenite resistant strain has been correlated to over-expression of topoisomerase II and substantiated by differential inhibition of enzyme activity in wild type and arsenite resistant L. donovani.

Altered expression, polymerisation and cellular distribution of alpha-/beta-tubulins and apoptosis-like cell death in arsenite resistant Leishmania donovani promastigotes.

Studies in mammalian systems have shown specific affinity of arsenite for tubulin proteins. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which is a 13-fold increase in resistance compared to the wild type. Data presented in this study shows decreased expression of alpha- and beta-tubulin in wild type L. donovani promastigotes on exposure to NaAsO2 from 0.0016 to 5.0 microM (IC50 in the wild type strain) in a dose-dependent manner. alpha- and beta-tubulins in the resistant strain show decreased expression levels only at 65.0 microM NaAsO2 (IC50 in the resistant strain). Treatment with respective IC50 concentrations of NaAsO2 caused alterations in tubulin polymerisation dynamics and deregulated the cellular distribution of the microtubules in wild type and resistant strains. The NaAsO2-induced cell death exhibited characteristics of apoptosis-like DNA laddering and fragmentation in both the affected wild type and resistant cells. However, poly(ADP-ribose)polymerase cleavage was evident in the wild type strain but not in the resistant strain.

Overexpression and increased DNA topoisomerase II-like enzyme activity in arsenite resistant Leishmania donovani.

Western immunoblot analyses of whole cell lysates probed with a human specific monoclonal anti-topoisomerase IIalpha antibody identified a 190 kDa protein over expressed in the arsenite resistant Leishmania donovani strain. The crude nuclear extract of the resistant strain showed higher topoisomerase II-like enzyme activity. suggesting a possible regulatory role of putative topoisomerase II in arsenite resistant Leishmania.

Microtubules: dynamics, drug interaction and drug resistance in Leishmania.

Microtubules are cytoskeletal polymers essential for the survival of all eukaryotes. These proteins are the proposed cellular targets of many anticancerous, antifungal and antihelminthic drugs. Sufficient differences exist between the microtubules of kinetoplastid parasites like Leishmania and humans to explore the selective targeting of these proteins for therapeutic purposes. This review describes the basic structure of microtubules and its dynamics in general, with specific insights into leishmanial microtubules, the salient features of microtubule-drug interactions including the specificity of certain drugs for parasitic microtubules. Chemotherapy against leishmanial parasites is failing because of the emergence of drug resistant strains. The possible mechanisms of resistance to antimicrotubule agents along with insights into the role of microtubules in mediating drug resistance in Leishmania are discussed.

Resistance to arsenite modulates expression of beta- and gamma-tubulin and sensitivity to paclitaxel during differentiation of Leishmania donovani.

Differentiation of Leishmania donovani promastigotes into infectious amastigotes is accompanied by differential tubulin gene expression. Tubulin is one of the proposed targets of clinically useful antileishmanial agents and its expression is known to alter due to drug resistance. In this study, beta- and gamma-tubulin expression under various stages of differentiation was measured in an in vitro generated arsenite-resistant L. donovani strain. Results showed higher constitutive expression of beta-tubulin in the arsenite-resistant promastigotes and amastigotes compared with the wild-type. beta-Tubulin expression in the resistant promastigotes increased on paclitaxel treatment. Significant differences in gamma-tubulin expression were observed only between the amastigotes, but not between promastigotes, of wild-type and resistant strains. Paclitaxel did not produce any significant change in the expression profile of gamma-tubulin in either of the strains, neither before nor after differentiation. Data suggest that the beta- and gamma-tubulin expression and the response to paclitaxel is affected due to arsenite resistance.