Reactive Oxygen Species Production and Mitochondrial Dysfunction Contribute to Quercetin Induced Death in Leishmania amazonensis

BACKGROUND: Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC(50) for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential. CONCLUSIONS/SIGNIFICANCE: The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function

Tolerance to drug-induced cell death favours the acquisition of multidrug resistance in Leishmania

The control of the protozoan parasite Leishmania relies on few drugs with unknown cellular targets and unclear mode of action. Several antileishmanials, however, were shown to induce apoptosis in Leishmania and this death mechanism was further studied in drug-sensitive and drug-resistant Leishmania infantum. In sensitive parasites, antimonials (SbIII), miltefosine (MF) and amphotericin B (AMB), but not paromomycin (PARO), triggered apoptotic cell death associated with reactive oxygen species (ROS). In contrast, Leishmania mutants resistant to SbIII, MF or AMB not only failed to undergo apoptosis following exposure to their respective drugs, but also were more tolerant towards apoptosis induced by other antileishmanials, provided that these killed Leishmania via ROS production. Such tolerance favored the rapid acquisition of multidrug resistance. PARO killed Leishmania in a non-apoptotic manner and failed to produce ROS. PARO resistance neither protected against drug-induced apoptosis nor provided an increased rate of acquisition of resistance to other antileishmanials. However, the PARO-resistant mutant, but not SbIII-, MF- or AMB-resistant mutants, became rapidly cross-resistant to methotrexate, a model drug also not producing ROS. Our results therefore link the mode of killing of drugs to tolerance to cell death and to a facilitated emergence of multidrug resistance. These findings may have fundamental implications in the field of chemotherapeutic interventions

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Antimonial-induced increase in intracellular Ca<SUP>2+</SUP> through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani Amastigotes

The capability of the obligate intracellular parasites like Leishmania donovani to survive within the host cell parasitophorous vacuoles as nonmotile amastigotes determines disease pathogenesis, but the mechanism of elimination of the parasites from these vacuoles are not well understood. By using the anti-leishmanial drug potassium antimony tartrate, we demonstrate that, upon drug exposure, intracellular L. donovani amastigotes undergo apoptotic death characterized by nuclear DNA fragmentation and externalization of phosphatidylserine. Changes upstream of DNA fragmentation included generation of reactive oxygen species like superoxide, nitric oxide, and hydrogen peroxide that were primarily concentrated in the parasitophorous vacuoles. In the presence of antioxidants like N-acetylcysteine or Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an inhibitor of inducible nitric-oxide synthase, a diminution of reactive oxygen species generation and improvement of amastigote survival were observed, suggesting a close link between drug-induced oxidative stress and amastigote death. Changes downstream to reactive oxygen species increase involved elevation of intracellular Ca2+ concentrations in both the parasite and the host that was preventable by antioxidants. Flufenamic acid, a non-selective cation channel blocker, decreased the elevation of Ca2+ in both the cell types and reduced amastigote death, thus establishing a central role of Ca2+ in intracellular parasite clearance. This influx of Ca2+ was preceded by a fall in the amastigote mitochondrial membrane potential. Therefore, this study projects the importance of flufenamic acid-sensitive non-selective cation channels as important modulators of antimonial efficacy and lends credence to the suggestion that, within the host cell, apoptosis is the preferred mode of death for the parasites

THE EFFECT OF METFORMIN MONOTHERAPY ON BIOCHEMICAL PARAMETERS ASSOCIATED WITH DIABETES MELLITUS, KIDNEY, CARDIAC, LIVER, THYROID, AND REPRODUCTIVE ORGAN FUNCTIONS IN PRE- AND POST-MENOPAUSE WOMEN WITH TYPE 2 DIABETES MELLITUS

 Objective: The aim of this study was to find out the alterations in biochemical parameters associated with the functioning of kidney, cardiac, liver, thyroid, and reproductive organs in pre- and post-menopause Type 2 diabetes mellitus (T2DM) women treated with metformin monotherapy (MMT) and to select a set of biochemical tests as routine to evaluate the functions of the above organs at regular intervals of time based on the levels of various biochemical parameters during a period of 1 year at 6 and 12 months.Method: A total of 100 T2DM patients (50 pre- and 50 post-menopause women) who visited Apollo Speciality Hospitals Vanagaram, Sugar Clinic, and who were on treatment with MMT and 100 (50 pre- and 50 post-menopause age-matched non-diabetic women) were enrolled for this study. Fully automated analyzers and reagents and controls were used for all the assays to ensure the validity of the results obtained. GraphPad online calculator was used to calculate t and p values.Results: MMT for menopause women with T2DM had improved the diabetic parameters, and the levels of fasting plasma glucose (FPG), postprandial plasma glucose (PPPG), and glycosylated hemoglobin (HbA1c) have dropped from 179.48 mg/dL, 270.8 mg/dL, and 8.75% at diagnosis to 135.36, 199.45, and 7.05, respectively, after 12 months of MMT. In the case of post-menopause, the corresponding levels were 180.6, 263.2, and 8.85 to 133.79, 189.06, and 6.68%, respectively, and all the five organ functions were also not altered as the parameters tested for each organ function returned to normal. The drop in all the parameters levels was significant compared to the levels at diagnosis and the p values ranged from &lt;0.01 to&lt;0.0001.Conclusion: Metformin treatment has shown very good improvement in the progressive reductions in the levels of FPG, PPPG, and HbA1c as well as maintaining the functions of kidney, cardiac, liver, thyroid, and reproductive organs to near normal as per the associations of these organ-specific parameters before and after 6 and 12 months of MMT

THE EFFECT OF METFORMIN MONOTHERAPY ON BIOCHEMICAL PARAMETERS ASSOCIATED WITH DIABETES MELLITUS, KIDNEY, CARDIAC, LIVER, THYROID, AND REPRODUCTIVE ORGAN FUNCTIONS IN PRE- AND POST-MENOPAUSE WOMEN WITH TYPE 2 DIABETES MELLITUS

 Objective: The aim of this study was to find out the alterations in biochemical parameters associated with the functioning of kidney, cardiac, liver, thyroid, and reproductive organs in pre- and post-menopause Type 2 diabetes mellitus (T2DM) women treated with metformin monotherapy (MMT) and to select a set of biochemical tests as routine to evaluate the functions of the above organs at regular intervals of time based on the levels of various biochemical parameters during a period of 1 year at 6 and 12 months.Method: A total of 100 T2DM patients (50 pre- and 50 post-menopause women) who visited Apollo Speciality Hospitals Vanagaram, Sugar Clinic, and who were on treatment with MMT and 100 (50 pre- and 50 post-menopause age-matched non-diabetic women) were enrolled for this study. Fully automated analyzers and reagents and controls were used for all the assays to ensure the validity of the results obtained. GraphPad online calculator was used to calculate t and p values.Results: MMT for menopause women with T2DM had improved the diabetic parameters, and the levels of fasting plasma glucose (FPG), postprandial plasma glucose (PPPG), and glycosylated hemoglobin (HbA1c) have dropped from 179.48 mg/dL, 270.8 mg/dL, and 8.75% at diagnosis to 135.36, 199.45, and 7.05, respectively, after 12 months of MMT. In the case of post-menopause, the corresponding levels were 180.6, 263.2, and 8.85 to 133.79, 189.06, and 6.68%, respectively, and all the five organ functions were also not altered as the parameters tested for each organ function returned to normal. The drop in all the parameters levels was significant compared to the levels at diagnosis and the p values ranged from &lt;0.01 to&lt;0.0001.Conclusion: Metformin treatment has shown very good improvement in the progressive reductions in the levels of FPG, PPPG, and HbA1c as well as maintaining the functions of kidney, cardiac, liver, thyroid, and reproductive organs to near normal as per the associations of these organ-specific parameters before and after 6 and 12 months of MMT

In Vitro Antileishmanial Activity of Aloe Vera Leaf Exudate: A Potential Herbal Therapy in leishmaniasis

Aloe vera has wide spread use in health products, and despite several reports on the whole plant and inner gel, little work has been performed on the leaf exudate. Our aim was to evaluate the in vitro efficacy of Aloe vera leaf exudate (AVL) in leishmaniasis. Irrespective of the disease manifestation, promastigotes from strains responsible for cutaneous, mucocutaneous, and visceral leishmaniasis were susceptible to AVL and their IC50 ranged from 100 to 180 μg/ml. In axenic amastigotes cultured from a L. donovani strain 2001 responsible for visceral leishmaniasis, the IC50 was 6.0 μg/ml. AVL caused activation of host macrophages evident by an increased release of members of reactive oxygen species that was attenuated by preincubation with free radical scavengers. Collectively, our data indicates that AVL, via its direct leishmanicidal activity which can be further enhanced by activation of host macrophages, isan effective antileishmanial agent meriting further pharmacological investigations