Dihydroartemisinin, an active metabolite of artemisinin, interferes with Leishmania braziliensis mitochondrial bioenergetics and survival.

Leishmaniasis is one of the most neglected parasitic infections of the world and current therapeutic options show several limitations. In the search for more effective drugs, plant compounds represent a powerful natural source. Artemisinin is a sesquiterpene lactone extracted from Artemisia annua L. leaves, from which dihydroartemisinin (DQHS) and artesunic acid (AA)/artesunate are examples of active derivatives. These lactones have been applied successfully on malaria therapy for decades. Herein, we investigated the sensitivity of Leishmania braziliensis, one of the most prevalent Leishmania species that cause cutaneous manifestations in the New World, to artemisinin, DQHS, and AA. L. braziliensis promastigotes and the stage that is targeted for therapy, intracelular amastigotes, were more sensitive to DQHS, showing EC50 of 62.3 ± 1.8 and 8.9 ± 0.9 µM, respectively. Cytotoxicity assays showed that 50% of bone marrow-derived macrophages cultures were inhibited with 292.8 ± 3.8 µM of artemisinin, 236.2 ± 4.0 µM of DQHS, and 396.8 ± 6.7 µM of AA. The control of intracellular infection may not be essentially attributed to the production of nitric oxide. However, direct effects on mitochondrial bioenergetics and H2O2 production appear to be associated with the leishmanicidal effect of DQHS. Our data provide support for further studies of artemisinin and derivatives repositioning for experimental leishmaniasis.

Cruzioseptins, antibacterial peptides from Cruziohyla calcarifer skin, as promising leishmanicidal agents.

Screenings of natural products have significantly contributed to the discovery of novel leishmanicidal agents. In this study, three known cruzioseptins-antibacterial peptides from Cruziohyla calcarifer skin-were synthesized and evaluated against promastigotes and amastigotes stages of Leishmania (L.) amazonensis and L. (V.) braziliensis. EC50 ranged from 9.17 to 74.82 µM, being cruzioseptin-1 the most active and selective compound, with selectivity index > 10 for both promastigotes and amastigotes of L. (V.) braziliensis. In vitro infections incubated with cruzioseptins at 50 µM showed up to ∼86% reduction in the amastigote number. Cruzioseptins were able to destabilize the parasite's cell membrane, allowing the incorporation of a DNA-fluorescent dye. Our data also demonstrated that hydrophobicity and charge appear to be advantageous features for enhancing parasiticidal activity. Antimicrobial cruzioseptins are suitable candidates and alternative molecules that deserve further in vivo investigation focusing on the development of novel antileishmanial therapies.

Potential use of 13-mer peptides based on phospholipase and oligoarginine as leishmanicidal agents.

Phospholipase A2 toxins present in snake venoms interact with biological membranes and serve as structural models for the design of small peptides with anticancer, antibacterial and antiparasitic properties. Oligoarginine peptides are capable of increasing cell membrane permeability (cell penetrating peptides), and for this reason are interesting delivery systems for compounds of pharmacological interest. Inspired by these two families of bioactive molecules, we have synthesized two 13-mer peptides as potential antileishmanial leads gaining insights into structural features useful for the future design of more potent peptides. The peptides included p-Acl, reproducing a natural segment of a Lys49 PLA2 from Agkistrodon contortrix laticinctus snake venom, and its p-AclR7 analogue where all seven lysine residues were replaced by arginines. Both peptides were active against promastigote and amastigote forms of Leishmania (L.) amazonensis and L. (L.) infantum, while displaying low cytotoxicity for primary murine macrophages. Spectrofluorimetric studies suggest that permeabilization of the parasite's cell membrane is the probable mechanism of action of these biomolecules. Relevantly, the engineered peptide p-AclR7 was more active in both life stages of Leishmania and induced higher rates of ethidium bromide incorporation than its native template p-Acl. Taken together, the results suggest that short peptides based on phospholipase toxins are potential scaffolds for development of antileishmanial candidates. Moreover, specific amino acid substitutions, such those herein employed, may enhance the antiparasitic action of these cationic peptides, encouraging their future biomedical applications.

Melatonin and Docosahexaenoic Acid Decrease Proliferation of PNT1A Prostate Benign Cells via Modulation of Mitochondrial Bioenergetics and ROS Production.

Prostate cancer development has been associated with changes in mitochondrial activity and reactive oxygen species (ROS) production. Melatonin (MLT) and docosahexaenoic acid (DHA) have properties to modulate both, but their protective role, mainly at early stages of prostate cancer, remains unclear. In this study, the effects of MLT and DHA, combined or not, on PNT1A cells with regard to mitochondria bioenergetics, ROS production, and proliferation-related pathways were examined. Based on dose response and lipid accumulation assays, DHA at 100 µM and MLT at 1 µM for 48 h were chosen. DHA doubled and MLT reduced (40%) superoxide anion production, but coincubation (DM) did not normalize to control. Hydrogen peroxide production decreased after MLT incubation only (p < 0.01). These alterations affected the area and perimeter of mitochondria, since DHA increased whereas MLT decreased, but such hormone has no effect on coincubation. DHA isolated did not change the oxidative phosphorylation rate (OXPHOS), but decreased (p < 0.001) the mitochondrial bioenergetic reserve capacity (MBRC) which is closely related to cell responsiveness to stress conditions. MLT, regardless of DHA, ameliorated OXPHOS and recovered MBRC after coincubation. All incubations decreased AKT phosphorylation; however, only MLT alone inhibited p-mTOR. MLT increased p-ERK1/2 and, when combined to DHA, increased GSTP1 expression (p < 0.01). DHA did not change the testosterone levels in the medium, whereas MLT alone or coincubated decreased by about 20%; however, any incubation affected AR expression. Moreover, incubation with luzindole revealed that MLT effects were MTR1/2-independent. In conclusion, DHA increased ROS production and impaired mitochondrial function which was probably related to AKT inactivation; MLT improved OXPHOS and decreased ROS which was related to AKT/mTOR dephosphorylation, and when coincubated, the antiproliferative action was related to mitochondrial bioenergetic modulation associated to AKT and ERK1/2 regulation. Together, these findings point to the potential application of DHA and MLT towards the prevention of proliferative prostate diseases.

Environmentally Realistic Doses of Cadmium as a Possible Etiologic Agent for Idiopathic Pathologies.

Cadmium is a heavy metal of increasing environmental concern that has long been associated to several human pathological processes. Recent population surveys have correlated cadmium non-occupational exposure to widespread idiopathic pathologies. Food and tobacco are reported to be the main exposure sources of cadmium to the general population, as phosphate fertilizers are rich in such a metal, thus contaminating the crops. Although its mechanisms of toxicity are not a consensus in the literature, it is well established that reactive oxygen species play a key role in this process, leading to the oxidation of several biological molecules. We have therefore assessed whether three environmentally realistic doses of cadmium alter the oxidative status of Wistar rat testis and eventually result in histological damages. Our results show that even the lowest environmental dose of cadmium was able to disturb the endogenous antioxidant system in Wistar testis, although an increase in lipid peroxidation was observed only within the group exposed to the highest environmental dose. Despite that no remarkable morphological changes were observed in any group, significant alterations in blood vessel lumen were reported for some cadmium-exposed animals, suggesting that endothelium is one of the primary targets involved in cadmium toxicity.

Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability.

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.

Redox metabolism in Trypanosoma cruzi: functional characterization of tryparedoxins revisited.

Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to various thiol proteins. In Trypanosoma cruzi, two genes coding for TXN-like proteins have been identified: TXNI, previously characterized as a cytoplasmic protein, and TXNII, a putative tail-anchored membrane protein. In this work, we performed a comparative functional characterization of T. cruzi TXNs. Particularly, we cloned the gene region coding for the soluble version of TXNII for its heterologous expression. The truncated recombinant protein (without its 22 C-terminal transmembrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione, or dihydrolipoamide to various acceptors, including methionine sulfoxide reductases and peroxiredoxins. The results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in the redox scenario for T. cruzi.

Trypanosoma cruzi MSH2: Functional analyses on different parasite strains provide evidences for a role on the oxidative stress response.

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei.

Cytotoxicity of goniothalamin enantiomers in renal cancer cells: involvement of nitric oxide, apoptosis and autophagy.

Goniothalamin is a styryllactone synthesized by plants of the genus Goniothalamus. The biological activities of this molecule, particularly its anti-protozoan, anti-fungal, and larvicidal properties, have received considerable attention. In this work, we investigated the action of the natural and synthetic enantiomers (R)-goniothalamin (1) and (S)-goniothalamin (ent-1) on cell viability, nitric oxide synthase (NOS) expression and activity, and the expression of selected proteins involved in apoptosis and autophagy in renal cancer cells. Both compounds were cytotoxic and decreased the mitochondrial function of renal cancer cells. However, the enantiomers differentially affected the expression/activity profiles of some signaling pathway mediators. Ent-1 (4 nM) was more potent than 1 (6.4 microM) in inhibiting constitutive NOS activity (54% and 59% inhibition, respectively), and both enantiomers decreased the protein expression of neuronal and endothelial NOS, as assessed by western blotting. Ent-1 and 1 caused down-regulation of Ras and TNFR1 and inhibition of protein serine/threonine phosphatase 2A (PP2A). Compound 1 markedly down-regulated Bcl2, an anti-apoptotic protein, and also induced PARP cleavage. Despite inducing an expressive down-regulation of Bax, ent-1 was also able to induce PARP cleavage. These results suggest that these compounds caused apoptosis in renal cancer cells. Interestingly, ent-1 enhanced the expression of LC3, a typical marker of autophagy. NFkappaB was down-regulated in 1-treated cells. Overall, these results indicate that the anti-proliferative activity of the two enantiomers on renal cancer cells involved distinct signaling pathways, apoptosis and autophagy as dominant responses towards 1 and ent-1, respectively.

A promising action of riboflavin as a mediator of leukaemia cell death.

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at microM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.