Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines.

BACKGROUND: Lung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PURPOSE: This study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. METHODS: TBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. RESULTS: In silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. CONCLUSION: These findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Larval excretion/secretion of dipters of Lucilia cuprina species induces death in promastigote and amastigote forms of Leishmania amazonensis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Larval excretion/secretion (ES) of the larvae of flies of the Calliphoridae family has microbicidal activity against Gram-positive and Gram-negative bacteria, in addition to some species of Leishmania. Our study aimed at assessing the in vitro efficacy of Lucilia cuprina larval ES against the promastigote and amastigote forms of Leishmania amazonensis, elucidating possible microbicidal mechanisms and routes of death involved. Larval ES was able to inhibit the viability of L. amazonensis at all concentrations, induce morphological and ultrastructural changes in the parasite, retraction of the cell body, roughness of the cytoplasmic membrane, leakage of intracellular content, ROS production increase, induction of membrane depolarization and mitochondrial swelling, the formation of cytoplasmic lipid droplets and phosphatidylserine exposure, thus indicating the possibility of apoptosis-like death. To verify the efficacy of larval ES on amastigote forms, we performed a phagocytic assay, measurement of total ROS and NO. Treatment using larval ES reduced the percentage of infection and the number of amastigotes per macrophage of lineage J774A.1 at all concentrations, increasing the production of ROS and TNF-α, thus indicating possible pro-inflammatory immunomodulation and oxidative damage. Therefore, treatment using larval ES is effective at inducing the death of promastigotes and amastigotes of L. amazonensis even at low concentrations.

4-nitrochalcone exerts leishmanicidal effect on L. amazonensis promastigotes and intracellular amastigotes, and the 4-nitrochalcone encapsulation in beeswax copaiba oil nanoparticles reduces macrophages cytotoxicity.

The Leishmaniasis treatment currently available involves some difficulties, such as high toxicity, variable efficacy, high cost, therefore, it is crucial to search for new therapeutic alternatives. Over the past few years, research on new drugs has focused on the use of natural compounds such as chalcones and nanotechnology. In this context, this research aimed at assessing the in vitro leishmanicidal activity of free 4-nitrochalcone (4NC) on promastigotes and encapsulated 4NC on L. amazonensis-infected macrophages, as well as their action mechanisms. Free 4NC was able to reduce the viability of promastigotes, induce reactive oxygen species production, decrease mitochondrial membrane potential, increase plasma membrane permeability, and expose phosphatidylserine, in addition to altering the morphology and lowering parasite cellular volume. Treatment containing encapsulated 4NC in beeswax-copaiba oil nanoparticles (4NC-beeswax-CO Nps) did not alter the viability of macrophages. Furthermore, 4NC-beeswax-CO Nps reduced the percentage of infected macrophages and the number of amastigotes per macrophages, increasing the production of reactive oxygen species, NO, TNF-α, and IL-10. Therefore, free 4NC proved to exert anti-promastigote effect, while 4NC-beeswax-CO Nps showed a leishmanicidal effect on L. amazonensis-infected macrophages by activating the macrophage microbicidal machinery.

Diethyldithiocarbamate encapsulation reduces toxicity and promotes leishmanicidal effect through apoptosis-like mechanism in promastigote and ROS production by macrophage.

The use of compounds from natural or synthetic sources and nanotechnology may represent an alternative to develop new drugs for the leishmaniasis treatment. DETC is an inhibitor of the SOD1 enzyme, which leads to increased ROS production, important for the elimination of Leishmania. Thus, our objective was to assess the leishmanicidal in vitro effect of free Diethydithiocarbamate (DETC) and DETC loaded in beeswax-copaiba oil nanoparticles (DETC-Beeswax-CO Nps) on L. amazonensis forms and elucidate the possible mechanisms involved in the parasite death. DETC-Beeswax-CO Nps presented size below 200 nm, spherical morphology, negative zeta potential, and high encapsulation efficiency. Free DETC reduced the viability of promastigotes and increase ROS production, lower the mitochondrial membrane potential, cause phosphatidylserine exposure, and enhance plasma membrane permeability, in addition to promoting morphological changes in the parasite. Free DETC proved toxic in the assessment of toxicity to murine macrophages, however, the encapsulation of this compound was able to reduce these toxic effects on macrophages. DETC-Beeswax-CO Nps exerted anti-amastigote effect by enhancing the production of ROS, superoxide anion, TNF-α, IL-6, and reduced IL-10 in macrophages. Therefore, free DETC induces antipromastigote effect by apoptosis-like; and DETC-Beeswax-CO Nps exerted anti-leishmanial effect due to pro-oxidant and pro-inflammatory response.

Grandiflorenic acid promotes death of promastigotes via apoptosis-like mechanism and affects amastigotes by increasing total iron bound capacity.

BACKGROUND: American tegumentary leishmaniasis (ATL) is a zoonotic disease caused by protozoa of the genus Leishmania. The high toxicity, high costs and resistance of some strains to current drugs has prompted the search for therapeutic alternatives for the management of this disease. Sphagneticola trilobata is a plant that has diterpenes as main constituents, including grandiflorenic acid (GFA) that has antiinflammatory, antiprotozoal, antibacterial and antinociceptive activity. PURPOSE: The aim of our study was to determine the effect of GFA on both the promastigotes and the amastigotes of Leishmania amazonensis. METHODS: Isolation by chromatographic methods and chemical identification of GFA, then evaluation of the in vitro leishmanicidal activity of this compound against Leishmania amazonensis promastigotes and L. amazonensis infected peritoneal Balb/c macrophages, as well its action and microbicide mechanisms. RESULTS: GFA treatment significantly inhibited the proliferation of promastigotes. This antiproliferative effect was accompanied by morphological changes in the parasite with 25 nM GFA. Afterwards, we investigated the mechanisms involved in the death of the protozoan; there was an increase in the production of reactive oxygen species (ROS), phosphatidylserine exposure, permeabilization of the plasma membrane and decreased mitochondrial depolarization. In addition, we observed that the treatment caused a reduction in the percentage of infected cells and the number of amastigotes per macrophage, without showing cytotoxicity in low doses to peritoneal macrophages and sheep erythrocytes. GFA increased IL-10 and total iron bound to transferrin in infected macrophages. Our results showed that GFA treatment acts on promastigote forms through an apoptosis-like mechanism and on intracellular amastigote forms, dependent of regulatory cytokine IL-10 modulation with increase in total iron bound to transferrin. CONCLUSION: GFA showed in vitro antileishmanial activity on L. amazonensis promastigotes forms and on L. amazonensis-infected macrophages.

Dehydroabietic acid isolated from Pinus elliottii exerts in vitro antileishmanial action by pro-oxidant effect, inducing ROS production in promastigote and downregulating Nrf2/ferritin expression in amastigote forms of Leishmania amazonensis.

Dehydroabietic acid (DHA) is one of the main constituents of the resin that have antiprotozoal activity against Leishmania spp., but the leishmanicidal mechanism is unknown. The objective of the study was to investigate in vitro the leishmanicidal activity of the natural compound DHA against intracellular and extracellular forms of L. amazonensis and the mechanism of action involved. The antileishmanial activity of DHA was evaluated in vitro against promastigote forms of L. amazonensis by counting in Neubauer chamber. The morphological changes were observed by scanning electron microscopy and cell death mechanism by fluorescence assay using 2',7'-dichlorofluorescein diacetate probe (H2DCFDA), tetramethylrhodamine ethyl ester (TMRE), annexin-V and propidium iodide (PI). The antiamastigote effect was observed by counting the number of amastigotes per macrophage and percentage of infected cells. In addition, reactive oxygen species (ROS) production, nitric oxide (NO), cytokines, free iron and total iron-binding capacity (TIBC), expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and ferritin were evaluated. DHA inhibited the proliferation of promastigotes at all times tested. The compound (IC50, 40 ±â€¯0.1458 µg/mL) altered the morphology of the promastigote forms, caused mitochondrial depolarization, induced ROS production, increased phosphatidylserine exposure and caused loss of plasma membrane integrity. DHA also reduced the number of amastigotes and the percentage of infected macrophages by increasing ROS production, free iron and TIBC, and also caused downregulation of Nrf2 and ferritin expression. DHA was effective in the elimination of L. amazonensis both in its promastigote forms by apoptosis-like mechanisms and intracellular form by ROS production.