Hydrogen Peroxide Generation as an Underlying Response to High Extracellular Inorganic Phosphate (Pi) in Breast Cancer Cells.

According to the growth rate hypothesis (GRH), tumour cells have high inorganic phosphate (Pi) demands due to accelerated proliferation. Compared to healthy individuals, cancer patients present with a nearly 2.5-fold higher Pi serum concentration. In this work, we show that an increasing concentration of Pi had the opposite effect on Pi-transporters only in MDA-MB-231 when compared to other breast cell lines: MCF-7 or MCF10-A (non-tumoural breast cell line). Here, we show for the first time that high extracellular Pi concentration mediates ROS production in TNBC (MDA-MB-231). After a short-time exposure (1 h), Pi hyperpolarizes the mitochondrial membrane, increases mitochondrial ROS generation, impairs oxygen (O2) consumption and increases PKC activity. However, after 24 h Pi-exposure, the source of H2O2 seems to shift from mitochondria to an NADPH oxidase enzyme (NOX), through activation of PKC by H2O2. Exogenous-added H2O2 modulated Pi-transporters the same way as extracellular high Pi, which could be reversed by the addition of the antioxidant N-acetylcysteine (NAC). NAC was also able to abolish Pi-induced Epithelial-mesenchymal transition (EMT), migration and adhesion of MDA-MB-231. We believe that Pi transporters support part of the energy required for the metastatic processes stimulated by Pi and trigger Pi-induced H2O2 production as a signalling response to promote cell migration and adhesion.

Acanthamoeba castellanii phosphate transporter (AcPHS) is important to maintain inorganic phosphate influx and is related to trophozoite metabolic processes.

Acanthamoeba castellanii is a free-living amoeba and the etiological agent of granulomatous amoebic encephalitis and amoebic keratitis. A. castellanii can be present as trophozoites or cysts. The trophozoite is the vegetative form of the cell and has great infective capacity compared to the cysts, which are the dormant form that protect the cell from environmental changes. Phosphate transporters are a group of proteins that are able to internalize inorganic phosphate from the extracellular to intracellular medium. Plasma membrane phosphate transporters are responsible for maintaining phosphate homeostasis, and in some organisms, regulating cellular growth. The aim of this work was to biochemically characterize the plasma membrane phosphate transporter in A. castellanii and its role in cellular growth and metabolism. To measure inorganic phosphate (Pi) uptake, trophozoites were grown in liquid PYG medium at 28 °C for 2 days. The phosphate uptake was measured by the rapid filtration of intact cells incubated with 0.5 µCi of 32Pi for 1 h. The Pi transport was linear as a function of time and exhibited Michaelis-Menten kinetics with a Km = 88.78 ± 6.86 µM Pi and Vmax = 547.5 ± 16.9 Pi × h-1 × 10-6 cells. A. castellanii presented linear phosphate uptake up to 1 h with a cell density ranging from 1 × 105 to 2 × 106 amoeba × ml-1. The Pi uptake was higher in the acidic pH range than in the alkaline range. The oxygen consumption of living trophozoites increased according to Pi addition to the extracellular medium. When the cells were treated with FCCP, no effect from Pi on the oxygen flow was observed. The addition of increasing Pi concentrations not only increased oxygen consumption but also increased the intracellular ATP pool. These phenomena were abolished when the cells were treated with FCCP or exposed to hypoxia. Together, these results reinforce the hypothesis that Pi is a key nutrient for Acanthamoeba castellanii metabolism.

Neutrophil properties in healthy and Leishmania infantum-naturally infected dogs.

Visceral leishmaniasis is a chronic disease that affects humans and dogs as well. Dogs, the domestic reservoir of Leishmania, play a central role in the transmission of visceral leishmaniasis, the most severe form of this disease. Neutrophils are the most abundant leukocytes in blood and interact with the parasite after infection. Here, we evaluate the effector properties of neutrophils from healthy and naturally Leishmania infantum-infected dogs. Our results showed that the parasite induced neutrophil extracellular trap (NET) release from neutrophils in both groups. Additionally, phagocytosis and NETs contributed differently to parasite killing by neutrophils from healthy and infected animals, and IFN-γ, IL-8, IL-4 and TNF-α production by neutrophils from both groups were differentially modulated by the parasite. Our results contribute to a better understanding of the complex role played by neutrophils in canine visceral leishmaniasis, which may favor the development of more effective therapies.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment.

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ±â€¯0.1674 mM Pi and Vmax = 198.6 ±â€¯10.23 Pi × h-1 × mg protein-1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

Resveratrol is active against Leishmania amazonensis: in vitro effect of its association with Amphotericin B.

Resveratrol is a polyphenol found in black grapes and red wine and has many biological activities. In this study, we evaluated the effect of resveratrol alone and in association with amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that resveratrol possesses both antipromastigote and antiamastigote effects, with 50% inhibitory concentrations (IC50s) of 27 and 42 µM, respectively. The association of resveratrol with AMB showed synergy for L. amazonensis amastigotes, as demonstrated by the mean sums of fractional inhibitory index concentration (mean ΣFIC) of 0.483, although for promastigotes, this association was indifferent. Treatment with resveratrol increased the percentage of promastigotes in the sub-G0/G1 phase of the cell cycle, reduced the mitochondrial potential, and showed an elevated choline peak and CH2-to-CH3 ratio in the nuclear magnetic resonance (NMR) spectroscopy analysis; all these features indicate parasite death. Resveratrol also decreased the activity of the enzyme arginase in uninfected and infected macrophages with and without stimulation with interleukin-4 (IL-4), also implicating arginase inhibition in parasite death. The anti-Leishmania effect of resveratrol and its potential synergistic association with AMB indicate that these compounds should be subjected to further studies of drug association therapy in vivo.

Interaction of the monoxenic trypanosomatid Blastocrithidia culicis with the Aedes aegypti salivary gland.

Blastocrithidia culicis is a monoxenous trypanosomatid that inhabits mosquitoes. Although its life cycle in these insects has not been described, we recently demonstrated that B. culicis colonizes the Aedes aegypti digestive tract and is also able to reach the mosquito's haemocoel. In this work, we describe the interaction of B. culicis with the A. aegypti salivary gland in vitro and in vivo. Ultrastructural analysis reveals different steps of the invasion process, beginning with the insertion of the B. culicis anterior flagellum into the basal lamina of the gland and ending with the protozoan inside the salivary gland acini compartment. Carbohydrates are involved in the initial adhesion of B. culicis cells to the salivary glands, as demonstrated by protozoan binding inhibition assays and fluorescent lectin labeling of the trypanosomatid-salivary gland interaction. B. culicis is able to survive after incubation in vitro in the mosquito haemolymph, and trypanosomatid binding to salivary glands was confirmed by the injection of radioactively labeled protozoa into the mosquito haemocoel. These results suggest that salivary gland invasion could be part of the B. culicis life cycle in A. aegypti, raising the possibility that B. culicis can be transmitted by these mosquitoes.

Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of Leishmaniasis.

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PS(POS)) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PS(POS) metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNEL(POS) promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PS(POS) and PS-negative (PS(NEG)) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PS(NEG) promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PS(POS) apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.

Development of a ligand blot assay using biotinylated live cells.

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid-sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions.