Iron-saturated bovine lactoferrin: a promising chemopreventive agent for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a tumor with minimal chance of cure due to underlying liver diseases, late diagnosis, and inefficient treatments. Thus, HCC treatment warrants the development of additional strategies. Lactoferrin (Lf) is a mammalian multifunctional iron-binding glycoprotein of the innate immune response and can be found as either a native low iron form (native-Lf) or a high iron form (holo-Lf). Bovine Lf (bLf), which shares many functions with human Lf (hLf), is safe for humans and has several anticancer activities, including chemotherapy boost in cancer. We found endogenous hLf is downregulated in HCC tumors compared with normal liver, and decreased hLf levels in HCC tumors are associated with shorter survival of HCC patients. However, the chemoprotective effect of 100% iron saturated holo-bLf on experimental hepatocarcinogenesis has not yet been determined. We aimed to evaluate the chemopreventive effects of holo-bLf in different HCC models. Remarkably, a single dose (200 mg kg-1) of holo-bLf was effective in preventing early carcinogenic events in a diethylnitrosamine induced HCC in vivo model, such as necrosis, ROS production, and the surge of facultative liver stem cells, and eventually, holo-bLf reduced the number of preneoplastic lesions. For an established HCC model, holo-bLf treatment significantly reduced HepG2 tumor burden in xenotransplanted mice. Finally, holo-bLf in combination with sorafenib, the advanced HCC first-line treatment, synergistically decreased HepG2 viability by arresting cells in the G0/G1 phase of the cell cycle. Our findings provide the first evidence suggesting that holo-bLf has the potential to prevent HCC or to be used in combination with treatments for established HCC.

A model of alcoholic liver disease based on different hepatotoxics leading to liver cancer.

The worst-case scenario related to alcoholic liver disease (ALD) arises after a long period of exposure to the harmful effect of alcohol consumption along with other hepatotoxics. ALD encompasses a broad spectrum of liver-associated disorders, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Based on the chronic administration of different hepatotoxics, including ethanol, sucrose, lipopolysaccharide, and low doses of diethylnitrosamine over a short period, here we aimed to develop a multiple hepatotoxic (MHT)-ALD model in the mouse that recapitulates the human ALD-associated disorders. We demonstrated that the MHT-ALD model induces ADH1A and NXN, an ethanol metabolizer and a redox-sensor enzyme, respectively; promotes steatosis associated with the induction of the lipid droplet forming FSP27, inflammation identified by the infiltration of hepatic neutrophils-positive to LY-6G marker, and the increase of MYD88 level, a protein involved in inflammatory response; and stimulates the early appearance of cellular senescence identified by the senescence markers SA-ß-gal activity and p-H2A.XSer139. It also induces fibrosis associated with increased desmin, a marker of hepatic stellate cells whose activation leads to the deposition of collagen fibers, accompanied by cell death and compensatory proliferation revealed by increased CASP3-mediated apoptosis, and KI67- and PCNA-proliferation markers, respectively. It also induces histopathological traits of malignancy and the level of the HCC marker, GSTP1. In conclusion, we provide a useful model for exploring the chronological ALD-associated alterations and stages, and addressing therapeutic approaches.

DCLK1 is Overexpressed and Associated with Immune Cell Infiltration in Hepatocellular Carcinoma.

Recent research has shown that Doublecortin-like kinase 1 (DCLK1) is overexpressed in different types of cancer. It has recently been described as a cancer stem cells (CSCs) marker, is associated with carcinogenesis, and positively correlates with infiltration of multiple immune cell types in some cancers. However, studies focused on assessing DCLK1 expression in HCC are limited, and the role of DCLK1 in HCC tumor immunity remains to be determined. In this study, we used a modified model of the resistant hepatocyte (MRHM) to evaluate DCLK1 expression in HCC. Furthermore, DCLK1 expression in HCC was analyzed using TIMER 2.0, UALCAN, GEPIA, GEO, and HPA web-based tools. Correlations between DCLK1 expression and clinicopathological factors in patients were analyzed using the UALCAN web-based tool. Finally, correlations between DCLK1 and immune infiltrates were investigated using the TIMER 2.0 and TISIDB web-based tools. The results showed that DCLK1 is significantly overexpressed during progression of the HCC carcinogenic process in the MRHM. DCLK1 is overexpressed in HCC according to multiple publics web-based tools, and its overexpression is associated with cancer stage. Furthermore, DCLK1 expression was correlated with infiltration levels of multiple immune cells, immunomodulatory factors, immunoinhibitors, MHC molecules, chemokines, receptors, and immune cell-specific markers. These results suggest that DCLK1 is a potential prognostic biomarker that determines cancer progression and correlates with immune cell infiltration in HCC.

The CD44std and CD44v9 subpopulations in non-tumorigenic invasive SNU-423 cells present different features of cancer stem cells.

Hepatocellular carcinoma (HCC) is a type of liver cancer, in which CD44 isoforms have been proposed as markers to identify cancer stem cells (CSCs). However, it is unclear what characteristics are associated with CSCs that exclusively express CD44 isoforms. The objective of the present study was to determine the expression of CD44 isoforms and their properties in CSCs. Analysis of transcriptomic data from HCC patient samples identified CD44v8-10 as a potential marker in HCC. In SNU-423 cells, CD44 expression was detected in over 99% of cells, and two CD44 isoforms, namely, CD44std and CD44v9, were identified in this cell line. CD44 subpopulations, including both CD44v9+ (CD44v9) and CD44v9- (CD44std) cells, were obtained by purification using a magnetic cell separation kit for human CD44v9+ cancer stem cells. CD44v9 cells showed greater potential for colony and spheroid formation, whereas CD44std cells demonstrated significant migration and invasion capabilities. These findings suggested that CD44std and CD44v9 may be used to identify features in CSC populations and provide insights into their roles in HCC.

EP300 as a Molecular Integrator of Fibrotic Transcriptional Programs.

Fibrosis is a condition characterized by the excessive accumulation of extracellular matrix proteins in tissues, leading to organ dysfunction and failure. Recent studies have identified EP300, a histone acetyltransferase, as a crucial regulator of the epigenetic changes that contribute to fibrosis. In fact, EP300-mediated acetylation of histones alters global chromatin structure and gene expression, promoting the development and progression of fibrosis. Here, we review the role of EP300-mediated epigenetic regulation in multi-organ fibrosis and its potential as a therapeutic target. We discuss the preclinical evidence that suggests that EP300 inhibition can attenuate fibrosis-related molecular processes, including extracellular matrix deposition, inflammation, and epithelial-to-mesenchymal transition. We also highlight the contributions of small molecule inhibitors and gene therapy approaches targeting EP300 as novel therapies against fibrosis.

Hemoglobin uptake and utilization by human protozoan parasites: a review.

The protozoan disease is a major global health concern. Amoebiasis, leishmaniasis, Chagas disease, and African sleeping sickness affect several million people worldwide, leading to millions of deaths annually and immense social and economic problems. Iron is an essential nutrient for nearly all microbes, including invading pathogens. The majority of iron in mammalian hosts is stored intracellularly in proteins, such as ferritin and hemoglobin (Hb). Hb, present in blood erythrocytes, is a very important source of iron and amino acids for pathogenic microorganisms ranging from bacteria to eukaryotic pathogens, such as worms, protozoa, yeast, and fungi. These organisms have developed adequate mechanisms to obtain Hb or its byproducts (heme and globin) from the host. One of the major virulence factors identified in parasites is parasite-derived proteases, essential for host tissue degradation, immune evasion, and nutrient acquisition. The production of Hb-degrading proteases is a Hb uptake mechanism that degrades globin in amino acids and facilitates heme release. This review aims to provide an overview of the Hb and heme-uptake mechanisms utilized by human pathogenic protozoa to survive inside the host.

Endocytosis, signal transduction and proteolytic cleaving of human holotransferrin in Entamoeba histolytica.

Amoebiasis is a parasitic infection of the human large intestine caused by Entamoeba histolytica; this disease mainly affects people from developing countries. To survive, this primitive protozoan has a high demand for iron, and it uses host iron proteins upon invasion. Transferrin (Tf) is a plasma iron-binding protein that transports and delivers iron to all cells. Iron-loaded Tf (holoTf) in humans can support the proliferation of amoebae in vitro by binding to an amoebic TfR (EhTfR), and amoebae endocytose it inside clathrin-coated vesicles. In this study, it was found that EhTfR phosphorylation is required for human holoTf endocytosis by E. histolytica. Once this complex is endocytosed, human holoTf could be degraded with a nutritional purpose by cysteine proteases. HoloTf endocytosis initiates the activation of the mitogen-activated protein kinases (MAPKs) and focal adhesion kinase (FAK) pathways, which induce cell proliferation with phosphoinositide 3-kinase (PI-3 K) and Ca2+ involvement. In the first minutes after holoTf is endocytosed, several proteins are phosphorylated including transketolase, enolase, L-myo-inositol-1-phosphate synthase and phosphoglucomutase, which control carbohydrate metabolism, and heat shock protein-70. The study of these proteins and their signal transduction pathways could be useful for developing future therapies.

Road to stemness in hepatocellular carcinoma.

Carcinogenic process has been proposed to relay on the capacity to induce local tissue damage and proliferative repair. Liver has a great regeneration capacity and currently, most studies point towards the dominant role of hepatocytes in regeneration at all levels of liver damage. The most frequent liver cancer is hepatocellular carcinoma (HCC). Historical findings originally led to the idea that the cell of origin of HCC might be a progenitor cell. However, current linage tracing studies put the progenitor hypothesis of HCC origin into question. In agreement with their dominant role in liver regeneration, mature hepatocytes are emerging as the cell of origin of HCC, although, the specific hepatocyte subpopulation of origin is yet to be determined. The relationship between the cancer cell of origin (CCO) and cancer-propagating cells, known as hepatic cancer stem cell (HCSC) is unknown. It has been challenging to identify the definitive phenotypic marker of HCSC, probably due to the existence of different cancer stem cells (CSC) subpopulations with different functions within HCC. There is a dynamic interconversion among different CSCs, and between CSC and non-CSCs. Because of that, CSC-state is currently defined as a description of a highly adaptable and dynamic intrinsic property of tumor cells, instead of a static subpopulation of a tumor. Altered conditions could trigger the gain of stemness, some of them include: EMT-MET, epigenetics, microenvironment and selective stimulus such as chemotherapy. This CSC heterogeneity and dynamism makes them out reach from therapeutic protocols directed to a single target. A further avenue of research in this line will be to uncover mechanisms that trigger this interconversion of cell populations within tumors and target it.

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1.

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 µM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa.

Iron is the fourth most abundant element on Earth and the most abundant metal in the human body. This element is crucial for life because almost all organisms need iron for several biological activities. This is the case with pathogenic organisms, which are at the vanguard in the battle with the human host for iron. The latest regulates Fe concentration through several iron-containing proteins, such as transferrin. The transferrin receptor transports iron to each cell that needs it and maintains it away from pathogens. Parasites have developed several strategies to obtain iron as the expression of specific transferrin receptors localized on plasma membrane, internalized through endocytosis. Signal transduction pathways related to the activation of the receptor have functional importance in proliferation. The study of transferrin receptors and other proteins with action in the signaling networks is important because these proteins could be used as therapeutic targets due to their specificity or to differences with the human counterpart. In this work, we describe proteins that participate in signal transduction processes, especially those that involve transferrin endocytosis, and we compare these processes with those found in T. brucei, T. cruzi, Leishmania spp., and E. histolytica parasites.

Identification of phosphatidylcholine transfer protein-like in the parasite Entamoeba histolytica.

Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells.

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors.

The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.

Host-parasite interaction: parasite-derived and -induced proteases that degrade human extracellular matrix.

Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina). The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa.

Iron-saturated lactoferrin and pathogenic protozoa: could this protein be an iron source for their parasitic style of life?

Iron is an essential nutrient for the survival of pathogens inside a host. As a general strategy against microbes, mammals have evolved complex iron-withholding systems for efficiently decreasing the iron accessible to invaders. Pathogens that inhabit the respiratory, intestinal and genitourinary tracts encounter an iron-deficient environment on the mucosal surface, where ferric iron is chelated by lactoferrin, an extracellular glycoprotein of the innate immune system. However, parasitic protozoa have developed several mechanisms to obtain iron from host holo-lactoferrin. Tritrichomonas fetus, Trichomonas vaginalis, Toxoplasma gondii and Entamoeba histolytica express lactoferrin-binding proteins and use holo-lactoferrin as an iron source for growth in vitro; in some species, these binding proteins are immunogenic and, therefore, may serve as potential vaccine targets. Another mechanism to acquire lactoferrin iron has been reported in Leishmania spp. promastigotes, which use a surface reductase to recognize and reduce ferric iron to the accessible ferrous form. Cysteine proteases that cleave lactoferrin have been reported in E. histolytica. This review summarizes the available information on how parasites uptake and use the iron from lactoferrin to survive in hostile host environments.

Use and endocytosis of iron-containing proteins by Entamoeba histolytica trophozoites.

Iron is essential for nearly all organisms; in mammals, it is part of proteins such as haemoglobin, and it is captured by transferrin and lactoferrin. Transferrin is present in serum, and lactoferrin is secreted by the mucosa and by neutrophils at infection sites, as a host iron-withholding response, sequestering iron away from invading microorganisms. Additionally, all cells contain ferritin, which sequesters iron when its intracellular levels are increased, detoxifying and preventing damage. Liver ferritin contains 50% of iron corporal reserves. During evolution, pathogens have evolved diverse strategies to obtain iron from their hosts in order to survive. The protozoan Entamoeba histolytica invades the intestinal mucosa, causing dysentery, and the trophozoites often travel to the liver producing hepatic abscesses; thus, intestine and liver proteins could be important iron supplies for E. histolytica. We found that E. histolytica trophozoites can grow in both ferrous and ferric iron, and that they can use haemoglobin, holo-transferrin, holo-lactoferrin, and ferritin as in vitro iron sources. These proteins supported the amoeba growth throughout consecutive passages, similarly to ferric citrate. By confocal microscopy and immunoblotting, iron-binding proteins were observed specifically bound to the amoeba surface, and they were endocytosed, trafficked through the endosomal/lysosomal route, and degraded by neutral and acidic cysteine-proteases. Transferrin and ferritin were mainly internalized through clathrin-coated vesicles, and holo-lactoferrin was mainly internalized by caveola-like structures. In contrast, apo-lactoferrin bound to membrane lipids and cholesterol, inducing cell death. The results suggest that in vivo trophozoites secrete products that can destroy enterocytes, erythrocytes, and hepatocytes, releasing transferrin, haemoglobin, ferritin, and other iron-containing proteins, which, together with lactoferrin derived from neutrophils and acinar cells, could be used as abundant iron supplies by amoebas.

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles.

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.

A quantitative competitive PCR method to determine the parasite load in the brain of Toxoplasma gondii-infected mice.

Efficacy of vaccine candidates against toxoplasmosis may be expressed in terms of reduction in cyst number in brains of animals vaccinated and then challenged with a cyst-forming strain of Toxoplasma gondii, compared to non-vaccinated animals. Cyst number generally has been determined by microscopic examination of brain homogenate samples, a technique which has a low sensitivity and is time-consuming. Here we describe a quantitative competitive PCR method, which allows quantifying T. gondii DNA in brain samples. The method uses a primer pair, which allows the amplification of a 301 bp fragment of the 35-fold repeated T. gondii B1 gene and an internal standard (non-homologous competitor) derived from phage lambda, which can be amplified using the same primers and whose size and G/C content are similar to that of the B1 target sequence. The method is sensitive (as few as 10 parasites can be quantified), reproducible, and is not affected by the presence of DNA extracted from mouse brain by means of a simple and rapid technique. It is suitable to quantify the parasite load in the brain of infected mice and to evaluate efficacy of toxoplasmosis vaccine candidates.