Bacteroides fragilis derived metabolites, identified by molecular networking, decrease Salmonella virulence in mice model.

In the gut microbiota, resident bacteria prevent pathogens infection by producing specific metabolites. Among bacteria belonging to phylum Bacteroidota, we have previously shown that Bacteroides fragilis or its cell-free supernatant inhibited in vitro Salmonella Heidelberg translocation. In the present study, we have analyzed this supernatant to identify bioactive molecules after extraction and subsequent fractionation using a semi-preparative reversed-phase Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS). The results indicated that only two fractions (F3 and F4) strongly inhibited S. Heidelberg translocation in a model mimicking the intestinal epithelium. The efficiency of the bioactive fractions was evaluated in BALB/c mice, and the results showed a decrease of S. Heidelberg in Peyer's patches and spleen, associated with a decrease in inflammatory cytokines and neutrophils infiltration. The reduction of the genus Alistipes in mice receiving the fractions could be related to the anti-inflammatory effects of bioactive fractions. Furthermore, these bioactive fractions did not alter the gut microbiota diversity in mice. To further characterize the compounds present in these bioactive fractions, Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS) data were analyzed through molecular networking, highlighting cholic acid (CA) and deoxycholic acid. In vitro, CA had inhibitory activity against the translocation of S. Heidelberg by significantly decreasing the expression of Salmonella virulence genes such as sipA. The bioactive fractions also significantly downregulated the flagellar gene fliC, suggesting the involvement of other active molecules. This study showed the interest to characterize better the metabolites produced by B. fragilis to make them means of fighting pathogenic bacteria by targeting their virulence factor without modifying the gut microbiota.

Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect.

Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines. Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer.

Synthesis and biological properties of Quilamines II, new iron chelators with antiproliferative activities.

To selectively target tumor cells expressing an overactive Polyamine Transport System (PTS), we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, derived from the lead compound HQ1-44, which we named Quilamines II. The structures of four new antiproliferative agents were developed. They differ in the size of the linker (HQ0-44 and HQ2-44) or in the nature of the linker (HQCO-44 and HQCS-44) between a hydroxyquinoline moiety (HQ) and a homospermidine (44) chain, the best polyamine vector. The Quilamines II were obtained after 6 to 9 steps by Michael addition, peptide linkage, and reductive amination or by using the Willgerodt-Kindler reaction. The biological evaluation of these second-generation Quilamines showed that modifying the size of the linker increased the selectivity of these compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0-44 and HQ2-44 highlighted their marked antiproliferative nature on several cancerous cell lines as well as a differential activity on nontransformed cells (fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of electrostatic interaction. We also demonstrated that the HCT116 cell line, originating from a human colon adenocarcinoma, was the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity of HQ1-44 could explain its higher antiproliferative efficiency.

Iron-mediated effect of alcohol on hepatocyte differentiation in HepaRG cells.

The development of alcoholic liver diseases depends on the ability of hepatocyte to proliferate and differentiate in the case of alcohol-induced injury. Our previous work showed an inhibitory effect of alcohol on hepatocyte proliferation. However, the effect of alcohol on hepatocyte differentiation has not yet been precisely characterized. In the present study, we evaluated the effect of alcohol on hepatocyte differentiation in relationship with changes of iron metabolism in HepaRG cells. This unique bipotent human cell line can differentiate into hepatocytes and biliary epithelial cells, paralleling liver development. Results showed that alcohol reduced cell viability, total protein level and enhanced hepatic enzymes leakage in differentiated HepaRG cells. Moreover, it caused cell enlargement, decreased number of hepatocyte and expression of C/EBPα as well as bile canaliculi F-actin. Alcohol increased expression of hepatic cell-specific markers and alcohol-metabolizing enzymes (ADH2, CYP2E1). This was associated with a lipid peroxidation and an iron excess expressed by an increase in total iron content, ferritin level, iron uptake as well as an overexpression of genes involved in iron transport and storage. Alcohol-induced hepatoxicity was amplified by exogenous iron via exceeding iron overload. Taken together, our data demonstrate that in differentiated hepatocytes, alcohol reduces proliferation while increasing expression of hepatic cell-specific markers. Moreover, iron overload could be one of the underlying mechanisms of effect of alcohol on the whole differentiation process of hepatocytes.

Involvement of polyamines in iron(III) transport in human intestinal Caco-2 cell lines.

Natural polyamines such as putrescine (Put), spermidine (Spd), and spermine (Spm), which are present in the human diet in large amounts, associated with their active transporter, are assumed to play a role in non-heme iron uptake and iron bioavailability from nutrients. Enterocytes and hepatocytes play pivotal roles in the regulation of body iron homeostasis. In this study, we report the effects of natural polyamines on iron transport in the Caco-2 cell line. In enterocyte-like Caco-2 cells, polyamines did not significantly modulate the transepithelial iron flux across the cell monolayer cultured on permeable membranes. In contrast, Spd, Spm, and to a lesser extent, Put were shown to activate Caco-2 cell iron uptake and to induce an increase in the ferritin level. This iron co-transport in enterocytes, which involved an interaction between iron and polyamine then cell uptake of the polyamine-iron complexes by the polyamine transport system, was more pronounced in proliferating than in differentiated Caco-2 cells. Moreover, it was observed at physiological concentrations of both polyamines and iron. It could thus play a role in the rapid renewal of enterocytes. These data suggest the involvement of polyamines as components of the pool of transferrin-independent iron-chelating vectors. Further investigations are needed to demonstrate their biological relevance in physiological situations.

Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation.

Iron chelation in tumoral cells has been reported as potentially useful during antitumoral treatment. Our aim was to develop new polyaminoquinoline iron chelators targeting tumoral cells. For this purpose, we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, which we named Quilamines, based on an 8-hydroxyquinoline (8-HQ) scaffold linked to linear polyamine vectors. These were designed to target tumor cells expressing an overactive polyamine transport system (PTS). A set of Quilamines bearing variable polyamine chains was designed and assessed for their ability to interact with iron. Quilamines were also screened for their cytostatic/cytotoxic effects and their selective uptake by the PTS in the CHO cell line. Our results show that both the 8-HQ moiety and the polyamine part participate in the iron coordination. HQ1-44, the most promising Quilamine identified, presents a homospermidine moiety and was shown to be highly taken up by the PTS and to display an efficient antiproliferative activity that occurred in the micromolar range. In addition, cytotoxicity was only observed at concentrations higher than 100 µM. We also demonstrated the high complexation capacity of HQ1-44 with iron while much weaker complexes were formed with other cations, indicative of a high selectivity. We applied the density functional theory to study the binding energy and the electronic structure of prototypical iron(III)-Quilamine complexes. On the basis of these calculations, Quilamine HQ1-44 is a strong tridentate ligand for iron(III) especially in the form of a 1:2 complex.

Ethanol effect on cell proliferation in the human hepatoma HepaRG cell line: relationship with iron metabolism.

BACKGROUND: Alcoholism increases the risk of cirrhosis and/or hepatocellular carcinoma development. Iron, like ethanol, modulates the cell growth. However, the relationship between alcohol and iron toward hepatocyte proliferation has not been clearly elucidated. The purpose of this study was to evaluate, in the human HepaRG cell line model, the impact of ethanol on hepatocyte proliferation in relation to modulations of iron metabolism and the protective effect of iron metabolism manipulation by chelators in alcohol liver diseases. METHODS: The human hepatoma HepaRG cell line model was used. Cell viability was determined by measuring succinate dehydrogenase activity, total protein level by the Bradford method. DNA synthesis was evaluated by [(3)H]-methyl thymidine incorporation. Cytotoxicity was studied by release of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in culture medium and apoptosis by measuring caspase 3/7 activity. Gene expression was analyzed by RT-qPCR. Total iron, soluble transferrin receptor, and ferritin levels were, respectively, measured by colorimetrical, immuno-nephelometrical, and immuno-turbidimetrical methods. Intracellular iron uptake and accumulation was examined by radionuclide (55)Fe (III) measurement and Perls staining. RESULTS: Results showed that ethanol decreased all the parameters associated with HepaRG cell proliferation (cell viability, total protein levels, and DNA synthesis) in a dose- and time-dependent manner. This effect was accompanied by cytotoxicity and apoptosis as evaluated by a significant increase in extracellular enzymes (LDH, AST, ALT) and caspase 3/7 activity, respectively. Ethanol exposure was accompanied by an increased cellular iron uptake, together with increased expression of genes involved in iron transport and storage such as l-ferritin, Divalent Metal transporter 1, transferrin, transferrin receptor 1, and ceruloplasmin. Ethanol impact was intensified by iron-citrate and decreased by iron chelators when added to the culture medium. CONCLUSIONS: The results indicated that (i) ethanol-induced iron metabolism dysfunction could be one of the underlying mechanisms of ethanol antiproliferative effect and (ii) exogenous iron may accentuate ethanol hepatoxicity. These data suggest that iron metabolism manipulation by chelators may be a useful therapeutic approach in alcohol-related liver diseases.

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG.

Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited (55)Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.

Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein.

The lack of an appropriate in vitro infection system for the major human pathogen hepatitis B virus (HBV) has prevented a molecular understanding of the early infection events of HBV. We used the novel HBV-infectible cell line HepaRG and primary human hepatocytes to investigate the interference of infection by HBV envelope protein-derived peptides. We found that a peptide consisting of the authentically myristoylated N-terminal 47 amino acids of the pre-S1 domain of the large viral envelope protein (L protein) specifically prevented HBV infection, with a 50% inhibitory concentration (IC50) of 8 nM. The replacement of myristic acid with other hydrophobic moieties resulted in changes in the inhibitory activity, most notably by a decrease in the IC50 to picomolar concentrations for longer unbranched fatty acids. The obstruction of HepaRG cell susceptibility to HBV infection after short preincubation times with the peptides suggested that the peptides efficiently target and inactivate a receptor at the hepatocyte surface. Our data both shed light on the molecular mechanism of HBV entry into hepatocytes and provide a basis for the development of potent hepadnaviral entry inhibitors as a novel therapeutic concept for the treatment of hepatitis Beta.

The hepatitis C virus NS2 protein is an inhibitor of CIDE-B-induced apoptosis.

Chronic hepatitis C virus (HCV) infection frequently leads to liver cancer. To determine the viral factor(s) potentially involved in viral persistence, we focused our work on NS2, a viral protein of unknown function. To assign a role for NS2, we searched for cellular proteins that interact with NS2. Performing a two-hybrid screen on a human liver cDNA library, we found that NS2 interacted with the liver-specific pro-apoptotic CIDE-B protein. Binding specificity of NS2 for CIDE-B was confirmed by cell-free assays associated with colocalization studies and coprecipitation experiments on human endogenous CIDE-B. CIDE-B, a member of the novel CIDE family of apoptosis-inducing factors, has been reported to show strong cell death-inducing activity in its C-terminal domain. We show that this CIDE-B killing domain is involved in the NS2 interaction. NS2 binding was sufficient to inhibit CIDE-B-induced apoptosis because an NS2 deletion mutant unable to interact with CIDE-B in vitro lost its capacity to interfere with CIDE-B cell death activity. Although it has been reported that CIDE-B-induced apoptosis is characterized by mitochondrial localization, the precise apoptotic mechanism remained unknown. Here, we show that CIDE-B induced cell death in a caspase-dependent manner through cytochrome c release from mitochondria. Furthermore, we found that NS2 counteracted the cytochrome c release induced by CIDE-B. In vivo, the CIDE-B protein level was extremely low in adenovirus-infected transgenic mice expressing the HCV polyprotein compared with that in wild-type mice. We suggest that NS2 interferes with the CIDE-B-induced death pathway and participates in HCV strategies to subvert host cell defense.

Infection of a human hepatoma cell line by hepatitis B virus.

Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.