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

International audienceTumor 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 cance

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 (IC(50)) 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 IC(50) 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 Β

TUMORAL VECTORIZATION OF NEW IRON CHELATORS FOR ANTIPROLIFERATIVE ACTIVITY: BIOLOGICAL PROPERTIES OF POLYAMINOQUINOLINES

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THE POLYAMINOQUINOLINE HQ1-44, A NEW IRON CHELATOR VECTORIZED BY POLYAMINE INHIBITS THE TUMOR GROWTH IN XENOGRAFTED IMUNODEPRESSED MICE

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Modulation of ethanol effect on hepatocyte proliferation by polyamines.

International audienceAn occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol

Les anesthésiques locaux stimulent l’apoptose des cellules de carcinome hépatocellulaire

National audienceIntroduction Le carcinome hépatocellulaire (CHC) est un cancer fréquent et agressif avec des options thérapeutiques limitées. Le principal traitement pour le CHC, à un stade précoce, est la chirurgie malgré un risque élevé de récidive (70 % après 5 ans). Des études rétrospectives ont montré que l’administration d’anesthésiques locaux (Als) lors de la chirurgie du cancer pourrait réduire la récidive du cancer. Dans des travaux précédents nous avions montré que les Als inhibaient la croissance des cellules de CHC HuH7 in vitro avec un mécanisme différent en fonction du type d’Als, la ropivacaïne induisait un blocage du cycle cellulaire en phase G2 alors que la lidocaïne n’avait pas d’effets spécifiques. Dans ce travail nous avons cherché à comprendre le(s) mécanisme(s) par lequel(s) les Als inhibent la croissance des cellules de CHC. Matériel et méthodes Afin de confirmer les résultats observés sur la lignée HuH7, une autre lignée de cellules de CHC à la croissance plus lente, la lignée cellulaire HepaRG au stade de cellules progénitrices, a été cultivée avec ou sans différentes doses de lidocaïne (10-3Molaire [M] à 10-4M) et ropivacaïne (10-3M à 10-4M). Des gènes d’intérêts (cycline A2, cycline B1, CDK1, APC, HRK, MKI67) ont été étudiés par Rt-qPCR. Des tests fonctionnels d’apoptose, basé sur l’activité caspase 3, et de sénéscence ont été réalisés pour étudier le devenir des cellules. Résultats Sur la lignée HepaRG, la ropivacaïne diminuait le taux d’ARNm de cyclines B1 (protéine clé de la progression en phase G2 du cycle cellulaire) (p < 0,05) mais n’avait pas d’effet sur cycline A2 et CDK1 (protéines impliquées dans la transition G2–M). Les Als augmentaient le taux d’ARNm de APC (p < 0,05) et de HRK (protéine pro-apoptotique) (p < 0,05) et diminuaient le taux d’ARNm de MKI67 (marqueur de prolifération cellulaire) (p < 0,05). La Lidocaïne augmentait le taux d’activité de la caspase 3 et le taux d’apoptose des cellules HepaRG aux dose de 10-3 M et 10-4M (p < 0,01), la ropivacaïne augmentait mais de façon plus modeste (p < 0,05) l’activité caspase. Aucun des Als n’avait d’action sur la sénéscence (Fig. 1). Discussion Il existe un effet et un mécanisme d’action spécifique de chaque Als sur les cellules cancéreuses de CHC : La lidocaïne induit une augmentation de l’apoptose dans les cellules de CHC in vitro ; la ropivacaïne inhibe spécifiquement la croissance des cellules CHC in vitro par blocage du cycle cellulaire. Ces résultats expérimentaux encourageants suggèrent un bénéfice à l’administration d’Als lors de la chirurgie carcinologique. Ils doivent être confirmés par des études clinique

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

International audienceNatural 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

Synthesis and Biological Properties of Quilamines II, New Iron Chelators with Antiproliferative Activities

International audienceTo 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

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

International audienceBACKGROUND: 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.

International audienceTwo 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