RESUMO
Liver cancer ranks among the most prevalent malignancies globally and stands as a leading cause of cancer-related mortality. Numerous isothiazolone derivatives and analogues have been synthesized and investigated for their potential as anticancer agents; however, limited data exist regarding their efficacy against liver cancer. In the present study, two nitrophenyl-isothiazolones, the 5-benzoyl-2-(4-nitrophenyl)isothiazol-3(2H)-one (IsoA) and the 2-(4-nitrophenyl)isothiazol-3(2H)-one (IsoB), were preliminarily investigated for their cytotoxicity against hepatoma human (Huh7) cells as a liver cancer model and Immortalized Human Hepatocytes (IHHs) as a model of non-cancerous hepatocytes. IsoB, derived from IsoA after removal of the benzoyl moiety, demonstrated the highest cytotoxic effect against Huh7 cells with CC50 values of 19.3 µΜ at 24 h, 16.4 µΜ at 48 h, and 16.2 µΜ at 72 h of incubation, respectively. IsoB also exhibited selective toxicity against the liver cancerous Huh7 cells compared to IHH cells, reinforcing its role as a potent and selective anticancer agent. Remarkably, the cytotoxicity of IsoB was higher when compared with the standard chemotherapeutical agent 5-fluorouracil (5-FU), which also failed to exhibit higher toxicity against the liver cancerous cell lines. Moreover, IsoB-treated Huh7 cells presented a noteworthy reduction in mitochondrial membrane potential (ΔΨm) after 48 and 72 h, while mitochondrial superoxide levels showed an increase after 24 h of incubation. The molecular mechanism of the IsoB cytotoxic effect was also investigated using RT-qPCR, revealing an apoptosis-mediated cell death along with tumor suppressor TP53 overexpression and key-oncogene MYCN downregulation.
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Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania. This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as "viral and symbiotic interaction," "actin cytoskeleton organisation" and "apoptosis," which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival.
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Although the multiplicative and growth-arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re-entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1-/- parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1-/- parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic-enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1-/- growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity.
Assuntos
Ciclo Celular , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas de Protozoários/fisiologia , Animais , DNA de Protozoário/genética , Feminino , Deleção de Genes , Técnicas de Inativação de Genes , Aptidão Genética , Gotículas Lipídicas/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Morfogênese , Quinases DyrkRESUMO
Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host, parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp. affect their host are not fully understood. Herein, proteomic alterations of primary, bone marrow-derived BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, applying a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (combined total of 6189 mouse proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface. SIGNIFICANCE: Little is known on proteome changes that occur in primary macrophages after Leishmania donovani infection. This study describes a SILAC-based quantitative proteomics approach to characterise changes of bone marrow-derived macrophages infected with L. donovani promastigotes for 72 h. With the application of SILAC and the use of SAX and GEL fractionation methods, we have tested new routes for proteome quantification of primary macrophages. The protocols developed here can be applicable to other diseases and pathologies. Moreover, this study sheds important new light on the "proteomic reprogramming" of infected macrophages in response to L. donovani promastigotes that affects primary metabolism, cellular catabolic processes, and lysosomal/vacuole organisation. Thus, our study reveals key molecules and processes that act at the host/pathogen interface that may inform on new immuno- or chemotherapeutic interventions to combat leishmaniasis.
Assuntos
Leishmania donovani , Macrófagos , Proteômica , Animais , Leishmania donovani/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Proteoma , Proteínas de ProtozoáriosRESUMO
The study of the MeOH extract of the leaves of Campylospermum excavatum led to the isolation of a nitrile glucoside, named campyloside C (1) and an original derivative of ochnaflavone, 7-O-methylochnaflavone (2), along with three known biflavonoids, amentoflavone, sequoiaflavone, and sotetsuflavone (3 - 5). The linkage site of the sub-units of 2 was confirmed by chemical correlation, after semi-synthesis of a trimethoxylated derivative of ochnaflavone (2a). The structures of these compounds as well as their relative and absolute configurations were assigned by 1D- and 2D-NMR experiments, HR-ESI-MS and Electronic Circular Dichroism (ECD) calculations. A low-pass J filter HMBC experiment was performed in order to define the configuration of the double bond of 1. All of the biflavonoids were evaluated against protozoan parasites. Amentoflavone moderately inhibited the promastigote form of Leishmania infantum.
Assuntos
Biflavonoides/química , Glucosídeos/química , Nitrilas/química , Ochnaceae/química , Animais , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Biflavonoides/isolamento & purificação , Biflavonoides/farmacologia , Linhagem Celular , Dicroísmo Circular , Flavonoides/síntese química , Flavonoides/química , Glucosídeos/isolamento & purificação , Leishmania/efeitos dos fármacos , Leishmania/fisiologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Espectroscopia de Ressonância Magnética , Camundongos , Conformação Molecular , Ochnaceae/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Jungia sellowii (Asteraceae) is a shrub that grows in Southern Brazil and polar extract of its leaves presents anti-inflammatory properties. Cyperane, guaiane, nortrixane, and trixane sesquiterpene types were reported as the main metabolites in Jungia species. This work aims to describe the isolation and identification of sesquiterpenes in the leaves of J. sellowii using liquid-liquid partition and centrifugal partition chromatography. Thus, the crude extract of fresh leaves of J. sellowii was partitioned with hexane, dichloromethane, ethyl acetate and butanol, respectively. The butanol fraction was then subjected to a selected ternary system optimized for the CPC (centrifugal partition chromatography): ethyl acetate-ethanol-water (9:2:10, v/v/v). The separation was carried out isocratically at a flow rate of 25 mL/min at 1200 rpm, affording seven fractions A to G. TLC of fractions B, C and F displayed a single spot corresponding to three new glycosylated sesquiterpenoids. Their structures were established by using spectroscopic data in comparison to those reported in the literature. Furthermore, the isolates were evaluated for their leishmanicidal and cytotoxic effects. No cytotoxic effect was observed against the three cancer cell lines (HL60, JURKAT and REH), but compound 1 showed a weak antiprotozoal activity. Liquid-liquid partition and CPC turned to be a versatile technique of glycoside purification which is environmentally friendly and requires a limited amount of organic solvents.
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Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.
Assuntos
Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Leishmania donovani/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Fosfatase Ácida/genética , Animais , Linhagem Celular , Sequência Conservada , Genes de Protozoários , Células HeLa , Humanos , Leishmania/enzimologia , Leishmania/genética , Leishmania/patogenicidade , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Macrófagos/parasitologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , VirulênciaRESUMO
The environmental carcinogen benzo[a]pyrene (B[a]P) after being metabolised by cytochrome P450 enzymes forms DNA adducts. This abnormal situation induces changes in the cell cycle, DNA damage, chromosomal and mitotic aberrations, all of which may be related to carcinogenesis. In order to further investigate the mechanistic basis of these effects, HepG2 cells were treated with 3µM B[a]P for various time periods, followed by further incubation in the absence of B[a]P for up to 192h. B[a]P treatment led initially to S-phase arrest followed by recovery and subsequent induction of G2/M arrest, indicating activation of the corresponding DNA damage checkpoints. Immunofluorescence-based studies revealed accumulation of B[a]P-induced DNA adducts and chromosomal damage which persisted beyond mitosis and entry into a new cycle, thus giving rise to a new round of activation of the S-phase checkpoint. Prolonged further cultivation of the cells in the absence of B[a]P resulted in high frequencies of various abnormal mitotic events. Abrogation of the B[a]P-induced S-phase arrest by the Chk1 inhibitor UCN-01 triggered a strong apoptotic response but also dramatically decreased the frequency of mitotic abnormalities in the surviving cells, suggesting that events occurring during S-phase arrest contribute to the formation of delayed mitotic damage. Overall, our data demonstrate that, although S-phase arrest serves as a mechanism by which the cells reduce their load of genetic damage, its prolonged activation may also have a negative impact on the balance between cell death and heritable genetic damage.
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Benzo(a)pireno/farmacologia , Carcinógenos Ambientais/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Mitose , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/genética , Dano ao DNA , Células Hep G2 , Humanos , Mitose/efeitos dos fármacos , Mitose/genética , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Overexpression of Leishmania histone H1 (LeishH1) was previously found to cause a promastigote-to-amastigote differentiation handicap, deregulation of cell-cycle progression, and loss of parasite infectivity. The aim of this study was to identify changes in the proteome of LeishH1 overexpressing parasites associated with the avirulent phenotype observed. 2D-gel electrophoresis analysis revealed only a small protein subset of differentially expressed proteins in the LeishH1 overexpressing promastigotes. Among these was the chaperone HSP83, known for its protective role in Leishmania drug-induced apoptosis, which displayed lower translational rates. To investigate if the lower expression levels of HSP83 are associated with the differentiation handicap, we assayed the thermostability of parasites by subjecting them to heat-shock (25°Câ37°C), a natural stress-factor occurring during stage differentiation. Heat-shock promoted apoptosis to a greater extent in the LeishH1 overexpressing parasites. Interestingly, these parasites were not only more sensitive to heat-shock but also to drug-induced [Sb(III)] cell-death. In addition, the restoration of HSP83 levels re-established drug resistance, and restored infectivity to LeishH1 overexpressing parasites in the murine J774 macrophage model. Overall, this study suggests that LeishH1 levels are critical for the parasite's stress-induced adaptation within the mammalian host, and highlights the cross-talk between pathways involved in drug resistance, apoptosis and virulence.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico/biossíntese , Histonas/metabolismo , Leishmania donovani/patogenicidade , Biossíntese de Proteínas , Proteínas de Protozoários/biossíntese , Fatores de Virulência/biossíntese , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional , Endocitose , Histonas/genética , Temperatura Alta , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/efeitos da radiação , Macrófagos/parasitologia , Camundongos , Proteoma/análise , Proteínas de Protozoários/análise , Estresse Fisiológico , TemperaturaRESUMO
Visceral leishmaniasis is the most severe form of leishmaniases affecting millions of people worldwide often resulting in death despite optimal therapy. Thus, there is an urgent need for the development of effective anti-infective vaccine(s). In the present study, we evaluated the prophylactic value of bone marrow-derived dendritic cells (BM-DCs) pulsed with the Leishmania (L.) infantum histone H1. We developed fully mature BM-DCs characterized by enhanced capacity of IL-12 production after ex vivo pulsing with GST-LeishH1. Intravenous administration of these BM-DCs in naive BALB/c mice resulted in antigen-specific spleenocyte proliferation and IgG1 isotype antibody production and conferred protection against experimental challenge with L. infantum independently of CpG oligonucleotides (ODNs) co-administration. Protection was associated with a pronounced enhancement of parasite-specific IFNγ-producing cells and reduction of cells producing IL-10, whereas IL-4 production was comparable in protected and non-protected mice. The polarization of immune responses to Th1 type was further confirmed by the elevation of parasite-specific IgG2a/IgG1 ratio in protected mice. The above data indicate the immunostimulatory capacity of Leishmania histone H1 and further support its exploitation as a candidate protein for vaccine development against leishmaniasis.
Assuntos
Antígenos de Protozoários/imunologia , Células Dendríticas/imunologia , Histonas/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos , Células da Medula Óssea/citologia , Proliferação de Células , Feminino , Histonas/administração & dosagem , Imunidade Celular , Imunoglobulina G/sangue , Injeções Intravenosas , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-12/imunologia , Interleucina-4/imunologia , Leishmania infantum/imunologia , Leishmania infantum/patogenicidade , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos/imunologia , Baço/imunologia , Baço/parasitologiaRESUMO
Inhibition of Trypanosoma brucei and Leishmania spp. sirtuins has shown promising antiparasitic activity, indicating that these enzymes may be used as targets for drug discovery against trypanosomatid infections. In the present work we carried out a virtual screening focused on the C pocket of Sir2 from Trypanosoma cruzi. Using this approach, the best ligand found was nicotinamide. In vitro tests confirmed the anti-T. cruzi activity of nicotinamide on epimastigote and trypomastigote forms. Moreover, treatment of T. cruzi-infected macrophages with nicotinamide caused a significant reduction in the number of amastigotes. In addition, alterations in the mitochondria and an increase in the vacuolization in the cytoplasm were observed in epimastigotes treated with nicotinamide. Analysis of the complex of Sir2 and nicotinamide revealed the details of the possible ligand-target interaction. Our data reveal a potential use of TcSir2 as a target for anti-T. cruzi drug discovery.
Assuntos
Doença de Chagas/parasitologia , Niacinamida/farmacologia , Sirtuínas/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Sobrevivência Celular , Doença de Chagas/tratamento farmacológico , Descoberta de Drogas , Humanos , Ligantes , Macrófagos/parasitologia , Modelos Moleculares , Proteínas de Protozoários , Alinhamento de Sequência , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologiaRESUMO
The Hepatitis C virus (HCV) NS4B protein, a multispanning endoplasmic reticulum (ER) membrane protein, generates intracellular rearrangements of ER-derived membranes, essential for HCV replication. In this study, we characterized NS4B elements involved in the process of targeting, association and retention in the ER membrane. We investigated the localization and membrane association of a number of C- or N-terminal NS4B deletions expressed as GFP chimeras by biochemical and fluorescence microscopy techniques. A second set of GFP-NS4B chimeras containing the plasma membrane ecto-ATPase CD39 at the C-terminus of each NS4B deletion mutant was used to further examine the role of N-terminal NS4B sequences in ER retention. Several structural elements, besides the first two transmembrane domains (TMs), within the NS4B N-terminal half (residues 1-130) were found to mediate association of the NS4B-GFP chimeras with ER membranes. Both TM1 and TM2 are required for ER anchoring and retention but are not sufficient for ER retention. Sequences upstream of TM1 are also required. These include two putative amphipathic alpha-helices and a Leucine Rich Repeat-like motif, a sequence highly conserved in all HCV genotypes. The N-terminal 55peptidic sequence, containing the 1st amphipathic helix, mediates association of the 55N-GFP chimera with cellular membranes including the ER, but is dispensable for ER targeting of the entire NS4B molecule. Importantly, the C-terminal 70peptidic sequence can associate with membranes positive for ER markers in the absence of any predicted TMs. In conclusion, HCV NS4B targeting and retention in the ER results from the concerted action of several NS4B structural elements.