RESUMO
Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.
Assuntos
Interferon gama/imunologia , Melanoma/imunologia , Monócitos/imunologia , Metástase Neoplásica/patologia , Neoplasias Cutâneas/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Microambiente Tumoral , Animais , Diferenciação Celular , Células Dendríticas/imunologia , Homeostase , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Monócitos/patologia , Análise de Sequência de RNA , Análise de Célula Única , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , TranscriptomaRESUMO
Immune responses are outcomes of complex molecular machinery which occur inside the cells. Unravelling the cellular mechanisms induced by immune stimulating molecules such as glycans and determining their structure-function relationship are therefore important factors to be assessed. With this viewpoint, the present study identifies the functional receptor binding unit of a well characterized heteroglycan and also delineates the cellular and molecular processes that are induced upon heteroglycan binding to specific cell surface receptors in immune cells. The heteroglycan was acid hydrolysed and it was revealed that 10-30 kDa fractions served as the functional receptor binding unit of the molecule. Increasing the size of 10-30 kDa heteroglycan showed prominent immune activity. The whole soluble heteroglycan was also conjugated with hyperbranched dendrimers so as to generate a particulate form of the molecule. Dectin-1 and TLR2 were identified as the major receptors in macrophages that bind to particulate as well as soluble form of the heteroglycan and subsequently caused downstream signaling molecules such as NF-κß and MAPK to get activated. High levels of 1L-1ß and IL-10 mRNA were observed in particulate heteroglycan treated macrophages, signifying that increasing the size and availability of the heteroglycan to its specific receptors is pertinent to its biological functioning. Upregulated expression of PKC and iNOS were also noted in particulate heteroglycan treated RAW 264.7 cells than the soluble forms. Taken together, our results indicate that biological functions of immunomodulatory heteroglycan are dependent on their size and molecular weight. J. Cell. Biochem. 117: 1580-1593, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Material Particulado/toxicidade , Polissacarídeos/toxicidade , Animais , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Proteína Quinase C/genética , Proteína Quinase C/imunologia , Células RAW 264.7 , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologiaRESUMO
BACKGROUND: Newer strategies for augmenting immune responses of pharmacologically active glucans may serve to improve the medicinal potential of these biomolecules. With this aim, the present work was focused on generating targeted high molecular size glucan particles with magnified immune response activity. METHODS: Heteroglucans were conjugated with PAMAM dendrimers using a Schiff base reductive amination reaction to generate a polytethered molecule with multiple glucan motifs. The modulated construct was characterized by FTIR, TEM, (1)H NMR and dynamic light scattering (DLS) methods. Effects of conjugated glucans were examined in RAW 264.7 macrophage cells as well as in S-180 murine tumor models. RESULTS: Dendrimer-conjugated glucans were found to exhibit a two-fold increase in immune stimulation in comparison to unconjugated glucans. This may be corroborated by the predominant enhancement in immunological functions such as nitric oxide production, ROS generation and immune directed tumor inhibition in murine models. Immune cell surface markers (CD4, CD8, CD19, MHC-II) and cytokine levels were also found to be highly up-regulated in the splenocytes of mice subjected to particulate glucan administration. Our study also demonstrated that conjugated glucan treatment to RAW 264.7 cells strongly enhanced the phosphorylation of two downstream signalling molecules of the mitogen activated protein kinase (MAPKs) family: p38 and MEK1/2 relative to single glucans thereby relating molecular mechanisms with enhanced immune stimulation. CONCLUSIONS AND GENERAL SIGNIFICANCE: The results obtained thus support that particulate format of soluble heteroglucan will thereby improve its functionality and identify leads in therapeutic competence.
Assuntos
Adjuvantes Imunológicos , Materiais Biocompatíveis , Dendrímeros , Macrófagos/imunologia , Polissacarídeos , Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos CD/imunologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Citocinas/imunologia , Dendrímeros/química , Dendrímeros/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/citologia , Camundongos , Polissacarídeos/química , Polissacarídeos/farmacologia , Espécies Reativas de Oxigênio/imunologiaRESUMO
We report for the first time the use of perylene-3-ylmethanol fluorescent organic nanoparticles as a drug delivery system. In the present system, perylene-3-ylmethanol nanoparticles performed four important roles: (i) "nanocarriers" for drug delivery; (ii) "phototriggers" for the drug release; (iii) fluorescent chromophores for cell imaging; and (iv) detectors for real time-monitoring of drug release. In vitro biological studies revealed that the newly developed perylene-3-ylmethanol nanoparticles exhibit good biocompatibility and cellular uptake as well as efficient photoregulated anticancer drug release ability. Such fluorescent organic nanoparticles may open up new perspectives for designing a new class of promising photoresponsive nanocarriers for drug delivery.
Assuntos
Antineoplásicos/administração & dosagem , Clorambucila/administração & dosagem , Preparações de Ação Retardada/química , Corantes Fluorescentes/química , Nanopartículas/química , Perileno/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Preparações de Ação Retardada/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Nanopartículas/ultraestrutura , Neoplasias/tratamento farmacológico , Perileno/metabolismo , FotóliseRESUMO
There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.
Assuntos
Linfócitos do Interstício Tumoral , Melanoma , Animais , Linfócitos T CD8-Positivos , Diferenciação Celular , Humanos , Melanoma/genética , Melanoma/terapia , Camundongos , Receptor de Morte Celular Programada 1RESUMO
Dendritic cells (DCs) are specialized immune sentinels that play key role in maintaining immune homeostasis by efficiently regulating the delicate balance between protective immunity and tolerance to self. Although DCs respond to maturation signals present in the surrounding milieu, multiple layers of suppression also co-exist that reduce the infringement of tolerance against self-antigens. These tolerance inducing properties of DCs are governed by their origin and a range of other factors including distribution, cytokines, growth factors, and transcriptional programing, that collectively impart suppressive functions to these cells. DCs directing tolerance secrete anti-inflammatory cytokines and induce naïve T cells or B cells to differentiate into regulatory T cells (Tregs) or B cells. In this review, we provide a detailed outlook on the molecular mechanisms that induce functional specialization to govern central or peripheral tolerance. The tolerance-inducing nature of DCs can be exploited to overcome autoimmunity and rejection in graft transplantation.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Tolerância Imunológica , Animais , Apresentação de Antígeno/imunologia , Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Diferenciação Celular , Apresentação Cruzada/imunologia , Citocinas/metabolismo , Células Dendríticas/classificação , Células Dendríticas/citologia , Regulação da Expressão Gênica , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Homeostase , Humanos , Imunomodulação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Especificidade de Órgãos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Vacinas/imunologiaRESUMO
The present study delves into a combined bio-nano-macromolecular approach for bone tissue engineering. This approach relies on the properties of an ideal scaffold material imbued with all the chemical premises required for fostering cellular growth and differentiation. A tannic acid based water dispersible hyperbranched polyurethane is fabricated with bio-nanohybrids of carbon dot and four different peptides (viz. SVVYGLR, PRGDSGYRGDS, IPP, and CGGKVGKACCVPTKLSPISVLYK) to impart target specific in vivo bone healing ability. This polymeric bio-nanocomposite is blended with 10 wt% of gelatin and examined as a non-invasive delivery vehicle. In vitro assessment of the developed polymeric system reveals good osteoblast adhesion, proliferation, and differentiation. Aided by this panel of peptides, the polymeric bio-nanocomposite exhibits in vivo ectopic bone formation ability. The study on in vivo mineralization and vascularization reveals the occurrence of calcification and blood vessel formation. Thus, the study demonstrates carbon dot/peptide functionalized hyperbranched polyurethane gel for bone tissue engineering application.
Assuntos
Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Engenharia Tecidual , Materiais Biocompatíveis/administração & dosagem , Regeneração Óssea/efeitos dos fármacos , Carbono/química , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Humanos , Nanocompostos/química , Osteoblastos/efeitos dos fármacos , Peptídeos/administração & dosagem , Peptídeos/química , Poliuretanos/administração & dosagem , Poliuretanos/química , Taninos/química , Alicerces Teciduais/químicaRESUMO
With the increase in cancer progression, alternatives in the medicinal field with minimal side effects need to be ascertained. In this context, for the first time novel glucans/glycans isolated from the mycelia and fruit body of Pleurotus ostreatus have been compared for their exquisite property as immunoceuticals. Glucans from both the sources displayed immunological functions which include lymphocyte proliferation, macrophage activation (nitric oxide production, ROS generation, phagocytosis, TNF-α production) as well as macrophage and NK cell mediated cytotoxicity. In vivo studies with Dalton's Lymphoma mice tumor model further enumerated the immune enhancing and tumor regression potential of the two glucan molecules. Highest tumor inhibition of about 75% and 71.4% were observed at 20mg/kg of mycelia and fruit body glucan/glycan treatments. A concomitant increase in the survival period of glucan treated tumor bearing mice was found to be primarily associated with immune boosting and apoptosis of cancerous cells. Both the glucan molecules exhibited similar degree of immune response at the systemic level with only subtle amount of differences in two dimensional in vitro cultures. Efficacy of glucans/glycans as immunomodulators may thereby provide decisive leads in strengthening the immune system along with other therapies.
Assuntos
Glucanos/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Linfoma/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Pleurotus , Animais , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Carpóforos/isolamento & purificação , Glucanos/isolamento & purificação , Humanos , Imunomodulação , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Melanoma Experimental , Camundongos , Micélio/isolamento & purificação , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Active polysaccharides isolated from various fungal sources have been implicated to stimulate immune response against various pathogens as well as self anomalies such as cancer. Therefore, the nuanced approach presented in our work was to blend polysaccharides derived from Pleurotus ostreatus with biocompatible ferrite nanoparticles and thereafter investigate the enhanced immune functionality of the polysaccharide-nanoparticle composite. A Schiff base reductive amination reaction occurred between the aldehyde group of the polysaccharide and the amine group of the nanoparticles in the presence of a strong reducing agent such as sodium cyanoborohydride to form a stable amide bond between the two conjugating molecules. The multifaceted conjugate was characterized by physiochemical techniques such as electron microscopy, FTIR, VSM and DLS measurements. This particulate form of the polysaccharide showed a marked escalation in the production of free radicals such as reactive oxygen and nitrogen species in murine macrophages as compared to the soluble form. Animal based experiments demonstrated a reduction in tumor volume and augmentation in the proliferation of splenocytes in particulate or conjugated polysaccharide treated mice. Furthermore, molecular signaling studies showed a high upregulation in p-p38 and p-MEK molecules in particulate polysaccharide treated RAW264.7 cells suggesting a cellular downstream mechanistic regulation behind the immunostimulative response.
Assuntos
Nanopartículas/química , Polissacarídeos/química , Vacinas/imunologia , Animais , Extratos Celulares , Linhagem Celular , Proliferação de Células , Separação Celular , Sobrevivência Celular , Glicoconjugados/química , Hidrodinâmica , Immunoblotting , Fígado/metabolismo , Linfócitos/patologia , Camundongos , Microscopia de Força Atômica , Nanopartículas/ultraestrutura , Neoplasias/patologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espalhamento de Radiação , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Baço/metabolismo , Análise de SobrevidaRESUMO
The therapeutic benefits of glycans have garnered much attention over the last few decades with most studies being reported in 2D cultures or in animal models. The present work is therefore aimed to assess the effects of an immunomodulatory heteroglycan in a 3D milieu. Briefly, HT29 tumor spheroids were incubated with THP-1 macrophages at 1:1 ratio in a culture medium supplemented with immune stimulants such as heteroglycans or LPS. Spheroidal distortion, migration of tumor cells from the periphery of the spheroids and 46% of higher macrophage invasiveness was noted in heteroglycan-treated co-cultures with respect to control cultures. Histological sections of the treated co-cultures revealed the presence of high apoptotic tumor cells in the spheroidal periphery. CD11c and CD68 staining further suggested the predominance of macrophages in the vicinity of the apoptotic tumor cells. Such an in vitro created tissue system may thereby confirm the effectiveness of heteroglycan in activating the immune cells to exhibit tumor cytotoxic properties.
Assuntos
Imunomodulação , Macrófagos/imunologia , Polissacarídeos/imunologia , Esferoides Celulares/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células HT29 , Humanos , Macrófagos/citologia , Camundongos , Modelos Biológicos , Pleurotus/química , Polissacarídeos/química , Polissacarídeos/isolamento & purificaçãoRESUMO
A water soluble fucogalactan (PS-II) of an average molecular weight â¼1.2×10(5) Da was isolated from the aqueous extract of an edible mushroom Macrolepiota dolichaula. It was composed of fucose, galactose and 3-O-methyl galactose in a molar ratio of nearly 1:4:1. Structural characterization of PS-II was carried out using total hydrolysis, methylation analysis, Smith degradation, and 1D/2D NMR experiments. These results indicated that the proposed repeating unit of the PS-II had a backbone chain consisting of four (1â6)- linked α-d-Galp residues, one residue methylated at O-3, and another one substituted at O-2 by (1â2)-α-d-Galp residue, which is terminated with a α-l-Fucp moiety. The PS-II exhibited the antioxidant properties in different in vitro test systems, and also showed in vitro macrophage activation in RAW 264.7 cell line as well as splenocyte and thymocyte activation in mouse cell culture medium.
Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Agaricales/química , Antioxidantes/química , Antioxidantes/farmacologia , Galactanos/química , Galactanos/farmacologia , Adjuvantes Imunológicos/isolamento & purificação , Animais , Antioxidantes/isolamento & purificação , Sequência de Carboidratos , Galactanos/isolamento & purificação , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Células RAW 264.7 , Baço/efeitos dos fármacos , Baço/imunologia , Timócitos/efeitos dos fármacos , Timócitos/imunologiaRESUMO
A water soluble ß-glucan having molecular weight â¼2×10(5)Da was isolated from hot water extract of the fruit bodies of an edible mushroom Entoloma lividoalbum (Kühner & Romagn) Kubicka. This polysaccharide (ELPS) contains (1â3,6)-ß-D-Glcp, (1â3)-ß-D-Glcp, (1â6)-ß-D-Glcp, and terminal ß-D-Glcp moieties in a molar ratio of nearly 1:1:3:1. Chemical and spectroscopic analysis showed that the backbone of glucan consists of three (1â6)-ß-D-glucopyranosyl and two (1â3)-ß-D-glucopyranosyl residues, out of which one (1â3)-ß-D-glucopyranosyl moiety was branched at O-6 with a terminal ß-D-glucopyranosyl residue. This ß-glucan exhibited macrophage, splenocyte, and thymocyte stimulations. It possesses promising antioxidant activities as evidenced from its hydroxyl and superoxide radical scavenging activities and reducing properties.
Assuntos
Antioxidantes/química , Basidiomycota/metabolismo , beta-Glucanas/química , Agaricales , Antioxidantes/isolamento & purificação , Basidiomycota/química , Sequência de Carboidratos , Carpóforos/química , Carpóforos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Superóxidos/química , beta-Glucanas/isolamento & purificação , beta-Glucanas/farmacologiaRESUMO
A water soluble pectic polysaccharide (PS) isolated from the aqueous extract of the green fruits of Momordica charantia contains D-galactose and D-methyl galacturonate in a molar ratio of nearly 1:4. It showed splenocyte, thymocyte as well as macrophage activations. Moreover, it exhibited potent antioxidant activities. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and 1D and 2D NMR studies, the structure of the repeating unit of the pectic polysaccharide was established as: [Formula: see text].
Assuntos
Frutas/química , Momordica charantia/química , Pectinas/química , Pectinas/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/isolamento & purificação , Adjuvantes Imunológicos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Sequência de Carboidratos , Proliferação de Células/efeitos dos fármacos , Radical Hidroxila/química , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Pectinas/isolamento & purificação , Baço/citologia , Baço/efeitos dos fármacos , Timócitos/citologia , Timócitos/efeitos dos fármacosRESUMO
Anticancer and immunostimulatory properties of tryptic digest peptides of Abrus precatorius agglutinin protein (10kDAGP) have already been reported. Here attempt has been made to further validate anticancer properties of 10kDAGP peptides in Ehrlich's ascites carcinoma (EAC) and B16 melanoma (B16M) bearing mice models and to analyze 10kDAGP by anion exchange chromatography and RP-HPLC for obtaining the bioactive fraction from the total peptide pool. 10kDAGP treatment decreased the tumour pack volume by â¼82% for EAC and 58.8% for B16M. It also showed increase in ex vivo proliferation of splenocyte and thymocyte isolated from tumour bearing mice and increase in TNF-α and Interferon-γ in splenocyte culture supernatant. From chromatographic analysis it was found that anionic peptide fraction may be responsible for anti-proliferative activities of 10kDAGP. As most anticancer peptides are cationic in nature, further studies regarding bioactivity of anionic peptide fraction may lead to novel anticancer peptides and pathways of action.
Assuntos
Abrus , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma de Ehrlich/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Peptídeos/uso terapêutico , Aglutininas/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Ehrlich/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Células HeLa , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/farmacologia , Fitoterapia , Baço/citologia , Timo/citologia , Carga Tumoral/efeitos dos fármacosRESUMO
Multifunctional mesoporous silica-coated superparamagnetic manganese ferrite (MnFe2O4) nanoparticles (M-MSN) were synthesized and evaluated for targeted drug delivery and magnetic resonance imaging (MRI) applications. MnFe2O4 nanoparticles were prepared by solvothermal route and were silica-coated by surface silylation using sol-gel reactions. Subsequently, silylation was done using (3-aminopropyl)triethoxysilane in presence of a surfactant (CTAB), followed by selective etching of the surfactant molecules that resulted in amine-functionalized superparamagnetic nanoparticles (NH2-MSN). Further modification of the surface of the NH2-MSN with targeting (folate) or fluorescent (RITC) molecules resulted in M-MSN. The formation of the M-MSN was proved by several characterization techniques viz. XRD, XPS, HRTEM, FESEM, VSM, BET surface area measurement, FTIR, and UV-Vis spectroscopy. The M-MSN were loaded with anticancer drug Doxorubicin and the efficacy of the DOX loaded M-MSN was evaluated through in vitro cytotoxicity, fluorescence microscopy, and apoptosis studies. The in vivo biocompatibility of the M-MSN was demonstrated in a mice-model system. Moreover, the M-MSN also acted as superior MRI contrast agent owing to a high magnetization value as well as superparamagnetic behavior at room temperature. These folate-conjugated nanoparticles (FA-MSN) exhibited stronger T2-weighted MRI contrast towards HeLa cells as compared to the nanoparticles without folate conjugation, justifying their potential importance in MRI based diagnosis of cancer. Such M-MSN with a magnetic core required for MRI imaging, a porous shell for carrying drug molecules, a targeting moeity for cancer cell specificity and a fluorescent molecule for imaging, all integrated into a single system, may potentially serve as an excellent material in biomedical applications.
Assuntos
Antibióticos Antineoplásicos , Materiais Revestidos Biocompatíveis , Doxorrubicina , Sistemas de Liberação de Medicamentos , Compostos Férricos , Compostos de Manganês , Nanopartículas/química , Dióxido de Silício , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Compostos Férricos/química , Compostos Férricos/farmacologia , Células HeLa , Humanos , Imageamento por Ressonância Magnética , Masculino , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Camundongos , Dióxido de Silício/química , Dióxido de Silício/farmacologiaRESUMO
10kDAGP, a tryptic digest of Abrus precatorius lectin 'Agglutinin' is known to induce apoptosis by mitochondria-dependent pathways in human cervical cancer (HeLa) cells. The present study was focused on deciphering the detailed molecular mechanism of apoptosis induction in vitro by 10kDAGP and also its in vivo therapeutic efficacy. For in vivo model, HeLa cell encapsulated hollow fiber was implanted in Swiss Albino mice and treated with 10kDAGP. Our results showed that 10kDAGP was able to enter the cell within a span of 20min and co-localized with mitochondria after 90min. of incubation. A drastic loss of mitochondrial membrane potential was noted within 6h of 10kDAGP administration along with an increase in ROS generation. ROS further led to symptoms of early apoptosis by deregulating Akt (Protein Kinase B) and activating c-Jun N-terminal Kinase (JNK), p38 Mitogen Activated Protein Kinase (MAPK), p53, and autophagy starting from â¼8h of incubation. Besides in vitro conditions, 10kDAGP activated JNK to mediate cancer cell killing in vivo. Therefore, 10kDAGP can be an excellent therapeutic agent as it can act through different ways in the cellular system. Future studies are directed to screen out active peptides from the pool of peptides and to study whether the mode of action is in synergistic way or in individual forms.
Assuntos
Apoptose/efeitos dos fármacos , Peptídeos/farmacologia , Lectinas de Plantas/farmacologia , Abrus , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peptídeos/química , Lectinas de Plantas/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A water soluble ß-glucan (PS) with an average molecular weight â¼1.95 × 10(5)Da was isolated from the alkaline extract of ectomycorrhizal edible mushroom, Russula albonigra (Krombh.) Fr. and found to consist of terminal, (1 â 3)-, (1 â 6)-, and (1 â 3,6)-linked ß-D-glucopyranosyl moieties in a ratio of nearly 1:2:2:1. The structure of this PS was elucidated on the basis of total hydrolysis, methylation analysis, Smith degradation, partial hydrolysis, and 1D/2D NMR experiments. On the basis of these experiments, the repeating unit of the PS was found to contain a backbone of three (1 â 6)-ß-D-glucopyranosyl residues, one of which was branched at O-3 position with the side chain consisting of two (1 â 3)-ß-D-glucopyranosyl and a terminal ß-D-glucopyranosyl residue. This PS showed in vitro macrophage activation by NO production as well as splenocytes and thymocytes proliferation. Moreover, it also exhibited potent antioxidant activities.
Assuntos
Adjuvantes Imunológicos/farmacologia , Agaricales/química , Antioxidantes/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , beta-Glucanas/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/isolamento & purificação , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Sequência de Carboidratos , Proliferação de Células , Células Cultivadas , Hidrólise , Radical Hidroxila/antagonistas & inibidores , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Óxido Nítrico/biossíntese , Solubilidade , Superóxidos/antagonistas & inibidores , Timócitos/citologia , Timócitos/efeitos dos fármacos , Água , beta-Glucanas/química , beta-Glucanas/isolamento & purificaçãoRESUMO
Targeted and efficient delivery of therapeutics to tumor cells is one of the key issues in cancer therapy. In the present work, we report a temperature and pH dual responsive core-shell nanoparticles comprising smart polymer shell coated on magnetic nanoparticles as an anticancer drug carrier and cancer cell-specific targeting agent. Magnetite nanoparticles (MNPs), prepared by a simple coprecipitation method, was surface modified by introducing amine groups using 3-aminopropyltriethoxysilane. Dual-responsive poly(N-isopropylacrylamide)-block-poly(acrylic acid) copolymer, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, was then attached to the amine-functionalized MNPs via EDC/NHS method. Further, to accomplish cancer-specific targeting properties, folic acid was tethered to the surface of the nanoparticles. Thereafter, rhodamine B isothiocyanate was conjugated to endow fluorescent property to the MNPs required for cellular imaging applications. The nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction (SAED), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), thermogravimetric analysis (TGA), zeta potential, vibrating sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS) measurements, and FTIR, UV-vis spectral analysis. Doxorubicin (DOX), an anticancer drug used for the present study, was loaded into the nanoparticles and its release behavior was subsequently studied. Result showed a sustained release of DOX preferentially at the desired lysosomal pH and temperature condition. The biological activity of the DOX-loaded MNPs was studied by MTT assay, fluorescence microscopy, and apoptosis. Intracellular-uptake studies revealed preferential uptake of these nanoparticles into cancer cells (HeLa cells) compared to normal fibroblast cells (L929 cells). The in vitro apoptosis study revealed that the DOX-loaded nanoparticles caused significant death to the HeLa cells. These nanoparticles were capable of target specific release of the loaded drug in response to pH and temperature and hence may serve as a potential drug carrier for in vivo applications.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Acrilamidas , Resinas Acrílicas , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacocinética , Portadores de Fármacos/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Polímeros , TemperaturaRESUMO
A water soluble heteroglycan isolated from hot aqueous extract of fruit bodies of an edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula edodes consists of d-glucose, d-mannose, and d-galactose residues in a molar ratio of nearly 1:1:1. This polysaccharide showed macrophage, splenocyte, and thymocyte activation. On the basis of sugar hydrolysis, methylation, periodate oxidation study, and NMR studies (1H, 13C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC), the structure of the repeating unit of the polysaccharide was established as: -->6)-α-D-Galp-(1-->6)-α-D-Glcp-(1-->2<--1ß-D-Manp.
Assuntos
Quimera , Pleurotus/química , Polissacarídeos/química , Cogumelos Shiitake/química , Sequência de Carboidratos , Espectroscopia de Ressonância MagnéticaRESUMO
Poly(ethylene glycol) stabilized colloidal silver nanoparticles were prepared using the reductive potency of the aqueous extract of Thuja occidentalis leaves under ambient conditions. The nanoparticles were well dispersed within a narrow size spectrum (7-14 nm) and displayed characteristic surface plasmon resonance peak at around 420 nm and Bragg's reflection planes of fcc structure. MTT assay revealed the dose-dependent cytocompatibility and toxicity of the nanoparticles with the L929 normal cell line. On the other hand, the antiproliferative action of the nanoparticles was evaluated on HeLa cell (cancerous cells) line. Fluorescence and phase contrast microscopic imaging indicated the appearance of multinucleate stages with aggregation and nuclear membrane disruption of the HeLa cells post treatment with the nanoparticles. The interaction at the prokaryotic level was also assessed via differential antibacterial efficacy against Staphylococcus aureus (MTCC 3160) and Escherichia coli (MTCC 40). Under these perspectives, it is also necessary to observe the environmental impact of the prepared silver nanoparticles. Hence, the dose dependent toxicity of silver nanoparticles was evaluated upon the earthworm species Eisenia fetida. Neither the survival nor the reproduction was affected by the addition of silver nanoparticles up to 1000 ppm. Thus these 'green' silver nanoparticles have promising potential as future materials.