RESUMEN
The highly pathogenic avian influenza H5N1 virus continues to spread globally in domestic poultry with sporadic transmission to humans. The possibility for its rapid transmission to humans raised global fears for the virus to gain capacity for human-to-human transmission to start a future pandemic. Through direct contact with infected poultry, it caused the largest number of reported cases of severe disease and death in humans of any avian influenza strains. For pandemic preparedness, use of safe and effective vaccine adjuvants and delivery systems to improve vaccine efficacy are considered imperative. We previously demonstrated CaPtivate's proprietary CaP nanoparticles (CaPNP) as a potent vaccine adjuvant/delivery system with ability to induce both humoral and cell-mediated immune responses against many viral or bacterial infections. In this study, we investigated the delivery of insect cell culture-derived recombinant hemagglutinin protein (HA) of A/H5N1/Vietnam/1203/2004 virus using CaPNP. We evaluated the vaccine immunogenicity in mice following two intramuscular doses of 3 µg antigen combined with escalating doses of CaPNP. Our data showed CaPNP-adjuvanted HA(H5N1) vaccines eliciting significantly higher IgG, hemagglutination inhibition, and virus neutralization titers compared to non-adjuvanted vaccine. Among the four adjuvant doses that were tested, CaPNP at 0.24% final concentration elicited the highest IgG and neutralizing antibody titers. We also evaluated the inflammatory response to CaPNP following a single intramuscular injection in guinea pigs and showed that CaPNP does not induce any systemic reaction or adverse effects. Current data further support our earlier studies demonstrating CaPNP as a safe and an effective adjuvant for influenza vaccines.
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Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Fosfatos de Calcio/administración & dosificación , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Nanopartículas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Femenino , Cobayas , Vacunas contra la Influenza/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Vacunas de Subunidad/administración & dosificaciónRESUMEN
Peroxidasin (PXDN), a human homolog of Drosophila PXDN, belongs to the family of heme peroxidases and has been found to promote oxidative stress in cardiovascular tissue, however, its role in prostate cancer has not been previously elucidated. We hypothesized that PXDN promotes prostate cancer progression via regulation of metabolic and oxidative stress pathways. We analyzed PXDN expression in prostate tissue by immunohistochemistry and found increased PXDN expression with prostate cancer progression as compared to normal tissue or cells. PXDN knockdown followed by proteomic analysis revealed an increase in oxidative stress, mitochondrial dysfunction and gluconeogenesis pathways. Additionally, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics confirmed that PXDN knockdown induced global reprogramming associated with increased oxidative stress and decreased nucleotide biosynthesis. We further demonstrated that PXDN knockdown led to an increase in reactive oxygen species (ROS) associated with decreased cell viability and increased apoptosis. Finally, PXDN knockdown decreased colony formation on soft agar. Overall, the data suggest that PXDN promotes progression of prostate cancer by regulating the metabolome, more specifically, by inhibiting oxidative stress leading to decreased apoptosis. Therefore, PXDN may be a biomarker associated with prostate cancer and a potential therapeutic target.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Estrés Oxidativo , Peroxidasa/metabolismo , Neoplasias de la Próstata/metabolismo , Apoptosis , Línea Celular Tumoral , Gluconeogénesis , Humanos , Masculino , Metabolómica , Neoplasias de la Próstata/patología , Proteómica , PeroxidasinaRESUMEN
Studies have shown that High mobility group A2 (HMGA2), a non-histone protein, can promote epithelial-mesenchymal transition (EMT), which plays a critical role in prostate cancer progression and metastasis. Interestingly, full-length or wild-type HMGA2 and truncated (lacking the 3'UTR) HMGA2 isoforms are overexpressed in several cancers. However, there are no studies investigating the expression and differential roles of WT vs truncated HMGA2 isoforms in prostate cancer. Immunohistochemical staining of prostate tissue microarray revealed low membrane expression in normal epithelial prostate cells, and that expression increased with tumor grade as well as a switch from predominantly cytoplasmic HMGA2 in lower tumor grades, to mostly nuclear in high grade and bone metastatic tissue. LNCaP cells stably overexpressing wild-type HMGA2 displayed nuclear localization of HMGA2 and induction of EMT associated with increased Snail, Twist and vimentin expression compared to LNCaP Neo control cells, as shown by immunofluorescence and western blot analyses. This was associated with increased cell migration on collagen shown using boyden chamber assay. Conversely, LNCaP cells overexpressing truncated HMGA2 showed cytoplasmic HMGA2 expression that did not induce EMT yet displayed increased cell proliferation and migration compared to LNCaP Neo. Both wild-type and truncated HMGA2 increased levels of phospho-ERK, and interestingly, treatment with U0126, MAPK inhibitor, antagonized wild-type HMGA2-mediated EMT and cell migration, but did not affect truncated HMGA2-mediated cell proliferation or migration. Therefore, although both wild-type and truncated HMGA2 may promote prostate tumor progression, wild-type HMGA2 acts by inducing EMT via MAPK pathway.
Asunto(s)
Transición Epitelial-Mesenquimal , Proteína HMGA2/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Masculino , Metástasis de la NeoplasiaRESUMEN
Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling.
Asunto(s)
Proteínas Inhibidoras de la Diferenciación/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Anilidas/farmacología , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Ciclohexanos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones SCID , Proteínas de Neoplasias/metabolismo , Fenotipo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gold nanoparticles (AuNPs) have been chemically functionalized onto multiwalled carbon nanotubes (MWCNT) through a metallopolymer linker-bis (2,2':6'2"-terpyridine) ruthenium(II)-connected diblock poly(N-isopropyacryamide). A "nano-snowflower" pattern was formed by self-assembly MWCNT-AuNP nanocomposite with anti-DNP IgE antibody. MWCNT-AuNP nanohybrid has unique biocompatibility and electronic current-voltage properties. This nanohybrid shows the potential application for IgE biosensor to diagnose cancer cells. We represent a step towards building complex electronic circuits response by providing molecular recognition properties.
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Anticuerpos Monoclonales/inmunología , Oro/química , Inmunoensayo/instrumentación , Inmunoglobulina E/inmunología , Nanopartículas del Metal/química , Rutenio/química , Adsorción , Anticuerpos Monoclonales/química , Conductometría/instrumentación , Conductividad Eléctrica , Diseño de Equipo , Análisis de Falla de Equipo , Inmunoglobulina E/análisis , Ensayo de Materiales , Nanocompuestos/química , Nanocompuestos/ultraestructura , Tamaño de la Partícula , Unión Proteica , Propiedades de SuperficieRESUMEN
To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias Óseas/prevención & control , Neoplasias de la Mama/patología , Catepsina L/antagonistas & inhibidores , Extractos Vegetales/farmacología , Neoplasias de la Próstata/patología , Factores de Transcripción/antagonistas & inhibidores , Vitis/química , Animales , Anticarcinógenos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias Óseas/secundario , Catepsina L/biosíntesis , Catepsina L/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimioprevención/métodos , Femenino , Humanos , Células MCF-7 , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Osteoclastos/citología , Osteogénesis/efectos de los fármacos , Osteoprotegerina/farmacología , Extractos Vegetales/uso terapéutico , Ligando RANK/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción de la Familia Snail , Factores de Transcripción/biosíntesisRESUMEN
Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Núcleo Celular/enzimología , Transición Epitelial-Mesenquimal/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Factores de Transcripción/fisiología , Animales , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas , Factores de Transcripción de la Familia SnailRESUMEN
Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the Western world. The transition of androgen-dependent PCa to castration-resistant (CRPC) is a major clinical manifestation during disease progression and presents a therapeutic challenge. Our studies have shown that genetic ablation of inhibitor of differentiation 4 (Id4), a dominant-negative helix loop helix protein, in mice results in prostatic intraepithelial neoplasia lesions and decreased Nkx3.1 expression without the loss of androgen receptor (Ar) expression. ID4 is also epigenetically silenced in the majority of PCa. However, the clinical relevance and molecular pathways altered by ID4 inactivation in PCa are not known. This study investigates the effect of loss of ID4 in PCa cell lines on tumorigenicity and addresses the underlying mechanism. Stable silencing of ID4 in LNCaP cells (L-ID4) resulted in increased proliferation, migration, invasion, and anchorage-independent growth. An increase in the rate of tumor growth, weight, and volume was observed in L-ID4 xenografts compared with that in the LNCaP cells transfected with nonspecific short hairpin RNA (L+ns) in noncastrated mice. Interestingly, tumors were also observed in castrated mice, suggesting that loss of ID4 promotes CRPC. RNA sequence analysis revealed a gene signature mimicking that of constitutively active AR in L-ID4, which was consistent with gain of de novo steroidogenesis. Prostate-specific antigen expression as a result of persistent AR activation was observed in L-ID4 cells but not in L+ns cells. The results demonstrate that ID4 acts as a tumor suppressor in PCa, and its loss, frequently observed in PCa, promotes CRPC through constitutive AR activation.
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Adenocarcinoma/metabolismo , Proteínas Inhibidoras de la Diferenciación/fisiología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Testosterona/biosíntesis , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia sin Enfermedad , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de la Próstata Resistentes a la Castración/mortalidad , Neoplasias de la Próstata Resistentes a la Castración/patología , Carga Tumoral , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Biofunctional block copolymers are becoming increasingly attractive materials as active components in biosensors and other nanoscale electronic devices. We have described two different classes of block copolymers with biofuctional properties. Biofunctionality for block copolymers is achieved through functionalization with appropriate biospecific ligands. We have synthesized block copolymers of electroactive poly(3-decylthiophene) and 2-hydroxyethyl methacrylate by atom transfer radical polymerization. The block copolymers were functionalized with the dinitrophenyl (DNP) groups, which are capable of binding to Immunoglobulin E (IgE) on cell surfaces. The block copolymers were shown to be redox active. Additionally, the triblock copolymer of α, ω-bi-biotin (poly(ethylene oxide)-b-poly (styrene)-b-poly(ethylene oxide)) was also synthesized to study their capacity to bind fluorescently tagged avidin. The surface-active property of the poly(ethylene oxide) block improved the availability of the biotin functional groups on the polymer surfaces. Fluorescence microscopy observations confirm the specific binding of biotin with avidin.
RESUMEN
BACKGROUND: Inhibitor of differentiation 4 (Id4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer. Id4 is expressed in the normal prostate where its expression is also regulated by androgens. In this study we investigated the effect of loss of Id4 (Id4-/-) on adult prostate morphology. METHODS: Histological analysis was performed on prostates from 6-8 weeks old Id4-/-, Id4+/- and Id4+/+ mice. Expression of Id1, Sox9, Myc, androgen receptor, Akt, p-Akt, Pten and Nkx3.1 was investigated by immunohistochemistry. Androgen receptor binding on NKX3.1 promoter was studied by chromatin immuno-precipitation. Id4 was either over-expressed or silenced in prostate cancer cell lines DU145 and LNCaP respectively followed by analysis of PTEN, NKX3.1 and Sox9 expression. RESULTS: Id4-/- mice had smaller prostates with fewer tubules, smaller tubule diameters and subtle mPIN like lesions. Levels of androgen receptor were similar between wild type and Id4-/- prostate. Decreased NKX3.1 expression was in part due to decreased androgen receptor binding on NKX3.1 promoter in Id4-/- mice. The increase in the expression of Myc, Sox9, Id1, Ki67 and decrease in the expression of PTEN, Akt and phospho-AKT was associated with subtle mPIN like lesions in Id4-/- prostates. Finally, prostate cancer cell line models in which Id4 was either silenced or over-expressed confirmed that Id4 regulates NKX3.1, Sox9 and PTEN. CONCLUSIONS: Our results suggest that loss of Id4 attenuates normal prostate development and promotes hyperplasia/dysplasia with subtle mPIN like lesions characterized by gain of Myc and Id1 and loss of Nkx3.1 and Pten expression. One of the mechanisms by which Id4 may regulate normal prostate development is through regulating androgen receptor binding to respective response elements such as those on NKX3.1 promoter. In spite of these complex alterations, large neoplastic lesions in Id4-/- prostates were not observed suggesting the possibility of mechanisms/pathways such as loss of Akt that could restrain the formation of significant pre-cancerous lesions.
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Proteínas de Homeodominio/metabolismo , Proteínas Inhibidoras de la Diferenciación/deficiencia , Fosfohidrolasa PTEN/metabolismo , Próstata/crecimiento & desarrollo , Neoplasia Intraepitelial Prostática/metabolismo , Receptores Androgénicos/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Humanos , Proteínas Inhibidoras de la Diferenciación/metabolismo , Masculino , Ratones , Ratones Noqueados , Fenotipo , Fosforilación , Próstata/patología , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
BACKGROUND: The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol-enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream-regulated gene 1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. METHODS: PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72h, followed by trypan blue dye-exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. RESULTS: Physiological doses (16microM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol-enrichment promoted cell growth (P<0.05), while phytosterols significantly induced growth-suppression (P<0.05) and apoptosis. Cell cycle analysis showed that contrary to cholesterol, phytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1 (cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. CONCLUSIONS: Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling may reveal potential targets for cancer prevention and/or chemotherapeutic intervention. Sterol regulation of NDRG1 transcription suggests its potential as biomarker for prediction of neoplasms that would be responsive to chemoprevention by phytosterols.