RESUMEN
In the previous study, we originally developed cancer stem cells (CSCs) models from mouse induced pluripotent stem cells (miPSCs) by culturing miPSCs in the conditioned medium of cancer cell lines, which mimiced as carcinoma microenvironment. However, the molecular mechanism of conversion in detail remains to be uncovered. Microarray analysis of the CSCs models in this study revealed Dsg2, one of the members of the desmosomal cadherin family, was up-regulated when compared with the original miPSCs. Moreover, the expression of key factors in Wnt/ß-catenin signaling pathway were also found up-regulated in one of the CSCs models, named miPS-LLCcm. An autocrine loop was implied between Dsg2 and Wnt/ß-catenin signaling pathway when miPSCs were treated with Wnt/ß-catenin signaling pathway activators, Wnt3a and CHIR99021, and when the CSCs model were treated with inhibitors, IWR-1 and IWP-2. Furthermore, the ability of proliferation and self-renewal in the CSCs model was markedly decreased in vitro and in vivo when Dsg2 gene was knocked down by shRNA. Our results showed that the Wnt/ß-catenin signaling pathway is activated by the up-regulation of Dsg2 expresssion during the conversion of miPSCs into CSCs implying a potential mechanism of the tranformation of stem cells into malignant phenotype.
Asunto(s)
Desmogleína 2 , Células Madre Pluripotentes Inducidas , Células Madre Neoplásicas , Vía de Señalización Wnt , Animales , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Células Madre Neoplásicas/metabolismo , Regulación hacia Arriba/genética , Vía de Señalización Wnt/genética , Desmogleína 2/genética , Desmogleína 2/metabolismo , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.
Asunto(s)
Neoplasias , Células Madre Neoplásicas , Diferenciación Celular , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/farmacología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismoRESUMEN
The growth of cancer tissue is thought to be considered driven by a small subpopulation of cells, so-called cancer stem cells (CSCs). CSCs are located at the apex of a hierarchy in a cancer tissue with self-renewal, differentiation and tumorigenic potential that produce the progeny in the tissue. Although CSCs are generally believed to play a critical role in the growth, metastasis, and recurrence of cancers, the origin of CSCs remains to be reconsidered. We hypothesise that, chronic diseases, including obesity and diabetes, establish the cancer-inducing niche (CIN) that drives the undifferentiated/progenitor cells into CSCs, which then develop malignant tumours in vivo. In this context, a CIN could be traced to chronic inflammation that involves long-lasting tissue damage and repair after being exposed to factors such as cytokines and growth factors. This must be distinguished from the cancer microenvironment, which is responsible for cancer maintenance. The concept of a CIN is most important for cancer prevention as well as cancer therapy.
Asunto(s)
Neoplasias , Diferenciación Celular , Humanos , Inflamación/patología , Neoplasias/patología , Células Madre Neoplásicas/patología , Microambiente TumoralRESUMEN
Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.
Asunto(s)
Antineoplásicos , Células Madre Pluripotentes Inducidas , Neoplasias , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , NADPH Oxidasas/metabolismo , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Compuestos OnioRESUMEN
The epidermal growth factor receptor (EGFR) was first tyrosine kinase receptor linked to human cancers. EGFR or ERBB1 is a member of ERBB subfamily, which consists of four type I transmembrane receptor tyrosine kinases, ERBB1, 2, 3 and 4. ERBBs form homo/heterodimers after ligand binding except ERBB2 and consequently becomes activated. Different signal pathways, such as phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), RAS/RAF/MEK/ERK, phospholipase Cγ and JAK-STAT, are triggered by ERBB activation. Since ERBBs, through these pathways, regulate stemness and differentiation of cancer stem cells (CSCs), their roles in CSC tumorigenicity have extensively been investigated. The hyperactivation of ERBBs and its downstream pathways stimulated by either genetic and/or epigenetic factors are frequently described in many types of human cancers. Their dysregulations make cells acquiring CSC characters such as survival, tumorigenicity and stemness. Because of the roles in tumor growth and progress, ERBBs are considered to be one of the drug targets as cancer treatment strategy. In this chapter, we will summarize the structure, function and roles of ERBB subfamily along with their relative pathways regulating the stemness and tumorigenicity of CSCs. Finally, we will discuss the targeting therapy strategies of cancer along with ERBBs in addition to some challenges and future perspectives.
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Neoplasias , Fosfatidilinositol 3-Quinasas , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Receptor ErbB-2/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias/genética , Neoplasias/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , FosforilaciónRESUMEN
Many tumors are resistant to conventional cancer therapies because a tumor is composed of heterogeneous cell population. Especially, subpopulation of cancer stem cells, which have self-renewal and differentiation properties and responsible for the tumor initiation, is generally considered resistant to chemo-, radio-, and immune therapy. Understanding the mechanism of drug resistance in cancer stem cells should lead to establish more effective therapeutic strategies. Actually, different molecular mechanisms are conceivable for cancer stem cells acquiring drug resistance. These mechanisms include not only cytoplasmic signaling pathways but also the intercellular communications in the tumor microenvironment. Recently, a great deal of successful reports challenged to elucidate the mechanisms of drug resistance and to develop novel treatments targeting cancer stem cells.
Asunto(s)
Resistencia a Antineoplásicos , Neoplasias , Humanos , Neoplasias/patología , Transformación Celular Neoplásica/patología , Diferenciación Celular , Células Madre Neoplásicas/patología , Microambiente TumoralRESUMEN
Cancer stem cells (CSCs) are small subpopulation sharing similar properties like normal stem such as self-renewal and differentiation potential to direct tumor growth. Last few years, scientists considered CSCs as the cause of phenotypic heterogeneity in diverse cancer types. Also, CSCs contribute to cancer metastasis and recurrence. The cellular and molecular regulators influence on the CSCs' phenotype changing their behaviors in different stages of cancer progression. CSC markers play significance roles in cancer diagnosis and characterization. We delineate the cross-talks between CSCs and the tumor microenvironment that supports their intrinsic properties including survival, stemness, quiescence and their cellular and molecular adaptation. An insight into the markers of CSCs specific to organs is described.
Asunto(s)
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/patología , Células Madre Neoplásicas/patología , Diferenciación Celular , Fenotipo , Microambiente Tumoral/genéticaRESUMEN
Cancer stem cells (CSCs) are responsible for cancer initiation, drug resistance, and aggressive tumor phenotypes. Our lab has established a novel method to induce CSCs from induced pluripotent stem (iPS) cells in a microenvironment mimicking chronic inflammation. The converted cells acquired CSC characteristics and developed malignant tumors. Recently, we demonstrated that nonmutagenic chemical inhibitors accelerated the conversion of mouse iPS (miPS) cells into CSCs. Here, we investigated the effects of AZD-6244, a MEK1/2-specific inhibitor, on the conversion of iPS cells into CSCs. The miPS cells were cultured for one week in the presence of the conditioned medium (CM) of Lewis lung carcinoma (LLC) cells and AZD-6244, PD0325901, a pan-MEK inhibitor, or GDC-0879, a B-Raf inhibitor. As a result, AZD-6244 enhanced the conversion of iPS cells into CSCs and upregulated AKT phosphorylation as same as GDC-0879 and PD0325901. The converted cells maintained their self-renewal ability and stemness gene expression. The expression of the CSC markers CD24, CD44 and CD133 was higher in the cells cultured with MAPK inhibitors than in those cultured without MAPK inhibitors. Moreover, converted cells gained migration and invasion abilities assessed by in vitro assays. Therefore, the inhibition of MEK1/2 was found to be critical for the conversion of normal stem cells into CSCs in the tumor-inducing microenvironment.
RESUMEN
Angiogenesis is generally involved in tumor growth and metastasis. Cancer stem cells (CSCs) are considered to facilitate the angiogenesis. Therefore, CSCs could be the effective targets to stop angiogenesis. Recently, our group successfully generated CSC models from induced pluripotent stem cells (iPSCs) in the presence of conditioned medium derived from cancer derived cells. These novel model CSCs has been characterized by highly tumorigenic, angiogenic and metastatic potentials in vivo. The angiogenic potential of CSCs has been explained by the expression of both angiogenic factors and their receptors implying the angiogenesis in autocrine manner. In this protocol we optimized the method to evaluate tumor angiogenesis with the CSC model, which was described effective to assess sorafenib as an antiangiogenic drug, on chick chorioallantoic membrane (CAM) assay. Our results demonstrate that CSCs developed from iPSCs and CAM assay are a robust and cost-effective tool to evaluate tumor angiogenesis with CSCs. Collectively, CSCs in CAM assay could serve as a very useful model for the screening of potential therapeutic agents targeting tumor angiogenesis.
Asunto(s)
Neoplasias , Neovascularización Patológica , Sorafenib/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Células Madre NeoplásicasRESUMEN
Metformin exhibits anti-cancer activities in various types of tumours while it is prescribed as the first-line drug for type 2 diabetes. Since new evidence has recently suggested that metformin could target cancer stem cells (CSCs) and prevent their recurrence, repositioning of metformin could be considered as a candidate for anti-CSC agent. In this study, we assessed the effect of metformin on the cancer stem cells developed from induced pluripotent stem cells. As the result, metformin significantly suppressed the self-renewal ability of CSCs when assessed by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell counting methods exhibiting the IC50 as approximately 20 mM, which suppressed tube formation by CSCs on Matrigel reducing the angiogenic potential of CSCs. Cell cycle analysis showed that metformin reduced the percentage of cells in the S phase increasing the percentage of cells in G0/G1 phase. Moreover, the tumorigenicity of CSCs was found to be attenuated when the cells were injected with metformin. From these results, we concluded that metformin could be promising for targeted therapy by repositioning the widely available drugs with safety. SIGNIFICANCE OF THE STUDY: Metformin could target CSCs and prevent their recurrence, repositioning of metformin could be considered as a candidate for the anti-CSC agent. In this paper, we assessed the effect of metformin on the CSCs developed from induced pluripotent stem cells. Here, we show that metformin suppresses the self-renewal and tube formation abilities of CSCs. We also show that metformin reduces the percentage of cells in the S phase increasing the percentage of cells in G0/G1 phase. Moreover, the tumorigenicity of CSCs was found to be attenuated when grafted in vivo after treatment with metformin.
Asunto(s)
Antineoplásicos/farmacología , Autorrenovación de las Células/efectos de los fármacos , Metformina/farmacología , Modelos Biológicos , Células Madre Pluripotentes/citología , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales CultivadasRESUMEN
Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Ligadas a GPI/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Proteínas de Neoplasias/inmunología , Teratocarcinoma/tratamiento farmacológico , Neoplasias Testiculares/tratamiento farmacológico , Secuencia de Aminoácidos , Anticuerpos Monoclonales Humanizados/inmunología , Antineoplásicos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Humanos , Masculino , Homología de Secuencia , Teratocarcinoma/inmunología , Teratocarcinoma/metabolismo , Teratocarcinoma/patología , Neoplasias Testiculares/inmunología , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Liver cancer is the second most common cause of cancer-related death. Every type of tumours including liver cancer contains cancer stem cells (CSCs). To date, the molecular mechanism regulating the development of liver CSCs remains unknown. METHODS: In this study, we tried to generate a new model of liver CSCs by converting mouse induced pluripotent stem cells (miPSCs) with hepatocellular carcinoma (HCC) cell line Huh7 cells conditioned medium (CM). miPSCs treated with CM were injected into the liver of BALB/c nude mice. The developed tumours were then excised and analysed. RESULTS: The primary cultured cells from the malignant tumour possessed self-renewal capacity, differentiation potential and tumorigenicity in vivo, which were found rich in liver cancer-associated markers as well as CSC markers. CONCLUSIONS: We established a model of liver CSCs converting from miPS and showed different stages of stemness during conversion process. Our CSC model will be important to assess the molecular mechanisms necessary to develop liver CSCs and could help in defeating liver cancer.
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Carcinogénesis/efectos de los fármacos , Carcinoma Hepatocelular/genética , Medios de Cultivo Condicionados/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neoplasias Hepáticas/genética , Animales , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Células Madre Neoplásicas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The surface reactivity of gold nanoparticles (AuNPs) is receiving attention as a radiosensitizer of cancer cells for radiation therapy and/or as a drug carrier to target cells. This study demonstrates the potential of DNA-AuNPs (prepared by mixing calf thymus DNA with HAuCl4 solution) as a radiosensitizer of human glioma cells that have cancer stem cell (CSC)-like properties, to reduce their survival. CSC-like U251MG-P1 cells and their parental glioblastoma U251MG cells are treated with a prepared DNA-AuNP colloid. The radiosensitivity of the resultant AuNP-associated cells are significantly enhanced. To reveal the mechanism by which survival is reduced, the generation of reactive oxygen species (ROS), apoptosis induction, or DNA damage in the cells is assayed using the fluorescent dye DCFDA, annexin V-FITC/PI, and foci formation of γ-H2AX, respectively. X-ray irradiation with administration of AuNPs overcomes the radioresistance of U251MG-P1 cells. It does not induce ROS generation or apoptosis in the cells but enhances the number of abnormal nuclei with abundant γ-H2AX foci, which is judged as cell death by mitotic catastrophe. The AuNP association with the cells effectively induces mitotic catastrophe in x-ray-irradiated CSC-like cells, implicating that DNA-AuNPs might be a promising tool to develop an efficient radiosensitizer against CSC.
Asunto(s)
ADN/administración & dosificación , Glioma/radioterapia , Oro/administración & dosificación , Nanopartículas del Metal/administración & dosificación , Células Madre Neoplásicas/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Anexinas/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Colorantes Fluorescentes/administración & dosificación , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Glioma/metabolismo , Histonas/metabolismo , Humanos , Mitosis/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Paclitaxel (PTX) is one of the front-line drugs approved for the treatment of ovarian cancer. However, the application of PTX is limited due to the significant hydrophobicity and poor pharmacokinetics. We previously reported target-directed liposomes carrying tumor-selective conjugated antibody and encapsulated glycosylated PTX (gPTX-L) which successfully overcome the PTX limitation. The tubulin stabilizing activity of gPTX was equivalent to that of PTX while the cytotoxic activity of gPTX was reduced. In human ovarian cancer cell lines, SK-OV-3 and OVK18, the concentration at which cell growth was inhibited by 50% (IC50) for gPTX range from 15â»20 nM, which was sensitive enough to address gPTX-L with tumor-selective antibody coupling for ovarian cancer therapy. The cell membrane receptor CD44 is associated with cancer progression and has been recognized as a cancer stem cell marker including ovarian cancer, becoming a suitable candidate to be targeted by gPTX-L therapy. In this study, gPTX-loading liposomes conjugated with anti-CD44 antibody (gPTX-IL) were assessed for the efficacy of targeting CD44-positive ovarian cancer cells. We successfully encapsulated gPTX into liposomes with the loading efficiency (LE) more than 80% in both of gPTX-L and gPTX-IL with a diameter of approximately 100 nm with efficacy of enhanced cytotoxicity in vitro and of convenient treatment in vivo. As the result, gPTX-IL efficiently suppressed tumor growth in vivo. Therefore gPTX-IL could be a promising formulation for effective ovarian cancer therapies.
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Receptores de Hialuranos/metabolismo , Terapia Molecular Dirigida , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Paclitaxel/uso terapéutico , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Glicosilación , Humanos , Liposomas/ultraestructura , Neoplasias Ováricas/patología , Paclitaxel/farmacologíaRESUMEN
Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90ß, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.
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Carcinoma de Células Escamosas/patología , Vesículas Extracelulares/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Metástasis Linfática/patología , Neoplasias de la Boca/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Molécula de Adhesión Celular Epitelial/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Estimación de Kaplan-Meier , Proteoma/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Liver fibrosis is a dynamic procedure that results from an irregularity between fibrogenesis and fibrolysis. After time this procedure can lead to cirrhosis of the liver. Liver fibrosis and cirrhosis assessment is very important for both therapeutic decisions and prognostic evaluations. In this study, we tried to use serum ferritin (SF) together with five fibrosis tests (Age-Platelet index (API), aspartate aminotransferase to alanine aminotransferase ratio (AAR), AST to platelet ratio index (APRI), Fibrosis 4 score (FIB-4), and fibro-quotient (Fibro-Q)) to assess liver fibrosis and cirrhosis and estimate possible correlation between inflammation and SF. METHODS: This study was carried out on eighty-eight patients infected with HCV and twenty healthy subjects as a control. Complete blood count (CBC), aspartate aminotransferase (AST), alanine aminotransferase (ALT), antiHCV antibody, detection of HCV RNA by real-time PCR, and serum ferritin (SF) were assessed. Then API, ARR, APRI, FIB-4, and Fibro-Q were calculated. Different fibrosis stages (mild fibrosis stage (F1), moderate fibrosis stage (F2), severe fibrosis stage (F3), cirrhotic stage (F4)) were assessed using transient elastography by Fibro Scan®. RESULTS: FIB-4 index was significantly elevated (p < 0.01) with the progression of liver fibrosis at F1, F2, F3, and F4 when compared to healthy control group. The APRI score elevation between F0 and F3 and between F0 and F4 was significant (p < 0.01). SF was elevated in all fibrosis stages and significantly (p < 0.01) at F3 and F4 compared to controls. CONCLUSIONS: APRI coupled with SF should be the best reliable biomarkers for liver cirrhosis. Simultaneously, from our data SF involved in all stages of inflammation. Therefore, down regulation of ferritin in the early stage of fibrosis should be helpful in decreasing the inflammatory effect of ferritin.
Asunto(s)
Biomarcadores/sangre , Ferritinas/sangre , Anticuerpos contra la Hepatitis C/sangre , Cirrosis Hepática/sangre , ARN Viral/sangre , Adulto , Alanina Transaminasa/sangre , Aspartato Aminotransferasas/sangre , Plaquetas/metabolismo , Fibrosis , Hepacivirus/genética , Hepacivirus/inmunología , Hepacivirus/fisiología , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/virología , Persona de Mediana Edad , Recuento de Plaquetas , ARN Viral/genética , Curva ROCRESUMEN
T cell-deficient mice such as nude mice are often used to generate tumor xenograft for the development of anticancer agents. However, the functionality of the other immune cells including macrophages, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) in the xenograft are largely unknown. Macrophages and dendritic cells (DCs) acquire functionally distinct properties in response to various environmental stimuli; the interaction of these cells with MDSCs in tumor microenvironments regulates cancer progression. Nude mice are less likely to reject human cancer cells because of major histocompatibility complex (MHC) mismatches. The tumor microenvironment in a xenograft, comprising human and mouse cells, exhibits more complex bidirectional signaling and function than that of allograft. Here, we evaluated the differences of myeloid cells between them. Plasma interferon-γ and interleukin-18 concentrations in the xenograft tumor model after lipopolysaccharide (LPS) administration were significantly higher than those in the allograft tumor model. MHC class I, II, and CD80 expression levels were increased in CD11b⺠and MDSC populations after LPS administration in the spleen of a xenograft tumor model but not in that of an allograft tumor model. Additionally, the number of CD80- and mannose receptor C type 1 (MRC1)-expressing cells was decreased upon LPS administration in the tumor of the xenograft tumor. These results suggest that functions of macrophages and DCs are sustained in the xenograft, whereas their functions in response to LPS were suppressed in the allograft. The findings will encourage the consideration of the effects of myeloid cells in the xenograft for drug development.
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Citocinas/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Aloinjertos , Animales , Línea Celular Tumoral , Femenino , Citometría de Flujo , Células HT29 , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cripto-1 is a glycophosphatidylinositol (GPI) anchored signaling protein of epidermal growth factor (EGF)-Cripto-1-FRL1-Cryptic (CFC) family and plays a significant role in the early developmental stages and in the different types of cancer cells, epithelial to mesenchymal transition and tumor angiogenesis. Previously, we have developed cancer stem cells (miPS-LLCcm) from mouse iPSCs by culturing them in the presence of conditioned medium of Lewis Lung Carcinoma (LLC) cells for four weeks. Nodal and Cripto-1 were confirmed to be expressed in miPS-LLCcm cells by quantitative reverse transcription PCR (rt-qPCR) implying that Cr-1 was required in maintaining stemness. To investigate the biological effect of adding exogenous soluble CR-1 to the cancer stem cells, we have prepared a C-terminally truncated soluble form of recombinant human CR-1 protein (rhsfCR-1), in which the GPI anchored moiety was removed by substitution of a stop codon through site-directed mutagenesis. rhsfCR-1 effectively suppressed the proliferation and sphere forming ability of miPS-LLCcm cells in a dose-dependent manner in the range of 0 to 5 µg/mL, due to the suppression of Nodal-Cripto-1/ALK4/Smad2 signaling pathway. Frequency of sphere-forming cells was dropped from 1/40 to 1/69 by rhsfCR-1 at 1 µg/mL. Moreover, rhsfCR-1 in the range of 0 to 1 µg/mL also limited the differentiation of miPS-LLCcm cells into vascular endothelial cells probably due to the suppression of self-renewal, which should reduce the number of cells with stemness property. As demonstrated by a soluble form of exogenous Cripto-1 in this study, the efficient blockade would be an attractive way to study Cripto-1 dependent cancer stem cell properties for therapeutic application.
Asunto(s)
Autorrenovación de las Células , Proteínas Ligadas a GPI/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/citología , Animales , Diferenciación Celular , Línea Celular , Humanos , Ratones , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteína Smad2/metabolismoRESUMEN
We recently have established a successful xenograft model of human glioblastoma cells by enriching hyaluronic acid-dependent spheroid-forming populations termed U251MG-P1 cells from U251MG cells. Since U251MG-P1 cells have been confirmed to express CD44 along with principal stemness marker genes, OCT3/4, SOX2, KLF4 and Nanog, this CD44 expressing population appeared to majorly consist of undifferentiated cells. Evaluating the sensitivity to anti-cancer agents, we found U251MG-P1 cells were sensitive to doxorubicin with IC50 at 200 nM. Although doxorubicin has serious side-effects, establishment of an efficient therapy targeting undifferentiated glioblastoma cell population is necessary. We previously designed a chlorotoxin peptide fused to human IgG Fc region without hinge sequence (M-CTX-Fc), which exhibited a stronger growth inhibitory effect on the glioblastoma cell line A172 than an original chlorotoxin peptide. Combining these results together, we designed M-CTX-Fc conjugated liposomes encapsulating doxorubicin and used U251MG-P1 cells as the target model in this study. The liposome modified with M-CTX-Fc was designed with a diameter of approximately 100-150 nm and showed high encapsulation efficiency, adequate loading capacity of anticancer drug, enhanced antitumor effects demonstrating increasing uptake into the cells in vitro; M-CTX-Fc-L-Dox shows great promise in its ability to suppress tumor growth in vivo and it could serve as a template for targeted delivery of other therapeutics.
Asunto(s)
Doxorrubicina/análogos & derivados , Glioblastoma/genética , Receptores de Hialuranos/genética , Proteínas Recombinantes de Fusión , Venenos de Escorpión/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Receptores de Hialuranos/metabolismo , Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G , Concentración 50 Inhibidora , Factor 4 Similar a Kruppel , Metaloproteinasa 2 de la Matriz , Ratones , Polietilenglicoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications.