RESUMO
Metallothioneins (MTs) are small cysteine-rich proteins which are involved in e.g. metal homeostasis, metal detoxification and protection against oxidative stress. In addition, several MTs have been shown to regulate expression of proangiogenic growth factors like vascular endothelial growth factor. Detailed information about the expression and regulation of specific MT isoforms in endothelial cells (EC) is limited. We therefore performed extensive mRNA expression profiling of all known human MTs in EC. We found that the basal endothelial expression is restricted to MT1E, MT1X, MT2A, and MT3. Physiological activation of EC by exposure to serum increased the expression of MT1E and MT2A and induced the expression of MT1M. Furthermore, exposure to zinc or copper induced the expression of most MT1 isoforms, while hypoxia specifically increased the expression of MT1E, MT1M, MT1X, and MT3. Finally, knockdown of the dominant MT isoform in EC, i.e. MT2A, resulted in decreased proliferation and sprouting as well as in increased migration of human umbilical vein EC. Together, these findings provide a link between MTs and angiogenesis.
Assuntos
Células Endoteliais/metabolismo , Metalotioneína/biossíntese , Metalotioneína/fisiologia , Movimento Celular/efeitos dos fármacos , Cobre/farmacologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Zinco/farmacologiaRESUMO
Background: The etiology of capsular contracture (CC), the most common complication following breast augmentation, remains unclear. Chronic, fibrotic inflammation resulting in excessive fibrosis has been proposed as a potential mechanism. Objectives: In this study, we aimed to investigate the relation between biomarkers that are associated with inflammation and fibrosis and the severity of CC. Methods: Fifty healthy females were categorized into 3 groups: females with no-to-mild CC (Baker 1-2; n = 15), females with severe CC (Baker 3-4; n = 20), and a control group awaiting breast augmentation (n = 15). We assessed 5 biomarkers (galectin-1 [Gal-1], interferon-ß [INF-ß], interferon-γ [INF-γ], interleukin-6 [IL-6], and tumor necrosis factor-α [TNF-α]) in breast implant capsules and serum samples. Results: No significant differences in intracapsular cytokine levels were observed between the Baker 1-2 and the Baker 3-4 groups, as the levels were generally low and, in some cases, almost undetectable. In the blood samples, no significant differences in Gal-1, INF-γ, IL-6, or TNF-α levels were found within the 3 groups. We identified significantly increased levels of INF-ß (P = .009) in the blood samples of females with severe CC, driven mainly by 3 extremely high values. Conclusions: The cytokines assessed in this study did not reflect the degree of CC among females with silicone breast implants. However, 3 females with severe CC, who all had prolonged silicone exposure, showed extremely elevated levels of INF-ß in their serum samples. This possible association between prolonged silicone exposure and systemic inflammation in some females should be further investigated.
RESUMO
Angiogenesis is a complex multi-step process involving various activities of endothelial cells. These activities are influenced in vivo by environmental conditions like interactions with other cell types and the microenvironment. Galectins play a role in several of these interactions and are therefore required for proper execution of in vivo angiogenesis. This chapter describes a method to study galectins during physiologic and pathophysiologic angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay.
Assuntos
Galectinas , Neovascularização Patológica , Neovascularização Fisiológica , Animais , Bioensaio , Galinhas , Membrana Corioalantoide , Células Endoteliais , Galectinas/fisiologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologiaRESUMO
The growth of new blood vessels is a key event in many (patho) physiological processes, including embryogenesis, wound healing, inflammatory diseases, and cancer. Neovascularization requires different, well-coordinated actions of endothelial cells, i.e., the cells lining the luminal side of all blood vessels. Galectins are involved in several of these activities. In this chapter, we describe methods to study galectins in three key functions of endothelial cells during angiogenesis, i.e., endothelial cell migration, endothelial cell sprouting, and endothelial cell network formation.
Assuntos
Células Endoteliais , Galectinas , Neovascularização Fisiológica , Movimento Celular , Células Endoteliais/fisiologia , Galectinas/antagonistas & inibidores , Galectinas/fisiologia , HumanosRESUMO
Galectins are versatile glycan-binding proteins involved in immunomodulation. Evidence suggests that galectins can control the immunoregulatory function of cytokines and chemokines through direct binding. Here, we report on an inverse mechanism in which chemokines control the immunomodulatory functions of galectins. We show the existence of several specific galectin-chemokine binding pairs, including galectin-1/CXCL4. NMR analyses show that CXCL4 binding induces changes in the galectin-1 carbohydrate binding site. Consequently, CXCL4 alters the glycan-binding affinity and specificity of galectin-1. Regarding immunomodulation, CXCL4 significantly increases the apoptotic activity of galectin-1 on activated CD8+ T cells, while no effect is observed in CD4+ T cells. The opposite is found for another galectin-chemokine pair, i.e., galectin-9/CCL5. This heterodimer significantly reduces the galectin-9 induced apoptosis of CD4+ T cells and not of CD8+ T cells. Collectively, the current study describes an immunomodulatory mechanism in which specific galectin-chemokine interactions control the glycan-binding activity and immunoregulatory function of galectins.
Assuntos
Quimiocina CXCL5/metabolismo , Galectina 1/metabolismo , Galectinas/metabolismo , Imunomodulação , Fator Plaquetário 4/metabolismo , Polissacarídeos/metabolismo , Humanos , Células JurkatRESUMO
BACKGROUND: Improvement of radiotherapy efficacy requires better insight in the dynamic responses that occur during irradiation. Here, we aimed to identify the molecular responses that are triggered during clinically applied fractionated irradiation. METHODS: Gene expression analysis was performed by RNAseq or microarray analysis of cancer cells or xenograft tumors, respectively, subjected to 3-5 weeks of 5 × 2 Gy/week. Validation of altered gene expression was performed by qPCR and/or ELISA in multiple cancer cell lines as well as in pre- and on-treatment biopsies from esophageal cancer patients ( NCT02072720 ). Targeted protein inhibition and CRISPR/Cas-induced gene knockout was used to analyze the role of type I interferons and cGAS/STING signaling pathway in the molecular and cellular response to fractionated irradiation. RESULTS: Gene expression analysis identified type I interferon signaling as the most significantly enriched biological process induced during fractionated irradiation. The commonality of this response was confirmed in all irradiated cell lines, the xenograft tumors and in biopsies from esophageal cancer patients. Time-course analyses demonstrated a peak in interferon-stimulated gene (ISG) expression within 2-3 weeks of treatment. The response was accompanied by a variable induction of predominantly interferon-beta and/or -lambda, but blocking these interferons did not affect ISG expression induction. The same was true for targeted inhibition of the upstream regulatory STING protein while knockout of STING expression only delayed the ISG expression induction. CONCLUSIONS: Collectively, the presented data show that clinically applied fractionated low-dose irradiation can induce a delayed type I interferon response that occurs independently of interferon expression or STING signaling. These findings have implications for current efforts that aim to target the type I interferon response for cancer treatment.
Assuntos
Neoplasias Esofágicas/radioterapia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Interferon Tipo I/genética , Proteínas de Membrana/genética , Animais , Astrocitoma/genética , Astrocitoma/imunologia , Astrocitoma/metabolismo , Astrocitoma/radioterapia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/radioterapia , Fracionamento da Dose de Radiação , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/metabolismo , Feminino , Células HT29 , Humanos , Imunidade/efeitos da radiação , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: Effective combination treatments with fractionated radiotherapy rely on a proper understanding of the dynamic responses that occur during treatment. We explored the effect of clinical fractionated radiotherapy on the development and timing of radioresistance in tumor cells. METHODS AND MATERIALS: Different colon (HT29/HCT116/COLO320/SW480/RKO) and high-grade astrocytoma (D384/U-251MG) cancer cell lines were treated for 6 weeks with daily fractions of 2 Gy, 5 days per week. Clonogenic survival was determined throughout the treatment period. In addition, the radiosensitivity of irradiated and non-irradiated was compared. Finally, the effect of different dose fractions on the development of radioresistance was determined. RESULTS: All cell lines developed radioresistance within 2-3 weeks during fractionated radiotherapy. This was characterized by the occurrence of a steady state phase of clonogenic survival. In U-251MG cells this was accompanied by increased cell senescence and stemness. After recovering from six weeks of treatment, the radiosensitivity of fractionally irradiated and non-irradiated cells was similar. Including transient radioresistance, described as (α/ß)-(d+1), as a factor in the classic LQ model resulted in a perfect fit with the experimental data observed during fractionated radiotherapy. This was confirmed when different dose fractions were applied. CONCLUSIONS: Fractionated irradiation of cancer cells in vitro following clinical radiation schedules induces a reversible radioresistance response. This adaptive response can be included in the LQ model as a function of the dose fraction and the alpha/beta-ratio of a given cell line. These findings warrant further investigation of the mechanisms and clinical relevance of adaptive radioresistance.
Assuntos
Neoplasias , Tolerância a Radiação , Sobrevivência Celular , HumanosRESUMO
PURPOSE: Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. EXPERIMENTAL DESIGN: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9-mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry. RESULTS: Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist. CONCLUSIONS: We conclude that the intrinsic, caspase-9-mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Apoptose/fisiologia , Caspase 9/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Inibidores de Caspase , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Etoposídeo/farmacologia , Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/genética , Compostos de Fenilureia/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
The growth of new blood vessels is a key event in many (patho)physiological processes, including embryogenesis, wound healing, inflammatory diseases, and cancer. Neovascularization requires different, well-coordinated actions of endothelial cells, i.e., the cells lining the luminal side of all blood vessels. Galectins are involved in several of these activities. In this chapter we describe methods to study galectins and galectin inhibition in three key functions of endothelial cells during angiogenesis, i.e., endothelial cell migration, endothelial cell sprouting, and endothelial cell network formation.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Galectinas/antagonistas & inibidores , Galectinas/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , HumanosRESUMO
Angiogenesis is a complex multi-process involving various activities of endothelial cells. These activities are influenced in vivo by environmental conditions like interactions with other cell types and the microenvironment. Galectins play a role in several of these interactions and are therefore required for proper execution of in vivo angiogenesis. In this chapter we describe a method to study galectins and galectin inhibitors during physiologic and pathophysiologic angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay.
Assuntos
Galinhas , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Galectinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Membrana Corioalantoide/citologia , Células Endoteliais , Galectinas/antagonistas & inibidores , Galectinas/química , SolubilidadeRESUMO
Clinical outcome in patients with primary nodal diffuse large B-cell lymphomas (DLBCLs) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including X-linked inhibitor of apoptosis protein (XIAP). XIAP suppresses apoptosis through inhibiting active caspase-3, caspase-7, and caspase-9. In this study, we investigated to see if the small-molecule XIAP antagonist 1396-12 induces cell death in cultured lymphoma cells of patients with DLBCL. Treatment with this XIAP antagonist resulted in relief of caspase-3 inhibition and in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy-refractory and -responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal-center B cells from healthy donors. XIAP antagonist-sensitive samples were characterized by high expression levels of XIAP, relatively low expression levels of Bcl-2, and by constitutive caspase-9 activation. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in cultured DLBCL cells and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers, suggesting the possibility of predefining patients most likely to benefit from XIAP antagonist therapy.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 9/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Compostos de Anilina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Tonsila Palatina/citologia , Compostos de Fenilureia/farmacologia , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Resistance to chemotherapy in therapy-refractory diffuse large B-cell lymphomas (DLBCL) is related to inhibition of the intrinsic apoptosis pathway. Human soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (hsTRAIL/Apo2L) induces apoptosis via the alternative, death-receptor mediated apoptosis pathway and might be an effective alternative form of therapy for these lymphomas. This study investigated whether hsTRAIL/Apo2L could actually induce apoptosis in isolated lymphoma cells of DLBCL biopsies of patients with chemotherapy-refractory DLBCL. Twelve out of a total of 22 DLBCL samples were sensitive to hsTRAIL/Apo2L. These sensitive lymphomas included seven clinically chemotherapy-refractory lymphomas. Furthermore, hsTRAIL/Apo2L induced apoptosis in DLBCL cells and in B-cell lines that showed high expression levels of inhibitors of the intrinsic apoptosis pathway: Bcl-2 and/or X-linked inhibitor of apoptosis (XIAP). hsTRAIL/Apo2L-sensitive lymphoma cells showed expression of the TRAIL receptors R1 and/or R2 and absence of R3 and R4. We conclude that hsTRAIL/Apo2L induced apoptosis in a subpopulation of chemotherapy-refractory nodal DLBCL and that disruption of the intrinsic apoptosis-mediated pathway and expression of Bcl-2 and XIAP did not confer resistance to hsTRAIL/Apo2L-induced apoptosis in DLBCL. Thus, based on our results, further exploration of hsTRAIL/Apo2L as an alternative treatment for patients with chemotherapy-refractory DLBCL should be considered.