RESUMEN
Human Klotho gene has many known functions such as anti-aging and anti-tumor, and decreased expression of this gene causes malignant formations in most types of cancer, including colon cancer. Interacting with TRAIL death receptors (DR4 and DR5) induces an apoptotic effect in cancer treatments by reducing the proliferation of cancer cells. The present study aimed to investigate downstream effect of overexpression of Klotho gene, which is known to have an antitumor effect on resistant human colon cancer cells, by examining its action on TRAIL death and decoy (DcR1 and DcR2) receptors for the first time. For this purpose, upregulation of human Klotho gene was achieved with CRISPR/Cas9-mediated system in resistant human colon cancer Caco-2 cells. To determine the effect of upregulation of Klotho gene on cancer cells evaluations with flow cytometry, WST-8, qRT-PCR, ELISA, and immunohistochemical analysis were performed. Then, Klotho gene was knocked out and its apoptotic effect was tested to find out whether it is due to overexpression of Klotho gene or not. Our results indicate that overexpression of Klotho gene in Caco-2 cells via CRISPR/Cas9-sensitized TRAIL death receptor DR4 suppresses the proliferation of cells by leading to apoptosis. Thus, this study conducted on apoptosis-resistant colon cancer cells may bring new insights about the role of Klotho gene in colon cancer.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Klotho/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Apoptosis , Células CACO-2 , Neoplasias del Colon/genética , Humanos , Transducción de Señal/fisiologíaRESUMEN
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) initiates the extrinsic apoptotic pathway through formation of the death-inducing signaling complex (DISC), followed by activation of effector caspases. TRAIL receptors are composed of death receptors (DR4 and DR5), decoy receptors (DcR1 and DcR2), and osteoprotegerin. Among them, only DRs activate apoptotic signaling by TRAIL. Since the levels of DR expressions are higher in cancer cells than in normal cells, TRAIL selectively activates apoptotic signaling pathway in cancer cells. However, multiple mechanisms, including down-regulation of DR expression and pro-apoptotic proteins, and up-regulation of anti-apoptotic proteins, make cancer cells TRAIL-resistant. Therefore, many researchers have investigated strategies to overcome TRAIL resistance. In this review, we focus on protein regulation in relation to extrinsic apoptotic signaling pathways via ubiquitination. The ubiquitin proteasome system (UPS) is an important process in control of protein degradation and stabilization, and regulates proliferation and apoptosis in cancer cells. The level of ubiquitination of proteins is determined by the balance of E3 ubiquitin ligases and deubiquitinases (DUBs), which determine protein stability. Regulation of the UPS may be an attractive target for enhancement of TRAIL-induced apoptosis. Our review provides insight to increasing sensitivity to TRAIL-mediated apoptosis through control of post-translational protein expression. [BMB Reports 2019; 52(2): 119-126].
Asunto(s)
Apoptosis/fisiología , Enzimas Desubicuitinizantes/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/fisiología , Humanos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Receptores Señuelo del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis via death receptor (DR) 4 or DR5 preferentially in cancer cells, and not in normal cells with relatively high decoy receptor expression. However, multiple mechanisms in cancer cells induce resistance to DRs-mediated apoptosis. Therefore, understanding of molecular mechanisms for resistance to DRs-mediated apoptosis can find the strategy to increase sensitivity. Although multiple proteins are involved in resistance to DRs-mediated apoptosis, we focus on modulation of DR5 to overcome resistance. Here, we discuss regulation of DR5 expression or activation by epigenetic modification, transcription factor at the transcriptional levels, micro RNA and RNA-binding proteins at the post-transcriptional levels, and ubiquitination and glycosylation at the post-translational levels. In addition, we also mention about relationship between localization of DR5 and death signaling activation. The purpose of this review is to help understand relationship between regulatory mechanisms of DR5 and resistance to TRAIL or DRs-targeted agonist monoclonal antibodies, and to develop innovative anti-cancer therapies through regulation of DR5 signaling.
Asunto(s)
Apoptosis/fisiología , Biomarcadores de Tumor/metabolismo , Neoplasias/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Transducción de Señal/fisiología , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
The discovery that the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis of cancer cells without causing toxicity in mice has led to the in-depth study of pro-apoptotic TRAIL receptor (TRAIL-R) signalling and the development of biotherapeutic drug candidates that activate TRAIL-Rs. The outcome of clinical trials with these TRAIL-R agonists has, however, been disappointing so far. Recent evidence indicates that many cancers, in addition to being TRAIL resistant, use the endogenous TRAIL-TRAIL-R system to their own advantage. However, novel insight on two fronts - how resistance of cancer cells to TRAIL-based pro-apoptotic therapies might be overcome, and how the pro-tumorigenic effects of endogenous TRAIL might be countered - gives reasonable hope that the TRAIL system can be harnessed to treat cancer. In this Review we assess the status quo of our understanding of the biology of the TRAIL-TRAIL-R system - as well as the gaps therein - and discuss the opportunities and challenges in effectively targeting this pathway.
Asunto(s)
Neoplasias/etiología , Neoplasias/terapia , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Humanos , Neoplasias/patología , Transducción de SeñalRESUMEN
In the process of cancer spreading, different modes of invasion exist. One is expansive invasion, in which a group of cancer cells gradually expands along with cancer cell proliferation. Invasion of cancer cells is also modified by their interaction with stromal cells including cancer-associated fibroblasts (CAFs). Cancer cells co-invade with CAFs, and invasion by CAFs frequently precede invasion by cancer cells, which indicates CAF-led cancer cell invasion. Here, we show that CAFs induce apoptosis in gastric cancer cells, which prevented expansive invasion by cancer cells and instead facilitated CAF-led invasion. Death receptor 4 and activation of caspase-8 in cancer cells mediated cancer cell apoptosis induced by CAFs, which was dependent on contact between cancer cells and CAFs. Apoptotic cancer cells in turn released apoptotic vesicles and stimulated invasion of CAFs. Accordingly, cancer cells followed the migrating CAFs. Treatment with a caspase inhibitor, ZVAD, or forced expression of a death domain fragment in cancer cells prevented cancer cell apoptosis induced by CAFs and increased expansive invasion by cancer cells in extracellular gel invasion assays, while the rate of cancer cell invasion led by CAFs was decreased. Death domain-fragment expression also prevented intramural invasion by gastric cancer cells in the stomach. Because CAF-led invasion is characterized by the movement of individual cancer cells away from the tumour, adequate cancer cell apoptosis may promote cancer dissemination.
Asunto(s)
Apoptosis , Fibroblastos Asociados al Cáncer/fisiología , Invasividad Neoplásica , Neoplasias Gástricas/patología , Animales , Caspasa 8/fisiología , Comunicación Celular , Línea Celular Tumoral , Vesículas Extracelulares/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiologíaRESUMEN
Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/EhsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.
Asunto(s)
Apoptosis/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Tolerancia a Radiación/genética , Elementos de Respuesta/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Células A549 , Caspasa 3/metabolismo , Hipoxia de la Célula/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/genética , Hipoxia/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
Since their identification more than 20 years ago, the death receptors CD95, TRAILR1, and TRAILR2 have been intensively studied with respect to their cell death-inducing activities. These receptors, however, can also trigger a variety of cell death-independent cellular responses reaching from the activation of proinflammatory gene transcription programs over the stimulation of proliferation and differentiation to induction of cell migration. The cell death-inducing signaling mechanisms of CD95 and the TRAIL death receptors are well understood. In contrast, despite the increasing recognition of the biological and pathophysiological relevance of the cell death-independent activities of CD95, TRAILR1, and TRAILR2, the corresponding signaling mechanisms are less understood and give no fully coherent picture. This review is focused on the cell death-independent activities of CD95 and the TRAIL death receptors and addresses mainly three questions: (a) how are these receptors linked to noncell death pathways at the molecular level, (b) which factors determine the balance of cell death and cell death-independent activities of CD95 and the TRAIL death receptors at the cellular level, and (c) what are the consequences of the cell death-independent functions of these receptors for their role in cancer and inflammatory diseases.
Asunto(s)
Muerte Celular/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Receptor fas/fisiología , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Activación Enzimática , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
The TNF-related apoptosis inducing ligand TRAIL is a member of the TNF superfamily that has been firstly studied and evaluated for its anti-cancer activity, and the insights into its biology have already led to the identification of several TRAIL-based anticancer strategies with strong clinical therapeutic potentials. Nonetheless, the TRAIL system is far more complex and it can lead to a wider range of biological effects other than the ability of inducing apoptosis in cancer cells. By virtue of the different receptors and the different signalling pathways involved, TRAIL plays indeed a role in the regulation of different processes of the innate and adaptive immune system and this feature makes it an intriguing molecule under consideration in the development/progression/treatment of several immunological disorders. In this context, central nervous system represents a peculiar anatomic site where, despite its "status" of immune-privileged site, both innate and adaptive inflammatory responses occur and are involved in several pathological conditions. A number of studies have evaluated the role of TRAIL and of TRAIL-related pathways as pro-inflammatory or protective stimuli, depending on the specific pathological condition, confirming a twofold nature of this molecule. In this light, the aim of this review is to summarize the main preclinical evidences of the potential/involvement of TRAIL molecule and TRAIL pathways for the treatment of central nervous system disorders and the key suggestions coming from their assessment in preclinical models as proof of concept for future clinical studies.
Asunto(s)
Enfermedades del Sistema Nervioso Central/fisiopatología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Enfermedad de Alzheimer/fisiopatología , Animales , Biomarcadores/metabolismo , Isquemia Encefálica/fisiopatología , Supervivencia Celular , Trastornos del Conocimiento/fisiopatología , Humanos , Inflamación/fisiopatología , Esclerosis Múltiple/fisiopatología , Neoplasias/fisiopatología , Transducción de Señal , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super family found in recent years, which widely exists in the body tissues and participates in the immune regulation, immune stability, and immune surveillance of the human body. The TRAIL receptor is expressed in the surface of a variety of cells. Recent studies show that TRAIL induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. Its anti-tumor activity and safety have been widely recognized. The development of prostate cancer is regulated by the mechanisms of cell apoptosis. TRAIL can induce the apoptosis of prostate cancer cells, and therefore has a great application value in the treatment of prostate cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis , Neoplasias de la Próstata/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Proteínas Reguladoras de la Apoptosis , Humanos , Masculino , Glicoproteínas de Membrana , Neoplasias de la Próstata/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfaRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, is a promising new strategy for the treatment of cancer. However, aberrant PI3K/Akt/mTOR survival signaling may confer TRAIL resistance by altering the balance between pro- and anti-apoptotic proteins. In the present study, we showed that the Akt/mTOR inhibitor RAD001 (everolimus) induced cell death in a dose-dependent manner and enhanced TRAIL-induced apoptosis in human leukemic Jurkat T cells, which show PI3K/Akt/mTOR pathway activation and basal expression levels of death receptor (DR) 5 (TRAIL-R2). Investigation of the effect of RAD001 treatment on the expression of TRAIL receptors (TRAIL-Rs) in Jurkat T cells showed that RAD001 significantly upregulated DR5 by up to 51.22%, but not other TRAIL-Rs such as DR4 (TRAIL-R1), decoy receptor (DcR) 1 (TRAIL-R3), and DcR2 (TRAIL-R4). Pretreatment with DR5:Fc chimera abrogated the RAD001-induced increase of TRAIL cytotoxicity, indicating that the upregulation of DR5 by RAD001 plays a role in enhancing the susceptibility of Jurkat T cells to TRAIL. Our results indicate that combination treatment with RAD001 and TRAIL may be a novel therapeutic strategy in leukemia.
Asunto(s)
Antineoplásicos/farmacología , Leucemia/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Sirolimus/análogos & derivados , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Regulación hacia Arriba/efectos de los fármacos , Apoptosis/fisiología , Relación Dosis-Respuesta a Droga , Everolimus , Humanos , Células Jurkat , Leucemia/fisiopatología , Sirolimus/farmacologíaRESUMEN
Despite improvements in detection, surgical approaches and systemic therapies, breast cancer remains typically incurable once distant metastases occur. High expression of TRAIL-R2 was found to be associated with poor prognostic parameters in breast cancer patients, suggesting an oncogenic function of this receptor. In the present study, we aimed to determine the impact of TRAIL-R2 on breast cancer metastasis. Using an osteotropic variant of MDA-MB-231 breast cancer cells, we examine the effects of TRAIL-R2 knockdown in vitro and in vivo. Strikingly, in addition to the reduced levels of the proliferation-promoting factor HMGA2 and corresponding inhibition of cell proliferation, knockdown of TRAIL-R2 increased the levels of E-Cadherin and decreased migration. In vivo, these cells were strongly impaired in their ability to form bone metastases after intracardiac injection. Evaluating possible underlying mechanisms revealed a strong downregulation of CXCR4, the receptor for the chemokine SDF-1 important for homing of cancers cells to the bone. In accordance, cell migration towards SDF-1 was significantly impaired by TRAIL-R2 knockdown. Conversely, overexpression of TRAIL-R2 upregulated CXCR4 levels and enhanced SDF-1-directed migration. We therefore postulate that inhibition of TRAIL-R2 expression could represent a promising therapeutic strategy leading to an effective impairment of breast cancer cell capability to form skeletal metastases.
Asunto(s)
Neoplasias Óseas/secundario , Carcinoma/secundario , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Neoplasias de la Mama Triple Negativas/patología , Animales , Neoplasias Óseas/genética , Cadherinas/biosíntesis , Cadherinas/genética , Carcinoma/genética , División Celular , Línea Celular Tumoral , Quimiocina CXCL12/fisiología , Quimiotaxis , Femenino , Proteína HMGA2/biosíntesis , Proteína HMGA2/genética , Xenoinjertos , Humanos , Ratones , Ratones SCID , Terapia Molecular Dirigida , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Especificidad de Órganos , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Animales , Apoptosis/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Modelos Biológicos , Invasividad Neoplásica/genética , Pronóstico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
The emergence during evolution of two tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, receptor-1/DR4 and -2/DR5, able to induce apoptosis has raised the question whether they differ in function and regulation, which is of key importance for selecting either DR4 or DR5 selective pro-apoptotic agents for cancer treatment. In this review we found practically no information regarding possible differences in DR4 and DR5 function based on structural differences. On the other hand, a panel of different DR4 or DR5 selective pro-apoptotic agonists have been developed that were explored for efficacy in different tumour types in a large number of studies. Leukemic cells appear mainly sensitive for DR4-induced apoptosis, contrasting the situation in other tumour types that show heterogeneity in receptor preference and, in some cases, a slight overall preference for DR5. Both receptors were found to mediate intracellular stress-induced apoptosis, although this is most frequently reported for DR5. Interestingly, DR5 was also found to transmit non-apoptotic signalling in resistant tumour cells and recently nuclear localization and a role in microRNA maturation has been described. DR4 expression is most heavily regulated by promoter methylation, intracellular trafficking and post-translational modifications. DR5 expression is predominantly regulated at the transcriptional level, which may reflect its ability to respond to cellular stressors. It will be important to further increase our understanding of the mechanisms determining TRAIL receptor preference in order to select the appropriate TRAIL receptor selective agonists for therapy, and to develop novel strategies to enhance apoptosis activation in tumours.
Asunto(s)
Neoplasias/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Secuencia de Aminoácidos , Apoptosis , Humanos , Datos de Secuencia Molecular , Neoplasias/metabolismo , Neoplasias/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
ER stress induces apoptosis through DR5, which is regulated by the UPR factors PERK and IRE1α.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Respuesta de Proteína Desplegada , Animales , HumanosRESUMEN
Protein folding by the endoplasmic reticulum (ER) is physiologically critical; its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here, we report that unmitigated ER stress promoted apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, which allowed time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Respuesta de Proteína Desplegada , Animales , Caspasas , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/metabolismo , Células HCT116 , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Factor de Transcripción CHOPRESUMEN
PURPOSE: Piperlongumine (PL) has been shown to selectively induce apoptotic cell death in cancer cells via reactive oxygen species (ROS) accumulation. In this study, we characterized a molecular mechanism for PL-induced cell death. METHODS: Cell viability and cell death were assessed by MTT assay and Annexin V-FITC/PI staining, respectively. ROS generation was measured using the H2DCFDA. Small interfering RNA (siRNA) was used for suppressing gene expression. The mRNA and protein expression were analyzed by RT-PCR and Western blot analysis, respectively. RESULTS: We found that PL promotes C/EBP homologous protein (CHOP) induction, which leads to the up-regulation of its targets Bim and DR5. Pretreatment with the ROS scavenger N-acetyl-cysteine abolishes the PL-induced up-regulation of CHOP and its target genes, suggesting an essential role for ROS in PL-induced CHOP activation. The down-regulation of CHOP or Bim with siRNA efficiently attenuates PL-induced cell death, suggesting a critical role for CHOP in this cell death. Furthermore, PL potentiates TRAIL-induced cytotoxicity in breast cancer cells by upregulating DR5, as DR5 knockdown abolished the sensitizing effect of PL on TRAIL responses. CONCLUSIONS: Overall, our data suggest a new mechanism for the PL-induced cell death in which ROS mediates CHOP activation, and combination treatment with PL and TRAIL could be a potential strategy for breast cancer therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Dioxolanos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factor de Transcripción CHOP/metabolismo , Proteínas Reguladoras de la Apoptosis/fisiología , Proteína 11 Similar a Bcl2 , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiologíaRESUMEN
The cytokine TNF-related apoptosis-inducing ligand (TRAIL) and its cell membrane receptors constitute an elaborate signaling system fulfilling important functions in immune regulation and tumor surveillance. Activation of the death receptors TRAILR1 and TRAILR2 can lead to apoptosis, whereas TRAILR3 and TRAILR4 are generally referred to as decoy receptors, which have been shown to inhibit TRAIL-induced apoptosis. The underlying molecular mechanisms, however, remain unclear. Alike other members of the TNF receptor superfamily, TRAIL receptors contain a pre-ligand binding assembly domain (PLAD) mediating receptor oligomerization. Still, the stoichiometry of TRAIL receptor oligomers as well as the issue of whether the PLAD mediates only homotypic or also heterotypic interactions remained inconclusive until now. Performing acceptor-photobleaching FRET studies with receptors 1, 2, and 4, we demonstrate interactions in all possible combinations. Formation of dimers was shown by chemical cross-linking experiments for interactions of TRAILR2 and heterophilic interactions between the two death receptors or between either of the death receptors and TRAILR4. Implications of the demonstrated receptor-receptor interactions on signaling were investigated in suitable cellular models. Both apoptosis induction and activation of the transcription factor NFκB were significantly reduced in the presence of TRAILR4. Our experimental data combined with mathematical modeling show that the inhibitory capacity of TRAILR4 is attributable to signaling-independent mechanisms, strongly suggesting a reduction of signaling competent death receptors through formation heteromeric receptor complexes. In summary, we propose a model of TRAIL receptor interference driven by PLAD-mediated formation of receptor heterodimers on the cell membrane.
Asunto(s)
Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Transducción de Señal , Apoptosis , Dimerización , Células HeLa , HumanosRESUMEN
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (TRAIL-R) play important roles in immune regulation and cancer cell death. Although TRAIL has been shown to induce chemokine release in various tumour cells, the function of TRAIL-R in the development of colitis and colitis-associated carcinogenesis has not been explored. In this study, we found that TRAIL-R-deficient mice exhibited a higher incidence of colitis and colitis-associated cancer than that of wild-type (WT) mice, and TRAIL-R expression was down-regulated in WT mice that were fed dextran sulphate sodium. Chemokines, including CCL2 and CXCL1, were highly expressed in the serum and inflammatory colon tissues of TRAIL-R(-/-) mice compared with WT mice, and TRAIL-R(-/-) mice showed a marked infiltration of immune cells during colitis. Hyperactivation of Janus kinase and nuclear factor-κB in colon epithelial cells was also observed, which correlated with the severity of colonic inflammation in TRAIL-R(-/-) mice. These data suggest that TRAIL-R plays a protective role in chemical-induced colon injury and negatively regulates mucosal immune responses.
Asunto(s)
Colitis/prevención & control , Neoplasias del Colon/etiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Animales , Movimiento Celular , Quimiocina CCL2/análisis , Quimiocina CXCL1/análisis , Colitis/inducido químicamente , Colitis/complicaciones , Sulfato de Dextran , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/deficienciaRESUMEN
BACKGROUND & AIMS: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS: Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS: TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS: Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.
Asunto(s)
Apoptosis/fisiología , Carcinoma Ductal Pancreático/metabolismo , MicroARNs/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/metabolismo , Humanos , Neoplasias Renales/metabolismo , Neoplasias Pulmonares/metabolismo , Ratones , Ratones SCID , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiologíaRESUMEN
Crystal-cell interaction has been reported as one of the most crucial steps in urinary stone formation. Hyperoxaluria-induced apoptotic changes in renal tubular epithelial cells is the end-stage of this interaction. We aimed to evaluate the possible pathways responsible in the induction of apoptosis within the involved cells by assessing the receptoral expression of three different pathways. 16 male Spraque-Dowley rats were divided into two groups: Group 1 (n:8) received only distilled water; Group 2 (n:8) received 0.75 % ethylene glycol (EG) in their daily water to induce hyperoxaluria for 2 weeks. After 24 h urine collection, all animals were euthenized and right kidneys were removed and fixed for immunohistochemical evaluation. Oxalate and creatinine levels (in 24 h-urine) and FAS, tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor-2 expressions (in tissue) have been assessed. In addition to TNF (p = 0.0007) expression; both FAS (p = 0.0129 ) and FASL (p = 0.032) expressions significantly increased in animals treated with EG. The expressions of TRAIL (p = 0.49) and TRAIL-R2 (p = 0.34) receptors did not change statistically after hyperoxaluria induction. Although a positive correlation with cytokine expression density and 24 h-urinary oxalate expression (mg oxalate/mg creatinine) has been assessed with TNF (p = 0.04, r = 0.82), FAS (p = 0.05, r = 0.80), FAS-L (p = 0.04, r = 0.82); no correlation could be demonstrated between TRAIL and TRAIL R2 expressions. Our results indicate that apoptosis induced by oxalate is possibly mediated via TNF and FAS pathways. However, TRAIL and TRAIL-R2 seemed to have no function in the cascade. Correlation with urinary oxalate levels did further strengthen the findings.