RESUMEN
Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.
Asunto(s)
Apoptosis , Proteolisis , Proteína p53 Supresora de Tumor , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Proteolisis/efectos de los fármacos , Animales , Ratones , Línea Celular Tumoral , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Regulación hacia Arriba/efectos de los fármacos , Unión ProteicaRESUMEN
BACKGROUND: Breast cancer (BC) is the most prevalent malignant disease affecting women globally. PANoptosis, a novel form of cell death combining features of pyroptosis, apoptosis, and necroptosis, has recently gained attention. However, its precise function in BC and the predictive values of PANoptosis-related genes remain unclear. METHODS: We used the expression data and clinical information of BC tissues or normal breast tissues from public databases, and then successfully developed and verified a BC PANoptosis-related risk model through a combination of univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and Kaplan-Meier (KM) analysis. A nomogram was constructed to estimate survival probability, and its accuracy was assessed using calibration curves. RESULTS: Among 37 PANoptosis-related genes, we identified 4 differentially expressed genes related to overall survival (OS). Next, a risk model incorporating these four PANoptosis-related genes was established. Patients were stratified into low/high-risk groups based on the median risk score, with the low-risk group showing better prognoses and higher levels of immune infiltration. Utilizing the risk score and clinical features, we developed a nomogram to predict 1-, 3- and 5-year survival probability. X-linked inhibitor of apoptosis protein (XIAP) emerged as a potentially risky factor with the highest hazard ratio. In vitro experiments demonstrated that XIAP inhibition enhances the antitumor effect of doxorubicin through the PANoptosis pathway. CONCLUSION: PANoptosis holds an important role in BC prognosis and treatment.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Oncogenes/genética , Doxorrubicina , Apoptosis/genéticaRESUMEN
PURPOSE: Oral Squamous Cell Carcinoma (OSCC) is the 6th most common cancer worldwide. It is generally aggressive and closely associated with chemoresistance and poor survival. There is accumulating evidence for the involvement of inhibitors of apoptosis proteins (IAPs), including IAP1 and XIAP, in mediating chemotherapy resistance in OSCC. Various strategies for targeting IAPs have been designed and tested in recent years and several small molecule IAP inhibitors are in clinical trials as monotherapies as well as in combination with radiotherapy and chemotherapy. The purpose of this study was to evaluate and compare the efficacy and biological activity of three IAP inhibitors both as stand-alone and sensitising agents to cisplatin in a preclinical model of squamous cell carcinoma of the tongue. METHODS: Cisplatin-sensitive SCC4 and -resistant SCC4cisR cells were utilised in this study. Apoptosis was evaluated by flow cytometric analysis of Annexin V/Propidium Iodide-stained cells. Expression of IAP proteins was determined by western blotting and knockdown of cIAP1, livin and XIAP was conducted by transfection of cells with siRNA. RESULTS: We establish for the first time the therapeutic efficacy of the Smac mimetic, BV6 and the XIAP inhibitor Embelin, for OSCC. Both of these IAP targeting agents synergistically enhanced cisplatin-mediated apoptotic cell death in resistant cells which was mediated in part by depletion of XIAP. In addition, knockdown of XIAP using siRNA enhanced cisplatin-mediated cell death, demonstrating the importance of targeting XIAP in this sensitisation. CONCLUSION: These findings provide pre-clinical evidence that IAP inhibition may be a valuable therapeutic option in OSCC.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de la Boca , Humanos , Cisplatino/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Línea Celular Tumoral , Neoplasias de la Boca/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Proteínas Portadoras , ARN Interferente PequeñoRESUMEN
Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by progressive loss of dopamine-producing neurons from the substantia nigra region of the brain. Mitochondrial dysfunction is one of the major causes of oxidative stress and neuronal cell death in PD. E3 ubiquitin ligases such as Parkin (PRKN) modulate mitochondrial quality control in PD; however, the role of other E3 ligases associated with mitochondria in the regulation of neuronal cell death in PD has not been explored. The current study investigated the role of TRIM32, RING E3 ligase, in sensitization to oxidative stress-induced neuronal apoptosis. The expression of TRIM32 sensitizes SH-SY5Y dopaminergic cells to rotenone and 6-OHDA-induced neuronal death, whereas the knockdown increased cell viability under PD stress conditions. The turnover of TRIM32 is enhanced under PD stress conditions and is mediated by autophagy. TRIM32 translocation to mitochondria is enhanced under PD stress conditions and localizes on the outer mitochondrial membrane. TRIM32 decreases complex-I assembly and activity as well as mitochondrial reactive oxygen species (ROS) and ATP levels under PD stress. Deletion of the RING domain of TRIM32 enhanced complex I activity and rescued ROS levels and neuronal viability under PD stress conditions. TRIM32 decreases the level of XIAP, and co-expression of XIAP with TRIM32 rescued the PD stress-induced cell death and mitochondrial ROS level. In conclusion, turnover of TRIM32 increases during stress conditions and translocation to mitochondria is enhanced, regulating mitochondrial functions and neuronal apoptosis by modulating the level of XIAP in PD.
Asunto(s)
Apoptosis , Neuronas Dopaminérgicas , Mitocondrias , Estrés Oxidativo , Enfermedad de Parkinson , Especies Reactivas de Oxígeno , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/genética , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Rotenona/farmacología , Transporte de Proteínas , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Línea Celular Tumoral , Oxidopamina/farmacología , Autofagia , Adenosina Trifosfato/metabolismo , Supervivencia Celular/genéticaRESUMEN
X-linked inhibitor of apoptosis protein (XIAP) deficiency is an inborn error of immunity (IEI). Allogeneic hematopoietic cell transplantation (HCT) is currently the only curative therapy available for XIAP deficiency. Granulomatous and lymphocytic interstitial lung disease (GLILD) is a common immune-related lung complication of IEIs. We present a 6-year-old boy with XIAP deficiency and GLILD. Computed tomography showed lung nodes but no symptoms. Before HCT, GLILD was not managed with immunosuppressive therapy, because he was asymptomatic. The HCT procedure was subsequently performed. The post-HCT course was uneventful; follow-up computed tomography on day 46 showed nodules had disappeared. HCT could potentially ameliorate GLILD like other inflammatory processes associated with the underlying IEIs.
Asunto(s)
Inmunodeficiencia Variable Común , Enfermedades Genéticas Ligadas al Cromosoma X , Trasplante de Células Madre Hematopoyéticas , Enfermedades Pulmonares Intersticiales , Trastornos Linfoproliferativos , Masculino , Humanos , Niño , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Enfermedades Pulmonares Intersticiales/etiología , Enfermedades Pulmonares Intersticiales/terapia , Trastornos Linfoproliferativos/terapia , Trastornos Linfoproliferativos/complicaciones , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunodeficiencia Variable Común/complicacionesRESUMEN
X-linked inhibitor of apoptosis protein (XIAP) deficiency causes refractory inflammatory bowel disease. The XIAP protein plays a pivotal role in the pro-inflammatory response through the nucleotide-binding oligomerization domain-containing signaling pathway that is important in mucosal homeostasis. We analyzed the molecular mechanism of non-synonymous pathogenic variants (PVs) of XIAP BIR2 domain. We generated N-terminally green fluorescent protein-tagged XIAP constructs of representative non-synonymous PVs. Co-immunoprecipitation and fluorescence cross-correlation spectroscopy showed that wild-type XIAP and RIP2 preferentially interacted in live cells, whereas all non-synonymous PV XIAPs failed to interact properly with RIP2. Structural analysis showed that various structural changes by mutations, such as hydrophobic core collapse, Zn-finger loss, and spatial rearrangement, destabilized the two loop structures (174-182 and 205-215) that critically interact with RIP2. Subsequently, it caused a failure of RIP2 ubiquitination and loss of protein deficiency by the auto-ubiquitination of all XIAP mutants. These findings could enhance our understanding of the role of XIAP mutations in XIAP-deficient inflammatory bowel disease and may benefit future therapeutic strategies.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Proteína Inhibidora de la Apoptosis Ligada a X , Humanos , Proteínas Fluorescentes Verdes , Homeostasis , Enfermedades Inflamatorias del Intestino/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
BACKGROUND: The contribution of genetic factors to the severity of adult hemophagocytic lymphohistiocytosis (HLHa) remains unclear. OBJECTIVE: We sought to assess a potential link between HLHa outcomes and HLH-related gene variants. METHODS: Clinical characteristics of 130 HLHa patients (age ≥ 18 years and HScore ≥ 169) and genotype of 8 HLH-related genes (LYST, PRF1, UNC13-D, STX11, STXBP2, RAB27A, XIAP, and SAP) were collected. A total of 34 variants found in only 6 genes were selected on the basis of their frequency and criteria predicted to impair protein function. Severity was defined by refractory disease to HLH treatment, death, or transfer to an intensive care unit. RESULTS: HLHa-associated diseases (ADs) were neoplasia (n = 49 [37.7%]), autoimmune/inflammatory disease (n = 33 [25.4%]), or idiopathic when no AD was identified (n = 48 [36.9%]). Infectious events occurred in 76 (58.5%) patients and were equally distributed in all ADs. Severe and refractory HLHa were observed in 80 (61.5%) and 64 (49.2%) patients, respectively. HScore, age, sex ratio, AD, and infectious events showed no significant association with HLHa severity. Variants were identified in 71 alleles and were present in 56 (43.1%) patients. They were distributed as follows: 44 (34.4%), 9 (6.9%), and 3 (2.3%) patients carrying 1, 2, and 3 variant alleles, respectively. In a logistic regression model, only the number of variants was significantly associated with HLHa severity (1 vs 0: 3.86 [1.73-9.14], P = .0008; 2-3 vs 0: 29.4 [3.62-3810], P = .0002) and refractoriness (1 vs 0: 2.47 [1.17-5.34], P = .018; 2-3 vs 0: 13.2 [2.91-126.8], P = .0003). CONCLUSIONS: HLH-related gene variants may be key components to the severity and refractoriness of HLHa.
Asunto(s)
Linfohistiocitosis Hemofagocítica , Adulto , Humanos , Adolescente , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/terapia , Alelos , Genotipo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Oral squamous cell carcinoma (OSCC), which accounts for 90% of all oral cancers, has become a public health crisis worldwide. despite advances in therapeutic interventions, the prognosis remains poor for advanced-stage OSCC. In this study, we investigate the anticancer activity and the mode of action of hellebrigenin in human OSCC. The findings demonstrated that hellebrigenin exerted cytotoxic effects in OSCC cells through cell cycle arrest at the G2/M phase and downregulation of cell cycle-related proteins (cyclins A2, B1 and D3, Cdc2, CDK4 and CDK6). Moreover, hellebrigenin caused activation of PARP and caspase 3, 8 and 9, followed by downregulation of antiapoptotic proteins (Bcl-2 and Bcl-xL) and upregulation of pro-apoptotic proteins (Bax and Bak). The hellebrigenin treatment also increased Fas, DR5, DcR2 and DcR3 expressions in oral cancer cells, indicating the compound causes oral cancer cell apoptosis through both intrinsic and extrinsic pathways. Regarding upstream signalling, hellebrigenin was found to reduce the phosphorylation of ERK, p38, and JNK, indicating that hellebrigenin triggers caspase-mediated apoptosis by downregulating MAPK signalling pathway. Finally, the human apoptosis array findings revealed that hellebrigenin specifically suppressed the expression of XIAP to execute its pro-apoptotic activities. Taken together, the study suggests that hellebrigenin can act as a potent anticancer compound in human OSCC.
Asunto(s)
Bufanólidos , Carcinoma de Células Escamosas , Neoplasias de la Boca , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Transducción de Señal , Apoptosis/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Triptolide (TP) is extracted from the traditional Chinese medicine Tripterygium wilfordii Hook. F. (TWHF). Its severe toxic side effects, especially hepatotoxicity, have limited the clinical application of TP-related drugs. In this study, we investigated the mechanism of the hepatotoxic effects of TP from the perspective that TP inhibited the expression of the pro-survival protein X-linked inhibitor of apoptosis protein (XIAP) and enhanced FasL-mediated apoptosis of hepatocytes. TP and CD95/Fas antibody (Jo-2) were administered by gavage to C57BL/6 mice for 7 consecutive days. After co-administration of TP and Jo-2, mouse livers showed large areas of necrosis and apoptosis and significantly increased Caspase-3 activity. KEGG pathway enrichment analysis indicated that TP may cause the development of liver injury through the apoptotic signaling pathway. Proteinprotein interaction networks showed that XIAP played an essential role in this process. TP reduced the protein expression of XIAP after combination treatment with Jo-2/FasL in vivo/in vitro. TP and FasL co-stimulation significantly increased microRNA-137 (miR-137) levels in AML12 cells, while inhibition of miR-137 expression induced a rebound in XIAP protein expression. In conclusion, TP presensitizes hepatocytes and enhances the sensitivity of hepatocytes to the Fas/FasL pathway by inhibiting the protein expression of XIAP, leading to hepatocyte apoptosis.
Asunto(s)
MicroARNs , Proteína Inhibidora de la Apoptosis Ligada a X , Ratones , Animales , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/farmacología , Ratones Endogámicos C57BL , Hígado/metabolismo , Hepatocitos , Apoptosis , MicroARNs/metabolismoRESUMEN
XIAP, the most potent mammalian inhibitor of apoptosis protein (IAP), critically restricts developmental culling of sympathetic neuronal progenitors, and is correspondingly overexpressed in most MYCN-amplified neuroblastoma tumors. Because apoptosis-related protein in the TGFß signaling pathway (ARTS) is the only XIAP antagonist that directly binds and degrades XIAP, we evaluated the preclinical effectiveness and tolerability of XIAP antagonism as a novel targeting strategy for neuroblastoma. We found that antagonism of XIAP, but not other IAPs, triggered apoptotic death in neuroblastoma cells. XIAP silencing induced apoptosis while overexpression conferred protection from drug-induced apoptosis. From a screen of IAP inhibitors, first-in-class ARTS mimetic A4 was most effective against high-risk and high XIAP-expressing neuroblastoma cells, and least toxic toward normal liver- and bone marrow-derived cells, compared with pan-IAP antagonists. On target engagement assays and nuclear magnetic resonance spectroscopy, A4 was observed to degrade rather than inhibit XIAP, catalyzing rapid degradation of XIAP through the ubiquitin-proteasome pathway. In MYCN-amplified neuroblastoma patient-derived xenografts, A4 significantly prolonged survival as a single agent, and demonstrated synergism with standard-of-care agents to reduce their effective required doses 3- to 6-fold. Engagement and degradation of XIAP by ARTS mimetics is a novel targeting strategy for neuroblastoma that may be especially effective against MYCN-amplified disease with intrinsically high XIAP expression. First-in-class ARTS mimetic A4 demonstrates preclinical efficacy and warrants further development and study. SIGNIFICANCE: XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.
Asunto(s)
Neuroblastoma , Proteína Inhibidora de la Apoptosis Ligada a X , Animales , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Apoptosis , Neuroblastoma/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/metabolismo , Mamíferos/metabolismoRESUMEN
Ehrlichia chaffeensis has evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported the E. chaffeensis tandem repeat protein (TRP)120 effector functions as a Notch ligand mimetic and a ubiquitin ligase that degrades the nuclear tumor suppressor, F-box and WD repeat domain-containing 7, a negative regulator of Notch. The Notch intracellular domain (NICD) is known to inhibit apoptosis primarily by interacting with X-linked inhibitor of apoptosis protein (XIAP) to prevent degradation. In this study, we determined that E. chaffeensis activation of Notch signaling increases XIAP levels, thereby inhibiting apoptosis through both the intrinsic and executioner pathways. Increased NICD and XIAP levels were detected during E. chaffeensis infection and after TRP120 Notch ligand mimetic peptide treatment. Conversely, XIAP levels were reduced in the presence of Notch inhibitor DAPT. Cytoplasmic and nuclear colocalization of NICD and XIAP was observed during infection and a direct interaction was confirmed by co-immunoprecipitation. Procaspase levels increased temporally during infection, consistent with increased XIAP levels; however, knockdown (KD) of XIAP during infection significantly increased apoptosis and Caspase-3, -7, and -9 levels. Furthermore, treatment with SM-164, a second mitochondrial activator of caspases (Smac/DIABLO) antagonist, resulted in decreased procaspase levels and increased caspase activation, induced apoptosis, and significantly decreased infection. In addition, RNAi KD of XIAP also decreased infection and significantly increased apoptosis. Moreover, ectopic expression of TRP120 HECT Ub ligase catalytically defective mutant in HeLa cells decreased NICD and XIAP levels and increased caspase activation compared to HeLa cells with functional HECT Ub ligase catalytic activity (TRP120-WT). This investigation reveals a mechanism whereby E. chaffeensis modulates Notch signaling to stabilize XIAP and inhibit apoptosis.
Asunto(s)
Ehrlichia chaffeensis , Ehrlichiosis , Humanos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Células HeLa , Ligandos , Apoptosis , Caspasas , Ehrlichia chaffeensis/genéticaRESUMEN
BACKGROUND: The X-linked inhibitor of apoptosis (XIAP) protein is encoded by the XIAP gene and is critical for multiple cell responses and plays a role in preventing cell death. XIAP mutations are associated with several diseases, primarily including hemophagocytic lymphohistiocytosis and inflammatory bowel disease (IBD). We report the clinical features and results associated with hemizygous mutation of the XIAP gene in a young male with Crohn's disease complicated with acute heart failure.This 16-year-old patient ultimately died of heart failure. CASE PRESENTATION: A young male of 16 years of age was initially diagnosed with Crohn's disease based on evidences from endoscopic and histological findings. Although supportive care, anti-infective drugs and biologics were administered consecutively for 11 months, his clinical manifestations and laboratory indices (patient's condition) did not improved. Additionally, the patient exhibited a poor nutritional status and sustained weight loss. Subsequently, acute heart failure led to the exacerbation of the patient's condition. He was diagnosed with wet beriberi according to thiamine deficiency, but the standard medical therapy for heart failure and thiamine supplementation did not reverse the adverse outcomes. Comprehensive genetic analysis of peripheral blood-derived DNA revealed a novel hemizygous mutation of the XIAP gene (c.1259_1262 delACAG), which was inherited from his mother. CONCLUSION: A novel XIAP mutation (c.1259_1262 delACAG) was identified in this study. It may be one of the potential pathogenic factors in Crohn's disease and plays an important role in the progression of heart failure. Additionally, thiamine deficiency triggers a vicious cycle.
Asunto(s)
Enfermedad de Crohn , Insuficiencia Cardíaca , Deficiencia de Tiamina , Masculino , Humanos , Adolescente , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/genética , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/genética , Pérdida de Peso , Apoptosis , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
INTRODUCTION: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be an effective apoptosis inducer due to its selectivity for tumor cells. However, many cancer cells, especially metastatic cancer cells, often exhibit resistance to TRAIL because their apoptotic pathway is impaired or their pro-survival pathway is overactivated. TRAIL resistance is the main obstacle to current TRAIL therapy. Nowadays, ceramide analogs represent a new class of potential anticancer agents. Therefore, we hypothesized that disrupting pro-survival signaling with ceramide analogs would increase TRAIL-mediated apoptosis. METHODS: MTT assay and flow cytometry were conducted to evaluate the synergistic effect of ceramide analog 5cc on TRAIL in metastatic colon cancer cells. Western blot was used to detect signaling proteins affected by 5cc. RNA interference was performed to analyze the effects of specific gene on 5cc-enhanced apoptosis. RESULTS: Ceramide analog 5cc markedly enhanced TRAIL-induced apoptosis evidenced by increased propidium iodide/annexin V double-positive cells and PARP cleavage in SW620 and LS411N cells. At the molecular level, 5cc significantly reduced the expression of anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through the activation of the c-Jun n-terminal kinase (JNK) pathway which is critically involved in sensitizing tumor cells to TRAIL/5cc combination. JNK-silenced cells exhibited a significant reversal of TRAIL/5cc-mediated apoptosis. CONCLUSION: Our data demonstrated that ceramide analog 5cc overcomes TRAIL resistance by enhancing JNK activation and repressing XIAP expression in metastatic colon cancer cells.
Asunto(s)
Neoplasias del Colon , Proteína Inhibidora de la Apoptosis Ligada a X , Humanos , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/farmacología , Ceramidas/farmacología , Ligandos , Línea Celular Tumoral , Apoptosis , Neoplasias del Colon/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
Chemotherapeutic drugs are used to treat almost all types of cancer, but the intended response, i.e., elimination, is often incomplete, with a subset of cancer cells resisting treatment. Two critical factors play a role in chemoresistance: the p53 tumour suppressor gene and the X-linked inhibitor of apoptosis (XIAP). These proteins have been shown to act synergistically to elicit cellular responses upon DNA damage induced by chemotherapy, yet, the mechanism is poorly understood. This study introduces a mathematical model characterising the apoptosis pathway activation by p53 before and after mitochondrial outer membrane permeabilisation upon treatment with the chemotherapy Doxorubicin (Dox). "In-silico" simulations show that the p53 dynamics change dose-dependently. Under medium to high doses of Dox, p53 concentration ultimately stabilises to a high level regardless of XIAP concentrations. However, caspase-3 activation may be triggered or not depending on the XIAP induction rate, ultimately determining whether the cell will perish or resist. Consequently, the model predicts that failure to activate apoptosis in some cancer cells expressing wild-type p53 might be due to heterogeneity between cells in upregulating the XIAP protein, rather than due to the p53 protein concentration. Our model suggests that the interplay of the p53 dynamics and the XIAP induction rate is critical to determine the cancer cells' therapeutic response.
Asunto(s)
Proteína p53 Supresora de Tumor , Proteína Inhibidora de la Apoptosis Ligada a X , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/fisiología , Muerte Celular , Doxorrubicina/farmacología , Línea Celular TumoralRESUMEN
XIAP is an endogenous inhibitor of cell death and inactivating mutations of XIAP are responsible for X-linked lymphoproliferative disease (XLP-2) and primary immunodeficiency, but the mechanism(s) behind these contradictory outcomes have been unclear. We report that during infection of macrophages and dendritic cells with various intracellular bacteria, XIAP restricts cell death and secretion of IL-1ß but promotes increased activation of NFκB and JNK which results in elevated secretion of IL-6 and IL-10. Poor secretion of IL-6 by Xiap-deficient antigen presenting cells leads to poor expansion of recently activated CD8 T cells during the priming phase of the response. On the other hand, Xiap-deficient CD8 T cells displayed increased proliferation and effector function during the priming phase but underwent enhanced contraction subsequently. Xiap-deficient CD8 T cells underwent skewed differentiation towards short lived effectors which resulted in poor generation of memory. Consequently Xiap-deficient CD8 T cells failed to provide effective control of bacterial infection during re-challenge. These results reveal the temporal impact of XIAP in promoting the fitness of activated CD8 T cells through cell extrinsic and intrinsic mechanisms and provide a mechanistic explanation of the phenotype observed in XLP-2 patients.
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Interleucina-6 , Trastornos Linfoproliferativos , Humanos , Muerte Celular , Trastornos Linfoproliferativos/genética , Macrófagos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Memoria Inmunológica , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.
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Cobre , Metaloproteínas , Cobre/química , Metaloproteínas/metabolismo , Unión Proteica , Ubiquitinas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Zinc/metabolismo , Dominios RING Finger/fisiologíaRESUMEN
Perturbations in autophagy, apoptosis and differentiation have greatly affected the progression and therapy of acute myeloid leukaemia (AML). The role of X-linked inhibitor of apoptosis (XIAP)-related autophagy remains unclear in AML therapeutics. Here, we found that XIAP was highly expressed and associated with poor overall survival in patients with AML. Furthermore, pharmacologic inhibition of XIAP using birinapant or XIAP knockdown via siRNA impaired the proliferation and clonogenic capacity by inducing autophagy and apoptosis in AML cells. Intriguingly, birinapant-induced cell death was aggravated in combination with ATG5 siRNA or an autophagy inhibitor spautin-1, suggesting that autophagy may be a pro-survival signalling. Spautin-1 further enhanced the ROS level and myeloid differentiation in THP-1 cells treated with birinapant. The mechanism analysis showed that XIAP interacted with MDM2 and p53, and XIAP inhibition notably downregulated p53, substantially increased the AMPKα1 phosphorylation and downregulated the mTOR phosphorylation. Combined treatment using birinapant and chloroquine significantly retarded AML progression in both a subcutaneous xenograft model injected with HEL cells and an orthotopic xenograft model injected intravenously with C1498 cells. Collectively, our data suggested that XIAP inhibition can induce autophagy, apoptosis and differentiation, and combined inhibition of XIAP and autophagy may be a promising therapeutic strategy for AML.
Asunto(s)
Leucemia Mieloide Aguda , ARN Interferente Pequeño , Proteína p53 Supresora de Tumor , Humanos , Apoptosis , Autofagia , Diferenciación Celular , Línea Celular Tumoral , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , ARN Interferente Pequeño/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoAsunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Enfermedad Injerto contra Huésped , Linfohistiocitosis Hemofagocítica , Proteína Inhibidora de la Apoptosis Ligada a X , Humanos , Lactante , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/terapia , Recurrencia , Terapia Recuperativa , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
OBJECTIVE: Non-small cell lung cancer (NSCLC) is recognized as one of the primary causes of global cancer-related mortality. Long noncoding RNAs (lncRNAs) participate in NSCLC cell progression. This study probed the potential mechanism of lncRNA small nucleolar RNA host gene 12 (SNHG12) in cisplatin (DDP)-resistance in NSCLC cells. METHODS: The intracellular expressions of SNHG12, miR-525-5p, and XIAP were examined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Afterwards, small interfering RNAs (siRNAs) of SNHG12, microRNA (miR)-525-5p inhibitor, and X-linked inhibitor of apoptosis (XIAP) pcDNA3.1 were transfected into NSCLC cells. Subsequently, changes in half-maximal (50%) inhibitory concentration (IC50) of NSCLC cells to DDP were detected through the cell counting kit-8 (CCK-8) method. NSCLC proliferative ability and apoptosis rate were determined with the help of colony formation and flow cytometry assays. The subcellular localization of SNHG12 was analyzed by nuclear/cytosol fractionation assay and binding relationships between miR-525-5p and SNHG12 or XIAP were analyzed via dual-luciferase reporter gene assay. Furthermore, rescue experiments were designed to detect the effects of miR-525-5p and XIAP on NSCLC sensitivity to DDP. RESULTS: SNHG12 and XIAP were up-regulated in NSCLC cells while miR-525-5p was down-regulated. After DDP treatment and SNHG12 repression, NSCLC proliferative ability was decreased whereas apoptosis rate was increased, and NSCLC sensitivity to DDP was enhanced. Mechanically, SNHG12 repressed miR-525-5p expression, and miR-525-5p could targeted inhibit XIAP transcription level. miR-525-5p repression or XIAP overexpression reduced NSCLC sensitivity to DDP. CONCLUSION: SNHG12 was overexpressed in NSCLC cells and promoted XIAP transcription by repressing miR-525-5p expression, enhancing DDP-resistance in NSCLC cells.