RESUMEN
Vascular endothelial cell growth factor (VEGF) is a key regulator of vascular permeability. Herein we aim to understand how acute and chronic exposures of VEGF induce different levels of vascular permeability. We demonstrate that chronic VEGF exposure leads to decreased phosphorylation of VEGFR2 and c-Src as well as steady increases of nitric oxide (NO) as compared to that of acute exposure. Utilizing heat-inducible VEGF transgenic zebrafish (Danio rerio) and establishing an algorithm incorporating segmentation techniques for quantification, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, was shown to play essential roles in both acute and chronic vascular permeability in cultured human cells, zebrafish model, and Miles assay. Taken together, our data reveal acute and chronic VEGF exposures induce divergent signaling pathways and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis.
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Background: Thymoquinone (TQ) has potential anti-inflammatory, immunomodulatory and anticancer effects but its clinical use is limited by its low solubility, poor bioavailability and rapid clearance. Aim: To enhance systemic bioavailability and tumor-specific toxicity of TQ. Materials & methods: Cationic liposomal formulation of TQ (D1T) was prepared via ethanol injection method and their physicochemical properties, anticancer effects in orthotopic xenograft pancreatic tumor model and pharmacokinetic behavior of D1T relative to TQ were evaluated. Results: D1T showed prominent inhibition of pancreatic tumor progression, significantly greater in vivo absorption, approximately 1.5-fold higher plasma concentration, higher bioavailability, reduced volume of distribution and improved clearance relative to TQ. Conclusion: Encapsulation of TQ in cationic liposomal formulation enhanced its bioavailability and anticancer efficacy against xenograft pancreatic tumor.
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Liposomas , Benzoquinonas , Disponibilidad Biológica , Línea Celular Tumoral , Humanos , SolubilidadRESUMEN
Glioblastoma multiforme (GBM) is a highly proliferative and locally invasive cancer with poor prognosis and a high recurrence rate. Although anti-VEGF (vascular endothelial growth factor) therapy offers short-term benefit to GBM patients, this approach fails as the tumor develops into a more invasive and drug-resistant phenotype and ultimately recurs. Recently, both glioma stemlike cells (GSCs) and brain tumor-initiating cells (BTICs) have been implicated in GBM recurrence and its resistance to therapy. We observed that patient-derived GBM cells expressing shRNAs of VEGF or neuropilin-1 (NRP-1) attenuate cancer stem cell markers, inhibit the tumor-initiating cell's neurosphere-forming capacity, and migration. Furthermore, both VEGF and NRP-1 knockdown inhibit the growth of patient-derived GBM xenografts in both zebrafish and mouse models. Interestingly, NRP-1-depleted patient-derived GBM xenografts substantially prolonged survival in mice compared to that of VEGF depletion. Our results also demonstrate that NRP-1 ablation of patient-derived GBM cells improves the sensitivity of TMZ and enhances the overall survival of the respective tumor-bearing mice. This improved outcome may provide insight into the inhibition of GBM progression and effective treatment strategies by targeting NRP-1 in addition to chemotherapy and radiotherapy.
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Resistencia a Antineoplásicos/genética , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Neuropilina-1/deficiencia , Neuropilina-1/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transformación Celular Neoplásica , Glioblastoma/patología , Humanos , Ratones , Fenotipo , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as "SBI-183," suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Renales/tratamiento farmacológico , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/uso terapéutico , Animales , Femenino , Humanos , Ratones , Ratones SCIDRESUMEN
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3ß (GSK-3ß) is an emerging target in human malignancies including PDAC.Experimental Design: Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence. RESULTS: 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage. CONCLUSIONS: These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.
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Adenocarcinoma/tratamiento farmacológico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Proteínas Nucleares/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Humanos , Indoles/farmacología , Maleimidas/farmacología , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , GemcitabinaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic cancer with limited treatment options. There is an urgent need for tools that monitor therapeutic responses in real time. Drugs such as gemcitabine and irinotecan elicit their therapeutic effect in cancer cells by producing hydrogen peroxide (H2O2). In this study, specific DNA-wrapped single-walled carbon nanotubes (SWCNT), which precisely monitor H2O2, were used to determine the therapeutic response of PDAC cells in vitro and tumors in vivo. Drug therapeutic efficacy was evaluated in vitro by monitoring H2O2 differences in situ using reversible alteration of Raman G-bands from the nanotubes. Implantation of the DNA-SWCNT probe inside the PDAC tumor resulted in approximately 50% reduction of Raman G-band intensity when treated with gemcitabine versus the pretreated tumor; the Raman G-band intensity reversed to its pretreatment level upon treatment withdrawal. In summary, using highly specific and sensitive DNA-SWCNT nanosensors, which can determine dynamic alteration of hydrogen peroxide in tumor, can evaluate the effectiveness of chemotherapeutics. SIGNIFICANCE: A novel biosensor is used to detect intratumoral hydrogen peroxide, allowing real-time monitoring of responses to chemotherapeutic drugs.
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Técnicas Biosensibles/métodos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Monitoreo de Drogas/métodos , Peróxido de Hidrógeno/análisis , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Técnicas Biosensibles/instrumentación , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Desoxicitidina/farmacología , Monitoreo de Drogas/instrumentación , Femenino , Humanos , Peróxido de Hidrógeno/metabolismo , Irinotecán/farmacología , Luminiscencia , Ratones SCID , Nanotubos de Carbono , Neoplasias Pancreáticas/metabolismo , Espectrometría Raman , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Neuropilin-1 (NRP1), a non-tyrosine kinase receptor, is overexpressed in many cancers including pancreatic and lung cancers. Inhibition of NRP1 expression, however, has differing pro-tumor vs. anti-tumor effects, depending on the cancer types. To understand the differential role of NRP1 in tumorigenesis process, we utilized cells from two different cancer types, pancreatic and lung, each containing either wild type KRAS (KRAS wt) or mutant KRAS (KRAS mt). Inhibition of NRP1 expression by shRNA in both pancreatic and lung cancer cells containing dominant active KRAS mt caused increased cell viability and tumor growth. On the contrary, inhibition of NRP1, in the tumor cells containing KRAS wt showed decreased tumor growth. Importantly, concurrent inhibition of KRAS mt and NRP1 in the tumor cells reverses the increased viability and leads to tumor inhibition. We found that NRP1 shRNA expressing KRAS mt tumor cells caused increased cell viability by decreasing SMAD2 phosphorylation. Our findings demonstrate that the effects of NRP1 knockdown in cancer cells are dependent on the genetic status of KRAS.
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Carcinogénesis/genética , Neuropilina-1/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones SCID , Proteínas Mutantes/metabolismo , Oncogenes , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Immunological strategies to treat pancreatic cancer offer new therapeutic approaches to improve patient outcomes. Understanding alterations in the immune systems of pancreatic cancer patients will likely lead to advances in immunotherapy for the disease. We profiled peripheral blood leukocytes from pancreatic cancer patients (n = 22) and age-matched controls (n = 20) using flow cytometry. Immune profiling of pancreatic cancer patients identified phenotypic changes in various immune cell populations, including a population of immunosuppressive monocytes (CD14+HLA-DRlo/neg), which were shown to be increased in these patients. There was a correlation between the levels of CD14+ monocytes and the levels of CD14+HLA-DRlo/neg monocytes in peripheral blood from pancreatic cancer patients. HLA-DR downregulation of monocytes was shown to occur through pancreatic cancer-derived exosome interactions with monocytes. In an in vitro model, exosomes from patient-derived xenograft cell lines and patient plasma decreased HLA-DR expression on CD14+ monocytes. Additionally, tumor-derived exosomes caused immune suppression in monocytes through altered STAT3 signaling, induction of arginase expression, and reactive oxygen species. These findings provide novel insights into the mechanisms that govern immunosuppression in pancreatic cancer. Understanding monocyte-exosome interactions could lead to novel immunotherapies for this disease.
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Oncogenic BRAFV600E mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAFV600E-mutant colorectal cancers, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small-molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAFV600E-mutant CRCs. Mol Cancer Ther; 15(12); 3015-27. ©2016 AACR.
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Apoptosis/efectos de los fármacos , Apoptosis/genética , Azetidinas/farmacología , Neoplasias Colorrectales/genética , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Alelos , Animales , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND OBJECTIVES: New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. DESIGN: Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. RESULTS: In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. CONCLUSIONS: PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.
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Adipocitos/metabolismo , Adrenomedulina/metabolismo , Exosomas/metabolismo , Lipólisis , Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Endocitosis/fisiología , Glicerol/metabolismo , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores de Adrenomedulina/antagonistas & inhibidores , Receptores de Adrenomedulina/metabolismo , Grasa Subcutánea/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVES: To further elucidate the anticancer mechanisms of metformin against pancreatic cancer, we evaluated the inhibitory effects of metformin on pancreatic tumorigenesis in a genetically engineered mouse model and investigated its possible anti-inflammatory and antiangiogenesis effects. METHODS: Six-week-old LSL-Kras;Trp53 mice (10 per group) were administered once daily intraperitoneally with saline (control) for 1 week or metformin (125 mg/kg) for 1 week (Met_1wk) or 3 weeks (Met_3wk) before tumor initiation. All mice continued with their respective injections for 6 weeks after tumor initiation. Molecular changes were evaluated through quantitative polymerase chain reaction, immunohistochemistry, and Western blotting. RESULTS: At euthanasia, pancreatic tumor volume in the Met_1wk (median, 181.8 mm) and Met_3wk (median, 137.9 mm) groups was significantly lower than those in the control group (median, 481.1 mm; P = 0.001 and 0.0009, respectively). No significant difference was observed between the Met_1wk and Met_3wk groups (P = 0.51). These results were further confirmed using tumor weight and tumor burden measurements. Furthermore, metformin treatment decreased the phosphorylation of nuclear factor κB and signal transducer and activator of transcription 3 as well as the expression of specificity protein 1 transcription factor and several nuclear factor κB-regulated genes. CONCLUSIONS: Metformin may inhibit pancreatic tumorigenesis by modulating multiple molecular targets in inflammatory pathways.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Metformina/farmacología , FN-kappa B/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Factor de Transcripción STAT3/antagonistas & inhibidores , Carga Tumoral/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Western Blotting , Esquema de Medicación , Femenino , Inmunohistoquímica , Inyecciones Intraperitoneales , Masculino , Metformina/uso terapéutico , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , Distribución Aleatoria , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: Neuropilin-1 (NRP-1) is a multidomain membrane receptor involved in angiogenesis and development of neuronal circuits, however, the role of NRP-1 in cardiovascular pathophysiology remains elusive. APPROACH AND RESULTS: In this study, we first observed that deletion of NRP-1 induced peroxisome proliferator-activated receptor γ coactivator 1α in cardiomyocytes and vascular smooth muscle cells, which was accompanied by dysregulated cardiac mitochondrial accumulation and induction of cardiac hypertrophy- and stress-related markers. To investigate the role of NRP-1 in vivo, we generated mice lacking Nrp-1 in cardiomyocytes and vascular smooth muscle cells (SM22-α-Nrp-1 KO), which exhibited decreased survival rates, developed cardiomyopathy, and aggravated ischemia-induced heart failure. Mechanistically, we found that NRP-1 specifically controls peroxisome proliferator-activated receptor γ coactivator 1 α and peroxisome proliferator-activated receptor γ in cardiomyocytes through crosstalk with Notch1 and Smad2 signaling pathways, respectively. Moreover, SM22-α-Nrp-1 KO mice exhibited impaired physical activities and altered metabolite levels in serum, liver, and adipose tissues, as demonstrated by global metabolic profiling analysis. CONCLUSIONS: Our findings provide new insights into the cardioprotective role of NRP-1 and its influence on global metabolism.
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Cardiomiopatías/metabolismo , Insuficiencia Cardíaca/metabolismo , Isquemia Miocárdica/metabolismo , Neuropilina-1/metabolismo , Animales , Homeostasis , Ratones Noqueados , Proteínas de Microfilamentos , Mitocondrias Cardíacas/metabolismo , Proteínas Musculares , Músculo Liso Vascular/metabolismo , Miocitos Cardíacos/metabolismo , PPAR gamma/metabolismo , Receptor Cross-Talk , Receptor Notch1/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic ß-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches ß cells remote from the cancer to induce ß-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to ß cells and impair insulin secretion. EXPERIMENTAL METHODS: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 ß cells and human islets, and demonstrated the mechanism of exosome internalization into ß cells. We studied the interaction between ß-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in ß cells was shown. RESULTS: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered ß cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on ß cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. ß cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. CONCLUSIONS: Pancreatic cancer causes paraneoplastic ß-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response.
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Adrenomedulina/metabolismo , Diabetes Mellitus/etiología , Exosomas/metabolismo , Células Secretoras de Insulina/metabolismo , Neoplasias Pancreáticas/complicaciones , Western Blotting , Antígeno CA-19-9/metabolismo , Humanos , Microscopía Confocal , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Síndromes Paraneoplásicos/etiología , Síndromes Paraneoplásicos/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Autofagia , Exosomas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Transporte Biológico , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Expresión Génica , Glucosa/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patología , Estrés Fisiológico , Enzimas Activadoras de Ubiquitina/metabolismo , GemcitabinaRESUMEN
Endoglin, a 180-kDa disulfide-linked homodimeric transmembrane receptor protein mostly expressed in tumor-associated endothelial cells, is an endogenous binding partner of GAIP-interacting protein, C terminus (GIPC). Endoglin functions as a coreceptor of TßRII that binds TGFß and is important for vascular development, and consequently has become a compelling target for antiangiogenic therapies. A few recent studies in gastrointestinal stromal tumor (GIST), breast cancer, and ovarian cancer, however, suggest that endoglin is upregulated in tumor cells and is associated with poor prognosis. These findings indicate a broader role of endoglin in tumor biology, beyond angiogenic effects. The goal of our current study is to evaluate the effects of targeting endoglin in pancreatic cancer both in vitro and in vivo. We analyzed the antiproliferative effect of both RNAi-based and peptide ligand-based inhibition of endoglin in pancreatic cancer cell lines, the latter yielding a GIPC PDZ domain-targeting lipopeptide with notable antiproliferative activity. We further demonstrated that endoglin inhibition induced a differentiation phenotype in the pancreatic cancer cells and sensitized them against conventional chemotherapeutic drug gemcitabine. Most importantly, we have demonstrated the antitumor effect of both RNAi-based and competitive inhibitor-based blocking of endoglin in pancreatic cancer xenograft models in vivo. To our knowledge, this is the first report exploring the effect of targeting endoglin in pancreatic cancer cells.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Receptores de Superficie Celular/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos CD/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Sinergismo Farmacológico , Endoglina , Humanos , Ligandos , Masculino , Ratones , Ratones SCID , Terapia Molecular Dirigida , Neoplasias Pancreáticas/patología , Péptidos/farmacología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Distribución Aleatoria , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina , Neoplasias PancreáticasRESUMEN
Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell-cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases, including type II diabetes, Alzheimer disease, bipolar disorder, inflammation, and cancer. Consequently, it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacologic and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent antiproliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The antiproliferative activity is most likely caused by G(0)-G(1) and G(2)-M phase arrest as evident from cell-cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. In addition, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft renal cell carcinoma tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells.
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Diferenciación Celular/fisiología , Glucosa/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias Renales/metabolismo , Animales , Autofagia/efectos de los fármacos , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Indoles/química , Indoles/farmacología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Masculino , Maleimidas/química , Maleimidas/farmacología , Ratones Desnudos , Microscopía Confocal , Estructura Molecular , Interferencia de ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
VEGF/vascular permeability factor (VEGF/VPF or VEGF-A) is a pivotal driver of cancer angiogenesis that is a central therapeutic target in the treatment of malignancy. However, little work has been devoted to investigating functions of VEGF that are independent of its proangiogenic activity. Here, we report that VEGF produced by tumor cells acts in an autocrine manner to promote cell growth through interaction with the VEGF receptor neuropilin-1 (NRP-1). Reducing VEGF expression by tumor cells induced a differentiated phenotype in vitro and inhibited tumor forming capacity in vivo, independent of effects on angiogenesis. Autocrine activation of tumor cell growth was dependent on signaling through NRP-1, and Ras was determined to be a critical effector signaling molecule downstream of NRP-1. Our findings define a novel function for VEGF in dedifferentiation of tumor cells expanding its role in cancer beyond its known proangiogenic function.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/genética , Desdiferenciación Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/genética , Células MCF-7 , Ratones , Ratones Desnudos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neuropilina-1/genética , Neuropilina-1/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transducción de Señal , Transfección , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The Na(+)/H(+) exchanger regulatory factor-2 (NHERF-2) is an integral component of almost all endothelial cells (ECs), yet its endothelial function is not known. Here, we found that NHERF-2, is a key regulator of endothelial homeostasis because NHERF-2-silenced ECs proliferate at a much higher rate even in the absence of mitogens such as VEGF compared with control ECs. We further show that the hyperproliferation phenotype of NHERF-2-silenced EC is because of an accelerated cell cycle that is probably caused by a combination of the following factors: increased cytoplasmic calcium, increased expression of c-Myc, increased expression of cyclin D1, and reduced expression of p27. Using an experimental mouse model of human hemangioma, we found that the endothelial neoplasms derived from NHERF-2-silenced cells were much larger in volume than those derived from control cells. Thus, NHERF-2 is a negative regulator of endothelial proliferation and may have important roles in endothelial homeostasis and vascular modeling.