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RATIONALE: Early neurological deterioration (END) within 72 h of stroke onset is associated with poor prognosis. Optimizing hydration might reduce the risk of END. AIMS: This study aimed to determine in acute ischemic stroke patients if enhanced hydration versus standard hydration reduced the incidence of major (primary) and minor (secondary) END, as well as whether it increased the incidence of early neurological improvement (secondary), at 72 h after admission. SAMPLE SIZE ESTIMATE: A total of 244 participants per arm. METHODS AND DESIGN: A prospective, double-blinded, multicenter, parallel-group, randomized controlled trial conducted at four hospitals from April 2014 to July 2020, with data analyzed in August 2020. The sample size estimated was 488 participants (244 per arm). Ischemic stroke patients with measurable neurological deficits of onset within 12 h of emergency department presentation and blood urea nitrogen/creatinine (BUN/Cr) ratio ⩾ 15 at point of admission were enrolled and randomized to 0.9% sodium chloride infusions of varying rates-enhanced hydration (20 mL/kg body weight, one-third given via bolus and remainder over 8 h) versus standard hydration (60 mL/h for 8 h), followed by maintenance infusion of 40-80 mL/h for the subsequent 64 h. The primary outcome measure was the incidence of major END at 72 h after admission, defined as an increase in National Institutes of Health Stroke Scale of ⩾ 4 points from baseline. RESULTS: Overall, 487 participants were randomized (median age 67 years; 287 females). At 72 h, 7 (2.9%) in the enhanced hydration arm and 5 (2.0%) in the standard hydration developed major END (p = 0.54). The incidence of minor END and early neurological improvement did not differ between treatment arms. CONCLUSION AND RELEVANCE: Enhanced hydration did not reduce END or improve short-term outcomes in acute ischemic stroke. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02099383, https://clinicaltrials.gov/study/NCT02099383).
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Glioblastoma (GBM) is a malignant brain tumor, commonly treated with temozolomide (TMZ). Upregulation of A disintegrin and metalloproteinases (ADAMs) is correlated to malignancy; however, whether ADAMs modulate TMZ sensitivity in GBM cells remains unclear. To explore the role of ADAMs in TMZ resistance, we analyzed changes in ADAM expression following TMZ treatment using RNA sequencing and noted that ADAM17 was markedly upregulated. Hence, we established TMZ-resistant cell lines to elucidate the role of ADAM17. Furthermore, we evaluated the impact of ADAM17 knockdown on TMZ sensitivity in vitro and in vivo. Moreover, we predicted microRNAs upstream of ADAM17 and transfected miRNA mimics into cells to verify their effects on TMZ sensitivity. Additionally, the clinical significance of ADAM17 and miRNAs in GBM was analyzed. ADAM17 was upregulated in GBM cells under serum starvation and TMZ treatment and was overexpressed in TMZ-resistant cells. In in vitro and in vivo models, ADAM17 knockdown conferred greater TMZ sensitivity. miR-145 overexpression suppressed ADAM17 and sensitized cells to TMZ. ADAM17 upregulation and miR-145 downregulation in clinical specimens are associated with disease progression and poor prognosis. Thus, miR-145 enhances TMZ sensitivity by inhibiting ADAM17. These findings offer insights into the development of therapeutic approaches to overcome TMZ resistance.
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Neoplasias Encefálicas , Glioblastoma , MicroARNs , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Línea Celular Tumoral , MicroARNs/metabolismo , Regulación hacia Abajo , Neoplasias Encefálicas/patología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Proteína ADAM17/genética , Proteína ADAM17/metabolismoRESUMEN
Among the different types of oral cancer, >90% of cases are oral squamous cell carcinoma (OSCC). 5fluorouracil (5FU) is a commonly used treatment for OSCC, but cells typically display resistance to the drug. Propofol, an intravenous anesthetic agent, exhibits certain anticancer effects, including the inhibition of cancer cell proliferation, migration and invasion. Secreted proteins, such as growth factors and cytokines are involved in cancer development and progression, but the effect of propofol on secreted proteins in OSCC is not completely understood. An MTT assay, flow cytometry and western blotting were performed to determine the anticancer effects of propofol. The secretion profile of OSCC was determined using an antibody array, and clinical importance was assessed using the Gene Expression Profiling Interactive Analysis database. The results were verified by performing reverse transcriptionquantitative PCR (RTqPCR) and western blotting. 5FUresistant cells were established to determine the role of the gene of interest in drug resistance. The results demonstrated that propofol decreased cell viability and promoted cell apoptosis. The antibody array results showed that propofol attenuated the secretion of multiple growth factors. The bioinformatics results indicated that amphiregulin (AREG) was expressed at significantly higher levels in cancer tissues, which was also related to poor prognosis. The results of RTqPCR and western blotting revealed that propofol decreased AREG expression. Pretreatment with exogenous recombinant AREG increased EGFR activation and conferred propofol resistance. Moreover, the results indicated that the expression and activation of AREG was also related to 5FU resistance, but propofol ameliorated 5FU drug resistance. Therefore, the present study suggested that propofol combination therapy may serve as an effective treatment strategy for OSCC.
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Anfirregulina/química , Carcinoma de Células Escamosas/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Propofol/farmacología , Anfirregulina/genética , Anfirregulina/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Humanos , Hipnóticos y Sedantes/farmacología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Células Tumorales CultivadasRESUMEN
Drug resistance and disease recurrence are important contributors for the poor prognosis of glioblastoma multiforme (GBM). Temozolomide (TMZ), the standard chemotherapy for GBM treatment, can methylate DNA and cause the formation of double-strand breaks (DSBs). X-ray repair cross complementing 5 (XRCC5), also known as Ku80 or Ku86, is required for the repair of DSBs. The present study identified novel determinants that sensitize cells to TMZ, using an array-based short hairpin (sh)RNA library. Then, cBioportal, Oncomine, and R2 databases were used to analyze the association between gene expression levels and clinical characteristics. Subsequently, lentiviral shRNA or pCMV was used to knockdown or overexpress the gene of interest, and the effects on TMZ sensitivity were determined using a MTT assay and western blot analysis. TMZ-resistant cells were also established and were used in in vitro and in vivo experiments to analyze the role of the gene of interest in TMZ resistance. The results indicated that XRCC5 was effective in enhancing TMZ cytotoxicity. The results from the bioinformatics analysis revealed that XRCC5 mRNA expression levels were associated with clinical deterioration and lower overall survival rates. In addition, XRCC5 knockdown could significantly increase TMZ sensitivity in GBM cells, while XRCC5 overexpression caused the cancer cells to be resistant to TMZ. Both the in vivo and in vitro experiments showed that TMZ treatment could induce expression of XRCC5 in TMZ-resistant cells. Taken together these findings suggested that XRCC5 could be a promising target for GBM treatment and could also be used as a diagnostic marker for refractory GBM.
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Imaging evidence for the effect of rehydration on cerebral perfusion and brain ischemia has never been proposed in the literature. This study aimed to test the hypothesis that early rehydration treatment can improve cerebral perfusion and decrease infarct volume, consequently reducing mortality of dehydrated stroke animals. METHODS: Thirty dehydrated experimental rats were randomly assigned to either a rehydration or control group after middle cerebral artery occlusion (MCAO). Diffusion-weighted imaging and dynamic contrast enhancement perfusion imaging were performed at 30 min and 6 h after MCAO using a 9.4T MR imaging scanner to measure the infarct volume and brain perfusion. RESULTS: The survival rates after the first MRI scan were 91.7% for the rehydration group and 58.3% for the control group (p = 0.059). The survival rates after the second MRI scan were 66.7% for the rehydration group, and 8.3% of the control group survived (p = 0.003). The infarct volume of the rehydration group was significantly smaller than control group at 30 min after MCAO (p = 0.007). The delay time and time to maximum were significantly shorter in the rehydration group at 30 min (p = 0.004 and 0.035, respectively). CONCLUSIONS: The findings suggest that early rehydration therapy can decrease the infarct volume, shorten the delay time of cerebral perfusion, and increase survival of dehydrated ischemic-stroke rats. This preliminary study provided imaging evidence that more intensive early hydration therapies and reperfusion strategies may be necessary for acute stroke patients with dehydrated status.
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Glioblastoma multiforme (GBM) is the most common type of malignant brain tumor. The present standard treatment for GBM has not been effective; therefore, the prognosis remains dramatically poor and prolonged survival after treatment is still limited. The new therapeutic strategies are urgently needed to improve the treatment efficiency. Doxorubicin (Dox) has been widely used in the treatment of many cancers for decades. In recent years, with the advancement of delivery technology, more and more research indicates that Dox has the opportunity to be used in the treatment of GBM. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor (EGFR), has been reported to have oncogenic effects in many cancer cell types and is implicated in drug resistance. However, the biological function and molecular mechanism of AREG in Dox treatment of GBM are still unclear. Here, we demonstrate that knockdown of AREG can boost Dox-induced endoplasmic reticulum (ER) stress to trigger activation in both autophagy and apoptosis in GBM cells, ultimately leading to cell death. To explore the importance of AREG in the clinic, we used available bioinformatics tools and found AREG is highly expressed in GBM tumor tissues that are associated with poor survival. In addition, we also used antibody array analysis to dissect pathways that are likely to be activated by AREG. Taken together, our results revealed AREG can serve as a potential therapeutic target and a promising biomarker in GBM.
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Anfirregulina/genética , Apoptosis , Autofagia , Neoplasias Encefálicas/metabolismo , Estrés del Retículo Endoplásmico , Glioblastoma/metabolismo , Anfirregulina/metabolismo , Antineoplásicos/toxicidad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Doxorrubicina/toxicidad , Glioblastoma/genética , Glioblastoma/patología , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Chondrosarcoma is the second most common form of bone cancer and is characterized by its ability to produce an extracellular matrix of the cartilage. High-grade chondrosarcoma is highly aggressive and can metastasize to other parts of the body. Chondrosarcoma is resistant to both conventional chemotherapy and radiotherapy; hence, the current main treatment is still surgical resection. Doxorubicin (Dox) has been shown to significantly improve patient survival compared with untreated chondrosarcoma. However, for patients with metastasis, surgical resection alone can hardly treat them. In addition, drug resistance is one of the leading causes of death in patients with chondrosarcoma. Secreted proteins can mediate cell-cell interactions in the cancer microenvironment, which may be associated with the development of drug resistance. In the present study, chondrosarcoma cells were treated with Dox, the conditioned medium was then collected and changes in secreted proteins were analyzed using the antibody array. Results showed that the Dox-treated group had the highest secretion of basic fibroblast growth factor (bFGF), indicating the effect of bFGF on Dox sensitivity in chondrosarcoma. Furthermore, lentiviral-mediated knockdown and treatment of exogenous recombinant protein were employed to further investigate the effect of bFGF on Dox resistance. Results demonstrated that bFGF can promote the expression of X-ray repair cross-complementing protein 5 (XRCC5), leading to Dox resistance. Secreted bFGF is likely to be detected in serum, in addition to being a biomarker for predicting Dox resistance, the combination of Dox and bFGF/XRCC5 blockers may be a new therapeutic strategy to improve the efficacy of Dox in future.
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Antibióticos Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Condrosarcoma/tratamiento farmacológico , Doxorrubicina/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Autoantígeno Ku/genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Condrosarcoma/genética , Condrosarcoma/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Chronic kidney disease (CKD) has become a major issue in long-term healthcare. It is caused by recurrent kidney injury, which is possible induced by dehydration and heat stress. Therefore, it is important to access the dehydration diagnosis on fields. Conventional instruments for assessing dehydration from blood and urine samples are expensive and time-consuming. These disadvantages limit their applications in high-risk groups susceptible to kidney disease. To address this unmet need, this study presents a portable miniaturized device for dehydration diagnosis with clinical saliva samples. With co-plane coating-free gold electrodes, the dehydration diagnosis was achieved with a saliva specimen at low volumes (50-500 µL). To examine the characteristics, the developed device was assessed by using standard conductivity solutions and the examined variation was <5%. To validate the use for field applications, saliva samples were measured by the developed device and the measured results were compared with standard markers of serum osmolality (N = 30). These data indicate that the measured saliva conductivity is consistent with serum osmolality. And it shows significant difference between healthy adults and healthy farmers (p < 0.05), who typically suffer high risks of CKD. Based on this work, the proposed device and measurement offer a useful method to diagnosis dehydrations and indicate possible potential for CKD.
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Deshidratación/diagnóstico , Saliva/química , Conductividad Eléctrica , Técnicas Electroquímicas/instrumentación , Electrodos , Diseño de Equipo , Humanos , Riñón/patología , Insuficiencia Renal Crónica/diagnósticoRESUMEN
Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite advances in surgery, radiotherapy and chemotherapy, the overall 5year survival rate of patients with OSCC has not significantly improved. In addition, the prognosis of patients with advancedstage OSCC remains poor. Therefore, it is necessary to develop novel therapeutic modalities. Vincristine (VCR), a naturally occurring vinca alkaloid, is a classical microtubuledestabilizing agent and is widely used in the treatment of a number of cancers. Despite the proven antitumor benefits of VCR treatment, one of the major reasons for the failure of treatment is drug resistance. Changes in the tumor microenvironment are responsible for crosstalk between cells, which may facilitate drug resistance in cancers; secreted proteins may promote communication between cancer cells to induce the development of resistance. To identify the secreted proteins involved in VCR resistance, conditioned media was obtained, and an antibody array was conducted to screen a comprehensive secretion profile between VCRresistant (SASVCR) and parental (SAS) OSCC cell lines. The results showed that amphiregulin (AREG) was highly expressed and secreted in SASVCR cells. Pretreatment with exogenous recombinant AREG markedly increased drug resistance against VCR in OSCC cells, as assessed by an MTT assay. Colony formation, MTT and western blot assays were performed to investigate the effects of AREG knockdown on VCR sensitivity. The results indicated that AREG expression can regulate VCR resistance in OSCC cells; overexpression of AREG increased VCR resistance in parental cells, whereas AREG knockdown decreased the VCR resistance of resistant cells. In addition, it was also demonstrated that the glycogen synthase kinase3ß pathway may be involved in AREGinduced VCR resistance. These findings may provide rationale to combine VCR with blockade of AREGrelated pathways for the effective treatment of OSCC.
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Anfirregulina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Resistencia a Antineoplásicos , Neoplasias de la Boca/metabolismo , Anfirregulina/genética , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Medios de Cultivo Condicionados/farmacología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Pronóstico , Transducción de Señal , Regulación hacia Arriba , Vincristina/farmacologíaRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is the most severe type of primary brain tumor with a high mortality rate. Although extensive treatments for GBM, including resection, irradiation, chemotherapy and immunotherapy, have been tried, the prognosis is still poor. Temozolomide (TMZ), an alkylating agent, is a front-line chemotherapeutic drug for the clinical treatment of GBM; however, its effects are very limited because of the chemoresistance. Valproic acid (VPA), an antiepileptic agent with histone deacetylase inhibitor activity, has been shown to have synergistic effects with TMZ against GBM. The mechanism of action of VPA on TMZ combination therapy is still unclear. Accumulating evidence has shown that secreted proteins are responsible for the cross talking among cells in the tumor microenvironment, which may play a critical role in the regulation of drug responses. METHODS: To understand the effect of VPA on secreted proteins in GBM cells, we first used the antibody array to analyze the cell culture supernatant from VPA-treated and untreated GBM cells. The results were further confirmed by lentivirus-mediated knockdown and exogenous recombinant administration. RESULTS: Our results showed that amphiregulin (AR) was highly secreted in VPA-treated cells. Knockdown of AR can sensitize GBM cells to TMZ. Furthermore, pretreatment of exogenous recombinant AR significantly increased EGFR activation and conferred resistance to TMZ. To further verify the effect of AR on TMZ resistance, cells pre-treated with AR neutralizing antibody markedly increased sensitivity to TMZ. In addition, we also observed that the expression of AR was positively correlated with the resistance of TMZ in different GBM cell lines. CONCLUSIONS: The present study aimed to identify the secreted proteins that contribute to the modulation of drug response. Understanding the full set of secreted proteins present in glial cells might help reveal potential therapeutic opportunities. The results indicated that AR may potentially serve as biomarker and therapeutic approach for chemotherapy regimens in GBM.
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Anfirregulina/metabolismo , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Neuroglía/efectos de los fármacos , Temozolomida/farmacología , Ácido Valproico/farmacología , Anfirregulina/genética , Anticuerpos Bloqueadores/farmacología , Biomarcadores de Tumor , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Técnicas de Silenciamiento del Gen , Humanos , Lentivirus/genéticaRESUMEN
The brain-derived neurotrophic factor (BDNF) is vital in the neural differentiation of neural stem/progenitor cells, and together may have therapeutic potential for neural regeneration. In this study, a multiplexed polybutylcyanoacrylate nanoparticle (PBCA NP) delivery platform was constructed, incorporating either surface-adsorbed or encapsulated BDNF for the induction of neural differentiation in induced pleuripotent stem cells (iPSCs), where tween 80 (T80) and superparamagnetic iron oxide (SPIO) were added for central nervous system (CNS) targeting and magnetic resonance (MR) image tracking, respectively. Both methods by which the BDNF was carried resulted in loading efficiencies greater than 95%. The nanoparticle-mediated delivery of BDNF resulted in neural differentiation of iPSCs detected on immunofluorescence staining as early as 7 days, with enhanced differentiation efficiency by 1.3-fold compared to the control on flow cytometry; the delivery system of surface-adsorbed BDNF gave rise to cells that had the best neural development than the encapsulated formulation. T80-coating disrupted the in vitro bloodâ»brain barrier model with a corresponding 1.5- to two-fold increase in permeability. SPIO-loaded PBCA NPs exhibited a concentration-dependent, rapid decay in signal intensity on the phantom MR experiment. This study demonstrates the versatility of the PBCA NP, and the surface-adsorption of BDNF is the preferred method of delivery for the differentiation of iPSCs.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Enbucrilato/farmacología , Células Madre Pluripotentes Inducidas/citología , Nanopartículas/química , Neuronas/citología , Adsorción , Animales , Barrera Hematoencefálica/metabolismo , Muerte Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Impedancia Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Imagen por Resonancia Magnética , Ratones , Modelos Biológicos , Nanopartículas/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Tamaño de la Partícula , Fantasmas de Imagen , Ratas , Electricidad Estática , Propiedades de SuperficieRESUMEN
Background: Dehydration is common among ischemic stroke patients and is associated with early neurological deterioration and poor outcome. This study aimed to test the hypothesis that dehydration status is associated with decreased cerebral perfusion and aggravation of ischemic brain injury. Methods: Diffusion-weighted imaging and arterial spin labeling perfusion MR imaging were performed on rats with middle cerebral artery occlusion (MCAO) by using a 9.4T MR imaging scanner to measure the volume of infarction and relative cerebral blood flow (rCBF) after infarction. Twenty-five rats were assigned to either a dehydration group or normal hydration group, and dehydration status was achieved by water deprivation for 48 h prior to MCAO. Results: The volume of the infarction was significantly larger for the dehydration group at the 4th h after MCAO (p = 0.040). The progression in the infarct volume between the 1st and 4th h was also larger in the dehydration group (p = 0.021). The average rCBF values of the contralateral normal hemispheres at the 1st and the 4th h were significantly lower in the dehydration group (p = 0.027 and 0.040, respectively). Conclusions: Our findings suggested that dehydration status is associated with the progression of infarct volume and decreases in cerebral blood flow during the acute stage of ischemic stroke. This preliminary study provided an imaging clue that more intensive hydration therapies and reperfusion strategies are necessary for the management of acute ischemic stroke patients with dehydration status.
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Chondrosarcoma is characterized by the ability to produce extracellular matrix of cartilage. Curative surgical resection becomes difficult when high-grade chondrosarcoma metastasizes to other parts of the body. Doxorubicin (Dox) has been widely used clinically to treat many different types of cancers, including chondrosarcomas. However, chondrosarcoma is often resistant to chemotherapy and radiotherapy. New systemic treatment regimens are needed to improve the prognosis. Amphiregulin (AR) has been reported to be involved in cancer metastasis and drug resistance. The aim of the present study was to investigate the role of AR on cell migration and Dox sensitivity. To gain insight into the mechanisms used by AR to mediate its effects, we generated two chondrosarcoma cell lines, migration-prone JJ012 and Dox-resistant SW1353, to analyze the relationship between AR levels and cell functions. AR was highly expressed and secreted in mobile and drug-resistant cells. Next, we used the lentivirus-mediated RNAi system to knock down AR expression and found that AR silencing was significantly attenuated in cell migration and drug resistance. In addition, we used exogenous recombinant AR (r-AR) to confirm cell function and we introduced antibody array analysis to investigate pathways of AR activation. The results showed that AR can promote migratory activity and Dox resistance through activation of the MAPK pathway. Whereas the inhibition of the MAPK pathway has the potential to inhibit chondrosarcoma cell migration, p38 inhibition, and ERK activation may render cells more sensitive to Dox. These findings suggest that combining AR with MAPK blockade may effectively treat chondrosarcoma.
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Anfirregulina/metabolismo , Neoplasias Óseas/metabolismo , Condrosarcoma/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Condrosarcoma/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas , Regulación hacia ArribaRESUMEN
In this study, we demonstrated that temozolomide (TMZ) and propyl gallate (PG) combination enhanced the inhibition of migration in human U87MG glioma cells. PG inhibited the TMZ-induced reactive oxygen species (ROS) generation. The mitochondrial complex III and NADPH oxidase are two critical sites that can be considered to regulate antimigration in TMZ-treated U87MG cells. PG can enhance the antimigration effect of TMZ through suppression of metalloproteinase-2 and metalloproteinase-9 activities, ROS generation, and the NF-κB pathway and possibly provide a novel prospective strategy for treating malignant glioma.
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Dacarbazina/análogos & derivados , Glioma/tratamiento farmacológico , Galato de Propilo/farmacología , Apoptosis , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dacarbazina/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Glioma/patología , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , TemozolomidaRESUMEN
In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was "QAMXXMM-E/G/M-S/G-A/G/F-XG". We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.
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Éter de Dihematoporfirina/administración & dosificación , Metionina/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Fotoquimioterapia , Línea Celular Tumoral , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Péptidos/metabolismo , ProteómicaRESUMEN
Brain derived neurotrophic factor (BDNF) can induce neural differentiation in stem cells and has the potential for repair of the nervous system. In this study, a polysorbate 80-coated polybutylcyanoacrylate nanocarrier (PS80 PBCA NC) was constructed to deliver plasmid DNAs (pDNAs) containing BDNF gene attached to a hypoxia-responsive element (HRE-cmvBDNF). The hypoxia-sensing mechanism of BDNF expression and inductiveness of the nano-formulation on mouse induced pluripotent stem cells (iPSCs) to differentiate into neurons following hypoxia was tested in vitro with immunofluorescent staining and Western blotting. The HRE-cmvBDNF appeared to adsorb onto the surface of PS80 PBCA NC, with a resultant mean diameter of 92.6 ± 1.0 nm and zeta potential of -14.1 ± 1.1 mV. HIF-1α level in iPSCs was significantly higher in hypoxia, which resulted in a 51% greater BDNF expression when transfected with PS80 PBCA NC/HRE-cmvBDNF than those without hypoxia. TrkB and phospho-Akt were also elevated which correlated with neural differentiation. The findings suggest that PS80 PBCA NC too can be endocytosed to serve as an efficient vector for genes coupled to the HRE in hypoxia-sensitive cells, and activation of the PI3/Akt pathway in iPSCs by BDNF is capable of neural lineage specification.
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Factor Neurotrófico Derivado del Encéfalo/genética , Diferenciación Celular , Enbucrilato/química , Células Madre Pluripotentes Inducidas/citología , Nanopartículas/química , Neuronas/citología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipoxia de la Célula , Línea Celular , Enbucrilato/efectos adversos , Vectores Genéticos/administración & dosificación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Polisorbatos/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN/administración & dosificación , Elementos de RespuestaRESUMEN
Spontaneous intracerebral hemorrhage (ICH) is associated with high rates of mortality and morbidity. Thus, the identification of novel therapeutic agents for preventing strokes and attenuating poststroke brain damage is crucial. Dexamethasone (DEX) is used clinically to reduce edema formation in patients with spinal cord injury and brain tumors. In this study, we sought to elucidate the effects of DEX treatment on apoptosis and inflammation following ICH in rats. A high dose of DEX (15 mg/kg) was administered immediately following ICH induction and again 3 days later. The inflammatory and apoptotic responses in the rat brains were evaluated by using hematoxylin-eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling, Nissl, and neurofilament-H staining. Levels of phosphorylated neurofilaments and apoptosis-related proteins such as B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-3, and P53 were analyzed by Western blotting. This study shows that rats without ICH that received DEX treatment had a fourfold higher expression of Bcl-2 than sham-operated rats. ICH causes an increase in Bax, cleaved caspase-3, and P53 proteins from 4 hr to 7 days following ICH induction. In comparison with the ICH rats, the ICH/DEX rats showed significantly decreased apoptotic cell death and increased neuron survival and maintained neurofilament integrity in the perihematomal region. DEX increased the Bcl-2/Bax ratio and lowered the expression of cleaved caspase-3 at 12 hr and 5 days. The ICH rats were accompanied by activation of the inflammatory response, and DEX treatment modulated the expression of a variety of cell types and then decreased ICH-induced apoptosis.
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Antiinflamatorios/uso terapéutico , Apoptosis/efectos de los fármacos , Encéfalo/patología , Hemorragia Cerebral/complicaciones , Dexametasona/uso terapéutico , Encefalitis , Neuronas/efectos de los fármacos , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Antiinflamatorios/farmacología , Encéfalo/efectos de los fármacos , Complejo CD3/metabolismo , Fragmentación del ADN/efectos de los fármacos , Dexametasona/farmacología , Modelos Animales de Enfermedad , Encefalitis/tratamiento farmacológico , Encefalitis/etiología , Encefalitis/patología , Masculino , Proteínas de Neurofilamentos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The involvement of brain-derived neurotrophic factor (BDNF) in regulating neuronal survival during neuron differentiation, growth, and maturation, and during the regeneration of injured nerve cells, has already been documented. In experimental Parkinson's disease, chronic exposure to cigarette smoke increased BDNF levels and survival of dopaminergic neurons. BDNF is also elevated in traumatic brain injury (TBI), where it is potentially involved in post-injury repair and regeneration. The aim of this study was to investigate the effects of chronic exposure to cigarette smoke on BDNF expression and apoptosis in rats with TBI. Three groups of rats were compared: rats with TBI after chronic exposure to cigarette smoke, rats with TBI and no exposure to cigarette smoke, and sham-operated rats. BDNF mRNA expression in the hippocampus increased from 2 to 24 h after TBI, and chronic exposure to cigarette smoke upregulated TBI-induced BDNF mRNA elevation at 0, 2, 4, 12, and 24 h after head injury. The BDNF protein levels generally corresponded to the mRNA levels in the hippocampal region. Compared to the TBI group without smoke exposure, chronic cigarette smoke exposure in rats inhibited the decrease of the Bcl-2/Bax ratio and reduced P53 expression and apoptosis 24 h after TBI. In addition, neuronal damage in the parietal and cingulate cortex 7 days after TBI was less extensive in rats exposed to cigarette smoke. In conclusion, although chronic exposure to cigarette smoke is a risk factor for myocardial and pulmonary disease, cigarette smoke exposure increases BDNF expression after TBI and thereby can play a neuroprotective role.
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Lesiones Encefálicas/metabolismo , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Nicotiana , Humo/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Enfermedad Crónica , Colorantes , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
Malignant pleural effusion (MPE) obtained from lung adenocarcinoma may contain potentially useful biomarkers for detection of lung cancer. In this study, we used a removal system for high-abundance proteins followed by one-dimensional SDS-PAGE combined with nano-LC-MS/MS to generate a comprehensive MPE proteome data set with 482 nonredundant proteins. Next, we integrated the MPE proteome and secretome data sets from three adenocarcinoma cell lines, with a view to identifying potential PE biomarkers originating from malignant cells. Four potential candidates, alpha-2-HS-glycoprotein (AHSG), angiogenin, cystatin-C, and insulin-like growth factor-binding protein 2, (IGFBP2), were isolated for preclinical validation using ELISA. Both AHSG and IGFBP2 levels were increased in lung patients with MPE (n = 68), compared to those with nonmalignant pleural effusion (n = 119). Notably, the IGFBP2 level was higher in MPE, compared with that in benign diseases (bacteria pneumonia and tuberculosis pleuritis), and significantly associated with malignancy, regardless of the cancer type. Our data additionally support an extracellular function of IGFBP2 in migration in lung cancer cells. These findings collectively suggest that the adenocarcinoma MPE proteome provides a useful data set for malignancy biomarker research.
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Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/metabolismo , Derrame Pleural Maligno/metabolismo , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Biología Computacional/métodos , Bases de Datos Factuales , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias Pulmonares/diagnóstico , Masculino , Persona de Mediana Edad , Derrame Pleural Maligno/patología , alfa-2-Glicoproteína-HS/metabolismoRESUMEN
BACKGROUND: The molecular mechanism of hemorrhagic stroke is unclear, and the identification of therapeutic agents for attenuating post-stroke brain damage remains an unresolved challenge. Dexamethasone (DEX) is used clinically to treat spinal cord injury and brain tumor patients by reducing edema formation, but has produced conflicting results in stroke management. METHODS: In this study, intracerebral hemorrhage (ICH) was induced in rats by intracranial stereotactic injection of collagenase into the caudate nucleus. DEX was given immediately and 3 days after ICH. The expression of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), nuclear factor (NF)-κB, and IκB were analyzed by Western blotting, and perihematomal edema formation was evaluated by magnetic resonance imaging. RESULTS: The results showed that ICH caused an increase of ICAM-1 and MMP-9 expression from 4 h to 7 days, which was inhibited following the administration of DEX. The perihematomal edema volume in ICH rats was high, with two peak periods at 12 h and 3 days, which was also reduced in DEX-treated groups. Furthermore, the administration of DEX not only maintained IκB in cytoplasm, but also decreased NF-κB elevation in the nucleus at 3 and 5 days in ICH rats. CONCLUSIONS: In conclusion, these data show that DEX successfully reduced post-stroke brain edema by decreasing MMP-9 and ICAM-1 levels, partially through the IκB/NF-κB signaling pathway. The timing of DEX administration in relation to the onset of brain injury may be critical.