RESUMO
BACKGROUND AND PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) commonly causes neuropathic pain, but its pathogenesis remains unclear, and effective therapies are lacking. Naringenin, a natural dihydroflavonoid compound, has anti-inflammatory, anti-nociceptive and anti-tumour activities. However, the effects of naringenin on chemotherapy-induced pain and chemotherapy effectiveness remain unexplored. EXPERIMENTAL APPROACH: Female and male mouse models of chemotherapy-induced pain were established using paclitaxel. Effects of naringenin were assessed on pain induced by paclitaxel or calcitonin gene-related peptide (CGRP) and on CGRP expression in dorsal root ganglia (DRG) and spinal cord tissue. Additionally, we examined peripheral macrophage infiltration, glial activation, c-fos expression, DRG neuron excitability, microglial M1/M2 polarization, and phosphorylation of spinal NF-κB. Furthermore, we investigated the synergic effect and related mechanisms of naringenin and paclitaxel on cell survival of cancer cells in vitro. KEY RESULTS: Systemic administration of naringenin attenuated paclitaxel-induced pain in both sexes. Naringenin reduced paclitaxel-enhanced CGRP expression in DRGs and the spinal cord, and alleviated CGRP-induced pain in naïve mice of both sexes. Naringenin mitigated macrophage infiltration and reversed paclitaxel-elevated c-fos expression and DRG neuron excitability. Naringenin decreased spinal glial activation and NF-κB phosphorylation in both sexes but influenced microglial M1/M2 polarization only in females. Co-administration of naringenin with paclitaxel enhanced paclitaxel's anti-tumour effect, impeded by an apoptosis inhibitor. CONCLUSION AND IMPLICATIONS: Naringenin's anti-nociceptive mechanism involves CGRP signalling and neuroimmunoregulation. Furthermore, naringenin facilitates paclitaxel's anti-tumour action, possibly involving apoptosis. This study demonstrates naringenin's potential as a supplementary treatment in cancer therapy by mitigating side effects and potentiating efficacy of chemotherapy.
Assuntos
Antineoplásicos Fitogênicos , Peptídeo Relacionado com Gene de Calcitonina , Flavanonas , Paclitaxel , Transdução de Sinais , Animais , Paclitaxel/farmacologia , Flavanonas/farmacologia , Flavanonas/administração & dosagem , Flavanonas/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Masculino , Feminino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Analgésicos/farmacologia , Camundongos Endogâmicos C57BL , Humanos , Linhagem Celular Tumoral , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Dor/tratamento farmacológico , Dor/metabolismo , Dor/induzido quimicamenteRESUMO
Gastric cancer (GC) with pulmonary metastasis is one of the deadliest diseases in the world; however, the underlying pathological mechanisms and potential therapeutic targets remain to be elucidated. As exosomes play indispensable roles in the formation of premetastatic niches (PMN) and cancer metastasis. Therefore, investigating the underlying mechanisms of exosome-mediated pulmonary metastasis of GC may shed new light on identifying novel therapeutic targets for GC treatment. GC-derived exosomes were isolated from the conditioned medium of mouse forestomach carcinoma (MFC) cell line. The effects of MFC-derived exosomes on pulmonary macrophage polarization were analyzed by reverse- transcription polymerase chain reaction and flow cytometry. Expression of PD-L1 and other proteins was evaluated by Western blot. Exosomal microRNAs (miRNAs) were analyzed by microarray. GC-derived exosomes (GC-exo) accumulated in high numbers in the lungs and were ingested by macrophages. The extracellular-signal-regulated kinase (ERK) signaling pathway was activated by GC-exo, inducing macrophage immunosuppressive-phenotype differentiation and increased PD-L1 expression. miRNA-sequencing identified 130 enriched miRNAs in GC-exo. Among the enriched miRNAs, miR-92a-3p plays a major role in activating ERK signaling via inhibition of PTEN expression. In addition, inhibiting ERK signaling with PD98059 significantly reduced the expression of PD-L1 in macrophages and, therefore, reversed the immunosuppressive PMN and inhibited the colonization of GC cells in the lungs. This study identified a novel mechanism of GC-exo mediated PD-L1 expression in lung macrophages that facilitates lung PMN formation and GC pulmonary metastasis, which also provided a potential therapeutic target for GC with pulmonary metastasis treatment.
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Exossomos , Neoplasias Pulmonares , MicroRNAs , Neoplasias Gástricas , Animais , Camundongos , Neoplasias Gástricas/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Exossomos/metabolismo , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Macrófagos/metabolismo , Neoplasias Pulmonares/metabolismoRESUMO
Functional analysis of immune subtypes in hepatocellular carcinoma has attracted much attention due to its advantages in solving some optimization problems. At present, the research on the immune subtype of hepatocellular carcinoma is still in its infancy, and the high stability of its system still has problems. Based on fuzzy logic and evolutionary algorithms, this paper constructs a Mate analysis of the optimization problem of immune subtypes and dynamic optimization problems of hepatocellular carcinoma. The model conducts in-depth analysis and research on the biological immune subtype system, solving the problems of reliable information processing and body defense. Tested with existing test functions, very competitive results were achieved. The simulation results show that the improved algorithm based on data statistics has global search ability, the solution accuracy reaches 0.931, and the stability reaches 88.1%.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Algoritmos , Simulação por Computador , Lógica Fuzzy , HumanosRESUMO
BACKGROUND: MUC5AC was recently identified to play important roles in the proliferation and metastasis of malignant mucinous lung tumor cells. Resveratrol (Res), a natural compound with anticancer effects in lung cancer cells, has been reported to inhibit mucin production in airway epithelial cells. This study aimed to investigate the inhibitory effect of Res on MUC5AC expression in lung mucinous adenocarcinoma cells and the potential mechanisms. METHODS: Mucus-producing A549 human lung carcinoma cells were used to test the effects of Res on SPDEF and MUC5AC expression. Gene and protein expression was assessed by real-time quantitative PCR (qPCR), immunofluorescence and western blotting assays. SPDEF lentivirus was used to upregulate SPDEF expression levels in mucus-producing A549 human lung carcinoma cells. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. RESULTS: Res decreased MUC5AC expression in an SPDEF-dependent manner in mucus-producing A549 human lung carcinoma cells, and this change was accompanied by decreased ERK expression and AKT pathway activation. Moreover, SPDEF was found to be overexpressed in lung adenocarcinoma (LUAD), especially in mucinous adenocarcinoma. In-vitro functional assays showed that overexpression of SPDEF reduced the chemosensitivity of A549 cells to cisplatin (DDP). In addition, Res treatment increased A549 cell chemosensitivity to DDP by inhibiting the SPDEF-MUC5AC axis. CONCLUSION: Our results indicate that the SPDEF-MUC5AC axis is associated with DDP sensitivity, and that Res decreases SPDEF and MUC5AC expression by inhibiting ERK and AKT signaling in A549 cells, which provides a potential pharmacotherapy for the prevention and therapeutic management of mucinous adenocarcinoma.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Mucina-5AC/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/metabolismo , Resveratrol , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mucina-5AC/genética , Resveratrol/farmacologiaRESUMO
BACKGROUND: Polygonum cuspidatum is a Chinese medicine commonly used to treat phlegm-heat asthma. However, its anti-asthmatic active ingredients and mechanism are still unknown. The aim of this study was to predict the active ingredients and pathways of Polygonum cuspidatum and to further explore the potential molecular mechanism in asthma by using network pharmacology. METHODS: The active ingredients and their targets related to Polygonum cuspidatum were seeked out with the TCM systematic pharmacology analysis platform (TCMSP), and the ingredient-target network was constructed. The GeneCards, DrugBank and OMIM databases were used to collect and screen asthma targets, and then the drug-target-disease interaction network was constructed with Cytoscape software. A target protein-protein interaction (PPI) network was constructed using the STRING database to screen key targets. Finally, GO and KEGG analyses were used to identify biological processes and signaling pathways. The anti-asthmatic effects of Polygonum cuspidatum and its active ingredients were tested in vitro for regulating airway smooth muscle (ASM) cells proliferation and MUC5AC expression, two main symptoms of asthma, by using Real-time PCR, Western blotting, CCK-8 assays and annexin V-FITC staining. RESULTS: Twelve active ingredients in Polygonum cuspidatum and 479 related target proteins were screened in the relevant databases. Among these target proteins, 191 genes had been found to be differentially expressed in asthma. PPI network analysis and KEGG pathway enrichment analysis predicted that the Polygonum cuspidatum could regulate the AKT, MAPK and apoptosis signaling pathways. Consistently, further in vitro experiments demonstrated that Polygonum cuspidatum and resveratrol (one active ingredient of Polygonum cuspidatum) were shown to inhibit ASM cells proliferation and promoted apoptosis of ASM cells. Furthermore, Polygonum cuspidatum and resveratrol inhibited PDGF-induced AKT/mTOR activation in ASM cells. In addition, Polygonum cuspidatum decreased H2O2 induced MUC5AC overexpression in airway epithelial NCI-H292 cells. CONCLUSION: Polygonum cuspidatum could alleviate the symptoms of asthma including ASM cells proliferation and MUC5AC expression through the mechanisms predicted by network pharmacology, which provides a basis for further understanding of Polygonum cuspidatum in the treatment of asthma.
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Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Fallopia japonica/química , Mucina-5AC/antagonistas & inibidores , Animais , Antiasmáticos/química , Asma/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Medicina Tradicional Chinesa , Estrutura Molecular , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Benzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression. METHODS: The human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus. RESULTS: We found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation. CONCLUSION: Here, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.
Assuntos
Poluentes Atmosféricos/toxicidade , Benzo(a)pireno/toxicidade , Células Epiteliais/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mucina-5AC/genética , Mucina-5AC/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Sistema Respiratório/citologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND Loss of the epithelial barrier is characterized by a reduction in E-cadherin expression and is a hallmark of asthma. Qi-xian decoction (QXT) is a Chinese medicinal formula that has been used to effectively treat asthma. This study aimed to investigate the effect of QXT on E-cadherin expression in human lung epithelial 16HBE cells and ovalbumin-challenged mice and to explore the underlying molecular mechanism. MATERIAL AND METHODS Ovalbumin (OVA)-induced mice were used as a model of asthma. Real-time PCR and Western blotting were utilized to examine mRNA and protein levels. Lung tissue reactive oxygen species (ROS) levels were evaluated using dichloro-dihydro-fluorescein diacetate (DCFH-DA). Serum superoxide dismutase (SOD) and the total antioxidant capacity (TAOC) were measured via enzyme-linked immunosorbent assay (ELISA)-based analyses. 16HBE cells were utilized to explore the effect of QXT or hydrogen peroxide (H2O2) on the expression of E-cadherin in vitro. RESULTS We found that QXT treatment increased E-cadherin expression and decreased extracellular-signal-regulated kinase (ERK) phosphorylation levels in the lung tissues of OVA-challenged mice. QXT also downregulated ROS levels and increased serum SOD and TAOC levels in OVA-challenged mice. In vitro studies demonstrated that increased ROS generation induced by H2O2 resulted in decreased E-cadherin expression levels in 16HBE cells, which was attenuated by inhibition of ERK signaling. Moreover, the H2O2-induced downregulation of E-cadherin expression, increased ROS generation, and ERK activation in 16HBE cells were restored by treatment with QXT water or ethanol extract. CONCLUSIONS These data demonstrate that one mechanism by which QXT protects against asthma is to restore E-cadherin expression in vivo and in vitro by inhibiting ROS-mediated ERK activation.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pulmão/efeitos dos fármacos , Ovalbumina/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Asma/metabolismo , Caderinas/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos BALB C , FosforilaçãoRESUMO
PURPOSE: Arl4c is overexpressed in several cancer tissues and is involved in cancer development. Nevertheless, the exact mechanism that regulates Arl4c expression in lung cancer has not been fully elucidated. The aim of this study was to investigate the regulatory mechanism of Arl4c and to explore potential chemotherapeutic drugs targeting Arl4c. METHODS: Immunohistochemistry was used to examine Arl4c expression levels in human lung adenocarcinoma cancer specimens. Protein expression was detected by western blot. Overexpression of Arl4c-Flag protein was used to detect the ubiquitination of Arl4c. A short interfering RNA against Arl4c was used for gene silencing. RESULTS: Arl4c was overexpressed in lung cancer tissues, and knockdown of Arl4c expression by siRNA decreased lung cancer A549 and 95-D cell proliferation. In addition, Arl4c expression was downregulated via inhibition of the AKT pathway in A549 and 95-D cells, whereas exposure to benzo (a) pyrene (a carcinogen in smoke) increased Arl4c expression in 16HBE cells via AKT activation. Finally, we found that chemotherapy drug hydroxycamptothecin (HCPT) could decrease Arl4c expression levels by inhibiting the activation of the AKT pathway in A549 and 95-D cells. Moreover, accumulation of ubiquitinated Arl4c protein was increased by HCPT and LY294002 (an AKT inhibitor) treatment whereas the proteasome inhibitor MG-132 attenuated the inhibitory effect of HCPT and LY294002 on Arl4c expression. CONCLUSION: Here, we highlighted the AKT pathway as an important regulatory pathway for Arl4c expression in lung cancer cells and identified HCPT as a promising drug for lung adenocarcinoma treatment that functioned by targeting Arl4c expression.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células A549 , Western Blotting , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Leupeptinas/farmacologia , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fumar/efeitos adversosRESUMO
Excessive mucin production in the airway may contribute to airway inflammatory diseases. Curcumin has been reported to prevent mucin 5AC (MUC5AC) production in human airway epithelial cells; however, the molecular targets of curcumin involved in regulating MUC5AC expression have remained elusive. The present study aimed to elucidate the molecular mechanisms by which curcumin regulates MUC5AC production, utilizing the NCIH292 human airway epithelial cell line featuring MUC5AC hypersecretion. Curcumin was able to counteract the endothelial growth factor (EGF)stimulated mRNA and protein expression of MUC5AC. In addition, curcumin treatment prevented EGFinduced phosphorylation of EGF receptor (EGFR) as well as the downstream AKT and signal transducer and activator of transcription 3 (STAT3), while inhibition of PI3K and STAT3 signaling significantly attenuated the expression of MUC5AC that was induced by EGF. Furthermore, EGFinduced increases in the levels of phosphorylated STAT3 in the nuclear fraction were inhibited by curcumin and PI3K inhibitors. In addition, treatment with curcumin significantly decreased MUC5AC and EGFR expression in a timedependent manner under basal conditions. These results demonstrated that curcumin inhibited MUC5AC protein expression in NCIH292 cells under basal conditions as well under EGF stimulation. This inhibition was accompanied by decreased activation of the EGFR/AKT/STAT3 pathway and reduced EGFR expression, which indicated that curcumin may have a dual role in interfering with the EGFR signaling pathway and inhibiting mucin expression in human airway epithelial cells.
Assuntos
Curcumina/farmacologia , Receptores ErbB/metabolismo , Mucina-5AC/biossíntese , Transdução de Sinais , Linhagem Celular Tumoral , Cromonas/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Humanos , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Lung squamous cell carcinoma (LSCC) is a common histological lung cancer subtype, but unlike lung adenocarcinoma, limited therapeutic options are available for treatment. Curcumin, a natural compound, may have anticancer effects in various cancer cells, but how it may be used to treat LSCC has not been well studied. Here, we applied curcumin to a human NCI-H292 LSCC cell line to test anticancer effects and explored underlying potential mechanisms of action. Curcumin treatment inhibited NCI-H292 cell growth and increased FOXA2 expression in a time-dependent manner. FOXA2 expression was decreased in LSCC tissues compared with adjacent normal tissues and knockdown of FOXA2 increased NCI-H292 cells proliferation. Inhibition of cell proliferation by curcumin was attenuated by FOXA2 knockdown. Moreover inhibition of STAT3 pathways by curcumin increased FOXA2 expression in NCI-H292 cells whereas a STAT3 activator (IL-6) significantly inhibited curcumin-induced FOXA2 expression. Also, SOCS1 and SOCS3, negative regulators of STAT3 activity, were upregulated by curcumin treatment. Thus, curcumin inhibited human NCI-H292 cells growth by increasing FOXA2 expression via regulation of STAT3 signaling pathways.
RESUMO
BACKGROUND/AIMS: A growing number of studies have demonstrated that the activity and expression level of sirtuin-1 (SIRT1) are decreased in asthma patients; however, the mechanisms underlying decreased SIRT1 expression and function are still not completely understood. Interleukin (IL)-6 plays important roles in inflammation during allergic asthma. In this study, we examined whether loss of SIRT1 activity regulated the expression of IL-6 and further verified the underlying mechanisms. METHODS: The human airway epithelial cell line 16HBE was used to test the effects of the SIRT1 inhibitor (salermide) on expression of IL-6. IL-6 mRNA and protein expression were assessed with real-time polymerase chain reaction (PCR), immunochemistry, and ELISA. OVA-challenged mice were used as an asthma model to investigate the effect of SIRT1 activation on IL-6 and relative Akt phosphorylation level. RESULTS: We found that inhibition of SIRT1 increased IL-6 mRNA and protein levels in a time-dependent manner, which was accompanied by increased Akt pathway activation in 16HBE cells. Furthermore activation of Akt showed upregulated expression of the IL-6 protein whereas Akt inhibitor, LY294002 or Akt siRNA significantly inhibited SIRT1-regulated IL-6 expression. Conversely, activation of SIRT1 inhibited Akt activation and IL-6 expression in an asthmatic mice model and 16HBE cells. CONCLUSION: Our results indicate the potential role of SIRT1 in regulating inflammation by modulation of IL-6 expression in an Akt-dependent manner during allergic asthma.
Assuntos
Asma/metabolismo , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuína 1/metabolismo , Animais , Asma/tratamento farmacológico , Western Blotting , Linhagem Celular , Cromonas/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-6/genética , Pulmão/citologia , Morfolinas/farmacologia , Naftóis/farmacologia , Fenilpropionatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Estilbenos/uso terapêuticoRESUMO
BACKGROUND: CLPTM1L (Cleft lip and palate transmembrane protein 1-like) has been previously shown to be overexpressed in lung cancer and is involved in regulating cisplatin sensitivity. In this study, we assessed the relationship between CLPTM1L expression and prognosis of lung cancer in patients and explored its role in regulating cell migration and invasion. METHODS: Immunohistochemistry was used to examine CLPTM1L expression levels on a tissue microarray containing 73 sets of human lung cancer specimens with adjacent normal tissue. The correlation between CLPTM1L expression and patient survival was analysed by the Kaplan-Meier method. In addition, CLPTM1L-knockdown lung cells were used to investigate the effect of CLPTM1L on cell migration and invasion in vitro. RESULTS: The results of immunohistochemical analysis showed that CLPTM1L was overexpressed in lung cancer tissues compared with adjacent normal tissues. Kaplan-Meier analyses revealed that patients with high CLPTM1L expression showed poorer survival than those with low CLPTM1L expression. In addition, in vitro studies revealed that the knockdown of CLPTM1L expression in 95-D lung cancer cells suppressed cell migration and invasion. Further, the loss of CLPTM1L resulted in decreased matrix metalloproteinase-2 (MMP-2) expression in these cells. CONCLUSION: Our data demonstrate that CLPTM1L overexpression can predict poor prognosis in patients with lung cancer and suggest that CLPTM1L might be associated with the regulation of cell migration and invasion.
Assuntos
Movimento Celular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/biossíntese , Proteínas de Membrana/biossíntese , Invasividade Neoplásica/genética , Proteínas de Neoplasias/biossíntese , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Previous in vitro studies have demonstrated that resveratrol is able to significantly inhibit the upregulation of mucin 5AC (MUC5AC), a major component of mucus; thus indicating that resveratrol may have potential in regulating mucus overproduction. However, there have been few studies regarding the resveratrolmediated prevention of MUC5AC overproduction in vivo, and the mechanisms by which resveratrol regulates MUC5AC expression have yet to be elucidated. In the present study, an ovalbumin (OVA)challenged murine model of asthma was used to assess the effects of resveratrol treatment on mucus production in vivo. The results demonstrated that resveratrol significantly inhibited OVAinduced airway inflammation and mucus production. In addition, the mRNA and protein expression levels of MUC5AC were increased in the OVAchallenged mice, whereas treatment with resveratrol significantly inhibited this effect. The expression levels of murine calciumactivated chloride channel (mCLCA)3, an important key mediator of MUC5AC production, were also reduced following resveratrol treatment. Furthermore, in vitro studies demonstrated that resveratrol significantly inhibited human (h)CLCA1 and MUC5AC expression in a dosedependent manner. These results indicated that resveratrol was effective in preventing mucus overproduction and MUC5AC expression in vivo, and its underlying mechanism may be associated with regulation of the mCLCA3/hCLCA1 signaling pathway.
Assuntos
Asma/tratamento farmacológico , Asma/metabolismo , Mucina-5AC/metabolismo , Muco/metabolismo , Estilbenos/farmacologia , Estilbenos/uso terapêutico , Animais , Asma/genética , Asma/patologia , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos BALB C , Mucina-5AC/genética , Pneumonia/complicações , Pneumonia/tratamento farmacológico , Pneumonia/patologia , ResveratrolRESUMO
OBJECTIVE: To prepare streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion proteins (SA/hI-TAC) and evaluate its biological activity. METHODS: pET24a-SA-hI-TAC/pET21a-hI-TAC-SA plasmids were constructed and expressed in BL21. SA-hI-TAC and hI-TAC-SA fusion proteins were purified by Ni-NTA affinity chromatography, refolded by dialysis and identified by Western blotting. The bifunctionality of the fusion proteins (biotin-binding function and hI-TAC activity) was analyzed by flow cytometry and lymphocyte chemotaxis experiment, respectively. RESULTS: SA-hI-TAC/hI-TAC-SA fusion proteins were expressed at about 12% and 25% of the total bacterial protein, respectively. The two fusion proteins had a purity of about 85% and 90% after purification, and their purity reached 98% after purification with S-100 gel filtration chromatography. Both of the fusion proteins were efficiently immobilized on the surface of biotinylated mouse bladder cancer MB49 cells (91.3% for SA-hI-TAC and 98.8% for hI-TAC-SA). SA/hI-TAC induced lymphocyte chemotaxis in a dose-dependent manner, and hI-TAC-SA showed a stronger chemotactic effect than SA-hI-TAC. CONCLUSIONS: We successfully obtained SA/hI-TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.
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Quimiocina CXCL11/química , Interferons/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Animais , Biotinilação , Western Blotting , Vacinas Anticâncer , Linhagem Celular Tumoral , Cromatografia de Afinidade , Humanos , Camundongos , Plasmídeos , EstreptavidinaRESUMO
Resveratrol, a natural polyphenolic compound known for its antioxidative and antiinflammatory effects, exerts antiasthmatic effects, although the mechanism underlying these effects remains elusive. The phosphatase and tensin homology deleted on chromosome ten gene (PTEN) is involved in the pathogenesis of asthma, and PTEN overexpression in asthmatic mice improved asthma symptoms. To investigate whether the antiasthmatic mechanisms of resveratrol correlated with the upregulation of PTEN expression, an ovalbumin (OVA)-induced murine asthma model was used to determine the effectiveness of resveratrol treatment. PTEN mRNA and protein expression was assessed with real-time polymerase chain reaction (PCR) and immunochemistry. To determine whether airway remodeling occurred, the inner airway wall, mucous layer, and smooth muscle areas were each determined using an image analysis system. The lung epithelial cell line 16HBE was used to study the regulation of PTEN expression levels by resveratrol in vitro. Our data demonstrated that resveratrol inhibited OVA-induced airway inflammation and airway remodeling in asthmatic mice. PTEN expression was decreased in the murine asthma model, although the expression of PTEN was restored following treatment with resveratrol. Correlation efficiency analysis showed that PTEN expression was associated with the degree of airway remodeling. Further in vitro studies demonstrated that the inhibition of Sirtuin 1 (SIRT1) activity by a SIRT1 inhibitor and RNA interference decreased PTEN protein expression, while resveratrol attenuated the decreases in PTEN expression induced by the SIRT1 inhibitor. These data suggest the mechanism of the antiasthmatic effects of resveratrol in an OVA-induced murine asthma model, which resulted in the upregulation of PTEN via SIRT1 activation.
Assuntos
Antiasmáticos/farmacologia , Asma/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Sirtuína 1/genética , Estilbenos/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos BALB C , Ovalbumina , PTEN Fosfo-Hidrolase/genética , Resveratrol , Estilbenos/uso terapêutico , Regulação para CimaRESUMO
CLPTM1L is believed to be associated with lung cancer. However, there is little information regarding its expression and function. Here using immunohistochemistry, we found that CLPTM1L expression was markedly increased in lung cancer tissues relative to normal tissues, especially in lung adenocarcinoma. CLPTM1L expression was not found to be associated with stages, smoking status, lymph node metastasis, or T lymphocyte infiltration but with differentiation stage. We found CLPTM1L to be enriched in the mitochondrial compared with plasma membrane protein extracts. CLPTM1L-EGFP transfection showed that the molecule product was expressed in cytoplasm and indicated the mitochondrial localization stained with mitochondrial marker MitoTracker. CLPTM1L transferred lung cancer cell line 95-D showed no growth inhibition or cell apoptosis, but it did show inhibited sensitivity to cis-diamminedichloroplatinum(II) (cisplatin, CDDP). Knockdown of CLPTM1L by RNAi did not interfere with cell proliferation but it did increase cell sensitivity to CDDP and activation of caspase-9 and caspase-3/7. These data indicate CLPTM1L is a mitochondria protein and that it may be associated with anti-apoptotic mechanism which affects drug-resistance in turn.
Assuntos
Adenocarcinoma/metabolismo , Apoptose , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas de Neoplasias/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Análise de Sequência de DNARESUMO
BACKGROUND: "Phosphatase and tensin homolog deleted on chromosome 10" (PTEN) is mostly considered to be a cancer-related gene, and has been suggested to be a new pathway of pathogenesis of asthma. The purpose of this study was to investigate the effects of the glucocorticoid, dexamethasone, on PTEN regulation. METHODS: OVA-challenged mice were used as an asthma model to investigate the effect of dexamethasone on PTEN regulation. Immunohistochemistry was used to detect expression levels of PTEN protein in lung tissues. The human A549 cell line was used to explore the possible mechanism of action of dexamethasone on human PTEN regulation in vitro. A luciferase reporter construct under the control of PTEN promoter was used to confirm transcriptional regulation in response to dexamethasone. RESULTS: PTEN protein was found to be expressed at low levels in lung tissues in asthmatic mice; but the expression was restored after treatment with dexamethasone. In A549 cells, human PTEN was up-regulated by dexamethasone treatment. The promoter-reporter construct confirmed that dexamethasone could regulate human PTEN transcription. Treatment with the histone deacetylase inhibitor, TSA, could increase PTEN expression in A549 cells, while inhibition of histone acetylase (HAT) by anacardic acid attenuated dexamethasone-induced PTEN expression. CONCLUSIONS: Based on the data a new mechanism is proposed where glucocorticoids treat asthma partly through up-regulation of PTEN expression. The in vitro studies also suggest that the PTEN pathway may be involved in human asthma.
Assuntos
Asma/tratamento farmacológico , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Glucocorticoides/uso terapêutico , PTEN Fosfo-Hidrolase/metabolismo , Animais , Asma/metabolismo , Linhagem Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , PTEN Fosfo-Hidrolase/genética , Transdução de SinaisRESUMO
BACKGROUND: Intravesical administration is an important treatment against superficial bladder cancer and CD40L is essential for the protective anti-tumor immunity. In situ gene therapy with CD40L was demonstrated to successfully inhibit tumor cell growth in the orthotopic mouse model of bladder cancer. In the present study, we prepared streptavidin (SA)-tagged sCD40L and developed a novel immunotherapy for superficial bladder cancer based on the strong interaction between streptavidin and biotin. MATERIAL AND METHODS: The SA-sCD40L fusion protein was expressed in E. coli and purified on the Ni-NTA column. After refolding with dialysis, the bi-function of the fusion protein was determined by flow cytometric analysis for streptaidin-mediated surface modification of MB49 bladder cancer cells and a mouse B cell CD40L-dependent proliferation assay. The mouse orthotopic model of MB49 superficial bladder cancer was used to evaluate the efficacy of SA-sCD40L immunotherapy. RESULTS: The SA-sCD40L fusion protein exhibited both full biotin-binding property and CD40L bioactivity. After intravesical instillation, the SA-sCD40L bi-functional fusion protein was durably immobilized on the biotinylated mucosal surface of bladder wall for up to four days. The SA-sCD40L treatment significantly prolonged the survival of MB49 tumor-bearing mice and cured 50% of mice with MB49 superficial bladder cancer without significant adverse effects. In addition, more tumor-infiltrating CD4(+)or CD8(+) T cells were observed in SA-sCD40L-treated group. CONCLUSION: Intravesical immobilization of SA-sCD40L elicited a strong and long-lasting immunity against the MB49 bladder cancer.