RESUMO
Pam3CSK4 activates Toll-like receptors 2 and 1 (TLR1/2), which recognize mainly molecules from gram-positive pathogens. The effect of Pam3CSK4 on various cytokine and chemokine expression in cultured human uveal melanocytes (UM) has not been studied systematically. The purpose of this study was to investigate the mechanistic expressions of seven cytokines and chemokines of interleukin- (IL-) 6, IL-10, MCP-1 (CCL-2), CXCL-1 (GRO-α), CXCL-8 (IL-8), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in UM. These cytokines are reported to be increased in intraocular fluids or tissues of the patients with endophthalmitis and non-infectious uveitis, as well as in various experimental animal uveitic models in the literature. Flow cytometry was used to measure the effects of Pam3CSK4 on the expression of TLR1/2 in UM. ELISA and Real-time PCR analysis were used to estimate the ability of Pam3CSK4 to elevate these cytokines and chemokines levels in conditioned media and cell lysates of UM, respectively. Flow cytometry measured and compared the phosphorylated MAPK pathway and activated NF-κB signals pathway in UM, treated with and without Pam3CSK4. ELISA analysis tested the effect of various signal inhibitors (ERK1/2, JNK1/2, p38 and NF-κB) on Pam3CSK4-induced IL-6 levels in cultured UM. The role of TLR2 in Pam3CSK4-induced acute anterior uveitis in experimental mouse model was tested in TLR2 knockout (TLR2 KO) mice and their wild-type C57Bl/6 controls. Pam3CSK4 increased the expression of TLR1/2 proteins in cultured UM. Pam3CSK4 significantly elevated the IL-6, MCP-1, CXCL-1, CXCL-8 protein, and mRNA levels in cultured UM, but not IL-10, TNF-α, or IFN-γ. Pam3CSK4 activated NF-κB, ERK, JNK, and p38 expression. Pam3CSK4-induced expression of IL-6 was decreased by NF-κB, ERK, INK, and p38 inhibitors; especially the NF-κB inhibitor, which can completely block the IL-6 stimulation. Intravitreal injection of Pam3CSK4 induced acute anterior uveitis in C57Bl/6 mice, this effect was significantly reduced in TLR2 KO mice. TLR1/2 plays an important role against invading pathogens, especially gram-positive bacteria; but an excessive reaction to molecules from gram-positive bacteria may promote non-infectious uveitis. UM can produce IL-6, MCP-1, CXCL-1, and CXCL-8, and are one of the target cells of TNF-α and IFN-γ. TLR-2 inhibitors might have a beneficial effect in the treatment of certain types of uveitis and other ocular inflammatory-related diseases and warrant further investigation.
Assuntos
Uveíte Anterior , Uveíte , Humanos , Animais , Camundongos , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 1 Toll-Like/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Citocinas/metabolismo , Melanócitos/metabolismo , Quimiocinas/metabolismo , Uveíte/metabolismo , Uveíte Anterior/metabolismoRESUMO
Autophagy dysfunction is one of the common causes of tumor formation and plays an important role in uveal melanoma (UM). However, little is known about the regulatory mechanisms of autophagy in UM. Here, we show that PTK6 can promote the proliferation, migration, and invasion of UM cells by inhibiting autophagy. SOCS3 can inhibit the proliferation, migration, and invasion of UM cells. Overexpression of SOCS3 can partially rescue the PTK6-induced promotion of UM cell proliferation, migration, and invasion. Mechanistically, PTK6 can bind to SOCS3, and SOCS3 can downregulate the expression of PTK6. Furthermore, PTK6 can upregulate the phosphorylation of mTOR to inhibit autophagy. Taken together, our findings demonstrate the functions of PTK6 and SOCS3 in UM cells and targeting the SOCS3-PTK6 signaling axis might be a novel and promising therapeutic strategy for patients with UM.
Assuntos
Apoptose , Transformação Celular Neoplásica , Humanos , Fosforilação , Linhagem Celular Tumoral , Proliferação de Células , Carcinogênese , Serina-Treonina Quinases TOR/metabolismo , Autofagia , Movimento Celular , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismoRESUMO
RNA 5-methylcytosine (m5C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m5C in UM. Initially, we identified significantly elevated global RNA m5C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m5C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m5C-RIP-qPCR, and luciferase assay identified ß-Catenin (CTNNB1) as a direct target of NSUN2-mediated m5C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m5C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis.
Assuntos
RNA , Neoplasias Uveais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Metilação de DNA , Humanos , Melanoma , Metiltransferases/genética , RNA/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologiaRESUMO
Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.
Assuntos
Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Diglicerídeos/farmacologia , Melanócitos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Úvea/citologia , Animais , Anticorpos Neutralizantes/farmacologia , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Injeções Intravítreas , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Uveíte/induzido quimicamente , Uveíte/metabolismoRESUMO
As a noninvasive therapeutic technique, photodynamic therapy (PDT) has attracted numerous research interests for cancer therapy. Nevertheless, the residual photosensitizers (PSs) still produce reactive oxygen species (ROS) and damage normal cells under sunlight after PDT, which limits their practical application in clinic. Herein, the authors propose a self-degradable type-I PS based on conjugated polymer, which is composed of aggregation-induced emission (AIE) and imidazole units. Due to the effective conjugated skeleton and unique AIE properties, thus-obtained polymers can effectively generate superoxide radical (O2-⢠) through the type-I process under light irradiation, which is ideal for hypoxic tumors treatment. Intriguingly, under light irradiation, O2-⢠produced by the conjugated polymers can further lead to the self-degradation of the polymer to form nontoxic micro-molecules. It not only helps to resolve the potential phototoxicity problems of residual PSs, but also can accelerate the metabolism of the conjugated polymers to avoid the potential biotoxicity of drug accumulation. This work develops a self-degradable type-I PS, which can turn off the generation of ROS in time after PDT, providing a novel strategy to balance the PDT effect and postoperative safety.
Assuntos
Neoplasias/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/metabolismo , Polímeros/metabolismo , Complicações Pós-Operatórias/prevenção & controle , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos TestesRESUMO
Purpose: Lipopolysaccharide (LPS) can activate Toll-like receptor 4 (TLR4) and increase the expression of CXCL1 and CXCL2, the potent neutrophils chemoattractants, in various cell types. These effects have not been previously reported in the uveal melanocytes. This study was designed to investigate the effects of LPS on the activation of TLR4 and expression of CXCL1/CXCL2 in cultured human uveal melanocytes and the relevant signal pathways.Methods: Effects of LPS on the expression of TLR4 were tested using real-time PCR, flow cytometry and fluorescence immunostaining. Effects of LPS-induced expression/secretion of CXCL1/CXCL2 were studied using real-time PCR in cell lysates and ELISA in conditioned media of cultured uveal melanocytes. Activated NF-κB and phosphorylated MAPK signals were tested in cells with and without LPS treatment using flow cytometry. Effects of various signal inhibitors on p38, ERK1/2, JNK1/2 and NF-κB on the secretion of CXCL1/CXCL2 were tested by ELISA. The effects of neutralized antibodies of CXCL1/CXCL2 on the severity of LPS-induced uveitis were tested in a mouse model.Results: LPS stimulation increased the expression of TLR4 mRNA and protein in culture uveal melanocytes. Constitutive secretion of CXCL1/CXCL2 was detected in uveal melanocytes and was significantly increased dose- and time-dependently by LPS stimulation. LPS mainly increased the activated NF-κB and phosphorylated JNK1/2. LPS-induced expression of CXCL1/CXCL2 was blocked by NF-κB and JNK1/2 inhibitors. The severity of LPS-induced uveitis was significantly inhibited by neutralizing antibody to CXCL1/CXCL2Conclusions: This is the first report on the LPS-induced expression of CXCL1 and CXCL2 by uveal melanocytes via the activation of TLR4. These results suggest that uveal melanocytes may play a role in the immune reaction that eliminates the invading pathogens. Conversely, an excessive LPS-induced inflammatory reaction may also lead to the development of inflammatory ocular disorders, such as non-infectious uveitis.
Assuntos
Quimiocina CXCL1/metabolismo , Lipopolissacarídeos/farmacologia , Melanócitos/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Úvea/citologia , Animais , Anticorpos Neutralizantes/farmacologia , Células Cultivadas , Quimiocina CXCL1/imunologia , Quimiocina CXCL2/imunologia , Quimiocina CXCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Melanócitos/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Uveais/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinase Induzida por NF-kappaBRESUMO
The development of multifunctional photosensitizers (PSs) with aggregation-induced emission (AIE) properties plays a critical role in promoting the progress of the photodynamic therapy (PDT). In this work, a multifunctional PS (named DSABBT NPs) with AIE activity has been designed and prepared to carry out ultrafast staining, excellent two-photon bioimaging, and high-efficiency image-guided PDT. Simply, DSABBT with AIE characteristic was synthesized by one-step Schiff reaction of 4-(diethylamino)-salicylaldehyde (DSA) and 4,7-bis(4-aminophenyl)-2,1,3-benzothiadiazole (BBT). Then, DSABBT and DSPE-PEG2000-cRGD generate nanoparticles (NPs) easily in an ultrapure water/tetrahydrofuran mixture through a facile nanoprecipitation at room temperature. We found that DSABBT NPs exhibit bright solid-state fluorescence with large stokes shifts (180 nm) and two-photon absorption cross-section (1700 GM). Importantly, DSABBT NPs exhibited excellent ability of ultrafast staining and two-photon imaging, which can readily label suborganelles by subtly shaking the living cells for 5 s under mild conditions. Moreover, DSABBT NPs displayed high singlet oxygen (1O2) generation capacity and remarkable image-guided PDT efficiency. Therefore, DSABBT NPs can act as the promising candidate for multifunctional PSs, which can destroy cancer cells and block malignant tumor growth via the production of reactive oxygen species upon irradiation conditions. These outcomes provide us with a selectable strategy for developing multifunctional theranostic systems.
Assuntos
Antineoplásicos/farmacologia , Corantes Fluorescentes/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Microscopia de Fluorescência por Excitação Multifotônica , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Estrutura Molecular , Imagem Óptica , Tamanho da Partícula , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Propriedades de SuperfícieRESUMO
N6 -methyladenosine (m6 A) is a novel epitranscriptomic marker that contributes to regulating diverse biological processes through controlling messenger RNA metabolism. However, it is unknown if m6 A RNA methylation affects uveal melanoma (UM) development. To address this question, we probed its function and molecular mechanism in UM. Initially, we demonstrated that global RNA m6 A methylation levels were dramatically elevated in both UM cell lines and clinical specimens. Meanwhile, we found that METTL3, a main m6 A regulatory enzyme, was significantly increased in UM cells and specimens. Subsequently, cycloleucine (Cyc) or METTL3 targeted small interfering RNA was used to block m6 A methylation in UM cells. We found that Cyc or silencing METTL3 significantly suppressed UM cell proliferation and colony formation through cell cycle G1 arrest, as well as migration and invasion by functional analysis. On the other hand, overexpression of METTL3 had the opposite effects. Furthermore, bioinformatics and methylated RNA immunoprecipitation-quantitative polymerase chain reaction identified c-Met as a direct target of m6 A methylation in UM cells. In addition, western blot analysis showed that Cyc or knockdown of METTL3 downregulated c-Met, p-Akt, and cell cycle-related protein levels in UM cells. Taken together, our results demonstrate that METTL3-mediated m6 A RNA methylation modulates UM cell proliferation, migration, and invasion by targeting c-Met. Such a modification acts as a critical oncogenic regulator in UM development.
Assuntos
Melanoma/genética , Proteínas Proto-Oncogênicas c-met/genética , RNA Neoplásico/genética , Neoplasias Uveais/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Epigênese Genética , Técnicas de Silenciamento de Genes , Humanos , Melanoma/metabolismo , Melanoma/patologia , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Regulação para Cima , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologiaRESUMO
Purpose: Uveal melanoma (UM) is the most frequent metastatic ocular tumor in adults. Therapeutic intervention remains ineffective since none of the novel procedures used to treat this disease increased survival rates. To deal with this limitation, additional studies are required to clarify its pathogenesis. The current study focused on describing how epigenetic modulation by miR-142-3p affects changes in some cellular functions underlying UM pathogenesis. Methods and results: Microarray analysis identified 374 miRNAs which were differentially expressed between UM cells and uveal melanocytes. miR-142-3p was one of the 10 most downregulated miRNAs. Quantitative RT-PCR analysis confirmed that miR-142-3p expression levels were significantly decreased in both UM cell lines and clinical specimens. The results of the MTS, clone formation, scratch wound, transwell assays, and in vivo biofluorescence imaging showed that miR-142-3p overexpression significantly inhibited cell proliferation, migration, and invasiveness. Nevertheless, miR-142-3p did not affect cell apoptotic activity or sensitivity to doxorubicin. Cell cycle and EdU analysis showed that miR-142-3p overexpression induced G1/G2 cell cycle arrest and reduced DNA synthesis in UM cells. Microarray analysis showed that miR-142-3p mainly regulates the TGFß signaling pathway, and those in which MAPK and PI3K-Akt are constituents. Functional interactions between miR-142-3p and CDC25C, TGFßR1, GNAQ, WASL, and RAC1 target genes were confirmed based on the results of the luciferase reporter assay and Western blot analysis. CDC25C or RAC1 downregulation is in agreement with cell cycle arrest and DNA synthesis disorder induction, while downregulation of TGFßR1, GNAQ, WASL, or RAC1 accounts for declines in cell migration. Conclusion: miR-143-3p is a potential therapeutic target to treat UM since overriding its declines in expression that occur in this disease reversed the pathogenesis of this disease. Such insight reveals novel biomarker for decreasing UM vitality and for improved tracking of tumor progression.
RESUMO
Butein is a chalcone, a flavonoid that is widely biosynthesized in plants. Butein has been identified to possess varied pharmacological activity and is extractable from traditional Chinese medicinal herbs, therefore applicable for disease treatment. Recently, in vitro and in vivo studies have shown that butein may induce apoptotic cell death in various human cancer cells. In this study we investigated the apoptotic effect of butein and the underlying mechanisms in human cervical cancer cells. Two cell lines, C-33A and SiHa cells, were treated with butein at different dosages for different durations. The effect of butein on cell viability was assessed by MTT assay, which revealed that butein exerted cytotoxicity in both cervical cancer cells in a dose- and time-dependent fashion. Apoptotic pathway-related factors in the butein-treated cervical cancer cells were then examined. JC-1 flow cytometry, cytochrome c assay, and caspase activity assays demonstrated that butein disturbed mitochondrial transmembrane potential, and increased cytosolic cytochrome c levels and caspase activities in both cervical cancer cells. Western blot analysis revealed that butein downregulated anti-apoptotic protein Bcl-xL and led to proteolytic cleavage of poly (ADP-ribose) polymerase. In addition, butein decreased expressions of the inhibitor of apoptosis (IAP) proteins, including X-linked IAP, survivin, and cellular IAP-1. The findings of this study suggest that butein can decrease cervical cancer cell viability via a pro-apoptotic effect, which involves inhibition of the IAP proteins and activation of both extrinsic and intrinsic pro-apoptotic pathways. Therefore, butein may be applicable for cervical cancer treatment.
RESUMO
Matrix metalloproteinase (MMP)-8 is the most potent MMP for degrading collagen type-1 and plays an important role in inflammatory reactions and tissue remolding processes. MMP-8 is expressed mainly by polymorphonuclear leukocytes and is not expressed constitutively by most non-leukocytes. We studied the constitutive and TNF-α-induced expression of MMP-8 in cultured human uveal melanocytes (UM) and the relevant signal pathways involved. Conditioned media and cells were collected from UM and other cell types. MMP-8 proteins and mRNA were measured using ELISA kit, western blot and real time RT-PCR, respectively. Phosphorylated p38 MAPK, ERK1/2, and JNK1/2 were measured by ELISA kit and western blot. Very high levels of MMP-8 proteins and mRNA were detected in the conditioned media and cell lysates in 11 UM cell lines and three uveal melanoma cell lines cultured without serum, but not in media and cell lysates from other ocular resident cells or 12 malignant cell lines from other tissues, with exception of cutaneous melanoma cells. TNF-α moderately increased MMP-8 mRNA and protein levels in a dose- and time-dependent manner, accompanied by a significant increase of phosphorylated JNK1/2 and ERK1/2 in cell lysates. ERK1/2 (U0126) and JNK1/2 (SP600125) inhibitors significantly blocked TNF-α-induced and constitutive expression of MMP-8 in UM. This is the first report on the expression and secretion of MMP-8 by UM and uveal melanoma cells. The data suggest that UM may play a role in the remolding process and pathogenesis of inflammatory-related diseases in the eye via secretion of MMP-8.
Assuntos
Regulação da Expressão Gênica/fisiologia , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 8 da Matriz/metabolismo , Melanócitos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Úvea/citologia , Adulto , Idoso , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Melanócitos/metabolismo , Pessoa de Meia-Idade , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma.
Assuntos
Apoptose/efeitos dos fármacos , Flavonoides/farmacologia , Melanoma/patologia , Mitocôndrias/efeitos dos fármacos , Neoplasias Uveais/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Flavonóis , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Germline RB1 mutations strongly predispose humans to cone precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRß2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRß1. TRß2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRß2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRß1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRß2 counteracts TRß1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838-50. ©2017 AACR.
Assuntos
Proliferação de Células/genética , Proteína do Retinoblastoma/fisiologia , Proteínas Quinases Associadas a Fase S/genética , Receptores beta dos Hormônios Tireóideos/fisiologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Mutação em Linhagem Germinativa , Células HCT116 , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteína do Retinoblastoma/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Ativação Transcricional/genética , Células Tumorais CultivadasRESUMO
PURPOSE: To identify an effective method to prevent myopia progression by characterizing the regulation of matrix metalloproteinase- (MMP-) 2 expression and its secretion from scleral fibroblasts and retinal pigment epithelium (RPE) cells by miR-29a. METHODS: The effects of miR-29a on the growth of scleral fibroblasts and RPE cells were assessed using the cell counting kit-8. The changes in MMP-2 mRNA levels in scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor were measured by quantitative PCR. Enzyme-linked immunosorbent assays were used to determine the changes in MMP-2 secretion from scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor. RESULTS: The miR-29a mimics or inhibitor did not significantly alter the growth of scleral fibroblasts or RPE cells at 24, 48, or 72 hours after transfection. MMP-2 mRNA levels were significantly decreased in scleral fibroblasts and RPE cells transfected with the miR-29a mimics. The secretion of MMP-2 by scleral fibroblasts and RPE cells was significantly decreased in cells transfected with the miR-29a mimics. CONCLUSIONS: Suppression of scleral fibroblast and RPE cell expression and secretion of MMP-2 by miR-29a can be used as a therapeutic target for the prevention and treatment of myopia.
Assuntos
Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Esclera/metabolismo , Linhagem Celular , Humanos , Metaloproteinase 2 da Matriz/genética , MicroRNAs/genéticaRESUMO
PURPOSE: Previous studies of the relationship between visual acuity (VA) and ocular dominance have produced conflicting results. We hypothesized that (1) the discrepancies were related mostly to sample size and interocular visual acuity difference (IOVAD); (2) in large samples of individuals with marked IOVADs, the eye with the better uncorrected distance visual acuity (UDVA) would be dominant. These hypotheses were tested in a large group of myopic patients. METHODS: This prospective study of cycloplegic refraction involved 2045 myopic refractive surgery candidates. Patients with amblyopia or strabismus were excluded. Ocular dominance was assessed using the hole-in-the-card test. RESULTS: In 2045 patients, the dominant eye had significantly better UDVA (p = 0.028) and was less astigmatic (p = 0.000) than the nondominant eye. In 426 patients with marked interocular difference in the UDVA (≥0.2 logMAR), the dominant eye not only had significant UDVA (p = 0.022) but also significantly less myopic (p = 0.028) and had a shorter axial length (AL; p = 0.001). In patients with smaller differences in UDVA (0.1 logMAR, n = 411) or no difference (n = 1208), the dominant and nondominant eyes did not differ significantly with respect to UDVA, myopic power, and AL (p > 0.05). CONCLUSIONS: The present study showed that the dominant eyes had significantly better UDVA than the nondominant eyes, especially in individuals with marked differences in UDVA. These results supported our hypothesis regarding the relationship between better VA and ocular dominance.
Assuntos
Dominância Ocular/fisiologia , Miopia/fisiopatologia , Refração Ocular , Acuidade Visual , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miopia/cirurgia , Estudos Prospectivos , Procedimentos Cirúrgicos Refrativos , Testes Visuais , Adulto JovemRESUMO
PURPOSE: To study the relationships between matrix metalloproteinases (MMP)-2, MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3 aqueous humor levels in patients with high myopia or cataract. MATERIALS AND METHODS: MMPs and TIMPs protein levels in 65 aqueous humor samples collected from patients with high myopia or cataract during cataract or clear lens extraction surgery were measured with the Luminex xMAP Technology. The relationship between MMPs and TIMPs levels was analyzed with Spearman's correlation test. RESULTS: MMP-2 levels, but not MMP-3 levels, were increased in the aqueous humor from high-myopia patients. Levels of TIMP-1, -2, and -3 were positively and very significantly correlated with the MMP-2 levels (TIMP-1: r=0.626, p < 0.001; TIMP-2: r = 0.545, p < 0.001; TIMP-3: r = 0.439, p < 0.001). TIMP-2 and-3 levels did not significantly correlate with MMP-3 levels (TIMP-2: r = 0.175, p > 0.05; TIMP-3: r = 0.127, p > 0.05) and TIMP-1 levels only marginally correlated with MMP-3 levels (r = 0.278, 0.01< P < 0.05). CONCLUSIONS: Compared to the present findings with the relationship of MMPs and TIMPs in other fields of medicine, our results are consistent with the homeostasis hypothesis that the increase of TIMPs serves as a compensation reaction to inhibit the excessive degradation caused by the increase of MMPs and limits the development of myopia.
Assuntos
Humor Aquoso/enzimologia , Catarata/enzimologia , Metaloproteinases da Matriz/metabolismo , Miopia Degenerativa/enzimologia , Inibidores Teciduais de Metaloproteinases/metabolismo , Adulto , Comprimento Axial do Olho/patologia , Feminino , Humanos , Imunoensaio/métodos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade , Estatística como Assunto , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
BACKGROUND: Hypoxia is the most important stimulus leading to up-regulation of vascular endothelial growth factor (VEGF) in the retina via elevation of hypoxia-inducible factors-1α (HIF-1α) protein. The purpose of this study was to test the effects of melatonin on the expression of VEGF and HIF-1α in the cultured human retinal pigment epithelial (RPE) cells under normoxia and hypoxia. METHOD: An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 ) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without melatonin under normoxia and hypoxia. The protein and RNA levels of VEGF and HIF-1α were measured by ELISA kits and RT-PCR, respectively. RESULT: Hypoxia induced a significant increase of expression and secretion of VEGF and accumulation of HIF-1α protein in RPE cells (P < 0.05). Melatonin at 10-5 to 10-8 M significantly inhibited hypoxia-induced expression, the secretion of VEGF and the accumulation of HIF-1α protein (P < 0.05), but not affected expression of VEGF and HIF-1α under normoxia (P > 0.05). CONCLUSION: This study suggests that melatonin may have potential value in the prevention and treatment of various retinal diseases associated with increase of VEGF, vascular leakage and angiogenesis.
Assuntos
Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/metabolismo , Melatonina/farmacologia , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Idoso , Antioxidantes/farmacologia , Sobrevivência Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/genética , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
AIM: To examine the genetic profile of primary uveal melanoma (UM) as compared to UM in immune escape. METHODS: Dendritic cells (DC) loaded with lysates of UM cells of high metastatic potential were used to stimulate CTLs(CTLs). When CTLs co-cultured with the UM cells, most UM cells could be eliminated. Survival UM cells grew slowly and were considered to be survival variants and examined by a microarray analysis. These differential genes were analyzed further with Venn Diagrams and functions related to immune escape. We additionally examined transcriptional changes of manually selected survival variants of UM cells and of clinical UM samples by quantitative real-time polymerase chain reaction (qRT-PCR), and analyzed the correlation of these expressions and patients' survival. RESULTS: Gene expression analyses revealed a marked up-regulation of SLAMF7 and CCL22 and a significant down-regulation of KRT10, FXYD3 and ABCC2. The expression of these genes in the relapsed UM was significantly greater than those in primary UM. UM patients with overexpression of these genes had a shorter survival period as compared with those of their underexpression. CONCLUSION: Gene expression, in particular of SLAMF7, CCL22, KRT10, FXYD3 and ABCC2, differed between primary UM cells and survival variants of UM cells.
RESUMO
As increases in hepatocyte growth factor/scatter factor (HGF/SF) induce retinal pigment epithelial (RPE) migration and proliferation into the vitreous cavity and contribute to proliferative vitreoretinopathy (PVR) development, we determined if changes in miR-182 expression affect such behavioral changes. We found that miR-182 expression was less in PVR clinical samples than in primary RPE cells whereas c-Met was upregulated. Ectopic miR-182 inhibited RPE cell proliferation, cell cycle, and migration. Bioinformatic analysis identified c-Met as a miR-182 target, which was confirmed with the luciferase reporter assay. Transfection of miR-182 into RPE cells induced c-Met downregulation, which led to reduced cell proliferation and migration through declines in p-Akt formation. MiR-182 downregulation along with c-Met upregulation in PVR tissues suggest that these two opposing effects play important roles in PVR development. As ectopic miR-182 expression suppressed RPE cell proliferation and migration, strategies to selectively upregulate miR-182 expression in a clinical setting may provide a novel option to treat this disease.
Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/metabolismo , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-met/genética , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/genética , Adulto , Movimento Celular , Células Cultivadas , Regulação para Baixo , Humanos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Regulação para Cima , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologiaRESUMO
BACKGROUND: Norcantharidin, a modified pure compound from blister beetles, was previously demonstrated to induce apoptosis of cancer cells. This study investigated its anti-cancer activity in prostate cancer cells and the mechanisms involved. METHODS: Two human prostate cancer cell lines, 22Rv1 and Du145, were treated with norcantharidin at concentrations ranging from 3 to 30µg/ml. Cytotoxic effect of norcantharidin was determined by use of the 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) assay. The effects of apoptosis were evaluated by cell death assay, Caspase-3, -8, -9 activity and cytochrome c release. The apoptotic related protein expressions (Bcl-2 family and inhibitor of apoptosis proteins) were determined using western blotting. RESULTS: An MTT assay revealed that norcantharidin induced cytotoxicity against both prostate cancer cells in dose- and time-dependent manners. Treatment with norcantharidin at 3µg/ml or higher significantly increased oligonucleosomal formation with concomitant appearance of PARP cleavage, implicating the induction of apoptosis. Norcantharidin intrinsically elevated cytosolic cytochrome c levels and activated caspase-3, -8, and -9. Extrinsically, it upregulated the expression of not only the death receptors Fas and DR5 in 22Rv1 cells, but also of RIP and TRADD adaptor proteins in Du145 cells. Mechanistically, norcantharidin increased ratios of pro-/anti-apoptotic proteins and decreased expression of IAP family member proteins, including cIAP1 and survivin, regardless of the distinct status of androgen receptor expression in both cells. CONCLUSIONS: Norcantharidin exhibited cytotoxicity against 22Rv1 and Du145 prostate cancer cells by inducing both intrinsic and extrinsic apoptotic pathways and could thus potentially be a remedy for prostate cancer.