RESUMO
BACKGROUND: Intravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis. METHODS: Quantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. RESULTS: tRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-ß1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. CONCLUSIONS: Concurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy.
Assuntos
Inibidores da Angiogênese , Anti-Inflamatórios , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Humanos , RNA de Transferência/metabolismo , RNA de Transferência/genética , Camundongos Endogâmicos C57BL , Proliferação de Células/efeitos dos fármacos , Neovascularização de Coroide/patologia , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/metabolismo , Masculino , Oftalmopatias/tratamento farmacológico , Oftalmopatias/patologia , Oftalmopatias/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Neovascularização Patológica , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Retinopatia Diabética/metabolismo , Camundongos , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
As a natural flavone, apigenin is abundantly present in vegetables, fruits, oregano, tea, chamomile, wheat sprout and is regarded as a major component of the Mediterranean diet. Apigenin is known to inhibit proliferation in different cancer cell lines by inducing G2/M arrest, but it is unclear whether this action is predominantly imposed on G2 or M phases. In this study, we demonstrate that apigenin arrests prostate cancer cells at G2 phase by flow cytometric analysis of prostate cancer cells co-stained for phospho-Histone H3 and DNA. Concurrently, apigenin also reduces the mRNA and protein levels of the key regulators that govern G2-M transition. Further analysis using chromatin immunoprecipitation (ChIP) confirmed the diminished transcriptional activities of the genes coding for these regulators. Unravelling the inhibitory effect of apigenin on G2-M transition in cancer cells provides the mechanistic understanding of its action and supports the potential for apigenin as an anti-cancer agent.
RESUMO
Cancer recurrence poses a significant challenge. At the cellular level, recurrence takes place as a result of reactivation of dormant cancer cells residing at G0 phase. The aim of the study was to identify compounds that can trap prostate and lung cancer cells in G0 phase from a new Chinese herb recipe, Astringent recipe, consisting of Radix Paeoniae Alba, Agrimonia pilosa Ledeb, Fructus Mume, Fritillaria thunbergii Miq., Ganoderma Lucidum Karst, and Astragalus membranaceus (Fisch.) Bunge. Astringent recipe impeded cell cycle progression in prostate and lung cancer cells by rounding them up at G0 phase by flow cytometric analysis of cancer cells stained with Hoechst 33342 and Pyronin Y, respectively, for DNA and RNA. The anti-cancer efficacy of the recipe was found to be attributable to Agrimonia pilosa Ledeb. Further study established that agrimol B, a polyphenol derived from Agrimonia pilosa Ledeb, contributed to the activity of the herb. The action of agrimol B on the cancer cells was likely derived from its effect on c-MYC, SKP2 and p27 by immunoblotting and immunofluorescence. Oral administration of Agrimonia pilosa Ledeb or agrimol B reduced growth of prostate cancer cell xenograft in animal. In conclusion, Agrimol B can enrich for prostate and lung cancer cells in G0 state and influence key regulators that govern G0 status.
Assuntos
Agrimonia , Antineoplásicos Fitogênicos/farmacologia , Butanonas/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Carga Tumoral/efeitos dos fármacos , Células A549 , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Butanonas/isolamento & purificação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/fisiologia , Relação Dose-Resposta a Droga , Ácido Elágico/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenóis/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Carga Tumoral/fisiologiaRESUMO
Epigenetic alterations occur in many physiological and pathological processes. N6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic mRNAs. However, the role of m6A modification in pathological angiogenesis remains elusive. In this study, we showed that the level of m6A modification was significantly upregulated in endothelial cells and mouse retinas following hypoxic stress, which was caused by increased METTL3 levels. METTL3 silencing or METTL3 overexpression altered endothelial cell viability, proliferation, migration, and tube formation in vitro. METTL3 knockout in vivo decreased avascular area and pathological neovascular tufts in an oxygen-induced retinopathy model and inhibited alkali burn-induced corneal neovascularization. Mechanistically, METTL3 exerted its angiogenic role by regulating Wnt signaling through the m6A modification of target genes (e.g., LRP6 and dishevelled 1 [DVL1]). METTL3 enhanced the translation of LRP6 and DVL1 in an YTH m6A RNA-binding protein 1 (YTHDF1)-dependent manner. Collectively, this study suggests that METTL3-mediated m6A modification is an important hypoxic stress-response mechanism. The targeting of m6A through its writer enzyme METTL3 is a promising strategy for the treatment of angiogenic diseases.
Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Regulação da Expressão Gênica , Metiltransferases/metabolismo , Neovascularização Patológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Biomarcadores , Suscetibilidade a Doenças , Inativação Gênica , Humanos , Hipóxia/complicações , Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Neovascularização Patológica/metabolismo , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Via de Sinalização WntRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd. (H) and Scutellaria barbata D.Don (S) are ancient anti-cancer Chinese herb medicines. When combined, known as HS, it is one of the most commonly prescribed Chinese Medicines for cancer patients today in China. AIM OF THE STUDY: The prevention of disease progression is a dominant concern for the growing number of men with prostate cancer. The purpose of this work is to evaluate the action and mode of action of Chinese Medicine recipe HS in inhibiting prostate cancer progression in preclinical models. METHODS: Effects of HS were analyzed in prostate cancer cell lines by evaluating proliferation, cell cycle profile, DNA damage and key regulators responsible for G2 to M phase transition. The transcriptional activities of these regulators were determined by RT-PCR and ChIP. The efficacy of HS in vitro was validated in an animal model. RESULTS: HS treatment was observed to reduce DNA content and accumulated prostate cancer cells at the G2/M phase. Immunolabeling for phospho-Histone H3 in association with nocodazole to capture mitotic cells confirmed that HS impeded G2 to M transition. After excluding DNA damage-induced G2 arrest, it was revealed that HS reduced expression of Cyclin B1, CDK1, PLK1 and Aurora A at both protein and mRNA levels, with concomitant reduction of H3K4 tri-methylation at their promoter-regions. Animals that received oral administration of HS with a dosage relevant to clinical application showed reduced tumor volume and weight with a reduction of Cyclin B1, CDK1, PLK1 and Aurora A protein levels. CONCLUSIONS: HS acts by impeding the G2 to M transition of prostate cancer cells. It is likely that the mode of action is transcriptionally suppressing proteins governing mitotic entry, without eliciting significant DNA damage.
Assuntos
Antineoplásicos Fitogênicos , Proteínas de Ciclo Celular/genética , Ciclo Celular/efeitos dos fármacos , Hedyotis , Extratos Vegetais , Neoplasias da Próstata , Scutellaria , Animais , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Medicina Tradicional Chinesa , Camundongos Nus , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Transcrição GênicaRESUMO
Chitosan-silk biocomposite films with nanofibrous structures have been prepared by facile solution casting of chitosan and silk co-dissolved in formic acid. The morphology, structure and mechanical properties of the chitosan-silk biocomposite were characterized by SEM, FTIR, TG-DSC, and mechanical testing. The results demonstrate that the prepared biocomposite films with a chitosan-silk ratio of 3:1 shows a high tensile strength of 97.8â¯MPa, a strain at break of 10.8% and a Young's modulus of 3.5â¯GPa, indicating its high strength and elasticity. Also, the preliminary cell culture experiment demonstrated the ideal biocompatibility of chitosan-silk composite films. As a result the superior mechanical properties of this composite film can be attributed to the silk nanofibrils and chitosan self-assembled nanofibers, and the strong hydrogen bonding interaction between the silk nanofibril and chitosan nanofibers. The specific nanostructure, enhanced mechanical properties, and biocompatibility make the biocomposite films a promising material for applications in biomedical devices.
Assuntos
Quitosana/química , Nanofibras/química , Seda/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Quitosana/farmacologia , Módulo de Elasticidade , Formiatos/química , Ligação de Hidrogênio , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Resistência à TraçãoRESUMO
Proliferative vitreoretinopathy (PVR) is one of the major challenges in retinal surgery, which occurs in the patient with complex retinal surgery or penetrating eye injury. Circular RNAs (circRNAs) have emerged as important regulators in many biological processes and disease development. However, the characterization and function of circRNAs in PVR remains elusive. In this study, we identified 91 dysregulated circRNAs in the epiretinal membranes (ERMs) of PVR patients. We further investigated the expression pattern of circ_0043144. circ_0043144 was significantly up-regulated in the vitreous samples and the corresponding serum samples of the patients with PVR. circ_0043144 expression was significantly down-regulated after PVR operation. In vitro studies revealed that circ_0043144 was involved in the regulation of the proliferation, migration and secretion ability of ARPE-19 cells, which is critical for ERM formation. Collectively, this study indicates that circRNAs are potential regulators of the pathogenesis of PVR. circ_0043144 is a promising prognostic and diagnostic indicator for PVR diseases.
Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , RNA/metabolismo , Vitreorretinopatia Proliferativa/genética , Vitreorretinopatia Proliferativa/metabolismo , Células Cultivadas , Membrana Epirretiniana/genética , Membrana Epirretiniana/metabolismo , Membrana Epirretiniana/patologia , Humanos , RNA Circular , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/patologiaRESUMO
Cancer cell repopulation through cell cycle re-entry by quiescent (G0 ) cell is thought to be an important mechanism behind treatment failure and cancer recurrence. Facilitates Chromatin Transcription (FACT) is involved in DNA repair, replication and transcription by eviction of histones or loosening their contact with DNA. While FACT expression is known to be high in a range of cancers, the biological significance of the aberrant increase is not clear. We found that in prostate and lung cancer cells FACT mRNA and protein levels were low at G0 compared to the proliferating state but replenished upon cell cycle re-entry. Silencing of FACT with Dox-inducible shRNA hindered cell cycle re-entry by G0 cancer cells, which could be rescued by ectopic expression of FACT. An increase in SKP2, c-MYC and PIRH2 and a decrease in p27 protein levels seen upon cell cycle re-entry were prevented or diminished when FACT was silenced. Further, using mVenus-p27K- infected cancer cells to measure p27 degradation capacity, we confirm that inhibition of FACT at release from quiescence suppressed the p27 degradation capacity resulting in an increased mVenus-p27K- signal. In conclusion, FACT plays an important role in promoting the transition from G0 to the proliferative state and can be a potential therapeutic target to prevent prostate and lung cancer from progression and recurrence.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Elongação da Transcrição/metabolismo , Células A549 , Carbazóis/farmacologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Células PC-3 , Neoplasias da Próstata/genética , Fase de Repouso do Ciclo Celular/genética , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/genéticaRESUMO
Antibody-based therapy is an effective strategy for treating ocular angiogenesis. However, short-acting efficacy and poor treatment compliance usually occurs in clinical practices. Thus, it is required to develop a drug delivery system to improve the bioavailability and decrease the toxicity of anti-angiogenic antibody. Bevacizumab is a recombinant humanized monoclonal antibody against vascular endothelial growth factor (VEGF). In this study, bevacizumab was encapsulated into poly (lactide-co-glycolide) (PLGA) nanoparticles. PLGA encapsulation could prolong the residency of bevacizumab in the vitreous and aqueous humor and produce long-lasting drug concentrations. Bevacizumab-encapsulated PLGA had no significant cytotoxicity and tissue toxicity effect in vitro and in vivo. In vitro studies showed that bevacizumab-encapsulated PLGA was more effective than bevacizumab in inhibiting VEGF-mediated endothelial cell proliferation, migration and tube formation. In vivo studies showed that bevacizumab-encapsulated PLGA enhanced the anti-angiogenic efficiency of bevacizumab for treating corneal neovascularization and retinal neovascularization. Thus, bevacizumab-encapsulated PLGA could increase the bioavailability and decrease the toxicity of bevacizumab during ocular angiogenesis therapy.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Bevacizumab/administração & dosagem , Neovascularização da Córnea/tratamento farmacológico , Neovascularização Retiniana/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/toxicidade , Animais , Humor Aquoso/metabolismo , Bevacizumab/farmacologia , Bevacizumab/toxicidade , Disponibilidade Biológica , Neovascularização da Córnea/patologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Corpo Vítreo/metabolismoRESUMO
Radiation therapy is used to treat cancer by radiation-induced DNA damage. Despite the best efforts to eliminate cancer, some cancer cells survive irradiation, resulting in cancer progression or recurrence. Alteration in DNA damage repair pathways is common in cancers, resulting in modulation of their response to radiation. This article focuses on the recent findings about molecules and pathways that potentially can be targeted to sensitize prostate cancer cells to ionizing radiation, thereby achieving an improved therapeutic outcome.
Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Neoplasias da Próstata/radioterapia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/efeitos da radiação , Aurora Quinases/efeitos da radiação , Ciclo Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem/efeitos da radiação , Quinases Ciclina-Dependentes/efeitos da radiação , Ciclinas/efeitos da radiação , Proteínas de Choque Térmico HSP90/efeitos da radiação , Histona Desacetilases/efeitos da radiação , Humanos , Receptores de Hialuronatos/efeitos da radiação , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos da radiação , Masculino , Mutação/efeitos da radiação , Proteína NEDD8/efeitos da radiação , Recidiva Local de Neoplasia/etiologia , Recidiva Local de Neoplasia/radioterapia , Neoplasia Residual , Células-Tronco Neoplásicas/efeitos da radiação , Fosfatidilinositol 3-Quinases/efeitos da radiação , Poli(ADP-Ribose) Polimerases/efeitos da radiação , Proteínas Proto-Oncogênicas c-met/efeitos da radiação , Tolerância a Radiação , Radiação Ionizante , Receptores Androgênicos/efeitos da radiação , Serina-Treonina Quinases TOR/efeitos da radiação , Proteína GLI1 em Dedos de Zinco/efeitos da radiaçãoRESUMO
BACKGROUND/AIMS: Pterygium is a common ocular surface disease with an unknown etiology and threatens vision as it invades into the cornea. Circular RNAs (circRNAs) are a novel class of RNA transcripts that participate in several physiological and pathological processes. However, the role of circRNAs in pathogenesis of pterygium remains largely unknown. METHODS: Genome-wide circRNA expression profiling was performed to identify pterygium -related circRNAs. GO analysis, pathway analysis, and miRNA response elements analysis was performed to predict the function of differentially expressed circRNAs in pterygium. MTT assays, Ki67 staining, Transwell assay, Hoechst 33342 staining, and Calcein-AM/PI staining were performed to determine the effect of circRNA silencing on pterygium fibroblast and epithelial cell function. RESULTS: Approximately 669 circRNAs were identified to be abnormally expressed in pterygium tissues. GO analysis demonstrated that the host genes of differentially expressed circRNAs were targeted to extracellular matrix organization (ontology: biological process), cytoplasm (ontology: cellular component), and protein binding (ontology: molecular function). Pathway analysis showed that dysregulated circRNAs-mediated regulatory networks were mostly enriched in focal adhesion signaling pathway. Notably, circ_0085020 (circ-LAPTM4B) was shown as a potential biomarker for pterygium. circ_0085020 (circ-LAPTM4B) silencing affected the viability, proliferation, migration, and apoptosis of pterygium fibroblast and epithelial cells in vitro. CONCLUSIONS: This study provides evidence that circRNAs are involved in the pathogenesis of pterygium and might constitute promising targets for the therapeutic intervention of pterygium.
Assuntos
Células Epiteliais , Fibroblastos , Estudo de Associação Genômica Ampla , Pterígio , RNA , Biomarcadores/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pterígio/genética , Pterígio/metabolismo , Pterígio/patologia , RNA/biossíntese , RNA/genéticaRESUMO
VEGF-induced neovascularization plays a pivotal role in corneal neovascularization (CoNV). The current study investigated the potential effect of ginsenoside Rh2 (GRh2) on neovascularization. In HUVECs, pretreatment with GRh2 largely attenuated VEGF-induced cell proliferation, migration, and vessel-like tube formation in vitro. At the molecular level, GRh2 disrupted VEGF-induced VEGF receptor 2 (VEGFR2)-Grb-2-associated binder 1 (Gab1) association in HUVECs, causing inactivation of downstream AKT and ERK signaling. Gab1 knockdown (by targeted short hairpin RNA) similarly inhibited HUVEC proliferation and migration. Notably, GRh2 was ineffective against VEGF in Gab1-silenced HUVECs. In a mouse cornea alkali burn model, GRh2 eyedrops inhibited alkali-induced neovascularization and inflammatory cell infiltrations in the cornea. Furthermore, alkali-induced corneal expression of mRNAs/long noncoding RNAs in cornea were largely attenuated by GRh2. Overall, GRh2 inhibits VEGF-induced angiogenic effect via inhibiting VEGFR2-Gab1 signaling in vitro. It also alleviates angiogenic and inflammatory responses in alkali burn-treated mouse corneas.-Zhang, X.-P., Li, K.-R., Yu, Q., Yao, M.-D., Ge, H.-M., Li, X.-M., Jiang, Q., Yao, J., Cao, C. Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.
Assuntos
Anti-Inflamatórios/farmacologia , Neovascularização da Córnea/tratamento farmacológico , Ginsenosídeos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anti-Inflamatórios/uso terapêutico , Córnea/efeitos dos fármacos , Córnea/metabolismo , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/metabolismo , Ginsenosídeos/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Gelo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Endogâmicos ICR , Fosfoproteínas/metabolismo , Fator A de Crescimento do Endotélio Vascular/toxicidade , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular dysfunction is a hallmark of ischemic, cancer, and inflammatory diseases, contributing to disease progression. Circular RNAs (circRNAs) are endogenous non-coding RNAs, which have been reported to be abnormally expressed in many human diseases. In this study, we used retinal vasculature to determine the role of circular RNA in vascular dysfunction. We revealed that cZNF609 was significantly up-regulated upon high glucose and hypoxia stress in vivo and in vitro. cZNF609 silencing decreased retinal vessel loss and suppressed pathological angiogenesis in vivo. cZNF609 silencing increased endothelial cell migration and tube formation, and protected endothelial cell against oxidative stress and hypoxia stress in vitro. By contrast, transgenic overexpression of cZNF609 showed an opposite effects. cZNF609 acted as an endogenous miR-615-5p sponge to sequester and inhibit miR-615-5p activity, which led to increased MEF2A expression. MEF2A overexpression could rescue cZNF609 silencing-mediated effects on endothelial cell migration, tube formation, and apoptosis. Moreover, dysregulated cZNF609 expression was detected in the clinical samples of the patients with diabetes, hypertension, and coronary artery disease. Intervention of cZNF609 expression is promising therapy for vascular dysfunction.
Assuntos
Retinopatia Diabética/prevenção & controle , Células Endoteliais/fisiologia , Inativação Gênica , RNA/antagonistas & inibidores , Retina/fisiologia , Animais , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/prevenção & controle , RNA CircularRESUMO
Vascular remodeling is an important pathological feature of hypertension, leading to increased vascular resistance and reduced compliance. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of EC and VSMC function. Herein, we determined whether long noncoding RNA-growth arrest-specific 5 (GAS5) is involved in hypertension-related vascular remodeling. We revealed that GAS5 knockdown aggravated hypertension-induced microvascular dysfunction as shown by increased retinal neovascularization and capillary leakage. GAS5 regulated the remodeling of arteries, including caudal arteries, carotid arteries, renal arteries, and thoracic arteries. GAS5 was mainly expressed in ECs and VSMCs, and its expression was significantly downregulated in hypertension. GAS5 knockdown affected endothelial activation, endothelial proliferation, VSMC phenotypic conversion, and EC-VSMC communication in vivo and in vitro. Mechanistically, GAS5 regulated EC and VSMC function through ß-catenin signaling. This study identified GAS5 as a critical regulator in hypertension and demonstrated the potential of gene therapy and drug development for treating hypertension.
Assuntos
Hipertensão/fisiopatologia , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno/genética , Remodelação Vascular/genética , beta Catenina/metabolismo , Animais , Proliferação de Células/genética , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Hipertensão/genética , Distribuição Aleatória , Ratos , Ratos Endogâmicos SHR , Sensibilidade e Especificidade , Transdução de Sinais , Estatísticas não Paramétricas , TransfecçãoRESUMO
The continuous activation of the mitogen-activated protein kinase signaling cascade, typified by the BRAFV600E mutation, is one of the key alterations in melanoma. Accordingly, two BRAF inhibitors (BRAFi), vemurafenib and dabrafenib are utilized to treat melanoma and resulted in an excellent clinical outcome. However, the clinical success is not long-lasting, and the BRAFi resistance and disease progression inevitably occurs in nearly all patients. Endoplasmic reticulum stress-induced unfolded protein response and autophagy have emerged as potential pro-survival mechanisms adopted by melanoma cells in response to BRAFi. In this review, we discuss the role of unfolded protein response and autophagy that are implicated in the development of BRAFi-resistant melanoma and the corresponding strategy aiming at overcoming the intractable clinical problem.
Assuntos
Autofagia , Resistencia a Medicamentos Antineoplásicos , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Progressão da Doença , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Humanos , Imidazóis/uso terapêutico , Indóis/uso terapêutico , Sistema de Sinalização das MAP Quinases , Mutação , Oximas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/uso terapêutico , Resultado do Tratamento , VemurafenibRESUMO
Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.
Assuntos
Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Fosfolipases A2 do Grupo IV/metabolismo , Neoplasias da Próstata/patologia , Animais , Benzoatos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Xenoenxertos , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/enzimologia , Sulfonamidas/farmacologiaRESUMO
p27(Kip1) is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27(Kip1) allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27(Kip1) protein levels, in particular nuclear expressed p27(Kip1), is implicated in both disease progression and poor prognosis in a variety of cancers. p27(Kip1) expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. However, regulation of p27(Kip1) protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27(Kip1) at Thr(187) and Ser(10) is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser(10) phosphorylated p27(Kip1) is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27(Kip1) in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27(Kip1) localization and/or block its degradation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Progressão da Doença , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Haploinsufficient inactivating phosphatase and tensin homolog (Pten) mutations cause Cowden syndrome, an autosomal dominant risk genotype for hormone dependent reproductive cancers. As androgen actions mediated via the androgen receptor (AR) supports uterine growth and may modify uterine cancer risk, we hypothesized that a functional AR may increase PTEN inactivation induced uterine cancer. To test the hypothesis, we compared the PTEN knockout (PTENKO) induced uterine pathology in heterozygous PTENKO and combined heterozygous PTEN and complete AR knockout (PTENARKO) female mice. PTENKO induced uterine pathology was significantly reduced by AR inactivation with severe macroscopic uterine pathology present in 21% of PTENARKO vs 46% of PTENKO at a median age of 45 weeks. This could be due to reduced stroma ERα expression in PTENARKO compared to PTENKO uterus, while AR inactivation did not modify PTEN or P-AKT levels. Unexpectedly, while progesterone (P4) is assumed protective in uterine cancers, serum P4 was significantly higher in PTENKO females compared to WT, ARKO, and PTENARKO females consistent with more corpora lutea in PTENKO ovaries. Serum testosterone and ovarian estradiol were similar between all females. Hence, our results demonstrated AR inactivation mediated protection against PTENKO induced uterine pathology and suggests a potential role for antiandrogens in uterine cancer prevention and treatment.
Assuntos
Androgênios/farmacologia , PTEN Fosfo-Hidrolase/fisiologia , Receptores Androgênicos/fisiologia , Neoplasias Uterinas/etiologia , Animais , Feminino , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
Aberrant increase in protein kinase B (AKT) phosphorylation (pAKT), due to a gain-of-function mutation of phosphatidylinositol-3-kinase (PI3K) or loss-of-function mutation or deletion of phosphatase and tensin homolog (PTEN), is a common alteration in prostate cancer and associated with poor prognosis. Cytosolic phospholipase A2α (cPLA2α) is a lipid modifying enzyme by catalyzing the hydrolysis of arachidonic acid from membrane phospholipid. The released arachidonic acid and its metabolites contribute to survival and proliferation of prostate cancer cells. In this mini-review, we summarize the relationship between pAKT and cPLA2α in prostate cancer cells. There was a concordant increase in pAKT and cPLA2α levels in prostate tissue of prostate epithelial-specific PTEN-knockout mice compared to PTEN-wild type mice. Restoration of PTEN expression or inhibition of PI3K action decreased cPLA2α expression in PTEN-mutated or deleted prostate cancer cells. An increase in AKT phosphorylation elevated, whereas inhibition of AKT phosphorylation diminished, cPLA2α protein levels. pAKT had no influence on cPLA2α expression at mRNA levels but stabilized cPLA2α at protein levels by protecting it from degradation. Conversely, an induction of cPLA2α expression led to an increase in pAKT levels in PTEN-mutated or deleted prostate cancer cells, while silencing of cPLA2α expression or pharmacological blocking cPLA2α action decreased pAKT levels. The diminishment of pAKT by either genetic silencing or pharmacological blocking of cPLA2α was mitigated by the addition of arachidonic acid. The stimulatory effect of arachidonic acid on pAKT levels was lessened by inhibiting the production of arachidonic acid metabolites. These studies have revealed a link between oncogenic pathway and lipid metabolism and provided potential molecular targets for treating prostate cancer.
Assuntos
Fosfolipases A2 do Grupo IV/biossíntese , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , Transdução de Sinais/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Fosfolipases A2 do Grupo IV/genética , Humanos , Masculino , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The activation of RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade by v-raf murine sarcoma viral oncogene homolog B1 (BRAF)(V600E) mutation is a key alteration in melanoma. Although BRAF inhibitor (BRAFi) has achieved remarkable clinical success, the positive response to BRAFi is not sustainable, and the initial clinical benefit is eventually barred by the development of resistance to BRAFi. There is growing evidence to suggest that endoplasmic reticulum (ER) stress-induced autophagy could be a potential pro-survival mechanism that contributes to genesis of melanoma and to the resistance to BRAFi. ER stress-induced autophagy is an evolutionarily conserved membrane process. By degrading and recycling proteins and organelles via the formation of autophagous vesicles and their fusion with lysosomes, the autophagy plays a key role in homeostasis as well as pathological processes. In this review, we examine the autophagy phenomenon in melanocytic nevus, primary and metastatic melanoma, and its significance in BRAFi-resistant melanoma.