RESUMO
Fatty acid synthase (FAS) is a central enzyme in lipogenesis and transcriptionally activated in response to feeding and insulin signaling. The transcription factor USF is required for the activation of FAS transcription, and we show here that USF phosphorylation by DNA-PK, which is dephosphorylated by PP1 in response to feeding, triggers a switch-like mechanism. Under fasting conditions, USF-1 is deacetylated by HDAC9, causing promoter inactivation. In contrast, feeding induces the recruitment of DNA-PK to USF-1 and its phosphorylation, which then allows recruitment of P/CAF, resulting in USF-1 acetylation and FAS promoter activation. DNA break/repair components associated with USF induce transient DNA breaks during FAS activation. In DNA-PK-deficient SCID mice, feeding-induced USF-1 phosphorylation/acetylation, DNA breaks, and FAS activation leading to lipogenesis are impaired, resulting in decreased triglyceride levels. Our study demonstrates that a kinase central to the DNA damage response mediates metabolic gene activation.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Insulina/metabolismo , Acetilação , Animais , Linhagem Celular Tumoral , Ingestão de Alimentos/fisiologia , Jejum/metabolismo , Ácido Graxo Sintases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos SCID , Fosforilação , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fatores Estimuladores Upstream/metabolismoRESUMO
BACKGROUND: More than half of the colorectal cancer (CRC) patients will develop liver metastasis that underlies the cancer mortality. In the hepatic tumor microenvironment, the interplay between CRC cells and hepatic stellate cells (HSCs), and the activation of HSCs to become carcinoma-associated fibroblasts (CAFs) will further promote the cancer development. Nevertheless, the critical signaling molecule that involved in these processes remains unknown, which hinders the development of effective therapeutic agents for the treatment of metastatic CRC (mCRC). METHODS: Conditioned medium system and co-cultured system were used to examine the interplay between CRC cells and HSCs. Luminex liquid suspension chip detection and enzyme-linked immunosorbent assay were used to screen for the mediators in the conditioned medium that facilitated the CRC-HSCs interplay and HSCs-to-CAFs differentiation. Cell and animal models were used to examine whether brevilin A inhibited CRC liver metastasis via the VEGF-IL6-STAT3 axis. RESULTS: In the CRC-HSCs interplay, CRC promoted HSCs-to-CAFs differentiation by releasing vascular endothelial growth factor (VEGF); and HSCs released interleukin 6 (IL6) that activated signal transducer and activator of transcription 3 (STAT3) in the CRC and hence increased the cancer metastatic potential. The functions of the VEGF-IL6-STAT3 axis in the HSCs-CRC interplay were further validated by VEGF recombinant protein and IL6 neutralizing antibody. More importantly, brevilin A, an active compound isolated from Centipeda minima (L.) A. Br. et Aschers, targeted the VEGF-IL6-STAT3 axis in the CRC-HSCs interplay, hence significantly inhibited colorectal liver metastasis and cancer growth both in vitro and in vivo. CONCLUSIONS: We are the first to demonstrate brevilin A possesses potent anti-mCRC effect by targeting the VEGF-IL6-STAT3 axis in the CRC-HSCs interplay. Our findings not only support the development of brevilin A as a novel therapeutic agent for mCRC treatment, but also pave the path for the development of other VEGF-IL6-STAT3 targeting therapeutic strategies.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Hepáticas , Neoplasias Retais , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Interleucina-6/metabolismo , Células Estreladas do Fígado/metabolismo , Fator de Transcrição STAT3/metabolismo , Meios de Cultivo Condicionados , Neoplasias Hepáticas/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as well as PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.
Assuntos
Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/fisiologia , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Transativadores/fisiologia , Adipogenia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/citologia , Animais , Resposta ao Choque Frio , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Canais Iônicos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Desenvolvimento Muscular , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Termogênese , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteína Desacopladora 1RESUMO
Our previous study found that the combination of halofuginone (HF) and artemisinin (ATS) synergistically arrest colorectal cancer (CRC) cells at the G1/G0 phase of the cell cycle; however, it remains unclear whether HF-ATS induces cell death. Here we report that HF-ATS synergistically induced caspase-dependent apoptosis in CRC cells. Specifically, both in vitro and in vivo experiments showed that HF or HF-ATS induces apoptosis via activation of caspase-9 and caspase-8 while only caspase-9 is involved in ATS-induced apoptosis. Furthermore, we found HF or HF-ATS induces autophagy; ATS can't induce autophagy until caspase-9 is blocked. Further analyzing the crosstalk between autophagic and caspase activation in CRC cells, we found autophagy is essential for activation of caspase-8, and ATS switches to activate capase-8 via induction of autophagy when caspase-9 is inhibited. When apoptosis is totally blocked, HF-ATS switches to induce autophagic cell death. This scenario was then confirmed in studies of chemoresistance CRC cells with defective apoptosis. Our results indicate that HF-ATS induces cell death via interaction between apoptosis and autophagy in CRC cells. These results highlight the value of continued investigation into the potential use of this combination in cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Neoplasias Colorretais/patologia , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Artemisininas/uso terapêutico , Autofagia/efeitos dos fármacos , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Piperidinas/uso terapêutico , Quinazolinonas/uso terapêutico , Receptor Cross-TalkRESUMO
Breast cancer (BC) is one of the most common cancers diagnosed and the leading cause of cancer-related death in women. Although there are first-line treatments for BC, drug resistances and adverse events have been reported. Given the incidence of BC keeps increasing, seeking novel therapeutics is urgently needed. Fucoxanthin (Fx) is a dietary carotenoid commonly found in seaweeds and diatoms. Both in vitro and in vivo studies show that Fx and its deacetylated metabolite fucoxanthinol (Fxol) inhibit and prevent BC growth. The NF-κB signaling pathway is considered the major pathway contributing to the anti-proliferation, anti-angiogenesis and pro-apoptotic effects of Fx and Fxol. Other signaling molecules such as MAPK, MMP2/9, CYP and ROS are also involved in the anti-cancer effects by regulating the tumor microenvironment, cancer metastasis, carcinogen metabolism and oxidation. Besides, Fx also possesses anti-obesity effects by regulating UCP1 levels and lipid metabolism, which may help to reduce BC risk. More importantly, mounting evidence demonstrates that Fx overcomes drug resistance. This review aims to give an updated summary of the anti-cancer effects of Fx and summarize the underlying mechanisms of action, which will provide novel strategies for the development of Fx as an anti-cancer therapeutic agent.
Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , NF-kappa B/metabolismo , Transdução de Sinais , Microambiente Tumoral , Xantofilas/farmacologia , Xantofilas/uso terapêuticoRESUMO
Resveratrol is well known to exhibit vascular relaxant and antihypertensive effects. In this study, we determined the effects of resveratrol on the modulation of cytosolic [Ca] level and adenosine 5'-triphosphate-induced Ca release from the sarcoplasmic reticulum (SR) in rat aortic smooth muscle cells (ASMCs) and explored its underlying mechanisms. In this article, cytosolic [Ca] and SR [Ca] in ASMCs were determined by Fluo-4/acetoxymethyl and Mag-Fluo-4/acetoxymethyl respectively. Resveratrol (20, 50, and 100 µM) caused a rapid and substantial reduction in cytosolic [Ca] in ASMCs bathed in normal Hank's Balanced Salt Solution or Ca-free Hank's Balanced Salt Solution. Pretreatment with resveratrol reduced adenosine 5'-triphosphate-induced SR Ca release and SR Ca content. In the cells bathed in Na-free physiological saline, which favors the reverse mode of the Na-Ca exchanger (NCX), resveratrol induced an increase in cytosolic [Ca] and SR [Ca]. However, its effect on cytosolic [Ca] was inhibited by the selective NCX inhibitor, SEA0400. Our findings suggest that resveratrol reduces cytosolic [Ca] and SR [Ca] in ASMCs in normal physiological saline, which might be, at least in part, mediated by the NCX.
Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Resveratrol/farmacologia , Trocador de Sódio e Cálcio/agonistas , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Regulação para Baixo , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos Sprague-Dawley , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
BACKGROUND: The effects of short-term high fat diets on physiology are elusive and the molecular changes following fat overconsumption remain largely unknown. In this study, we aimed to evaluate exercise capacity in mice fed with a high fat diet (HFD) for 3 days and investigate the molecular mechanisms in the early response to high-fat feeding. METHODS: Exercise capacity was assessed by weight-loaded swimming test in mice fed a control diet (10 kcal% fat) or a HFD (60 kcal% fat) for 3 days. Global gene expression of ten important tissues (brain, heart, liver, spleen, lung, kidney, stomach, duodenum, skeletal muscle and blood) was analyzed using RNA Sequencing. RESULTS: A HFD for just 3 days can induce 71% decrease of exercise performance prior to substantial weight gain (P <0.01). Principle component analysis revealed that differential gene expression patterns existed in the ten tissues. Out of which, the brain, spleen and lung were demonstrated to have more pronounced transcriptional changes than other tissues. Biological process analysis for differentially expressed genes in the brain, spleen and lung showed that dysregulation of peripheral and central immune response had been implicated in the early stage of HFD exposure. Neurotransmission related genes and circulatory system process related genes were significantly down-regulated in the brain and lung, respectively. CONCLUSIONS: Our findings provide new insights for the deleterious effects of high-fat feeding, especially revealing that the lung maybe as a new important target attacked by short-term high-fat feeding.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Condicionamento Físico Animal/fisiologia , Transcriptoma , Animais , Sangue/metabolismo , Peso Corporal , Encéfalo/fisiologia , Pulmão/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Baço/fisiologiaRESUMO
Oxidative stress-induced myocardial apoptosis is a key step in the pathogenesis of ischemic heart disease. Calycosin is a phytoestrogen extracted from Radix astragali. In this study, we examined the effects and mechanisms of calycosin on oxidative stress-induced myocardial apoptosis. Molecular docking showed that calycosin can couple into binding site of ERα and ß. Pretreatment with calycosin increased the expression levels of ERα and ß. In H9C2 cells, H2O2 reduced cell viability and induced apoptosis, however, calycosin diminished the effects of H2O2 in a dose-dependent manner. Pretreatment with ICI 182,780, an estrogen receptor inhibitor, negated the protective effect of calycosin against H2O2-induced apoptosis. In addition, Akt phosphorylation was upregulated by calycosin mono treatment and downregulated by co-treatment with calycosin and ICI 182,780. These data demonstrated that calycosin exhibits anti-apoptotic effects by activating ERα/ß and enhancing Akt phosphorylation in cardiomyocytes.
Assuntos
Apoptose/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Isoflavonas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/química , Isoflavonas/química , Modelos Moleculares , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Although toxic, the Chinese medicinal herb Xanthii Fructus (XF) is commonly used to treat traditional Chinese medicine (TCM) symptoms that resemble cold, sinusitis and arthritis. According to TCM theory, stir-baking (a processing method) can reduce the toxicity and enhance the efficacy of XF. METHODS: Cytotoxicities of raw XF and processed XF (stir-baked XF, SBXF) were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in normal liver derived MIHA cells. Nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression were measured by the Griess reagent and quantitative real-time PCR, respectively. The chemical profiles of XF and SBXF were compared using an established ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method. RESULTS: SBXF was less toxic than XF in MIHA cells. Both XF and SBXF had anti-inflammatory effects as demonstrated by their abilities to reduce nitric oxide production as well as inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effects of SBXF were more potent than that of XF. By comparing the chemical profiles, we found that seven peaks were lower, while nine other peaks were higher in SBXF than in XF. Eleven compounds including carboxyatractyloside, atractyloside and chlorogenic acid corresponding to eleven individual changed peaks were tentatively identified by matching with empirical molecular formulae and mass fragments, as well as literature data. CONCLUSION: Our study showed that stir-baking significantly reduced the cytotoxicity and enhanced the anti-inflammatory effects of XF; moreover, with a developed ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry method we differentiated XF and SBXF by their chemical profiles. Further studies are warranted to establish the relationship between the alteration of chemical profiles and the changes of medicinal properties caused by stir-baking.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Xanthium/química , Animais , Anti-Inflamatórios não Esteroides/química , Antineoplásicos Fitogênicos/química , Culinária , Citotoxinas/química , Citotoxinas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/química , Frutas/química , Humanos , Lipopolissacarídeos , Camundongos , Células Tumorais CultivadasRESUMO
BACKGROUND: Melanoma is notorious for its propensity to metastasize, which makes treatment extremely difficult. Receptor tyrosine kinase c-Met is activated in human melanoma and is involved in melanoma progression and metastasis. Hepatocyte growth factor (HGF)-mediated activation of c-Met signaling has been suggested as a therapeutic target for melanoma metastasis. Quercetin is a dietary flavonoid that exerts anti-metastatic effect in various types of cancer including melanoma. In a previous report, we demonstrated that quercetin inhibited melanoma cell migration and invasion in vitro, and prevented melanoma cell lung metastasis in vivo. In this study, we sought to determine the involvement of HGF/c-Met signaling in the anti-metastatic action of quercetin in melanoma. METHODS: Transwell chamber assay was conducted to determine the cell migratory and invasive abilities. Western blotting was performed to determine the expression levels and activities of c-Met and its downstream molecules. And immunoblotting was performed in BS(3) cross-linked cells to examine the homo-dimerization of c-Met. Quantitative real-time PCR analysis was carried out to evaluate the mRNA expression level of HGF. Transient transfection was used to overexpress PAK or FAK in cell models. Student's t-test was used in analyzing differences between two groups. RESULTS: Quercetin dose-dependently suppressed HGF-stimulated melanoma cell migration and invasion. Further study indicated that quercetin inhibited c-Met phosphorylation, reduced c-Met homo-dimerization and decreased c-Met protein expression. The effect of quercetin on c-Met expression was associated with a reduced expression of fatty acid synthase. In addition, quercetin suppressed the phosphorylation of c-Met downstream molecules including Gab1 (GRB2-associated-binding protein 1), FAK (Focal Adhesion Kinase) and PAK (p21-activated kinases). More importantly, overexpression of FAK or PAK significantly reduced the inhibitory effect of quercetin on the migration of the melanoma cells. CONCLUSIONS: Our findings suggest that suppression of the HGF/c-Met signaling pathway contributes to the anti-metastatic action of quercetin in melanoma.
Assuntos
Movimento Celular/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Quercetina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Quinases Ativadas por p21/metabolismoRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis and kills cancer cells but not normal cells. However, TRAIL resistance due to low level of TRAIL receptor expression is widely found in cancer cells and hampers its development for cancer treatment. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are urgently needed. We investigated whether tanshinones, the major bioactive compounds of Salvia miltiorrhiza (danshen), can up-regulate TRAIL receptor expression. Among the major tanshinones being tested, cryptotanshinone (CT) showed the best ability to induce TRAIL receptor 2 (DR5) expression. We further showed that CT was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. CT-induced DR5 induction was not cell type-specific, as DR5 induction was observed in other cancer cell types. DR5 knockdown abolished the enhancing effect of CT on TRAIL responses. Mechanistically, induction of the DR5 by CT was found to be p53-independent but dependent on the induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). Knockdown of CHOP abolished CT-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, CT-induced ROS production preceded up-regulation of CHOP and DR5 and consequent sensitization of cells to TRAIL. Interestingly, CT also converted TRAIL-resistant lung A549 cancer cells into TRAIL-sensitive cells. Taken together, our results indicate that CT can potentiate TRAIL-induced apoptosis through up-regulation of DR5.
Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Fenantrenos/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salvia miltiorrhiza/química , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Colorectal cancer (CRC) is the third most common cause of cancer-related morbidity worldwide, with an estimated of 1.85 million new cases and 850,000 deaths every year. Nevertheless, the current treatment regimens for CRC have many disadvantages, including toxicities and off-targeted side effects. STAT3 (signal transducer and activator of transcription 3) has been considered as a promising molecular target for CRC therapy. Brevilin A, a sesquiterpene lactone compound rich in Centipedae Herba has potent anticancer effects in nasopharyngeal, prostate and breast cancer cells by inhibiting the STAT3 signaling. However, the anti-CRC effect of brevilin A and the underlying mechanism of action have not been fully elucidated. In this study, we aimed to investigate the involvement of STAT3 signaling in the anti-CRC action of brevilin A. Here, HCT-116 and CT26 cell models were used to investigate the anti-CRC effects of brevilin A in vitro. HCT-116 cells overespressing with STAT3 were used to evaluate the involvement of STAT3 signaling in the anti-CRC effect of brevilin A. Screening of 49 phosphorylated tyrosine kinases in the HCT-116 cells after brevilin A treatment was performed by using the human phospho-receptor tyrosine kinase (phospho-RTK) array. Results showed that brevilin A inhibited cell proliferation and cell viability, induced apoptosis, reduced cell migration and invasion, inhibited angiogenesis, lowered the protein expression levels of phospho-Src (Tyr416), phospho-JAK2 (Y1007/1008) and phospho-STAT3 (Tyr705), and inhibited STAT3 activation and nuclear localization. Brevilin A also significantly reduced the protein expression levels of STAT3 target genes, such as MMP-2, VEGF and Bcl-xL. More importantly, over-activation of STAT3 diminished brevilin A's effects on cell viability. All these results suggest that brevilin A exerts potent anti-CRC effects, at least in part, by inhibiting STAT3 signaling. Our findings provide a strong pharmacological basis for the future exploration and development of brevilin A as a novel STAT3-targeting phytotherapeutic agent for CRC treatment.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a common difficult disease with a high disability rate. Siegesbeckia orientalis L. (SO), a Chinese medicinal herb that is commonly used for treating RA in clinical practice. While, the anti-RA effect and the mechanisms of action of SO, as well as its active compound(s) have not been elucidated clearly. AIM OF THE STUDY: We aim to explore the molecular mechanism of SO against RA by using network pharmacology analysis, as well as the in vitro and in vivo experimental validations, and to explore the potential bioactive compound(s) in SO. METHODS: Network pharmacology is an advanced technology that provides us an efficient way to study the therapeutic actions of herbs with the underlying mechanisms of action delineated. Here, we used this approach to explore the anti-RA effects of SO, and then the molecular biological approaches were used to verify the prediction. We first established a drug-ingredient-target-disease network and a protein-protein interaction (PPI) network of SO-related RA targets, followed by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Further, we used lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and vascular endothelial growth factor-A (VEGFA)-induced human umbilical vein endothelial cell (HUVEC) models, as well as adjuvant-induced arthritis (AIA) rat model to validate the anti-RA effects of SO. The chemical profile of SO was also determined by using the UHPLC-TOF-MS/MS analysis. RESULTS: Network pharmacology analysis highlighted inflammatory- and angiogenesis-related signaling pathways as promising pathways that mediate the anti-RA effects of SO. Further, in both in vivo and in vitro models, we found that the anti-RA effect of SO is at least partially due to the inhibition of toll like receptor 4 (TLR4) signaling. Molecular docking analysis revealed that luteolin, an active compound in SO, shows the highest degree of connections in compound-target network; moreover, it has a direct binding to the TLR4/MD-2 complex, which is confirmed in cell models. Besides, more than forty compounds including luteolin, darutoside and kaempferol corresponding to their individual peaks were identified tentatively via matching with the empirical molecular formulae and their mass fragments. CONCLUSION: We found that SO and its active compound luteolin exhibit anti-RA activities and potently inhibit TLR4 signaling both in vitro and in vivo. These findings not only indicate the advantage of network pharmacology in the discovery of herb-based therapeutics for treating diseases, but also suggest that SO and its active compound(s) could be developed as potential anti-RA therapeutic drugs.
Assuntos
Artrite Reumatoide , Asteraceae , Medicamentos de Ervas Chinesas , Humanos , Animais , Ratos , Simulação de Acoplamento Molecular , Luteolina/farmacologia , Luteolina/uso terapêutico , Sigesbeckia , Receptor 4 Toll-Like , Fator A de Crescimento do Endotélio Vascular , Farmacologia em Rede , Espectrometria de Massas em Tandem , Artrite Reumatoide/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêuticoRESUMO
cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 µm 8-Br-cAMP, a cell-permeable analog of cAMP, elicited [Ca(2+)](i) increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced [Ca(2+)](i) increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.
Assuntos
AMP Cíclico/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células Mesangiais/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Canais de Cátion TRPC/metabolismo , Substituição de Aminoácidos , Animais , Cálcio/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/genética , Inibidores Enzimáticos/farmacologia , Glucagon/farmacologia , Células HEK293 , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Células Mesangiais/citologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Mutação de Sentido Incorreto , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
Acidosis, regardless of hypoxia involvement, is recognized as a chronic and harsh tumor microenvironment (TME) that educates malignant cells to thrive and metastasize. Although overwhelming evidence supports an acidic environment as a driver or ubiquitous hallmark of cancer progression, the unrevealed core mechanisms underlying the direct effect of acidification on tumorigenesis have hindered the discovery of novel therapeutic targets and clinical therapy. Here, chemical-induced and transgenic mouse models for colon, liver and lung cancer were established, respectively. miR-7 and TGF-ß2 expressions were examined in clinical tissues (n = 184). RNA-seq, miRNA-seq, proteomics, biosynthesis analyses and functional studies were performed to validate the mechanisms involved in the acidic TME-induced lung cancer metastasis. Our data show that lung cancer is sensitive to the increased acidification of TME, and acidic TME-induced lung cancer metastasis via inhibition of miR-7-5p. TGF-ß2 is a direct target of miR-7-5p. The reduced expression of miR-7-5p subsequently increases the expression of TGF-ß2 which enhances the metastatic potential of the lung cancer. Indeed, overexpression of miR-7-5p reduces the acidic pH-enhanced lung cancer metastasis. Furthermore, the human lung tumor samples also show a reduced miR-7-5p expression but an elevated level of activated TGF-ß2; the expressions of both miR-7-5p and TGF-ß2 are correlated with patients' survival. We are the first to identify the role of the miR-7/TGF-ß2 axis in acidic pH-enhanced lung cancer metastasis. Our study not only delineates how acidification directly affects tumorigenesis, but also suggests miR-7 is a novel reliable biomarker for acidic TME and a novel therapeutic target for non-small cell lung cancer (NSCLC) treatment. Our study opens an avenue to explore the pH-sensitive subcellular components as novel therapeutic targets for cancer treatment.
RESUMO
TRPC1 (transient receptor potential canonical 1) is a Ca(2+)-permeable cation channel involved in diverse physiological function. TRPC1 may associate with other proteins to form a signaling complex, which is crucial for channel function. In the present study, we investigated the interaction between TRPC1 and large conductance Ca(2+)-sensitive K(+) channel (BK(Ca)). With the use of potentiometric fluorescence dye DiBAC(4)(3), we found that store-operated Ca(2+) influx resulted in membrane hyperpolarization of vascular smooth muscle cells (VSMCs). The hyperpolarization was inhibited by an anti-TRPC1 blocking antibody T1E3 and 2 BK(Ca) channel blockers, charybdotoxin and iberiotoxin. These data were confirmed by sharp microelectrode measurement of membrane potential in VSMCs of intact arteries. Furthermore, T1E3 treatment markedly enhanced the membrane depolarization and contraction of VSMCs in response to several contractile agonists including phenylephrine, endothelin-1, and U-46619. In coimmunoprecipitation experiments, an antibody against BK(Ca) alpha-subunit [BK(Ca)(alpha)] could pull down TRPC1, and moreover an anti-TRPC1 antibody could reciprocally pull down BK(Ca)(alpha). Double-labeling immunocytochemistry showed that TRPC1 and BK(Ca) were colocalized in the same subcellular regions, mainly on the plasma membrane, in VSMCs. These data suggest that, TRPC1 physically associates with BK(Ca) in VSMCs and that Ca(2+) influx through TRPC1 activates BK(Ca) to induce membrane hyperpolarization. The hyperpolarizing effect of TRPC1-BK(Ca) coupling could serve to reduce agonist-induced membrane depolarization, thereby preventing excessive contraction of VSMCs to contractile agonists.
Assuntos
Sinalização do Cálcio , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Barbitúricos , Membrana Celular/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Isoxazóis , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Masculino , Potenciais da Membrana , Microeletrodos , Microscopia Confocal , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPC/genética , Transfecção , Vasoconstrição , Vasoconstritores/farmacologiaRESUMO
BACKGROUND/AIMS: ATP can activate several Ca(2+) influx channels in vascular endothelial cells. For example, it stimulates TRPC channels via capacitative and noncapacitative Ca(2+) entry (CCE and non-CCE, respectively) mechanisms; it also directly acts on P2X purinoceptors, resulting in Ca(2+) influx. In the present study, we tested the hypothesis that cyclic nucleotide-gated (CNG) channels also contribute to ATP-induced non-CCE. METHODS: Two selective inhibitors of CNG channels, L-cis-diltiazem and LY-83583, and CNGA2-specific siRNA were used to study the involvement of CNGA2 in ATP-induced non-CCE in endothelial cells. Ca(2+) influx was studied using Ca(2+)-sensitive fluorescence dyes Fluo-3 and Fluo-4. RESULTS/CONCLUSION: L-cis-diltiazem and LY-83583 markedly reduced ATP-induced non-CCE in 3 types of endothelial cells including the H5V endothelial cell line, the primary cultured bovine aortic endothelial cells and the endothelial cells within isolated mouse aortic strips. The CNGA2-specific siRNA also reduced the ATP-induced non-CCE in H5V endothelial cells. The Ca(2+) influx was inhibited by Rp-8-CPT-cAMPS, MDL-12330A, SQ-22536 and MRS-2179, but not by ODQ or NF-157. Taken together, the present study demonstrated that CNGA2 channels contribute to ATP-induced non-CCE in vascular endothelial cells. It is likely that ATP acts through P2Y(1)receptors and adenylyl cyclases to stimulate CNGA2.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Células Endoteliais/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Aminoquinolinas/farmacologia , Compostos de Anilina , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Bovinos , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Diltiazem/farmacologia , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Iminas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Antagonistas do Receptor Purinérgico P2 , Interferência de RNA , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1 , Tionucleotídeos/farmacologia , Fatores de Tempo , Transfecção , Vasodilatação , XantenosRESUMO
Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. The pathogenesis of NAFLD is complex. Frontline western medicines only ameliorate the symptoms of NAFLD. On the contrary, the uniqueness of Chinese medicine in its interpretation of NAFLD and the holistic therapeutic approach lead to a promising therapeutic efficacy. Recent studies reveal that the gut-liver axis and adipose tissue-liver axis play important roles in the development of NAFLD. Interestingly, with advanced technology, many herbal formulae are found to target the gut-liver axis and adipose tissue-liver axis and resolve the inflammation in NAFLD. This is the first review summarizes the current findings on the Chinese herbal formulae that target the two axes in NAFLD treatment. This review not only demonstrates how the ancient wisdom of Chinese medicine is being interpreted by modern pharmacological studies, but also provides valuable information for the further development of the herbal-based treatment for NAFLD.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Microbioma Gastrointestinal/efeitos dos fármacos , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologiaRESUMO
Heart, liver, and kidney, which are known as the essential organs for metabolism, possess the unique ability to regulate the proliferation function of the body against injury. Silibinin (SB), a natural polyphenolic flavonoid extracted from traditional herb Silybum marianum L., has been used to protect hepatocytes. Whether SB can regulate mitochondrial fission in normal cells and the underlying mechanisms remain unclear. Here, we showed that SB markedly promoted cell proliferation by facilitating G1/S transition via activating dynamin-related protein 1 (Drp1), which in turn mediated mitochondrial fission in these normal cells. SB dose-dependently increased the mitochondrial mass, mtDNA copy number, cellular adenosine triphosphate production, mitochondrial membrane potential, and reactive oxygen species in normal cells. Furthermore, SB dose-dependently increased the expression of Drp1. Blocking Drp1 abolished SB-induced mitochondrial fission. In conclusion, we demonstrate that SB promotes cell proliferation through facilitating G1/S transition by activating Drp1-mediated mitochondrial fission. This study suggests that SB is a potentially useful herbal derivative for the daily prevention of various diseases caused by impaired mitochondrial fission.
Assuntos
Dinaminas/metabolismo , Fase G1/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Fase S/efeitos dos fármacos , Silibina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Túbulos Renais/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
Cervical cancer is the fourth leading cancer type and the second most common gynecological malignancy among women worldwide. Silibinin (SB), a chief bioactive natural polyphenolic flavonoid of Silybum marianum L., has been used clinically for its hepatocyte protective effects. It also has anticancer effects via the induction of apoptosis and cell cycle arrest. However, the effects of SB on cervical cancer cells through mitochondrial fission have not been studied. Here, we showed that SB markedly suppressed cervical cell proliferation by inducing G2/M cell cycle arrest via the activation of dynamin-related protein 1 (Drp1), which in turn mediated the mitochondrial fission dysfunction both in vitro and in vivo. SB decreased the ATP content, mitochondrial membrane potential, and mtDNA copy number, as well as reduced the reactive oxygen species levels in cervical cells. Furthermore, SB induced excessive mitochondrial fragmentation and reduced tubule formation. Further study showed that knockdown of Drp1 abolished the SB-induced G2/M cell cycle arrest in cervical cancer cells by inhibiting the mitochondrial fission pathway. More importantly, SB inhibited Hela cell growth in vivo model. In conclusion, we are the first to demonstrate that SB induces cervical cancer cell G2/M cell cycle arrest by activating Drp1-dependent mitochondrial fission dysfunction. This study suggests the strategy of inducing Drp1-dependent mitochondrial fission for cervical cancer prevention and treatment.