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1.
Chem Biodivers ; 17(2): e1900473, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31961474

RESUMO

Veratrum plant contains a family of compounds called steroidal alkaloids which have been previously reported to cause DNA damage and blood pressure decrease in vivo. In this study, the antihypertensive effects and DNA damage in brain cells of 12 steroidal alkaloids separated from Veratrum plant were all evaluated to develop a relationship among chemical structure, antihypertensive activity and neurotoxicity by utilization of chemical principal component analysis (PCA) and hierarchical cluster analysis (HCA). Twelve steroidal alkaloids markedly reduced high blood pressure of hypertensive mice and also similarly induced varying degrees of DNA single-strand breaks in mouse cerebellum and cerebral cortex after oral administration. On the basis of the PCA and HCA results, it was suggested that the 3-carboxylic esters and benzene group play a core role in the DNA damage of brain cells, while more hydroxy groups in the A-ring and B-ring structure of jervine-type alkaloid led to stronger antihypertensive activity. The primary structure, activity and neurotoxicity relationship were discussed briefly.


Assuntos
Anti-Hipertensivos/química , Alcaloides de Veratrum/química , Veratrum/química , Administração Oral , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Análise por Conglomerados , Dano ao DNA/efeitos dos fármacos , Camundongos , Extratos Vegetais/química , Análise de Componente Principal , Relação Estrutura-Atividade , Veratrum/metabolismo , Alcaloides de Veratrum/farmacologia
2.
Mol Pharmacol ; 97(1): 23-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707356

RESUMO

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.


Assuntos
Sítios de Ligação/genética , Bioensaio/métodos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Compostos de Boro/química , Cinamatos/farmacologia , Descoberta de Drogas/métodos , Técnicas de Inativação de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Luciferases/química , Morfolinas/farmacologia , Nanoestruturas/química , Purinas/farmacologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
3.
Fitoterapia ; 137: 104281, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31381957

RESUMO

Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.


Assuntos
Alcaloides/química , Esteroides/química , Veratrum/química , Alcaloides/isolamento & purificação , Proteínas Hedgehog/antagonistas & inibidores , Idaho , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Componentes Aéreos da Planta/química , Rizoma/química , Estações do Ano , Esteroides/isolamento & purificação , Alcaloides de Veratrum
4.
Biomed Pharmacother ; 117: 109059, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31207578

RESUMO

Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing the anti-proliferation effect of doxorubicin (DOX). We demonstrated that the synergistic mechanism was related to the intracellular accumulation of DOX via modulating ABCB1 transportation. Jervine did not affect the expression of ABCB1 in mRNA nor protein levels. However, jervine increased the ATPase activity of ABCB1 and possibly served as a substrate of ABCB1. The molecular docking results indicated that jervine was bound to a closed ABCB1 conformation and blocked drug entrance to the central binding site at the transmembrane domain. The present study identifies jervine acts as a substrate of ABCB1, and has potential to be developed as a novel and potent chemotherapy sensitizer used for patients developing multidrug resistance.


Assuntos
Doxorrubicina/farmacologia , Teratogênios/toxicidade , Alcaloides de Veratrum/toxicidade , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Células MCF-7 , Estrutura Secundária de Proteína , Especificidade por Substrato/efeitos dos fármacos , Teratogênios/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia
5.
Nature ; 571(7764): 279-283, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31168089

RESUMO

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Oxisteróis/química , Receptor Smoothened/química , Receptor Smoothened/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Ligantes , Modelos Moleculares , Oxisteróis/metabolismo , Receptor Patched-1/metabolismo , Conformação Proteica , Transdução de Sinais , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
6.
J Enzyme Inhib Med Chem ; 34(1): 789-798, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30871382

RESUMO

In this study, we investigated whether jervine (J) could prevent gastrointestinal (GI) side effects of abdominopelvic radiotherapy (RT) in Wistar-Albino female rats. Rats were divided into five groups: control (C), J only (J), J administered at 5 mg/kg/days for 7 days, RT only (RT), J before RT (J + RT), J administered for seven days before RT, J both before and after RT (J + RT + J), and J administered for 7 days before RT and after RT for 3 days. The weights of rats were measured on the 1st, 7th, and 10th days of the study. Rats were sacrificed to obtain tissues from the liver and intestine, which was followed by taking blood samples intracardially. In addition, the tissues were stained with pyruvate dehydrogenase (PDH) immunohistochemically. In our study, J supplementation markedly reduced weight loss, and histopathological, immunohistochemical, biochemical results suggest that J had a protective effect on GI toxicity following RT.


Assuntos
Fármacos Gastrointestinais/uso terapêutico , Lesões por Radiação/patologia , Lesões por Radiação/prevenção & controle , Alcaloides de Veratrum/uso terapêutico , Animais , Fármacos Gastrointestinais/química , Fármacos Gastrointestinais/farmacologia , Ratos , Ratos Wistar , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia
7.
Med Sci Monit ; 25: 1518-1525, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30807555

RESUMO

BACKGROUND Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth. MATERIAL AND METHODS Gli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression. RESULTS Gli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P<0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h (F=214.57), 24 h (F=76.832), 48 h (F=236.90), and 72 h (F=164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant (P<0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 µM, (13.37±1.42)% at 5.0 µM, (22.3±2.92)% at 10.0 µM, and (33.57±1.75)% at 20.0 µM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent. CONCLUSIONS Gli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.


Assuntos
Alcaloides de Veratrum/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , China , Progressão da Doença , Regulação para Baixo , Neoplasias Esofágicas/tratamento farmacológico , Feminino , Humanos , Metástase Linfática , Masculino , Recidiva Local de Neoplasia/patologia , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismo
8.
Biomed Chromatogr ; 33(9): e4518, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30805953

RESUMO

The prominent stromal compartment surrounds pancreatic ductal adenocarcinoma and protects the tumor cells from chemo- or radiotherapy. We hypothesized that our nano formulation carrying cyclopamine (CPA, stroma modulator) and paclitaxel (PTX, antitumor agent) could increase the permeation of PTX through the stromal compartment and improve the intratumoral delivery of PTX. In the present study a sensitive, reliable UPLC-MS/MS method was developed and validated to quantify PTX and CPA simultaneously in mouse whole blood, pancreas, liver and spleen samples. Docetaxel was used as the internal standard. The method demonstrated a linear range of 0.5-2000 ng/mL for whole blood and tissue homogenates for both PTX and CPA. The accuracy and precision of the assay were all within ±15%. Matrix effects for both analytes were within 15%. Recoveries from whole blood, liver, spleen and pancreas homogenates were 92.7-105.2% for PTX and 72.8-99.7% for CPA. The stability was within ±15% in all test biomatrices. The validated method met the acceptance criteria according to US Food and Drug Administration regulatory guidelines. The method was successfully applied to support a pharmacokinetic and biodistribution study for PTX and CPA in mice biomatrices.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Paclitaxel/análise , Espectrometria de Massas em Tandem/métodos , Alcaloides de Veratrum/análise , Animais , Limite de Detecção , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Paclitaxel/química , Paclitaxel/farmacocinética , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Reprodutibilidade dos Testes , Distribuição Tecidual , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacocinética , Alcaloides de Veratrum/uso terapêutico
9.
Phytomedicine ; 55: 191-199, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668429

RESUMO

BACKGROUND: Veratrum, hellebore is an important plant species of the Liliaceae family and jervine is the characteristic steroidal alkaloid constituent of Veratrum album. PURPOSE: In the current study, anti-inflammatory and antioxidant effects of jervine isolated from NH4OH-benzene extract of V. album rhizomes were investigated on CAR induced paw edema in rats. METHODS/STUDY DESIGN: In inflammatory study, 50, 100, 200 and 400  mg/kg doses of jervine, 25  mg/kg doses of DIC and IND were orally administered, and the volume of the foots were measured up to their knee arthrosis by plethismometer. After one hour of the oral administration of the all treatments, 0.1 ml of CAR solution (1%) was injected into the foot of the all rat groups and the volume of the foots were measured during 5 h after CAR injection. GPx, SOD, GR, MPO, CAT enzymes activities and GSH, LPO levels of the supernatants of paw homogenates and inflammation biomarkers such as TNF-α and IL-1ß in the rats serums were also estimated. RESULTS: According to the present results, jervine exerted 50.4-73.5% anti-inflammatory effects in carrageenan induced paw edema. Inflammation biomarkers such as TNF-α, IL-1ß and MPO that increased by CAR injection were suppressed by the administrations of all doses of jervine, IND and DIC. In all paw tissues, LPO levels as indicator of oxidative tissue damage were found to be high in CAR-treated group and it was found to be decreased in all doses of jervine. CONCLUSION: Jervine, DIC and IND reduced the negative effects of CAR due to increasing effects on the SOD, CAT, GSH, GPx and GR antioxidants.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Alcaloides de Veratrum/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Antioxidantes/metabolismo , Carragenina/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Enzimas/metabolismo , Inflamação/tratamento farmacológico , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Extratos Vegetais/química , Ratos Sprague-Dawley , Rizoma/química , Fator de Necrose Tumoral alfa/metabolismo , Veratrum/química , Alcaloides de Veratrum/administração & dosagem , Alcaloides de Veratrum/isolamento & purificação
10.
Basic Clin Pharmacol Toxicol ; 124(6): 660-669, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30548093

RESUMO

Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Proteínas Hedgehog/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Alcaloides de Veratrum/farmacologia , Animais , Isquemia Encefálica/patologia , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Camundongos Endogâmicos C57BL , Ocludina/metabolismo , Receptores Patched/metabolismo , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo , Acidente Vascular Cerebral/patologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
11.
Int J Mol Med ; 43(2): 830-838, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535481

RESUMO

The present study aimed to investigate the effects of astragaloside IV on osteoblast­like cell proliferation and migration, in addition to the underlying signaling pathway. In order to observe the effect on proliferation, a Cell Counting Kit­8 assay and flow cytometry were used. To detect cell migration ability, cell scratch and Transwell cell migration assays were performed. The RNA and protein expression levels of hedgehog signaling molecules, including Sonic hedgehog (SHH) and GLI family zinc finger 1 (GLI1), were examined by reverse transcription­quantitative polymerase chain reaction and western blot analyses. To inhibit the hedgehog signaling pathway, cyclopamine was used. Astragaloside IV, at a dosage of 1x10­2 µg/ml in MG­63 cells and 1x10­3 µg/ml in U­2OS cells, resulted in the enhanced proliferation and migration of cells, and the gene expression levels of the SHH and GLI1 were significantly increased. The combination of astragaloside IV and cyclopamine reduced MG­63 and U­2OS cell proliferation and migration, and inhibited the gene expression of SHH and GLI1. Astragaloside IV enhanced the proliferation and migration of human osteoblast­like cells through activating the hedgehog signaling pathway. The results of the present study provide a rational for the mechanistic link in astragaloside IV promoting the proliferation and migration of osteoblasts via the hedgehog signaling pathway.


Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Proteínas Hedgehog/metabolismo , Osteoblastos , Saponinas/farmacologia , Triterpenos/farmacologia , Linhagem Celular Tumoral , Antagonismo de Drogas , Humanos , Terapia de Alvo Molecular , Osseointegração/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo
12.
Biomed Chromatogr ; 33(1): e4377, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30187929

RESUMO

In this study, a sensitive hydrophilic interaction liquid chromatography (HILIC) method was developed to determine pseudojervine (PJV), veratrosine (VTS), jervine (JV), veratramine (VTM), veramarine (VA) and veratroylzygadenine (VTG) in rat plasma. Separations were carried out using LC-MS/MS with a Chrom Matrix HP amide column (5 m, 10 cm × 3.0 mm i.d.). The mobile phases were (A) 0.01 mm formic acid and (B) acetonitrile. Good linearity was found for all analytes (R2  > 0.995) in the concentration range from 5 to 1000 µg/L with LLOQ at 5 µg/L for VTM and VTS; and from 1 to 1000 µg/L with LLOQ at 1 µg/L for PJV, JV, VA and VTG. Accuracy of the assay varied from 90.5 to 108.1%. The extraction recovery and matrix effect of six analytes ranged from 72.2 to 95.5% and from 79.2 to 98.4%. According to the stability test, six analytes in rat plasma were stable during the analysis process. On the basis of validation of the assay, the pharmacokinetics of the six steroid alkaloids were investigated after oral administration of Lilu extracts to rats.


Assuntos
Cromatografia Líquida/métodos , Alcaloides de Veratrum/sangue , Alcaloides de Veratrum/farmacocinética , Animais , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Esteroides/sangue , Esteroides/química , Esteroides/farmacocinética , Alcaloides de Veratrum/química
13.
Colloids Surf B Biointerfaces ; 174: 467-475, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497008

RESUMO

Although layered double hydroxides (LDH) have been listed as promising nanomaterials in human healthcare, very little has been achieved on osteoblast inflammatory signaling. Thus, osteoblasts were challenged with two LDHs (Mg2Al-Cl and Zn2Al-Cl, at 0.002 mg/mL) up to 24 h, establishing an acute inflammatory mechanism, as well as identifying whether Sonic hedgehog (Shh) signaling has an influence. Functional experiments were performed by previously treating (2 h) semiconfluent osteoblast cultures with cyclopamine molecule (cyc), a widely used Shh inhibitor. Considering inflammasome complex, the asc1 gene was significantly up-expressed in response to Zn2Al-Cl - LDHs, as well as the nrlp3 gene. By treating the osteoblast with cyc, the asc1 gene presented an even higher profile. Our results found a down-modulation of major pro-inflammatory cytokines-related genes, when tnfα and il1ß were significantly down-modulated in response to LDHs. Conversely, anti-inflammatory cytokines were up-modulated considering the same experimental procedures. Except the il6, the other il13, il10, and tgfß genes were up modulated. Additionally, Shh signaling seems to modulate this repertory as both the il13 and il10 genes were significantly up-modulated when the Shh signaling was inhibited. Altogether, our results reveal for the first time the exigency of Shh-dependent anti-inflammatory signals in LDH-induced osteoblast responses.


Assuntos
Proteínas Hedgehog/metabolismo , Hidróxidos/farmacologia , Mediadores da Inflamação/metabolismo , Inflamação/imunologia , Osteoblastos/imunologia , Alcaloides de Veratrum/farmacologia , Diferenciação Celular , Células Cultivadas , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Hidróxidos/química , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Alcaloides de Veratrum/química
14.
Toxicol Appl Pharmacol ; 364: 77-82, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578886

RESUMO

Hedgehog (Hh) pathway hyperactivation has been observed in various tumors, including breast cancer, and Hh pathway inhibitors have demonstrated antitumor activity in breast cancer. The tumor microenvironment (TME) has been shown to play an important role in modulating cancer cell drug sensitivity, but the TME response to Hh pathway inhibitors is unclear. In the current study, we observed increased TME infiltration of macrophages in breast cancer tissue, and specifically, M2 polarized macrophages after neoadjuvant chemotherapy. Furthermore, we observed an enhanced tolerance to Hh pathway inhibitors in MDA-MB-231 cells after co-culturing with M2 macrophages. In addition, we demonstrated that Hh pathway inhibition significantly induced IL6 expression, and validated that the tolerance to Hh pathway inhibitors was IL6-dependent. This study demonstrates a role of macrophages in Hh pathway inhibition resistance and a role of macrophage-derived IL6 in this resistance of breast cancer cells to Hh inhibition. These data indicate that antagonizing IL6 together with Hh pathway inhibitors may be a novel therapeutic strategy for breast cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteínas Hedgehog/antagonistas & inibidores , Interleucina-6/metabolismo , Macrófagos/metabolismo , Comunicação Parácrina , Alcaloides de Veratrum/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cocultura , Feminino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Macrófagos/patologia , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Células THP-1 , Microambiente Tumoral , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
15.
Free Radic Biol Med ; 129: 582-599, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30347228

RESUMO

Hh/Gli1 cascade as well as Gsk3ß-Gli1 crosstalk play crucial role in estrogen-dependent progression of endometrial hyperplasia (EH). However, the underlying mechanisms involved in progression of disease still remain unclear. In the present study, we explored the role of Hh signaling in protection of endometrial hyperplasial cells against oxidative stress and the underlying mechanism involved therein. EH cells were found to be more resistant towards H2O2-induced oxidative stress (IC50: ~ 3×) as compared with normal endometrial cells. Estrogen (E2) pre-treatment followed by cytotoxic dose of H2O2, almost rescued the EH cells from apoptosis and caused the increased expression of downstream Shh signaling molecules i.e., Smo, Ptch and Gli1. Whereas pretreatment with cyclopamine was not able to curtail H2O2-induced effects indicating that estrogen protects these cells via activation of Shh pathway. Further, H2O2-induced ROS and lipid peroxidation alongwith decreased activities of antioxidant enzymes glutathione peroxidase and superoxide dismutase were found to be reversed in EH cells pre-exposed to E2 or rShh. The rShh suppressed H2O2-induced cell death and caused attenuation of mitochondrial apoptotic mediators and prevented disruption in mitochondrial morphology and mitochondrial membrane potential in EH cells. The functional blockage of signaling by Shh siRNA or Gli1siRNA led to significantly increased expression of mitochondrial fission protein dynamin-like GTPase (Drp1). The H2O2-treated EH cells showed diminished Gli1 and increased Drp1 expression, concurrent with reduced p-Drp1-(serine637). Whereas rShh pre-treated EH cells presented normal mitochondrial dynamics with dense, long networks of mitochondria alongwith nuclear accumulation of Gli1 and the decreased expression of Drp1. Overall, our results implicated that Shh signaling modulates antioxidant defense system and stabilizes mitochondrial dynamics by suppressing Drp1 protein which maintains survival of EH cells against oxidative stress.


Assuntos
Hiperplasia Endometrial/genética , Células Epiteliais/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas Hedgehog/genética , Proteínas Associadas aos Microtúbulos/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteína GLI1 em Dedos de Zinco/genética , Adulto , Animais , Estudos de Casos e Controles , Progressão da Doença , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patologia , Hiperplasia Endometrial/cirurgia , Endométrio/metabolismo , Endométrio/patologia , Endométrio/cirurgia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Estrogênios/farmacologia , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Histerectomia , Peroxidação de Lipídeos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/metabolismo
16.
Cell Physiol Biochem ; 50(4): 1346-1360, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30355933

RESUMO

BACKGROUND/AIMS: Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1. METHODS: NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit. RESULTS: Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1. CONCLUSION: We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury.


Assuntos
Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Animais , Benzamidas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Naftóis/farmacologia , Receptor Patched-1/metabolismo , Resveratrol , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/química , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismo , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
17.
Int J Oncol ; 53(6): 2445-2457, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30272371

RESUMO

Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinicopathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Regulação para Cima , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Estadiamento de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prognóstico , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Regulação para Cima/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/farmacologia
18.
ACS Nano ; 12(10): 9881-9893, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30231203

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most difficult cancers to treat. It is refractory to most existing therapies, including immunotherapies, due to the presence of an excessive desmoplastic stroma, which restricts penetration of drugs and cytotoxic CD8+ T cells. Stromal modulation has shown promising results in the enhancement of immune checkpoint blockade treatment in PDAC. We demonstrate here effective stromal modulation by a polymeric micelle-based nanoformulation to codeliver a sonic hedgehog inhibitor (cyclopamine, abbreviated as CPA) and a cytotoxic chemotherapy drug (paclitaxel, abbreviated as PTX). The formulation, M-CPA/PTX, modulated the PDAC stroma by increasing the intratumoral vasculature density, which then promoted the tumor infiltration by cytotoxic CD8+ T cells without depletion of tumor-restraining α-smooth muscle action-positive fibroblasts and type I collage in the stroma. The combination of M-CPA/PTX and the PD-1 checkpoint blockade significantly prolonged animal survival in an orthotopic murine PDAC model as well as a genetically engineered mouse model of PDAC. The superior antitumor efficacy was mediated by enhanced tumor infiltration of CD8+ T cells without concomitant infiltration of suppressive regulatory T cells or myeloid-derived suppressor cells and by the coordinated action of PTX and interferon-gamma. Our results demonstrate that stroma-modulating nanoformulations are a promising approach to potentiate immune checkpoint blockade therapy of pancreatic cancer.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Paclitaxel/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Células Estromais/imunologia , Alcaloides de Veratrum/farmacologia , Animais , Antineoplásicos/administração & dosagem , Carcinoma Ductal Pancreático/imunologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/imunologia , Células Estromais/patologia , Alcaloides de Veratrum/administração & dosagem
19.
Cell Prolif ; 51(6): e12500, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30151845

RESUMO

OBJECTIVES: The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of Shh in skin-derived precursors (SKPs), which have the capacity for multipotency and self-renewal, is not yet clear. The present study investigated the effects of the Shh signalling pathway on the proliferation and self-renewal of murine SKPs (mSKPs). METHODS: The Shh signalling pathway was activated by treatment with purmorphamine (Shh agonist) or recombinant Shh in mSKPs. Cyclopamine (Shh antagonist) or GANT-61 (Gli inhibitor) was used to inhibit the pathway. Western blot, qPCR, and immunofluorescence were used to analyse the expression of genes related to self-renewal, stemness, epithelial-mesenchymal transition (EMT) and the Shh signalling pathway. In addition, cell proliferation and apoptosis were examined. RESULTS: Inhibiting the Shh signalling pathway reduced mSKP proliferation and sphere formation, but increased apoptosis. Activating this signalling pathway produced opposite results. The Shh signalling pathway also controlled the EMT phenotype in mSKPs. Moreover, purmorphamine recovered the self-renewal and proliferation of aged mSKPs. CONCLUSION: Our results suggest that the Shh signalling pathway has an important role in the proliferation, self-renewal and apoptosis of mSKPs. These findings also provide a better understanding of the cellular mechanisms underlying SKP self-renewal and apoptosis that allow more efficient expansion of SKPs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Morfolinas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Purinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Pele/citologia , Alcaloides de Veratrum/metabolismo
20.
Organogenesis ; 14(3): 147-157, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30102120

RESUMO

To investigate the molecular mechanism underlying taurine-stimulated proliferation and differentiation of cochlear neural stem cells (NSCs) and potential involvement of Sonic Hedgehog (Shh) pathway. The NSCs were characterized with immunofluorescence stained with nestin antibody. Cell viability was determined by MTT assay. The relative proliferation was measured by BrdU incorporation assay. The morphologic index was measured under light microscope. The relative protein level was determined by immunoblotting. Here we presented our findings that taurine stimulated proliferation and neurite outgrowth of NSCs, which was completely abolished by Shh inhibitor cyclopamine. In addition, cyclopamine antagonized taurine's effect on glutamatergic and GABAergic neuron population via suppressing expressions of Ptc-1, Smo and Gli-1. Our data supported the critical role of Shh pathway underlying the protective effect of taurine on auditory neural system.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cóclea/citologia , Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/citologia , Transdução de Sinais , Gânglio Espiral da Cóclea/citologia , Taurina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Gânglio Espiral da Cóclea/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia
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