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1.
Comput Math Methods Med ; 2022: 8289548, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35785141

RESUMO

Background: Chinese Materia Medica and Jiangsu New Medical College record that Radix Veratri root is Liliaceae Veratrum taliense Loses. f. and the root of Veratrum stenophyllum Diels. According to traditional Chinese medicine (TCM) example, Radix Veratri is a Liliaceae plant Veratrum taliense. Another literature pointed out that the aliases of Veratrum taliense and Veratrum angustifolia are both Radix Veratri, and their effects are basically the same. The main active ingredient of Veratrum is veratramine, of which veratramine and Jervine are higher in content, reaching 24.60% and 21.28% of the total alkaloids, respectively. Veratrum alkaloids are both toxic and effective ingredients. In addition to its good clinical efficacy, attention should also be paid to its pharmacokinetic characteristics in vivo. It is particularly important to study the pharmacokinetic characteristics of veratramine and Jervine in vivo. Objective: The goal of this study was to develop a simple and effective method for measuring veratramine and Jervine in rat plasma at the same time. This method was used to study the pharmacokinetic characteristics of veratramine and Jervine in the alcohol extract of Radix Veratri in rats, to provide a reasonable basis for the clinical use of Radix Veratri. Methods: Eighteen SD rats were randomly assigned into three groups, half male and half female, and were given 0.04 g/kg, 0.08g/kg, and 0.16 g/kg Radix Veratri alcohol extract, respectively. Blood samples were collected at different time points and were analyzed by LC-MS/MS after protein precipitation. Bullatine was set as the internal standard; the plasma samples were extracted with ethyl acetate. After the sample was processed, acetonitrile-10 mM ammonium acetate, whose pH was adjusted to 8.8 with ammonia water, was taken as the mobile phase. Veratramine quantitative ion pair was 410.1⟶295.1m/z, Jervine quantitative ion pair was 426.2⟶114.1m/z, and Bullatine B (IS) quantitative ion pair was 438.2⟶420.1m/z. In the positive ion mode, the multireaction monitoring (MRM) mode was used to determine the blood concentration of veratramine and Jervine. DAS 3.3.0 was used to calculate the relevant pharmacokinetic parameters. Results: Veratramine had a good linear relationship in the concentration range of 0.0745~18.2 ng/mL, and that of Jervine was 1.11~108 ng/mL. The correlation coefficient r of three consecutive batches of the standard curve was greater than 0.995. Veratramine's lower quantification limit was 0.745 ng/mL, Jervine's was 1.11 ng/mL, and precision and accuracy were both less than 15%. The accuracy of veratramine was between 88.96% and 101.85%, and the accuracy of Jervine was between 92.96% and 104.50%. This method was adopted for the pharmacokinetic study of alcohol extracts of Radix Veratri. The results showed that only C max of veratramine female rats did not show linear kinetic characteristics in the dose range of Radix Veratri alcohol extract from 0.04 g/kg to 0.16 g/kg. For AUC0-t and C max of veratramine and Jervine, it could not determine whether the Radix Veratri alcohol extract showed linear kinetic characteristics within the dosage range of 0.04 g/kg~0.16 g/kg. Veratramine and Jervine showed obvious gender differences in the absorption and elimination stages. The absorption rate of veratramine and Jervine by male mice was about 10 times higher than that of female mice, and the elimination rate of male mice is about 20 times lower than that of female mice. It was suggested that the clinical application of the steroidal alkaloids veratramine and Jervine in Radix Veratri required rational use of drugs based on gender. Conclusion: An LC-MS/MS analysis method suitable for the pharmacokinetic study of veratramine and Jervine in Radix Veratri in SD rats was established to provide a basis for in vivo pharmacokinetic studies. The pharmacokinetic characteristics of veratramine and Jervine in the alcohol extract of Radix Veratri were significantly different in female and male rats. During the clinical use of Radix Veratri, it should pay close attention to the obvious gender differences that may occur after the medication.


Assuntos
Alcaloides , Veratrum , Animais , Cromatografia Líquida , Feminino , Humanos , Masculino , Camundongos , Extratos Vegetais , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Veratrum/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacocinética
2.
Toxins (Basel) ; 14(7)2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35878228

RESUMO

Jervine, protoveratrine A (proA), and protoveratrine B (proB) are Veratrum alkaloids that are presented in some remedies obtained from Veratrum lobelianum, such as Veratrum aqua. This paper reports on a single-center pilot cardiotoxic mechanism study of jervine, proA, and proB in case series. The molecular aspects were studied via molecular dynamic simulation, molecular docking with cardiac sodium channel NaV1.5, and machine learning-based structure-activity relationship modeling. HPLC-MS/MS method in combination with clinical events were used to analyze Veratrum alkaloid cardiotoxicity in patients. Jervine demonstrates the highest docking score (-10.8 kcal/mol), logP value (4.188), and pKa value (9.64) compared with proA and proB. Also, this compound is characterized by the lowest calculated IC50. In general, all three analyzed alkaloids show the affinity to NaV1.5 that highly likely results in cardiotoxic action. The clinical data of seven cases of intoxication by Veratrum aqua confirms the results of molecular modeling. Patients exhibited nausea, muscle weakness, bradycardia, and arterial hypotension. The association between alkaloid concentrations in blood and urine and severity of patient condition is described. These experiments, while primary, confirmed that jervine, proA, and proB contribute to cardiotoxicity by NaV1.5 inhibition.


Assuntos
Alcaloides , Veratrum , Alcaloides/toxicidade , Cardiotoxicidade , Humanos , Simulação de Acoplamento Molecular , Projetos Piloto , Espectrometria de Massas em Tandem , Alcaloides de Veratrum/farmacologia
3.
Life Sci ; 288: 120170, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34826438

RESUMO

AIMS: Antitumor effects of veratramine in prostate and liver cancers has been investigated, but it is still unclear whether veratramine can be used as an effective therapeutic agent for glioma. The aim of this study was to evaluate the potential pharmacological mechanism of veratramine in glioma. MAIN METHODS: Using four types of human glioblastoma cell lines, including A172, HS-683, T98G, and U-373-MG the dose-dependent antitumor effect of veratramine was evaluated. The cytotoxicity and cell proliferation were examined by CCK-8, and cell proliferation was further confirmed by anchorage-independent colony formation assay. The cell cycle distribution and apoptotic rate was assessed by flow cytometry, and apoptosis was further evaluated by apoptosis assay. The migration and invasiveness capacity were analyzed by using transwell. Protein and mRNA levels of related factors were determined by western blotting and RT-qPCR, respectively. KEY FINDINGS: Veratramine markedly induced apoptosis, suppressed the cell proliferation via the cell cycle G0/G1 phase arrest, and reduced the capacity for the migration and invasion in human glioblastoma multiforme cell lines. Moreover, veratramine was sufficient to affect the phosphatidylinositol-3-kinase/serine-threonine kinase/mechanistic target of rapamycin signaling pathway and its downstream Mdm2/p53/p21 pathway in human glioblastoma cell lines. SIGNIFICANCE: Antitumor effects of veratramine in suppression of glioma progression was mediated by the regulation of PI3K/Akt/mTOR and Mdm2/p53/p21 signaling pathway.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alcaloides de Veratrum/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
4.
Neurol Res ; 44(4): 318-330, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34592910

RESUMO

OBJECTIVE: Ischemic stroke is a major cause of death in the global population, with a high disability and mortality rate. Lack of regenerative ability is considered to be the fundamental cause. This study aims to determine the effect of Shh pathway, which mediates regenerative signaling in response to CNS injury, on myelin repair and Olig1 expression in focal ischemic lesions in the rat. METHODS: A model of middle cerebral artery occlusion (MCAO) was established using the intraluminal suture method where the middle cerebral artery (MCA) was restricted for 120 min. Cyclopamine, a specific inhibitor of Shh, or saline was administered 12h after MCAO surgery and lasted for 7d. After MCA occlusion, male Sprague-Dawley rats were randomly allocated to cyclopamine- or saline-treated groups. A group of no-injection animals after MCAO were used as control. The Shh signaling pathway, myelinogenesis-related factor MBP and Olig1 were tested using immunohistochemistry and RT-PCR assay. RESULTS: The levels of Shh and its component Gli1 were elevated from 1d up to 14d following ischemia, indicating that the Shh-Gli1 axis was broadly reactivated. Treatment with cyclopamine can partially block the Shh signaling pathway, prevent myelin repair, and decrease the Olig1 expression following ischemic stroke. CONCLUSION: That blockade of Shh signaling concurrently with the creation of a lesion aggravated ischemic myelin damage, probably via its downstream effects on Olig1 transcription. Shh plays a contributory role during regeneration in the CNS, thereby providing promising new therapeutic strategies to assist in recovery from ischemic stroke.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Hedgehog/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/efeitos dos fármacos , Masculino , Regeneração Nervosa/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína GLI1 em Dedos de Zinco/efeitos dos fármacos
5.
J Anal Toxicol ; 46(1): e42-e47, 2022 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33559680

RESUMO

Veratrum poisonings are described in the toxicology literature as multiple Veratrum species grow in different parts of the Northern Hemisphere and are occasionally ingested by mistake. Veratrum toxicity is attributed to the steroidal alkaloids contained in all parts of the plant. In Russia, Veratrum poisonings are more common since there is an over-the-counter Veratrum lobelianum-based tincture, Veratrum Aqua (VA), which is topically used for the treatment of lice infestation. Despite its toxicity, VA is misused in traditional medicine as a remedy for alcohol use disorder. We describe four cases of VA poisoning that occurred in Moscow, Russia. Three main V. lobelianum alkaloids (jervine, protoveratrine A (proA) and protoveratrine B) were determined in patient plasma and urine samples using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Here, we describe a novel validated LC-MS-MS method for jervine and proA quantification. A simple and rapid liquid-liquid extraction with methyl tert-butyl ether was utilized for analyte extraction. Chromatographic separation was achieved using a Poroshell 120 EC-C18 column, and the total run time was 14 min. The lower limit of quantification was 0.1 ng/mL for jervine and proA in both plasma and urine. Biological samples were obtained upon hospital admission and during treatment, thus enabling to get a better understanding of the alkaloid elimination profile. Upon admission, plasma concentrations of jervine (concentration range: 0.10-5.01 ng/mL) prevailed over proA (concentration range: 0-0.67 ng/mL). At this time, proA already reached maximum concentrations in urine (concentration range: 0.15-37.70 ng/mL). Maximum concentrations of jervine in urine were observed 24 h after admission (concentration range: 0.10-9.55 ng/mL). In all cases, plasma concentrations of Veratrum alkaloids correlated with condition severity. Since none of the patients confirmed VA intake, instrumental analysis was the basis for the definitive diagnosis of VA poisoning.


Assuntos
Alcaloides , Veratrum , Cromatografia Líquida , Humanos , Espectrometria de Massas , Alcaloides de Veratrum
6.
Int J Mol Med ; 49(5)2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35266008

RESUMO

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU­SKI43, cyclopamine and GLI­antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786­O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription­quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma­associated zinc finger protein (GLI)1­3, BCL2 apoptosis regulator (BCL2), MYC proto­oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786­O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU­SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU­SKI43 appear to be potential anti­renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias Renais/genética , Fator A de Crescimento do Endotélio Vascular , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/genética
7.
Int J Mol Sci ; 22(19)2021 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-34639065

RESUMO

BACKGROUND: Basal cell carcinoma is one of the most common types of non-melanoma skin cancers, which can be locally destructive despite low-rate metastasis. Surgery is the treatment of choice, but it lacks of efficacy on advanced cases. Hedgehog pathway inhibitors are a class of drugs providing a new therapeutic option for patients affected by advanced disease. Besides systemic therapy, such as vismodegib and sonidegib, also topical inhibitors have been developed. Patidegib is able to decrease tumor burden, reducing the adverse effects induced by systemic targeted therapies. METHODS: We performed comprehensive research to summarize the use of patidegib in advanced and recurrent aggressive basal cell carcinomas. Only English language human studies were included in the search. RESULTS: Seven trials reported the application of patidegib. Both topical and systemic patidegib demonstrated safety, tolerability, and efficacy in naïve patients with stage II and III basal cell carcinomas, while stage IV disease and not-naïve patients did not show any benefit. CONCLUSION: Unlike systemic Hedgehog pathway inhibitors, patidegib 2% gel is not associated with systemic adverse effects and allows a better patient management. Considering the multidisciplinary management of neoplasia, in the era of precision medicine, it is mandatory to confide in pharmacogenomics to obtain personalized combined or sequential therapies.


Assuntos
Antineoplásicos/uso terapêutico , Dermatologia , Proteínas Hedgehog/metabolismo , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos de Bifenilo , Ensaios Clínicos como Assunto , Dermatologia/métodos , Humanos , Prognóstico , Piridinas , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Resultado do Tratamento , Alcaloides de Veratrum
8.
PLoS One ; 16(9): e0257395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34534256

RESUMO

Sensory experience modulates proliferation, differentiation, and migration of oligodendrocyte progenitor cells (OPCs). In the mouse primary visual cortex (V1), visual deprivation-dependent modulation of OPCs has not been demonstrated. Here, we demonstrate that undifferentiated OPCs developmentally peaked around postnatal day (P) 25, and binocular enucleation (BE) from the time of eye opening (P14-15) elevated symmetrically-divided undifferentiated OPCs in a reversible G0/G1 state even more at the bottom lamina of the cortex by reducing maturing oligodendrocyte (OL) lineage cells. Experiments using the sonic hedgehog (Shh) signaling inhibitor cyclopamine in vivo suggested that Shh signaling pathway was involved in the BE-induced undifferentiation process. The undifferentiated OPCs then differentiated within 5 days, independent of the experience, becoming mostly quiescent cells in control mice, while altering the mode of sister cell symmetry and forming quiescent as well as maturing cells in the enucleated mice. At P50, BE increased mature OLs via symmetric and asymmetric modes of cell segregation, resulting in more populated mature OLs at the bottom layer of the cortex. These data suggest that fourth postnatal week, corresponding to the early critical period of ocular dominance plasticity, is a developmentally sensitive period for OPC state changes. Overall, the visual loss promoted undifferentiation at the early period, but later increased the formation of mature OLs via a change in the mode of cell type symmetry at the bottom layer of mouse V1.


Assuntos
Proteínas Hedgehog/genética , Oligodendroglia/citologia , Células-Tronco/citologia , Córtex Visual/fisiologia , Animais , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Proliferação de Células , Olho , Camundongos , Camundongos Endogâmicos C57BL , Modelos Estatísticos , Neurogênese , Células Precursoras de Oligodendrócitos/metabolismo , Transdução de Sinais , Alcaloides de Veratrum/farmacologia , Córtex Visual/metabolismo
9.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34417306

RESUMO

In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of Gli1, a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., Science 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.


Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas Hedgehog/metabolismo , Tamoxifeno/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Gliose/metabolismo , Proteínas Hedgehog/genética , Camundongos , Camundongos Transgênicos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Medula Espinal/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia
10.
Pharm Biol ; 59(1): 974-985, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34348563

RESUMO

CONTEXT: Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties. OBJECTIVE: To investigate the protective effect and mechanism of EDT in ulcerative colitis (UC). MATERIALS AND METHODS: UC model was induced by 3% dextran sulphate sodium (DSS) solution for 7 days, meanwhile, EDT and Smoothened (Smo) inhibitor cyclopamine (Cyc) were intraperitoneally injected. In the first experiment, C57BL/6 mice divided into blank control, DSS, DSS + EDT (20 or 40 mg/kg) groups. In second experiment, added Cyc (5 mg/kg) and EDT + Cyc groups. All mice were sacrificed on day 8. Disease activity index (DAI), colon length and colon histology as well as MDA levels, SOD, and GSH-Px activities were measured. The expression of Sonic hedgehog (Shh), Patched, Smo, glioblastoma-1, zonula occludens-1 (ZO-1), occludin, cleaved caspase 3, Bax and Bcl-2 in colon was detected using RT-PCR and Western blotting. RESULTS: After EDT treatment, compared with the DSS group, DAI (2.33 ± 0.516 vs. 3.67 ± 0.516), colon shortening (5.27 ± 0.476 vs. 4.53 ± 0.528 cm) and histological score (6.67 ± 1.211 vs. 12 ± 1.265) was significantly decreased. EDT also reduced inflammation, oxidative stress and apoptosis in colon. Additionally, EDT increased the expression of the tight junction proteins ZO-1 (35%) and occludin (66.3%). Mechanistically, EDT upregulated the Shh signalling pathway. However, Cyc-mediated inhibition of the Shh pathway partially abolished the effects of EDT. DISCUSSION AND CONCLUSIONS: These results indicate EDT attenuates DSS-induced colitis by activating the Shh pathway. Further clinical trials are needed to demonstrate its efficacy on UC.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Colo/patologia , Flavanonas/farmacologia , Proteínas Hedgehog/metabolismo , Animais , Colite Ulcerativa/induzido quimicamente , Colo/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Sulfato de Dextrana/farmacologia , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia
11.
Nature ; 595(7868): 596-599, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234347

RESUMO

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.


Assuntos
/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Linhagem Celular , Feminino , Humanos , Corpos de Inclusão , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus Sincicial Respiratório Humano/fisiologia , Fatores de Transcrição , Proteínas Virais
12.
Hematology ; 26(1): 518-528, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34314648

RESUMO

OBJECTIVE: Hypomethylating agents (HMAs) have been reported to target the Sonic Hedgehog (Shh) signaling pathway in myelodysplastic syndrome (MDS). However, the synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine in MUTZ-1 cell lines remains lacking. METHODS: We used a CCK-8 assay to detect the in-vitro proliferation rate of MUTZ-1 cell lines. Besides, the Annexin V-FITC/PI double staining flow cytometry was utilized to detect the apoptosis rate and cell cycle changes. The expression levels of mRNA were quantified by using qRT-PCR, and the western blot was employed to detect the expression of proteins. RESULTS: We found that the single-agent jervine or decitabine can significantly inhibit the proliferation rate of MUTZ-1 cell lines, and this inhibitory effect is time-dependent and concentration-dependent. The combined intervention of the jervine and decitabine can more significantly inhibit cell proliferation, induce cell apoptosis, and block the G1 phase of the cell cycle. The combined intervention of the two drugs significantly reduced Smo and G1i-1 mRNA expression in MUTZ-1 cells. Furthermore, after combining both of the drug treatments, the proteins levels of Smo, G1i-1, PI3K, p-AKT, Bcl2, and Cyclin Dl were significantly downregulated, and Caspase-3 is upregulated, indicating that jervine with its combination of decitabine might be effective for controlling the proliferation, apoptosis, and cell cycle. CONCLUSION: The Smo inhibitor jervine and its combination with decitabine have a synergistic effect on the proliferation, cell cycle, and apoptosis of MUTZ-1 cells, and its mechanism may be achieved by interfering with the Shh signaling pathway.


Assuntos
Decitabina/farmacologia , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
13.
Nat Commun ; 12(1): 3919, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168128

RESUMO

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.


Assuntos
Receptores Frizzled/química , Receptores Frizzled/metabolismo , Receptor Smoothened/química , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Compostos de Boro/química , Microscopia Crioeletrônica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores Frizzled/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Fosfoproteínas/metabolismo , Conformação Proteica , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/metabolismo
14.
Cancer Med ; 10(13): 4510-4521, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34076346

RESUMO

BACKGROUND: The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood. METHODS: We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well-known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK-8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT-PCR and Western Blot. RESULTS: We found up-regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low-dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells. CONCLUSIONS: Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer.


Assuntos
Morte Celular Autofágica/fisiologia , Carcinoma Epitelial do Ovário/metabolismo , Movimento Celular/fisiologia , Proteínas Hedgehog/metabolismo , Neoplasias Ovarianas/metabolismo , Proteína Sequestossoma-1/metabolismo , Animais , Morte Celular Autofágica/efeitos dos fármacos , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Cloroquina/farmacologia , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regulação para Cima , Alcaloides de Veratrum/farmacologia
15.
Neurochem Int ; 146: 105042, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33838160

RESUMO

In the adult brain, sonic hedgehog acts on cerebral microvascular endothelial cells to stabilize the blood-brain barrier. The expression of sonic hedgehog by astrocytes is altered during brain injury, and this change has been shown to affect permeability of blood-brain barrier. However, much remains unknown about the regulation of astrocytic sonic hedgehog production. Our results showed that endothelin-1 reduced sonic hedgehog mRNA expression and extracellular protein release in mouse cerebral cultured astrocytes, but had no effect in bEnd.3, a mouse brain microvascular endothelial-derived cell line. The effect of endothelin-1 on astrocyte sonic hedgehog expression was suppressed by an ETB antagonist BQ788, but was unchanged by the ETA antagonist FR139317. In cultured astrocytes and bEnd.3, endothelin-1 did not affect the expression of the sonic hedgehog receptor-related molecules, patched-1 and smoothened. In an animal model of traumatic brain injury, fluid percussion injury on the mouse cerebrum increased the expression of sonic hedgehog, patched-1, and smoothened. Repeated administration of BQ788 enhanced sonic hedgehog expression at 5 days after fluid percussion injury. Histochemical examination revealed sonic hedgehog expression in glial fibrillary acidic protein-positive astrocytes in the cerebrum after fluid percussion injury. Administration of exogenous sonic hedgehog and BQ788 suppressed Evans blue extravasation, an indicator of blood vessel permeability, induced by fluid percussion injury. The effects of BQ788 on fluid percussion injury-induced Evans blue extravasation were reduced by the administration of jervine, a sonic hedgehog inhibitor. Altogether, these results suggest that endothelin-1 down-regulates astrocytic sonic hedgehog to promote disruption of the blood-brain barrier during traumatic brain injury.


Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Endotelina-1/farmacologia , Proteínas Hedgehog/metabolismo , Receptor de Endotelina B/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Lesões Encefálicas Traumáticas/patologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Proteínas Hedgehog/antagonistas & inibidores , Masculino , Camundongos , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Alcaloides de Veratrum/farmacologia
16.
Cell Death Dis ; 12(3): 259, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707419

RESUMO

Chronic myeloid leukemia (CML) patients with complex chromosomal translocations as well as non-compliant CML patients often demonstrate short-lived responses and poor outcomes on the current therapeutic regimes using Imatinib and its variants. It has been derived so far that leukemic stem cells (LSCs) are responsible for Imatinib resistance and CML progression. Sonic hedgehog (Shh) signaling has been implicated in proliferation of this Imatinib-resistant CD34(+) LSCs. Our work here identifies the molecular mechanism of Shh-mediated mutation-independent Imatinib resistance that is most relevant for treating CML-variants and non-compliant patients. Our results elucidate that while Shh can impart stemness, it also upregulates expression of anti-apoptotic protein-Bcl2. It is the upregulation of Bcl2 that is involved in conferring Imatinib resistance to the CD34(+) LSCs. Sub-toxic doses of Bcl2 inhibitor or Shh inhibitor (<

Assuntos
Antineoplásicos/farmacologia , Cromossomos Humanos , Resistencia a Medicamentos Antineoplásicos , Proteínas Hedgehog/metabolismo , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tiazóis/farmacologia , Regulação para Cima , Alcaloides de Veratrum/farmacologia
17.
Biochem Cell Biol ; 99(5): 587-595, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33677985

RESUMO

Cervical cancer is one of the leading causes of mortality amongst women in developing countries, and resistance to therapy is the main reason for treatment failure. Recent advances suggest that cancer stem cells (CSCs) are critically involved in regulating the chemo-resistant behavior of cervical cancer cells. In our study, cells with the CSC phenotype were isolated, and we examined the expression levels of stem cell markers and genes associated with epithelial-mesenchymal transition (EMT) using different assays. However, the cells with the CSC phenotype could not be cultured for further cytotoxicity studies, so we established a model of CSC in cervical cancer cells. We performed siRNA-mediated knockdown of E-cadherin in these cells, and studied them for EMT-associated stem-cell-like properties. We also performed dose-dependent cell viability assays using clinically relevant drugs such as cisplatin, cyclopamine, and GANT58 to analyze the drug resistant behavior of these cancer cells. We found that knockdown of E-cadherin induces EMT in cervical cancer cells, imparting stem-cell like characteristics along with enhanced tumorsphere formation, cell migration, invasiveness, and drug resistance. This is the first study to establish a CSC model in cervical cancer cells by knockdown of E-cadherin, which can be used to develop anti-cancer therapies.


Assuntos
Caderinas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias do Colo do Útero/metabolismo , Antineoplásicos/farmacologia , Caderinas/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Tiofenos/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Alcaloides de Veratrum/farmacologia
18.
J Orthop Surg Res ; 16(1): 160, 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637095

RESUMO

OBJECTIVE: This study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in order to investigate the role and significance of the Hh signal in fluoride-induced bone injury. METHODS: Healthy Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, the fluorosis group (F Group), the fluoride + blocker group (F + Cycl group: rats were treated with fluoride + cyclopamine), and the fluoride + blocker control group (F + DMSO group). After 6 months of intervention, the urinary fluoride content of rats in each group was detected. The primary osteoblasts of rats were selected for cell experiment, and the experiment was carried out after the cells were passaged from the second to the fourth generation. RESULTS: The proliferation rate of primary rat osteoblasts presented time-affected and dose-affected relationships in a short time under treatment with a low dose of sodium fluoride (NaF), but the proliferation of osteoblasts was inhibited by long-term and high-dose NaF exposure. In the F group, the alkaline phosphatase (ALP) activity of osteoblasts increased gradually. The ALP activity was lower in the F + Cycl group than in the F group, and there was no significant difference between the F + DMSO group and F group. With the increase in fluoride exposure, the expression of Hh signal factors and osteogenic-related factor proteins increased gradually. The expressions of Indian hedgehog (Ihh), smoothened (Smo), Glioma-associated oncogene homolog (Gli) 2, and Runt-related transcription factor 2 (Runx2)in the F + Cycl group increased with the dose of fluoride but they were significantly inhibited compared with the F group. Compared with the control group, the content of urinary fluoride in the F group was significantly higher (P < 0.05), but there was no significant change in urinary fluoride content in the F + Cycl group and the F + DMSO group. Compared with the control group, the serum bone alkaline phosphatase (BALP) contents of rats in the other groups increased after 6 months' intake of fluoride water (P < 0.05). After drug blocking, the serum BALP content in the F + Cycl group was lower than that in the F + DMSO group (P < 0.05). The BALP content in the F + DMSO group was similar to that in the F group: it did not decrease. The mRNA expressions of Ihh, Smo, Gli2, and Runx2 in bone tissue of the F group were significantly higher than those in the control group (P < 0.05). After cyclopamine blocking, the expressions decreased (P < 0.05), but the differences between the F + DMSO group and F group were not statistically significant. CONCLUSION: Hh signal plays an important role in fluoride-induced bone injury. The effective inhibition of cyclopamine is expected to be a new target for the treatment of skeletal damage caused by fluorosis.


Assuntos
Doenças Ósseas/induzido quimicamente , Doenças Ósseas/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/genética , Fluoreto de Sódio/efeitos adversos , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Osteoblastos/fisiologia , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Fatores de Tempo , Alcaloides de Veratrum/efeitos adversos
19.
Drug Des Devel Ther ; 14: 5667-5678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33380787

RESUMO

BACKGROUND AND PURPOSE: Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix (ECM). Inhibition of HSC activation may be an effective treatment. Since various pathways control HSC activation, a combination of drugs with different mechanisms may be more effective than monotherapy. METHODS: Here, we prepared liposomes loaded with curcumin and cyclopamine to inhibit HSC activation. We systematically analyzed the physicochemical characteristics of liposomes loaded with the two drugs, as well as their effects on HSC proliferation, activation and collagen production on gene, protein and cellular levels. RESULTS: The prepared liposomes helped solubilize both drugs, contributing to their uptake by cells. Liposomes loaded with both drugs inhibited cell proliferation, migration and invasion, as well as induced more apoptosis and perturbed the cell cycle more than the free combination of both drugs in solution or liposomes loaded with either drug alone. Liposomes loaded with both drugs strongly suppressed HSC activation and collagen secretion. CONCLUSION: Our results suggest that liposome encapsulation can increase the uptake of curcumin and cyclopamine as well as the synergism between them in anti-fibrosis. This approach shows potential for treating hepatic fibrosis.


Assuntos
Curcumina/farmacologia , Cirrose Hepática/tratamento farmacológico , Alcaloides de Veratrum/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Lipossomos/química , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Ratos
20.
Biomed Pharmacother ; 132: 110898, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33113432

RESUMO

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the superior mucosal epithelium of the nasopharynx. However, effective therapies for NPC are still required. Reducing Hedgehog signaling pathway has been shown to suppress tumor growth. In this study, we attempted to explore whether Jervine (JV), an inhibitor of Hedgehog signaling, had anti-cancer effects on NPC, and the underlying mechanisms. Our findings showed that JV treatments markedly reduced the proliferation of NPC cells in a dose- and time-dependent manner. Cell cycle arrest in G2/M phase was significantly enhanced by JV, along with evident DNA damage. Moreover, JV treatment effectively induced apoptosis in NPC cells through improving Caspase-3 activation. Furthermore, ROS production and mitochondrial impairments were detected in JV-incubated NPC cells with elevated releases of Cyto-c from mitochondria. JV also dramatically triggered autophagy through blocking AKT/mTOR and increasing AMPK signaling pathways. Intriguingly, we showed that JV-induced apoptosis was mainly via an autophagy-dependent manner. In addition, the expression levels of SHH, PTCH1, SMO and GLI1 were markedly suppressed in NPC cells, demonstrating the hindered Hedgehog signaling. Importantly, we found that JV-induced apoptosis and autophagy were closely associated with the blockage of Hedgehog signaling. Our in vivo studies confirmed the anti-cancer effects of JV on NPC through inducing autophagy, as evidenced by the markedly reduced tumor growth rate and weight without side effects and toxicity. Taken together, JV may be a promising and effective agent for human NPC treatment through repressing Hedgehog signaling pathway and inducing autophagic cell death.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Alcaloides de Veratrum/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
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