RESUMEN
Herein, we used isotopic formaldehyde and sodium cyanoborohydride via reductive amination to label two methyl groups on primary amine to arrange the standards (h2-formaldehyde-modified) and internal standards (ISs, d2-formaldehyde-modified) of tryptophan and its metabolites, such as serotonin (5-hydroxytryptamine) and 5-hydroxytryptophan. These derivatized reactions with a high yield are very satisfactory for manufacturing standards and ISs. This strategy will generate one or two methyl groups on amine to create different mass unit shifts with 14 vs. 16 or 28 vs. 32 in individual compounds for biomolecules with amine groups. In other words, multiples of two mass units shift are created using this derivatized method with isotopic formaldehyde. Serotonin, 5-hydroxytryptophan, and tryptophan were used as examples to demonstrate isotopic formaldehyde-generating standards and ISs. h2-formaldehyde-modified serotonin, 5-hydroxytryptophan, and tryptophan are standards to construct calibration curves, and d2-formaldehyde-modified analogs such as ISs spike into samples to normalize the signal of each detection. We utilized multiple reaction monitoring modes and triple quadrupole mass spectrometry to demonstrate the derivatized method suitable for these three nervous biomolecules. The derivatized method demonstrated a linearity range of the coefficient of determinations between 0.9938 to 0.9969. The limits of detection and quantification ranged from 1.39 to 15.36 ng/mL.
Asunto(s)
5-Hidroxitriptófano , Triptófano , 5-Hidroxitriptófano/metabolismo , Triptófano/metabolismo , Serotonina/metabolismo , Aminación , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Formaldehído/química , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Histamine is an organic nitrogenous compound that acts as a neurotransmitter in the uterus, spinal cord, and brain and is involved in local immune responses. In this study, we developed a fast and simple derivatization method based on reductive amination that can be used to quantify histamine by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Histamine isotope analogs were synthesized via reductive amination. Histamine was modified with H2-formaldehyde to form N-dimethylated histamine to act as a standard or with D2-formaldehyde to form N-dimethylated histamine-d4 to act as an internal standard. Using this method, we achieved a limit of detection of 3.6 ng/mL, a limit of quantification of 7.9 ng/mL, and a linear calibration curve with a coefficient of determination (R2) of 0.9987. Furthermore, the intra-day relative standard deviations ranged from 0.9% to 3.7% and the inter-day relative standard deviations ranged from 2.0% to 17.6%. After derivatization, N-dimethylated histamine showed 382.5% signal enhancement compared to unmodified histamine in mass spectrometry detection. To demonstrate the applicability of this method for biological samples, we utilized standard addition method to quantify histamine in fetal bovine serum and achieved a recovery of 86.7%.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Histamina/química , Espectrometría de Masas en Tándem/métodos , Aminación , Animales , Bovinos , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Albúmina Sérica Bovina/químicaRESUMEN
HIGHLIGHTS: A reductive amination-assisted method was used to synthesize standards and internal standards of ractopamine and salbutamol. Standard and internal standard analogs were fabricated by isotopic formaldehydes and sodium cyanoborohydride. A quantitative method of modified ractopamine and salbutamol was successfully validated. The reductive amination-assisted method enhances the signal for MS detection.
Asunto(s)
Agonistas Adrenérgicos beta/análisis , Albuterol , Aminación , Fenetilaminas , Espectrometría de Masas en TándemRESUMEN
Growth differentiation factor-10 (GDF10), commonly referred as BMP3b, is a member of the transforming growth factor-ß (TGF-ß) superfamily. GDF10/BMP3b has been considered as a tumor suppressor, however, little is known about the molecular mechanism of its roles in tumor suppression in oral cancer. Clinical significance of GDF10 downregulation in oral squamous cell carcinoma (OSCC) was evaluated using three independent cohorts of OSCC patients. The molecular mechanisms of GDF10 in the suppression of cell survival, cell migration/invasion and epithelial-mesenchymal transition (EMT) were investigated by using oral cancer cell lines. The present study shows that GDF10 is downregulated during oral carcinogenesis, and GDF10 expression is also an independent risk factor for overall survival of OSCC patients. Overexpression of GDF10 attenuates cell proliferation, transformation, migration/invasion, and EMT. GDF10-inhibited EMT is mediated by ERK signaling but not by typical TGF-ß signaling. In addition, overexpression of GDF10 promotes DNA damage-induced apoptosis and sensitizes the response to all-trans retinoic acid (ATRA) and camptothecin (CPT). Intriguingly, the expression of GDF10 is induced by type III TGF-ß receptor (TGFBR3) through TGF-ß-SMAD2/3 signaling. Our findings suggest that TGFBR3 is an upstream activator of GDF10 expression and they share the same signaling to inhibit EMT and migration/invasion. These results support that GDF10 acts as a hinge to collaborate with TGFBR3 in the transition of EMT-MET program. Taken together, we illustrated the clinical significance and the molecular mechanisms of tumor-suppressive GDF10 in OSCC.
Asunto(s)
Carcinoma de Células Escamosas/patología , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Factor 10 de Diferenciación de Crecimiento/metabolismo , Neoplasias de la Boca/patología , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Factor 10 de Diferenciación de Crecimiento/genética , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Pronóstico , Proteoglicanos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Análisis de SupervivenciaRESUMEN
HYPOTHESES: Epithelial-mesenchymal transition (EMT) and invasion play a critical role in cancer progression and metastasis. We have shown that low E-cadherin and high Twist expression are significantly correlated with prognostic survival prediction in oral squamous cell carcinoma (OSCC). This study aimed to determine the anti-invasive effect of curcumin on the expression of matrix metalloproteinases (MMPs) and of EMT regulators in OSCC. METHODS: SCC-25 cells were treated with curcumin, and cell proliferation, invasion, and expression of MMPs and EMT regulators were assessed for cell viability by trypan blue exclusion, for invasion by Matrigel invasion chamber, and for EMT regulators and MMP changes in the levels of proteins by immunoblotting. RESULTS: Our data showed that curcumin treatment not only decreased the expression of MMP-2 and MMP-9 to inhibit invasiveness in oral cancer but also modulated the expression of EMT markers, such as Snail, Twist, and E-cadherin, and induced p53 expression that is crucial to EMT repression. CONCLUSION: Curcumin has the potential to become an adjunctive regimen for the prevention of cancer progression and metastasis in oral cancer.
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Carcinoma de Células Escamosas/tratamiento farmacológico , Curcumina/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Cadherinas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Neoplasias de la Boca/patología , Invasividad Neoplásica , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Aberration in DNA replication is a major cause to genome instability that is a hallmark of cancer cells. Cell division cycle 6 (Cdc6) and c-Myc have a critical role in the initiation of DNA replication. However, whether their interaction induces epithelial-mesenchymal transition (EMT) and promotes tumorigenesis in in vivo animal model remains unclear. Since using zebrafish as a cancer model has been restricted by the late onset of tumorigenesis and extreme difficulty in transformation on skin, we tried to establish a novel non-melanoma skin model in zebrafish to study their role in tumorigenesis. A stable transgenic zebrafish was created by using tol2 transposon, in which cdc6 and c-myc were co-overexpressed in epidermis driven by a skin-specific krt4 promoter. Intriguingly, co-overexpression of cdc6 and c-myc in transgenic zebrafish skin triggered tumor-like transformation, apoptosis attenuation, genomic instability, and EMT, hallmarks of malignant tumorigenesis. Our findings and other characteristics of zebrafish, including optical clarity and small molecule treatment, provide the future utility of this model for easy and non-invasive detection and for identification of new anti-cancer drug.
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Proteínas de Ciclo Celular/genética , Transición Epitelial-Mesenquimal/genética , Genes myc , Inestabilidad Genómica , Proteínas Nucleares/genética , Animales , Animales Modificados Genéticamente , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Pez CebraRESUMEN
Immune escape is a characteristic of cancer progression, but its underlying molecular mechanism is still poorly understood. An immunomodulatory protein, indoleamide 2,3-dioxygenase (IDO), is induced by gamma-interferon (IFN-gamma) in several immune cells; those cells are observed in cancer cell microenvironment and can enhance immune escape. Previous studies show that IDO is expressed in the process of tumor formation and associated with cancer cell immune tolerance. By locally degrading tryptophan, IDO inhibits the proliferation of T lymphocytes and induces T cell apoptosis, leading to suppression of T cell response. In this study, (-)-epigallocatechin-3-gallate (EGCG), the major constituent of green tea, is found to significantly inhibit the expression of IDO in human oral cancer cell lines. EGCG suppresses the induction of IDO at transcriptional level. Activation of STAT1 is discovered to play an important role in regulating IDO expression by IFN-gamma. The study results demonstrate that EGCG can inhibit translocation of STAT1 into nucleus in IFN-gamma-stimulated human oral cancer cells. In addition, both tyrosine and serine phosphorylation of STAT1 are revealed to be suppressed by EGCG. Moreover, phosphorylation of PKC-delta, JAK-1, and JAK-2, which are the upstream event for the activation of STAT1, are also inhibited by EGCG in IFN-gamma-stimulated human oral cancer cells. These data show that EGCG inhibited IDO expression by blocking the IFN-gamma-induced JAK-PKC-delta-STAT1 signaling pathway. This study indicates that EGCG is a potential drug for immune and target therapy to enhance cancer therapy by increasing antitumor immunity.
Asunto(s)
Catequina/análogos & derivados , Regulación hacia Abajo/efectos de los fármacos , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Neoplasias de la Boca/metabolismo , Transducción de Señal/efectos de los fármacos , Catequina/farmacología , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Interferón gamma/inmunología , Quinasas Janus/genética , Quinasas Janus/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/enzimología , Neoplasias de la Boca/inmunología , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismoRESUMEN
The interferon (IFN) antagonists of Japanese encephalitis virus (JEV) proteins contribute to the JE pathogenesis. Most flavivirus non-structural (NS) proteins correlate with virus-induced inflammation and immune escape. NS4A proteins of West Nile virus and dengue type 2 virus have been demonstrated to inhibit IFN signaling. In this study, JEV NS4A without the C-terminal 2K domain has been demonstrated to partially block activation of an IFN-stimulated response element (ISRE)-based cis-reporter by IFN-alpha/beta. In addition, JEV NS4A significantly inhibited the phosphorylation levels of STAT1 and STAT2, but not TYK2 in the IFN-treated cells. Moreover, the N-terminus of a RNA helicase DDX42 protein identified using a phage display human brain cDNA library have been demonstrated to specifically bind to JEV NS4A in vitro using a co-immunoprecipitation assay. The interaction between JEV NS4A and RNA helicase DDX42 showed partial co-localization in human medulloblastoma TE-671 cells by confocal microscopy. Importantly, the expression of N-terminal DDX42 is able to overcome JEV-induced antagonism of IFN responses. Therefore, these results show that JEV NS4A without the C-terminal 2K domain is associated with modulation of the IFN response and the interaction of JEV NS4A with RNA helicase DDX42 could be important for JE pathogenesis.