RESUMO
Bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1) is an innate immunity defense protein. Our previous studies proved its antibacterial and antiviral effects, but its role in fungi remains unknown. The study aimed to identify antifungal peptides (AFP) derived from BPIFA1, and three antimicrobial peptides (AMP1-3) were designed. The antifungal effects were proved by growth inhibition assay. AMP3 activity was confirmed by germ tube growth experiment and XTT assay. Its effects on cell wall and membrane of Candida albicans were assessed by tannic acid and Annexin V-FITC/PI double staining, respectively. Additionally, scanning electron microscope (SEM) and transmission electron microscopy (TEM) were used for morphological and ultrastructural observation. The expression of ALS1, EAP1, and SUN41 was tested by qPCR. Ultimately, three AMPs could fight against C. albicans in vitro, and AMP3 was highly effective. It functioned by destroying the integrity of cell wall and normal structure of cell membrane. It also inhibited biofilm formation of C. albicans. In addition, AMP3 down-regulated the expression of ALS1, EAP1, and SUN41, those are known to be involved in virulence of C. albicans. Altogether, the study reported successful development of a novel AFP, which could be used as a new strategy for antifungal therapy.
Assuntos
Antifúngicos , Candida albicans , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Peptídeos Antimicrobianos , Biofilmes , Candida albicans/metabolismo , Glicoproteínas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Fosfoproteínas/metabolismo , alfa-Fetoproteínas/metabolismo , alfa-Fetoproteínas/farmacologiaRESUMO
Gastric cancer mainly metastasizes via lymphatic vessels. Thus, it is critical to identify efficacious chemopreventive agents for lymphangiogenesis. The present study was undertaken to explore the effects of rosiglitazone (ROSI) on the growth and lymphangiogenesis of human gastric cancer. We established a model of gastric cancer by subcutaneously inoculating the human gastric cancer cell line SGC-7901 into nude mice. Mice were randomly divided into 4 groups and each group received a different agent by oral gavage. The control group received normal saline and treatment groups received different doses of ROSI once every 2 days. The growth of the tumor in vivo was assessed by measuring tumor volume. After 42 days, the mice were sacrificed and the tumors were removed. H&E staining was used to observe the histomorphological features; immunohistochemistry staining for lymphatic vessel density (LVD) was used to evaluate tumor lymphangiogenesis, RT-PCR was performed to determine the mRNA expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 (VEGFR-3), and western blotting was used to detect the protein expression of VEGF-C and VEGFR-3. Compared with the control group, all treatment groups had smaller tumor volume and higher tumor growth inhibitory rate every day. The number of typical tumor cells in the control group was higher compared to that in the treatment groups, and the highest level of LVD was found in the control group. Furthermore, both the expression of VEGF-C and VEGFR-3 mRNA and proteins in the control group were significantly higher compared to those in the treatment groups. Markedly, these changes were correlated in a dose-dependent manner with ROSI. These results demonstrated that, through simultaneously blocking the expression of VEGF-C and VEGFR-3, ROSI suppresses lymphangiogenesis. This may represent a powerful therapeutic approach for controlling gastric cancer cell growth and metastasis.
Assuntos
Linfangiogênese/efeitos dos fármacos , Metástase Linfática/patologia , Neoplasias Gástricas/tratamento farmacológico , Tiazolidinedionas/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , RNA Mensageiro/biossíntese , Rosiglitazona , Neoplasias Gástricas/patologia , Fator C de Crescimento do Endotélio Vascular/biossíntese , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
OBJECTIVES: Phenylalanine (Phe) and tyrosine (Tyr) are the most reliable indicators for the diagnosis of phenylketonuria (PKU). The purpose of this study is to establish a simple and rapid method for the determination of Phe and Tyr in peripheral capillary blood from newborns and children by high performance liquid chromatography with ultraviolet detection (HPLC-UV). METHODS: Peripheral blood specimens were taken from newborns or children by heel stick or skin puncture on the fingers. Blood samples were deproteinized by equal volume of 5% perchloric acid. Amino acid separation was carried out on a Hypersil C8 column. The mobile phase containing 5% acetonitrile (v/v) was run at a flow rate of 1.5 mL/min. Phe and Tyr were determined in the ultraviolet detector at 210 nm. RESULTS: The determination was within 10.0 min. The linear range was from 6.0 to 1512.0 µmol/L for Phe and 5.5 to 1250.0 µmol/L for Tyr. Although Phe and Tyr levels from peripheral blood samples were relatively lower than those from serum or plasma, the data showed a good agreement between them. CONCLUSION: The method we developed is simple, rapid and convenient. It is useful for screening and diagnosis of PKU in newborns and children.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fenilalanina/sangue , Tirosina/sangue , Raios Ultravioleta , Anticoagulantes/farmacologia , Calibragem , Criança , Pré-Escolar , Feminino , Saúde , Humanos , Lactente , Recém-Nascido , Masculino , Padrões de Referência , Temperatura , Fatores de TempoRESUMO
Both breast cancer resistance protein (BCRP, ABCG2) and apoptosis-related molecules are involved in the development of multidrug resistance (MDR) in cancer cells. However, the association of BCRP with apoptosis-related molecules in the development of MDR is unknown. In this study, we investigated the changes in expression levels of BCRP, Survivin, p53, Bcl-2, Bcl-xL or Bax in cultured MCF-7 and MCF-7/5-FU cells, and explored whether these changes affected the expressions of BCRP. Our data showed that the protein and mRNA expression levels of BCRP, Survivin and Bcl-2 were significantly higher in MCF-7/5-FU cells than in MCF-7 cells, while p53 expression lower in MCF-7/5-FU cells than in MCF-7 cells. Knockdown of Survivin or Bcl-2 in MCF-7/5-FU cells and overexpression of Survivin in MCF-7 cells revealed that Survivin had significant association with BCRP expression. Luciferase reporter gene assays showed that Survivin up-regulated BCRP expression at transcriptional level and this response was mediated through NF-κB(p50) pathway. However, may be due to the physical interaction between p53 and Survivin, p53 directly affected Survivin-regulated BCRP expressions. Interestingly, we found that Survivin would suppress p53 expression. Furthermore, our data revealed that Survivin itself had no apparent effect on NF-κB(p50) and BCRP expression when knockdown of p53 in MCF-7 cells; whereas p53 exerted significant inhibitory action on these when knockdown of Survivin. In conclusion, through down regulation of p53 expression, Survivin attenuates the suppressing effect of p53 on NF-κB(p50) expression and then enhances BCRP expression. This may represent a novel strategy for reversal of BCRP drug transporter activity to modulate MDR in cancer cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Neoplasias da Mama/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Subunidade p50 de NF-kappa B/biossíntese , Proteínas de Neoplasias/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Células MCF-7 , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Survivina , Transcrição Gênica , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: To describe a simple, rapid, and sensitive HPLC method for simultaneous determination of TRP and KYNA in human serum. DESIGN AND METHOD: Samples were deproteinized by perchloric acid. KYNA was detected at 344 nm of excitation wavelength and 404 nm of emission wavelength, TRP was detected at 254 nm and 404 nm, with a total run time of 13 min per sample. RESULTS: Standard curves of 0.49 micromol/L to 196 micromol/L of TRP were linear. Inter-day and intra-day coefficient of variations were 3.31% and 4.14%, respectively. Average recovery was 104.43%. Detection limit was 0.001 micromol/L. The linearity of the assay was maintained from 1.5 nmol/L to 2093 nmol/L of KYNA. Inter-day and intra-day CVS were 3.20% and 4.27%, respectively. Average recovery was 101.19%. Detection limit was 0.05 nmol/L. CONCLUSION: The developed HPLC method is simple, convenient and can be applied in the diagnosis of related diseases.