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1.
J Proteomics ; 161: 1-10, 2017 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-28391045

RESUMO

The cyclic AMP (cAMP) response element binding protein 1 (CREB1) is a promising target for cancer therapy. Here, we report that luteolin, a natural product, inhibits the expression of CREB1 at the transcriptional level and blocks epithelial-to-mesenchymal transition (EMT) of colorectal cancer cells. Treatment of colorectal cancer cells with luteolin induced mesenchymal-to-epithelial transition, reduced the expressions of mesenchymal markers and inhibited cell mobility in vitro. Through comparison of the proteomic profile of HCT-116 cells with and without luteolin treatment, we identified 366 differentially expressed proteins. Bioinformatics analysis revealed that downregulation of CREB1 plays a central role in this process. Immunoblot analysis verified that the protein levels of CREB1 and its downstream target genes were decreased in luteolin-treated cells. Moreover, forced expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells, suggesting the important role of CREB1 in this process. Furthermore, luciferase reporter assay and examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX) revealed that luteolin inhibits the expression of CREB1 at the transcriptional level. In summary, our results demonstrated that suppressing the expression of CREB1 is crucial in the mechanism-of-action of luteolin inhibiting EMT of colorectal cancer cells. SIGNIFICANCE: It is no doubt that understanding the mechanism-of-action of natural products at the molecular level is important for their translational application. Proteomics is a powerful platform to explore the effects of natural products on the cells. In this study, we compared the proteomic profile of HCT-116 colorectal cancer cells with and without luteolin treatment to investigate the mechanism-of-action of luteolin against colorectal cancer cells. Subsequent bioinformatics analysis revealed that CREB1 could be one of the main targets of luteolin against colorectal cancer cells. Downregulation of CREB1 by luteolin affects glucagon signaling pathway and cAMP signaling pathway. The proteomics findings were verified with mechanistic analyses. We first identified that luteolin decreased the mRNA and protein levels of CREB1 and its downstream target genes. We then found that luteolin inhibits CREB1 expression at the transcriptional level by real-time PCR and luciferase reporter assay which confirmed by examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX). Finally, we generated CREB1-overexpressing stable cell line and showed that ectopic expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells and restored the expression levels of CREB1 target genes in colorectal cancer cells, and thereby demonstrated the critical role of CREB1 in the mechanism-of-action of luteolin against colorectal cancer. In summary, we revealed a novel mechanism-of-action of luteolin against colorectal cancer cell by the combination of proteomics discovery and mechanistic analyses.


Assuntos
Neoplasias Colorretais/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Luteolina/farmacologia , Proteômica/métodos , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Biologia Computacional/métodos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos
2.
Biomed Pharmacother ; 89: 108-115, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28219049

RESUMO

Pituitary tumor transforming gene-1 (PTTG1) has been suggested to serve as an oncogene in several types of human tumors, but little is known about the biological function of PTTG1 in colorectal cancer. PTTG1 mRNA and protein expressions in colorectal cancer tissues and cell lines were measured by qRT-PCR, western blot or immunohistochemistry. The association between PTTG1 protein expression and clinicopathological features was analyzed. The function of PTTG1 on colorectal cancer cell proliferation and metastasis were explored through MTT, colony formation, migration and invasion assays. In our results, PTTG1 mRNA and protein expressions were increased in colorectal cancer tissues and cell lines compared with normal colonic tissues and colon epithelial cell line. PTTG1 overexpression positively associated with clinical stage, T classification, N classification, M classification and differentiation. The univariate and multivariate analyses suggested PTTG1 overexpression was an independent poor prognostic factor for colorectal cancer patients. The in vitro experiments showed knocking down PTTG1 inhibited colorectal cancer growth and metastasis. In conclusion, PTTG1 is an independent prognostic factor and acts as an oncogene in colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Securina/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/sangue , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Securina/biossíntese , Securina/genética , Ensaio Tumoral de Célula-Tronco
3.
Hepatobiliary Pancreat Dis Int ; 14(1): 90-5, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25655296

RESUMO

BACKGROUND: Pancreaticobiliary maljunction is a high risk factor of pancreatitis and biliary tract cancer. How this maljunction affects the liver remains obscure. This study aimed to examine the effects of pancreaticobiliary maljunction on the liver, pancreas and gallbladder in a cat model. METHODS: A model of choledocho-pancreatic side-to-side ductal anastomosis was created in ten cats. Before the procedure, a small piece of tissue from the liver, pancreas and gallbladder was collected as a control. The common channel formation was checked by cholecystography. The livers, pancreases and gallbladders of these cats were harvested for histological examination. The expression of proliferating cell nuclear antigen in the gallbladder was examined with immunohistochemistry. RESULTS: Seven of the 10 cats survived for 6 months after surgery. The color of the liver was darker in the PBM model than the control specimen, with nodules on the surface. Histological examination showed ballooning changes and inflammatory infiltrations and the histopathological score increased significantly (P<0.05). Also, mitochondria swelling and lipid droplet in cytoplasm were observed under an electron microscope. The pancreas also appeared darker in the PBM model than the control specimen and dilated pancreatic ducts were found in three cats. Histopathological examination revealed vascular proliferation and inflammatory infiltration with numerous neutrophils. Gallbladder epithelial cells were featured by expanded endoplasmic reticulum, increased intercellular space and cellular nucleus deformation. The positive cells of proliferating cell nuclear antigen were increased significantly (P<0.05). CONCLUSION: The present study demonstrated that pancreaticobiliary maljunction can lead to the injuries of the liver, pancreas and gallbladder.


Assuntos
Anormalidades do Sistema Digestório/patologia , Vesícula Biliar/patologia , Fígado/patologia , Pâncreas/patologia , Animais , Biomarcadores/metabolismo , Gatos , Proliferação de Células , Anormalidades do Sistema Digestório/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/patologia , Células Epiteliais/patologia , Vesícula Biliar/metabolismo , Vesícula Biliar/cirurgia , Vesícula Biliar/ultraestrutura , Fígado/cirurgia , Fígado/ultraestrutura , Mitocôndrias Hepáticas/patologia , Dilatação Mitocondrial , Infiltração de Neutrófilos , Pâncreas/cirurgia , Pâncreas/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo
4.
Oncol Rep ; 33(4): 1707-16, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25682742

RESUMO

MicroRNA-182 (miR-182) is significantly downregulated in human gastric tissue samples. Overexpression of miR-182 suppresses the proliferation and colony formation of gastric cancer cells. However, new aspects of the mechanism are still emerging in gastric cancer. ANUBL1, also known as ZFAND4 (zinc finger, AN1-type domain 4), its roles are scarely reported in cancer. In this study, we not only showed that ANUBL1 as an oncogene was upregulated and could promote proliferation of SGC-7901 cells, but also demonstrated that its over-expression led to a strong decrease of miR-182 expression and expression of ANUBL1 was in turn directly downregulated by miR-182, thereby establishing a negative feedback loop between miR-182 and ANUBL1. The elucidation of the mechanisms of miR-182 targeting ANUBL1 in gastric cancer helps us to further understand the mechanism of gastric cancer initiation and progression.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , RNA Neoplásico/fisiologia , Neoplasias Gástricas/genética , Regiões 3' não Traduzidas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Ciclo Celular , Replicação do DNA , Retroalimentação Fisiológica , Feminino , Genes Reporter , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA/genética , Interferência de RNA , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transfecção , Ensaio Tumoral de Célula-Tronco
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