RESUMEN
Objective:To discuss the effect of royal jelly acid(10-HDA)on the proliferation and migration of the human colon cancer SW620 cells based on the network pharmacology,and to clarify its related molecular mechanism.Methods:The active ingredients such as 10-HDA and their corresponding targets were retrieved by using the keyword"royal jelly"from the Traditiomal Chinese Medicine Systems Pharmacology(TMSCP)Database and the Traditiomal Chinese Medicine Integrated Database(TCMID);the small molecule targets were predicted by the Swiss Target Prediction Database.The GeneCards Database and the Online Mendelian Inheritance in Man(OMIM)Database were used to obtain the targets with the keyword"Colon Cancer";the protein-protein interaction(PPI)network was constructed by using the String Database and Cytoscape 3.8.0 Software to screen the core targets;the Gene Ontology(GO)function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis were analyzed by Metascape Database;the specific ingredient 10-HDA was screened for the in vitro activity experiments.The human colon cancer SW620 cells with good growth status were divided into control group and different doses(1,5,10,15,and 20 mmol·L-1)of 10-HDA groups.The viabilities of the cells in various groups were detected by MTT method and the survival rates of the cells were calculated.The SW620 cells were divided into control group,low dose(5 mmol·L-1)of 10-HDA group,middle dose(10 mmol·L-1)of 10-HDA group,and high dose(15 mmol·L-1)of 10-HDA group;Hoechst33342 staining method was used to observe the morphology of the cells in various groups;cell scratch test was used to detect the scratch healing rates of the cells in various groups;flow cytometry was used to detect the percentages of the cells at different cell cycles in various groups;biochemical method was used to detect the activities of total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)in the cells in various groups;Western blotting method was used to detect the expression levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cysteine-containing aspartate proteolytic enzyme-3(Caspase-3),cysteine-containing aspartate proteolytic enzyme-9(Caspase-9),glycogen synthase kinase 3β(GSK3β),β-catenin,and cyclin D1 proteins in the cells in various groups.Results:Six active ingredients of royal jelly were screened out by the TCMSP Database,and 28 core targets of 10-HDA in the treatment of colon cancer were obtained.The GO function enrichment analysis mainly included the signaling pathways such as cell proliferation and apoptosis.The KEGG signaling pathway enrichment analysis included the cell cycle,prostate cancer,cell senescence,and p53 signaling pathways;the GSK3β/β-catenin signaling pathway was closely related to the cell cycle.Compared with control group,the viabilities of the cells in 5,10,15,and 20 mmol·L-110-HDA groups were decreased in a dose-dependent manner(P<0.05 or P<0.01),the numbers of apoptotic cells in different doses of 10-HDA groups were significantly increased,and the scratch healing rates of the cells were significantly decreased(P<0.05 or P<0.01);the percentages of the cells at S phase in middle and high doses of 10-HDA groups were significantly increased(P<0.05 or P<0.01),the activities of T-AOC and SOD in the cells in different doses of 10-HDA groups were significantly decreased(P<0.05 or P<0.01).Compared with control group,the expression level of Bcl-2 protein in the cells in low dose of 10-HDA group was significantly decreased(P<0.01),and the expression level of GSK3β protein was significantly increased(P<0.05);compared with control group,the expression levels of Bax,Caspase-3,Caspase-9,and GSK3β proteins in the cells in middle and high doses of 10-HDA groups were significantly increased(P<0.05 or P<0.01),and the expression levels of Bcl-2,β-catenin,and CyclinD1 proteins were significantly decreased(P<0.01).Conclusion:10-HDA can significantly inhibit the proliferation and migration of the colon cancer cells and promote the apoptosis and oxidation levels of the colon cancer cells,and its mechanism may be related to the activation of the GSK3β/β-catenin signaling pathway.
RESUMEN
Objective: To explore the antitumor effect of oxymatrine and detect the mechanism involved. Methods: The Anti-proliferative effects of oxymatrine in human colon adenocarcinoma SW620 cells were assessed using MTT assay. SW620 cells treated with oxymatrine were assessed with Hoechst 33258 staining and cell cycle distribution assay was performed by flow cytometry. The quantitative real-time PCR assay was used to evaluate the expression of p16, cell cycle-related cyclinD1 and CDK4 mRNA at the genetic level. To investigate the molecular mechanisms underlying alterations in cell apoptosis, the proteins p16, cell cycle-related cyclinD1, and CDK4 were determined by Western blotting analysis. Results: Oxymatrine could significantly inhibit the growth of SW620 cells compared with the control group, the IC50 was 4.02 μmol/L. Its anticancer activity was related to the alteration in expression of p16, cyclinD1, and CDK4 (P<0.05, 0.01). Conclusion: These results suggest that oxymatrine produces the obvious antitumor effects on SW620 cells in vitro, induces the cell arrest in G1 phase which is related to the regulation on the protein expression of p16, cyclinD1, and CDK4.
RESUMEN
Objective: To investigate the effects of phospholipase C epsilon-1 (PLCE1 ) over-expression on migration, cell cycle and apoptosis of human colon cancer cells. Methods: The SW620 cells overexpressing PLCE1 were constructed through lipofection. Three groups were designed as follows: parent group (without transfection), control group (transfected with empty plasmid containing green fluorescent protein) and experimental group (transfected with pcDNA-DEST53-PLCE1 plasmid). The expression levels of PLCE1 mRNA and protein in SW620 cells were detected by real-time fluorogenic quantitative-PCR (RFQ-PCR) and Western blotting, respectively. The effect of PLCE 1 over-expression on migation ability of SW620 cells was detected by Transwell chamber assay. The cell cycle distribution and apoptosis rate of SW620 cells were detected by flow cytometry (FCM). The apoptosis was analyzed by using DNA ladder method. Results: The migration ability of SW620 colon cancer cells was inhibited by over-expression of PLCE 1. The numbers of migrated cells in the parent, control and experimental groups were 32.60±2.42, 32.20±3.25 and 8.80±1.72, respectively, and the difference among three groups was significant (P < 0.01). The over-expression of PLCE1 prolonged phase G1 and induced apoptosis. Conclusion: PLCE1 over-expression can inhibit the migration ability of colon cancer cells and induce their apoptosis. PLCE1 over-expression can reduce the malignant degree of colon cancer cells, and this gene may be a new antioncogene related to colon cancer. Copyright© 2011 by TUMOR.
RESUMEN
Objective To inhibit the expression of transcription factor special protein 1(Sp1) through RNA interference (RNAi) technique and to investigate its impact on the proliferation ability of colorectal cancer cell line SW620. Methods The recombinant plasmid of Sp1 RNAi (pGenesil-1-Sp1) was constructed and transfected into SW620 cells by Lipofectamine. The transfcction efficiency was observed under fluorescence confocal microscopy. Expression levels of Sp1 mRNA and protein from SW620 after transfection were examined by real time PCR and Western blot respectively, after transduction of the recombinant plasmid into the SW620. The proliferation ability of SW620 cell line was evaluated by MTT assay. Results The expression plasmid (pGenesil-1-Sp1) against Sp1 was successfully constructed, recombinant vectors could reduce the expressions of Sp1 mRNA and protein in SW620, the ratio of inhibition of the expression of Sp1 mRNA and protein was 68.47 % and 73.82 % in 48th hour respectively. Compared with the control group, the difference was significant (P <0.05). MTT showed that the proliferation ability of SW620 cell was degraded. Conclusion Silencing Sp1 gene by the RNAi technology can actively inhibit the proliferation of SW620 cell. The successful application of Spl SiRNA extends the list of available therapeutic modalitics in the treatment of human colon cancer.