RESUMO
Objective To investigate the effect of gambogic acid (GA) on invasion in human gastric carcinoma SGC-7901 cells and its possible mechanism. Methods Cell counting kit-8(CCK-8) assay was performed to detect the effects of GA, inhibitor of nuclear factor kappa-B kinase( IKK) 16 and 5-fluorouracil (5-FU) on cell activity of GES-1 and SGC-7901 cells. Cell invasion was assessed with Transwell invasion assay. Western blotting was used to analyze the protein levels of vimentin, matrix metalloproteinase 2 ( MMP-2) and MMP-9 and protein phosphorylation of IKKα and p65. Results The cell activity was significantly decreased in SGC-7901 cells treated with GA in a dose-dependent manner with a half inhibiton concentration(IC50) value of 1. 89 μmol/L. But GA had no significant influence on cell viability of GES-1 cells. Meanwhile, 5-FU reduced the cell activity of GES-1 and SGC-7901 cells with IC50values of 7.36 μmol/L and 199.57 μmol/L respectively. Low-dose GA and IKK 16 impaired separately the ability of invasion in SGC-7901 cells, and down-regulated the protein levels of MMP-2, MMP-9 and vimentin, and inhibited phosphorylation of IKKot and p65, while a stronger inhibition was showed when the combination of GA and IKK16 was used. Conclusion Low-dose GA might inhibit invasion of SGC-7901 cells via IKKot/p65 signaling pathway.
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Objective To investigate the reversal effect and molecular mechanisms of NF-κB inhibitor parthenolide (PTL) on insulin resistance ( IR). Methods HepG2 cells were treated with insulin at different concentrations and time points, the glucose consumption of HepG2 cells was measured via glucose oxidase method to determine the best concentrations and time for establishing insulin-induced insulin resistance on HepG2 cells. After modeling, different concentrations of PTL were added in cells for 24 h for determining cell activity and glucose-consumption. Q-PCR was used to detect the expression of NF-κB and IκBα mRNA in cells, and western blot was used to detect the expression of protein IκBα. Results The best reaction time and concentration for insulin inducing resistance of HepG2 cells were 24 h and at 10 μg·mL-1 . The optimum acting dose of PTL was 20 μmol·L-1 . NF-κB activity was significantly reduced (P<0. 05), IκBα degradation was significantly inhibited (P<0. 05) compared to HepG2 cells with insulin resistance upon intervention on insulin resistance HepG2 cells by PTL. Conclusion PTL can inhibit IκBα degradation and disassociation of it from NF-κB, which in turn improves insulin resistance, providing theo-retical basis for preventing and treating diabetes with PTL.
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Objective: To establish a human hepatic cancer cell line- HepG2 with stable, simultaneous knockdown of three nuclear factor-kappaB inhibitors (IKappa;Bα, IκBβ, and IκBε) by RNA interference using pcDNA™ 6.2-miR-IκBα-IκBβ-IκBε co-targeting IκBα, IκBβ, and IκBε genes, so as to lay a foundation for future study. Methods: We designed and constructed three interfering plasmids pcDNA™ 6.2-miR-IκBα (1,2,3) targeting human IκBα gene; the three vectors were transfected into HepG2 cells separately and the most effective interfering fragment was identified by RT-PCR and Western blotting analysis. Similarly, we also constructed and selected the most effective interfering vectors targeting IκBβ and IκBε. Then we chained the three most effective interfering fragments (miR-IκBα, miR-IκBβ, and miR-IκBε) into pcDNA™ 6.2-miR vector to construct pcDNA™ 6.2-miR-IκBα-IκBβ-IκBε vector, which was used to transfect HepG2 cells and the transfectants were selected by G418. The expression of IκBα, IκBβ, and IκBε in the transfectants was examined by Western blotting analysis. Results: We successfully constructed the pcDNA™ 6.2-miR-IκBα- IκBε-IκBε vector. Western blotting analysis showed that, compared with the normal HepG2 cells and those transfected with pcDNA™ 6.2-miR-Neg vector, the protein expression of IκBα, IκBβ, and IκBε was stably down-regulated in cells transfected with pcDNA™ 6.2-miR-IκBα-IκBβ-IκBε vector. Conclusion: We have successfully established a HepG2 cell line with simultaneous knockdown of IκBα, IκBβ, and IκBε, paving a way for future study.