Hangyu Li1, Yan Li, Dan Liu, Hongzhi Sun, Jingang Liu. 1. Department of General Surgery, Shengjing Hospital Affiliated to China Medical University, China Medical University, Shenyang.
Abstract
BACKGROUND/AIMS: The molecular mechanisms of celastrol on hepatocellular carcinoma (HCC) cells migration and invasion ability is the major problem that prompted the study. METHODS: We first evaluated the effect of celastrol on migration and invasion ability of HepG2 cells using transwell migration and matrigel invasion assays. Next, we assessed the effect of celastrol on NF-κB transcriptional activity in hepatocellular carcinoma cells using western blot and luciferase reporter assay. We also performed real-time PCR to measure miR-224, MMP-2 and MMP-9 expression. Western blot was used to measure protein expression of MMP-2 and MMP-9. Furthermore, we used miR-224 inhibitor to evaluate whether down-regulation of miR-224 expression can affect MMP-2 and MMP-9 expression. The binding ability of p65/NF-κB on the miR-224 promote has been assessed by chromatin immunoprecipitation and quantitative real-time PCR (ChIP-qPCR). Finally, we evaluated the effect of miR-224 on celastrol-induced anti-tumor activity using miR-224 precursor. RESULTS: Celastrol significantly impaired migration and invasion of HepG2 cells and inhibited the activation of NF-κB and Akt in dose-dependent manner. IGF (the strong stimulator of Akt) inhibited the transcriptional activity of NF-κB in cells treated with celastrol. Besides, celastrol efficiently decreased the expression of miR-224 and protein expression of MMP-2 and MMP-9. ChIP-qPCR showed that p65/NF-κB binding to the miR-224 promoter sharply decreased after exposure to celastrol in time-dependent manner. Furthermore, inhibition of miR-224 expression can decrease MMP-9 protein level. Most importantly, miR-224 precursor can reverse the effect of celastrol on impairment of migration and invasion in HepG2 and Huh7 cells. CONCLUSION: Celastrol treatment inhibits migration and invasion of HCC cell and that the effect is partly due to NF-κB regulating miR-224 expression.
BACKGROUND/AIMS: The molecular mechanisms of celastrol on hepatocellular carcinoma (HCC) cells migration and invasion ability is the major problem that prompted the study. METHODS: We first evaluated the effect of celastrol on migration and invasion ability of HepG2 cells using transwell migration and matrigel invasion assays. Next, we assessed the effect of celastrol on NF-κB transcriptional activity in hepatocellular carcinoma cells using western blot and luciferase reporter assay. We also performed real-time PCR to measure miR-224, MMP-2 and MMP-9 expression. Western blot was used to measure protein expression of MMP-2 and MMP-9. Furthermore, we used miR-224 inhibitor to evaluate whether down-regulation of miR-224 expression can affect MMP-2 and MMP-9 expression. The binding ability of p65/NF-κB on the miR-224 promote has been assessed by chromatin immunoprecipitation and quantitative real-time PCR (ChIP-qPCR). Finally, we evaluated the effect of miR-224 on celastrol-induced anti-tumor activity using miR-224 precursor. RESULTS:Celastrol significantly impaired migration and invasion of HepG2 cells and inhibited the activation of NF-κB and Akt in dose-dependent manner. IGF (the strong stimulator of Akt) inhibited the transcriptional activity of NF-κB in cells treated with celastrol. Besides, celastrol efficiently decreased the expression of miR-224 and protein expression of MMP-2 and MMP-9. ChIP-qPCR showed that p65/NF-κB binding to the miR-224 promoter sharply decreased after exposure to celastrol in time-dependent manner. Furthermore, inhibition of miR-224 expression can decrease MMP-9 protein level. Most importantly, miR-224 precursor can reverse the effect of celastrol on impairment of migration and invasion in HepG2 and Huh7 cells. CONCLUSION:Celastrol treatment inhibits migration and invasion of HCC cell and that the effect is partly due to NF-κB regulating miR-224 expression.
Authors: Christopher R Bodle; Duncan I Mackie; Michael P Hayes; Josephine H Schamp; Michael R Miller; Michael D Henry; Jonathan A Doorn; Jon C D Houtman; Michael A James; David L Roman Journal: J Nat Prod Date: 2017-06-16 Impact factor: 4.050
Authors: Jason M Aliotta; Mandy Pereira; Sicheng Wen; Mark S Dooner; Michael Del Tatto; Elaine Papa; Laura R Goldberg; Grayson L Baird; Corey E Ventetuolo; Peter J Quesenberry; James R Klinger Journal: Cardiovasc Res Date: 2016-03-14 Impact factor: 10.787