Inhibition Of Celastrol On Hypoxia-mediated Angiogenesis Via HIF-1α-Sema4D Pathway

Part One Influence of Celastrol on angiogenesisinduced by hypoxia in vitroAIM:To study the effect of Celastrol on hypoxia-induced angiogenesis and the possible mechanism.METHODS:Human endothelial (EA.hy 926) and cancer cells (HepG2 and A549) were treated with indicated concentrations of Celastrol under hypoxia conditions for 6 or 16 hours. The inhibition rate of both endothelial and cancer cells were detected by MTT. Tube formation and transwell migration were performed to study the inhibitory effect of Celastrol on hypoxia-induced angiogenesis by endothelial cells. FITC-phalloidin and Transwell invasion assay was used to determine the inhibitory effect of Celastrol on invasion ability of hypoxic cancer cells. Real-time PCR was used to test the inhibitory effect of Celastrol on Sema4D mRNA expression. Western blot and ELISA assay were performed to detect the inhibitory effect of Celastrol on the expression of Sema4D membrance and secreted protein.RESULTS:Celastrol displayed potent inhibition of hypoxia-induced angiogenic and metastasic activity shown by decreasing the proliferation of both endothelial and cancer cells under hypoxia, blocking hypoxia-induced migration and tube formation of endothelial cells, and inhibiting cytoskeletal arrangement and invasion ability of cancer cells in a concentration-dependent manner. Moreover, Celastrol decreased Sema4D expression at both mRNA and protein levels.CONCLUSION:Celastrol inhibits hypoxia-induded angiogenesis. The down-regulation of Sema4D mRNA and protein expression might play a key role in the anti-angiogenesis effect of Celastrol. Part Two Influence of Celastrol on HIF-la pathwayAIM:To study the effect of Celastrol on hypoxia-induced HIF-1αand PI3K/Akt pathway.METHODS:The nuclear HIF-1αprotein expression of cancer cells were detected by Immunofluorescence and Western blot. Real-time PCR was used to test the inhibitory effect of Celastrol on HIF-la mRNA expression. Real-time PCR and ELISA assay were performed to detect the inhibitory effect of Celastrol on the level of VEGF mRNA and protein. Real-time PCR was used to test the inhibitory effect of Celastrol on CA9 mRNA expression. Western blot assay were performed to detect the inhibitory effect of Celastrol on the level of Akt/pAkt protein.RESULTS:Celastrol decreased HIF-1αmRNA levels under both normoxia and hypoxia and inhibited hypoxia-induced accumulation of nuclear HIF-1αprotein. Meanwhile, inhibition of nuclear HIF-la protein levels were accompanied by a reduction in the transcriptional activity of HIF-1αtarget genes, including VEGF and CA9. In addition, the inhibitory effect of Celastrol on HIF-1αprotein was partly due to its suppression of PI3K/Akt pathway.CONCLUSION:Celastrol inhibits HIF-1αpathway and the inhibitory effect of HIF-1αpathway might play a key role in the anti-Sema4D effect of Celastrol.