Molecular Mechanisms Of Imperatoria In Anti-inflammation And Kamebakaurin In Anti-tumour

Aim:NF-?B and HIF-1? involved in inflammation,tumor cell proliferation,apoptosis and angiogenesis and a variety of biological processes.Imperatorin and Kamebakaurin were isolated from Angelica dahurica and Isodonexcia(Maxin.)Hara of changbai mountain.We used NF-?B and HIF-1? signal as the target of drug action to study the molecular mechanism of anti-inflammation and anti-tumorigenesis of Imperatorin and Kamebakaurin,which provided a theoretical basis for the development of anti-inflammatory and anti-tumor drugs.Methods:(1)The effect of imperatorin about transcriptional activity of NF-?B and cell viability were detected by luciferase reporter assay and MTT assay;The expression of p65 and COX-2,cyclin D1,MMP-9,VEGF,IL-6,Bcl-xL,p-IKK?/?,p-Akt,caspases-8,PARP were detected by immunofluorescence and immunoblotting assay;RT-qPCR and ELISA were used to detect the effect of imperatorin on Bcl-xL mRNA,IL-6 mRNA,TNF-? mRNA and MMP-9,IL-6 in cell secretion;Apoptosis and ROS were used to detect the effect of imperatorin on cell apoptosis and intracellular ROS.(2)The effect of kamebakaurin about transcriptional activity of NF-?B and cell viability were detected by luciferase reporter assay and MTT assay;The expression of HIF-1? protein in tumor cells was detected by immunoblotting and immunofluorescence assay;RT-PCR and ELISA were used to detect the effects of kamebakaurin on the expression of HIF-1? mRNA,VEGF mRNA,EPO mRNA and VEGF of cell secretions;Flow cytometry and tumor xenograft were used to detect the effects of kamebakaurin on the cell cycle and xenograft tumor model in vivo.Results:(1)Imperatorin significantly inhibited the TNF-?-induced expression of NF-?B target genes;Imperatorin promoted TNF-?-induced apoptosis and inhibited the activation of PI3K/Akt,the phosphorylation of IKK?/?,the degradation of I?B? and the nuclear transfer of p65 in HeLa cells;Imperatorin inhibited TNF-a-induced intracellular ROS levels.(2)Kamebakaurin showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines;kamebakaurin inhibited HIF-1? protein synthesis,without affecting the expression level of HIF-la mRNA or degradation of HIF-1? protein;kamebakaurin prevented hypoxia-induced expression of HIF-1 target genes for VEGF/EPO/GLUT1;The inhibitory effect of kamebakaurin on the expression of HIF-1? proteins,leading to a growth inhibition of HCT116 cells in a xenograft tumor model.Conclusions:(1)Imperatorin plays an anti-inflammatory role by inhibiting TNF-a induced ROS/PI3K/Akt/NF-?B signaling.(2)Kamebakaurin inhibited the expression of HIF-la in tumor cells and the growth of HCT116 cells in a xenograft tumor model.