Effects Of Compounds GA5 And GA6 On Inflammatory Microenvironment And Tumor Cell Invasion And Metastasis

Objective:In vitro study on the effects of compounds GA5 and GA6 on invasion and metastasis of tumor cells in inflammatory microenvironment.Methods:1.The effects of compounds GA5 and GA6 on the proliferation and survival of mouse mononuclear macrophage cell line RAW264.7 were detected by MTT assay.2.Lipid polysaccharide(LPS)was used to stimulate mouse mononuclear macrophage cell RAW264.7,and RAW264.7 inflammation model was established.3.To investigate the inhibitory effects of compounds GA5 and GA6 on the RAW264.7 inflammatory model,and to detect IL-6,IL-1?,COX-2,iNOS and TNF-? in the RAW264.7 inflammatory model by RQ-PCR;the secretion of IL-6 was detected by enzyme-linked immunosorbent assay(ELISA);the NO secretion was detected by NO detection kit method.4.Establish a non-contact tumor cell macrophage co-culture model in vitro and detect the migration,invasion and colony formation ability of tumor cells under co-culture conditions.Results:1?The results of MTT assay showed that the IC50 and 10%inhibitory concentration IC10 of compound GA5 and GA6 on mouse monocyte macrophage cell RAW264.7 for 24h were 12.95±0.07?M?2.78±0.8?M and14.311.1.6?M,5.1+0.66pM,respectively.The survival rates of GA5 and GA6 with compound concentration of 2?M,4?M and 6?M on mouse mononuclear macrophage cell RAW264.7 for 24h were 100%,98%and 93%,respectively.2.According to the morphology of mouse mononuclear macrophage cell RAW264.7,the inflammatory model was successfully induced by lipopolysaccharide(LPS)l?g/ml.3?In the RAW264.7 inflammatory model of compound GA5 and GA6,The results of RQ-PCR showed that IL-6 gene Transcription level was down-regulated by 49.8%,71.4%,90.3%under the action of GA5 concentration of 2?M,4?M and 6?M.Under the action of GA6 concentration of 2?M,4?M and 6?M,IL-6 gene Transcription level was down-regulated by 37.3%.48.1%,56.8%.Under the action of GA5 concentration of 2?M,4?M and 6?M,the gene transcription level of IL-1? was down-regulated by 51.6%,65%and 81.3%.Under the action of GA6 concentration of 2?M,4?M and 6?M,the gene Transcription level of IL-1? was down-regulated by 33.3%,48.2%and 53.4.%;Under the action of GA5 concentration of 2?M,4?M and 6?M,the gene transcription level of COX-2 was down-regulated by 29.9%,55.7%and 77.7%.Under the action of GA6 concentration of 2?M,4?M and 6?M,the gene Transcription level of COX-2 was down-regulated by 32.2%,62%and 81%;Under the action of GA5 concentration of 2?M,4?M and 6?M,the gene Transcription level of iNOS was down-regulated by 67%,78.1%and 84.8%.Under the action of GA6 concentration of 2?M,4?M and 6?M,the Transcription level of iNOS was down-regulated by 39.4%,50%and 73.7%.TNF-? gene Transcription has no significant effect;ELISA results showed that the Transcription levels of secretory factor IL-6 were down-regulated by 5.3%,76%,and 94.4%,respectively,under GA5 concentration of 2?M,4?M,and 6?M.The expression levels of secretory factor IL-6 decreased by 3%,49.7%and 96.7%under the action of GA6 concentration of 2?M,4?M and 6?M,respectively.The results of NO detection kit method showed that the expression levels of NO in the secretion of GA5 concentration of 2?M,4?M and 6?M were down-regulated by 23.8%,44.6%and 65.6%,respectively.The expression levels of NO in the GA6 concentration of 2?M,4pM and 6?M were down-regulated by 20.7.%,46.9%,63.5%;4?In vitro non-contact tumor cell macrophage co-culture model showed that compounds GA5,GA6 have a certain inhibitory capacity on tumor cell migration and invasion,and dose-dependent.Conclusions:1.The results of MTT assay showed that the compounds GA5 and GA6 had no significant effect on the survival rate of RAW264.7 at 2?M,4?M.2.Compounds GA5 and GA6 inhibit the inflammatory response of LPS-stimulated macrophage RAW264.7 by inhibiting the transcription of IL-6,IL-1?,Cox-2,and iNOS genes in inflammatory mediators and decreasing the secretion levels of IL-6 and NO.In turn,inhibition of migration and invasion of tumor cells mediated thereby.3.The inhibition of tumor cells by compounds GA5 and GA6 is related to the inhibition of inflammatory response.