| Lung cancer is the most common malignant tumor with higher occurrence rate and fatality rate. Surgery is the most effective therapeutic method for early stage of lung cancer, but many patients still are dead from lung cancer recurrence and metastasis after radical resection. The research mechanisms for lung cancer treatment, mainly focused on tumor cells, many studies have proved tumor inflammatory microenvironment could affect tumor cell metastasis. Fei-Liu-Ping ointment(FLP) is developed by GUANG AN MEN Hospital affiliated to China Academy of Chinese Medical Sciences. A large number of clinical trials have shown that FLP could improve patients quality of life, extend the tumors stable time. Experiments also show that FLP can significantly improve the immune function of the patients. We have in vivo results indicate that FLP decreases the expression of inflammation cytokines(IL1-β/IL-6、 TNF-a)apparently compared with the vehicle treatment and decreases NF-κB expression in solid tumor tissue compared to vehicle group. In addition, FLP also decreases the expression of MMP-9、N-cadherin and increases the expression of E-cadherin in solid tumor tissue. Based on the results in vivo, in vitro study will be done as follows:Objectives:1) To explore the effects of tumor inflammatory microenvironment on lung cancer invasion and metastasis;2) To explore mechanisms of Fei-Liu-Ping ointment regulating tumor inflammatory microenvironment to prevent lung cancer metastasis.Methods:1) To observe the effect of FLP on lung cancer cell proliferation by MTT;2) To establish co-culture system of A549cells and macrophage and to observe the effect of inflammatory microenvironment on A549cells and to observe what roles FLP plays on inflammatory microenvironment.3) To observe the effect of FLP on A549cell migration under co-culture conditions by wound healing and transwell;4) To observe the effect of FLP on A549cell invasion under co-culture conditions by transwell;5) To observe the effect of FLP on the expression of TNF-α、IL-1β、IL-6、TGF-β1MMP-2、MMP-9and uPA under co-culture conditions by ELISA;6)To observe the effect of FLP on A549cell gene expression of NF-κB and EMT related genes under co-culture conditions by RT-PCR; 7) To observe the effect of FLP on the A549cell protein expression of NF-κB and EMT related protein under co-culture conditions by Western blotting;8) To observe the effect of FLP on the A549cell NF-κB nuclear translocation;9) To observe the effect of FLP on the expression of E-cadherin、N-cadherin、Vimentin、 ZO-1、MMP-2and MMP-9by immunofluorescence;10) To observe the effect of FLP on the activation of MMP-2. MMP-9under co-culture conditions by gelatin zymography.Results:1) FLP treatment inhibits A549cell proliferation after24h, which is stronger after48h, and the inhibition rate decrease after72h. The FLPM group has the strongest inhibition roles after48h;2) A549cells develop into mesenchymal-like cells in co-culture conditions, which can been blocked by FLP in part;3) For wound healing experiment, under normal culture medium conditions, compared with blank group, in addition to the CTX group, the rest of FLP groups can reduce the migration distance (P<0.05); under the conditional culture medium conditions, the FLPM and FLPH groups can reduce the migration distance (P<0.05).4) For transwell migration experiment, compared with blank group, FLP treatment reduces the cell numbers of A549cells under co-culture conditions(P<0.05).5) For transwell invasion experiment, compared with blank group, FLP treatment reduces the cell numbers of A549cells under co-culture conditions(P<0.05).6) Under co-culture conditions, the concentration of TNF-α、IL-1β、IL-6、TGF-β1are increasing(P<0.05), but FLP reduces the concentration of TNF-α;7) Under co-culture conditions, the mRNA levels of CHUK、IKBKB、NFKBIA、RelA are significantly elevated(P<0.05); FLP treatment decreases the gene expression of CHUK、 IKBKB and Re1A, but elevates NFKBIA(P<0.05);8) Under co-culture conditions, the total protein levels of NF-κB p65、p-IKKα/β、p-IκBa increase in A549cells (P<0.05),IKKβ、IκBα protein level decrease(P<0.05), IKKa level has no changes; FLP reduces the expression of IKKα p-IKKα/β.p-1κBa(P<0.05), and elevates the expression of IκBa(P<0.05),and has no effect on IKKβ and NF-κB p65; The level of p-NF-KB p65in cytoplasm and the nucleus also increase(P<0.05), but FLP reduces both of them and the ratio of nucleus/cytoplasm;9) For NF-κB p65nuclear translocation experiment:compared with A549+NRS group, co-culture increases nuclear translocation(P<0.05); FLP decreases the nuclear translocation under co-culture conditions(P<0.05); compared with A549+NRS, A549+NRS+TNF-α increases nuclear translocation(P<0.05), BAY11-7082decreases the nuclear translocation (P<0.05); compared with co-culture+NRS group, co-culture+NRS+BAY11-7082also decreases the nuclear translocation(P<0.05);10) Under co-culture conditions, the mRNA expression of SNAIL1、ZEB1、CTNNB1、 FLRT1、CDH2、VIM increase(P<0.05),the expression of TJP1decrease(P<0.05),the expression of SNAIL1、CLDN1、CDH1has no changes; FLP reduces the expression of CTNNB1、FLRT1、CDH2(P<0.05) and elevates the expression of SNAIL2、TJP1、CLDN1、 CDH1、VIM(P<0.05),and has no effect on SNAIL1and ZEB1;11) Under co-culture conditions, the expression of Snail、N-cadherin、Vimentin of A549cells increase(P<0.05) and the expression of E-cadherin decrease(P<0.05), the expression of Slug、ZEB1has no changes; FLP reduces the expression of Snail、N-cadherin、 Vimentin(P<0.05) and elevates the expression of E-cadherin(P<0.05),and has no effect on Slug and ZEB1;12) Immunofluorescence experiment shows that E-cadherin expression on the cell membrane and cytoplasm and most of them expression on the cell membrane; N-cadherin expression on the cell membrane and cytoplasm and most of them expression in cytoplasm; Vimentin expression within the cytoplasm; ZO-1expression in cell junction, part of the cell membrane also positive staining. Under co-culture conditions and FLP intervention, FLP has effect on E-cadherin、N-cadherin and Vimentin but no effect on ZO-1.13) Under co-culture conditions, the mRNA expression of MMP-2、MMP-9、PLAU is elevated(P<0.05), but FLP treatment reduces the expression of these genes(P<0.05);14) Under co-culture conditions, the expression of MMP-2、MMP-9in A549cells is elevated(P<0.05), but FLP reduces the expression of these protein levels(P<0.05);15) Under co-culture conditions, the concentration of MMP-2、MMP-9、uPA all increase(P<0.05),but FLP treatment reduces the concentration of uPA(P<0.05);16) Under co-culture conditions, the activation of MMP-2、MMP-9increase (P<0.05),and FLP has no effect on them.Conclusions:1) FLP inhibits A549cell proliferation.2) FLP inhibits A549cell invasion and migration under co-culture conditions.3) FLP decreases the concentration of TNF-a and inhibits NF-κB p65nuclear translocation, which induces EMT;4) FLP decreases MMP-2、MMP-9expression in A549cells and decreases the concentration of uPA; but has no effect on the concentration of MMP-2、MMP-9and their activation. |
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