Curcumin Inhibits Cell Proliferation And Metastasis In NSCLC Through A Synergistic Effect Of The TLR4/MyD88 And EGFR Pathways

BackgroundLung cancer is the first high incidence of malignant tumor in China,local recurrence and distant metastasis have been the main factors affecting the prognosis of lung cancer.Searching for a drug with multiple effects to inhibit tumor proliferation and metastasis with few side effects is a key factor to further improve the prognosis of lung cancer.Numerous studies have shown that curcumin is a multipotent drug with antioxidant,antibacterial,anti-inflammatory and anticancer activities.Previous studies have found that curcumin inhibits lung cancer growth and is closely related to TOLL pathway and EGFR pathway,but the mechanism has not been elucidated so far,which is worthy of further studying.Objective1.To study the effects of curcumin on proliferation,invasion and migration of NSCLC;2.To study the mechanism of curcumin inhibites proliferation and metastasis of NSCLC cells;3.To clarify the role and mechanism of curcumin on TLR4/MyD88 pathway;4.Analysis of the expression characteristics and correlation of TLR4 and MyD88 in NSCLC tissues;the relevance between TLR4 and Myd88 were analyzed.Method1.Function in vitro experiment which is to investigate the effect of curcumin on the proliferation and metastasis of non-small cell lung cancer.(1)The effect of curcumin on the cell proliferation of NSCLC is detected by MTS test and plate cloning assay;(2)The effect of curcumin on cycle of NSCLC cells is detected by flow cytometry;(3)The effect of curcumin on the migration ability of NSCLC cells is detected by Transwell chamber migration assay.2.Concrete mechanism of curcumin on biological function of lung cancer cells(1)Western blotting is detected to the change of cell cycle associated protein and related transcription factor,and to clarify the molecular mechanism of curcumin regulating cell proliferation;(2)Western blotting is detected to the change of EMT associated protein,and clarify the molecular mechanism of curcumin regulating the metastasis of NSCLC cells.3.Curcumin regulates TLR4/MyD88 and EGFR pathwaysAfter Western blotting detection of curcumin and treatment of NSCLC cells,the expression of TLR4/MyD88 and EGFR are changed;subsequently to add the stimulation of EGF,and detect the expression changes of TLR4/MyD88 and EGFR at different time points.4.The high expression of TLR4/MyD88 in patients with NSCLC and its relationship with prognosisThe expression of immunohistochemical detection of TLR4 and MyD88 in non-small cell lung cancer(NSCLC)and lung benign lesions,and analyze the relationship of expression level with the clinical parameter and prognosis;The correlation between TLR4 and MyD88 was analyzed.Result1.Curcumin inhibites proliferation in vitro,cloning formation,invasion and metastasis of NSCLC cells.2.The expression of the cycling A1、A2、B1 and cyclin-dependent kinase inhibitors(CDK1)are down-regulated after the treatment of curcuminin,and blocked the cell cycle arrest in G2-M phase.At the same time,the expression of E-cadherin expression is up-regulated and the expression of while Vimentin,TCF8,Snail and Slug are down-regulated,suggesting that the process of EMT process is reversed.In addition,the expression of related transcription factor AP-1 family proteins such as c-Jun,c-Jun ser63,JunB,JunB ser259,c-fos and c-fos ser32 related to cell cycle,which related to the cell cycle and EMT regulation are all decreased.It is suggested that curcumin can induce G2-M phase arrest and EMT reverse in NSCLC through down-regulating the expression of AP-1 family protein.3.Curcumin inhibites the expression of TLR4,MyD88 and EGFR.After the stimualtion of EGF,it can reverse the inhibition of curcumin on TLR4 signaling pathway.4.The positive rate of TLR4 and MyD88 in NSCLC tissues are significantly higher than those in benign tissues(p<0.001).High expression of TLR4 is a poor prognosis factor for NSCLC patients;The expression of TLR4 is positively related to MyD88 in NSCLC.ConclusionCurcumin can inhibit the expression of AP-1 by regulating the interaction between TLR4/MyD88 and EGFR pathways,and then to regulate the expression of cyclin and epithelial-mesenchymal transition protein,so that to inhibit the proliferation and metastasis of non-small cell lung cancer cell.The high expression of TLR4/MyD88 in non-small cell lung cancer may be significantly related to prognosis of patients with NSCLC.