20(S)-Protopanaxadiol Ginsenosides InhibitA549 Cells Proliferation Through EGFR-MAPK Signaling Pathway

Ginseng has the effects of tonifying vitality,quenching thirst,tonifying and nourishing blood.It has a long medicinal history in China.The Food Safety Law of the People's Republic of China and New Resource Food Management Measures by the Ministry of Health in 2012 approved ginseng(artificially planted)as a new resource food,providing a new direction for the development and application of ginseng.Ginsenoside,as one of the main active components in ginseng,has a variety of pharmacological activities.After phosphorylation and activation of epidermal growth factor receptor(EGFR),it can regulate the process of cell proliferation,apoptosis,cycle and invasion.EGFR is overexpressed or abnormally activated in tumor cells,which induces the occurrence and development of tumor cells.Lung cancer ranks first in cancer mortality,and is mainly non-small cell lung cancer(NSCLC).Therefore,it is of great significance to screen compounds targeting EGFR to inhibit the proliferation of lung cancer cells from ginsenosides.This research provides new directions and ideas for the application of ginseng,and promotes the development of ginseng industry in Jilin Province.The main research contents and conclusions of this paper are as follows:(1)The homogeneous time-resolved fluorescence(HTRF)screening platform was established at the molecular level to screen highly active and selective EGFR-TKIs from ginsenosides.After optimizing ATP concentration,EGFR kinase concentration,and reaction time,the reaction conditions were determined as follows:EGFR kinase concentration 0.0041 ng/?L,reaction time 40 min and ATP concentration 1.41?M.Staurosporine was selected as the positive control to verify the accuracy of the screening platform.Finally,ginsenosides of 20(R)-Rg3,20(S)-Rg3,20(R)-Rh2,20(S)-Rh2,Rc,CK,20(R)-PPD,20(S)-PPD,20(R)-Rg2,F1,20(R)-PPT,20(S)-PPT,and pseudoginsenoside Rh2 were screened to inhibit the activity of EGFR kinase.Panaxadiol ginsenosides showed better inhibitory effect than protopanaxatriol ginsenosides.At the cell level,the anti-proliferative activity of ginsenosides screened by HTRF were tested.Based on A549 human NSCLC cell model,ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD with obvious inhibitory effect were finally selected by MTT assay.Meanwhile,MTT assay of MRC-5 human normal embryonic lung fibroblasts confirmed that ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD did not inhibit the growth and proliferation of normal lung cells.The inhibitory effects of ginsenosides20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD on IC₂₀,IC₄₀,and IC₆₀ of A549 cells were chosen for the further studies.These three ginsenosides belong to protopanaxadiol ginsenosides and are metabolites of each other.At the simulation level,through molecular docking and molecular dynamics simulation,it was confirmed that EGFR protein could stably bind to ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD through hydrophobic interaction and hydrogen bonding,and the binding stabilities sorted from high to low were 20(S)-Rg3,20(S)-PPD,and 20(S)-Rh2.(2)Ras-Raf-MEK-ERK cascade signal is one of the important components of MAPK pathway to regulate the physiological processes of cell proliferation,differentiation,apoptosis,migration,and invasion.In this study,real-time fluorescence quantitative PCR and Western blot were used to explore the effects of different concentrations of ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD on the expression levels of EGFR-MAPK signal pathway related m RNA and protein.The results showed that ginsenoside 20(S)-Rh2 could inhibit the phosphorylation of EGFR kinase Tyr1068,while ginsenosides 20(S)-Rg3 and 20(S)-PPD did not inhibit the phosphorylation of EGFR protein through Tyr1068.Ginsenoside 20(S)-Rg3 mainly inhibited Ras and Raf kinase activities.Ginsenoside 20(S)-Rh2 mainly inhibited the phosphorylation of B-Raf and Raf-1,then downregulated the expression of downstream target protein MEK and ERK.Ginsenoside 20(S)-PPD mainly inhibited the phosphorylation of B-Raf,then downregulated the phosphorylation of downstream target protein MEK and ERK.Therefore,ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD could inhibit the proliferation of A549 cells by inhibiting the activation of EGFR-MAPK signal pathway.The inhibitory effects of EGFR target protein were 20(S)-PPD,20(S)-Rh2,20(S)-Rg3,respectively.The cell migration was measured by cell scratch test.Cells were photographed after 0,6,12,and 24 hours of culture.The results showed that when treated with ginsenoside 20(S)-Rg3 for 24 hours,the healing rate of A549 cells decreased significantly compared with DMSO control group in a dose-dependent manner,from24.63%to 21.92%(24?M),19.66%(30?M),and 17.10%(37?M).When ginsenoside20(S)-Rh2 was treated for only 6 h,the healing rate of A549 cells decreased significantly compared with DMSO control group,only 6.76%(22?M)and 6.35%(35?M),respectively.When treated with ginsenoside 20(S)-PPD for 12 hours,the healing rate of A549 cells decreased from 17.00%to 9.37%(36?M),13.41%(41?M),and14.22%(46?M)compared with DMSO control group.In brief,ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD could inhibit cell migration.The inhibitory effect was time and concentration dependent,in brief,the inhibitory effect was better under the conditions of long time and high concentration.(3)The effects of ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD on A549cells apoptosis were detected by flow cytometry.The cells were stained with Annexin V-FITC and finally detected by flow cytometry.The flow detection data were analyzed by Flow Jo software.The results showed that after ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD treatment,the overall apoptosis rate increased,and mainly affected the number of late apoptotic cells.In addition,real-time fluorescence quantitative PCR and Western blot were used to explore the effects of ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD on the expression levels of m RNA and protein related to the apoptosis signal pathway.The results showed that ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD could promote A549 cell apoptosis by regulating the expression levels of key factors such as Caspase-9,Caspase-3,PARP1,STAT3,Bcl-XL,and Survivin.(4)The effects of ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD on A549cells cycle were detected by flow cytometry.The cells were stained with propidium iodide(PI)and finally detected by flow cytometry.The flow detection data were analyzed by Flow Jo software.The results showed that ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD could block the cell cycle in G₀/G₁ phase and inhibit the proliferation of A549 cells.The inhibitory effects on cell cycle sorted from high to low were 20(S)-PPD,20(S)-Rh2,20(S)-Rg3,respectively.In addition,real-time fluorescence quantitative PCR and Western blot were used to explore the effects of ginsenosides 20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD on the expression levels of m RNA and protein related to cell cycle signal pathway.The results showed that ginsenosides20(S)-Rg3,20(S)-Rh2,and 20(S)-PPD could regulate the expression levels of Cyclin,CDK,Cdc25,and other key factors in cell cycle signal pathway.The contents of complexes formed by Cyclin A2,Cyclin D1,Cyclin E1 and CDK protein were mainly downregulated.Therefore,the transformation of cell cycle to S phase was inhibited and cell cycle was blocked in G₀/G₁ phase.