Study On The Antitumor Material Basis Of Solanum Lyratum And Its Intervention With Exosomes To Inhibit Metastasis Of Non-small Cell Lung Cancer

Tumor metastasis is a great challenge in the clinical treatment of non-small cell lung cancer(NSCLC).The abnormal activation of epithelia-mesenchymal transition(EMT)of tumor cells is a key mechanism that promotes lung cancer metastasis.And tumor-derived exosomes(TDEs)play a very critical role in the process of EMT and cancer progression,as well as in invasion and metastasis of tumor cells.As the frontier and hotspot of tumor research,the formation process,structure and function of exosomes are closely related to membrane lipid raft.Solanum lyratum as a Chinese herb,has the function of clearing heat and detoxicating,and is often used to treat a variety of malignant tumors.S.lyratum is rich in a great many bioactive components,and the steroidal alkaloids contained in it have been found to have significant anti-tumor activity.Previous studies have shown that total alkaloids from S.lyratum(TAS)had significant anti-NSCLC effect.Meanwhile,it has been proved that the steroidal alkaloids from it had the unique anti-tumor mechanism of agglutinating cholesterol of lipid rafts,regulating the formation and function of tumor exosomes,and inhibiting tumor angiogenesis.On this basis,combined with the key role of TDEs in tumor metastasis,we proposed to carry out the research on the anti-tumor mechanism of the regulation of exosomes mediated EMT to inhibit NSCLC metastasis.This study is divided into two parts.Based on our previous studies,this study aims to make further research on the anti-tumor metastasis effect of TAS and its material basis.And combined with the close relationship between TDEs and lipid rafts of cell membrane,the anti-NSCLC metastasis of EMT mediated by steroidal alkaloids from S.lyratum was studied from the aspects of cell function and molecular mechanism.1.Study on the chemical constituents of the total alkaloids from S.lyratumObjective:To study the alkaloids of Solanum lyratum to clarify the anti-tumor material basis of TAS.Methods:ODS and silica gel column chromatography combined with HPLC were used to separate and purify the alkaloids.The structures of the isolated monomeric compounds were identified by UHPLC-ESI-Q-TOF-MS,infrared spectroscopy,1D and 2D NMR and available literatures.Results:Thirty-three monomers,including twenty-nine steroidal alkaloids(1-28,31),one phenylpropanamide(29),one isoquinoline alkaloid(30),and two aliphatic hydrocarbons(32,33),were isolated and identified from S.lyratum.Among them,compounds 1-16 were new compounds,all of which were steroidal alkaloids,named Solalyraine J,Solalyraine K,Solalyraine L,Solalyraine M,Solalyraine N,Solalyraine O,Solalyraine P,Solalyraine Q,Solalyraine R,Solalyraine S,Solalyraine T,Solalyraine U,Solalyraine V,Solalyraine W,Solalyraine X,Solalyraine Y,respectively.And compounds 30-33 were isolated from S.lyratum for the first time.2.Study on the inhibition of tumor metastasis by the intervention of steroidal alkaloids from&lyratum in A549 cell-derived exosomes.Objective:To investigate the molecular mechanism of steroidal alkaloids from S.lyratum interfering with TDEs to inhibit NSCLC metastasis.Methods:CCK8 assay was used to detect the effects of six neosteroidal alkaloids(GS1,GS2,GS3,GS4,GS5 and GS6)on the proliferation of human lung adenocarcinoma A549 cells.The exosomes of A549 cells were extracted by ultracentrifugation,and identified by Western blot,transmission electron microscopy and nano-particle size analysis.Migration,invasion and adhesion experiments in vitro were used to study the effects of steroidal alkaloids on invasion and metastasis of A549 cells.And the expression levels of N-cadherin,E-cadherin and Vimentin as well as p-JAK2,JAK2,p-STAT3 and STAT3 were detected by Western blot.Results:After 48 h of treatment,GS1,GS2,GS3,GS4,GS5 and GS6 showed a weak inhibitory effect on A549 cells,but there was an obvious tendency to restrain it.Therefore,GS2 was selected for subsequent experiments according to the previous results and literature reports.The exosomes of A549 cells isolated by ultracentrifugation were in the shape of concave hemispheric or saucer-like vesicles.The expression of CD9 and TSG101 were positive in TDEs.Before and after intervention with GS2,the particle size of TDEs secreted by A549 cells mainly ranged from 50 nm to 80 nm.After 48 h of treatment,the concentration of exosomes in the supernatant of A549 cells increased by 1.75 times compared with that without intervention.Compared with blank control and blank exosome groups,A549 cell-derived exosomes treated by GS2 significantly inhibited the migration and invasion effects(P<0.001,P<0.001),as well as adhesive ability of A549 cells(P<0.01).And GS2 significantly up-regulated the expression of E-cadherin(P<0.01),down-regulated it of N-cadherin and Vimentin(P<0.001,P<0.001),which were EMT marker proteins,and down-regulated the expression of p-JAK2 and p-STAT3(P<0.01,P<0.01).Conclusion:In vitro experiments showed that GS2 had the activity of inhibiting tumor metastasis.The intervention of GS2 made the exosomes of A549 cells have significant anti-metastasis activity in vitro,and may regulate exosomes to inhibit NSCLC metastasis by inhibiting the phosphorylation of JAK2/STAT3 signaling pathway and EMT process.