The Therapeutic Effect Of Silibinin On Leptomeningeal Metastasis-prone Cells From Lung Cancer

Objective: Leptomeningeal Metastasis(LM)is a typical metastatic tumor of the central nervous system,which is caused by distant solid tumor metastasis or the spread of malignant brain tumors.The primary lesion is often lung cancer,and adenocarcinoma is common histological type.Although many drugs have been used clinically,including the use of some molecularly targeted drugs,the prognosis of patients is still very poor so far.Silibinin belongs to flavonoids and is an important active ingredient in Silymarin seed extract.It has been used as a liver protection drug in clinical practice for a long time.Later studies have found that silibinin has a good effect on the inhibition of malignant tumors,and it is a natural anti-cancer drug with low toxic and side effects.Therefore,this study adopted silibinin as a drug for the treatment of LM,mainly to observe whether silibinin has effect on leptomeningeal metastasis-prone cells of lung cancer.In this experiment,the anti-tumor efficacy of silibinin was observed through the effects of silibinin on the viability,proliferation,cloning,migration,invasion and apoptosis of LeptoM-LLC cells in vitro,and certain experimental basis for subsequent drug research in mouse models.Methods:1.cellLewis lung carcinoma cell line(LLC)is a lung adenocarcinoma cell from mice.According to the methods of foreign scholars,LLC cells are used to establish lung adenocarcinoma cell line with leptomeningeal metastasis(LeptoM-LLC),which is presented by Memorial Sloan Kettering Cancer Center Laboratory in the United States.2.CCK-8 assay was used to detect cell activityLeptoM-LLC cells were inoculated into 96-well plates.After the cells grew to a certain density,blank group,control group and experimental group were set up,each group was set up with 6 rewells,and treated with silibinin at different concentrations and for different time periods.CCK-8 reagent was added,and the absorbance at 450 nm was measured with a microplate analyzer to show the cell activity.And choose the appropriate concentration for subsequent experiments.3.The cloning experimentsLeptoM-LLC was inoculated on the 6-well plate,the control group and the experimental group were set,and different concentrations of silibinin were given for intervention.When each clone reached more than 50 cells,paraformaldehyde was added to fix the cells and GIEMSA staining was performed.The number of clones in each group was observed.4.Migration and invasion experimentsLeptoM-LLC cells were inoculated in the upper chamber and the lower chamber was filled with subculture medium in culture plates using Transwell chamber.Different from the migration experiment,the invasion experiment required laying a layer of artificially reconstructed basement membrane on the polycarbonate membrane of the Transwell chamber,and then inoculating the cells into the upper Transwell chamber.The control group and the experimental group were set,each group had 3 replicates,and different concentrations of silibinin were given.To observe the ability of tumor cells to pass through polycarbonate membrane or polycarbonate membrane +artificial reconstruction of basement membrane under the action of silibinin,which reflects the ability of cell migration and invasion,respectively.5.Cell apoptosis was detected by TUNEL assayLeptoM-LLC cells were seeded on a 14 mm cell slide and placed in a24-well plate.After the cells adhered to the wall,the control group and the experimental group were set,each group had 3 replicates,and different concentrations of silibinin were given respectively.The TUNEL kit was used for cell patch fluorescence staining,and the apoptotic cell content in different groups was observed under fluorescence microscope,which showed the effect of TUNEL on cell apoptosis.Results:1.After treatment with silibinin for 24 h,48h and 72 h,the cell viability of LeptoM-LLC cells was gradually decreased,and the cell viability was gradually decreased with the increase of silibinin concentration,and the difference between the multiple groups was statistically significant(P<0.001).When the concentration of silibinin was 20μg/ml,the cell viability increased with time,and the difference was not statistically significant(P=0.242;P = 0.052).2.In the experimental group,40μg/ml and 80μg/ml of silibinin decreased the colony formation ability of LeptoM-LLC cells.With the increase of silibinin concentration,the colony formation ability of LeptoM-LLC cells decreased.The comparison of multiple groups was statistically significant(P<0.05).3.Compared with the control group,the number of cells passing through the microporous membrane decreased in the experimental group(40μg/ml,80μg/ml),and the number of LeptoM-LLC cells passing through the microporous membrane decreased gradually with the increase of the concentration of silibinin.Silibinin significantly reduced the migration ability of LeptoM-LLC cells.The comparison of multiple groups was statistically significant(P<0.05).In the invasion experiment,there were relatively more cells in the microporous membrane.The number of LeptoM-LLC cells in the experimental group(40μg/ml,80μg/ml)passing through the matrigel and microporous membrane was less than that in the control group,and the number of LeptoM-LLC cells passing through the matrigel and microporous membrane gradually decreased with the increase of the concentration of silibinin.Silibinin significantly reduced the invasive ability of LeptoM-LLC cells.The comparison of multiple groups was statistically significant(P<0.05).4.TUNEL staining microscopy showed red fluorescence in apoptosis.The LeptoM-LLC cells in the control group did not show red fluorescence,while the LeptoM-LLC cells in the experimental group treated with silibinin(40μg/ml)showed more obvious red fluorescence.Therefore,silibinin can promote the apoptosis of LeptoM-LLC cells.Conclusions:1.Silibinin inhibits the proliferation of LeptoM-LLC cells in a timedependent and concentration-dependent manner.2.Silibinin inhibits the migration and invasion of LeptoM-LLC cells in a concentration-dependent manner.3.Silibinin induces LeptoM-LLC cells apoptosis.