The Study Of The Effect Of Fingolimod On Proliferation,Invasion And Migration Of Chordoma And Related Mechanisms

Purpose: Chordoma is the most common primary malignant tumor in the spine,accounting for about 1-4% of all bone malignancies.Chordoma is not sensitive to traditional chemotherapy and radiation therapy,and there are many complications and high recurrence rate in Surgical treatment.Therefore,the treatment of chordoma requires new effective treatment strategies and methods.FTY720 is a new type immunosuppressant.A number of studies have shown that it has a good antitumor effect and can inhibit the proliferation and metastasis of various malignant tumors.However,there have been no reports of FTY720 in chordoma.This study explores the antitumor effect of FTY720 on chordoma and related mechanisms in vivo and in vitro,in order to find a new targeted therapy for chordoma treatment,and provides a theoretical basis for the clinical application of FTY720 in chordoma.Methods: In the in vitro test,the two most common sacral chordoma cell lines MUG-Chor1 and U-CH1 and human osteoblast cell line hFOB1.19 were used in this study.After these cell lines were treated with FTY720,the proliferation ability was analyzed by MTS proliferation detection experiments.The colony forming ability of MUG-Chor1 and U-CH1 was tested by colony formation experiments.The proliferation of two cell lines was further observed using EDU cell proliferation imaging technology.After MUG-Chor1 and U-CH1 were treated with FTY720,Cell migration experiments were used to observe the migration ability of the two cell lines.Transwell invasion experiments were used to detect the invasion ability of MUG-Chor1 and U-CH1.Further,the cell apoptosis rate was analyzed by flow cytometry using Annexin V-FITC / PI staining.RNase / PI staining was used to detect cell cycle progression of both cells.In order to study the mechanism of FTY720 antitumor effect,the expression of IL-6,p-STAT3 and STAT3 were detected by Western-blot.Further,Chordoma cells MUG-Chor1 and U-CH1 were given FTY720 at the same time under the condition of high expression of IL-6.The previous experimental methods were implemented to study the relationship between the IL-6 / STAT3 pathway and the antitumor effect of FTY720.In vivo experiments,6-week-old BALB /cfemale immunodeficiency nude mice were used as experimental animals,and chordoma cells U-CH1 were planted under the skin of nude mice.After the tumor grew to a certain size,FTY720 was injected into the tail vein of nude mice.After 3 weeks of feeding,the tumor volume and weight were calculated,and chordoma tissue sections were made to observe the morphological changes of cells.Western-blot method was used to detect the expression of IL-6,p-STAT3,and STAT3 in chordoma tissues of nude mice.Results: 1.The results of MTS experiments show that FTY720 can significantly reduce the survival rate of chordoma cells MUG-Chor1 and U-CH1 with obvious time dependence and dose dependence.In addition,colony formation experiments show that FTY720 is effective in decreasing the density and volume of MUG-Chor1 and U-CH1 chordoma cell colonies.The percentage of MUG-Chor1 and U-CH1 cells in the proliferating state decreased significantly after FTY720 treatment by Edu imaging.The above results indicate that FTY720 can inhibit chordoma cell proliferation.2.The results of cell migration experiments showed that the migration distance of MUG-Chor1 and U-CH1 cells was significantly reduced after FTY720 treatment.Transwell invasion experiments showed that the number of invaded cells of MUG-Chor1 and U-CH1 after FTY720 treatment was significantly less than that of the control group.The above results indicate that FTY720 inhibits the invasion and migration of chordoma cells.3.Flow cytometry was used to analyze the apoptosis rate and cell cycle progress using Annexin V-FITC/PI and RNase I/PI staining.It was found that after FTY720 treatment,the apoptosis rate of MUG-Chor1 and U-CH1 cells increased significantly,and the ratio of the two cells in the G0/G1 phase increased significantly after FTY720 treatment with the ratio in the S phase decreasing significantly.4.Western-blot results showed that after FTY720 treatment,the E-cadherin were significantly increased,while the expression of vimentin was significantly decreased,and the expression of the IL-6/STAT3 pathway was significantly reduced.Further,MUG-Chor1 and U-CH1 were treated with IL-6 and FTY720 together.Compared with FTY720 treatment alone,the survival rate,migration distance,and number of invading cells were significantly increased while the Apoptosis and cell cycle arrest are suppressed.These show that IL-6 is related to FTY720's inhibition of cell proliferation,invasion and migration,and FTY720's induction of apoptosis and cycle arrest.5.In animal experiments,chordoma cells were planted under the skin of nude mice.Compared with the control group,the volume and weight of chordoma tissues were significantly reduced in nude mice treated with FTY720,and The expression of IL-6/STAT3 signalling decreased significantly.Conclusion: 1.FTY720 can inhibit the proliferation,invasion and migration ability of chordoma cells.2.FTY720 inhibits the growth of chordoma cells by inducing cell apoptosis and cell cycle arrest.3.FTY720 inhibits the invasion and migration of chordoma cells by inhibiting the EMT process of chordoma cells.4.FTY720 can exert antitumor effect by inhibiting IL-6/STAT3 pathway in the treatment of chordoma.5.FTY720 can inhibit the growth of chordoma subcutaneously implanted tumors.