The Mechanism Of Tubeimoside-1 On The Proliferation And Metastasis In Glioblastoma Cells

Glioblastoma is the most common primary tumor in the brain.Glioblastoma(GBM,grade 4 glioma)is the most invasive human tumor,and there are few effective treatments [1-4].In previous studies,the mesenchymal to epidermal transition factor(MET)was found to play an important role in glioblastoma.Over activited and amplification of the MET gene occurred in many glioma patients [5].Therefore,the therapy target MET become heated and more and more drugs targeting MET gene in clinical trials or used for clinic treatment [6].In China,the use of Chinese medicine has always been a very important part of our disease treatment.However,due to the lack of research on the mechanism of many Chinese medicines,there is a lack of international recognition.Therefore,the research on the pharmacological mechanism of traditional Chinese medicine can provide an important scientific basis for the promotion of traditional Chinese medicine.As a major medical problem in the contemporary world,cancer has always threatened the health of human beings.In this study,the monomer of Chinese traditional herbs extracted from the traditional Chinese medicine rhizoma bolbostemmae tuber has played its anticancer activity in glioma.However,its specific mechanism is not perfect.Therefore,the specific mechanism of its anticancer effect can be further improved through the research of this subject.And provide a potential pathway for the treatment of glioma.The major findings of this research are as follows:1.The effects of tubeimoside-1 on proliferation of glioblastoma cellsWe obtained the appropriate concentration of drug-treated LN229 and U87 cells by consulting the previous research literature [7].The LN229 and U87 cells were then treated in a concentration corresponding to the design.After treated with tubeimoside-1 for 24 hours glioblastoma cells were observed by a microscope.As result,we found that the number of LN229 and U87 cells was significantly decreased as the concentration was increased.To further explore the effect of tubeimoside-1 on the proliferation of glioblastoma.We used the MTT assay to measure the survival curves of the above two cell lines.The results are consistent with the above results.From the experimental results above,we found that tubeimoside-1 could inhibit the proliferation of glioblastoma with increasing concentration gradient.2.Effect of tubeimoside-1 on cell cycle of glioblastoma cellsThe inhibition of cell proliferation by drugs is often reflected in the inhibition of the cell cycle.Therefore,we used the BrdU assay to examine the effect of tubeimoside-1 on DNA replication in glioblastoma cells.The results showed that the addition of tubeimoside-1 inhibited DNA replication in glioblastoma cells,performed by flow cytometry and Western Blot experiments.It was found that tubeimoside-1 blocked the cell cycle of LN229 and U87 in G2/M phase.From the above results,we found that tubeimoside-1 could arrest glioblastoma cells in the G2/M phase,thereby inhibiting cell proliferation.3.Effect of tubeimoside-1 on metastasis of glioblastoma cellsA major feature of glioblastoma is its strong metastatic ability [8].Therefore,we examined the migration and invasion abilities of LN229 and U87 cells after drug treatment by Wound-healing and Transwell experiments.It was found that the migration and invasion ability of glioblastoma cells was significantly inhibited after treatment with tubeimoside-1,and certified by Western Blot experiments which tested the proteins levels of MMP-7,MMP-2,Snail and Slug.It was found that tubeimoside-1 can significantly down-regulate the expression of the above proteins.From the results analysis,tubeimoside-1 could reduce the expression of migration-invasive-related proteins and inhibit the migration and invasion ability of cells.4.Effect of tubeimoside-1 on MET and its important downstream pathwaysIn order to further explore the related mechanism of the effect of tubeimoside-1 on the proliferation and migration of glioblastoma cells.We used the Western Blot assay to detect important proteins in related pathways.The results showed that tubeimoside-1 significantly inhibited the phosphorylation of AKT,ERK protein and inhibited the protein level of MET.As an important tyrosine receptor kinase,MET can significantly inhibit the activity of its downstream related pathways.Therefore,we overexpress MET in LN229 and U87 cells and treat it with drugs.As a result,we found that the above cell line overexpressing MET can significantly attenuate the inhibition of the proliferation and migration of the cells by the dosing.Moreover,the phosphorylation levels of AKT and ERK proteins also showed significant recovery.In order to explore the mechanism of the reduction of MET protein levels by tubeimoside-1,we used real-time PCR and IP experiments.It was found that the decrease in MET protein level was caused by an increase in the level of MET ubiquitination induced by tubeimoside-1.Based on the above experimental results,we found that tubeimoside-1 can down-regulate its protein level by increasing the ubiquitination level of MET,and ultimately affect the activity of MET downstream pathway to inhibit the proliferation and migration of glioblastoma cells.5.The effect of tubeimoside-1 on clonogenicity and tumorigenesis of mice with glioblastoma cells.To further verify the antitumor activity of tubeimoside-1,we designed a subcutaneous tumor formation experiment in mice.From the experimental results,we found that tubeimoside-1 can significantly inhibit the growth of subcutaneous tumors in mice.Furthermore,the analysis of the expression of the tumor growth marker Ki-67 was significantly inhibited in mice by immunohistochemistry,and the inhibition of MET protein levels was also demonstrated in vivo.From the above results,we found that tubeimoside-1 also has significant anti-tumor activity in mice.In summary,tubeimoside-1 can inhibit the proliferation and migration of glioblastoma cells by inducing ubiquitination degradation of MET.Furthermore,it is also possible to target MET in a mouse subendothelial tumor and to exercise its antitumor activity.Through the research of this subject,the anti-tumor activity mechanism of tubeimoside-1 is further clearfied,which can provide a new potential pathway for the treatment of target MET in glioblastoma.