Anti-cancer Effects Of Verbascoside On Glioma Cells And Its Underlying Mechanism

BackgroundGlioblastoma(GBM)is the most common primary CNS tumor,accounting for 51%of all gliomas.Glioblastoma is the most malignant glioma of grade IVdefined by WHO.For decades,it has been known that some gliomas of lower WHO grade can recur,progress,or transform into GBM.Even with aggressive surgical resections,along with recent advances in radiotherapy and chemotherapy,the prognosis for GBM patients remains dismal.New therapy method and drug need to be explored.The verbascoside is the main active ingredient in cistanche deserticola.Previous study has found that it has the pharmacological effects such as immunomodulation,anti-inflammatory and anti-oxidation.In recent years we found verbascoside can inhibit liver cancer,colon cancer,gastric cancer and some other tumors,but its role in glioma has not been reported.Moreover,it has been previously found that verbascoside enhances the expression of SHP-1 in activated macrophages.The SHP-1/STAT3 signaling pathway plays an important role in the development and progression of glioblastoma.Therefore,we wonder whether it can affect the expression of SHP-1 in glioblastoma and thus inhibit the glioblastoma.Purpose1.To investigate the effects of verbascoside on the proliferation,apoptosis,migration and angiogenesis of glioblastoma.2.To investigate the effect of verbascoside on SHP-1/STAT3 signaling pathway.3 To investigate whether verbascoside can enhance the anti-glioblastoma effect of temozolomide.4.To investigate whether verbascoside can inhibit the growth of gliomas in subcutaneous tumorigenesis model in nude mice.MethodsThe proliferation of glioblastoma was treated with different concentrations of verbascoside.MTT assay was used to detect the effect of VB on cell proliferation,and the inhibition rate was calculated.Apoptotic cells were detected by Annexin V-FITC/PI double staining cells and flow cytometry.Transwell was used to detect cell migration and invasion.Western blotting was used to detect the expression of SHP-1/STAT3 signaling pathway.And transfection of SHP-1 siRNA,we observed whether the verbascosid reverse the biologic behavior of glioblastoma and the expression of related proteins.Verbascoside was combined with temozolomide to study whether verbascoside can enhance the function of temozolomide against glioblastoma.Nude mice were transplanted into U87 tumor cells and then injected intraperitoneally with PBS and verbascoside to observe whether verbascoside would inhibited glioblastoma cell growth in vivo.Statistical analysis All data were expressed as mean ± standard deviation.SPSS 13.0 statistical software was used to test the positive and variance homogeneity of the measured results.The data are presented as the means ± SD.Statistical analyses were conducted using one way ANOVA.The level of significance a=0.05,P<0.05 was considered statistically significant.The student t-test was used to compare two independent groups’ means.Statistical significance was established at the P<0.05.Results1.Verbascoside inhibited cell proliferation and induced apoptosis in U87 and U251 cells.MTT assay was used to detect the effects of different concentrations and time of verbascoside on cell proliferation.It was found that verbascoside inhibited glioblatoma proliferation(P<0.05).And the drug concentration and time has an interaction effects.At the same time different concentrations and time of verbascoside promoted U87 and U251 apoptosis.Cells were treated by different concentrations of verbascoside and detected by transwell assay,and verbascoside attenuated the ability of cells to migrate into the lower chamber.Western blot analysis showed that inhibited STAT3 phosphorylation of glioblastoma and increased the expression of SHP-1.And verbascoside changes the expression of downstream molecules,the effect can be partially reversed by SHP-1 siRNA.Transfection of SHP-1 siRNA reverse the effect of verbascoside on glioblatoma proliferation,apoptosis and migration.Western blot analysis showed that the related protein had changed accordingly.2.Verbascoside enhanced the inhibitory effect of temozolomide on glioblastoma proliferation and promoted apoptosis.MTT assay showed that the inhibittory rate of was significantly higher than that of TMZ group.Flow cytometry showed that apoptosis rate of(TMZ+ VB)group was significantly higher than that of TMZ group.In the protein level,combination group can increase the expression of pro-apoptotic protein,reduce the expression of anti-apoptotic protein.3.Verbascoside inhibited the growth of xenografts in nude mice and affected the expression of related proteins,and had no effect on the weight of nude miceThe tumor volume of the VB group was smaller than that of the control group.And the quality of VB group was lower than that of control group.Nude mice had no difference in body weight.The protein was extracted to detect the proapoptotic protein and anti-apoptotic protein by western blot.conclusion1.Verbascoside can inhibit the phosphorylation of STAT3 by up-regulating the expression of SHP-1,which affects the expression of downstream target gene and inhibit the proliferation,migration and apoptosis of glioblastoma.Verbascoside also inhibits the growth of gliomas in subcutaneous of nude mice2.Verbascoside enhances the anti-glioblastoma effect of temozolomide.3.Verbascoside can inhibit the tumor grouth in nude mouse.