Anti-Tumor Activity Of Toosendanin And Its Underlying Mechanism Of Promoting Cell Apoptosis In Glioblastoma

Glioblastoma（GBM）is the most common primary malignant brain tumor in clinic.It is extremely prone to relapse and GBM is one of the poorest prognostic human tumors.According to the symptoms or pathological feature of the lesion,it can be classified as "headache","epilepsy","convulsions" or "amassment disease","carcinoma" in traditional Chinese medicine.The poor prognosis of GBM is mainly due to the high malignant degree of the tumor and resistance to conventional anti-cancer treatments.Therefore,it is urgently needed to discover new cytotoxic drugs.According to traditional Chinese medicine,brain is to "clear orifices".The pathogenesis of GBM is closely related to "Turbid Yin".TSN,proved to be able to penetrate the blood-brain barrier and the main active component of Melia toosendan Sieb.et Zucc.,which has been used in the treatment of multiple tumors.However,the relationship between TSN and GBM has not been reported.In this study,we evaluated the anti-cancer efficacy of TSN to glioma in vivo and in vitro.By using a panel of biological experiments,we found that TSN treatment readily reduces the tumor burden in the BALB/c-nu athymic nude mice.In addition,MTT assay,colony-forming assay,flow cytometry analysis and Western blot experiments showed that TSN treatment markedly inhibits U87 MG and C6 cell proliferation and induces apoptosis,with IC₅₀ concentration as low as 10 n M.Further pharmacological and molecular studies show that estrogen receptor β（ERβ）and functional p53 are the prominent targets for TSN.In conclusion,TSN is a new candidate drug for GBM.ERβ and functional p53 can be used as predictive biomarkers for TSN in GBM.Methods:1.At in vivo level,we used the xenograft model by seeding GBM cells in nude mice.The mice were fed with TSN or vehicle.At the end of experiment,the xenograft tumor was carefully isolated and the anti-cancer efficacy of TSN was evaluated by HE,IHC,TUNEL and Ki67 assay,respectively.2.At in vitro level,MTT assay,colony-formation experiment,flow cytometry analysis,Western blot were used to evaluate the cytotoxicity of TSN on U87 MG and C6 cell lines.3.To explore the mechanism which underlies the cytotoxicity of TSN in GBM,we tested the cytotoxicity of TSN in another GBM cell line,T98 G.MTT,flow cytometry and Western blot were used to determine the anti-cancer activity of TSN in T98 G cells.4.By screening the genetic background of indicated GBM cell lines,we wish to identify biomarkers that predict sensitivity of TSN.By using pharmacological and molecular approaches,we interfered ERβ signaling in U87 MG cells by either si RNA or PHTPP.We also rescued ERβ expression in the ERβ-deficient T98 G cells.The anti-cancer activity of TSN in these indicated cells was determined as we have described previously.5.To investigate the role of p53 in TSN’s anti-cancer efficacy,we knockdown endogenous expression of p53 in U87 MG cells,or overexpressed its expression in T98G/ERβ cells,respectively.Western blot and flow cytometry analysis were used to determine the cytotoxicity of TSN in the resultant cells.6.By using PHTPP/si ERβ inhibiting ERβ in U87 MG cells,the expression of p53 was detected after TSN treatment;In contrast,wild-type p53 was transfected into T98G/ERβ cells and the drug resistance of TSN was observed.Results:1.In comparison with vehicle control,TSN treatment readily reduced tumor burden,tumor weight and pathological scores（^*p < 0.05）.IHC staining experiments also showed TSN treatment inhibited cancer cell proliferation and promoted apoptosis（^*p < 0.05）.2.U87 MG and C6 cells were treated with increasing concentrations of TSN,MTT assay showed that the IC₅₀ value of TSN were 12 n M and 8 n M,respectively.Moreover,10 n M TSN readily promoted U87 MG and C6 cell apoptosis（^*p < 0.05）.3.In contrast to U87 MG and C6 cells,the ERβ-deficient T98 G cells were resistant to TSN,and the treatment failed to elicit anti-cancer activity（p > 0.05）.4.By interfering ERβ expression in U87 MG and C6 cells that express endogenous ERβ,we found that inhibition of the ERβ signaling antagonized TSN’s anti-cancer efficacy.However,overexpression of ERβ in the ERβ-deficient T98 G cells（T98G/ERβ）was not sufficient to elicit cytotoxicity after TSN treatment.5.si RNA-mediated knockdown of p53 in U87 MG cells antagonized TSN’s anti-cancer efficacy.In agreement with these findings,overexpression of functional p53 in the p53 mutant T98G/ERβ cells would enhance the anti-cancer efficacy of TSN.6.After using PHTPP/si ERβ inhibiting ERβ in U87 MG cells,the expression of p53 did not increase;In contrast,the sensitivity of T98G/ERβ/p53 cells to TSN was restored.Conclusion:Our study highlighted the anti-cancer efficacy of TSN in vitro and in vivo,regardless of species origin.These findings strongly indicated that TSN might be a candidate drug for GBM treatment.Biochemical and molecular experiments showed that TSN selectively kills ERβ-positive GBM cells,while TSN failed to elicit anti-cancer efficacy in ERα-positive GBM cells.In agreement with these findings,we anticipated that ERβ would be used as a biomarker for predicting the sensitivity of TSN in GBM patients.In addition,we found p53 as a pivotal molecule in mediating TSN’s cytotoxicity.Activation of the ERβ-p53 signaling would be responsible for the anti-cancer efficacy of TSN.Collectively,our study would promote further in-depth investigation of TSN in treating GBM patients in the clinic.