Fluvoxamine Inhibit Glioblastoma Multiforme(GBM) Cells Growing And Metastasis Via Down-regulating Cdc42 And Inhibiting F-actin Polymerization In Vitro

Objective:In this part of study,we researched on the human GBM cell and treated with different concentrations of fluvoxamine to observe the cell-morphology,growing and metastasis,moreover,to investigate the mechanism of the phenomenon.Methods:(1)In our research,different human GBM cells(U87PA,U251)were chosen and applied them to different concentration of fluvoxamine(10uM?15uM?25uM?35uM?45uM?50uM?60uM)and interacted with different time set(6h?12h?24?48h),which were used for "MTT Assay" firstly to test the drug toxicity range.(2)Then carried on the "Wound Healing Assay" with the aboved safe drug range(15?M,25?M)and different time set(Omin,12h,24h)to observe whether fluvoxamine can inhibit the cancer cells growing and metastasis.(3)Flow-Cytometer(FCM)Assay was carried out in different drug concentration(5?M,25?M)to observe influence of F-actin content in single cell.(4)U87 was used for immunochemistry assay,which means the F-actin in cells will be dyed with phaloidin-555,which featured targeting F-actin only and then made observation of cells in different concentrations of fluvoxamine(15uM,25uM,35uM);(5)we tested the qualitative analysis of some F-actin polymerization related small molecule Rho GTPase Family such as,RhoA,cdc42 and Racl via western blotting and to find out its expression changes.Results:(1)In MTT assay,different Fluvo concentrations(10uM?15uM?25uM?35uM?45uM?50uM?60uM)effected the U87 cell line somehow within drug applying time 6h-48h.Compared with control group,OD570nm of Fluvo group decreased and the difference was significant(P<0.05).Moreover,the results presented dose-effect and inhibition=time relationship.Notably,within 3 5uM was the best drug safe range in our reseach.(2)In the wound healing assay,the GBM cells growing and metastasis in the group with fluvoxamine were more than lower than the control group without fluvoxamine(including cells number and growing condition)and it was the positive correlation with drug concentration.(3)The FCM assay inferred that,the fluvoxamine inhibited GBM cells growing and moving by affecting cytoskeletal protein F-actin,which played a significant role in cancer cells metastasis.Actin is always in the balance of polymerization and de-polymerization in cells,we can tell that fluvoxamine suppressed the atcin-polymerization to inhibit the cancer cells growing and invasion.(4)In ICC Assay,the group applied with drug showed F-actin was decreasing and depolymerization,which is drug concentration dependent.Inferred that fluvoxamine could inhibit cancer cells activity and growth via hindering F-actin polymerization.(5)Western blotting provided a strong evidence qualitatively to infer that one of the Rho GTPase family molecule---cdc42 might be the target gene for being inhibited by Fluvoxamine and prevented cancer cells growth and invasion via actin de-polymerization.Conclusion:Fluvoxamine was capable of suppressing GBM cells growth and metastasis,effectively and moderately,via inhibiting F-actin polymerization and cdc42 gene might be the target of the mechanism by decreasing its gene expression.