The Mechanism Of Thalidomide And Temozolomide Inhibition On Adhesion, Invasion And Integrin αvβ3 Expression In Human Glioblastoma Cells

Background＆objectiveGlioblastoma(GBM) is the most common brain tumors and the most aggressive astrocytic tumor.The prognosis for patients with glioblastoma remains poor,even treated by surgery combined with radiation and chemotherapy,The median survival is less than 1 year.Temozolomide(TMZ),a second generation of alkylating drug is an effective cytotoxic agent for malignant brain tumours.Thalidomide(THD) is a derivative of glutamic acid,however the antitumor effection of thalidomide is not understood completely.It is postulated that THD could inhibit cell adhension and angiogenesis by reducing the expression of integrin avβ3 and av(35 and inhibiting vascularizion induced by vascular endothelial growth factor(VEGF).In our preliminary study,the combination of THD and TMZ in GBM treatment appears to be more effective than THD or TMZ used alone,which is also confirmed by clinical trials.In this study we aimed to investigate the inhibition mechanism in adhension and invasion of human glioblastoma LN229 cells treated by THD plus TMZ.MethodsCulture plates were coated with glioma-derived ECM proteins.LN229 cells were cultured in DMEM medium containing THD(100μg/ml),TMZ(l00μM),or THD+TMZ(100μg/ml+100μM),dissolvant control group contained DMSO with equal volum.An inverted microscope was applied for observation of cell growth..The determination of the percentage of integrin avβ3 positive LN229 cells and the mean fluorescence intensities(MFI) Sulforhodamine B assay was used to determined the survival rate of suspending cells.After cultured for 4 hr,the attached cells were digested with 0.25%trypsinase.1×10⁴cells were pelleted at 200xg for 5 min and the supernatant was decanted.After washed,cells were incubated at 4℃for 45 min with FITC-conjugated mouse anti-human integrin avβ3 or its isotype control (mouse IgGl) to identify integrin expression.The cells were washed with PBS containing 1%FBS and pelleted at 200xg for 5min,followed by flow cytometer analysis.The percentage of positive cells as well as the mean fluorescence intensities (MFI) were determined. Detection of inhibition effect on cells adhering to extracellular matrix(ECM) proteins after THD,TMZ or THD+TMZ treatment LN229 cells were harvested from monolayer cultures and placed into 96-well flat bottom plates coated with autologous ECM at a concentration of 5×10⁴ cells/ml(5,000 cells/well),then incubated overnight with medium containing THD(100μg/ml),TMZ(100μM) or THD+TMZ(100μg/ml+100μM).Cells in control group were incubated with medium containing DMSO with equal volum.After 4 hr incubation at 37℃,the medium containing nonattached cells was removed and the wells were rinsed with PBS. Attached cells were fixed in 10%glutaraldehyde for 30 min before staining with crystal violet(0.1%in H₂O).Spectrophotometric absorbance of the stained nuclei was quantified at 595 nm.The evaluation of cell invasion and migration ability The effect of THD and TMZ on the migration and invasion ability of LN229 cells was meatured by scratch model and transwell chamber invasion models.ResultLN229 cells in control group and TMZ group formed filopodia and adhered to other cells to form a cell-monolayer,while the other two groups trended to maintain round shape;The effect of THD and TMZ on expression of integrin av(33 in LN229 glioma cells Sulforhodamine B assay was used to determine the survival rate of suspending cells. Suspending cells was too few to detect in control and TMZ groups.The survival rate of suspending cells in THD and THD+TMZ groups were 93.5,%90.5%respectively. Both of them were above ninety percent.The percentage of the integrin avβ3 positive attached cells was decreased to 81.8%and 77.9%,and the mean fluorescence intensity(MFI) was decreased to 66.8%and 53.7%in THD and THD+TMZ group respectively compared to control group.The percentage of the integrin avβ3 positive suspending cells was decreased to 63.7%and 55.0%,and the MFI was decreased to 49.8%and 54.1%in THD and THD+TMZ group respectively compared to control group.In TMZ group,integrin avβ3 positive rate and MFI were 103.3%and 90.4%compared to control group. The effect of THD and TMZ treatment on glioma cell adhering to glioma ECM protein-coated plates Fewer crystal-violet stained cells attached on the plate after incubated with THD or THD+TMZ for 60 min,compared to those in control or TMZ group(P＜0.05).The optical density(OD) had no difference statistically between THD group and THD+TMZ group(P＞0.05),which reflected the number of attached cells.The adhesion-inhibiting rate of THD,TMZ and THD+TMZ was 25.23%, 0.88%and 26.83%respectively.The effect of THD and TMZ on LN229 cells invasion and migration In transwell chamber invasion model,less invaded cells were found on the membrane of transwell chamber in THD and THD+TMZ groups compared to other two groups(P＜0.05).In scratch model,the distance of cells migration in THD and THD+TMZ group was shorter than in control or TMZ group(P＜0.05).However,the difference of migration between control group and TMZ group was not statistically significant (P＞0.05).Conclusion:THD could inhibit cell adhension,invasion and migration,while TMZ has no such effect.The inhibition on cell adhension,invasion and migration caused by THD treatment might be ascribe to the reducing of the expression of integrin avβ3. No synergistic effect is found on cell adhension,invasion and migration under the THD treatment combine with TMZ.