The Research In Anti-tumor Activity Of The Novel GSK-3β Inhibitor-polypyridyl Ruthenium Complexes

Objective:Aim to screen the optimal polypyridyl ruthenium complexes with the best activity on human glioma SWO-38 cells. Further, to investigate the possible mechanism of the anti-tumor activity of chiral ruthenium complexes inhibiting GSK-3βactivity in vitro.Methods:1. To screen the optimal complexes with highest activity and lowest toxicity from eight kinds of polypyridyl ruthenium complexes with chiral separation by MTT colorimetric assay. And detecting the proliferation of the cells with different concentrations of the complexes. The location of complexes in the cells could be observed under the confocal laser scanning microscope.2. FITC-Annexin V/PI double labelling was used to detect the apoptosis of SWO-38 cells;3. Western blot was employed to detect the expression of total GSK-3β, p-GSK-3βSer9, total PTEN and p-PTEN ser380/Thr382/383 after the treatment of the optimal complexes, then analyzing the mechanism of the inhibition proliferation on SWO-38 cells.4. The expression of PTEN in SWO-38 cells was knocked down by target-specific siRNA of wild type PTEN. Cell proliferation was evaluated by MTT assay. And the changes of total PTEN, p-GSK-3βSer9 and total GSK-3βexpressions in SWO-38 cells were detected by western blot.Results:1. MTT assay showed A-[Ru(bpy)2 pyip]2+ could inhibit the growth of SWO-38 cells obviously, IC50 was 40.5±8.4μmol/L; Compared the dosing concentrations>10μmol/L groups with the control group, the proliferation rates of SWO-38 cells had some differences (P<0.05).2. Flow cytometry showed the complexes could significantly induce the apoptosis of SWO-38 cells, the early apoptosis rates were 2.3%(the control group) and 5%,10.6%,17%(the dosing concentrations groups with 10,30,50μmol/L).3. Western blot showed that there was no significant difference of the expressions of GSK-3βand PTEN proteins after the treatment of A-[Ru(bpy)2 pyip]2+, but the p-GSK-3βSer9 expression level of SWO-38 was higher and the p-PTEN Ser38O/Thr382/383 was lower compared with the control group.4. By MTT assay, the proliferational ability of SWO-38 cells in siRNA-PTEN+A-[Ru(bpy)2 pyip]2+group was significantly higher than the siRNA-control+A-[Ru(bpy)2 pyip]2+group (P<.05); Western blot revealed that there was no change of p-GSK-βSer9 expression in siRNA-control group and siRNA-PTEN group (P>0.05).Conclusion:1. A-[Ru(bpy)2 pyip]2+ possessed the feature of high activity and low toxicity which was picked out from the 8 ruthenium complexes, which was sensitive with SWO-38 cells and insensitive with human embryonic kidney cells.2. A-[Ru(bpy)2 pyip]2+ could induce the apoptosis of SWO-38 cells, which mainly localized in the cytoplasm.3. The anti-tumor mechanism of the ruthenium complexes A-[Ru(bpy)2 pyip]2+ was involved with the inactiviatity of GSK-3βprotein, and PTEN might be the downstream gene which contributed to the inhibition of SWO-38 cells.