| Objective:In this study,we used MG63/ADM cells which successfully established in the early stage by the treatment of adriamycin with high dose gap as the research object,to observe the expression of PTEN,p-Akt and Akt in MG63/ADM cells,to evaluate the effects of Pien Tze Huang(PZH)on MG63/ADM cells,and the impacts on PI3K/Akt Signal pathway,further in-depth piece of the relationship between inhibitory effect of PZH on proliferation of osteosarcoma cells and PI3K/Akt signaling pathway,further providing theoretical basis for clinical treatment of osteosarcoma.Method:1、The expressions of PTEN,Akt and p-Akt protein in MG63/ADM cells.Use MG63/ADM cells which successfully established as the research object,to detect the expressions of phosphatase and tensin homolog(PTEN).protein kinase B(Akt)、phospho-AKT(p-Akt)on MG63/ADM cells by Western blot.2.Effect of PZH on MG63/ADM cells and the impacts on the PI3K/Akt pathway.To detect the effects of proliferation of MG63/ADM cells after treated with PZH at different concentrations and action times by MTT method;To observe the effect of PZH on apoptosis of MG63/ADM cells in Hoechst 33342 staining under fluorescence microscope.The expressions of PTEN、Akt、p-Akt、PARP、Cleaved PARP on MG63/ADM cells which intervened by PZH were studied by Western blot.Result:1、Western blot showed that the expression of PTEN in MG63/ADM cells was significantly lower than that of MG63 cells and the expression of p-Akt protein in MG63/ADM cells was significantly higher than that of MG63 cells,Akt protein expression was not different significantly as compared with MG63/ADM cells and MG63 cells(P<0.05);the differences of Akt protein expression between the two is not obvious.These results suggest that the drug resistance of MG63/ADM cells may be related to the down regulation of PTEN expression and the level of Akt phosphorylation.2、Effect of PZH on proliferation of MG63/ADM cells:MTT shows PZH significantly inhibited the proliferation of MG63/ADM cells with a dose and time dependent manner;there are significant differences among the groups(P<0.01).That PZH inhibited the proliferation of MG63/ADM cells relate to the concentration and action time.3、Effect of PZH on apoptosis of MG63/ADM cells:MG63/ADM cells were treated with different concentrations of PZH for 48h,morphological changes of MG63/ADM cells were observed under fluorescence microscope after Hoechst 33342 staining,the nucleus of the control group showed an oval blue uniform fluorescence,and PZH groups showed condensation nuclei,crescent shaped change of apoptosis expression,especially the 1.2mg/mL group,the number of cells was significantly reduced,and the typical apoptotic bodies appeared in the nuclei of the remaining cells.That PZH can obviously promote the apoptosis of MG63/ADM cells.4、Effect of PTEN,Akt,p-Akt,PARP and Cleaved-PARP protein expression in MG63/ADM cells with PZH:Western Blot results showed that,after intervening by PZH for 48h,the expression level of PTEN increased with the increase of the concentration of PZH;The expression level of Akt protein was not obvious difference;The expression level of p-Akt decreased with the increase of the concentration of PZH;The expression of PARP protein decreased slightly;The expression level of Cleaved-PARP increased with the concentration of PZH that PZH can promote PARP cleavage.The expression of PTEN,Akt,p-Akt,PARP,Cleaved-PARP protein was significantly different from the blank control group(P<0.05).That PZH can increase the expression of PTEN,inhibit the phosphorylation of Akt protein(p-Akt)level,promote the cleavage of PARP protein,up-regulate expression of Cleaved-PARP,and promote the apoptosis of MG63/ADM cells.Conclusion1、The drug resistance to ADM may be related to PI3K/Akt kinase pathway activation in osteosarcoma.2、PZH can inhibit the proliferation and induce the apoptosis of MG63/ADM Cells,the mechamism may be associated with up-regulating the expression of PTEN and down-regulating the expression of p-Akt. |
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