Molecular Mechanism Of The Inhibitton Of Human K562 And U266 Cell Proliferation By PP-22

Objective: This study mainly investigates the proliferation inhibition effect and molecular mechanism induced by PP-22(a monomer of Paris polyphylla)on human K562 cells and U266 cells,in order to provide certain experiment basis for the development and utilization of PP-22,and to provide data for the development of new drugs for multiple myeloma and leukemia in clinical practice.Methods:1.MTT assay and Ed U kit were used to detect the effect of PP-22 on proliferation inhibition of human chronic myeloid leukemia K562 cells and myeloma U266 cells.2.Annexin V-FITC /PI double staining assay was used to detect the changes of apoptosis levels of K562 cells and U266 cells induced by PP-22.Hoechst 33258 staining was used to detect the morphological changes of tumor cells.3.Rhodamine 123 staining and JC-1 staining were applied to detect the influence of mitochondrial transmembrane potential on K562 and U266 cells.DHE staining was used to detect the changes of intracellular ROS in tumor cells.4.The effect of different concentrations of PP-22 on the cell cycle of K562 cells and U266 cells was determined by flow cytometry PI single staining.Western blotting was used to detect the expression level of p38 MAPK kinase signaling pathway and cyclin-related proteins induced by PP-22.5.To further study the mechanism of PP-22 on tumor cells,the expression levels of the apoptosis-related proteins of Caspase family and Bcl-2 family induced by PP-22 on human K562 cells and U266 cells were analyzed by western blotting.Then,using caspase inhibitor z-DVAD-fmk further explored the relationship between the proliferation inhibition and the mitochondrial apoptosis pathway.6.The expression levels of PI3K/AKT signaling pathway and endoplasmic reticulum stress(ER-stress)signaling pathway and the change of its downstream protein were detected by western blotting.For further exploring the relationship between the apoptosis of K562 cells and U266 cells induced by PP-22 and PI3K/AKT and ER-stress signaling pathways,a selective PI3 K inhibitor LY294002 and endoplasmic reticulum stress activator TM were used to inhibit and activate the expression of related signaling pathways,respectively.Result:1.The IC50 range of PP-22 for lymphoma Jurkat,A20,EL4,EB1 cells,leukemia K562,Molt 4,CCRF-CEM cells,and myeloma U266 cells at 24 h,48 h and 72 h was 5.04 to 14.44 ?M.The results of MTT assay showed that the inhibitory effect of PP-22 on tumor cells was dose-and time-dependent.Further,Ed U staining showed that PP-22 inhibted proliferation of K562 and U266 cells.2.Annexin V-FITC/PI double staining showed that the apoptosis rates of K562 cells treated PP-22 at different concentrations for 24 h were 6.39 ± 1.09 %,10.07 ±3.94 %,15.31 ± 4.20 %,81.5 ± 4.70 % in groups of 0 ?M?5 ?M?10 ?M?20 ?M.The apoptosis rates of U266 cells treated with various concentrations PP-22 in groups of 0 ?M,5 ?M,10 ?M and 20 ?M were 3.97 ± 0.37 %,9.66 ± 2.69 %,38.36± 1.31 %,71.92 ± 19.25 %,respectively.The results of Hoechst 33258 staining revealed that K562 cells and U266 cells treated with PP-22 showed increased staining,and nuclear showed shrinkage and lysis.3.JC-1 and Rho123 staining results showed that the mitochondrial membrane potential decreased significantly in K562 cells and U266 cells with increasing PP-22 concentration,and the level of ROS in tumor cells was significantly increased.4.Flow analysis showed that G1 phase cell cycle arrest occurred in K562 cells and U266 cells with the increase concentration of PP-22.Western blotting results showed that the expression levels of p-p38,p21 and p-p53 proteins were increased,and the expression levels of cdc25 A,cyclin E and CDK2 proteins were significantly decreased.5.The results of Western blotting showed that the apoptotic proteins Bax and Bim were significantly increased and the anti-apoptotic proteins Bcl-2 and Bcl-XL were significantly decreased after the treatment with PP-22 at 10?M on human K562 cells and U266 cells for 24 h.At the same time,the expressions levels of caspase-9,caspase-3 and PARP proteins were significantly decreased,while the expressions levels of cleaved-caspase-9,cleaved-caspase-3 and cleaved-PARP proteins were significantly increased.The expression of caspase and its cleaved proteins were significantly reversed after the use of a caspase inhibitor z-DEVD-fmk.The results indicated that PP-22 induced apoptosis in human K562 cells and U266 cells by the mitochondrial apoptosis pathway.6.The results of Western blotting showed that the expression levels of AKT,GSK-3?and their phosphorylated proteins in human K562 cells and U266 cells treated with PP-22 at 10 ?M were significantly decreased,and the same to the expressions levels of proteins GSK-3? and p-GSK-3?.The PI3 K inhibitor LY294002 could significantly inhibit the expression of AKT and its downstream signaling pathway.Meanwhile,the downstream proteins of the ER-stress signaling pathway,PERK,BIP and CHOP were significantly increased.TM,ER-stress activator,could significantly synergize activate the expression of proteins related to the ER-stress signaling pathway.Conclusion:1.PP-22 inhibited the proliferation of human lymphoma Jurkat,A20,EL4,EB1 cells,leukemia K562,Molt 4,CCRF-CEM cells,and myeloma U266 cells in a dose-and time-dependent manner.2.PP-22 induced the G1 phase cell cycle arrest in K562 and U266 cells by activating p38MAPK-p53-p21 and p38MAPK-cdc25 A signaling pathways.3.PP-22 could induce the apoptosis of K562 and U266 cells by inhibiting the PI3K/AKT signaling pathway,and activating the ER-stress signaling pathway to mediate mitochondrial apoptosis pathway.