Role Of Protein Tyrosine Phosphatase Shp2 In The Induced Differentiation Activity Of 4-Amino-2-Trifluoromethyl-Phenyl Retinate In K562 Cells

Protein tyrosine phosphatase (Shp2) is one of the earliest tyrosine phosphatase and is defined as a cancer gene. Participating in a variety of cell signal transduction pathways, Shp2 is closely related to the different types of leukemia and solid tumor. All trans retinoic acid (ATRA), as the representative of differentiation inducer, has been widely used in clinical treatment of acute promyelocytic leukemia. However, the side-effects accompanied with the long-term use of ATRA including retinoic acid syndrome (RAS) and resistance, restrict its wide application in clinic. Reforming the polar group at the end of the carbon chain and modifying the cyclohexene ring with ATRA as the lead compound,a series of new retinoic acid derivatives,were synthesized in our previous study. Result of our previous studies has demonstrated the anti-tumor effct of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR). Aim to investigate the anti-tumor effect of ATPR on K562 cells and explore the role of protein-tyrosine-phosphatase (Shp2) in the induction differentiation action, the pharmacokinetic effect of ATPR was observed, and a Shp2-silenced cell model was established via small interfering RNA(siRNA).Objective:To investigate the anti-tumor effect of ATPR on K562 cells and explore the role of protein-tyrosine-phosphatase Shp2 in the inductive differentiation action.Methods:1. The chronic granulocytic leukemia K562 cells were cultured and treated with ATPR with different concentration and the cell proliferation was detected via CCK-8 assay. The morphologic changes were observed via Wright-Giemsa staining, and the expression of an exclusive cell surface antigen CD235a and the distribution of cell cycle were observed using FCS. The mRNA and protein expressions of Shp2 were detected by Q-PCR and western blotting, respectively.2. Lipofectamine 2000 transfection reagent was used to transfect the small interfering RNA (siRNA) with FAM (Carboxyfluorescein) in K562 cells. The transfection efficiency of siRNA and the expression level of Shp2 protein were detected by fluorescence microscope and western blotting, respectively.3. K562 cells were divided into 6 groups including the control group (adding volume RPMI-1640), the ATRA group (10-5 mol/L, positive control), the ATPR group(10"5 mol/L), the siRNA-lipo group, the siRNA-lipo+ATRA group and the siRNA-lipo+ATPR group. The cell proliferation was detected via CCK-8 assay. The morphologic changes were observed via Wright-Giemsa staining, and thea expression of an exclusive cell surface antigen CD235a and the distribution of cell cycle were observed using FCS.4. K562 cells were divided into 6 groups including the control group (adding volume RPMI-1640), the ATRA group (10’5 mol/L, positive control), the ATPR group(10-5 mol/L), the siRNA-lipo group, the siRNA-lipo+ATRA group and the siRNA-lipo+ATPR group. mRNA and protein expressions of RARs were detected by Q-PCR and western blotting, respectively.Results:1. The proliferation of K562 cells was inhibited by treatment of ATPR (10-5～10-9 mol·L-1) in a dose and time-dependent manner. Results of Wright-Giemsa staining showed that ATPR treated K562 cells presented higher degree of mature and differentiation through Wright-Giemsa staining than the control ones. After treated with ATPR, the percentage of cells in Go/Gi phase and the expression level of the maturation specific cell surface marker CD235a were increased in K562 cells, while the expression of Shp2 was decreased.2. All the 3 Shp2 siRNAs synthesized in the present study could be transfected into K562 cells successfully. Detected through western blot, the expression of Shp2 protein in transfected K562 cells was lower than that in the controll group. When the proportion of PTPN-Homo-438 and lipo was 1:0.05, the expression of Shp2 protein was lowest.3. After the Shp2 was silenced, the proliferation of K562 cells in the siRNA-lipo group was lower than that in the control group. Compared with the control group, the morphologic changes was disappeared in the siRNA-lipo groups, The percentage of cells in Go/Gi phase in the siRNA-lipo cells was lower than in the control group, which was similar to the expression level of the maturation specific cell surface marker CD235a.4. After the Shp2 was silenced, the effect was compared between the siRNA-lipo+ATPR group and the ATPR group. The results of Q-PCR and western blot showed that the expression of RARa in the siRNA-lipo+ATPR group was increased, the expression of RARy was decreased, and there were no significant difference of RARp.Conclusion:1. The differentiation of K562 cells could be induced by ATPR.2. The silence cells model of Shp2 expression by PTPN-homo-438 in K562 cells is the perfect model.3. ATPR modulates Shp2 to exert induction differentiation function.