| Objective: Our study aimed to explore the antileukemia effects and molecular mechanisms of PI3 K inhibitor ZSTK474 in human myeloid leukemia cell lines K562,HL60 and resistant cell lines K562/A02 and HL60/ADR in vitro.On this basis,to further expolre the combined application of ZSTK474 with leukemia chemotherapy and the mechanism of ZSTK474 on reversing drug resistance.Our first systematic described of effect of ZSTK474 alone or combined with chemotherapy drugs on acute/chronic leukemia in vitro,provided a theoretical basis and a preliminary experimental basis for the development and application of PI3 K inhibitors as a new antileukemia drug.This paper is divided into three parts: First part is antileukemia of ZSTK474 in vitro;Second part studies the ZSTK474 combination with leukemia chemotherapy drugs;Third part is to explore the mechanism of ZSTK474 on reversing drug resistance.Methods:1、MTT method combined with soft agar colony formation assay was used to detect the antiproliferative activity of ZSTK474 against K562,HL60,K562/A02,HL60/ADR cells in vitro.2、Flow cytometry was used to detect the effects of ZSTK474 on the cell cycle and apoptosis of K562,HL60,K562/A02,HL60/ADR cells.3、Western-blot was conducted to evaluate the effect of ZSTK474 on cell cycle related protein phosphorylation and expression of p-Rb,cyclinD1 and p27,as well as phosphorylation of PI3K/Akt pathway related protein PDK-1,GSK-3β,Akt of K562,HL60,K562/A02,HL60/ADR cells.4、The effect of ZSTK474 on the expression of p27 mRNA in K562,HL60,K562/A02,HL60/ADR was evaluated by qRT-PCR assay.5、Chou and Talalay ’s method combined with CalcuSyn software was used to analyze combination index of ZSTK474 with chemotherapy drug in order to evaluate the effect of drug combination.6、ADR acculation,5-CFDA effluent combined with Western-blot methods were used to study the effect of ZSTK474 on MDR relative protein MDR1 and MRP1 expression and function.Results:1、MTT and soft agar cloning results showed that ZSTK474 inhibited K562,HL60,K562/A02,HL60/ADR cell proliferation in dose-dependent manner with the IC50 values of 4.69 μM,1.165 μM,7.57 μM,1.160 μM,respectively.2、PI staining and annexin-V/PI double staining combined with flow cytometry showed that ZSTK474 induced K562,HL60,K562/A02,HL60/ADR cell cycle arrest in the G1 phase without inducing apoptosis.3、The specific mechanism of ZSTK474 induced cell cycle arrest may be through the decreased of cell cycle related protein cyclin D1 expression and Rb phosphorylation,increased the expression level of p27 protein,while ZSTK474 decreased the phosphorylation level of PDK-1,Akt,GSK-3β in PI3K/Akt pathway of K562,HL60,K562/A02 and HL60/ADR.4、ZSTK474 increased the expression level of p27 mRNA of K562,HL60,K562/A02,HL60/ADR cells.5、ZSTK474 combined with IM against K562,K562/A02 cells,the CI value of ED50,ED75,ED90 was 0.37,0.39,0.54 and 0.08,016,0.39.In HL60,HL60/ADR cells,ZSTK474 combined with CY,the CI value of ED50,ED75,ED90 was 0.73,0.35,0.24 and 0.42,0.64,1.17.ZSTK474 combined with VCR,the CI value of ED50,ED75,ED90 was 0.76,1.08,1.67 and 0.25,0.70,2.03.ZSTK474 combined with HHRT,the the CI value of ED50,ED75,ED90 was 0.68,0.57,0.67 and 0.96,1.62,2.75.6、ZSTK474 increased ADR concentration in K562/A02 and HL60/ADR cells as well as 5-CFDA concentration in HL60/ADR cells.Western blot result showed that MDR1 decreased in both two cells but MRP1 was detected only in HL60/ADR but not K562/A02 cells.Conclusions:1、ZSTK474 inhibited K562,HL60,K562/A02,HL60/ADR cell proliferation activity and induced cell cycle arrest in G1 may be via PI3K/Akt pathway.2、ZSTK474 combined with antileukemia chemotherapy drug in our study against K562,HL60,K562/A02,HL60/ADR cells was synergistic effects in ED50。3、ZSTK474 reversed the drug resistance by inhibiting the expression and function of drug resistance relative protein MDR1 and/or MRP1. |
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