Effects Of Simvastatin On WT1/hDMP1 Gene Expression Profiles Of Human Acute Myeloid Leukemia Cells Lines

Objective: To investigate the effects of Simvastatin ( SV ) in combination with Chemotherapeutic drugs on the proliferation ,differentiation,apoptosis and WT1/hDMP1 gene expression profiles of human acute myeloid leukemia cell lines K562, NB4 and SHI-1.Methods: Human acute myeloid leukemia cell lines K562, NB4 and SHI-1 were incubated with Simvastatin solely or in combination with cytosine arabinoside(Ara-C)for K562 cells, ATRA for NB4 cells and phorbol 12-myristate 13- acetate (PMA)for SHI-1 cells respectively,taking K562,NB4,SHI-1 cells without any treatment as normal controls. Cells of different groups were collected at 24 h, 48 h and 72h after incubation for further detection.Morphological changes by Wright stain were performed.M'IT method was used to assay the growth inhibition rate and the flow cytometric assay was used to detect the cell differentiation markers of CD11b,CD11c, CD14 , myeloid lineage markers CD13,CD33 and early stage apoptosis ratio and cell necrosis ratio. Real-time quantitative reverse transcriptase polymerase chain reaction (real-time RT-PCR)was used to detect the WT1/hDMP1 gene expression levels.Results: (1)Simvastatin has the effects on inhibition of cell proliferation in human leukemia cell lines K562, NB4 and SHI-1. Synergistic cell proliferation inhibition was observed in K562 cells treated with simvastatin(15μmol/L)combining with Ara-C (20μmol/L), as well as in SHI-1 cells treated simvastatin(15μmol/L)combining with PMA (5ng/L). However, combination of simvastatin with ATRA (0.5μmol/L)did not synergistically inhibit proliferation in NB4 cells. (2)The flow cytometric analysis showed that treated with simvastatin solely, the expression levels of CD11b of NB4 cells and CD11c of SHI-1 cells gradually increased in a time and dose-dependent manner, morever, synergistic effects of the expression levels of CD11b of NB4 cells and CD11c of SHI-1 cells were found when treated simvastatin in combination with ATRA or PMA in each. (3)The results of the apoptosis assessed by annexin V–FITC/PI analysis showed that increasing the dose of simvastatin and prolongating the time of incubation with simvastatin resulted in higher apoptotic ratio of K562, NB4 and SHI-1 cells, as were in consistent with the inhibition ratio in the same manner. Meanwhile, simvastatin combining with Ara-C /PMA synergistically induced K562 /SHI-1 cells apoptosis. However no synergistic apoptosis was observed in NB4 cells after incubation with simvastatin combining with ATRA. (4)Real-time quantitative RT-PCR detection showed that the expression of WT1 gene gradually decreased in parallel with the increasing expression of hDMP1 gene reversely in K562 cells, NB4 cells and SHI-1 cells treated with simvastatin of different concentrations. Of 5, 10 and 15μmol/L simvastatins, the group of 15μmol/L simvastatin exhibited the most significant changes of WT1 and hDMP1 expression. Furthermore, the WT1 gene expression level in K562, NB4, and SHI-1 cells treated with simvastatin combining with Ara-C, ATRA and PMA respectively was lower than that treated with simvastatin solely, so was hDMP1 expression increase in reverse, indicating that simvastatin exhibited a synergistic effect in combination with chemotherapeutic dugs.Conclusion: Simvastatin exhibits effects of antiproliferation on acute myeloid leukemia cell lines ,induction of differentiation on NB4 cells and SHI-1 cells, pro-apoptosis, decreasing the expression of WT1 gene and increasing the expression of hDMP1 gene in a dose-dependent manner in vitro, which suggests that simvastatin plays a potential synergistic role in leukemia therapy.