The Effect Of Simvastatin On Acute Myeloid Leukemia

1. Effects of simvastatin on proliferation and apoptosis of NB4, HL-60 and SHI-1 cellsObjective To observe the effects of simvastatin on proliferation and apoptosis of NB4, HL-60 and SHI-1 cells.Methods MTT assay and trypan blue exclusion assay to evaluate proliferation of NB4, HL-60 and SHI-1 cells with treatment of various concentrations of simvastatin for 1 to 3 days. Annexin V antibody detection was used to evaluate cell apoptosis after treatment with simvastatin. Cell cycle parameters were determined by flow cytometry using propidium iodides. Amplex red assay, which measures the release of hydrogen peroxide upon cholesterol oxidation, and was used to measure total cholesterol levels in three cell lines. Quantitative, real-time RNA analysis ere performed to evaluate the expressions of HMG-CoA reductase (HMG-CoA R) and low density lipoprotein reductase (LDL R) gene.Results Compared with control group, after treatment with different concentrations of simvastatin for 1 to 3 days, the number of live cells decreased in different level, and in a dose and time-dependent manner. MTT assay clearly highlighted the different responses to simvastatin, NB4 and HL-60 were significantly more sensitive to simvastatin. Flow cytometric analysis used by Annexin V antibody indicated that the apoptotic rate increased after treatment with simvastatin. Cell cycle distribution assays showed that the percentage of G0/G1 phase cells elevated. We found that cholesterol levels were significantly increased after treatment with daunorubicin (DNR) and cytarabine (Ara-C) of three cell lines, while simvastatin could decrease the cholesterol level in all three cell lines. When combined treatment with simvastatin and chemotherapy, the cellular cholesterol level decreased significantly and the growth inhibition markedly increased. The expression of RNA levels of HMG-CoA R and LDL R were both increased after treatment with simvastatin, and in a time dependent manner.Conclusions Simvastatin could inhibit the growth of acute myeloid leukemia cells and induce them to apoptosis. Modulation of cellular cholesterol could affect the growth of AML cells.2. Effects of Simvastatin on the Expression of Multidrug Resistance Gene of SHI-1 CellsObjective To observe of simvastatin on the expression of multidrug resistance genes of SHI-1 cells.Methods Trypan blue exclusion assay to evaluate cell viability after treatment with simvastatin or cultured with serum free medium (SFM). Surface p-gp was measured using flow cytometry after culturing in serum free medium for 1 to 3 days, for treatment with various concentrations of simvastatin. The expressions of RNA of MDR-1 gene were evaluated by quantitative, real-time PCR. Amplex Red assay was used to measure the change of cellular cholesterol after culture in serum free medium. Chemosensitivity assays were performed after treatment with simvastatin of SHI-1 cells..Results Compared with control group, the growth of SHI-1 cells decreased when cultured in serum-free conditions, and in a time dependent manner. The growth inhibitory markedly increased when SHI-1 cells treatment with simvastatin and cultured in serum free medium. And p-gp was downregulated in SHI-1 cells cultured in serum free medium or treatment with simvastatin. The mRNA level of MDR1 gene also decreased after simvastatin treatment or serum free culture. While cellular cholesterol level increased when cells cultured in serum free medium. Total cellular cholesterol level treated with simvastatin decreased in serum free culture. Chemosensitivity assays found that preculture with simvastatin could increase the cytotoxic of DNR of SHI-1 cells.Conclusions Simvastatin and serum free culture could downregulate the expression of p-gp of SHI-1 cells. 3. Effects of simvastatin on a subset of primary CD34+ acute myeloid leukemiaObjective To observe the effects of simvastatin on human primary CD34+ acute myeloid leukemia and healthy donors.Methods After informed consent was given, bone marrow from 15 AML patients at diagnosis and 8 healthy donors were collected in this study. Mononuclear cells were obtained by centrifugation on Ficoll-hypaque medium, and primary AML cells from fifteen patients and normal cells from six healthy donors were purified using CD34 microbeads according to the instruction of the manufacturer. Normal CD34+, AML CD34+, and CD34- sorted subfractions were exposed to simvastatin. Results In primary sorted CD34+ cells, a heterogeneous response pattern wasobserved upon treatment with simvastatin when analyzing cell survival. A group of normal and abnormal responders were identified within the AML CD34+ subfraction when compared with normal CD34+ cells. This distinction was not observed within CD34- cells. When the CD34+ AML cells were exposed to simvastatin, a significant enhanced inhibitory effect was shown in normal AML responder group. The heterogeneity in AML responsiveness could not be explained by differences by differences in effects on cholesterol metabolism genes. Treatment with simvastatin could enhance the sensitivity of CD34+ cells to chemotherapy.Conclusions The results suggest that treatment with statins together with chemotherapy may be beneficial for a subset of AML patients.