Experimental Study for Effects of Simvastatin Alone and Combination with Daunorubicin on Proliferation and Apoptosis of SHI-1 Cells
|Keywords||simvastatin DNR SHI-1 cell cell proliferation apoptosis|
Objective To observe the effects of simvastatin alone and combination with daunorubicin on proliferation and apoptosis of acute monocytic leukemia cell line SHI-1. and to explore its possible mechanism of these effects.Methods (1) Acute monocytic leukemia cell line SHI-1 was cultured in vitro. (2)MTT colorimetric assay was used to determine the proliferation rate of cells, when SHI-1 cells were cultured in medium containing the simvastatin in different time and concentrations. (3)Experiment was divided into four groups:negative, 5 umol/L, 10umol/L, 20umol/L simvastatin group, The flow cytometry (FCM) method was used to study the apoptosis and cell cycle induced by simvastatin in SHI-1 cells; The expression of Bcl-2, Caspase-3 mRNA were determined by reverse transcriplase polymerase chain reaction (RT-PCR). The expression of Bcl-2, Caspase-3 protein levels were analyzed by Western blot. (4) Experiment was divided into two groups: DNR, Sim+DNR, the proliferation rate of cells was measured by trypan blue, The interaction between two agents was judged by combination index (CI), Cells were observed by inverted microscope,Flow cytometry was used for analysis of apoptosis and cell cycle.Results (1)MTT assay indicated that the inhibitory effect of simvastatin on the proliferation of SHI-1 cells was found in a time-and-does dependent manner. (2) Comparing with control group, the percentage of G0/G1 phase cells treated by simvastatin for 48 hours was elevated (P<0.05). 5umol/L simvastatin significantly blocked cell cycle progression and induce apoptosis at 48 hours. (3)RT-PCR assay showed that the expression of Bcl-2 mRNA was downregulated and caspase-3 mRNA was upregulated by simvastatin at 48 hours with concentration increased (P<0.05); Western blot showed that the expression of Bcl-2 protein level was downregulated and Caspase-3 protein was upregulated with concentration increased. (4)DNR significantly inhibited the growth of SHI-1 cells in a dose-and time-dependent way (P<0.05), the IC50 of 12h and 36h was 1.27umol/L and 0.27 umol/L. 5umol/L simvastatin synergistically augmented 0.1umol/L DNR cytotoxicity in SHI-1 cells (CI<1, P<0.05). Treated by simvastatin and DNR for 36h, cells became spare under inverted microscope. The size of sub-G1 peaks as well as apoptosis rate in combination are higher than DNR group.Conclusion The growth of SHI-1 cell in virtro is inhibited by simvastatin and in dose-and-time dependent manner. simvastatin also can induce cell apoptosis, arrest cell cycle at G0/G1 phase.Maybe its mechanism is through downgulating the expression of apoptosis-related gene bcl-2, upregulating the expression of caspase-3. A certain concentration of simvastatin synergistically augmented DNR cytotoxicity in SHI-1 cells.