Experimental Study For Effects Of Simvastatin Alone And Combination With Cytarabine On Proliferation And Apoptosis Of AML Cell Lines

Objective (1)To observe the effects of simvastatin on proliferation and apoptosis ofacute promyelocytic leukemia cell line NB4、HL60and acut myelocytic luekemia cell lineoriginated from Di Guglielmo syndrome KG1,and to analyse its mechanism of theseeffects.（2）To observe the effects of simvastatin combined with cytarabine on proliferationof acute leukemia cell line NB4、HL60and KG-1, analyze whether the combination ofthe two drugs has synergistic effect and detect the mechanisms of the effect.Methods （1）Cell Counting Kit assay was used to determine the proliferation rate ofcells,when NB4、 HL60、 KG1cells were cultured with different concentrationsimvastatin.Upon treatment with simvastatin,the half maximal inhibitory concentration（IC50）in the various cells lines was determined.Detect the proliferation rate of cell linesafter being cultred24hours with simvastatin alone(the IC50concentrations were usedduring the following experiments.)or in the presence of mevalonate,squalene,geranylgeranyl pyrophosphate salt (GGPP) and farnesy pyrophosphate salt (FPP).（2）Theflow cytometry (FCM) method was used to study the apoptpsis of cell lines cultured24or48hours with simvastatin alone or in the presence of mevalonate,squalene,GGPP andFPP.(3)The western blot was used to detect the change of RAS and RHO on cell membranewhich were cultured with simvastatin alone or in the presence ofmevalonate,squalene,GGPP and FPP.（4）In order to confirm whether the simvastatin canenhance sensibility of chemotherapy,we detected the proliferation and apoptosis of NB4、HL60and KG1cell lines cultured with simvstatin alone or combined with cell-toxic drugAra-c.Meanwihle,we also detect the change of P65/NF-κB in cell nucleus.Results (1)There is inhibitory effect of SIM on the proliferation of NB4、HL60、KG1 cell lines,and they are in a time-and-does dependent manner;But the sensitivity is differentduring all cell lines, NB4>HL60>KG1;When cultured cell lines with simvastatin alone orin the presence of different rescue drugs,the result demonstrate that mevalonate,GGPP andFPP could prevent SIM-induced cytotoxicity on three cell lines,but squalene could not.Theresult also indicated that the precvention of simvastatin-induced cytotoxicity of GGPP wasmore obviosuly than FPP.(2)The apoptosis of three cell lines can be induced bysimvastatin,and the effects are also in a time-and-does dependent manner. Comparing withthe group clutured with SIM alone,the apoptpsis rate of all cell lines reduced when theywere cultrued with SIM in the presence of mevalonate, GGPP and FPP(p<0.05),butsqualene did not have the effect(p>0.05).(3)The expression of RAS and RHO on cellmenbrance can be regulated-down by simvastatin,and the regulation can be more obviouslywhen the concentration was higher and time longer.The expression can be recovered indifferent drgree when mevalonate,GGPP and FPP were used to rescue the cytotoxicty ofsimvastatin,but squalene did not have the effect.(4)Comparing with control，SIM orARA-C alone,the proliferation rate of cell lines cultrued with both of SIM and ARA-Creduced significantly(p<0.05).Combination of SIM and ARA-C showed synergisticinhibition with Q value of1.4734、1.3799in NB4and HL60group,and in KG1groupshowed addtion inhibition with Q value of1.1057. Comparing with control，SIM orARA-C alone,the apoptosis rate of cell lines cultrued with both of SIM and ARA-Cincreased significantly(p<0.05). Comparing with control group,the P65/NF-κB in nucleuswere expressed much more in the group with ARA-C alone and much less in the groupwith SIM alone.When SIM combinated with ARA-C,the expression of P65/NF-κB innucleus reduced significantly than the control group or the group with ARA-Calone(p<0.05).Conclusion (1)SIM can inhibit the proliferation and induce the apotosis of NB4、HL60、KG1cell lines, the effect of SIM on three cell lines was in a time-and-doesdependent manner,and the sensitivity of SIM was different in the three cell lines（NB4>HL60>KG1）.(2) The cytotoxicty of simvastatin can be rescued in different drgree by mevalonate,GGPP and FPP,but squalene did not have the effect.(3) The expression ofRAS and RHO on cell menbrance decreased when cell lines were cultured withsimvastatin.The expression can be recovered in different drgree when cell lines culturedwith SIM in the presence of mevalonate,GGPP and FPP,but squalene did not have theeffect.(4)SIM can inhance the cytotoxicty of ARA-C on AML cell lines.The combinationof the two drugs showed synergy,and could reduce P65/NF-κB in cell nucleussignificantly than the group with ARA-C alone.