索拉非尼通过抑制细胞外调解蛋白激酶下游作用底物磷酸化ets样基因-1诱导肿瘤细胞凋亡的研究

The phosphorylation of ets-like gene-1 as a downstream substrate of extracellular mediator protein kinases

目的探讨索拉非尼对肿瘤细胞凋亡的影响以及涉及髓样细胞白血病-1(Mcl-1)下调的作用机制。方法采用噻唑蓝(MTT)法检测索拉非尼对肠嗜铬细胞(EC细胞,购自美国典型菌种保藏中心)增殖的影响,以确定索拉非尼的作用浓度和作用时间。将EC细胞分为4组:对照组、索拉非尼组、索拉非尼+短发夹RNA以下调荧光素酶的质粒(shLuc)组和索拉非尼+细胞外调解蛋白激酶下游作用底物磷酸化ets样基因-1(Elk-1)短发夹RNA(shElk-1)组。流式细胞仪检测各组细胞的凋亡率,蛋白质印迹法(Western blot)和实时定量聚合酶链反应(Real-time PCR)检测B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)、Elk-1、Mcl-1、蛋白激酶B(Akt)和糖原合成酶激酶3β(GSK3β)的蛋白及mRNA的相对表达量。多组间比较采用单因素方差分析。结果1、5和10 mol/L的索拉非尼作用后EC细胞的存活率分别为(67.42±5.08)%、(50.81±6.11)%和(38.49±5.41)%,显著低于0 mol/作用浓度(99.98±0.25)%(t1=5.148,t5=2.142,t10=1.764,P值均<0.05),差异有统计学意义。索拉非尼作用12、24和48 h后EC细胞的存活率分别为(65.41±4.61)%、(49.64±4.73)%和(42.49±5.02)%,显著低于0 h作用时间(99.99±0.18)%(t12=4.624,t24=1.856,t48=1.751,P值均<0.05);而与作用6 h比较,EC细胞的存活率差异无统计学意义(t6=1.266,P>0.05)。与对照组比较,索拉非尼组细胞凋亡率显著增加(t=12.235,P<0.05);与索拉非尼组比较,索拉非尼+shElk-1组细胞凋亡率显著增加(t=9.271,P<0.05);各组间bcl-2相对表达量、bax相对表达量和bcl-2/bax比值比较,差异无统计学意义(Fbcl-2=0.198,Fbax=0.541,Fbcl-2/bax=2.679,P值均>0.05)。与对照组比较,索拉非尼组细胞Elk-1蛋白的相对表达量差异无统计学意义(tElk-1蛋白=1.426,P>0.05),而Elk-1 mRNA、Mcl-1蛋白及mRNA的相对表达量显著降低(tElk-1 mRNA=2.266,tElk-1 mRNA=0.045,tMcl-1蛋白=2.774,tMcl-1 mRNA=2.987,P值均<0.05),差异均有统计学意义。与索拉非尼组比较,索拉非尼+shElk-1组细胞Elk-1蛋白及mRNA、Mcl-1蛋白及mRNA的相对表达量显著降低(tElk-1蛋白=5.340,tElk-1 mRNA=6.240,tMcl-1蛋白=6.248,tMcl-1 mRNA=5.217,P值均<0.05),差异均有统计学意义。与对照组比较,索拉非尼组细胞Akt蛋白及mRNA、GSK3β蛋白及mRNA的相对表达量显著降低(tAkt蛋白=3.579,tAkt mRNA=3.641,tGSK3β蛋白=2.694,tGSK3βmRNA=3.091,P值均<0.05);与索拉非尼组比较,索拉非尼+shElk-1组细胞Akt蛋白及mRNA、GSK3β蛋白及mRNA的相对表达量显著降低(tAkt蛋白=8.041,tAkt mRNA=6.342,tGSK3β蛋白=4.391,tGSK3βmRNA=8.327,P值均<0.05),差异均有统计学意义。结论索拉非尼可促进EC细胞凋亡,其机制可能与抑制Elk-1/Mcl-1和Akt/GSK3β通路有关。

Objective To explore the effect of sorafenib on tumor cell apoptosis and the mechanism involved in the down-regulation of myeloid cell leukemia-1(Mcl-1).Methods Methyl thiazolyl tetrazolium(MTT)method was used to detect the effect of sorafenib on the proliferation of EC cells.The concentration and duration of sorafenib have been determined.enterochromaffin cells(EC cells)were divided into 4 groups:control group,sorafenib group,sorafenib+shLuc group and sorafenib+shets-like gene-1(Elk-1)group.Flow cytometry was used to detect the apoptosis rate of cells in each group.Western blotting and real-time quantitative polymerase chain reaction(Real-time PCR)were used to detect the relative expression of B cell lymphoma/leukemia-2(bcl-2),bcl-2 associated X protein(bax),Elk-1,Mcl-1,protein kinase B(Akt)and glycogen synthase kinase 3β(GSK3β).Results The survival rates of EC cells after treatment with sorafenib at dosage of 1,5 and 10 mol/L were(67.42±5.08)%,(50.81±6.11)%and(38.49±5.41)%,which were significantly lower than that of 0 mol/L(99.98±0.25)%(t1=5.148,t5=2.142,t10=1.764,P<0.05).The survival rate of EC cells after 12,24 and 48 h of sorafenib treatment was(65.41±4.61)%,(49.64±4.73)%and(42.49±5.02)%,respectively,which was significantly lower than that of 0 h(99.99±0.18)%(t12=4.624,t24=1.856,t48=1.751,P<0.05),and there is no statistical difference in the survival rate of EC cells compared with 6 h Academic significance(t6=1.266,P>0.05).Compared with the control group,the cell apoptosis rate in the sorafenib group was significantly increased(t=12.235,P<0.05),compared with the sorafenib group,the cell apoptosis in the sorafenib+shElk-1 group The rate increased significantly(t=9.271,P<0.05),there was no significant difference in the relative expression of bcl-2,the relative expression of bax,and the ratio of bcl-2/bax between the groups(Fbcl-2=0.198,Fbax=0.541,Fbcl-2/bax=2.679,P>0.05).Compared with the control group,the relative expression of Elk-1 protein in the sorafenib group was not significantly different(tElk-1 protein=1.426,P>0.05),while Elk-1mRNA,Mcl-1 protein and mRNA was significantly reduced(tElk-1 mRNA=2.266,tElk-1 mRNA=0.045;tMcl-1 protein=2.774,tMcl-1 mRNA=2.987,P<0.05),Compared with the sorafenib group,the relative expression of Elk-1 protein and mRNA,Mcl-1 protein and mRNA in the sorafenib+shElk-1 group was significantly reduced(tElk-1 protein=5.340,tElk-1 mRNA=6.240,tMcl-1 protein=6.248,tMcl-1 mRNA=5.217,P both<0.05).Compared with the control group,the relative expression of Akt protein and mRNA,GSK3βprotein and mRNA in the sorafenib group was significantly reduced(tAkt protein=3.579,tAkt mRNA=3.641,tGSK3βprotein=2.694,tGSK3βmRNA=3.091,P<0.05),Compared with sorafenib group,Akt protein and mRNA,GSK3βin sorafenib+shElk-1 group the relative expression of protein and mRNA was significantly reduced(tAkt protein=8.041,tAkt mRNA=6.342,tGSK3βprotein=4.391,tGSK3βmRNA=8.327,P<0.05).Conclusion Sorafenib can promote the apoptosis of EC cells,and its mechanism may be related to the inhibition of Elk-1/Mcl-1 and Akt/GSK3βpathway.