基于Notch信号通路探讨β-榄香烯逆转肺腺癌耐药机制的研究

Study on the mechanism of β-elemene reversing drug resistance of lung adenocarcinoma based on Notch signaling pathway

目的基于Notch信号通路,探讨β-榄香烯逆转肺腺癌耐药的机制。方法选择对A549/DDP细胞无细胞毒性的β-榄香烯浓度处理A549/DDP细胞,设β-榄香烯组与对照组,两组细胞均给予不同浓度的DDP(0.25、0.5、1、2、4、8、16、32μg·mL^(-1))处理24h,MTT法检测细胞活力并计算IC_(50),流式细胞仪检测细胞凋亡,Western blotting检测凋亡相关蛋白(cleaved-caspase 3、cleaved-caspase 9)、耐药相关蛋白(P-gp、survivin、p21)及Notch信号通路蛋白(Notch1、Hes1、Hey2)的表达。A549/DDP细胞在β-榄香烯处理的基础上,联合1μg·mL^(-1)Notch信号通路激活剂rhNF-κB处理,比较两组的IC_(50),Western blotting检测P-gp以及Notch1、Hes1、Hey2的表达。结果当β-榄香烯浓度低于20μg·mL^(-1)时,其对A549/DDP细胞的抑制率小于10%,无细胞毒性。β-榄香烯组DDP的IC_(50)显著低于对照组(t=14.712,P<0.05),逆转倍数为4.452倍。β-榄香烯组细胞凋亡率以及凋亡相关蛋白cleavedcaspase 3和cleaved-caspase 9的表达水平均高于对照组,差异有统计学意义(P<0.05)。β-榄香烯组P-gp、survivin蛋白相对表达量低于对照组,而p21蛋白相对表达量高于对照组,差异均有统计学意义(P<0.05)。β-榄香烯组Notch1、Hes1、Hey2蛋白相对表达量均低于对照组,差异有统计学意义(P<0.05)。β-榄香烯+rhNF-κB组DDP的IC_(50)以及P-gp、Notch1、Hes1、Hey2蛋白表达水平均高于β-榄香烯组,差异有统计学意义(P<0.05)。结论β-榄香烯可通过抑制Notch信号通路促进A549/DDP细胞凋亡,并逆转肺腺癌耐药。

Objective To explore the mechanism of β-elemene reversing drug resistance of lung adenocarcinoma based on Notch signaling pathway.Methods A549/DDP cells were treated withβ-elemene in a concentration that was not cytotoxic to A549/DDP cells.Designed β-elemene group and control group.Cells in the two groups were given different concentrations (0.25,0.5,1,2,4,8,16,32μg·mL^(-1)) of DDP for 24 h.Cell viability and the IC_(50)were measured by MTT,and cell apoptosis were detected by flow cytometry.The apoptosis-related proteins (cleaved-caspase 3,cleaved-caspase 9),drug resistance-associated proteins (P-gp,survivin,p21) and Notch signaling pathway proteins (Notch1,Hes1,Hey2) were detected by Western blotting.After A549/DDP cells were treated withβ-elemene+1μg·mL^(-1) Notch signaling pathway activator rhNF-κB,compare the IC_(50)between the β-elemene group and β-elemene+rhNF-κB group,and detect the proteins P-gp,Notch1,Hes1 and Hey2 with Western blotting.Results When the concentration of β-elemene was lower than 20μg·mL^(-1),the inhibition rate of A549/DDP cells was lower than 10%,showing no cytotoxicity.The IC_(50)of DDP in A549/DDP cells inβ-elemene group was lower than that in control group (t=14.712,P<0.05),and the reversal fold was 4.452 times.The apoptosis rate in the β-elemene group was higher than in the control group,and the expression levels of apoptosis-related proteins (cleaved-caspase 3 and cleaved-caspase 9) were higher than those of the control group (P<0.05).The relative expression levels of P-gp and survivin proteins inβ-elemene group were lower than in control group,and the relative expression level of p21 protein was higher inβ-elemene group than in control group (P<0.05).The relative expression levels of Notch1,Hes1 and Hey2 proteins inβ-elemene group was lower than that in control group (P<0.05).The A549/DDP cells inβ-elemene+rhNF-κB group had a higher DDP IC_(50)than that in theβ-elemene group,and the expression levels of P-gp,Notch1,Hes1,Hey2 proteins were higher than those of theβ-elemene group (P<0.05).Conclusion β-elemene can promote apoptosis of A549/DDP cells by inhibiting Notch signaling pathway,so that can reverse the drug resistance of lung adenocarcinoma.