摘要
目的观察胆红素(bilirubin,Bil)对索拉非尼(sorafenib,Sor)治疗肝癌效果的影响,并探讨其可能机制。方法体外实验:用不同浓度的Sor(0、2、4、8、16、32μmol/L)、Bil(0、2.5、5、10、20、40μmol/L)及不同浓度Sor+Bil(20μmol/L)培养Huh7细胞系不同时间(24、48、72 h)后,通过CCK-8法检测细胞活力。采用Sor(4μmol/L)、Bil(20μmol/L)、Sor(4μmol/L)+Bil(20μmol/L)处理Huh7细胞,48 h后用流式细胞仪检测细胞周期,14 d后通过克隆形成实验检测药物对细胞增殖的影响。体内实验:取16只裸鼠构建Huh7细胞异种移植瘤模型,按S型抽样方法将裸鼠分为Con组、Bil组(25 mg·kg^(-1)·d^(-1))、Sor(15 mg·kg^(-1)·d^(-1))组、Bil(25 mg·kg^(-1)·d^(-1))+Sor(15 mg·kg^(-1)·d^(-1))组,每组各4只,并进行相应处理。通过Western blot和实时荧光定量PCR(RT-qPCR)检测各组过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor,PPARα)、肉毒碱棕榈酰基转移酶1A(carnitine palmityl transferase 1A,CPT1A)的表达量。加入CPT1A抑制剂乙莫克舍(etomoxir,Eto)后,通过CCK-8、克隆形成实验验证Bil削弱Sor对肝癌细胞的增殖抑制与PPARα/CPT1A的相关性。结果CCK-8结果显示,Sor对Huh7细胞的增殖抑制效应呈现剂量和时间依赖(P<0.05),Bil对Huh7细胞活力没有明显影响(P>0.05)。与单独Sor处理组相比,Bil+Sor组细胞增殖活力增加,细胞克隆数目较多(P<0.05),细胞在S期的数目减少(P<0.05)。体内实验结果显示:Sor组肿瘤体积和质量小于Con组(P<0.05),Bil+Sor组肿瘤体积和质量大于Sor组(P<0.05)。Western blot和RT-qPCR结果显示:与Sor组相比,Bil+Sor组PPARα、CPT1A的表达增多(P<0.05)。与Bil+Sor组相比,加入Eto后,Bil+Sor+Eto组细胞增殖活力降低(P<0.05),细胞克隆数目减少(P<0.05)。结论胆红素通过PPARα/CPT1A介导脂肪酸氧化削弱索拉非尼的抗肿瘤效果。
Objective To observe the effect of bilirubin(Bil)on sorafenib(Sor)in the treatment of liver cancer and explore the involved molecular mechanisms.Methods In vitro,Huh7 cells were treated with different concentration of Sor(0,2,4,8,16,32μmol/L),of Bil(0,2.5,5,10,20,40μmol/L)and of Sor(0,2,4,8,16,32μmol/L)+Bil(20μmol/L)for 24,48 and 72 h.Cell viability was detected by CCK-8 assay.Huh7 cells were treated with Sor(4μmol/L),Bil(20μmol/L)and Sor(4μmol/L)+Bil(20μmol/L),cell cycle was detected by flow cytometry after 48 h,and the effects of drugs on cell proliferation were detected by clone formation assay after 14 d.In vivo,16 mice bearing xenografts of Huh7 cells were divided into Con group,Bil group(25 mg·kg^(-1)·d^(-1)),Sor group(15 mg·kg^(-1)·d^(-1))and Bil(25 mg·kg^(-1)·d^(-1))+Sor(15 mg·kg^(-1)·d^(-1))group(n=4),according to the S type sampling method.The expression levels of peroxisome proliferator-activated receptor(PPARα)and carnitine palmityl transferase 1 A(CPT1 A)were detected by Western blotting and real time PCR.After etomoxir(Eto),the inhibitor of CPT1 A,was added,CCK-8 and clone formation assay were used to verify the mechanisms correlated with PPARα/CPT1 A.Results CCK-8 results showed that the inhibitory effect of Sor on Huh7 cells was in a dose and time-dependent manner(P<0.05),and Bil had no significant effect on Huh7 cells(P>0.05).Compared with Sor group,the cell proliferation in Bil+Sor group was increased(P<0.05),the relative cell colony formation rate was grown(P<0.05),and the number of cells in S phase was decreased(P<0.05).In vivo,results showed that the tumor volume and weight of Sor group were lower than that of Con group(P<0.05),and the tumor volume and weight of Bil+Sor group were higher than those of Sor group(P<0.05).Western blotting and RT-qPCR results showed that PPARαand CPT1 A expression levels were increased in Bil+Sor group compared with Sor group(P<0.05).After Eto was added,the cell proliferation and the relative cell clone formation rate of Bil+Sor+Eto group were
作者
谭君
张弛
董严
梁后杰
TAN Jun;ZHANG Chi;DONG Yan;LIANG Houjie(Department of Oncology,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2022年第16期1606-1612,共7页
Journal of Army Medical University
基金
国家自然科学基金青年科学基金项目(81803028)。
