抑制MCL-1可逆转PD-L1敲除引起的结直肠癌细胞对司美替尼化疗敏感性下降  

Inhibition of MCL-1 reverses poor chemosensitivity of colorectal cancer cells to selumetinib induced by PD-L1 knockout

作　　者：孙磊 洪楚原[1] 尹雪霞[1] 郭雄波 张实 钟北平 王国强[1] SUN Lei;HONG Chu-yuan;YIN Xue-xia;GUO Xiong-bo;ZHANG Shi;ZHONG Bei-ping;WANG Guo-qiang(The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China)

机构地区：[1]广州医科大学附属第二医院胃肠外科,广东广州510260

出　　处：《中国病理生理杂志》2022年第6期1008-1014,共7页Chinese Journal of Pathophysiology

基　　金：广州市属高校科研项目(No.1201630159);广州市科技计划市校联合项目(No.202102010062);广州医科大学附属第二医院精英人才配套项目(No.010M07060)。

摘　　要：目的:明确程序性死亡配体1(programmed death ligand-1,PD-L1)敲除是否可引起结直肠癌(colorectal cancer,CRC)细胞对司美替尼化疗敏感性下降,并探寻逆转PD-L1敲除引起的司美替尼化疗敏感性下降的方法。方法:人结肠癌RKO细胞及敲除PD-L1的RKO细胞接受司美替尼处理后,Western blot检测两组细胞中活化的胱天蛋白酶3(cleaved caspase-3,C-Casp-3)蛋白水平的差异,流式细胞术及细胞集落形成实验分别检测两组细胞的凋亡率及细胞存活率。沉默髓细胞白血病基因1(myeloid cell leukemia-1,MCL-1)或AZD5991联合司美替尼处理后,Western blot检测RKO及敲除PD-L1的RKO细胞中C-Casp-3蛋白水平,流式细胞术及细胞集落形成实验分别检测细胞的凋亡率及细胞存活率。司美替尼联合AZD5991处理小鼠结肠腺癌MC38细胞及沉默PD-L1的MC38细胞,Western blot检测细胞中C-Casp-3蛋白水平,流式细胞术及细胞集落形成实验分别检测细胞的凋亡率及细胞存活率。结果:Western blot结果发现,司美替尼可显著降低磷酸化细胞外信号调节激酶(phosphorylated extracellu⁃lar signal-regulated kinase,p-ERK)蛋白水平,敲除PD-L1可显著降低细胞中司美替尼诱导产生的C-Casp-3,而MCL-1 shRNA或联合使用AZD5991可显著增加细胞中司美替尼诱导产生的C-Casp-3。流式细胞术结果发现,敲除PD-L1显著降低了司美替尼诱导产生的细胞凋亡,而MCL-1 shRNA或联合使用AZD5991均可显著增加司美替尼诱导产生的细胞凋亡。细胞集落形成结果发现,敲除PD-L1显著提高了低浓度司美替尼条件下细胞的存活率,而MCL-1 shRNA或联合使用AZD5991均可显著降低细胞的存活率。更重要的是,MCL-1 shRNA或联合使用AZD5991均有效逆转了PD-L1敲除引起的C-Casp-3、细胞凋亡及细胞存活率的改变。结论:敲除PD-L1可引起CRC细胞对司美替尼化疗敏感性下降,沉默MCL-1基因或联合使用AZD5991可逆转PD-L1敲除引起的CRC细胞对司美替尼化�AIM:To determine the effects and mechanism of programmed death ligand-1(PD-L1)on chemo⁃sensitivity to selumetinib in colorectal cancer(CRC)cells.METHODS:After human colon carcinoma RKO cells and PD-L1 knockout RKO cells were treated with selumetinib,cleaved caspase-3(C-Casp-3)protein level was detected by Western blot,and the apoptosis rate and cell survival rate were tested by flow cytometry(FCM)and colony formation assay,respectively.The RKO and PD-L1 knockout RKO cells were treated with selumetinib after myeloid cell leukemia-1(MCL-1)knockdown,or co-treated with selumetinib and AZD5991,C-Casp-3 protein level was detected by Western blot,and the apoptosis rate and cell survival rate were tested by FCM and colony formation assay,respectively.After mouse colon adenocarcinoma MC38 cells and PD-L1 knockout MC38 cells were co-treated with selumetinib and AZD5991,C-Casp-3 protein level was detected by Western blot,and the apoptosis rate and cell survival rate were tested by FCM and colony formation assay,respectively.RESULTS:Western blot showed that selumetinib significantly decreased phosphorylated extracellular signal-regulated kinase(p-ERK)protein level.Knockout of PD-L1 significantly reduced selumetinib-in⁃duced C-Casp-3 protein level.MCL-1 shRNA or co-treatment with AZD5991 significantly increased selumetinib-induced C-Casp-3 protein level.The FCM results showed that knockout of PD-L1 significantly reduced the apoptosis induced by se⁃lumetinib,and MCL-1 shRNA or co-treatment with AZD5991 significantly increased apoptosis induced by selumetinib.Colony formation assay showed that knockout of PD-L1 significantly increased cell survival rate after treatment with low concentration of selumetinib,and MCL-1 shRNA or co-treatment with AZD5991 significantly reduced cell survival rate.More importantly,MCL-1 shRNA or co-treatment with AZD5991 eliminated the differences in C-Casp-3,apoptosis and cell survival rate caused by PD-L1 knockout.CONCLUSION:Knockout of PD-L1 decreases the chemosensitivity of CRC cells to selu

关 键 词：髓细胞白血病基因1 程序性死亡配体1 司美替尼 结直肠癌 化疗敏感性 

分 类 号：R363[医药卫生—病理学] R73-36[医药卫生—基础医学] R735.35