Lan R L, Huang F, Chen R Q, Wang Z, Chen J Y, Lin J A, Fu L X
Central Lab, First Affiliated Hospital of Fujian Medical University, Fuzhou, China 350005.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2018 Jul 7;53(7):524-529. doi: 10.3760/cma.j.issn.1673-0860.2018.07.008.
To investigate the effect of ubiquitous mitochondrial creatine kinase 1(CKMT1) on the sensitivity of human nasopharyngeal carcinoma cell line CNE-1 to DDP. CNE-1 cells were transiently transfected with CKMT1 overexpression (CKMT1) or empty vector (EV). The growth curve and DDP IC50 were developed by MTT assay, plate clone formation assay was performed by gradient concentration of DDP treatment, cell cycle and apoptosis were detected by flow cytometry, levels of apoptosis related protein Bax/Bcl-2/C-PARP and the transcription factor p-STAT3-Tyr705 were detected by Western Blot. The transfection efficiencies of CKMT1 and EV were more than 90% with a higher proliferation rate in the CKMT1-transfected cells. However, the CKMT1-transfected cells had a DDP IC50 of 2.76 μmol/L, which was significantly lower than that of 4.60 μmol/L in the EV-transfected cells (<0.01). With the treatment of certain concentration of DDP, the CKMT1-transfected cells had a lower clone formation rate, the cell cycle arrested more obviously in G2/M phase, and the apoptosis rate was higher (<0.01), with higher levels of Bax/C-PARP (<0.05 or <0.01), but lower levels of Bcl-2 (<0.01) and p-STAT3-Tyr705 (<0.01), compare with the EV-transfected cells. CKMT1 may inhibit the activation of STAT3, increasing the sensitivity of CNE-1 to chemotherapeutic drug DDP.
探讨泛在性线粒体肌酸激酶1(CKMT1)对人鼻咽癌细胞系CNE-1顺铂(DDP)敏感性的影响。将CNE-1细胞分别用CKMT1过表达载体(CKMT1)或空载体(EV)进行瞬时转染。采用MTT法绘制生长曲线并测定DDP的半数抑制浓度(IC50),通过不同梯度浓度DDP处理进行平板克隆形成实验,采用流式细胞术检测细胞周期及凋亡情况,运用蛋白质免疫印迹法检测凋亡相关蛋白Bax/Bcl-2/C-聚(ADP-核糖)聚合酶(C-PARP)及转录因子磷酸化信号转导子和转录激活子3(p-STAT3-Tyr705)的表达水平。CKMT1和EV的转染效率均超过90%,且CKMT1转染细胞增殖率更高。然而,CKMT1转染细胞的DDP IC50为2.76 μmol/L,显著低于EV转染细胞的4.60 μmol/L(<0.01)。经一定浓度DDP处理后,CKMT1转染细胞的克隆形成率较低,细胞周期在G2/M期阻滞更明显,凋亡率更高(<0.01),与EV转染细胞相比,Bax/C-PARP水平更高(<0.05或<0.01),而Bcl-2和p-STAT3-Tyr705水平更低(<0.01)。CKMT1可能抑制STAT3的激活,从而增加CNE-1对化疗药物DDP的敏感性。