·论著·

荜茇酰胺对人胃癌细胞株MKN45增殖、凋亡的影响及其机制研究

段超勤*邓超邹晓平#

南京大学医学院附属鼓楼医院消化科(210008)

背景：近年研究显示，荜茇酰胺(PL)在诸多恶性肿瘤中可通过升高活性氧(ROS)水平选择性杀伤肿瘤细胞，然而其对胃癌细胞的作用仍有待进一步研究。目的：探讨PL对人胃癌细胞株MKN45增殖、凋亡的影响及其可能机制。方法：以不同浓度PL以及caspase抑制剂、抗氧化剂单独或联合处理MKN45细胞，CCK-8法检测细胞活性，流式细胞术检测细胞周期、细胞凋亡和细胞内ROS水平，蛋白质印迹法检测凋亡相关蛋白XIAP、cleaved-caspase3、7、9、cleaved-PARP、p53及其下游靶基因p21、GADD45α、PUMA表达。结果：PL可剂量和时间依赖性地抑制MKN45细胞增殖，经PL处理的MKN45细胞G1期细胞比率、细胞凋亡率和细胞内ROS水平显著升高，抗凋亡蛋白XIAP表达下调，caspase依赖性凋亡途径、p53及其下游靶基因激活。抗氧化剂NAC或广谱caspase抑制剂Z-VAD-FMK预处理可逆转PL的促ROS生成以及增殖抑制和促凋亡作用。结论：PL能抑制MKN45细胞增殖，使细胞发生G1期阻滞，诱导细胞凋亡，其抗肿瘤效应的作用机制可能为升高肿瘤细胞内ROS水平，进而激活p53和caspase依赖性凋亡途径。

关键词荜茇酰胺；活性氧；肿瘤抑制蛋白p53；半胱氨酸天冬氨酸蛋白酶；细胞凋亡；胃肿瘤

Effect and Underlying Mechanism of Piperlongumine on Proliferation and Apoptosis of Human Gastric Cancer Cell Line MKN45

DUANChaoqin,DENGChao,ZOUXiaoping.DepartmentofGastroenterology,theAffiliatedDrumTowerHospitalofNanjingUniversityMedicalSchool,Nanjing(210008)

Correspondence to: ZOU Xiaoping, Email: zouxiaoping795@hotmail.com

Background: Recently, studies have shown that piperlongumine (PL) selectively killed cancer cells by elevating reactive oxygen species (ROS) in various cancers. However, the effect of PL on gastric cancer cells remained to be further studied. Aims: To investigate the effect of PL on proliferation and apoptosis of human gastric cancer cell line MKN45 and its underlying mechanism. Methods: MKN45 cells were treated with different doses of PL, caspase inhibitor, antioxidant, and their combinations, respectively. Cell viability was assessed by CCK-8 assay; cell cycle, apoptosis and intracellular ROS level were measured by flow cytometry; and Western blotting was employed to determine the expression of apoptosis-related proteins (XIAP, cleaved-caspase3, 7, 9 and cleaved-PARP), p53 and its downstream target genes (p21, GADD45α and PUMA). Results: PL inhibited the proliferation of MKN45 cells in a dose- and time-dependent manner. In MKN45 cells treated with PL, the proportion of cells in G1 phase, apoptotic rate and intracellular ROS level were significantly increased, the expression of inhibitor of apoptosis protein XIAP was down-regulated, and the caspase-dependent apoptosis pathway, p53 and its downstream target genes were activated. Pretreatment with antioxidant NAC or Z-VAD-FMK, a general caspase inhibitor could partially abolish the effect of PL on ROS production and its antitumor effect. Conclusions: PL can inhibit cell proliferation and induce cell cycle G1 phase arrest and apoptosis in MKN45 cells. Its antitumor effect may be associated with a ROS-mediated p53 activation and subsequent triggering of caspases cascade of cell apoptosis.

Key wordsPiperlongumine;Reactive Oxygen Species;Tumor Suppressor Protein p53;Caspases;

Apoptosis;Stomach Neoplasms

尽管胃癌发病率已从1975年位居世界第一位下降至2012年的世界第五位，但胃癌致死率仍居癌症相关死亡率的第三位[1]。大部分胃癌患者确诊时已属晚期，且半数以上的局部进展期胃癌患者术后可能复发，3年生存率低于40%[2]。近年来，随着免疫治疗、新辅助治疗、分子靶向药物等治疗方法的不断进步，胃癌患者的长期生存率有所提高。……