乏氧和去血清通过诱导自噬拮抗KAI1诱导凋亡  

Hypoxia and serum deprivation protected MiaPaCa-2 cells from KAI1-induced apoptosis through autophagy pathway activation in solid tumors

作　　者：吴春燕[1] 郭晓钟[1] 

机构地区：[1]解放军沈阳军区总医院消化内科,110016

出　　处：《中国临床实用医学》2016年第2期13-17,共5页China Clinical Practical Medicine

摘　　要：目的：观察乏氧和缺血是否通过诱导自噬拮抗肿瘤转移抑制基因KAI1/CD82（KAI1）的生长抑制作用。方法应用无KAI1表达的人胰腺癌细胞MiaPaCa2，通过感染带有KAI1目的基因的复制缺陷型腺病毒Ad5-KAI1使细胞表达KAI1蛋白，通过乏氧和去血清培养细胞模拟实体瘤内缺血、缺氧的微环境，根据培养条件不同，将人胰腺癌细胞株MiaPaCa-2分为4组：对照组（正常培养）、乏氧组、去血清组、乏氧去血清组。Western blot检测细胞LC3-Ⅱ/LC3-Ⅰ比值的改变。3-MA预处理阻断自噬，共聚焦显微镜观察自噬标志颗粒GFP-LC3颗粒。采用Annexin V-FITC/PI实验检测细胞增殖和凋亡的变化。结果 MiaPaCa2细胞不表达KAI1蛋白，感染Ad5-KAI1后表达KAI1蛋白，乏氧和去血清均显著促进LC3-Ⅱ/LC3-Ⅰ比值的增加和GFP-LC3融合质粒的增加，且均可被3-MA有效阻断。3-MA 预处理后感染Ad5-KAI1，4组细胞（含氧含血清正常培养的细胞、单纯去血清培养的细胞、单纯乏氧培养的细胞、联合去血清和乏氧培养的细胞）的Annexin V表达均显著增长，分别由70%、34%、28%和19%增至86%～90%。提示3-MA预处理可以阻断去血清和乏氧对KAI1引起的Annexin V表达增加的抑制作用。提示乏氧和去血清通过诱导自噬拮抗KAI1诱导的凋亡。结论乏氧和缺血清培养细胞通过诱导自噬拮抗KAI1诱导的凋亡，促进细胞生存。Objective KAI1 closely correlates with pancreatic cancer metastasis. There might be some factors that protect the cells from a proliferation inhibition by KAI1 in the solid tumors＇ microenvironment. Hypoxia and ischemia are the main characteristics of the microenvironment within solid tumors. Whether they affect the KAI1 inhibitory effects on cell proliferation is still unclear. Methods MiaPaCa-2 human pancreatic cancer cells do not express KAI1 protein. However, after being infected with Ad5-KAI1, they expressed KAI1 protein. We cultured them under hypoxic and serum-free conditions to simulate the solid tumor hypoxic-ischemic microenvironment. The cells were divided into the control, hypoxic, serum-free, and hypoxic with serum-free groups. The apoptosis were observed Annexin V-FITC/PI. The green fluorescent protein-labeled light chain 3 association with autophagosome membranes was detected by confocal microscopy. The ratio of LC3-Iito LC3-I expression level was detected by western blot. Pre-treatment of 3-MA was used to inhibit the autophagy. We,then observed whether the hypoxic and serum-free condi-tions could change the effect of KAI1 on cell survival and whether the pretreatment of 3-MA could inhibit the effect of hypoxic and serum-free conditions on KAI1 function. Results Hypoxia and serum-free media effectively reduced the apoptosis and proliferation inhibition caused by KAI1 and was beneficial to the cell survival. 3-MA pretreatment effectively blocked the protective effect of hypoxia and serum-free media on the cells by autophagy block. Conclusion Serum free media and hypoxia protected the MiaPaCa-2 cells from a KAI1-induced apoptosis via autophagy induction.

关 键 词：肿瘤转移抑制基因KAI1/CD82(KAI1) 人胰腺癌 乏氧 去血清 自噬 

分 类 号：R735[医药卫生—肿瘤]