三氧化二砷介导的人胃癌培养细胞的细胞周期阻滞及凋亡性细胞死亡
Cell cycle arrest and apoptotic cell death in cultured human gastric carcinoma cells mediated by arsenic trioxide.
作者信息
Shao Qin-Shu, Ye Zai-Yuan, Ling Zhi-Qiang, Ke Jin-Jing
机构信息
Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang Province, China.
出版信息
World J Gastroenterol. 2005 Jun 14;11(22):3451-6. doi: 10.3748/wjg.v11.i22.3451.
AIM
To investigate the effect of arsenic trioxide on human gastric cancer cell line MKN45 with respect to both cytotoxicity and induction of apoptosis in vitro.
METHODS
MKN45 cells were treated with arsenic trioxide (As2O3) at the concentration of 1, 5, and 10 micromol/L, respectively, for three successive days. Cell growth and proliferation were observed by cell counting and trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Morphologic changes were studied with light microscopy. Flow cytometry was used to assay cell DNA distribution and apoptotic cells were confirmed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and DNA electrophoresis.
RESULTS
The growth of MKN45 cells was significantly inhibited by As2O3 which was confirmed by colony-forming assay. After 7 d of culture with various concentrations of As2O3, colony-forming capacity of MKN45 cells decreased with As2O3 increment in comparison with that of control group. The inhibitory rate of colony-formation was 38.5%, 99.1%, and 99.5% when the concentration of As2O3 was 1, 5, and 10 micromol/L in culture medium, respectively. The cell number of a single colony in drug treatment groups was less than that of control group. The cell-killing rate of As2O3 to MKN45 cells was both dose- and time-dependent with an IC50 of (11.05+/-0.25) micromol/L. After incubation in 10 micromol/L As2O3 for 24 h, the cell-killing rate was 27.1%, and it was close to 50% after 48 h. The results showed that As2O3 induced time- and dose-dependent apoptosis in MKN45 cells, blocked at G2/M phase. The apoptotic peak (sub-G1 phase) appeared and cell apoptotic rate in MKN45 cells was 18.3-32.5% after treatment by 10 micromol/L As2O3 for 48 h. The percentage of G2/M cell of the experimental groups was 2.0-5.0 times than that of the control group. Gel electrophoresis of DNA from cells treated with each concentration of As2O3 for 48 h revealed a "ladder" pattern, indicating preferential DNA degradation at the internucleosomal, linker DNA sections. TUNEL also demonstrated strand breaks in DNA of MKN45 cells treated with As2O3, while control cells showed negative labeling.
CONCLUSION
As2O3 can induce apoptosis of human gastric carcinoma cells MKN45, which is the basis of its effectiveness. It shows great potential in the treatment of gastric carcinoma.
目的
研究三氧化二砷对人胃癌细胞系MKN45的体外细胞毒性及诱导凋亡作用。
方法
分别用1、5和10 μmol/L的三氧化二砷（As2O3）连续处理MKN45细胞3天。通过细胞计数和台盼蓝拒染法观察细胞生长和增殖情况。采用MTT法测定As2O3的细胞毒性。用光镜研究形态学变化。用流式细胞术检测细胞DNA分布，并用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）和DNA电泳法确认凋亡细胞。
结果
集落形成试验证实As2O3能显著抑制MKN45细胞的生长。用不同浓度的As2O3培养7天后，与对照组相比，MKN45细胞的集落形成能力随As2O3浓度的增加而降低。当培养基中As2O3浓度分别为1、5和10 μmol/L时，集落形成抑制率分别为38.5%、99.1%和99.5%。药物处理组单个集落中的细胞数少于对照组。As2O3对MKN45细胞的杀伤率呈剂量和时间依赖性，IC50为（11.05±0.25）μmol/L。在10 μmol/L As2O3中孵育24小时后，细胞杀伤率为27.1%，48小时后接近50%。结果表明，As2O3可诱导MKN45细胞发生时间和剂量依赖性凋亡，阻滞于G2/M期。经10 μmol/L As2O3处理48小时后，MKN45细胞出现凋亡峰（亚G1期），细胞凋亡率为18.3% - 32.5%。实验组G2/M期细胞百分比是对照组的2.0 - 5.0倍。对用各浓度As2O3处理48小时的细胞进行DNA凝胶电泳，显示出“梯形”图谱，表明在核小体间连接DNA片段处优先发生DNA降解。TUNEL法也证实As2O3处理的MKN45细胞DNA发生链断裂，而对照细胞呈阴性标记。
结论
As2O3可诱导人胃癌细胞MKN45凋亡，这是其发挥疗效的基础。它在胃癌治疗中显示出巨大潜力。
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