Chen Jiayu, Jin Xiaoyan, Chen Jie, Liu Chibo
Medical School of Taizhou University, Taizhou, Zhejiang, 318000, China.
Tumour Biol. 2013 Jun;34(3):1381-9. doi: 10.1007/s13277-013-0746-7. Epub 2013 Apr 12.
To study the antitumor effect of glycyrrhiza polysaccharide (GPS) on human hepatocellular carcinoma cells and its mechanism, GPS was extracted and identified with phenol-sulfuric acid assay, Limulus amebocytes lysate assay, gel permeation chromatography, and infrared spectroscopy analysis. To study its antitumor function, 4-5-week-old imprinting control region mice were subcutaneously implanted with H22 cells and intragastrically subjected to 1 ml GPS (25, 50, and 75 mg/kg/day), 150 mg/kg cyclophosphamide in a dose of 150 mg/kg, or equal volume of phosphate buffered saline as control. Tumor weights were detected 10 days later. Apoptosis of intraperitoneally cultured and GPS-treated H22 cells was identified by flow cytometry and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide. In vitro, the function of GPS on cell proliferation was applied on BEL7402 cells and confirmed by 4,6-diamidino-z-phenylindole staining. Assessment of the effect of GPS on P53 gene was analyzed by real-time PCR and Western blot, and the effects of GPS on phosphatidylinositol-3 kinase (PI3K), AKT, p-PI3K, and p-AKT were analyzed by Western blot. We extracted the GPS, and it dose-dependently inhibited the tumorigenicity of hepatocellular carcinoma cells in nude mice. GPS treatment resulted in a significant (P<0.05) dose-dependent increase in the number of apoptotic cells in vivo and a significant (P<0.05) dose-dependent decrease in hepatocellular carcinoma cell proliferation in vitro. GPS modified multiple key enzymes (p-PI3K, p-AKT, and P53) in P53/PI3K/AKT signaling pathways on DNA or protein levels. Taken together, we extracted the GPS successfully and our findings suggest that GPS functions as a tumor suppressor through influencing the P53/PI3K/AKT pathway in the carcinogenesis of hepatocellular carcinoma and may have therapeutic implications for the clinical management of hepatocellular carcinoma patients.
为研究甘草多糖（GPS）对人肝癌细胞的抗肿瘤作用及其机制，采用苯酚-硫酸法、鲎试剂法、凝胶渗透色谱法和红外光谱分析法对GPS进行提取与鉴定。为研究其抗肿瘤功能，将4-5周龄的印记控制区小鼠皮下接种H22细胞，分别灌胃给予1 ml GPS（25、50和75 mg/kg/天）、剂量为150 mg/kg的环磷酰胺150 mg/kg，或等体积的磷酸盐缓冲盐水作为对照。10天后检测肿瘤重量。通过流式细胞术和5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基-羰花青碘化物鉴定腹腔内培养并经GPS处理的H22细胞的凋亡情况。在体外，将GPS对细胞增殖的作用应用于BEL7402细胞，并通过4,6-二脒基-2-苯基吲哚染色进行确认。通过实时荧光定量PCR和蛋白质免疫印迹法分析GPS对P53基因的影响，通过蛋白质免疫印迹法分析GPS对磷脂酰肌醇-3激酶（PI3K）、AKT、p-PI3K和p-AKT的影响。我们提取了GPS，其剂量依赖性地抑制裸鼠体内肝癌细胞的致瘤性。GPS处理导致体内凋亡细胞数量显著（P<0.05）剂量依赖性增加，体外肝癌细胞增殖显著（P<0.05）剂量依赖性减少。GPS在DNA或蛋白质水平上修饰了P53/PI3K/AKT信号通路中的多种关键酶（p-PI3K、p-AKT和P53）。综上所述，我们成功提取了GPS，研究结果表明，GPS在肝癌发生过程中通过影响P53/PI3K/AKT途径发挥肿瘤抑制作用，可能对肝癌患者的临床治疗具有指导意义。
