骆驼蓬提取物β-咔啉类生物碱对SGC-7901及裸鼠移植瘤组织PI3K和AKT表达影响

目的新疆地道药材骆驼蓬有抗肿瘤作用。本课题组通过前期实验表明,其提取物β-咔啉类生物碱有诱导胃癌SGC-7901细胞凋亡的作用,但信号通路尚未明确。本研究拟探讨β-咔啉类生物碱对人胃癌SGC-7901细胞及裸鼠移植瘤组织中PI3K和AKT表达的影响。方法使用β-咔啉类生物碱及氟尿嘧啶体外干预SGC-7901细胞,使用蛋白质印迹及RT-PCR法检测PI3K和AKT蛋白及mRNA的表达。构建裸鼠移植瘤模型,使用不同剂量β-咔啉类生物碱及氟尿嘧啶行体外干预,使用TUNEL法检测凋亡,同时使用蛋白质印迹、RT-PCR、免疫组化法检测PI3K、AKT表达。结果在体外实验中,β-咔啉类生物碱组PI3K、p-PI3K mRNA表达为0.387±0.218、0.325±0.105,低于空白对照组的0.473±0.119和0.423±0.122及阳性对照组的1.003±0.101和1.001±0.201,F值分别为13.912和13.875,P值分别为0.006和0.056。阳性对照组AKT、p-AKT mRNA表达分别为0.679±0.061和0.621±0.043,低于空白对照组的1.001±0.065和1.021±0.037及β-咔啉类生物碱组的0.800±0.056和0.728±0.031,F值分别为8.036和49.747,P值分别为0.020和<0.001。空白对照组PI3K、p-PI3K蛋白表达分别为1.330±0.091和1.244±0.030,高于阳性对照组的1.321±0.194和1.006±0.082及β-咔啉类生物碱组的1.014±0.063和1.011±0.151,F值分别为5.837和5.439,P值分别为0.039和0.045。空白对照组AKT、p-AKT蛋白表达分别为0.934±0.037和0.903±0.129,高于阳性对照的0.887±0.041和0.788±0.054及β-咔啉类生物碱组的0.865±0.080和0.594±0.131,F值分别为11.531和5.973,P值分别为0.009和0.037。在体内实验中,β-咔啉类生物碱组与各组瘤质量相比较,F=4.659,P=0.009。在体内实验中,β-咔啉类生物碱高剂量组PI3K、p-PI3K mRNA表达分别为0.520±0.215和0.513±0.201,低于空白对照组的1.003±0.088和1.001±0.051及阳性对照组的0.517±0.146和0.521±0.212,F值分别为6.873和6.327,P值分别为0.006和0.005。β-咔啉类生物碱高剂量组AKT、p-AKT mRNA表达分别为0.765±0.121和0.732±0.101,低于空白对照组的1.001±0.047和1.004±0.032及阳性对照组的0.851±0.213和0.847±0.094,F值分别为1.227和1.319,P值分别为0.359和0.372。β-咔啉类生物碱高剂量组PI3K、p-PI3K蛋白表达分别为0.621±0.113和0.323±0.069,低于空白对照组的0.706±0.047和0.745±0.153及阳性对照组的0.631±0.105和0.550±0.243,F值分别为1.109和1.194,P值分别为0.409和0.377。β-咔啉类生物碱高剂量组AKT、p-AKT蛋白表达分别为0.435±0.067和0.400±0.118,低于空白对照组的0.536±0.098和0.807±0.161及阳性对照组的0.462±0.166和0.639±0.202,F值分别为5.946和0.417,P值分别为0.013和0.793。结论在人胃癌SGC-7901细胞及裸鼠移植瘤组织中,β-咔啉类生物碱可降低PI3K和AKT蛋白和基因的表达,PI3K/AKT通路上可能有β-咔啉类生物碱作用的靶点。

Effect of Peganum camel extract on expression of PI3 Kand AKTin gastric cancer cell SGC-7901 and xenograft tumor tissue of nude mice

OBJECTIVE Peganum camel is a genuine medicinal material in Xinjiang and has anti-tumor effect.Previous experiments showed that its extract beta-carbaline alkaloids could induce apoptosis of gastric cancer SGC-7901 cells,but the signal pathway was not clear.The aim of this study was to investigate the effects of beta carolinoid alkaloids on the expression of PI3 K and AKTin human gastric cancer SGC-7901 cells and nude mice xenografts.METHODS The SGC-7901 cells were treated with beta carolinoid alkaloids and fluorouracil in vitro.The expression of PI3 K and AKT protein and their mRNA were detected by Western blotting and RT-PCR.Nude mice transplanted tumor model was constructed.Different doses of beta carolinoid alkaloids and fluorouracil were used to intervene in vitro.Apoptosis was detected by TUNEL method.The expressions of PI3 Kand AKT were detected by Western blotting,RT-PCR and immunohistochemistry.RESULTS In vitro,the expression of PI3 K and p-PI3 K mRNA in beta carolinoid alkaloids group(0.387±0.218,0.325±0.105)was lower than that in blank control group(0.473±0.119,0.423±0.122)and positive control group(1.003±0.101,1.001±0.201;F=13.912,P=0.006;F=13.875,P=0.056).The expression of AKTand p-AKT mRNA in positive control group(0.679±0.061,0.621±0.043)was lower than that in blank control group(1.001±0.065,1.021±0.037)and beta carolinoid alkaloids group(0.800±0.056,0.728±0.031;F=8.036,P=0.020;F=49.747,P<0.001).The expression of PI3 K and p-PI3 K protein in blank control group(1.330±0.091,1.244±0.030)was higher than that in positive control group(1.321±0.194,1.006±0.082)and beta-carbaline alkaloids group(1.014±0.063,1.011±0.151;F=5.837,P=0.039;F=5.439,P=0.045).The expression of AKTand p-AKTprotein in blank control group(0.934±0.037,0.903±0.129)was higher than that in positive control(0.887±0.041,0.788±0.054)and beta-carbaline alkaloids group(0.865±0.080,0.594±0.131;F=11.531,P=0.009;F=5.973,P=0.037).In vivo experiments,the tumor weight of beta carolinoid alkaloids group and other group was(F=4.659,P=0.009).In vivo,the expression of PI3 K and p-PI3 K mRNA in the high dose group of beta carolinoid alkaloids(0.520±0.215,0.513±0.201)was lower than that in the blank control group(1.003±0.088,1.001±0.051)and the positive control group(0.517±0.146,0.521±0.212;F=6.873,P=0.006;F=6.327,P=0.005).The expression of AKTand p-AKT mRNA in the high dose group of beta carolinoid alkaloids(0.765±0.121,0.732±0.101)was lower than that in the blank control group(1.001±0.047,1.004±0.032)and the positive control group(0.851±0.213,0.847±0.094;F=1.227,P=0.359;F=1.319,P=0.372).The expression of PI3 Kand p-PI3 K protein in high dose group of beta carolinoid alkaloids(0.621±0.113,0.323±0.069)was lower than that in blank control group(0.706±0.047,0.745±0.153)and positive control group(0.631±0.105,0.550±0.243;F=1.109,P=0.409;F=1.194,P=0.377).The expression of AKTand p-AKT protein in high dose group of beta carolinoid alkaloids(0.435±0.067,0.400±0.118)was lower than that in blank control group(0.536±0.098,0.807±0.161)and positive control group(0.462±0.166,0.639±0.202;F=5.946,P=0.013;F=0.417,P=0.793).CONCLUSION In human gastric cancer SGC-7901 cells and nude mice transplanted tumor tissues,beta carolinoid alkaloids can reduce the expression of PI3 K and AKT proteins and genes.There may be a target of beta carolinoid alkaloids in PI3 K/AKTpathway.