藤黄茎皮中莽吉柿素对人胃癌HGC-27细胞的作用研究

Inhibitory Effect of Gartanin from Stem Barks of Garcinia mangostana on HGC-27 Cell Growth

目的:探讨藤黄树茎皮中氧杂环酮类成分莽吉柿素对人胃癌HGC-27细胞的生长抑制作用及潜在的作用机制。方法:采用噻唑蓝(MTT)染色实验检测莽吉柿素对HGC-27细胞的抑制作用。采用克隆形成实验评估莽吉柿素对HGC-27细胞增殖的影响。采用Hoechst染色实验研究莽吉柿素对HGC-27细胞形态变化的影响。采用蛋白免疫印迹法(Western blot)检测莽吉柿素给药后与凋亡过程、Neddylation修饰途径及丝裂原活化蛋白激酶(MAPK)信号通路相关的蛋白表达水平变化。采用裸鼠移植瘤模型进行莽吉柿素裸鼠体内抗肿瘤药效研究。结果:莽吉柿素给药后能够抑制HGC-27细胞的增殖能力和细胞集落形成。与对照组比较,莽吉柿素显著上调HGC-27细胞多聚ADP核糖聚合酶1(PARP1)和Bax蛋白表达(P<0.001),下调抗凋亡蛋白B细胞淋巴瘤-2(Bcl-2)的表达(P<0.001),发挥促进胃癌细胞凋亡的作用。莽吉柿素给药后HGC-27细胞中β淀粉样蛋白前体蛋白结合蛋白1(APPBP1)、泛素样修饰激活酶3(UBA3)和泛素结合酶E2M(UBC12)蛋白及MAPK信号通路蛋白c-Jun、c-Jun氨基端激酶(JNK)和p38的磷酸化水平均显著下调(P<0.001)。进一步的体内移植瘤动物实验结果显示,莽吉柿素给药后裸鼠肿瘤体积缩小18.7%,差异具有统计学意义(P<0.05)。与对照组比较,莽吉柿素组裸鼠体内与肿瘤细胞增殖相关的标志蛋白Ki67阳性细胞数明显减少,肿瘤组织中棕色细胞数量明显增加。结论:藤黄树茎皮中氧杂环酮类化学成分莽吉柿素在HGC-27细胞和裸鼠上均显示良好的抗胃癌活性,其作用机制可能与抑制Neddylation修饰途径以及抑制MAPK信号通路相关。

Objective:To investigate the inhibitory effect of gartanin,an oxacyclic ketone,in the stem barks of Garcinia mangostana on the growth of human gastric cancer HGC-27 cells and its underlying mechanism.Methods:MTT assay was used to detect the inhibitory effect of gartanin on HGC-27 cells.Clone formation assay was used to evaluate the effect of gartanin on the proliferation of HGC-27 cells.Hoechst staining was carried out to study the effect of gartanin on the morphological changes of HGC-27 cells.Western blot was performed to detect the changes in the expression levels of proteins related to apoptosis process,Neddylation modification pathway and mitogen activated protein kinase(MAPK)signaling pathway after gartanin treatment.In addition,the xenograft model in nude mice was used to study the anti-tumor efficacy of gartanin.Results:Gartanin could inhibit the proliferation and colony formation of HGC-27 cells.Compared with the conditions in the control group,gartanin significantly up-regulated poly ADP-ribose polymerase-1(PARP1)and Bax protein expression(P<0.001)and down-regulated anti-apoptotic protein human B-cell leukemia/lymphoma 2(Bcl-2)expression(P<0.001),which promoted the apoptosis of gastric cancer cells.The amyloid beta precursor protein binding protein 1(APPBP1),ubiquitin-like modifier activating enzyme 3(UBA3),and ubiquitin conjugating enzyme E2M(UBC12)proteins and phosphorylation levels of proteins related to the MAPK signaling pathway,such as c-Jun,c-Jun N-terminal kinase(JNK),and p38,were significantly down-regulated in HGC-27 cells after gartanin treatment(P<0.001).Further results from in vivo animal experiments on xenograft model showed that the tumor volume was reduced by 18.7%after gartanin treatment(P<0.05).Compared with the results in the control group,the gartanin group showed a decreased number of Ki67-positive cells associated with tumor cell proliferation and an increased number of brown cells in tumor tissues in the nude mice.Conclusion:Gartanin from the stem barks of G.mangostana shows good activity against gastric cancer on both HGC-27 cells and nude mice,and its mechanism of action may be related to the inhibition of Neddylation and the MAPK signaling pathway.