To investigate the effect of the jianpi-jiedu formula (JPJD) on the expression of angiogenesis-relevant genes in colon cancer. Methods: Crude extract was obtained from JPJD by water extract method. The effect of JPJD crude extract on colon cancer cell proliferation capacity was determined by MTT assays. The IC50 value was calculated by GraphPad Prism5 software. Affymetrix gene expression profiling chip was used to detect significant differences in expressions of genes after JPJD intervention, and pathway enrichment analysis was performed to analyze the differentially expressed genes. Ingenuity Pathway Analysis software was applied to analyze differentially expressed genes relevant to tumor angiogenesis based on mammalian target of rapamycin (mTOR) signaling pathway and then the network diagram was built. Western blot was used to verify the protein levels of key genes related to tumor angiogenesis. Results: JPJD crud extract inhibited the proliferation capacity in colon cancer cells. The IC50 values in 24, 48, and 72 hours after treatment were 13.060, 9.646 and 8.448 mg/mL, respectively. The results of chip showed that 218 genes significantly upgraded, and 252 genes significantly downgraded after JPJD treatment. Most of the genes were related to the function of biosynthesis, metabolism, cell apoptosis, antigen extraction, angiogenesis and so on. There were 12 differentially expressed angiogenesis genes. IPA software analysis showed that the JPJD downregulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), VEGF, vascular endothelial growth factor A (VEGFA), integrin subunit alpha 1 (ITGA1), cathepsin B (CTSB), and cathepsin S (CTSS) genes, while upregulated expressions of GAB2 and plasminogen activator, urokinase receptor (PLAUR) genes in the colorectal cancer cell. Western blot results demonstrated that JPJD obviously downregulated expressions of phospho-mTOR (P-mTOR), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α (HIF-1α), and VEGF proteins, while obviously upregulated the level of phospho-P53 (P-P53) protein. Conclusion: JPJD may inhibit colorectal tumor angiogenesis through regulation of the mTOR-HIF-1α-VEGF signal pathway.
目的：探讨健脾解毒方对大肠癌肿瘤血管形成相关基因表达的影响。方法：采用水提法制作健脾解毒方粗提取物， MTT法检测健脾解毒方粗提取物对大肠癌HT29细胞增殖的影响，GraphPad Prism 5 软件计算IC50(50% inhibitory concentration)值，Affymetrix基因表达谱芯片检测健脾解毒方干预后有显著差异表达的基因并进行路径富集分析，基于哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)信号通路利用IPA(Ingenuity Pathway Analysis)软件对肿瘤血管形成的相关差异表达基因进行生物信息学分析并构建网路关系图，Western 印迹法验证肿瘤血管形成相关差异表达的关键基因的蛋白表达水平。结果：健脾解毒方粗提取物能够抑制大肠癌HT29细胞增殖，处理24，48及72 h后的IC50值分别为13.060，9.646及8.448 mg/mL。Affymetrix基因表达谱芯片检测健脾解毒方干预后显著上调的基因218个，显著下调的基因252个，主要为生物合成、新陈代谢、细胞凋亡、抗原提取、血管生成等相关基因，其中肿瘤血管形成相关差异表达基因12个。IPA软件分析结果显示健脾解毒方干预后可使大肠癌中的鞘磷脂磷酸二酯酶3(sphingomyelin phosphodiesterase 3，SMPD3)，VEGF，血管内皮生长因子A(vascular endothelial growth factor A，VEGFA)，整合数α亚基1(integrin subunit alpha 1，ITGA1)，组织蛋白酶B(cathepsin B，CTSB)，组织蛋白酶S(cathepsin S，CTSS)等基因下调，GRB2相关结合蛋白2(GRB2 associated binding protein 2，GAB2)，PLAUR(plasminogen activator，urokinase receptor)等基因上调。Western印迹检测显示健脾解毒方能明显下调磷酸化mTOR(phospho-mTOR，P-mTOR)，信号转导和转录激活因子3(signal transducer and activator of transcription 3，STAT3)，缺氧诱导因子-1α(hypoxia inducible factor-1α，HIF-1α)和VEGF蛋白表达，明显上调磷酸化P53(phospho-P53，P-P53)蛋白表达。结论：健脾解毒方可能通过影响mTOR-HIF-1α-VEGF信号通路抑制大肠癌肿瘤血管形成，这可能是健脾解毒方治疗大肠癌的作用机制之一。.