基于结肠癌HCT116细胞三维培养模型的灵芝组分抗肿瘤活性研究  

Study on the Anti-tumor Activity of Components of Ganoderma Lucidum on the Three-dimensional Culture Model of Colorectal Cancer HCT116 Cells

作　　者：潘海涛 陈栋杰 张国亮 胡凌娟 王晓彤 仲怿 杨继鸿 李振皓 PAN Haitao;CHEN Dongjie;ZHANG Guoliang;HU Lingjuan;WANG Xiaotong;ZHONG Yi;YANG Jihong;LI Zhenhao(Zhejiang ShouXianGu Botanical Drug Institute Co.,Ltd.,Hangzhou 311100,China;Hangzhou Yuhang BoYu Intelligent Health Innovation Lab,Hangzhou 311100,China;Zhejiang ShouXianGu Pharmaceutical Co.,Ltd.,Wuyi 321200,China;Zhejiang Key Agricultural Enterprise Institute of ShouXianGu Rare Herb Products,Wuyi 321200,China)

机构地区：[1]浙江寿仙谷植物药研究院有限公司,杭州311100 [2]杭州市余杭区伯宇智慧健康研究院,杭州311100 [3]浙江寿仙谷医药股份有限公司,浙江武义321200 [4]寿仙谷珍稀药材产品省级重点农业企业研究院,浙江武义321200

出　　处：《中国现代应用药学》2023年第13期1795-1809,共15页Chinese Journal of Modern Applied Pharmacy

基　　金：浙江省科技计划重点研发项目(2019C02100)。

摘　　要：目的基于结肠癌HCT116细胞的二维(2D)和三维(3D)培养模型研究灵芝组分(Ganoderma components,Gc)的抗肿瘤作用。方法采用UPLC-Q-TOF-MS鉴定3种Gc的化学组成;以Matrigel基质胶为基质材料建立体外HCT116细胞3D培养模型,评价Gc1、Gc2、Gc3和5-氟尿嘧啶(5-fluorouracil,5-FU)对2D和3D培养的HCT116细胞增殖的影响,并在HCT116细胞3D培养模型中分析Gc3对周期、凋亡、耐药、脂代谢及5-FU抗肿瘤活性的影响;采用CCK-8法检测细胞活力;Real-time PCR检测细胞mRNA表达水平;Western blotting检测细胞蛋白表达水平;采用HPLC检测细胞内5-FU含量。结果从Gc1、Gc2和Gc3中分别鉴定出76,69和17个化合物;与2D培养相比,3D培养的HCT116细胞增殖率更低,周期促进蛋白CDK2、CDK4、CDK6及脂肪酸合成蛋白FASN、SREBP1的表达显著下调,周期抑制蛋白p21和p27、脂滴分解蛋白ATGL及药物抵抗基因ITGB1、CDH1、ABCB1、ABCC1 mRNA的表达水平显著上调;Gc1、Gc2、Gc3和5-FU作用48 h可呈浓度依赖性抑制2D和3D培养的HCT116细胞增殖,且Gc3的抑制作用显著强于Gc1和Gc2;在HCT116细胞3D培养模型中,Gc3可显著降低CDK2、CDK4、Bcl-xl、ATGL、LC3B的表达水平,升高p21、p27、Bax、Cleaved caspase-3、Cleaved PARP1的表达水平,过表达LC3B或ATGL均可减弱Gc3诱导的细胞毒性;Gc3还可显著抑制ITGB1、CDH1、ABCB1、ABCC1 mRNA的表达,提高细胞内5-FU的浓度,并加强5-FU的抗肿瘤活性。结论Gc3通过抑制细胞自噬和脂滴分解而诱导3D培养的HCT116细胞凋亡,并通过抑制ITGB1、CDH1、ABCB1、ABCC1 mRNA的表达加强5-FU的抗肿瘤活性。OBJECTIVE To investigate the anti-tumor effects of the components of Ganoderma lucidum(Gc)based on the two-dimensional(2D)and three-dimensional(3D)culture of colorectal cancer HCT116 cells.METHODS The chemical compositional of the three components was identified by UPLC-Q-TOF-MS.An in vitro 3D culture model of HCT116 cells was established by using Matrigel as the matrix material,and the effects of Gc1,Gc2,Gc3,and 5-fluorouracil(5-FU)on the proliferation of HCT116 cells in 2D and 3D culture models were evaluated,and the effects of Gc3 on cell-cycle,apoptosis,drug resistance,lipid metabolism,and 5-FU’s anti-tumor activity were evaluated.Cell viability was detected by CCK-8 assay.mRNA expression level of the cells was analyzed by Real-time PCR.Proteins expression level of the cells was analyzed by Western blotting.HPLC was used to detect the content of 5-FU in cells.RESULTS A total of 76,69,and 17 compounds were identified from Gc1,Gc2,and Gc3,respectively.Compared with 2D culture,the proliferation rate of HCT116 cells was decreased in the 3D culture model,and the expression of cell cycle-promoters CDK2,CDK4,CDK6,and fatty acid synthesizer FASN,SREBP1 were significantly down-regulated.On the contrary,the expression of cell cycle-suppressor p21,p27,and lipid droplet breakdown proteins ATGL and drug resistance gene ITGB1,CDH1,ABCB1,and ABCC1 mRNA were significantly up-regulated.Gc1,Gc2,Gc3 and 5-FU inhibited the proliferation of both 2D and 3D cultured HCT116 cells in a dose dependent manner after incubation for 48 h,and the inhibitory effect of Gc3 was significantly stronger than Gc1 and Gc2.Gc3 could not only reduce the expression of CDK2,CDK4,Bcl-xl,ATGL,and LC3B proteins,but also increase the expression of p21,p27,Bax,Cleaved caspase-3,and Cleaved PARP1 proteins,and overexpression of LC3B or ATGL attenuated Gc3-induced cytotoxicity in 3D cultured HCT116 cells.In addition,Gc3 significantly inhibited the expression of ITGB1,CDH1,ABCB1,and ABCC1 mRNA,and increased the intracellular 5-FU content,and enhanced the ant

关 键 词：灵芝 结肠癌 三维培养 脂解 脂噬 

分 类 号：R285.5[医药卫生—中药学]