| 引用本文: | 潘海涛,陈栋杰,张国亮,胡凌娟,王晓彤,仲怿,杨继鸿,李振皓.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[J].中国现代应用药学,2023,40(13):1795-1809. |
| PAN Haitao,CHEN Dongjie,ZHANG Guoliang,HU Lingjuan,WANG Xiaotong,ZHONG Yi,YANG Jihong,LI Zhenhao.Study on the Anti-tumor Activity of Components of Ganoderma Lucidum on the Three-dimensional Culture Model of Colorectal Cancer HCT116 Cells[J].Chin J Mod Appl Pharm(中国现代应用药学),2023,40(13):1795-1809. |
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| 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 |
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潘海涛1,2, 陈栋杰1,2, 张国亮2,3, 胡凌娟3, 王晓彤3,4, 仲怿1,2, 杨继鸿1,2, 李振皓1,2,3
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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
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| 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。 |
| 关键词: 灵芝 结肠癌 三维培养 脂解 脂噬 |
| DOI：10.13748/j.cnki.issn1007-7693.20222521 |
| 分类号:R285.5 |
| 基金项目:浙江省科技计划重点研发项目(2019C02100) |
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| Study on the Anti-tumor Activity of Components of Ganoderma Lucidum on the Three-dimensional Culture Model of Colorectal Cancer HCT116 Cells |
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PAN Haitao1,2, CHEN Dongjie1,2, ZHANG Guoliang2,3, HU Lingjuan3, WANG Xiaotong3,4, ZHONG Yi1,2, YANG Jihong1,2, LI Zhenhao1,2,3
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1.Zhejiang ShouXianGu Botanical Drug Institute Co., Ltd., Hangzhou 311100, China;2.Hangzhou Yuhang BoYu Intelligent Health Innovation Lab, Hangzhou 311100, China;3.Zhejiang ShouXianGu Pharmaceutical Co., Ltd., Wuyi 321200, China;4.Zhejiang Key Agricultural Enterprise Institute of ShouXianGu Rare Herb Products, Wuyi 321200, China
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| Abstract: |
| 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 anti-tumor activity. CONCLUSION Gc3 significantly inhibit the proliferation of 3D-cultured HCT116 cells by inhibiting cell autophagy and lipid droplet breakdown, and enhance the anti-cancer activity of 5-FU by inhibiting the expression of ITGB1, CDH1, ABCB1, and ABCC1 mRNA. |
| Key words: Ganoderma lucidum colorectal cancer three-dimensional culture lipolysis lipophagy |
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