基于结肠癌HCT116细胞三维培养模型的灵芝水提物抗肿瘤活性研究

Study on the Anti-Cancer Activity of Ganoderma lucidum Water Extract Based on the Three-Dimensional Culture Model of Colorectal Cancer HCT116 Cells

关键词:  灵芝  结肠癌  三维培养  E-cadherin  Integrin β1

Key words:  Ganoderma lucidum  colorectal cancer  three-dimensional culture  E-cadherin  integrin β1

摘要:

目的 采用结肠癌HCT116细胞的二维（2D）和三维（3D）培养模型研究灵芝水提物（Ganoderma lucidum water extract，GLWE）的抗肿瘤作用。方法 采用紫外-可见光分光光度法测定GLWE中灵芝多糖（Ganoderma lucidum polysaccharides，GLP）、三萜和甾醇的含量，HPLC分析GLP中水溶性单糖的组成和比例；以Matrigel基质胶为基质材料建立体外HCT116细胞3D培养模型，评价GLWE的抗肿瘤及辅助抗肿瘤活性；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。结果 GLWE中GLP和三萜及甾醇的含量分别为20.92%和7.18%，其中GLP主要由甘露糖、葡萄糖醛酸、葡萄糖、半乳糖、D-木糖、L-岩藻糖组成；GLWE和5-氟尿嘧啶（5-fluorouracil，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；与2D培养相比，3D培养的HCT116细胞中整合素β1（Integrin β1）和钙黏蛋白（E-cadherin）mRNA的表达显著升高，并对GLWE和5-FU的敏感性降低；在HCT116细胞3D培养模型中，GLWE既可降低CDK2、CDK4和Bcl-2蛋白的表达，升高p21、p27、cleaved caspase-3和cleaved PARP蛋白的表达，还可显著降低Integrin β1和E-cadherin mRNA的表达，并加强5-FU的抗肿瘤活性。结论 GLWE通过抑制细胞周期和诱导细胞凋亡而抑制3D培养的HCT116细胞增殖，并通过抑制Integrin β1和E-cadherin mRNA的表达增强5-FU的抗肿瘤活性。

Abstract:

OBJECTIVE To investigate the anti-cancer effects of Ganoderma lucidum water extract(GLWE) based on the two-dimensional(2D) and three-dimensional(3D) culture of colorectal cancer HCT116 cells.METHODS The content of Ganoderma lucidum polysaccharide(GLP), Ganoderma triterpenes and sterols in GLWE were determined by UV-visible spectrophotometry, and the composition and proportion of water-soluble monosaccharide in GLP were determined by HPLC. An 3D culture model of HCT116 cells in vitro was established using Matrigel as the matrix material, and the anti-cancer and adjuvant anti-cancer activities of GLWE were evaluated. Cell viability was detected by CCK-8 assay. mRNA expression level of the cells was analyzed by Real-time PCR. Western blotting was used to detect cell protein expression level. RESULTS The contents of GLP and Ganoderma triterpenes(contained sterols) in GLWE were 20.92% and 7.18%, respectively, and GLP was mainly comprised of mannose, glucuronic acid, glucose, galactose, D-xylose and L-fucose. GLWE and 5-fluorouracil(5-FU) inhibited the proliferation of both 2D and 3D cultured HCT116 cells in a dose dependent manner after incubation for 48 h. However, compared with the 2D culture, the expression of E-cadherin and Integrin β1 mRNA was significantly increased, and the sensitivity to GLWE and 5-FU was reduced in 3D cultured HCT116 cell. GLWE could not only reduce the expression of CDK2, CDK4 and Bcl-2 proteins, increase the expression of p21, p27, cleaved caspase-3 and cleaved PARP proteins, but also significantly reduce the expression of E-cadherin and Integrin β1 mRNA, and enhance the anti-cancer activity of 5-FU in 3D cultured HCT116 cell. CONCLUSION GLWE significantly inhibit the proliferation of 3D-cultured HCT116 cells by inhibiting cell cycle and inducing apoptosis, and enhanced the anti-cancer activity of 5-FU by inhibiting the expression of E-cadherin and Integrin β1 mRNA.