苍术（Thunb.）通过 ERK 信号级联抑制胆管癌细胞生长和增殖。
Suppression of Cholangiocarcinoma Cell Growth and Proliferation by Atractylodes lancea (Thunb) DC. through ERK-Signaling Cascade.
机构信息
Division of Parasitology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani, Thailand.
Center of Excellence in Molecular Biology and Pharmacology of Malaria and Cholangiocarcinoma, Thammasat University, Pathumthani, Thailand.
出版信息
Asian Pac J Cancer Prev. 2021 Nov 1;22(11):3633-3640. doi: 10.31557/APJCP.2021.22.11.3633.
OBJECTIVE
The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells.
MATERIALS AND METHODS
The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR.
RESULTS
The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell.
CONCLUSION
The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.
目的
本研究旨在探讨 AL 对 ERK 信号分子（ERK、p-ERK、细胞周期蛋白 D 和 eIF4B）的抑制作用以及对 CCA 细胞生长和增殖的影响。
材料与方法
采用 MTT 法测定三种 CCA 细胞系 CL-6、HuCCT1 和 HuH28 的活力。研究了 Ras/ERK 抑制剂在 AL 提取物存在下对蛋白表达的影响。用 AL 和/或 Ras/ERK 抑制剂处理后，从每种 CCA 细胞中提取蛋白，在 12.5% SDS-PAGE 上分离。通过 RT-PCR 分析 AL 暴露 48 和 72 小时与 0 小时（未暴露细胞）相比的 mRNA 表达。
结果
AL（MTT 法）的细胞毒活性效力约比标准药物 5-氟尿嘧啶高 3 倍。CL-6、HuCCT-1 和 HuH28 细胞系的 AL 的 IC50（抑制细胞生长 50%的浓度）分别为 29.77±6.64、35.45±4.96 和 35.32±6.69 µg/mL（均值+SD）。将细胞在无和有 Ras/ERK 抑制剂（salirasib 和 XMD8-92）的情况下分别用 IC50 的 AL 提取物孵育 0、12、24、48 和 72 小时。通过 Western blot 分析测定蛋白表达。结果表明，在所有 CCA 细胞类型中，AL 在暴露 48 和 72 小时时对 ERK 缺乏明显的抑制作用。另一方面，在所有 CCA 细胞类型中均观察到 p-ERK 表达的显著抑制。在所有细胞类型中，细胞周期蛋白 D 在暴露 72 小时时显著下调，而在不同细胞类型中具有不同的效力。eIF4B 的表达在 HuCCT-1 中明显抑制，在 CL-6 和 HuH28 细胞中略有抑制。实时 PCR 分析显示，在 HuCCT1 和 HuH28 细胞中，AL 暴露 72 小时后 ERK 显著下调，但在 CL-6 细胞中没有。
结论
ERK 信号级联和下游分子是 AL 在 CCA 中的潜在作用靶点。
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