Division of Anatomy, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand.
Research Unit in Nutraceuticals and Food Safety, Faculty of Medicine, Thammasat University, Pathumthani, 12120, Thailand.
Asian Pac J Cancer Prev. 2021 Jan 1;22(1):209-217. doi: 10.31557/APJCP.2021.22.1.209.
Many previous studies reported that fucoidan has antitumor activities. The objective of the present study was to determine the cytotoxic effects and related mechanisms of cell death induced by fucoidan extracted from Fucus vesiculosus on CL-6 cholangiocarcinoma cell.
CL-6 and OUMS cells were treated with 0, 100, 200, and 300 μg/mL of fucoidan. MTT assay was used to determine cytotoxicity. Flow cytometry-based assay was used to examine the distribution of apoptosis and cell cycle. The changes in nuclear morphology were determined using Hoechst 33,342 staining. Mitochondrial membrane potential (ΔΨm) was evaluated using the JC-1 kit. The apoptotic, anti-apoptotic, and cell cycle-related proteins study were examined by Western blot analysis.
The relative viable cell number of treated CL-6 cells was decreased but no effect was observed in OUMS normal cells. Furthermore, treated cells were arrested in the G0/G1 phase with down-regulation of cyclin D1 and CDK4. Annexin V/PI staining with flow cytometry analysis suggested that fucoidan could induce apoptosis in CL-6 cells. Western blot study revealed the up-regulation of apoptotic markers including Bax, cleaved PARP, cleaved caspase-3, but down-regulation of anti-apoptotic markers, cl-2. Moreover, fucoidan could induce nuclear fragmentation and chromatin condensation with alteration of ΔΨm. Conclusion: Fucoidan exerts antitumor properties against CL-6 cholangiocarcinoma cells illustrated by the induction of apoptosis and cell cycle arrest.
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许多先前的研究报告指出，褐藻糖胶具有抗肿瘤活性。本研究的目的是确定从泡叶藻中提取的褐藻糖胶对 CL-6 胆管癌细胞的细胞毒性作用及相关细胞死亡机制。
用 0、100、200 和 300μg/ml 的褐藻糖胶处理 CL-6 和 OUMS 细胞。MTT 法测定细胞毒性。采用流式细胞术检测细胞凋亡和细胞周期分布。用 Hoechst 33,342 染色检测核形态变化。用 JC-1 试剂盒评估线粒体膜电位（ΔΨm）。采用 Western blot 分析检测凋亡、抗凋亡和细胞周期相关蛋白的变化。
处理后的 CL-6 细胞相对活细胞数减少，但 OUMS 正常细胞无影响。此外，处理后的细胞被阻滞在 G0/G1 期，cyclin D1 和 CDK4 下调。用流式细胞术检测 Annexin V/PI 染色表明褐藻糖胶可诱导 CL-6 细胞凋亡。Western blot 研究表明，凋亡标志物 Bax、cleaved PARP、cleaved caspase-3 上调，抗凋亡标志物 cl-2 下调。此外，褐藻糖胶可诱导核碎裂和染色质浓缩，同时改变 ΔΨm。结论：褐藻糖胶对 CL-6 胆管癌细胞具有抗肿瘤作用，表现为诱导细胞凋亡和细胞周期阻滞。
