Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, 225001, PR China; The Key Laboratory of Syndrome Differentiation and Treatment of Gastric Cancer of the State Administration of Traditional Chinese Medicine, Yangzhou, 225001, PR China.
Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, 225001, PR China.
J Ethnopharmacol. 2023 Jan 30;301:115737. doi: 10.1016/j.jep.2022.115737. Epub 2022 Sep 27.
The traditional Chinese medicine herb Celastrus orbiculatus Thunb. is an important folk medicinal plant in China that has been used as an anti-inflammatory, antitumor, and analgesic in various diseases. Recent years, many studies have reported the significant effects of Celastrus orbiculatus Thunb. extract (COE) on gastric cancer. However, the specific mechanism by which COE regulates gastric cancer cytoskeleton remodeling and thus inhibits EMT has not yet been reported.
To study the effect and mechanism of COE in inhibiting the epithelial-mesenchymal transition (EMT) and metastasis of gastric cancer cells, laying an experimental foundation for the clinical application and further development of COE.
The high-content cell dynamic tracking system was used to continuously track the trajectory of cell movement in real time. Through the high-content data, the average movement distance and movement speed of the cells are calculated. Additionally, the dynamic images of the cell movement in the high-content imaging system are derived to analyze the impact of COE on the movement of gastric cancer cells. Cytoskeleton staining experiment was performed to detect the effect of COE on the assembly of gastric cancer cell cytoskeleton proteins. Western blot was employed to detect the changes of EMT and metastasis-related proteins in the gastric cancer cells treated by COE. The effect of COE on the key regulatory protein Cofilin-1 (CFL1) of cell movement was examined by Western blot and protein degradation experiment. The effect of COE on EMT and metastasis of the gastric cancer cells lacking CFL1 was assessed by a transwell assay. The in vivo inhibitory effect of COE on EMT and metastasis of gastric cancer was determined by the animal living image system. IHC assays were used to detect the levels of EMT-related proteins in COE reversal in vivo.
The results showed that the movement distance and average movement speed of gastric cancer cells after COE treatment were significantly lower than those of the control group. Cytoskeleton staining experiments revealed that COE can significantly change the distribution of skeletal proteins in gastric cancer cells. Additionally, COE treatment significantly reduced the expression of Matrix metalloproteinases (MMP-2, MMP-9) and other proteins. Furthermore, COE can significantly accelerate the degradation of CFL1 protein, and both COE treatment and CFL1 deletion can significantly inhibit EMT and metastasis of gastric cancer cells. Lastly, the number of peritoneal metastases of gastric cancer cells was significantly reduced in animals after COE treatment. COE can reverse the levels of EMT-related proteins while reducing the expression levels of CFL1 protein in vivo.
COE can significantly inhibit EMT and metastasis of gastric cancer cells in vivo and in vitro. This effect may be achieved by reducing the stability of CFL1 and inhibiting the assembly of actin in gastric cancer cells.
中国传统医学草药南蛇藤是中国一种重要的民间药用植物，已被用于治疗各种疾病的抗炎、抗肿瘤和镇痛。近年来，许多研究报告了南蛇藤提取物（COE）对胃癌的显著影响。然而，COE 调节胃癌细胞骨架重塑从而抑制上皮-间充质转化（EMT）的确切机制尚未报道。
研究 COE 抑制胃癌细胞上皮-间充质转化（EMT）和转移的作用及机制，为 COE 的临床应用和进一步开发奠定实验基础。
使用高通量细胞动态跟踪系统实时连续跟踪细胞运动轨迹。通过高通量数据，计算细胞的平均运动距离和运动速度。此外，从高通量成像系统的动态图像中分析 COE 对胃癌细胞运动的影响。进行细胞骨架染色实验，检测 COE 对胃癌细胞骨架蛋白组装的影响。采用 Western blot 检测 COE 处理后胃癌细胞中 EMT 和转移相关蛋白的变化。Western blot 和蛋白降解实验检测 COE 对细胞运动关键调节蛋白丝切蛋白-1（Cofilin-1，CFL1）的影响。通过 Transwell 测定 COE 对 CFL1 缺失的胃癌细胞 EMT 和转移的影响。动物活体成像系统检测 COE 对胃癌 EMT 和转移的体内抑制作用。免疫组化（IHC）检测 COE 在体内逆转 EMT 相关蛋白的水平。
结果表明，COE 处理后胃癌细胞的运动距离和平均运动速度明显低于对照组。细胞骨架染色实验显示，COE 可显著改变胃癌细胞骨架蛋白的分布。此外，COE 处理可显著降低基质金属蛋白酶（MMP-2、MMP-9）等蛋白的表达。此外，COE 可显著加速 CFL1 蛋白的降解，COE 处理和 CFL1 缺失均可显著抑制胃癌细胞的 EMT 和转移。最后，COE 处理后动物体内胃癌细胞腹膜转移数量明显减少。COE 可在体内逆转 EMT 相关蛋白水平，同时降低 CFL1 蛋白表达水平。
COE 可显著抑制体内外胃癌细胞 EMT 和转移。这种作用可能是通过降低 CFL1 的稳定性和抑制胃癌细胞中肌动蛋白的组装来实现的。
