Luo Zhi-Yi, Tian Qi, Cheng Niang-Mei, Liu Wen-Han, Yang Ye, Chen Wei, Zhang Xiang-Zhi, Zheng Xiao-Yuan, Chen Ming-Sheng, Zhuang Qiu-Yu, Zhao Bi-Xing, Liu Cong-Sheng, Liu Xiao-Long, Li Qin, Wang Ying-Chao
The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, China.
Fujian Pien Tze Huang Enterprise Key Laboratory of Natural Medicine Research and Development, Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd., Zhangzhou, Fujian Province, 363099, China.
Chin J Integr Med. 2024 Feb;30(2):115-124. doi: 10.1007/s11655-022-3533-8. Epub 2022 Aug 10.
To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism.
Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 10 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (10 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP).
PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP.
PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.
探讨片仔癀（PZH）对肝癌细胞迁移和侵袭的影响及其潜在分子机制。
采用细胞计数试剂盒-8（CCK-8）评估不同浓度PZH（0、0.2、0.4、0.6、0.8 mg/mL）与SMMC-7721、SK-Hep-1、C3A和HL-7702（6×10个细胞/孔）共孵育24小时后的细胞活力。分别进行Transwell实验、伤口愈合实验、CCK-8实验和Annexin V-FITC/PI染色，以研究PZH对SK-Hep-1和SMMC-7721细胞（SK-Hep-1细胞为650μg/mL，SMMC-7721细胞为330μg/mL）迁移、侵袭、增殖和凋亡的影响。在体内，通过尾静脉注射肝癌细胞构建肺转移小鼠模型，用于评估PZH的抗转移功能。将SK-Hep-1细胞（每只小鼠10个细胞/200μL）经尾静脉注射到B-NDG小鼠体内。共8只小鼠随机分为PZH组和对照组，每组4只。接种2天后，PZH组小鼠从异种移植后第2天至第17天给予PZH（250mg/kg，每日），对照组小鼠仅接受溶剂（PBS）。随后基于RNA测序进行转录组分析，以解读PZH的抗肿瘤机制。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法验证RNA测序结果。进行荧光素酶报告基因实验以检测Yes相关蛋白（YAP）的转录活性。
PZH处理在体外和体内均显著抑制肝癌细胞的迁移、侵袭和增殖，并促进其凋亡（P<0.01）。转录组分析表明，Hippo信号通路与PZH的抗转移功能相关。机制研究表明，PZH显著抑制血小板衍生生长因子受体β（PDGFRB）、YAP、结缔组织生长因子（CCN2）、N-钙黏蛋白、波形蛋白和基质金属蛋白酶2（MMP2）的表达（P<0.01）。同时，PZH处理在体外和体内均增强了YAP的磷酸化。此外，PZH在抑制YAP的转录活性中发挥作用。
PZH通过抑制PDGFRB/YAP/CCN2轴抑制肝癌细胞的迁移、侵袭和上皮-间质转化。
