Xu Menglong, Li Yongchao, Sun Wenqiang, Guan Haocheng, Shi Ruijun, Li Shuwei
Department of Biochemistry and Molecular Biology, College of Life Science and Technology, the State Key Laboratory Incubation Base for Conservation and Utilization of Bio-Resource in the Tarim Basin of the Xinjiang Production and Construction Corps, Tarim University, Alar, China.
J Gastrointest Oncol. 2025 Aug 30;16(4):1699-1710. doi: 10.21037/jgo-2025-185. Epub 2025 Aug 11.
Neochlorogenic acid (NCA), a naturally occurring polyphenolic compound, exhibits diverse biological activities. This study aimed to investigate the inhibitory effects of NCA on hepatocellular carcinoma (HCC) cells and elucidate its underlying molecular mechanisms.
The anti-proliferative activity of NCA on human HCC cell lines HepG2 and Huh-7 was assessed using the Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to analyze apoptosis and cell cycle distribution. Wound-healing assays were conducted to evaluate the effects of NCA on cell migration. Transcriptome sequencing was performed on NCA-treated and untreated Huh-7 cells to identify differentially expressed genes (DEGs) and signaling pathways. Western blot was used to validate the expression of key apoptosis-related proteins. experiments were carried out using a nude mouse xenograft model to assess the anti-tumor effects of NCA.
NCA significantly inhibited the proliferation of HepG2 and Huh-7 cells, with half maximal inhibitory concentration (IC) values of 345.5 and 231.8 µM at 24 hours, and 244.0 and 199.2 µM at 48 hours, respectively. Flow cytometry revealed that NCA induced apoptosis and G1 phase cell cycle arrest. Wound-healing assays demonstrated that NCA effectively suppressed HCC cell migration. Transcriptome analysis revealed 2,297 DEGs in NCA-treated Huh-7 cells (Padj<0.01), with 1,162 upregulated and 1,135 downregulated. Pathway enrichment analysis indicated significant enrichments in pathways related to "Alcoholism", "MicroRNAs in cancer", "Hepatocellular carcinoma", and "TGF-beta signaling pathway". Western blot confirmed the upregulation of pro-apoptotic proteins [BCL2-associated X protein (BAX); cysteinyl aspartate specific proteinase 3 (CASP3), BH3 interacting domain death agonist (BID), and cytochrome C (CYCS)] and downregulation of the anti-apoptotic protein B-cell lymphoma 2 (BCL2). , NCA treatment significantly inhibited tumor growth.
This study provides compelling evidence that NCA inhibits HCC cell growth and migration both and through the induction of apoptosis and cell cycle arrest. Transcriptomic analysis reveals that NCA induces widespread changes in transcriptional networks and metabolic pathways within HCC cells, highlighting its potential as a promising therapeutic strategy for HCC.
新绿原酸（NCA）是一种天然存在的多酚化合物，具有多种生物活性。本研究旨在探讨NCA对肝癌（HCC）细胞的抑制作用，并阐明其潜在的分子机制。
使用细胞计数试剂盒-8（CCK-8）检测法评估NCA对人肝癌细胞系HepG2和Huh-7的抗增殖活性。采用流式细胞术分析细胞凋亡和细胞周期分布。进行伤口愈合试验以评估NCA对细胞迁移的影响。对经NCA处理和未处理的Huh-7细胞进行转录组测序，以鉴定差异表达基因（DEG）和信号通路。蛋白质免疫印迹法用于验证关键凋亡相关蛋白的表达。使用裸鼠异种移植模型进行实验，以评估NCA的抗肿瘤作用。
NCA显著抑制HepG2和Huh-7细胞的增殖，24小时时半数最大抑制浓度（IC）值分别为345.5和231.8μM，48小时时分别为244.0和199.2μM。流式细胞术显示NCA诱导细胞凋亡和G1期细胞周期阻滞。伤口愈合试验表明NCA有效抑制HCC细胞迁移。转录组分析显示，经NCA处理的Huh-7细胞中有2297个DEG（Padj<0.01），其中1162个上调，1135个下调。通路富集分析表明，与“酒精中毒”、“癌症中的微小RNA”、“肝细胞癌”和“转化生长因子-β信号通路”相关的通路有显著富集。蛋白质免疫印迹法证实促凋亡蛋白[BCL2相关X蛋白（BAX）；半胱氨酸天冬氨酸特异性蛋白酶3（CASP3）、BH3相互作用结构域死亡激动剂（BID）和细胞色素C（CYCS）]上调，抗凋亡蛋白B细胞淋巴瘤2（BCL2）下调。此外，NCA处理显著抑制肿瘤生长。
本研究提供了有力证据，表明NCA在体内和体外均通过诱导细胞凋亡和细胞周期阻滞来抑制HCC细胞的生长和迁移。转录组分析表明，NCA诱导HCC细胞内转录网络和代谢途径发生广泛变化，突出了其作为HCC一种有前景的治疗策略的潜力。
