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. 2023 Aug 20;43(8):1297-1305.
doi: 10.12122/j.issn.1673-4254.2023.08.05.

[Dealcoholized red wine inhibits occurrence and progression of hepatocellular carcinoma possibly by inducing cell cycle arrest and apoptosis]

[Article in Chinese]
Y Lan  1 , K Wang  1 , Z Lan  1 , H Zhou  1 , J Sun  1
Affiliations

[Dealcoholized red wine inhibits occurrence and progression of hepatocellular carcinoma possibly by inducing cell cycle arrest and apoptosis]

[Article in Chinese]
Y Lan et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the inhibitory effect of dealcoholized red wine (DRW) on occurrence and progression of hepatocellular carcinoma (HCC) and explore its possible mechanisms.

Methods: Three HCC cell lines (Huh7, HepG2 and SKHep-1) treated with 5, 10, 25, 50 and 100 μL/mL DRW were examined for changes in proliferation and colony formation ability using CCK-8 assay and colony formation assay. A nude mouse model bearing subcutaneous HCC xenograft was used to test the effect of 300 μL/day DRW for 4 weeks on tumor growth. The inhibitory effect of 300 μL/day DRW for 6 weeks on tumor growth was also observed in a mouse model of chemically induced HCC by examining the tumor number, largest tumor diameter and the liver/body ratio. RNA-seq technique was used for transcriptome sequencing of Huh7 cells treated with DRW (75 μL/mL) for 48 h, and gene-set enrichment analysis (GSEA) was performed to identify the changes in genes and pathways. Flow cytometry assay was used to analyze the changes in cell cycle and apoptosis of the cells.

Results: DRW inhibited the proliferation of the HCC cell lines in a concentration-and time-dependent manner, and concentration-dependently inhibited colony formation of the cells. Treatment with DRW significantly reduced the volume of subcutaneous tumor xenograft in the tumor-bearing nude mice (P < 0.05), and lowered the number of tumors (P < 0.001), the largest tumor diameter (P < 0.05) and the liver/body ratio (P < 0.01) in mice with chemically induced HCC. RNA-seq showed that 634 genes were significantly up-regulated and 478 were down-regulated in Huh7 cells after treatment with DRW. Gene-set enrichment analysis revealed that DRW significantly down-regulated cell cycle-related pathways (E2F Targets, G2M Checkpoint and MYC Targets) and up-regulated apoptosis pathways. Flow cytometry assay showed that DRW induced cell cycle arrest in G1 phase and apoptosis of Huh7 cells.

Conclusion: DRW inhibits the occurrence and progression of HCC, and this effect is mediated possibly by inducing cell cycle arrest and apoptosis.

目的: 探讨脱醇红酒对肝细胞癌（HCC）发生发展的抑制作用，并初步探索其可能的机制。

方法: 不同浓度的脱醇红酒（0、5、10、25、50和100 μL/mL）处理人肝癌细胞（Huh7，HepG2和SK-Hep-1）后，采用CCK-8和克隆形成实验分别检测各组细胞的增值活性和克隆形成能力。构建裸鼠皮下瘤模型，分为实验组（脱醇红酒，300 μL/d）和对照组，干预4周后对比两组肿瘤体积。构建化学诱导小鼠肝癌模型，分为实验组（脱醇红酒，300 μL/d）和对照组，干预6周后对比两组肝脏肿瘤数量、最大肿瘤直径和肝体比。脱醇红酒（75 μL/mL）处理48 h后，利用RNA-seq对Huh7细胞转录组测序，基因集富集分析（GSEA）观察基因集及通路的变化；流式细胞术检测脱醇红酒（75 μL/mL）干预后Huh7细胞周期和凋亡变化。

结果: 在体外，脱醇红酒呈浓度和时间依赖性地抑制肝癌细胞的增殖活性，呈浓度依赖性地抑制其克隆形成能力。在体内，相比于对照组，脱醇红酒显著抑制实验组裸鼠皮下瘤的体积（P < 0.05），也能显著抑制实验组化学诱导肝癌小鼠的肝脏肿瘤数量（P < 0.001）、最大肿瘤直径(P < 0.05）和肝体比（P < 0.01）。RNA-seq结果显示，脱醇红酒干预后Huh7细胞内有634个基因上调和478个基因下调（|log2FC|≥2，Q≤0.05）。基因集富集分析发现，脱醇红酒能够诱导细胞周期通路相关基因集显著下调（包括E2F Targets、G2M Checkpoint、MYC Targets等）和细胞凋亡通路相关基因集上调；流式细胞检测结果表明，脱醇红酒的干预使Huh7细胞G1期阻滞，同时诱导细胞凋亡。

结论: 脱醇红酒对HCC的发生发展具有抑制作用，其机制可能与细胞周期和凋亡相关通路介导的肝癌细胞G1期阻滞和细胞凋亡有关。

Keywords: RNA-seq; apoptosis; cell cycle; dealcoholized red wine; hepatocellular carcinoma; polyphenol.

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Figures

图 1
图 1
DRW对肝癌细胞的增殖抑制作用 Inhibitory effect on dealcoholized red wine (DRW) on proliferation of HCC cells. A: Inhibition rate and A valve of Huh7 cells. B: Inhibition rate and OD valve of HepG2 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs DRW group.
图 2
图 2
DRW对肝癌细胞克隆形成数量的影响 Effect of dealcoholized red wine (DRW) on clone formation of HCC cells. A: Gross observation of the colonies. B: Colony number of Huh7 cells. C: Colony number of Sk-Hep-1 cells. ***P < 0.001 vs Control group.
图 3
图 3
DRW抑制裸鼠皮下荷瘤的生长 Dealcoholized red wine (DRW) inhibits subcutaneous HCC xenograft growth in nude mice. A: General observation of the tumors. B: Tumor growth curve of DRW and Control groups. *P < 0.05 vs DRW group.
图 4
图 4
DRW抑制化学诱导小鼠肝癌的发生发展 DRW inhibits occurrence and progression of chemically induced HCC in mice. A: Experimental design of DEN/CCL4-induced mouse model. B: General view of HCC in the two groups. C-E: Tumor number, largest tumor diameter and liver/body ratio in the control group (n=25) and DRW group (n=12). *P < 0.05, **P < 0.01, ***P < 0.001.
图 5
图 5
DRW诱导Huh7细胞周期G1期阻滞和细胞凋亡比例增加 DRW induces G1 phase arrest and apoptosis of Huh7 cells. A: Cell cycle distribution in DRW (75 and 150 μL/mL) and control groups. B: Flow cytometric analysis of cell apoptosis in DRW (75 and 150 μL/mL) and control groups. C: Percentage of cell population in G1, S and G2 phases in different groups. D: Cell apoptosis rates in different groups. ***P < 0.001.
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