Abstract: 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 SK-Hep-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 (60 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.

Key words: dealcoholized red wine, hepatocellular carcinoma, polyphenol, RNA-seq, cell cycle, apoptosis