Role of arbutin in the inhibition of FBXO5 in hepatocellular carcinoma

Abstract

Purpose: This work investigated the effect of FBXO5 in hepatocellular carcinoma (HCC) and the mechanism of action of arbutin in its inhibition.

Methods: FBXO5 mRNA and protein expressions in the tumor were assessed using TCGA, ICGC and HPA databases. Cox regression analysis and Kaplan-Meier survival curves were employed to assess the impact of FBXO5 on the survival outcomes of patients with HCC. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were used to investigate the biological function associated with FBXO5-related genes. The role of FBXO5 as oncogene and the inhibitory mechanism of arbutin were confirmed through western blotting (WB), reverse transcription quantitative polymerase chain reaction (RT-qPCR), and in vitro experiments such as scratch wound-healing migration assay, plate clone formation assay, and transwell migration assay.

Results: Patients with high FBXO5 expression showed shorted overall survival (OS), progression-free survival (PFS), disease-specific survival (DSS), and disease-free survival (DFS). FBXO5 was identified as an independent prognostic risk factor associated with the cell cycle. In vitro investigations indicated that FBXO5 facilitated HCC progression by modulating the cell cycle, while arbutin suppressed FBXO5 expression and regulated cell cycle dynamics.

Conclusion: FBXO5 is a potential diagnostic and prognostic biomarker for HCC, and arbutin may exert anticancer effects through the suppression of FBXO5 expression.

Keywords: Arbutin; Cell cycle; FBXO5; HCC.

Fig. 1

FBXO5 expression. a Pan-cancer expression of FBXO5. b Differential expression of FBXO5 in the tumor versus normal tissue in the TCGA-LIHC cohort. c Differential expression of FBXO5 in the tumor versus normal tissue in the GSE76427 cohort. d Differential expression of FBXO5 in the tumor versus normal tissue in the GSE64041 cohort. e Immunohistochemical staining of FBXO5 in normal liver tissue, as revealed by the HPA database. f Immunohistochemical staining of FBXO5 in HCC tissue, as revealed by the HPA database. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

Prognostic analysis of FBXO5. a Differential expression of FBXO5 across various age subgroups in the TCGA-LIHC cohort. b Differential expression of FBXO5 across different stages of HCC in the TCGA-LIHC cohort. c Differential expression of FBXO5 across T-stages of HCC in the TCGA- LIHC cohort. d-g Survival curves of OS, PFS, DFS, DSS in patients with high and low FBXO5 expression in the TCGA-LIHC cohort; h Correlation between FBXO5 expression and OS in the ICGC-LIRI-JP cohort. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

FBXO5-related functional enrichment analysis. a GSEA analysis revealing the enriched GO terms in the group with high FBXO5 expression. b GSVA scores indicating differences in hallmark pathway activity between the groups with high and low FBXO5 expression. c GSVA scores demonstrate a correlation between FBXO5 expression and hallmark pathway activities. d–f Kaplan–Meier survival plots illustrating a significant correlation between OS and GSVA scores of G2M_CHECKPOINT (d), E2F_TARGETS (e), and UNFOLDED_PROTEIN_RESPONSE (f)

Fig. 4

FBXO5 affects HCC cell proliferation and migration. a Impact of various interference fragments on FBXO5 mRNA expression in Hep-3B and SNU-449 cells by RT-qPCR. b Spontaneous fluorescent imaging of virus encapsulation. c FBXO5 mRNA expression in SNU449-FBXO5-OE cell lines by RT-qPCR. d Scratch wound-healing migration assay performed on Hep-3B and SNU-449 cells pre and post-interference. e Healing rate derived from the wound healing assays. f–g Plate clone formation assays and transwell migration assay performed on SNU-449 cells. h–i Quantification of the plate clone formation and transwell migration assay on SNU-449 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Arbutin reduces HCC cell proliferation and migration. a Cytotoxic effect of arbutin on Hep-3B and SNU-449 HCC cells by CCK8 assay. b Effect of different concentrations of arbutin on FBXO5 mRNA expression in Hep-3B and SNU-449 HCC cells by RT-qPCR. c Effect of different concentrations of arbutin Hep-3B and SNU-449 HCC cell migration by Scratch wound-healing migration assay. d Wound healing rate comparison before and after treatment. e Colony formation assay performed on Hep-3B and SNU-449 HCC cells after the treatment with arbutin. f Cell migration assay performed on Hep-3B and SNU-449 HCC cells. g Number of colonies before and after treatment. h Cell migration before and after treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Arbutin affects HCC by inhibiting FBXO5 expression. a Molecular docking of FBXO5 with arbutin. b FBXO5 mRNA expression in different treatment groups. c-d FBXO5 protein expression in different treatment groups. e–f Cell migration in different treatment groups, and their quantification. g-h Cell cycle results in different treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001