Schisandrin B downregulates exosomal fibronectin 1 expression to inhibit hepatocellular carcinoma growth

Abstract

Introduction: In recent years, natural compounds have attracted wide attention for the treatment of liver cancer due to their therapeutic potential and reduced toxicity. Among these, Schisandrin B (Sch B), a primary bioactive component derived from Schisandra chinensis, has shown notable antitumor activity; however, its specific mechanism remains unclear.

Methods: The effect of Sch B on the growth of hepatocellular carcinoma(HCC) cells were assessed using CCK-8 assay, colony formation assay and EdU assay, and apoptosis was detected by flow cytometry. The co-culture system of macrophages and HCC cells was established to detect the effect of Sch B on the cell viability and cell cycle changes of HCC cells in the co-culture system. Then, the migration of HCC cells in the co-culture system was studied using a subtoxic concentration of Sch B. Exosomes of the co-culture system with or without Sch B effect were collected for identification and protein spectrum analysis. The differential protein was analyzed by KEGG enrichment analysis and protein interaction network, which was verified by western blotting. Meanwhile, the expression changes of macrophage polarization markers were detected. Finally, the inhibitory effect of Sch B on HCC and the changes of FN1 were verified by in vivo experiments.

Results: Sch B inhibited HCC cell growth; moreover, it significantly suppressed HCC cell proliferation in the co-culture system and induced S-phase cell cycle arrest by downregulating CDK4, CDK2, and cyclin A2 while upregulating p27 Kip1. Additionally, Sch B inhibited the migration of HCC cells in the co-culture system.The differentially expressed protein fibronectin 1(FN1) in liver cancer patients was higher than that in healthy people. Moreover, after SchB treatment, the expression of FN1 protein in exosomes decreased and the macrophages exhibited M1 polarization. In vivo experiments also verified that Sch B inhibited HCC growth and downregulated the expression of FN1 protein in tumor tissues.

Conclusion: Sch B may inhibit the development of HCC by inhibiting the expression of exosomal FN1during interactions between macrophages and HCC cells.

Keywords: Schisandrin B; exosomes; fibronectin 1; hepatocellular carcinoma; tumor microenvironment.

FIGURE 1

Sch B inhibits HCC cell growth. (A) HCC cells were treated with various concentrations of Sch B, and cell viability was measured using the CCK-8 assay. (B–F) HCC cells were treated with Sch B (0, 20 and 40 μg/mL), and their proliferation abilities were measured using plate cloning experiment (BC) and a EdU assay (DE). HCC cell apoptosis was detected using flow cytometry (F). There sults are presented as the mean ± SD (n = 3). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 2

Sch B inhibits HCC cell growth in the co-culture system of macrophages and liver cancer cells. (A) Schematic diagram of the co-culture of HCC cells and macrophages. Sch B (0, 20, or 40 μg/mL) acted on the co-culture system, and HCC cell proliferation in the co-culture system was detected using cell counting (B), the EdU assay (C and D) and the colony formation assay (E). (F) Flow cytometry was used to assess the cell cycle and perform quantitative analysis of HCC cells in the co-culture system. (G) Western blotting was performed to measure CDK4, CDK2, cyclin A2, and p27 Kip1 protein expressions in HCC cells in the co-culture system. The results are presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

Sch B inhibits HCC cell migration in the co-culture system of macrophages and HCC cells. (A) M0 macrophages were treated with various concentrations of Sch B, and their cell viability was assessed using the CCK-8 assay. Sch B at concentrations of 0, 20, and 40 μg/mL was selected to treat the co-culture system, and the migration ability of HCC cells in the co-culture system was detected using the Transwell migration assay (B) and quantitative analysis (C).

FIGURE 4

Identification of exosomes in the co-culture system with Sch B. (A) Sch B-treated (0 and 20 μg/mL) co-culture system with experimental flow diagram for collecting exosomes. (B) Western blotting was performed to detect the expression of exosome surface marker proteins CD9 and Alix, and GAPDH was used as a negative control. (C) Particle size analysis of exosome diameters and concentrations. (D) TEM analysis of exosome morphology. N-exo represents the exosome of the control group and S-exo represents the exosome of the Sch B-treated group.

FIGURE 5

Proteomic analysis of exosomes in the Sch B-treated co-culture system. Exosome protein correlation analysis (A) and differential protein volcano map (B) of the control (N) and Sch B-treated (S) groups. (C) Bubble map of KEGG pathway enrichment analysis of downregulated differential proteins. (D) PPI analysis network map of differential proteins: the size and color depth of each circle represent the degrees. (E) Box plot of differential expression analysis of FN1 in patients with LIHC and healthy individuals. ****p < 0.0001. (F) Western blotting was performed to detect the expression of FN1 protein in exosomes. N-exo represents the exosome of the control group and S-exo represents the exosome of the Sch B-treated group (Sch B treatment concentration, 20 μg/mL). (G) Expression levels of TNF-α and Arg-1 in M0 macrophages in co-culture system. *p < 0.01.

FIGURE 6

Sch B inhibits FN1 expression in HCC cells. Subcutaneous tumor formation in nude mice. (A) Body weights of mice in different treatment groups. (B) Tumor images of the different treatment groups. (C) Tumor volumes and tumor weight. (D) Immunohistochemistry was performed to assess Ki67 expression. Immunofluorescence (E) and Western blotting (F) were used to detect FN1 expression in tumors. **p < 0.01, ****p < 0.0001. N represents the control group and S represents the Sch B-treated group.