Scoparone suppresses proliferation and cell cycle of hepatocellular carcinoma cells via inhibiting AKT/GSK-3β/cyclin D1 signaling pathway

Abstract

Background: Hepatocellular carcinoma (HCC) ranks as the sixth most prevalent cancer and the fourth leading cause of cancer-related mortality globally. Scoparone, a natural coumarin derivative primarily derived from Artemisia Capillaris Thunb, has demonstrated antitumor properties across various cancer types. However, its functions in HCC have not been clearly elucidated. This study aimed to investigate the antitumor effects of scoparone on the MHCC-97L and HCCC-9810 HCC cell lines.

Methods: Cell proliferation was assessed through viability and colony formation assays. Migration and invasion capabilities of the cells were evaluated by wound healing assays and Transwell assays. Additionally, transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted to uncover pathways linked to gene enrichment in the artemisinin treatment group. Western blotting and flow cytometry were utilized to analyze the expression of mechanistic proteins associated with artemisinin treatment in HCC.

Results: Our findings revealed that scoparone effectively inhibited the proliferation, migration, and invasion of HCC cells. The genes affected by scoparone treatment were predominantly enriched in pathways related to the cell cycle. Specifically, scoparone reduced the expression of genes such as CDK2, CDK3, CDK4, CDC25A, CCND1, and CCNE1, while it increased the expression of CDKN1A (p21). Furthermore, scoparone suppressed the levels of cell cycle-related proteins CDK2, CDK4, and cyclin D1, along with the signaling pathways involving p-AKT and p-GSK-3β. Notably, the inhibitory effects of scoparone on HCC cell proliferation were partially reversed by the AKT activator, SC79.

Conclusions: Scoparone inhibited HCC cell viability by targeting the AKT/GSK-3β/cyclin D1 pathway.

Keywords: AKT/GSK-3β/cyclin D1 pathway; Hepatocellular carcinoma (HCC); cell cycle; scoparone.

Figure 1

Scoparone suppresses HCC cell proliferation in vitro. (A) Scoparone at concentrations of 0, 50, 100, 150, 200, 250, 300, 350 µg/mL was applied to MHCC-97L, HCCC-9810, and L-02 cells for 24 and 48 hours. Cell viability was assessed using the CCK-8 assay. (B) The IC50 of scoparone for the three lines. (C) HCC cells were treated with 0, 50, 100, and 200 µg/mL scoparone for 48 hours and 0.01% crystal violet staining was used to evaluate the clonogenesis ability of cancer cells. (D) HCC cells were treated with 0, 100, and 200 µg/mL scoparone for 48 hours. The DNA replication status of the cells, as well as EdU positive cells stained with Hoechst 33342 (scale bars, 100 µm), was analyzed. Data were expressed as mean ± SD. The experiments were independently conducted three times. *, P<0.05; **, P<0.01 (vs. L-02). CCK-8, cell counting kit-8; EdU, 5-ethynyl-2'-deoxyuridine; HCC, hepatocellular carcinoma; IC50, half-maximal inhibitory concentration; Sco, scoparone; SD, standard deviation.

Figure 2

Scoparone suppresses HCC cell invasion and migration in vitro. (A,B) Scoparone inhibits the migration and invasion of HCC cells. After treating HCC cells with 0, 100, and 200 µg/mL scoparone for 48 hours, the wound healing assay (scale bars, 20 µm) and transwell assay (scale bars, 50 µm; staining methods: 0.2% crystal violet) were performed to detect changes in cell migration and invasion ability. (C) Scoparone inhibits the expression of MMP-2 and MMP-9 in HCC cells. The expression levels of MMP-2 and MMP-9 proteins were detected in HCC cells with treatment of 0, 50, 100, and 200 µg/mL scoparone for 48 hours. Data were expressed as mean ± SD. The experiments were independently conducted three times. *, P<0.05; **, P<0.01. HCC, hepatocellular carcinoma; MMP, matrix metalloproteinase; Sco, scoparone; SD, standard deviation.

Figure 3

Scoparone induces cell cycle arrest in HCC cells. (A) The number of upregulated and downregulated genes within the two cell lines. Cells were treated with 0 or 200 µg/mL scoparone for 48 hours and transcriptome sequencing was performed. (B) Differentially expressed genes collection between two cell lines. (C) KEGG pathway analysis of the differentially expressed genes showed the enrichment primarily in cell cycle-related pathways. (D) Cell cycle genes expression in both HCC cells with treatment 0 or 200 µg/mL scoparone for 48 hours. qRT-PCR was used to detected changes in the expression of cell cycle-related genes. (E) Scoparone induces cell cycle arrest in HCC cells. Flow cytometry analysis of cell cycle distribution in HCC cells treated with 0, 50, 100, and 200 µg/mL scoparone for 48 hours. Data were expressed as mean ± SD. The experiments were independently conducted three times. *, P<0.05; **, P<0.01. HCC, hepatocellular carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; mRNA, messenger RNA; PI, propidium iodide; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Sco, scoparone; SD, standard deviation.

Figure 4

Scoparone suppresses HCC cell proliferation via the AKT/GSK-3β/cyclin D1 signaling pathway. (A) HCC cells were treated with scoparone at concentrations of 0, 50, 100, and 200 µg/mL for 48 hours. Western blotting analysis revealed that scoparone inhibited the expression of cell cycle-related proteins. (B) After treatment with scoparone at 0, 50, 100, and 200 µg/mL for 48 hours, the expression levels of proteins in the AKT/GSK-3β/cyclin D1 signaling pathway in HCC cells were evaluated. The results demonstrated that scoparone suppressed the expression of these signaling pathway proteins. Data are expressed as mean ± SD. Conducted three independent experiments. *, P<0.05; **, P<0.01. HCC, hepatocellular carcinoma; Sco, scoparone; SD, standard deviation.

Figure 5

AKT activator SC79 partially reverses the inhibitory effect of scoparone on the proliferation of HCC cells. (A) SC79 partially reverses the inhibitory effect of scoparone on the proliferation of HCC cells. Cell viabilities were detected in control, SC79, scoparone, and SC79 + scoparone groups. (B) SC79 partially reverses the inhibitory effect of scoparone on DNA replication in HCC cells. DNA replications were detected in control, SC79, scoparone, and SC79 + scoparone groups (scale bars, 100 µm; staining methods: Hoechst 33342). Data were expressed as mean ± SD. The experiments were independently conducted three times. *, P<0.05; **, P<0.01 (vs. control); ^##, P<0.01 (vs. Sco). HCC, hepatocellular carcinoma; OD, optical density; Sco, scoparone; SD, standard deviation.