4-Methoxydalbergione Elicits Anticancer Effects by Upregulation of GADD45G in Human Liver Cancer Cells

Abstract

Background: 4-Methoxydalbergione (4MOD) is a flavonoid isolated from the heartwood of Dalbergia. Studies have demonstrated that 4MOD exerts anticancer activities on bladder cancer and astrocytoma. However, the anticancer activity of 4MOD in hepatocellular carcinoma (HCC) remains unknown. This study aims to examine its anticancer activities and mechanisms in human liver cancer cells.

Methods: CCK-8, colony forming, wound healing, transwell migration, and AnnexinV/PI assays were used to assess the anticancer effects of 4MOD in HCC cells. RNA sequencing (RNA-Seq) was selected to explore the possible mechanisms underlying the anti-HCC activity of 4MOD. The mRNA expression levels of target genes were verified through quantitative real-time PCR (qRT-PCR). A lentiviral shRNA interference technique was used to silence GADD45G expression. GADD45G knockdown was employed to confirm the crucial role of GADD45G in the 4MOD-mediatedanti-HCC effects.

Results: 4MOD inhibited HCC cells' proliferation and migration and promoted tumor cell apoptosis. RNA-Seq and qRT-PCR analyses revealed that 4MOD treatment increased GADD45G expression. Silencing GADD45G reversed 4MOD-mediated inhibition of proliferation, migration, and promotion of apoptosis.

Conclusions: Our findings show that 4MOD elicits anti-HCC effects by upregulating GADD45G expression and could be a valuable anticancer agent for liver cancer.

Figure 1

4MOD inhibits HCC proliferation. (a, b) Morphological changes of SK-HEP-1 and HuH-7 cells treated with different doses of 4MOD for 48 h (100x). (c, d) CCK-8 assay on SK-HEP-1 and HuH-7 cells treated with different doses of 4MOD at different times. (e, f) Colony formation assay on SK-HEP-1 and HuH-7 cells exposed to 0, 5 and 10 μM of 4MOD for 48 h. The histogram shows statistical analysis for the colony numbers. ^∗p <  0.05 and ^∗∗∗p < 0.001, compared with control group (0 μM).

Figure 2

4MOD inhibits HCC migration. (a, b) Wound healing assay on SK-HEP-1 and HuH-7 cells treated with or without 4MOD for 24 and 48 h (40x). The histogram shows statistical analysis for the scratch area. (c, d) Transwell migration assay on SK-HEP-1 cells and HuH-7 cells treated with or without 4MOD for 48 h (100x). The histogram shows statistical analysis for the migration cells. ns, p ≥ 0.05, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, compared with control group (0 μM).

Figure 3

4MOD induces apoptosis of HCC cells. (a) Flow cytometry on apoptosis rates of SK-HEP-1 cells exposed to 0, 20, and 40 μM of 4MOD for 48 h. The histogram shows statistical analysis for the apoptotic cells. (b) Flow cytometry on apoptosis rates of HuH-7 cells exposed to 0, 10, and 20 μM of 4MOD for 48 h. The histogram shows statistical analysis for the apoptotic cells. ns, p ≥ 0.05, ^∗∗∗p < 0.001, compared with 0 μM group.

Figure 4

Differential gene expression analysis between the 4MOD-treatment groups (20 μM) and control groups (0 μM) in SK-HEP-1 cells. (a) Volcano plot of DEGs. (b) Histogram of DEGs. (c) Heatmap of top 20 upregulated DEGs. (d) Heatmap of top 20 downregulated DEGs.

Figure 5

GO, KEGG and GSEA enrichment analyses of DEGs. (a) GO-BP annotations. (b) GO-CC annotations. (c) GO-MF annotations. (d) KEGG pathway enrichment analysis. (e) GSEA enrichment analysis.

Figure 6

Transcriptome validation by qRT-PCR. (a) Heatmap of DEGs involved in the regulation of MAPK pathway. GADD45G, the most significantly upregulated gene, was marked in the red box. (b) qRT-PCR validation on ten dysregulated genes involved in MAPK signaling pathway in SK-HEP-1 cells. (c) Expression levels of GADD45G were detected in 4MOD-treatment groups (10 μM) and control groups (0 μM) by qRT-PCR in HuH-7 cells. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001, compared with 0 μM group.

Figure 7

4MOD exerts anticancer effects by upregulating GADD45G expression in HCC. (a) The relative GADD45G mRNA expression was detected by qRT-PCR in shGADD45G-transfected cells. (b, c) SK-HEP-1 and HuH-7 cells were transfected with negative control (shNC) or shGADD45G, respectively. CCK8 was used to detect cell viability after treatment with or without 4MOD (20 μM) for 48 h. (d) SK-HEP-1 was transfected with shNC or shGADD45G, respectively. Wound healing assay was used to detect cell migration after treatment with or without 4MOD (20 μM) for 48 h. (e) HuH-7 was transfected with shNC or shGADD45G, respectively. Wound healing assay was used to detect cell migration after treatment with or without 4MOD (10 μM) for 48 h. (f) SK-HEP-1 were transfected with shNC or shGADD45G, respectively. Flow cytometry was used to detect cell apoptosis after treatment with or without 4MOD (40 μM) for 48 h. (g) HuH-7 was transfected with shNC or shGADD45G, respectively. Flow cytometry was used to detect cell apoptosis after treatment with or without 4MOD (20 μM) for 48 h. ^∗∗p < 0.01, ^∗∗∗p < 0.001.