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

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.


Introduction
As the predominant histological type of primary liver cancer, hepatocellular carcinoma (HCC) seriously endangers people's health. According to global cancer statistics 2020, there are about 906,000 new cases of HCC and 830,000 deaths worldwide every year [1]. Currently, despite progress in therapeutic approaches for liver cancer such as surgery, targeted therapy, and liver transplantation, the 5-year overall survival (OS) of advanced-stage HCC patients remains only 10% [2]. Resistance to chemotherapy remains a major cause of treatment failure in advanced-stage HCC [3]. Terefore, the development of more efective antiliver cancer drugs is of clinical signifcance.
Currently, many natural herbal products have been proven to exhibit anticancer efects and have received considerable attention [4][5][6][7]. Flavonoids, a complex class of bioactive compounds, are widely distributed in various plants, foods, and vegetables. Increasing evidence has demonstrated that favonoids exhibit signifcant antiinfammatory [8], anticancer [9], and antioxidant [10] activities. 4-Methoxydalbergione (4MOD) is a kind of favonoid isolated from the heartwood of Dalbergia, with anti-infammatory activities [11]. In addition, recent studies have revealed that 4MOD has anticancer efects on bladder cancer and astrocytoma [12,13]. However, the anticancer activity of 4MOD in HCC remains unknown.
Growth arrest and DNA damage are inducible. Te 45G (GADD45G), a member of the GADD45 family, participates in DNA damage response and cell growth arrest. Studies have confrmed that GADD45G is lowly expressed in esophageal squamous cell carcinoma, breast cancer, and acute myeloid leukemia [14][15][16]. It has also been reported that the upregulation of GADD45G expression induces the senescence of HCC cells [17]. Hence, GADD45G might be a novel target for HCC treatment.
Herein, we have characterized 4-Methoxydalbergione's anticancer efects on HCC cells' proliferation, migration, and apoptosis. In addition, the molecular mechanism of 4MOD's action against liver cancer was elucidated. We determined that 4MOD signifcantly inhibited human liver cancer cells' proliferation and migration, as well as promoting cell apoptosis through upregulation of GADD45G in vitro. . In addition, the morphological changes of cells exposed to different doses of 4MOD for 48 h were observed and photographed.

Colony Forming
Assay. 1 × 10 3 cells/well were planted overnight in 6-well plates (Corning, NY, USA). After treatment with or without 4MOD for 48 h, cells were incubated in a 5% CO 2 humidifed incubator for 2-3 weeks. When the colonies appeared, cells were fxed with 4% paraformaldehyde (Beyotime, Shanghai, China) and stained with crystal violet staining (Beyotime, Shanghai, China). An inverted microscope was used to count colonies.

Wound Healing
Assay. HCC cells were grown overnight in 6-well plates to 90% confuency. We scratched the cells with a 100 μL sterile pipette tip and further treated them with diferent concentrations of 4MOD. Te diferent drug concentrations were diluted in DMEM containing 1% FBS. Pictures were obtained at 0, 24, and 48 h after creating the scratch wound using an inverted microscope. Te area of scratches was measured using ImageJ software (MD, USA).

Transwell Migration Assay.
After 48 h of culture with or without 4MOD, 3 × 10 4 cells/100 μL were planted into the transwell upper chamber (Corning, NY, USA) with serumfree DMEM, and the lower chamber contained 20% FBS. After 24 h of incubation, cells were fxed with 4% paraformaldehyde and stained with crystal violet staining solution. Te cells at the bottom of the upper chamber were photographed at random. Te numbers of cells were analyzed by ImageJ software (MD, USA).
2.6. Flow Cytometry Assay for Apoptosis Analysis. Cells were grown overnight in 6-well plates to 50% confuency. After 48 h of culture with or without 4MOD, cells were harvested and stained with Annexin V/PI (KeyGEN BioTECH, Jiangsu, China) in accordance with the protocol of manufacture. Te cell apoptosis rate was detected by fow cytometry (Beckman Coulter, Miami, FL, USA). Te FlowJo 10 software (Tree Star, Inc. Ashland, OR) was used for data analysis.

RNA Extraction and RNA Sequencing (RNA-Seq).
SK-HEP-1 cells (1 × 10 6 cells/plate) were planted in 10 cm 2 plates (Corning, NY, USA) overnight and then treated with 0 μM or 20 μM of 4MOD for 24 h. Te total RNA was isolated from SK-HEP-1 cells using TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA). Te RNA-Seq was completed by Gene Denovo Biotechnology Co. (Guangzhou, China) through Illumina Novaseq6000 platform. For the diferentially expressed genes' (DEGs) identifcation, a false discovery rate (FDR) <0.05 and an absolute log2 fold change of >1 were set as cutof criteria. For the gene ontology (GO) enrichment analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA), FDR ≤0.05 was set as the threshold.

Transcriptome Validation by qRT-PCR.
After treatment with or without 4MOD for 24 hours, the total RNA was isolated from HCC cells using the TRIzol reagent kit. HiScript IIIRT SuperMix for qPCR (Vazyme, Nanjing, China) was used to obtain cDNA. Te cDNA products were amplifed using 2X Universal SYBR Green FAST qPCR Mix (ABclonal, Wuhan, China). Primer sequences used for quantitative real-time PCR (qRT-PCR) are exhibited in Table 1. Relative mRNA expression levels of target genes were analyzed by 2 −ΔΔCt method. GAPDH was used as the internal reference.

Statistical Analysis.
All experiments were repeated three times. Data were presented as mean ± standard deviation (SD) and analyzed by SPSS26.0 and GraphPad Prism 8.0 softwares. Te student t-test was used for statistical comparison between two groups. A one-way ANOVA was used for statistical comparison among multiple groups. p values < 0.05 were considered statistically signifcant.

4MOD Inhibits the Proliferation of HCC Cells.
To understand the cytotoxicity of 4MOD on HCC cells, morphological changes of cells exposed to diferent doses of 4MOD for 48 h were observed and photographed. We found that 4MOD could remarkably damage HCC cells. It was evident that 4MOD reduced cell numbers, destroyed cell structure, and made the cell round (  Table 2). In the following cell function experiments, we selected 5 μM, 10 μM, and 20 μM of 4MOD for treating SK-HEP-1 cells and 2.5 μM, 5 μM, and 10 μM for HuH-7 cells treatment. In addition, we further evaluated the inhibitory efect of 4MOD on HCC cells by a colony-forming assay. As expected, the results revealed that the proliferation of HCC cells was markedly inhibited by 4MOD treatment (Figures 1(e) and 1(f)). Taken together, 4MOD can signifcantly inhibit the proliferation of liver cancer in vitro.

4MOD Inhibits the Migration of HCC Cells.
To ascertain the efect of 4MOD on HCC cells' migration, wound healing, and transwell migration assays were performed. After 24 or 48 h of creating the scratch wound, 4MOD treatment signifcantly inhibited wound healing (Figures 2(a) and 2(b)).
Transwell migration experiments showed that the number of cells at the bottom of the upper chamber in 4MOD-treated groups was less than that in the 0 μM group after 24 h of seeding (Figures 2(c) and 2(d)). Wound healing and transwell migration experiments revealed that 4MOD could signifcantly inhibit the migration of liver cancer.

4MOD Induces Apoptosis of HCC Cells.
To observe whether apoptosis was related to the anticancer efects of 4MOD in HCC, we detected the apoptotic ratio using fow cytometry. We found that 4MOD could induce apoptosis in liver cancer cells. Te apoptosis rate of SK-HEP-1 cells without 4MOD treatment (0 μM group) was 6.14%, while the apoptosis rate of SK-HEP-1 cells with 4MOD treatment (40 μM group) was up to 50.9%. However, low concentration of 4MOD (20 μM) had no impact on SK-HEP-1 cells apoptosis (Figure 3(a)). Similarly, the apoptosis rate of the  (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. Te histogram shows statistical analysis for the colony numbers. * p < 0.05 and * * * p < 0.001, compared with control group (0 μM).   Journal of Healthcare Engineering 4MOD (20 μM) group (40.6%) was signifcantly higher than that of the control (0 μM) group (6.08%) and low concentration (10 μM) group (6.74%) in HuH-7 cells (Figure 3(b)).

Diferential Gene Expression Analysis.
To understand the potential anticancer mechanisms of 4MOD in SK-HEP-1 cells, we performed RNA-Seq on control cells and treated cells using the Illumina Novaseq6000 system. Upon analysis, a total of 600 DEGs (387 up-and 213 downregulated genes) were identifed between 4MOD-treated (20 μM) groups and the control groups (0 μM) (Figures 4(a) and 4(b)). Based on the absolute log2 fold change, the top 20 up and downregulated DEGs are shown in the heatmap (Figures 4(c) and 4(d)).

GO, KEGG, and GSEA Enrichment Analyses of DEGs.
To further uncover the molecular mechanisms of 4MOD against HCC, GO, KEGG, and GSEA, enrichment analyses were performed on all DEGs. GO biological processes (BP) annotations showed that the DEGs mainly participated in the regulation of multicellular organismal processes, positive regulation of multicellular organismal processes, and muscle structure development ( Figure 5(a)). GO cellular component (CC) annotations showed that the DEGs mainly focused on the extracellular matrix, the extracellular region part, and the transcription factor AP-1 complex ( Figure 5(b)). GO molecular function (MF) annotations showed that the DEGs were associated with transcription factor binding, DNAbinding transcription factor activity, RNA polymerase IIspecifc and signaling receptor binding ( Figure 5(c)). According to KEGG pathway enrichment analysis, DEGs were mainly enriched in the MAPK signaling pathway, IL-17 signaling pathway, and cytokine-cytokine receptor interaction ( Figure 5(d)). Te GSEA analysis showed that a number of DEGs were signifcantly related to MAPK signaling pathway ( Figure 5(e)).

Transcriptome Validation by qRT-PCR.
Considering that the MAPK pathway was the most signifcantly enriched pathway in this study, DEGs that participated in the regulation of the MAPK pathway were identifed. Te heatmap showed all of the DEGs involved in the regulation of the MAPK pathway, with GADD45G being the most signifcantly upregulated gene (Figure 6(a)). Ten of DEGs associated with MAPK pathway were further validated by qRT-PCR ( Figure 6(b)). In HuH-7 cells, qRT-PCR results also revealed that 4MOD upregulated GADD45G mRNA expression (Figure 6(c)).

Downregulation of GADD45G Rescues the Regulation of Proliferation, Migration, and Apoptosis by 4MOD in HCC
Cells. To explore whether 4MOD elicited anti-HCC activities by upregulating GADD45G expression in vitro, we transfected specifc shRNA sequences into HCC cells to knockdown GADD45G expression. Our qRT-PCR data showed that shGADD45G transfection signifcantly reduced GADD45G expression (Figure 7(a)). We frst examined whether downregulation of GADD45G could eliminate the inhibitory efects of 4MOD on HCC cell proliferation. Our CCK8 data revealed that GADD45G downregulation promoted cell viability in SK-HEP-1 and HuH-7 cells (Figures 7(b) and 7(c)). Te migration ability of HCC cells transfected with shGADD45G plus 4MOD was examined by a wound healing assay. As expected, downregulation of GADD45G reversed 4MOD-mediated reduction of migration activity in HCC cells (Figures 7(d) and 7(e)). In addition, we further verifed that 4MOD-induced apoptosis was partially inhibited by GADD45G downregulation (Figures 7(f ) and 7(g)).

Discussion
HCC is a common malignant neoplasm with high morbidity and mortality rates. Although a variety of chemotherapeutic drugs are widely used, the high incidence of drug resistance has become the main reason for chemotherapy failure [18]. Consequently, it is necessary to explore more efective drugs with low toxicity against HCC. Excessive proliferation, uncontrolled migration, and abnormal apoptosis are important malignant biological behaviors of tumor cells [19,20]. It is well known that several natural herbal products exhibit remarkably anticancer effects by inhibiting tumor cells proliferation, migration, and inducing apoptosis. For decades, 4MOD has been known as a natural favonoid compound with anti-infammatory activity. Recently, 4MOD has been shown to inhibit tumor cells growth and migration, as well as to promote apoptosis [12,13]. Mechanistically, 4MOD exerts anticancer efects on human bladder cancer through inactivating the AKT/ERK signaling pathway and inducing autophagy [12]. Nevertheless, the antitumor efects and mechanism of action of 4MOD against liver cancer remain unknown.
In this study, 4-Methoxydalbergione's anticancer efects and underlying mechanisms in HCC were explored. Our in vitro results showed that 4MOD repressed HCC cells proliferation and migration. Furthermore, fow cytometry analysis proved that high concentrations of 4MOD remarkably promoted tumor cell apoptosis. Terefore, our results demonstrated that 4MOD has anti-HCC activity and could be a valuable anticancer agent. However, it is necessary to validate these fndings in vivo.
Studies have shown that many natural herbal products exert anticancer activity by interacting with multiple target genes and various signaling pathways [21,22]. RNA-Seq analysis has become a mature tool for exploring the potential mechanisms of natural herbal products in cancer treatment and identifying candidate drug targets. In our study, RNA-Seq was performed to explore the anti-HCC molecular mechanism of 4MOD in vitro. In total, 600 target genes (387 up-and 213 down-regulated genes) were identifed based on diferential gene expression. Although these target genes need to be further validated, our study ofers a valuable research direction for future functional studies. Moreover, we performed GO, KEGG, and GSEA analyses to uncover the anticancer mechanisms of 4MOD. According to the results of KEGG enrichment analysis, multiple molecular pathways were altered in SK-HEP-1 cells upon 4MOD treatment, including the MAPK signaling pathway.

Journal of Healthcare Engineering
Te MAPK pathway is a common signaling pathway in human disease, including four main pathways: ERK, JNK, p38/MAPK, and ERK5 signaling [23,24]. Abnormal activation of the MAPK signaling pathway can result in various cancers, including HCC [25]. Our RNA-Seq and qRT-PCR results showed a variety of abnormal expression genes related to the MAPK pathway, including GADD45G. Tese genes may infuence the malignant biological behavior of HCC by regulating the activation of the MAPK pathway. GADD45G is a tumor suppressor gene, which is known to regulate cell proliferation through the MAPK pathway [16]. Evidence has shown that overexpression of GADD45G in tumor cells inhibits tumor proliferation and migration and promotes apoptosis [14][15][16]26]. Our RNA-seq results revealed that 4MOD was able to upregulate GADD45G mRNA expression. We hypothesized that knockdown of GADD45G might promote the malignant biological behavior of HCC cells. We further verifed that 4MOD could enhance GADD45G expression in HCC cell lines. After silencing the expression of GADD45G, the abilities of 4MOD to induce apoptosis and inhibit proliferation and migration were weakened in HCC cells. In short, 4MOD signifcantly inhibited the growth of human liver cancer cells in vitro by targeting GADD45G. However, our fndings cannot explain the specifc molecular  mechanism underlying 4MOD-mediated GADD45G upregulation. Future studies will be needed to explore the specifc molecular mechanisms involved.

Conclusions
Our results demonstrate for the frst time that 4MOD could suppress proliferation and migration and promote apoptosis by targeting GADD45G in HCC cells. Tese results provide novel directions for exploring the drug therapy of liver cancer, implying that 4MOD may be used as a new antitumor agent in the future.

Data Availability
Te experiment data used to support the fndings of this study are available from the corresponding author upon request. (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.