Introduction

Gastric cancer (GC) is the fifth most common cancer. In 2020, over one million new cases were diagnosed1. The incidence rates of GC vary widely by sex, race, and geographic location, with considerably higher incidence in Eastern Asia and in men1,2. Although great progress has been made in GC treatment including surgery, chemotherapy, radiotherapy and targeted therapy, the morbidity and mortality of GC remains high: GC is the fourth leading cause of cancer-related death and accounts for 769,000 deaths worldwide each year1,3. Most patients present with advanced-stage gastric cancer as the low early diagnosis rate, even though the 5-year survival rate of early gastric cancer can reach > 90%4. The median survival time of metastatic gastric cancer is less than 1 year5,6. Thus, chemotherapy such as oxaliplatin or cisplatin plus a fluoropyrimidine (5-fluorouracil, capecitabine or S-1) is a suitable method for most patients who are diagnosed in the advanced stage5. However, its application is limited due to drug resistance and side effects, including genotoxicity, nephrotoxicity and acute myelotoxicity7,8. Thus, it is urgent to develop novel drugs and/or new therapeutic combinations for patients with gastric cancer.

Natural products are valuable sources of therapeutic substances and a structural basis for novel drug development. Flavonoids are polyphenolic substances that are widely present in plants and have been proved to have multiple health-promoting properties9,10. Wogonin (5, 7-dihydroxy-8-methoxyflavone), a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, has been proposed to exert potent antitumor11, anti-inflammatory12,13and anti-allergic12,14activities. Previous studies revealed the mechanisms underlying the antitumor effects of wogonin in several tumors; these mechanisms include cell cycle arrest15,16, cell apoptosis induction17,18, tumor immunity promotion19and angiogenesis suppression20. Additionally, it has been well documented that wogonin potentiates the antineoplastic effects of traditional chemotherapy with less toxicity as an adjunct treatment or pretreatment21,22.

It has been reported that wogonin exerts anti-GC effects through different mechanisms11. Research has shown that wogonin can inhibit energy metabolism, cell proliferation and angiogenesis in SGC-7901 and A549 cells23. Another study demonstrated that wogonin induces immunity to GC cell vaccines by activating PI3K pathway elicited by ER stress-induced CRT/Annexin A1 translocation and HMGB1 release19,24. Moreover, wogonin was shown to potentiate the antitumor effects of oxaliplatin, paclitaxel and 5-fluorouracil in vivo and in vitro21,25,26. A wogonin-condensed Pt (IV) prodrug has been reported to reverse cisplatin resistance in human gastric cancer cells by attenuating casein kinase 2-mediated nuclear factor-κB pathways22. These studies indicate that wogonin is a promising new strategy in the treatment of GC, but the exact mechanism by which wogonin inhibits GC is still unclear.

In the present study, we investigated the effects of wogonin on GC cells. Our data indicated that wogonin significantly inhibited GC cell proliferation in vitro and tumor growth in vivo by inducing cell cycle arrest and cell apoptosis. Moreover, we also demonstrated that wogonin dramatically inhibited GC cell migration and invasion. The mechanistic study showed that wogonin exerted its antiproliferative effects by downregulating the JAK-STAT3 pathway. These findings may have implications for improving the treatment of GC.

Materials and methods

Cell culture and manipulation

The human gastric cancer cell lines MKN45, SGC-7901 and BGC-823 were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). These cells were maintained in RPMI 1640 medium (Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies, Carlsbad, CA, USA), 100 U/mL of penicillin and 100 mg/L of streptomycin in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C with 5% CO2. Wogonin powder was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China) and dissolved with DMSO (Sangon Biotech, Shanghai, China). MG132 was purchased from Sigma (St. Louis, MO, USA), Stattic was purchased from MedChemExpress (Monmouth Junction, NJ, USA), IL-6 was purchased from R&D Systems (Minneapolis, MN, USA) and Ruxolitinib was purchased from Selleck (Houston, USA).

Cell proliferation and colony formation assays

The cell proliferation was determined using the CCK-8 assay as described previously27. Briefly, MKN45, SGC-7901 and BGC-823 cells were respectively planted in a 96-well plate at a density of 1 × 104/well and cultured overnight. Then the cells were incubated with various concentrations (0, 30, 60, 90 and 120µM) of wogonin for 48 h and incubated with 30µM wogonin for different time (24, 48, 72 and 96 h). The cell viability was measured using Cell Counting Kit-8 (CCK8) (Beyotime Biotechnology, shanghai, China) at different time points following the manufacturer’s protocols. All experiments were performed at least in triplicate.

For colony formation assay, single-cell suspensions with 1000 cells were planted into each well of six-well plates and incubated overnight with complete RPMI1640 medium. Afterwards, the cells were incubated with various concentrations of wogonin and then further incubated at 37 °C for 10–15 days until colonies reached an average of > 50 cells in a colony. Colonies were fixed with 4% Paraformaldehyde and stained with 0.1% crystal violet for 15 min at room temperature. Then colonies were photographed under inverted microscope (CKX41, Olympus Life Science, Tokyo, Japan) and counted manually. The clone formation rate was determined as the number of colonies formed (> 50 cells) / number of cells seeded. All experiments were performed at least in triplicate.

EdU incorporation, cell cycle and apoptosis analyses

EdU (5-ethynyl-20-deoxyuridine) incorporation assays was performed using the Cell-Light™ EdU Apollo567 In Vitro Kit (Ribobio, Guangzhou, China) following the manufacturer’s protocols as previously described28,29. Briefly, cells were plated into 24-well plates overnight and exposed to various concentrations of wogonin for 48 h. Then EdU solutions were added to treated cells and incubated for 30 min. After fixation in 4% polyformaldehyde for another 30 min, cells were incubated with Apollo® staining mixture for 1 h. Then cells were stained the nuclear with DAPI for 10 min and were photographed by a fluorescence microscope BX51 equipped with a DP71 microscope digital camera (Olympus Life Science, Tokyo, Japan) subsequently. The rate of EdU-positive cells was calculated by imageJ v1.52 software (https://imagej.net/ij/index.html, Bethesda, MD, USA).

The cell cycle analysis was performed using a Becton Dickinson FACScan instrument (BD Biosciences, San Jose, CA) as previously described27. In brief, cells treated with various concentrations of wogonin for 48 h were collected and then fixed in 75% ethanol at 4 °C overnight. After being washed with PBS, fixed cells were incubated with propidium iodide (PI) staining buffer containing 50ug/mL PI and 20 ug/mL plus RNase A (Sigma, St. Louis, MO, USA) at room temperature for 30 min in dark. Then the cell cycle distribution was evaluated by flow cytometric analysis.

Flow cytometric analysis of apoptosis was detected using Annexin V-FITC/PI double staining (Kaiji, Nanjing, China) according to manufacturer’s instructions. Briefly, cells were collected using EDTA-free trypsin solution and washed with 1×PBS for twice. Then the cells were resuspended in 500 µL of binding buffer and stained with Annexin V-FITC for 10 min and PI for 5 min at room temperature in the dark. After being filtered, the samples were detected by the Becton Dickinson FACScan instrument (BD Biosciences, San Jose, CA).

TUNEL assays were detected using one-step TUNEL Apoptosis Assay Kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. After labeling, the slides were counterstained with DAPI and visualized under a fluorescence microscopy.

ROS and JC-1 assay

Intracellular ROS level was determined by Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, shanghai, China) according to the manufacture’s direction. Cells treated with various concentrations of wogonin were collected with EDTA-free trypsin solution and then resuspended with serum-free RPMI 1640 medium supplemented with DCFH-DA probe at concentration of 10µM. After 20 min of incubation at 37℃, the cells were rinsed with serum-free RPMI 1640 medium for three times and then evaluated by a Becton Dickinson FACScan instrument (BD Biosciences, San Jose, CA). Rosup diluted in serum-free RPMI 1640 medium was applied as positive control.

JC-1(5, 5′, 6, 6′-Tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide) assay kit (Beyotime Biotechnology, shanghai, China) was used to detection of mitochondrial membrane potential (MMP, ΔΨm) following the manufacturer’s directions. After incubation for 48 h in a 24-well plate, the cells treated with various concentrations of wogonin were incubated with JC-1 staining working solution at 37 °C with 5% CO2 for 20 min. Then the cells were washed with cold JC-1 staining buffer, and then were incubated with PBS and photographed under the fluorescence microscope BX51 equipped with a DP71 microscope digital camera (Olympus Life Science, Tokyo, Japan).

Tumor xenografts

The male BALB/c nude mice (3 weeks old) were obtained from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China) and housed in SPF environment. After 5-day for adjustment, about 8 × 106 cells suspended in 200ul PBS were injected subcutaneously into each mouse. After tumors were formed in the nude mice, ten mice with similar tumor volumes were selected and randomly divided into two groups (five mice in each group): the mice in wogonin group were injected with wogonin (60 mg/kg/d) intraperitoneally everyday for 12 days while the mice in control group were injected with same volume of the DMSO. The tumor volumes were measured and recorded once every 3 days. At the end of experiment, the mice were sacrificed and the tumor xenografts were removed and weighed. This study was performed in accordance with the ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of Shandong University and all procedures were performed in compliance with the institutional guidelines.

Wound-healing, transwell migration and invasion assays

Wound-healing, transwell migration and invasion assays were performed as previously described30,31. 106/well cells were planted into 24-well plates and incubated with complete RPMI1640 medium until about 90% confluency as a monolayer. A 200 µl pipette tip was used to make linear wounds in each monolayer well. Then the medium was replaced with the fresh serum-free RPMI1640 medium with various concentrations of wogonin. Scratched areas were selected randomly in each well and images at 0 h and 24 h were taken under an inverted microscope (CKX41, Olympus Life Science, Tokyo, Japan). Wound healing percentage was calculated using the formula: [(mean wound width-mean remaining width)/ mean wound width] × 100 (%).

Transwell migration and invasion assays were performed in 24-well transwell inserts (Corning, New York, USA) containing polycarbonate filters with 8-µm pores, with or without Matrigel (BD Biosciences, NJ, USA) as previously described29,32. Briefly, Matrigel was mixed with serum-free RIPM-1640 (1:6 ratio) in upper chambers, and incubated at 37 °C for 2 h. GC cells treated with various concentrations of wogonin were suspended in serum-free RPMI-1640 medium (4 × 106 cells/mL) and then planted into the upper chamber applied with Matrigel, while complete RPMI-1640 medium was placed into the lower chamber. The cells were incubated at 37 °C for 48 h, and then the cells on the upper surface of the membrane were removed with a cotton swab. The cells migrated to the lower surface were fixed with 4% paraformaldehyde for 30 min, stained by crystal violet for 15 min and then were counted under the inverted microscope (CKX41, Olympus Life Science, Tokyo, Japan) in five different views per well. The chambers without Matrigel were used for migration assay. All experiments were performed in triplicate.

RNA sequencing (RNA-seq) experiment and data analysis

Total RNA was extracted from SGC-7901 cells treated with the DMSO or wogonin. The RNA integrity numbers (RINs) were obtained using Agilent 2100 Bioanalyzer, RIN score > 8 is considered sufficient for sequencing library preparation. Libraries prepared using TruSeq Stranded mRNA prep kit (Illumina Inc. San Diego, USA) according to the manufacturer’s protocol. These pooled libraries were sequenced using the HiSeq 2000 (Illumina Inc. San Diego, CA, USA). Raw sequencing reads were QC checked using FastQC and trimmed with Cutadapt. Clean reads were mapped onto human reference genome GRCh38 using Hisat2 (v2.2.1). Stringtie was used for read counting and gene quantification. Gene count normalization and differential analysis were conducted using DESeq2 R package. Genes with a fold-change (FC) > 2 (in either direction) and false discovery rate (FDR) < 0.05 were identified as differentially expressed genes (DEGs). The ‘clusterProfiler’ R package were used for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis33,34,35 (www.kegg.jp/kegg/kegg1.html) of DEGs. The RNA sequencing raw data have been deposited in Genbank repository under accession number PRJNA 1,155,224.

RNA isolation and real-time quantitative PCR

Total RNA was isolated from GC cells treated with various concentrations of wogonin using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s directions and the RNA concentrations were measured by spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA). Real-time quantitative PCR (qRT-PCR) assays was performed as described previously36. Briefly, total RNA (2.5 µg) was reverse transcribed to cDNA using PrimeScript RT Reagent Kit (Takara Co, Otsu, Japan) with random primers according to the manufacturer’s protocols. The qRT-PCR reactions were undertaken on LightCycler 480 system (Roche, Mannheim, Germany) using SYBR Premix Ex Taq (Takara Co, Otsu, Japan). All experiments were performed at least in triplicate. Primer sequences used for qPCR are listed in Supplementary Table S1.

Protein extraction and western blotting

Total proteins were extracted from cells treated with various concentrations of wogonin as previously described37. Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, shanghai, China) were used to isolated nuclear proteins according to the manufacturer’s instructions. Western blotting analysis was performed as described previously37. The primary antibodies are listed in Supplementary Table S2.

Plasmids and luciferase assays

Luciferase reporter plasmid containing STAT3 responsive elements (pSTAT3-TA-luc) was purchased from Beyotime (Biotechnology, shanghai, China). For luciferase assays, GC cells were plated in 96-well plates and incubated with complete RPMI 1640 medium overninght and then the cells were co-transfected with STAT3 reporter construct and pRL-TK vector that provides constitutive expression of Renilla luciferase serving as an internal control with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. 6 h after transfection, cells were treated with various concentrations of wogonin in complete RPMI1640 medium for another 36 h. Then the Dual-Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, shanghai, China) was used to detect Firefly and Renilla luciferase activities, and then ratio of Firefly and Renilla luciferase activities can be used to calculate relative luciferase activity. All the experiments were performed in triplicate.

Statistical analysis

Statistical analyses were performed using GraphPad 7 by the Student t test or one way ANOVA. P < 0.05 was considered to be statistically significant. All results are presented as mean with SD.

Results

Wogonin inhibits the proliferation of GC cells

We first evaluated the effects of wogonin on the proliferation of three GC cell lines, SGC-7901, BGC-823 and MKN45. The results of CCK-8 assays showed that wogonin significantly inhibited the growth of these cell lines (Fig. 1A), and the inhibitory effect of wogonin was concentration-dependent (Fig. 1B). Moreover, wogonin treatment suppressed the colony formation ability of these cells (Fig. 1C). Furthermore, EdU incorporation assays were conducted to investigate the effect of wogonin on DNA replication in GC cells. Consistent with the results obtained from CCK-8 and colony formation assays, treatment with wogonin for 48 h decreased the percentage of EdU-positive cells dramatically (Fig. 1D and Supplementary Fig. S1), pointing out wogonin plays an inhibitory role on DNA replication in GC cells.

Then, to confirm the effects of wogonin on the proliferation of GC cells in vivo, we designed a xenograft model by injecting SGC-7901 cells into right lateral thigh of nude mice. As shown in Fig. 1E, wogonin significantly suppressed tumor growth. At the endpoint, the tumor volume and weight were both significantly reduced in the group receiving wogonin. Together, these results have shown that the growth of gastric cancer cells was suppressed significantly by wogonin, in vitro and in vivo.

Fig. 1
figure 1

Wogonin inhibits proliferation of GC cells. (A) Viability of SGC-7901, BGC-823 and MKN45 cells treated with or without 30µM wogonin was analyzed by CCK8 assay. (B) CCK8 assay of SGC-7901, BGC-823 and MKN45 cells treated with wogonin at different concentrations for 48 h. (C) Colony formation assays to assess the clone formation rate of SGC-7901, BGC-823 and MKN45 cells treated with wogonin at different concentrations. (D) The effect of wogonin on DNA replication was analyzed by EdU incorporation assays. (E) Tumor formation by SGC-7901 cells in nude mice. 200µL 8 × 106 SGC-7901 cells were injected subcutaneously into each mouse. Mice were randomly divided into 2 groups and were treated with wogonin (60 mg/kg) or DMSO (as control) per day for 12 d. Tumor volume was measured every 3 d by vernier caliper. At the end mice were sacrificed and the tumor weight were measured. *p < 0.05 vs. DMSO group, **p < 0.01 vs. DMSO group, ***p < 0.001 vs. DMSO group.

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Wogonin induces G0/G1 arrest and downregulates G1/S transition-related proteins

To identify the underlying mechanisms of the inhibitory effects of wogonin on GC cell proliferation, we utilized flow cytometric analysis to determine the cell cycle distribution of SGC-7901 and BGC-823 cells treated with wogonin for 48 h. The proportion of cells in G0/G1 phase was markedly increased in wogonin-treated SGC-7901 cells, and the effect of wogonin on the induction of G0/G1 phase arrest was concentration-dependent (Fig. 2A), suggesting that wogonin induces G0/G1 arrest in SGC-7901 cells. No obvious cell cycle change was observed in BGC-823 cells, and only a minor increase in the proportion of G2/M phase cells was observed after treatment with 90 µM wogonin (Fig. 2A).

Then the expression of cell cycle regulation-related proteins were evaluated. Wogonin induced to decrease the expression levels of p-RB, CDK6 and CDK4, which are required for the G1-S transition in cell cycle (Fig. 2B). No changes in the levels of total CDK2, Cyclin B1, Cyclin E and Cyclin D1 proteins were observed (Fig. 2B).

Fig. 2
figure 2

Wogonin disrupts the cell cycle of GC cells. (A) Cell cycle distribution of SGC-7901 and BGC-823 cells exposed to wogonin at different concentrations for 48 h was analyzed by flow cytometry. ***p < 0.001 vs. DMSO group. (B) Protein levels of cyclins and cell cycle regulatory proteins in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h were measured by western blotting.

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Wogonin induces the apoptosis of GC cells

Then, we investigated the role of wogonin on apoptosis in GC cells. Flow cytometric assays showed that treatment with wogonin for 48 h significantly increased the percentage of apoptotic cells in both SGC-7901 and BGC-823 cell lines (Fig. 3A). Similar results were obtained from TUNEL analysis (Fig. 3B). Activation of caspase, an intracellular cysteine proteolytic enzyme, serves as a marker of cell apoptosis. As expected, the expression level of cleaved-caspase 3 protein increased in wogonin-treated GC cells compared with untreated and DMSO-treated cells (Fig. 3C).

Mitochondrial dysfunction plays a central role in the induction of apoptosis and is closely related to changes in mitochondrial membrane permeability, which results in the release of apoptogenic factors. Therefore, JC-1 assays were employed to evaluate the effects of wogonin on mitochondrial membrane potential (∆Ψm), the loss of which is an important indicator of mitochondrial damage. The results showed that treatment with wogonin for 48 h dramatically decreased ∆Ψm in both SGC-7901 and BGC-823 GC cells (Fig. 3D). Mitochondria are the primary generators of reactive oxygen species (ROS), the accumulation of which could induce apoptosis by damaging DNA. We asked whether ROS could be associated with apoptosis induction by wogonin in GC cells. As shown in Fig. 3E, the levels of ROS were only slightly increased by wogonin treatment. Together, these results indicate that wogonin disrupts mitochondrial function and induces apoptosis in GC cells.

Fig. 3
figure 3

Wogonin induces increased apoptosis and ROS of GC cells. (A) Flow cytometric analysis of apoptosis of SGC-7901 and BGC-823 cells exposed to wogonin at different concentrations for 48 h. (B) TUNEL analysis of apoptosis of SGC-7901 and BGC-823 cells treated with 90 µM wogonin for 48 h. (C) Western blotting analysis to detect the levels of apoptosis regulating proteins of SGC-7901 and BGC-823 cells exposed to wogonin at different concentrations for 48 h. (D) Mitochondrial membrane potential analysis of SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h. (E) ROS levels in SGC-7901 and BGC-823 cells treated with wogonin for 48 h were presented through combination with DCFH-CA. *p < 0.05 vs. DMSO group, ***p < 0.001 vs. DMSO group.

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Wogonin inhibits the migration and invasion of GC cells

We next investigated the effect of wogonin on the migration and invasion of GC cells. Wound healing assays were performed, and the results showed that wogonin considerably reduced the migration rate of GC cells in a concentration-dependent manner (Fig. 4A). In addition, wogonin can decrease greatly the number of cells that migrated across the transwell membrane by transwell assays (Fig. 4B). We utilized matrigel invasion assays to determine the effect of wogonin on the invasion of GC cells. The results showed that treatment with wogonin significantly reduced the number cells invading the matrigel by approximately 50% (Fig. 4C). Taken together, these results indicate that the migration and invasion of GC cells were inhibited by wogonin.

Fig. 4
figure 4

Wogonin inhibits migration and invasion of GC cells. (A) SGC-7901 and BGC-823 cells were scraped with a pipette tip and then were treated with wogonin at different concentrations for 24 h. Then the percentages of closure wound were measured and calculated. (B-C) SGC-7901 and BGC-823 cells were treated with different concentrations of wogonin for 24 h and then transwell assays were applied to determine the ability of cell migration (B) and invasion (C). Then the migration rate (B) and invasion rate (C) were evaluated relative to the control group. ***p < 0.001 vs. DMSO group.

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Transcriptomic analysis of wogonin-treated GC cells

To elucidate the mechanisms underlying the regulatory effects of wogonin on GC cell proliferation and migration, we performed RNA-seq analysis to compare the transcriptomes of wogonin-treated GC cells and DMSO-treated GC cells. Because 90 µM wogonin led to a more obvious phenotype in GC cells in the above experiments, we chose this concentration for RNA-seq analysis. In total, 154 genes were downregulated and 166 genes were upregulated in the 90 µM wogonin treatment group compared to the DMSO treatment group (Fig. 5A). Similar results were obtained in the comparison of the blank control and 90 µM wogonin treatment groups (Supplementary Fig. S2A). The volcano plot for the DEGs is displayed in Fig. 5A. qPCR was performed to confirm the RNA-seq results (Fig. 5B). GO enrichment analysis was performed for the 320 DEGs. The most enriched GO Terms of p value are shown in Fig. 5C and Supplementary Fig. S2B.

In the molecular function category, GO function enrichment analyses showed that the DEGs were enriched in “binding”, “catalytic activity” and “signal transducer activity”. In the cellular component category, “cell part”, “organelle”, “membrane” and “membrane part” were the most enriched terms. “Cellular process”, “biological regulation”, “single-organism process” and “metabolic process” were the most highly enriched terms in the biological process category. Then, KEGG enrichment analysis was performed to further investigate the signaling pathways of the DEGs. DEGs were significantly enriched in tumor proliferation and metastasis related KEGG pathways, such as “p53 signal pathways”, “JAK-STAT signaling pathway”, and “FoxO signaling pathway” (Fig. 5D and Supplementary Fig. S2C). Taken together, these GO and KEGG pathway enrichment results could provide essential information for the investigation of wogonin in SGC-7901 GC cells.

Fig. 5
figure 5

Transcriptomic analysis of wogonin-treated GC cells compared to the DMSO treated cells. RNA-seq analysis and bioinformatic analysis of SGC-7901 cells treated with 90 µM of wogonin, compared with DMSO treated cells. (A) Volcano map for the DEGs. (B) qPCR was performed to confirm the RNA-seq results. (C) The most enriched GO Terms. (D) The KEGG enrichment analysis of the DEGs (www.kegg.jp/kegg/kegg1.html).

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Wogonin downregulates actived STAT3 in GC cells

To further explore the mechanisms of the antitumor effects of wogonin on GC cells, the activation of ERK, AKT and STAT3, which are known to be important for cell proliferation and are associated with the progression of various tumors, was examined. As shown in Fig. 6A and B, treatment with wogonin significantly decreased the levels of phosphorylated STAT3 and AKT but not the levels of total STAT3 and AKT, and the effects were dose-dependent. In contrast, neither total nor phosphorylated ERK protein levels were changed by wogonin treatment (Supplementary Fig. S3). It has been reported that the JAK-STAT3 signaling pathway played an important role in tumor proliferation and metastasis2,256. Given that wogonin decreased the level of phosphorylated STAT3 and that RNA-seq analysis showed that the DEGs associated with wogonin were significantly enriched in the “JAK-STAT signaling pathway” based on GO analysis (Fig. 5D and Fig. S2C), we next determined whether wogonin affected STAT3 function in GC cells. We transfected the p-STAT3-TA-luc reporter plasmid into SGC-7901 cells, and found out that STAT3 transcriptional activity was significantly decreased by wogonin using luciferase assays (Fig. 6C). Activated STAT3 is transported to the nucleus and induces the transcription of downstream target genes. We extracted nuclear and cytoplasmic fractions of SGC-7901 and BGC-823 cells separately, and analyzed the levels of STAT3 protein by western blotting. The results showed that the levels of nuclear STAT3 protein were decreased, while the levels of cytoplasmic STAT3 protein were increased in wogonin treated GC cells (Fig. 6D). Together, these results suggest that wogonin decreases the activity of STAT3 in GC cells.

Considering the important role of STAT3 activation in cancer, we sought to determine the correlation between STAT3 and wogonin-mediated proliferation inhibition in GC cells. Similar to treatment with wogonin, treatment with the STAT3 inhibitor Stattic also resulted in proliferation inhibition in GC cells (Fig. 6E). Importantly, the inhibitory activity of Stattic toward GC cells was enhanced by treatment with wogonin (Fig. 6E). These data suggest that wogonin exerts its antitumor activity at least partly by decreasing STAT3 activity.

To determine whether wogonin also has an inhibitory effect on STAT3 activation in other cancer cells, the levels of p-STAT3 in T24 (bladder cancer), A375 (melanoma cancer), MCF-7 (breast cancer) and B16 (murine melanoma cancer) cells treated with wogonin for 48 h were examined. In contrast to the results from GC cells, there was no change in p-STAT3 levels was observed in B16 cells, while treatment with wogonin significantly increased p-STAT3 levels in T24, A375 and MCF-7 cells (Supplementary Fig. S4), suggesting that the inhibitory activity of wogonin on STAT3 may depend on cancer type.

Fig. 6
figure 6

Wogonin inhibits STAT3 activation in GC cells. (A) Levels of total STAT3 and p-STAT3 in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h were analyzed by western blotting. (B) Levels of total AKT and p-AKT in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h were analyzed by western blotting. (C) SGC-7901 cells were transfected with STAT3 responsive luciferase reporter. After 24 h, cells were treated with wogonin at different concentrations for another 24 h and luciferase assays were performed (***p < 0.001 vs. DMSO group). (D) Levels of STAT3 protein in nucleus or cytoplasm of SGC-7901 and BGC-823 cells treated with 60 µM wogonin for 48 h were analyzed by western blotting. (E) CCK8 assay of wogonin treated SGC-7901 cells treated with or without STAT3 inhibitor stattic (***p < 0.001 vs. DMSO group).

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Wogonin inhibits JAK-STAT3 signaling in GC cells

STAT3 can be activated by a number of different cytokines and growth factors, such as IL-6. We then investigated whether wogonin antagonizes IL-6–mediated activation of STAT3 in GC cells. As shown in Fig. 7A, IL-6 significantly promoted STAT3 phosphorylation in SGC-7901 cells. However, wogonin effectively reversed the activation/phosphorylation of STAT3 induced by IL-6, suggesting that wogonin may inhibit cytokine-mediated activation of STAT3 in GC cells (Fig. 7A).

It is well established that Janus tyrosine kinase (JAK)/signal transducer activator of transcription 3 (STAT3) signaling is involved in a number of important biological processes, including cell proliferation, differentiation and apoptosis. Extracellular factors such as IL-6 bind to membrane receptors and trigger signaling pathways via JAKs, which in turn activate STAT3. To identify the mechanisms underlying the inhibition of STAT3 by wogonin, we detected the protein levels of JAKs in SGC-7901 and BGC-823 cells treated with wogonin. As shown in Fig. 7B, treatment with wogonin for 48 h significantly decreased the levels of total JAK1/2 and p-JAK1/2 proteins. Moreover, the effects of Ruxolitinib, a JAK1/2 inhibitor, on STAT3 activation inhibition were enhanced by wogonin (Fig. 7C). Together, those results suggest that wogonin perturbs JAK-STAT3 signaling in GC cells.

We next explored the potential mechanisms of JAK1/2 downregulation induced by wogonin. JAK1/2 mRNA levels were not decreased but were considerably elevated in wogonin-treated GC cells (Fig. 7D). Moreover, treatment with the proteasome inhibitor MG132 could not block the decrease in the levels of total JAK1/2 protein induced by wogonin (Fig. 7E). Together, these data demonstrate that downregulation of JAK1/2 protein expression may not be associated with transcription inhibition or accelerated proteasome-dependent degradation of JAK1/2.

Fig. 7
figure 7

Wogonin down-regulates expression of JAK1/JAK2 to inhibit activity of STAT3. (A) Levels of phosphorylated and total STAT3 in wogonin treated SGC-7901 cells treated with or without IL-6 stimulated for 48 h were analyzed by western blotting. (B) Levels of phosphorylated and total JAK1/2 in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h were analyzed by western blotting. (C) Levels of phosphorylated and total STAT3 in wogonin treated SGC-7901 cells with or without ruxolitinib stimulated for 48 h were analyzed by western blotting. (D) qPCR was performed to analyze JAK1 and JAK2 mRNA levels in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h (***p < 0.001 vs. DMSO group). (E) Levels of indicated proteins in SGC-7901 cells with wogonin treated for 48 h and then MG132 for another 6 h were analyzed by western blotting.

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Discussion

Treatment of GC has undergone a great change in the last decade4,38. Systemic chemotherapy, radiotherapy, surgery, immunotherapy, and targeted therapy all have proven efficacy in GC, and multidisciplinary treatment is paramount to treatment selection39. However, most patients ultimately experience cancer progression40. Therefore, the search for new antitumor agents that are more effective and less toxic is needed in the treatment of cancer. Wogonin, a naturally bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, has been used to treat allergic and inflammatory diseases41. In recent years, many studies have shown that wogonin can inhibit tumor growth, metastasis and angiogenesis in vivo and in vitro11,42. Importantly, wogonin shows no or low toxicity to normal cells and has no obvious toxicity in animals11. In this study, we demonstrated that wogonin significantly inhibited the proliferation of SGC-7901, BGC-823 and MKN45 cells in vitro. Subsequent in vivo experiments confirmed the antitumor effect of wogonin on GC xenografts in nude mice. We further showed that wogonin caused G1 cell cycle arrest, induced cell apoptosis, and inhibited the migration and invasion of GC cells. Previous studies have shown that wogonin inhibited proliferation and the energy metabolism of SGC7901 cells23. Another study showed that wogonin significantly promoted apoptosis of both SGC7901 and the mouse gastric cancer cell line (MFC). Moreover, this study also showed that wogonin could inhibit tumor growth and promote the recruitment of DC, T, and NK cells into tumor tissues in tumor xenograft models19. These data and the results of our findings suggest that wogonin has significant pharmacological effects in inhibiting the proliferation and metastasis of GC cells and is a potential natural drug for GC treatment.

We found that wogonin significantly decreased the protein levels of CDK4 and CDK6, which mediate cycle transition from G0/G1-phase to S-phase. Cyclin D-bound CDK4/6 could phosphorylates RB, leading to the release of E2F transcription factors and increase transcription of the E2F target genes including CCNE1/243. However, no change in the protein levels of cyclins were observed in wogonin treated GC cell lines. Gene expression involves multiple sequential steps which are highly regulated and coordinated. In addition to be regulated by CDKs, the expression of cyclins are also governed by other transcriptional and post-transcriptional regulation mechanisms such as miRNAs and ubiquitination mediated proteasomal degradation44. Therefore, decrease in CDKs expression may not result in reduction in protein levels of cyclins. It was reported that ectopic expression of RUNX3 in 786-O cells was associated with G1 phase arrest and reduction in protein levels of CDK2, CDK4 and p-RB, while the protein levels of cyclin D and cyclin E were not decreased45. Our findings suggest that wogonin may inhibit CDKs activity via downregulating CDK4/6 expression, through which wogonin caused G1 arrest in GC cells.

Transcriptome analysis is an important way to identify molecular targets of drugs in pharmacological analysis46,47. The results of transcriptome analysis showed that wogonin affected the JAK-STAT signaling pathway, which is closely related to the proliferation and metastasis of cancer cells48. We further confirmed the decrease in the phosphorylation level of STAT3 at tyrosine (Tyr) 705 in wogonin-treated GC cells, phosphorylation at this site is important for the maintenance of the transcriptional activity of STAT3 and its entry into the nucleus49,50. Indeed, STAT3 transcriptional activity was significantly decreased, and the level of nuclear STAT3 was also reduced in wogonin treated GC cells. These findings suggest that STAT3 may be an effective target by which wogonin inhibits the GC cell proliferation, metastasis and drug resistance.

STAT3 is a key member of the signal transducer and activator of transcription (STAT) family. STAT3 is involved in a variety of biological processes, including cell proliferation, survival, differentiation and angiogenesis51. In normal cells, STAT3 is instantaneously activated to transmit transcription signals of cytokines and growth factors from outside the cell to the nucleus. Constitutive activation of STAT3 occurs in more than 70% of human malignant tumors51,52. Hyperactivated STAT3 can promote the proliferation and metastasis of cancer cells and induce chemotherapy resistance. A number of experiments demonstrated that STAT3 was hyperactivated in many GC cell lines and that inhibition of STAT3 signaling inhibited cell proliferation and induced apoptosis51. The expression and phosphorylation level of STAT3 are considered to be closely related to the occurrence and development of GC and are considered to be the key targets to inhibit those processes51. The identification and development of novel drugs that can target deregulated STAT3 has become an attractive new way to overcome cancer.

Previous studies have shown that wogonin can inhibit the activation of STAT3 in several malignant tumors11,19. Wogonin has been reported to induce the senescence of MDA-MB-231 human breast cancer cells by inhibiting STAT3 activity53. Wogonin suppresses the migration of human alveolar adenocarcinoma cell A549 by inactivating STAT3 signaling pathway54. Wogonin suppresses IL-6-induced VEGF expression by inhibiting the IL-6R/JAK1/STAT3 signaling pathway in HUVECs55. Wogonin inhibits the phosphorylation of STAT3 to reduce the expression of B7H1 and MHC class I chain-associated protein A, enhances calreticulin on the cell membrane, and promotes tumor immunity in GC cells19. Therefore, STAT3 is a key target of wogonin in cancer cells. Together with a previous report that wogonin showed immune-enhancing activities by suppressing STAT3 phosphorylation to decrease the expression of B7H1 and MHC class I chain-related protein and upregulate CRT expression on the cell membrane in GC cells, these findings suggest that wogonin could be an attractive natural drug for GC therapy. In the present study, we found that wogonin decreased the level of phosphorylated STAT3 and inhibited STAT3 transcriptional activity in GC cells, suggesting that STAT3 may be an important target by which wogonin inhibits the proliferation, migration and invasion of GC cells. Interestingly, although wogonin treatment significantly increased p-STAT3 levels in T24, A375 and MCF-7 cells, no changes in p-STAT3 levels were observed in B16 cells, suggesting that the inhibitory activity of wogonin on STAT3 activation may depend on the type of cancer cells.

As an important signal transduction molecule, STAT3 can be phosphorylated and activated by extracellular signal factors such as IL6 and then enter the nucleus to play a transcriptional activation role participating in cell growth, differentiation, immune regulation and other processes. We found that wogonin treatment effectively reversed IL-6-induced STAT3 phosphorylation. Together these results indicate that wogonin downregulates the phosphorylated STAT3 levels but does not affect total STAT3 levels, indicating that wogonin can inhibit cytokine-mediated STAT3 activation in GC cells. In the tumor microenvironment, the response of STAT3 to cytokines is mediated by JAKs56,57. Previous studies have shown that wogonin inhibits IL-6-induced angiogenesis by regulating JAK/STAT3 signaling55. Wogonin reduces the generation of proinflammatory cytokines, such as IL-6 and tumor necrosis factor-α (TNF-α) in activated microglia by JAK1/3-STAT1/3 signaling pathway58. In this study, we found that wogonin reduced the protein expression level of JAK1/2 proteins, suggesting that wogonin may downregulate STAT3 by inhibiting JAKs.

Further studies showed that wogonin did not reduce the mRNA levels of JAK1/2, indicating that the regulation of the level of JAK1/2 protein by wogonin is not by inhibiting the transcription of the JAK1/2gene. Moreover, treatment with the proteasome inhibitor MG132 could not block the decrease in of JAK1/2 protein levels induced by wogonin, indicating that the decrease in JAK1/2 protein was not caused by excessive degradation. MicroRNAs (miRNAs) are a class of small noncoding RNAs that target the 3’-UTR of complementary RNAs and repress their expression through RNA degradation and/or translational repression to regulate various cellular activities including cell growth, differentiation, development, and apoptosis59,60. Accumulating studies have revealed that miRNAs interact with certain genes in JAK-STAT3 signaling pathway and participate in the occurrence and development of tumors including GC61. Previous studies have shown that miR-135a targets JAK2 to repress p-STAT3 activation, reduce cyclin D1 and Bcl-xL expression and inhibit GC cell proliferation62. MiRNA-216a inhibits migration and invasion of GC cells by downregulating JAK2/STAT3-mediated mesenchymal transition (EMT) process63. Berberine inhibits the proliferation of bladder cancer (BCa) cells by downregulating JAK1-STAT3 signaling through the regulation of miR-17-5p, which directly targets JAK1 and STAT3 to reduce their expression37. MiR-340 was found to inhibit GC cell proliferation through regulating SOCS3/JAK-STAT signaling pathway64. The present study shows that downregulation of JAK1/2 expression by wogonin is not mediated by the regulation of transcription or protein degradation which suggests that wogonin may inhibit JAK1/2 expression at the posttranscriptional level. Several previous studies have shown that wogonin can regulate miRNAs. Wogonin regulates the expression of miR-155 by NF-κB to promote Raji cell apoptosis65. Wogonin regulates the expression of miR-145 in neointimal formation in vitro and in vivo66. These studies show that wogonin can affect physiological processes by regulating miRNAs. These results suggest that wogonin may regulate the expression of JAK1/2 by affecting miRNA. In conclusion, we showed that wogonin effectively inhibits the proliferation, migration and invasion of GC cells by inhibiting the JAK-STAT3 signaling pathway, and that wogonin may regulate JAK1/2 at the posttranscriptional level. Our results provide supporting evidence for the clinical application of wogonin in GC treatment.