Wogonin Suppresses Proliferation, Invasion and Migration in Gastric Cancer cells via Targeting the JAK-STAT3 Pathway

Wogonin Suppresses Proliferation, Invasion and Migration in Gastric Cancer cells via Targeting the JAK-STAT3 Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Wogonin Suppresses Proliferation, Invasion and Migration in Gastric Cancer cells via Targeting the JAK-STAT3 Pathway Yang Song, Hui zhao, Runze Yu, Yang Zhang, Yongxin Zou, Xiaofei Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4461628/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Wogonin is a compound extracted from the medicinal plant Scutellaria baicalensis Geogi and has been found to exert antitumor activities in a variety of malignancies. However, the molecular mechanisms involved in the anti-gastric cancer (GC) effects of wogonin remain poorly understood. In the present study, we found that wogonin treatment inhibited the proliferation of GC cells, induced apoptosis and G0/G1 cell arrest, and suppressed the migration and invasion of SGC-7901 and BGC-823 cells in vitro. In addition, wogonin inhibited in vivo tumor growth in SGC-7901 xenograft mice. Transcriptomic analysis suggested that wogonin affected several signaling pathways closely related to tumor proliferation and metastasis, including the STAT3 signaling pathway. Further research indicated that wogonin may exert antitumor effects in GC cells by downregulating the JAK-STAT3 pathway. Altogether, our results demonstrate that wogonin exerts antitumor effects by perturbing JAK-STAT3 signaling in GC cells and that wogonin may be a potential therapeutic option for GC. Biological sciences/Cancer Biological sciences/Drug discovery Biological sciences/Molecular biology wogonin gastric cancer natural compounds STAT3 signaling pathways antitumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Gastric cancer (GC) is the fifth most common cancer. In 2020, over one million new cases were diagnosed [ 1 ]. The incidence rates of GC vary widely by sex, race, and geographic location, with considerably higher incidence in Eastern Asia and in men [ 1 , 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 year [ 1 , 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 year [ 5 , 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 stage [ 5 ]. However, its application is limited due to drug resistance and side effects, including genotoxicity, nephrotoxicity and acute myelotoxicity [ 7 , 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 properties [ 9 , 10 ]. Wogonin (5, 7-dihydroxy-8-methoxyflavone), a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi , has been proposed to exert potent antitumor [ 11 ], anti-inflammatory [ 12 , 13 ] and anti-allergic [ 12 , 14 ] activities. Previous studies revealed the mechanisms underlying the antitumor effects of wogonin in several tumors; these mechanisms include cell cycle arrest [ 15 , 16 ], cell apoptosis induction [ 17 , 18 ], tumor immunity promotion [ 19 ] and angiogenesis suppression [ 20 ]. Additionally, it has been well documented that wogonin potentiates the antineoplastic effects of traditional chemotherapy with less toxicity as an adjunct treatment or pretreatment [ 21 , 22 ]. It has been reported that wogonin exerts anti-GC effects through different mechanisms [ 11 ]. Research has shown that wogonin can inhibit energy metabolism, cell proliferation and angiogenesis in SGC-7901 and A549 cells [ 23 ]. 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 release [ 19 , 24 ]. Moreover, wogonin was shown to potentiate the antitumor effects of oxaliplatin, paclitaxel and 5-fluorouracil in vivo and in vitro [ 21 , 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 pathways [ 22 ]. 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. 2. Materials and Methods 2.1. 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% CO 2 . 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). 2.2. Cell proliferation and colony formation assays The cell proliferation was determined using the CCK-8 assay as described previously [ 56 ]. Briefly, MKN45, SGC-7901 and BGC-823 cells were respectively planted in a 96-well plate at a density of 1×10 4 /well and cultured overnight. Then the cells were incubated with various concentrations (0, 30, 60, 90 and 120µM) of wogonin for 48h and incubated with 30µM wogonin for different time (24, 48, 72 and 96h). 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 500 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 were large enough to be visualized. Colonies were fixed with 4% Paraformaldehyde and stained with 0.1% crystal violet for 15 min at room temperature. Then colonies were counted and photographed under inverted microscope (CKX41, Olympus Life Science, Tokyo, Japan). All experiments were performed at least in triplicate. 2.3. 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 described [ 54 , 55 ]. 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 image J software (Bethesda, MD, USA). The cell cycle analysis was performed using a Becton Dickinson FACScan instrument (BD Biosciences, San Jose, CA) as previously described [ 56 ]. 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. Cell 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). 2.4. 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) were used to detection of mitochondrial membrane potential (MMP, ΔΨm) following the manufacturer’s directions. After incubation for 48 hours 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% CO 2 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). 2.5. Tumor Xenografts Ten male BALB/c nude mice (3–4 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 8×10 6 cells suspended in 200ul PBS were injected subcutaneously into each mouse. Then nude mice were randomly divided into two groups (five mice in each group): the mice in wogonin group were injected with wogonin (60mg/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 approved by the Institutional Animal Care and Use Committee of Shandong University and all procedures were performed in compliance with the institutional guidelines (Animal Research Ethics approval number: LL-201601015). 2.6. Wound-healing, transwell migration and invasion assays Wound-healing, transwell migration and invasion assays were performed as previously described [ 57 , 58 ]. 10 6 /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 described [ 55 , 59 ]. 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 (4×10 6 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. 2.7. 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) analysis of DEGs. 2.8. 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 previously [ 60 ]. Briefly, a total of RNA (2.5 µg) were 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 1. 2.9. Protein extraction and western blot Total proteins was extracted from cells treated with various concentrations of wogonin as previously described [ 61 ]. Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, shanghai, China) were used to isolated nuclear proteins according to the manufacturer’s instructions. Western blot analysis was performed as described previously [ 61 ]. The primary antibodies are listed in Supplementary Table 2. 2.10. 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 hours after transfection, cells were treated with various concentrations of wogonin in complete RPMI1640 medium for another 36h. Then the Dual-Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, shanghai, China) were 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. 2.11. 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. 3. Results 3.1. 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. 1 A), and the inhibitory effect of wogonin was concentration-dependent (Fig. 1 B). Moreover, wogonin treatment suppressed the colony formation ability of these cells (Fig. 1 C). 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. 1 D and Figure 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. 1 E, wogonin significantly suppressed tumor growth. At the endpoint, the tumor volume and weight were both significantly reduced in the group receiving wogonin (Fig. 1 E). Together, these results have shown that the growth of gastric cancer cells was suppressed significantly by wogonin, in vitro and in vivo. 3.2. 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. 2 A), suggesting that wogonin induces G0/G1 arrest in SGC-7901 cells. However, 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. 2 A). 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. 2 B). No changes in the levels of total CDK2, Cyclin B1, Cyclin E and Cyclin D1 proteins were observed (Fig. 2 B). 3.3. Wogonin induces the apoptosis of GC cells Then, we investigated the role of wogonin on apoptosis in GC cells. According to the results of flow cytometric assays, the percentage of apoptotic SGC-7901 cells only slightly increased after treatment with 60 µM wogonin for 48 h, while this percentage was significantly increased in 90 µM wogonin-treated SGC-7901 cells (Fig. 3 A). However, no obvious change in the percentage of apoptotic BGC-823 cells was observed after treatment with wogonin for 48 h while this percentage was slight increased in BGC-823 cells treated with 90 µM wogonin (Fig. 3 A). 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. 3 B). 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. 3 C). 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. 3 D, 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. 3.4. 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. 4 A). In addition, wogonin can decrease greatly the number of cells that migrated across the transwell membrane by transwell assays (Fig. 4 B). 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. 4 C). Taken together, these results indicate that the migration and invasion of GC cells were inhibited by wogonin. 3.5. 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. 5 A). Similar results were obtained in the comparison of the blank control and 90 µM wogonin treatment groups (Figure S2 A). The volcano plot for the DEGs is displayed in Fig. 5 A. qPCR was performed to confirm the RNA-seq results (Fig. 5 B). GO enrichment analysis was performed for the 320 DEGs. The most enriched GO Terms of p value are shown in Fig. 5 C and Figure S2 B. 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. 5 D and Figure S2 C). Taken together, these GO and KEGG pathway enrichment results could provide essential information for the investigation of wogonin in SGC-7901 GC cells. 3.6. 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. 6 A and Fig. 6 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 (Figure S3). It has been reported that the JAK-STAT3 signaling pathway played an important role in tumor proliferation and metastasis [ 25 , 26 ]. 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. 5 D and Figure S2 C), 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. 6 C). Activated STAT3 is transported to the nucleus and induces the transcription of downstream target genes. Meanwhile, the expression level of nuclear STAT3 was lower in wogonin-treated GC cells (Fig. 6 D). 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. 6 E). Importantly, the inhibitory activity of Stattic toward GC cells was enhanced by treatment with wogonin (Fig. 6 E). 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 (Fig. 6 F), suggesting that the inhibitory activity of wogonin on STAT3 may depend on cancer type. 3.7. 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. 7 A, 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. 7 A). 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. 7 B, treatment with wogonin for 48 h significantly decreased the levels of total JAK1/2 proteins. Moreover, the effects of Ruxolitinib, a JAK1/2 inhibitor, on STAT3 activation inhibition were enhanced by wogonin (Fig. 7 C). 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. 7 D). Moreover, treatment with the proteasome inhibitor MG132 could not block the decrease in the levels of total JAK1/2 protein induced by wogonin (Fig. 7 E). 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. 4. Discussion Treatment of GC has undergone a great change in the last decade [4, 27]. Systemic chemotherapy, radiotherapy, surgery, immunotherapy, and targeted therapy all have proven efficacy in GC, and multidisciplinary treatment is paramount to treatment selection [28]. However, most patients ultimately experience cancer progression [29]. 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 diseases [30]. In recent years, many studies have shown that wogonin can inhibit tumor growth, metastasis and angiogenesis in vivo and in vitro [11, 31]. Importantly, wogonin shows no or low toxicity to normal cells and has no obvious toxicity in animals [11]. 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 induced DNA damage, G0/G1 cell cycle arrest and cyclin downregulation in GC cells. Moreover, wogonin treatment caused GC cell apoptosis and significantly increased the levels of apoptosis markers. The results of this study also showed that wogonin treatment significantly inhibited the migration and invasion of GC cells. These results 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. Transcriptome analysis is an important way to identify molecular targets of drugs in pharmacological analysis [32, 33]. 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 cells [34]. 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 nucleus [35, 36]. Indeed, STAT3 transcriptional activity was significantly decreased, and the level of nuclear STAT3 was also reduced in wogonin treated GC cells. These results 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 angiogenesis [37]. 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 tumors [37, 38]. 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 apoptosis [37]. 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 processes [37]. 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 tumors [11, 19]. Wogonin has been reported to induce the senescence of MDA-MB-231 human breast cancer cells by inhibiting STAT3 activity [39]. Wogonin suppresses the migration of human alveolar adenocarcinoma cell A549 by inactivating STAT3 signaling pathway [40]. Wogonin suppresses IL-6-induced VEGF expression by inhibiting the IL-6R/JAK1/STAT3 signaling pathway in HUVECs [41]. 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 cells [19]. 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 JAKs [42, 43]. Previous studies have shown that wogonin inhibits IL-6-induced angiogenesis by regulating JAK/STAT3 signaling [41]. 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 pathway [44]. 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 JAK1/2 gene. 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 apoptosis [45, 46]. 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 GC [47]. 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 proliferation [48]. MiRNA-216a inhibits migration and invasion of GC cells by downregulateding JAK2/STAT3-mediated mesenchymal transition (EMT) process [49]. 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 expression [50]. MiR-340 was found to inhibit GC cell proliferation through regulating SOCS3/JAK-STAT signaling pathway [51]. 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 apoptosis [52]. Wogonin regulates the expression of miR-145 in neointimal formation in vitro and in vivo [53]. 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 inhibites 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. Declarations Date availability The datasets generated during and/or analysed during the current study are available in the the corresponding author on reasonable request. Acknowledgements We thank the reviewers and also the authors of all references. Funding This research was funded by the Natural Science Foundation of Shandong Province for Youth (grants ZR 2020QC235). Author information Yang Song，Hui zhao and Runze Yu have contributed equally to this work. Authors and Affiliations Sch ool of Bioengineering, Qilu University of Technology, Shandong Academy of Sciences, Jinan 250353 , China Yang Song Department of Dermatology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, The First Clinical Medical College of Shandong University of Traditional Chinese Medicine, Shandong Provincial Hospital of Traditional Chinese Medicine, Jinan 250011, China Shuna Sun Advanced Medical Research Institute, Shandong University, Jinan 250100, China Hui zhao Department of Pulmonary and Critial Care Medicine, Affiliated Hospital of Shandong University of Traditional Chinese Medicine ， Jinan 250011, China Yang Zhang The Key Laboratory of Experimental Teratology, Ministry of Education and Department of Molecular Medicine and Genetics, School of Basic Medical Sciences, Shandong University, Jinan 250012, China Runze Yu, Yongxin Zou Breast and Thyroid Surgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, China Xiaofei Liu Contributions Conceptualizatin, S.S., X.L and Y.Z.; methodology, S.S., X.L and Y.Z.; writing-original draft, Y.S., H.Z.and R.Y; writing-review&editing, S.S., X.L and Y.Z.; formal analysis, S.S., X.L and Y.Z.; investigation, Y.S., H.Z.and Y.Z.; visualization, Y.S., H.Z. and R.Y; data curation, Y.S., H.Z.and Y.Z.; validation, H.Z. and R.Y; supervision, S.S., X.L and Y.Z.. All authors have read and agreed to the published version of the manuscript. Corresponding authors Correspondence to Shuna Sun, Xiaofei Liu or Yongxin Zou. Ethics declarations Conflict of interest The authors declare no conflicts of in-terest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Ethical approval The animal study protocol was approved by the Institutional Animal Care and Use Committee of Shandong University (protocol code LL-201601015, date 2 March 2016). Statement The authors confirmed that the present study was reported in accordance with AARIVE guidelines (https://arriveguidelines.org). Consent to participate Not applicable. Consent to publication Not applicable. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Supplementary Files SupplementaryFiguresandTables.pdf sourcedataofWB.pdf Cite Share Download PDF Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 07 Oct, 2024 Reviews received at journal 24 Sep, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviews received at journal 09 Sep, 2024 Reviewers agreed at journal 02 Sep, 2024 Reviews received at journal 27 Jun, 2024 Reviewers agreed at journal 27 Jun, 2024 Reviews received at journal 12 Jun, 2024 Reviewers agreed at journal 12 Jun, 2024 Reviewers invited by journal 12 Jun, 2024 Editor assigned by journal 12 Jun, 2024 Editor invited by journal 12 Jun, 2024 Submission checks completed at journal 12 Jun, 2024 First submitted to journal 22 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4461628","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":319900934,"identity":"d03c27b8-ee9d-40b0-9216-b90cc735f591","order_by":0,"name":"Yang Song","email":"","orcid":"","institution":"School of Bioengineering, Qilu University of Technology, Shandong Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Song","suffix":""},{"id":319900935,"identity":"95d9268a-b12a-4de8-a48c-deb1712670ff","order_by":1,"name":"Hui zhao","email":"","orcid":"","institution":"Advanced Medical Research Institute, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"zhao","suffix":""},{"id":319900936,"identity":"1d837602-42f7-48f1-98fa-0cfcecf03aa8","order_by":2,"name":"Runze Yu","email":"","orcid":"","institution":"The Key Laboratory of Experimental Teratology, Ministry of Education and Department of Molecular Medicine and Genetics, School of Basic Medical Sciences, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Runze","middleName":"","lastName":"Yu","suffix":""},{"id":319900938,"identity":"b761525f-d79e-45ec-b7dc-7d396624e73c","order_by":3,"name":"Yang Zhang","email":"","orcid":"","institution":"Department of Pulmonary and Critial Care Medicine, Affiliated Hospital of Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Zhang","suffix":""},{"id":319900939,"identity":"0902a258-669a-4888-8158-c00cbea233e6","order_by":4,"name":"Yongxin Zou","email":"","orcid":"","institution":"The Key Laboratory of Experimental Teratology, Ministry of Education and Department of Molecular Medicine and Genetics, School of Basic Medical Sciences, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yongxin","middleName":"","lastName":"Zou","suffix":""},{"id":319900940,"identity":"7b7fd3a9-4caa-4119-aed4-a692d4e5ae7f","order_by":5,"name":"Xiaofei Liu","email":"","orcid":"","institution":"Breast and Thyroid Surgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"Liu","suffix":""},{"id":319900941,"identity":"6c3b4430-8147-4bb7-a3f6-06ffb4e37c20","order_by":6,"name":"Shuna Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBAC+8OMDQcSKmyY+RkYDKBiCQT0HGc++ODDmTR2yQaglgNEaTnPlmw4s+0Qv8EBYrUwNvOYSfOcOSBtfP7wNukPNdsY+NlzDBh+7sCthZkZpKXijrHZjbQyiQPHbjNI9rwxYOw9g1sLG1jLmWfJZjd4zCQONtxmMLiRY8DM2IZbCw9IC2/b4frN/WcgWuwJaZFgBnv/MLMBQw7UFgkCWgyYIYHMLHEjrdjizLHbPBJnnhUc7MWnhf8gNCr7D2+8UVFzW46/PXnjg594tGB6DkQcIEHDKBgFo2AUjAIsAAC7FVT3fx3gxAAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Dermatology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shuna","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-05-22 14:31:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4461628/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4461628/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-81196-2","type":"published","date":"2024-12-28T15:57:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59257520,"identity":"16e334ce-7404-4074-a8fe-8cc9eb694e9a","added_by":"auto","created_at":"2024-06-28 09:04:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWogonin inhibits proliferation of GC cells. A.\u003c/strong\u003e Viability of SGC-7901, BGC-823 and MKN45 cells treated with or without 30μM wogonin was analyzed by CCK8 assay. \u003cstrong\u003eB.\u003c/strong\u003e CCK8 assay of SGC-7901, BGC-823 and MKN45 cells treated with wogonin at different concentrations for 48h. \u003cstrong\u003eC.\u003c/strong\u003e Colony formation assays to assess the clonogenicity of of SGC-7901, BGC-823 and MKN45 cells treated with wogonin at different concentrations. \u003cstrong\u003eD.\u003c/strong\u003e The effect of wogonin on DNA replication was analyzed by EdU incorporation assays. \u003cstrong\u003eE. \u003c/strong\u003eTumor formation by SGC-7901 cells in nude mice. 200μL 8×10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;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 were measured every 3 d by vernier caliper. At the end mice were sacrificed and the tumor weight were measured. *p\u0026lt;0.05 vs DMSO group, **p\u0026lt;0.01 vs DMSO group，***p\u0026lt;0.001 vs DMSO group.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/30dffc934412ea83bf076722.jpg"},{"id":59256938,"identity":"23351d39-9267-49b9-bb12-82dcde3bfaf0","added_by":"auto","created_at":"2024-06-28 08:56:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWogonin disrupts the cell cycle of GC cells. A.\u003c/strong\u003e Cell cycle distribution of SGC-7901 and BGC-823 cells exposed to wogonin at different concentrations for 48h was analyzed by flow cytometry. \u003cstrong\u003eB. \u003c/strong\u003eProtein levels of cyclins and cell cycle regulatory proteins in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48h were measured by western blot.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/f6f3e0b2d278fc4ff560f4bc.jpg"},{"id":59256935,"identity":"df33170b-451e-49b7-b124-967efe2e5e22","added_by":"auto","created_at":"2024-06-28 08:56:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWogonin induces increased apoptosis and ROS of GC cells. A. \u003c/strong\u003eFlow cytometric analysis of apoptosis of SGC-7901 and BGC-823 cells exposed to wogonin at different concentrations for 48h. \u003cstrong\u003eB. \u003c/strong\u003eWestern blot analysis to detect the levelsof apoptosis regulating proteins of SGC-7901 and BGC-823 cells exposed to wogonin at different concentrations for 48h. \u003cstrong\u003eC. \u003c/strong\u003eMitochondrial membrane potential analysis of SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48h. \u003cstrong\u003eD. \u003c/strong\u003eROS levels in SGC-7901 and BGC-823 cells treated with wogonin for 48h were presented through combination with DCFH-CA.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/80a6d526169856ae2afe5007.jpg"},{"id":59256934,"identity":"30eb47f0-42ce-4040-8c2d-366485ea9018","added_by":"auto","created_at":"2024-06-28 08:56:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":138427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWogonin inhibits migration and invasion of GC cells. A.\u003c/strong\u003e 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. \u003cstrong\u003eB-C.\u003c/strong\u003e 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\u0026lt;0.001 vs DMSO group.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/5db0fa0d8eb82f8ff841a728.jpg"},{"id":59256940,"identity":"de6e8f8a-63e8-4e89-8e1d-8ff4b2e9abd1","added_by":"auto","created_at":"2024-06-28 08:56:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of wogonin-treated GC cells compared to the DMSO treated cells. \u003c/strong\u003eRNA-seq analysis and bioinformatic analysis of\u003cstrong\u003e \u003c/strong\u003eSGC-7901 cells treated with 90 μM of wogonin, compared with DMSO treated cells. \u003cstrong\u003eA.\u003c/strong\u003e Volcano map for the DEGs.\u003cstrong\u003e B. \u003c/strong\u003eqPCR was performed to confirm the RNA-seq results. \u003cstrong\u003eC. \u003c/strong\u003eThe most enriched GO Terms. \u003cstrong\u003eD. \u003c/strong\u003eThe KEGG enrichment analysis of the DEGs.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/f8ef63ad8b013fb7eb169d17.jpg"},{"id":59256937,"identity":"ca039643-04f9-40a3-97a4-bab5833cd4c5","added_by":"auto","created_at":"2024-06-28 08:56:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":89087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWogonin inhibits STAT3 activation in GC cells. A. \u003c/strong\u003eLevels 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 blot. \u003cstrong\u003eB.\u003c/strong\u003e 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 blot. \u003cstrong\u003eC. \u003c/strong\u003eSGC-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\u0026lt;0.001 vs DMSO group).\u003cstrong\u003e D. \u003c/strong\u003eLevels \u003cstrong\u003eo\u003c/strong\u003ef total STAT3 in nucleus of SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h were analyzed by western blot. \u003cstrong\u003eE. \u003c/strong\u003eCCK8 assay of wogonin treated SGC-7901 cells\u003cstrong\u003e \u003c/strong\u003etreated with or without STAT3 inhibitor stattic (***p\u0026lt;0.001 vs DMSO group). \u003cstrong\u003eF.\u003c/strong\u003ep-STAT3 levels in T24, A375 and MCF-7 cells treated with wogonin were analyzed by western blot.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/de3cedc327365498327885dc.jpg"},{"id":59257522,"identity":"2d171abf-e453-49f8-92a1-dc93cc6ee098","added_by":"auto","created_at":"2024-06-28 09:04:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":76611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWogonin down-regulates expression of JAK1/JAK2 to inhibit activity of STAT3.\u003c/strong\u003e A. Levels of p-STAT3 in wogonin treated SGC-7901 cells treated with or without IL-6 stimulated for 48 h were analyzed by western blot. B. Levels of total JAK1 and JAK2 in SGC-7901 and BGC-823 cells treated with wogonin at different concentrations for 48 h were analyzed by western blot. C. Levels of p-STAT3 in wogonin treated SGC-7901 cells with or without ruxolitinib stimulated for 48 h were analyzed by western blot. 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\u0026lt;0.001 vs DMSO group). E. Levels of total JAK1 and JAK2 in SGC-7901 cells with wogonin treated for 48h and then MG132 for another 6 h were analyzed by western blot.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/1b3629d9da27151810cce063.jpg"},{"id":72640489,"identity":"f86c02b8-64a5-4e0b-9268-e4b9d4b8379a","added_by":"auto","created_at":"2024-12-30 16:05:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1735463,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/d0f62a4f-fbfd-4d3a-b14a-d52196dd9be0.pdf"},{"id":59256939,"identity":"af26fe1b-21c0-47de-b862-4e0e2073e185","added_by":"auto","created_at":"2024-06-28 08:56:29","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":774237,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresandTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/92bc89d3a68eb4f4396742f6.pdf"},{"id":59257521,"identity":"3ae0dc3f-1328-4760-8e77-04a8db45cab4","added_by":"auto","created_at":"2024-06-28 09:04:28","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":798497,"visible":true,"origin":"","legend":"","description":"","filename":"sourcedataofWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4461628/v1/48e530c3ce6350d50c7ba56d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Wogonin Suppresses Proliferation, Invasion and Migration in Gastric Cancer cells via Targeting the JAK-STAT3 Pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGastric cancer (GC) is the fifth most common cancer. In 2020, over one million new cases were diagnosed [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The incidence rates of GC vary widely by sex, race, and geographic location, with considerably higher incidence in Eastern Asia and in men [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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 year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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\u0026thinsp;\u0026gt;\u0026thinsp;90% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The median survival time of metastatic gastric cancer is less than 1 year [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 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 stage [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, its application is limited due to drug resistance and side effects, including genotoxicity, nephrotoxicity and acute myelotoxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, it is urgent to develop novel drugs and/or new therapeutic combinations for patients with gastric cancer.\u003c/p\u003e \u003cp\u003eNatural 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 properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Wogonin (5, 7-dihydroxy-8-methoxyflavone), a bioactive flavonoid isolated from the root of \u003cem\u003eScutellaria baicalensis Georgi\u003c/em\u003e, has been proposed to exert potent antitumor [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], anti-inflammatory [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and anti-allergic [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] activities. Previous studies revealed the mechanisms underlying the antitumor effects of wogonin in several tumors; these mechanisms include cell cycle arrest [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], cell apoptosis induction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], tumor immunity promotion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and angiogenesis suppression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, it has been well documented that wogonin potentiates the antineoplastic effects of traditional chemotherapy with less toxicity as an adjunct treatment or pretreatment [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been reported that wogonin exerts anti-GC effects through different mechanisms [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Research has shown that wogonin can inhibit energy metabolism, cell proliferation and angiogenesis in SGC-7901 and A549 cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 release [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, wogonin was shown to potentiate the antitumor effects of oxaliplatin, paclitaxel and 5-fluorouracil in vivo and in vitro [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 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 pathways [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell Culture and manipulation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe 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\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. 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\u0026amp;D Systems (Minneapolis, MN, USA) and Ruxolitinib was purchased from Selleck (Houston, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell proliferation and colony formation assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cell proliferation was determined using the CCK-8 assay as described previously [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Briefly, MKN45, SGC-7901 and BGC-823 cells were respectively planted in a 96-well plate at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well and cultured overnight. Then the cells were incubated with various concentrations (0, 30, 60, 90 and 120\u0026micro;M) of wogonin for 48h and incubated with 30\u0026micro;M wogonin for different time (24, 48, 72 and 96h). The cell viability was measured using Cell Counting Kit-8 (CCK8) (Beyotime Biotechnology, shanghai, China) at different time points following the manufacturer\u0026rsquo;s protocols. All experiments were performed at least in triplicate.\u003c/p\u003e \u003cp\u003eFor colony formation assay, single-cell suspensions with 500 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\u0026deg;C for 10\u0026ndash;15 days until colonies were large enough to be visualized. Colonies were fixed with 4% Paraformaldehyde and stained with 0.1% crystal violet for 15 min at room temperature. Then colonies were counted and photographed under inverted microscope (CKX41, Olympus Life Science, Tokyo, Japan). All experiments were performed at least in triplicate.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. EdU Incorporation, Cell Cycle and apoptosis Analyses\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEdU (5-ethynyl-20-deoxyuridine) incorporation assays was performed using the Cell-Light\u0026trade; EdU Apollo567 In Vitro Kit (Ribobio, Guangzhou, China) following the manufacturer\u0026rsquo;s protocols as previously described [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. 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\u0026reg; 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 image J software (Bethesda, MD, USA).\u003c/p\u003e\u003cp\u003eThe cell cycle analysis was performed using a Becton Dickinson FACScan instrument (BD Biosciences, San Jose, CA) as previously described [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In brief, cells treated with various concentrations of wogonin for 48 h were collected and then fixed in 75% ethanol at 4\u0026deg;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.\u003c/p\u003e\u003cp\u003e Cell apoptosis was detected using Annexin V-FITC/PI double staining (Kaiji, Nanjing, China) according to manufacturer\u0026rsquo;s instructions. Briefly, cells were collected using EDTA-free trypsin solution and washed with 1\u0026times;PBS for twice. Then the cells were resuspended in 500 \u0026micro;L 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).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. ROS and JC-1 Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIntracellular ROS level was determined by Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, shanghai, China) according to the manufacture\u0026rsquo;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 10\u0026micro;M. 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.\u003c/p\u003e \u003cp\u003eJC-1(5, 5\u0026prime;, 6, 6\u0026prime;-Tetrachloro-1, 1\u0026prime;, 3, 3\u0026prime;-tetraethyl-imidacarbocyanine iodide) assay kit (Beyotime Biotechnology, shanghai, China) were used to detection of mitochondrial membrane potential (MMP, ΔΨm) following the manufacturer\u0026rsquo;s directions. After incubation for 48 hours in a 24-well plate, the cells treated with various concentrations of wogonin were incubated with JC-1 staining working solution at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e 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).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Tumor Xenografts\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTen male BALB/c nude mice (3\u0026ndash;4 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\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells suspended in 200ul PBS were injected subcutaneously into each mouse. Then nude mice were randomly divided into two groups (five mice in each group): the mice in wogonin group were injected with wogonin (60mg/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 approved by the Institutional Animal Care and Use Committee of Shandong University and all procedures were performed in compliance with the institutional guidelines (Animal Research Ethics approval number: LL-201601015).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Wound-healing, transwell migration and invasion assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWound-healing, transwell migration and invasion assays were performed as previously described [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. 10\u003csup\u003e6\u003c/sup\u003e/well cells were planted into 24-well plates and incubated with complete RPMI1640 medium until about 90% confluency as a monolayer. A 200 \u0026micro;l 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] \u0026times; 100 (%).\u003c/p\u003e \u003cp\u003eTranswell migration and invasion assays were performed in 24-well transwell inserts (Corning, New York, USA) containing polycarbonate filters with 8-\u0026micro;m pores, with or without Matrigel (BD Biosciences, NJ, USA) as previously described [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Briefly, Matrigel was mixed with serum-free RIPM-1640 (1:6 ratio) in upper chambers, and incubated at 37\u0026deg;C for 2 h. GC cells treated with various concentrations of wogonin were suspended in serum-free RPMI-1640 medium (4\u0026times;10\u003csup\u003e6\u003c/sup\u003e 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\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. RNA sequencing (RNA-seq) experiment and data analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal 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\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026rsquo;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)\u0026thinsp;\u0026gt;\u0026thinsp;2 (in either direction) and false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were identified as differentially expressed genes (DEGs). The \u0026lsquo;clusterProfiler\u0026rsquo; R package were used for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis of DEGs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. RNA Isolation and real-time quantitative PCR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was isolated from GC cells treated with various concentrations of wogonin using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer\u0026rsquo;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 previously [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Briefly, a total of RNA (2.5 \u0026micro;g) were reverse transcribed to cDNA using PrimeScript RT Reagent Kit (Takara Co, Otsu, Japan) with random primers according to the manufacturer\u0026rsquo;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\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Protein extraction and western blot\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal proteins was extracted from cells treated with various concentrations of wogonin as previously described [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, shanghai, China) were used to isolated nuclear proteins according to the manufacturer\u0026rsquo;s instructions. Western blot analysis was performed as described previously [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The primary antibodies are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Plasmids and luciferase assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLuciferase 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\u0026rsquo;s instructions. 6 hours after transfection, cells were treated with various concentrations of wogonin in complete RPMI1640 medium for another 36h. Then the Dual-Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, shanghai, China) were 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStatistical analyses were performed using GraphPad 7 by the Student t test or one way ANOVA. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant. All results are presented as mean with SD.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Wogonin inhibits the proliferation of GC cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the inhibitory effect of wogonin was concentration-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, wogonin treatment suppressed the colony formation ability of these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), pointing out wogonin plays an inhibitory role on DNA replication in GC cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, wogonin significantly suppressed tumor growth. At the endpoint, the tumor volume and weight were both significantly reduced in the group receiving wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Together, these results have shown that the growth of gastric cancer cells was suppressed significantly by wogonin, in vitro and in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Wogonin induces G0/G1 arrest and downregulates G1/S transition-related proteins\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that wogonin induces G0/G1 arrest in SGC-7901 cells. However, 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 \u0026micro;M wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). No changes in the levels of total CDK2, Cyclin B1, Cyclin E and Cyclin D1 proteins were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Wogonin induces the apoptosis of GC cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThen, we investigated the role of wogonin on apoptosis in GC cells. According to the results of flow cytometric assays, the percentage of apoptotic SGC-7901 cells only slightly increased after treatment with 60 \u0026micro;M wogonin for 48 h, while this percentage was significantly increased in 90 \u0026micro;M wogonin-treated SGC-7901 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, no obvious change in the percentage of apoptotic BGC-823 cells was observed after treatment with wogonin for 48 h while this percentage was slight increased in BGC-823 cells treated with 90 \u0026micro;M wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMitochondrial 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Wogonin inhibits the migration and invasion of GC cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, wogonin can decrease greatly the number of cells that migrated across the transwell membrane by transwell assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Taken together, these results indicate that the migration and invasion of GC cells were inhibited by wogonin.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Transcriptomic analysis of wogonin-treated GC cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo 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 \u0026micro;M 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 \u0026micro;M wogonin treatment group compared to the DMSO treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar results were obtained in the comparison of the blank control and 90 \u0026micro;M wogonin treatment groups (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). The volcano plot for the DEGs is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. qPCR was performed to confirm the RNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). GO enrichment analysis was performed for the 320 DEGs. The most enriched GO Terms of p value are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the molecular function category, GO function enrichment analyses showed that the DEGs were enriched in \u0026ldquo;binding\u0026rdquo;, \u0026ldquo;catalytic activity\u0026rdquo; and \u0026ldquo;signal transducer activity\u0026rdquo;. In the cellular component category, \u0026ldquo;cell part\u0026rdquo;, \u0026ldquo;organelle\u0026rdquo;, \u0026ldquo;membrane\u0026rdquo; and \u0026ldquo;membrane part\u0026rdquo; were the most enriched terms. \u0026ldquo;Cellular process\u0026rdquo;, \u0026ldquo;biological regulation\u0026rdquo;, \u0026ldquo;single-organism process\u0026rdquo; and \u0026ldquo;metabolic process\u0026rdquo; 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 \u0026ldquo;p53 signal pathways\u0026rdquo;, \u0026ldquo;JAK-STAT signaling pathway\u0026rdquo;, and \u0026ldquo;FoxO signaling pathway\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Taken together, these GO and KEGG pathway enrichment results could provide essential information for the investigation of wogonin in SGC-7901 GC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Wogonin downregulates actived STAT3 in GC cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, 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 (Figure S3). It has been reported that the JAK-STAT3 signaling pathway played an important role in tumor proliferation and metastasis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 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 \u0026ldquo;JAK-STAT signaling pathway\u0026rdquo; based on GO analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC), 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Activated STAT3 is transported to the nucleus and induces the transcription of downstream target genes. Meanwhile, the expression level of nuclear STAT3 was lower in wogonin-treated GC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Together, these results suggest that wogonin decreases the activity of STAT3 in GC cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Importantly, the inhibitory activity of Stattic toward GC cells was enhanced by treatment with wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These data suggest that wogonin exerts its antitumor activity at least partly by decreasing STAT3 activity.\u003c/p\u003e \u003cp\u003eTo 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), suggesting that the inhibitory activity of wogonin on STAT3 may depend on cancer type.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Wogonin inhibits JAK-STAT3 signaling in GC cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSTAT3 can be activated by a number of different cytokines and growth factors, such as IL-6. We then investigated whether wogonin antagonizes IL-6\u0026ndash;mediated activation of STAT3 in GC cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, treatment with wogonin for 48 h significantly decreased the levels of total JAK1/2 proteins. Moreover, the effects of Ruxolitinib, a JAK1/2 inhibitor, on STAT3 activation inhibition were enhanced by wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Together, those results suggest that wogonin perturbs JAK-STAT3 signaling in GC cells.\u003c/p\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Moreover, treatment with the proteasome inhibitor MG132 could not block the decrease in the levels of total JAK1/2 protein induced by wogonin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTreatment of GC has undergone a great change in the last decade [4, 27]. Systemic chemotherapy, radiotherapy, surgery, immunotherapy, and targeted therapy all have proven efficacy in GC, and multidisciplinary treatment is paramount to treatment selection [28]. However, most patients ultimately experience cancer progression [29]. 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 \u003cem\u003eScutellaria baicalensis Georgi\u003c/em\u003e, has been used to treat allergic and inflammatory diseases [30]. In recent years, many studies have shown that wogonin can inhibit tumor growth, metastasis and angiogenesis in vivo and in vitro [11, 31]. Importantly, wogonin shows no or low toxicity to normal cells and has no obvious toxicity in animals [11]. 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 induced DNA damage, G0/G1 cell cycle arrest and cyclin downregulation in GC cells. Moreover, wogonin treatment caused GC cell apoptosis and significantly increased the levels of apoptosis markers. The results of this study also showed that wogonin treatment significantly inhibited the migration and invasion of GC cells. These results 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.\u003c/p\u003e\n\u003cp\u003eTranscriptome analysis is an important way to identify molecular targets of drugs in pharmacological analysis [32, 33]. 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 cells [34]. 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 nucleus [35, 36]. Indeed, STAT3 transcriptional activity was significantly decreased, and the level of nuclear STAT3 was also reduced in wogonin treated GC cells. These results suggest that STAT3 may be an effective target by which wogonin inhibits the GC cell proliferation, metastasis and drug resistance.\u003c/p\u003e\n\u003cp\u003eSTAT3 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 angiogenesis [37]. 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 tumors [37, 38]. 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 apoptosis [37]. 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 processes [37]. The identification and development of novel drugs that can target deregulated STAT3 has become an \u0026nbsp;attractive new way to overcome cancer. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that wogonin can inhibit the activation of STAT3 in several malignant tumors [11, 19]. Wogonin has been reported to induce the senescence of MDA-MB-231 human breast cancer cells by inhibiting STAT3 activity [39]. Wogonin suppresses the migration of human alveolar adenocarcinoma cell A549 by inactivating STAT3 signaling pathway [40]. Wogonin suppresses IL-6-induced VEGF expression by inhibiting the IL-6R/JAK1/STAT3 signaling pathway in HUVECs [41]. 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 cells [19]. 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 \u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eAs 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 JAKs [42, 43]. Previous studies have shown that wogonin inhibits IL-6-induced angiogenesis by regulating JAK/STAT3 signaling [41]. Wogonin reduces the generation of proinflammatory cytokines, such as IL-6 and tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;) in activated microglia by JAK1/3-STAT1/3 signaling pathway [44]. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther 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 \u003cem\u003eJAK1/2\u003c/em\u003e gene. 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\u0026rsquo;-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 apoptosis [45, 46]. 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 GC [47]. 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 proliferation [48]. MiRNA-216a inhibits migration and invasion of GC cells by downregulateding JAK2/STAT3-mediated mesenchymal transition (EMT) process [49]. 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 expression [50]. MiR-340 was found to inhibit\u0026ensp;GC\u0026ensp;cell proliferation through regulating SOCS3/JAK-STAT signaling pathway [51]. 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-\u0026kappa;B to promote Raji cell apoptosis [52]. Wogonin regulates the expression of miR-145 in neointimal formation in vitro and in vivo [53]. 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 inhibites 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDate availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available in the the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the reviewers and also the authors of all references.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science Foundation of Shandong Province for Youth (grants ZR 2020QC235).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Song，Hui zhao\u0026nbsp;and\u0026nbsp;Runze Yu\u0026nbsp;have contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSch\u003c/strong\u003e\u003cstrong\u003eool of Bioengineering, Qilu University of Technology, Shandong Academy of Sciences, Jinan 250353 , China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Song\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Dermatology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, The First Clinical Medical College of Shandong University of Traditional Chinese Medicine,\u0026nbsp;Shandong Provincial Hospital of Traditional Chinese Medicine, Jinan 250011, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShuna Sun\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdvanced Medical Research Institute, Shandong University, Jinan 250100, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHui zhao\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Pulmonary and Critial Care Medicine, Affiliated Hospital of Shandong University of Traditional Chinese Medicine\u003c/strong\u003e\u003cstrong\u003e，\u003c/strong\u003e\u003cstrong\u003eJinan 250011, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Zhang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Key Laboratory of Experimental Teratology, Ministry of Education and Department of Molecular Medicine and Genetics, School of Basic Medical Sciences, Shandong University, Jinan 250012, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRunze Yu,\u0026nbsp;Yongxin Zou\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBreast and Thyroid Surgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaofei Liu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualizatin, S.S., X.L and Y.Z.; methodology, S.S., X.L and Y.Z.; writing-original draft, Y.S., H.Z.and R.Y; writing-review\u0026amp;editing, S.S., X.L and Y.Z.; formal analysis, S.S., X.L and Y.Z.; investigation, Y.S., H.Z.and Y.Z.; visualization, Y.S., H.Z. and R.Y; data curation, Y.S., H.Z.and Y.Z.; validation, H.Z. and R.Y; supervision, S.S., X.L and Y.Z.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Shuna Sun, Xiaofei Liu\u0026nbsp;or\u0026nbsp;Yongxin Zou.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of in-terest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the Institutional Animal Care and Use Committee of Shandong University (protocol code LL-201601015, date 2 March 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirmed that the present study was reported in accordance with AARIVE guidelines (https://arriveguidelines.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Molecular cancer 2022, 21, 63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia Y, Chen S, Cui J, Wang Y, Liu X, Shen Y, Gong L, Jiang X, Wang W, Zhu Y \u003cem\u003eet al\u003c/em\u003e. Berberine suppresses bladder cancer cell proliferation by inhibiting JAK1-STAT3 signaling via upregulation of miR-17-5p. Biochemical pharmacology 2021, 188, 114575.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"wogonin, gastric cancer, natural compounds, STAT3 signaling pathways, antitumor","lastPublishedDoi":"10.21203/rs.3.rs-4461628/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4461628/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWogonin is a compound extracted from the medicinal plant \u003cem\u003eScutellaria baicalensis Geogi\u003c/em\u003e and has been found to exert antitumor activities in a variety of malignancies. However, the molecular mechanisms involved in the anti-gastric cancer (GC) effects of wogonin remain poorly understood. In the present study, we found that wogonin treatment inhibited the proliferation of GC cells, induced apoptosis and G0/G1 cell arrest, and suppressed the migration and invasion of SGC-7901 and BGC-823 cells in vitro. In addition, wogonin inhibited in vivo tumor growth in SGC-7901 xenograft mice. Transcriptomic analysis suggested that wogonin affected several signaling pathways closely related to tumor proliferation and metastasis, including the STAT3 signaling pathway. Further research indicated that wogonin may exert antitumor effects in GC cells by downregulating the JAK-STAT3 pathway. Altogether, our results demonstrate that wogonin exerts antitumor effects by perturbing JAK-STAT3 signaling in GC cells and that wogonin may be a potential therapeutic option for GC.\u003c/p\u003e","manuscriptTitle":"Wogonin Suppresses Proliferation, Invasion and Migration in Gastric Cancer cells via Targeting the JAK-STAT3 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-28 08:56:23","doi":"10.21203/rs.3.rs-4461628/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-07T05:54:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-24T05:16:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334527707893710159260227690414418695921","date":"2024-09-24T04:51:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-09T20:20:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181965808525407436935383799614061646194","date":"2024-09-02T16:35:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-27T16:59:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172359334416680463645980584081694785688","date":"2024-06-27T16:57:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-12T18:58:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4573135920938180849636374651032855598","date":"2024-06-12T18:52:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-12T18:47:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-12T18:44:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-12T17:37:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-12T17:23:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-22T14:30:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6af4d152-ee2f-494d-96a4-824b8a0e81f6","owner":[],"postedDate":"June 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33827745,"name":"Biological sciences/Cancer"},{"id":33827746,"name":"Biological sciences/Drug discovery"},{"id":33827747,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-12-30T15:59:19+00:00","versionOfRecord":{"articleIdentity":"rs-4461628","link":"https://doi.org/10.1038/s41598-024-81196-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-28 15:57:07","publishedOnDateReadable":"December 28th, 2024"},"versionCreatedAt":"2024-06-28 08:56:23","video":"","vorDoi":"10.1038/s41598-024-81196-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-81196-2","workflowStages":[]},"version":"v1","identity":"rs-4461628","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4461628","identity":"rs-4461628","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}