Wogonin Inhibits Colorectal Cancer Proliferation and Epithelial Mesenchymal Transformation by Suppressing Phosphorylation in the AKT Pathway

Introduction

Colorectal cancer (CRC) is currently the third most common cancer worldwide and the second leading cause of cancer-related death (Siegel et al., 2020). The clinical treatments of CRC are mainly surgery, radiotherapy, and chemotherapy. In recent years, the emergence of targeted therapeutic drugs and immunotherapy drugs has greatly enriched the methods to treat CRC and has achieved certain clinical effects. Although great progress has been made in the treatment of CRC, the drugs currently in clinical use are often accompanied by increased side effects and poor efficacy (Aoullay et al., 2020; Johdi and Sukor, 2020). Therefore, identifying new anticancer strategies in addition to chemotherapy, targeted therapy, and immunotherapy will become a future research direction to improve the survival of patients with CRC.

Wogonin, a flavonoid from the herbal medicine Scutellaria baicalensis, has been reported to exert unique antitumor activity on several cancer types. Yang et al. (2020) demonstrated that wogonin could induce cellular senescence in breast cancer by suppressing TXNRD2 expression. Wogonin also acts against ovarian cancer by blocking the cell cycle and thereby inhibiting cell proliferation (Ruibin et al., 2017; Zhao et al., 2019). In addition, in gastric cancer and in head and neck cancer, wogonin enhanced sensitivity to chemotherapy drugs and showed synergistic effects when used in combination with chemotherapy drugs (Kim et al., 2016; Hong et al., 2018). However, the role and mechanism of wogonin in CRC still need to be further explored.

The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently activated in various human cancers and has been considered a promising therapeutic target (Porta et al., 2014). PI3K/AKT and mTOR are two signaling pathways that are critical for cell proliferation and survival in physiological and pathological states. They are related to each other, and phosphorylation is an important form of activation. Phosphorylation of AKT activates downstream mTOR, thus playing a role (Aoki and Fujishita, 2017; Yi et al., 2020). Small molecule inhibitors targeting three major nodes of this pathway (PI3K, AKT, and mTOR) have been developed, and inhibitors aimed at these targets could demonstrate antitumor effects (LoRusso, 2016).

In this study, we investigated the effects of wogonin on the proliferation and invasion of CRC cells and revealed its potential mechanism of action through the inhibition of AKT signaling pathway phosphorylation with a phosphorylated protein microarray screen. In vitro and in vivo experiments verified that wogonin, as an AKT phosphorylation inhibitor, could have an anti-CRC effect.

Materials and Methods

Cell Lines and Reagents

Human CRC cell lines (SW620, RKO, SW480, HCT116, and HT29) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). HT29 and HCT116 cells were cultured in McCoy’s 5A (Gibco, Carlsbad, CA, USA), while SW480, SW620, and RKO cells were cultured in DMEM (Gibco, Carlsbad, CA, USA). All culture media contained 10% fetal bovine serum and 1% penicillin. All cell lines were maintained in a humidified atmosphere of 5% CO₂ at $37^{\circ }$C. Purified wogonin ($>98\%$) (#20489) was purchased from Shanghai Yuanye Biological Co., Ltd. A 50mM stock solution was made in dimethyl sulfoxide (DMSO) purchased from Solarbio Science & Technology Co., Ltd. The anti-Caspase-3 antibody (#9661), anti-AKT1 antibody (#2938), anti-phospho-AKT (Ser473) antibody (#4058), anti-phospho-AKT (Thr308) antibody (#13038), anti-mTOR antibody (#2972), anti-phospho-mTOR (Ser2448) antibody (#2971), and anti-Ki67 antibody (#12202) were obtained from Cell Signaling Technology, and all were diluted 1:1000 dilution for immunoblotting. Anti-actin (sc-1616, 1:5000 dilution), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (sc-2055, 1:5000 dilution), and HRP-conjugated anti-rabbit IgG (sc-2054, 1:5000 dilution) were purchased from Santa Cruz. Crystal violet was purchased from Sigma-Aldrich (St. Louis, MO, USA).

WST-1 Assay

Cells were seeded in 96-well plates at 5000–10,000 cells per well and treated with different concentrations of wogonin for 24h, 48h, or 72h. Then, the cell inhibition rate was evaluated using a WST-1 assay kit (Beyotime, Shanghai, China).

Crystal Violet Assay

Cells were seeded in 24-well plates at 5000–10,000 cells per well and treated with different concentrations of wogonin for 48h. After fixation and staining with crystal violet, the cells were imaged and counted.

Apoptosis Assay

SW620 and RKO cells were seeded in 6-well plates and incubated overnight followed by treatment with different concentrations of wogonin for 24h. A Pharmingen Annexin V/FITC Apoptosis Detection Kit I (BD, San Jose, California, USA) was utilized to detect cell death by flow cytometry.

Transwell Migration and Invasion Assays

A Transwell chamber (Corning, Kennebunk, ME, USA) was used for the migration assays, and a Transwell chamber precoated with Matrigel was used for the invasion assays. According to the protocol, $1\times 105$cells/ml were suspended in the upper chamber of a 24-well Transwell plate. Following a 2-h preincubation, the cells were treated with different concentrations of wogonin for 24h. Then, the cells were washed, fixed, and stained with crystal violet. Based on the crystal violet staining data, we calculated the migration and invasion rates by counting the cells in at least five random fields.

Molecular Docking

The crystal structures of proteins were retrieved from Protein Data Bank. The docking process was as follows: Discovery Studio Client was used to perform dehydration and hydrogenation of proteins. PyRx-0.8 and AutoDock Vina 39 were used for molecular docking, and PyMOL software for mapping.

Cellular Thermal Shift Assay–Western Blot

Briefly, the soluble protein lysate of SW620 cells was aliquoted into PCR tubes and treated with wogonin (200$\mu$M) or DMSO for 1h at RT prior to Cellular Thermal Shift Assay (CETSA) heat pulse. The solutions were heated at the indicated temperatures (35–$75^{\circ }$C) for 3min, followed by cooling at $4^{\circ }$C for 3min in a thermocycler (Applied Biosystems, USA). After centrifugation for 20min ($20,000\times g$, $4^{\circ }$C), the soluble supernatant was subject to Western blotting (WB).

Phospho-Antibody Array

Phosphoprotein profiling was performed with the cancer-signaling phospho-antibody microarray. The cancer-signaling phospho-antibody microarray (CSP100), which was designed and manufactured by Full Moon BioSystems, Inc. (Sunnyvale, CA), contained 269 antibodies. A total of 132 phosphorylation sites on 93 signaling proteins were detected. These signaling proteins are widely involved in the transduction of many tumor-related signaling pathways. Each of the antibodies had six replicates that were printed on coated glass microscope slides, along with multiple positive and negative controls. The antibody array experiment was performed by Wayne Biotechnology (Shanghai, China) according to their established protocol. Briefly, cell lysates obtained from SW620 cells treated with wogonin were biotinylated with an Antibody Array Assay Kit (Full Moon BioSystems, Inc.). The antibody microarray slides were first blocked in blocking solution for 30min at room temperature, rinsed with Milli-Q grade water for 3–5min, and dried with compressed nitrogen. The slides were then incubated with the biotin-labeled cell lysates ($\sim 100\mu$g of protein) in coupling solution at room temperature for 2h. The array slides were washed 4–5 times with $1\times$ Wash Solution and rinsed extensively with Milli-Q grade water before detection of bound biotinylated proteins using Cy3-conjugated streptavidin. The slides were scanned on a GenePix 4000 scanner, and the images were analyzed using GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). The fluorescence signal of each antibody was obtained from the fluorescence intensity of the antibody-stained regions. A ratio calculation was used to measure the extent of protein phosphorylation. The phosphorylation ratio was calculated as follows: phosphorylation $\mathrm{\text{ratio}}=\mathrm{\text{phospho}}$ value/nonphospho value. The total proteome ratios were standardized to $\beta$-actin.

WB Assay

SW620 and RKO cells were seeded in a 10cm dish and incubated overnight before treatment with wogonin. The cells were harvested and lysed in radio-immunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors (cOmplete mini EASYpack, Roche, Basel, Switzerland). The lysates were resolved on 10% sodium dodecyl sulfate–polyacrylamide using gel electrophoresis (SDS–PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Blot images were acquired with a GBOX Chemi XT4 System (Syngene, Cambridge, UK) and quantified using densitometry with GeneTools software (Syngene).

Immunofluorescence

SW620 and RKO cells were seeded on glass coverslips and treated with wogonin. After being washed three times with phosphate-buffered saline (PBS), the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 for 10min. The cells were then treated with blocking buffer, primary antibody, and secondary antibody, and finally, the nuclei were stained with DAPI. The cells were visualized using fluorescence microscopy.

Xenograft Tumor Model

BALB/c nude mice (male, 4–5 weeks old) were injected subcutaneously with $5\times 106$ SW620 cells or $5\times 106$ HT-29 cells. After the xenografts reached a volume of approximately 100mm³, the mice were randomly divided into three groups. Mice treated with PBS were considered the control group. Mice were intraperitoneally injected with low-dose wogonin 25mg/kg and high-dose wogonin 50mg/kg body weight per day, respectively. 5-Fluorouracil (5-Fu) was injected intraperitoneally at a dose of 20mg/kg body weight every other day. The entire treatment cycle lasted for three weeks, and the volumes of the tumors were estimated every three days using the following formula: tumor volume $({\mathrm{\text{mm}}}^3)=\mathrm{\text{length}}(\mathrm{\text{mm}})\ast \mathrm{\text{width}}{(\mathrm{\text{mm}})}^2∕2$. At the end of the experiment, the animals were sacrificed, and the tumor tissues were excised to further assess tumor weight and perform pathological staining.

Immunohistochemistry

Immunohistochemistry was performed on 5$\mu$m thick paraffin sections of tumor tissues. Briefly, the sections were deparaffinized and rehydrated followed by antigen retrieval using boiling 0.01M sodium citrate buffer (pH 6.0) for 10min. Then, the sections were incubated with 3% hydrogen peroxide for 10min, 5% bovine serum albumin for 1h, and primary antibodies at $4^{\circ }$C overnight. The sections were incubated with secondary antibodies after being washed three times with PBS. Finally, the DAB system was used to visualize the signal, and hematoxylin was used to stain the nucleus. Immunostaining images were captured using a microscope.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., CA, USA). Student’s t-test and one-way analysis of variance (ANOVA) were used to compare differences between groups as appropriate. Data are presented as the $\mathrm{\text{mean}}\pm \mathrm{\text{standard}}$ deviation (SD), and $p<0.05$ was considered statistically significant.

Results

Wogonin Inhibits Cell Proliferation in CRC Cells

To determine the cytotoxicity and inhibitory effects of wogonin (Fig. 1A) in CRC cells, SW620, RKO, SW480, HT29, and HCT116 cells were treated with different concentrations of wogonin ranging from 1$\mu$M to 500$\mu$M for the indicated time points. The WST-1 assay showed that wogonin increased the inhibition of CRC cell growth at 24h, 48h, and 72h (Figs. 1B–1D). Based on the IC${}_{50}$ values, we found that SW620 and RKO cells were more sensitive to wogonin than the other three cell lines. Therefore, we selected these two cell lines for follow-up studies. Moreover, through crystal violet staining, the inhibitory effects of wogonin on SW620 and RKO cells were more intuitively demonstrated (Figs. 1E and 1F). These results indicated that wogonin could effectively inhibit the proliferation of CRC cells.

Wogonin Promotes Apoptosis of CRC Cells

To investigate whether wogonin induces apoptosis in CRC cells, we first detected the apoptosis of SW620 and RKO cells using flow cytometry. The results showed that wogonin treatment increased the apoptotic population of both SW620 and RKO cells in a dose-dependent manner (Figs. 2A and 2B). To further confirm the apoptotic effects of wogonin on CRC cells, we estimated the expression level of cleaved caspase-3 using immunofluorescence. In both SW620 and RKO cells, after two doses of wogonin, cleaved caspase-3 expression was significantly promoted, especially in the high-dose group (Figs. 2C–2F). These results confirmed that wogonin treatment could induce apoptosis of CRC cells.

Wogonin Inhibits the Migration and Invasion of CRC Cells

Next, we conducted a Transwell assay to estimate the effects of wogonin on CRC cell migration and invasion. As a result, the cell migration and invasion abilities of SW620 and RKO cells were significantly inhibited after wogonin treatment (Figs. 3A and 3C). The results from three independent experiments are shown in Figs. 3B and 3D. To further substantiate the effect of wogonin on CRC cell migration and invasion, we also estimated the protein expression levels of E-cadherin, N-cadherin, and vimentin. As shown in Figs. 3E–3H, E-cadherin protein expression was upregulated by increasing concentrations of wogonin in both SW620 and RKO cells. The protein expression levels of both N-cadherin and vimentin were significantly reduced by wogonin treatment. These results demonstrated that wogonin could significantly inhibit the migration and invasion of CRC cells.

Wogonin Inhibits the Phosphorylation Level of the AKT Protein

Moreover, to further explore the molecular mechanism of wogonin, we used a phospho-antibody array and screened more than 200 molecules clustered in 12 cancer-related pathways and found that phosphorylation variation of the AKT protein at site Ser473 exhibited the highest variation in SW620 cells after treatment with wogonin (Fig. 4A). AKT is a Ser/Thr protein kinase that synergizes with phosphatidylinositol-dependent protein kinases to cause the translocation of AKT from the cytoplasm to the plasma membrane and promotes the phosphorylation of AKT at Ser473 and Thr308. Phosphorylation at Ser473 and/or Thr308 is necessary for AKT activation to affect tumor cell proliferation and invasion (Vincent et al., 2011; Wu et al., 2020). mTOR is an important downstream molecule affected by AKT, and the AKT/mTOR signaling pathway plays an important role in the proliferation and survival of normal and tumor cells (Duan et al., 2018; Li et al., 2020). Therefore, we conducted WB experiments to verify the phosphorylation levels of the AKT and mTOR proteins in SW620 and RKO cells after intervention with different concentrations of wogonin. As shown in Figs. 4B and 4C, wogonin inhibited the phosphorylation of the AKT and mTOR proteins in SW620 and RKO cells in a dose-dependent manner without changing the total protein expression levels. Finally, the expression level and phosphorylation level of the AKT protein were in accordance with the immunofluorescence assay results, which also confirmed the inhibitory effects of wogonin on AKT phosphorylation (Figs. 5A and 5B). Therefore, inhibition of phosphorylation in the AKT/mTOR signaling pathway may be one of the mechanisms by which wogonin exerts antitumor effects.

Wogonin Binds Directly to AKT

Next, we used molecular docking to predict the binding targets of wogonin to AKT. Molecular docking results showed that the binding sites of wogonin and AKT included Ser205 and Asp292, and the binding activity was $-$8.8kcal$\cdot$mol${}^{-1}$. This was subsequently visualized in PyMOL (Fig. 6A). In addition, cell lysates CETSA–WB assays were performed to support the direct interaction of wogonin with AKT (Figs. 6B or 6C). Protein extracts from SW620 cells were treated with wogonin (200$\mu$M) or DMSO and subjected to CETSA heat pulse, followed by soluble protein extraction and quantification. The results showed that wogonin exhibited a significant binding effect with AKT at $75^{\circ }$C (Fig. 6B). Then, we further reduced the temperature range, and it was found that wogonin and AKT showed good binding activity at $74^{\circ }$C, $76^{\circ }$C, and $78^{\circ }$C, of which $76^{\circ }$C was the most significant (Fig. 6C). Moreover, the binding ability of wogonin to AKT was dose-dependent at $76^{\circ }$C (Fig. 6D). Finally, we verified the phosphorylation level of Thr308 in SW620 treated with different concentrations of wogonin using WB. However, we found that wogonin had no effect on the phosphorylation level of Thr308 (Fig. S1). Collectively, these findings suggest that wogonin may bind directly to AKT.

Wogonin Inhibits AKT Phosphorylation and Produces Antitumor Effects in Vivo

To estimate the therapeutic potential of wogonin in vivo, we established nude mouse tumor models with SW620 cells. First, SW620 cells were subcutaneously injected into BALB/c nude mice. When the xenografts grew to approximately 100cm³, the nude mice were randomly assigned a drug intervention group. The animals were treated with low-dose wogonin (WL) 25mg/kg once daily, high-dose wogonin (WH) 50mg/kg once daily, and 5-Fu 20mg/kg once every other day. The body weights and tumor sizes were recorded every three days. Figures 7A–7C shows that wogonin treatment significantly reduced tumor size and weight, and the therapeutic effect of the high dose is more significant. From the body weight data, the body weights of mice in the 5-Fu treatment group decreased significantly. In contrast, wogonin intervention did not cause significant changes in the body weights of mice. In addition, high-dose wogonin had a better weight maintenance effect on mice (Fig. S2A). We then performed immunostaining of tumor tissue samples, and the results showed that the expression of Ki67 in tumor tissues from the WH treatment group was significantly downregulated, and the phosphorylation level of AKT was significantly decreased compared with the control group (Figs. 7D and 7E). We also established a subcutaneous transplant tumor model with HT-29 intestinal cancer cells and used wogonin for intervention, which gave the same results as those from the SW620 model. Wogonin inhibited tumor growth without affecting weight loss (Figs. 7F–7H and S2B).

Discussion

Wogonin is a small molecule compound extracted from S. baicalensis, and previous studies have shown that it has certain antitumor effects against a variety of tumor types (Polier et al., 2015; Zhao et al., 2019; Yang et al., 2020). In this research, we observed the anticancer effects of wogonin on CRC both in vitro and in vivo. Our results demonstrated that wogonin has a wide spectrum of effects on CRC cells by inhibiting cancer cell growth and invasion while also inducing CRC cell apoptosis.

Epithelial mesenchymal transformation (EMT) is widely considered an important physiological process that regulates the initial steps of cancer cell invasion and migration (Vucicevic et al., 2014; Bu and Chen, 2017; Zhao et al., 2019). The progression of tumor invasion and metastasis could be determined by monitoring the expression levels of some EMT-related markers. Among them, the expression level of E-cadherin in EMT-induced epithelial cells decreased, while the levels of N-cadherin and vimentin increased (Breyer et al., 2016; Wang et al., 2021). Recent studies have shown that some small molecule compounds that can inhibit cancer cell invasion and metastasis can also regulate the EMT process (Xu et al., 2018; Chen et al., 2020; Liu et al., 2021). Inhibition of the EMT process is one of the most important steps by which drugs inhibit tumor metastasis. Our small molecule data showed that wogonin inhibited the EMT process in CRC cells and prevented CRC cell invasion.

To understand the molecular mechanism of the anticancer effects of wogonin against CRC, we screened its potential pathway with a phospho-antibody array containing 21 tumor-related signaling pathways. Chip screening results showed that the phosphorylation levels of multiple signaling pathways changed to varying degrees after treatment with wogonin. A change of more than two-fold is generally considered meaningful, and the phosphorylation level of AKT was the most significant change. Therefore, we conducted a series of experiments to verify the inhibitory effects of wogonin on AKT phosphorylation, which fully confirmed that wogonin significantly inhibited the phosphorylation level of AKT at Ser473 without affecting the total expression level of AKT in CRC cells. It has been reported that the PI3K/AKT signaling pathways can mediate cell apoptosis, proliferation and metastasis in CRC (Danielsen et al., 2015; Narayanankutty, 2019; Xu et al., 2020). Phosphorylation of AKT is a key process in the activation of this signaling pathway, which in turn affects the activation of downstream signaling pathways. Therefore, finding effective AKT phosphorylation inhibitors is an effective means of cancer treatment. Here, we demonstrated that wogonin could reduce the phosphorylation of AKT1 in CRC cells, revealing for the first time that wogonin could play an antitumor role as a potential inhibitor of AKT phosphorylation. AKT/mTOR signaling is an important event in colorectal carcinogenesis. In addition, it plays significant roles in the metastatic initiation events of CRC (Porta et al., 2014; Narayanankutty, 2019). In cells, mTOR activity is controlled by positive and negative upstream regulators (Hay and Sonenberg, 2004). Positive regulators include growth factors and their receptors and vascular endothelial growth factor receptors and their ligands. The most important negative regulators of mTOR activity are phosphatase and tensin homolog (PTEN). Although there are many regulators, they are all similar in that they transmit signals to mTOR via PI3K/AKT (Shaw et al., 2004; Alzahrani, 2019). Therefore, we also detected changes in the mTOR signaling pathway and found that wogonin affected mTOR activity by inhibiting AKT1 phosphorylation.

Conclusions

In summary, our research revealed that wogonin might be a new AKT phosphorylation inhibitor and has the potential to inhibit cell proliferation and invasion and induce apoptosis in CRC cells. Our data elucidated the role and underlying mechanism of wogonin in CRC inhibition, which is expected to support the use of wogonin as a compound for CRC treatment.

Acknowledgments

This work was supported by the National Natural Science Foundation of China, Nos. 82104466, 82322076; Shanghai Frontier Research Base of Disease and Syndrome Biology of Inflammatory cancer transformation, No. 2021KJ03-12; Shanghai Rising-Star Program, No. 20QA1409300; and the Program for Young Eastern Scholar at Shanghai Institutions of Higher Learning, No. QD2019034. Health Science and Technology project of Shanghai Pudong New Area Health Commission (No. PW2021A-43, China), and Shanghai Pudong New Area Health System discipline leader Training Program (No. PWRd2021-21, China).

ORCID

Yujing Liu  https://orcid.org/0000-0002-9879-4413

Lu Lu  https://orcid.org/0000-0003-2777-5992

Peiqiu Cheng  https://orcid.org/0000-0002-4244-2224

Shengan Zhang  https://orcid.org/0000-0001-6560-8808

Yangxian Xu  https://orcid.org/0000-0001-8738-3291

Dan Hu  https://orcid.org/0000-0002-5331-3276

Guang Ji  https://orcid.org/0000-0003-0842-3676

Hanchen Xu  https://orcid.org/0000-0003-0441-7500

Supplementary Information