Evodiamine Inhibits Gastric Cancer Cell Proliferation via PTEN-Mediated EGF/PI3K Signaling Pathway

Aims In this study, the pharmacological effects and potential molecular mechanisms of evodiamine in treating gastric cancer (GC) were investigated. Methods GC cells lines of AGS and BGC-823 were treated with evodiamine at various concentrations for different times (24, 48, and 72 h). Inhibition of the proliferation of AGS and BGC-823 cells was assessed using a CCK-8 assay. The morphology of gastric cancer cells was detected by high-content screening (HCS). The apoptosis-inducing effect of evodiamine on AGS and BGC-823 cells was detected by flow cytometric analysis. Cell migration and invasion were detected by Transwell assay. The relative mRNA and protein expression levels of PTEN-mediated EGF/PI3K signaling pathways were investigated via RT-qPCR or western blotting, respectively. Results Evodiamine substantially inhibited AGS and BGC-823 cells proliferation in a dose- and time-dependent manner. Flow cytometric analysis revealed that evodiamine could induce apoptosis of AGS and BGC-823 cells in a dose-dependent manner. In addition, evodiamine inhibited AGS and BGC-823 cell migration and invasion. Mechanistically, the results demonstrated that evodiamine promoted the relative mRNA and protein expression of PTEN and decreased expression of EGF, EGFR, PI3K, AKT, p-AKT, and mTOR. Most importantly, evodiamine could effectively increase the mRNA and protein expression of PTEN and decrease the protein expression of EGF/PI3K pathway, indicating that evodiamine downregulated EGF/PI3K through the activation of PTEN pathway. Conclusion Evodiamine inhibited the directional migration and invasion of GC cells by inhibiting PTEN-mediated EGF/PI3K signaling pathway. These findings revealed that evodiamine might serve as a potential candidate for the treatment or prevention of GC.


Introduction
Gastric cancer (GC) is a malignant tumor originating from gastric mucosal epithelium. It is the fifth most common cancer as well as the third most lethal of cancer death worldwide [1]. Eastern Asian countries account for approximately half of gastric carcinoma globally. In view of the high incidence rate, gastric cancer is a major burden of society [2]. In China, the incidence and mortality rate of gastric carcinoma are far higher than the world average level, coupled with complex etiology, low survival rate, and poor prognosis, which has become the main factor restricting the diagnosis and treatment of gastric cancer [3]. erefore, there is a huge demand for promising agents and new therapies for the prevention and treatment of gastric cancer.
Plant-derived compounds are considerable source of anticancer drugs. In recent decades, the number of anticancer compounds has been extracted from natural sources [4]. Natural compounds and their derivatives isolated from Chinese herbal medicines are considered to be potential anticancer drugs and new adjuvants to improve the clinical efficacy of chemotherapeutic agents [5]. Evodiamine is a natural beta-carboline alkaloid extracted from the fruit of Euodia rutaecarpa (Wu-Zhu-Yu in Chinese). Traditionally, evodiamine has been commonly traditionally used to cure headache, amenorrhea, postpartum hemorrhage, and gastrointestinal diseases [6]. e multiple bioactive properties of evodiamine have been widely investigated, including antitumor [7], anti-inflammatory [8], antibacterial [9], neuroprotective [10], vasodilation [11], and antigastrointestinal motility [12]. Among them, the multitargeting molecule effect of evodiamine on the gastrointestinal is attractive. Studies have found that evodiamine has inhibitory effects on SGC-7901 cells, which is associated with apoptosis, autophagy, and cell cycle arrest at the G2/M phase in a dose-dependent manner [13]. Moreover, the potential mechanism is involving the downregulation of survivin and upregulation of caspase-3, -8, and -9 and altering the expression of caspase-3, Bax, and Bcl-2 [14,15]. In addition, evodiamine inhibited the Wnt/β-catenin signaling pathway to inhibit proliferation and stem cell properties of GCSCs and repressed the EMT [16]. e present findings suggest that evodiamine is an effective natural compound for the treatment of gastric cancer. However, the antitumor effect and potential mechanism of evodiamine on gastric cancer cells remain to be further elucidated. Phosphatase and tensin homolog (PTEN), a tumorsuppressor gene located on chromosome 10, is one of the key factors in the diagnosis of gastric cancer prognosis [17,18]. Studies have shown that PTEN gene inactivation is closely related to cell apoptosis, cell proliferation, cell migration, and cell metastasis, resulting in association with the progression and incidence rate of gastric cancer [19,20]. Conversely, patients with lower expression of PTEN protein have high probability in distant metastasis and advanced clinical stage of gastric cancer than in adjacent nontumor tissues [21]. e upregulation of epidermal growth factor (EGF), its receptor (EGFR), and ErbB2 protein in gastric mucosa plays an important role in the occurrence and development of gastric cancer [22]. EGF stimulates cell proliferation and migration by interacting with receptors and triggers epithelial cell signaling [23].
Phosphatidylinositol-3-kinase (PI3K) signaling pathway plays an important role in various aspects in terms of cell growth and cell survival by regulating cell cycle, differentiation, transcription, and apoptosis [24]. e imbalance of PI3K class I signaling pathway, whether through gene amplification or mutation, is closely related to the occurrence and development of a variety of cancers [25]. e genetic changes of proteins in this signaling pathway include PTEN and Akt [26]. e imbalance of PI3K pathway can induce a variety of downstream effectors, including mammalian target of rapamycin (mTOR). mTOR is a member of the PI3K-related kinase (PIKK) family. Its catalytic kinase domain is highly homologous to that of PI3K [27]. PI3K/ mTOR pathway is one of the most common activated signaling pathways in human tumors [28]. Previous study indicated that evodiamine-induced PC cell apoptosis by inhibiting PI3K/AKT and mitogen-activated protein kinase/ ERK and inhibiting the phosphorylation of signal transducer and activator of transcription activator 3 in human pancreatic cancer cells to inhibit autophagy [29], suggesting that evodiamine may be considered as a novel pancreatic cancer treatment. However, whether evodiamine plays an antigastric cancer effect by regulating EGF/PI3K signaling pathways remains to be further investigated. us, this study aimed to explore the effect of evodiamine on EGF/PI3K signaling pathways, which contributed to better understanding of the anticancer mechanisms of evodiamine.

Cell
Culture. Human AGS and BGC-823 cell lines were obtained from the Cell Resource Centre (IBMS, CAMS/ PUMC, Beijing, China). Cells were cultured in RPMI-1640 medium supplement 10% fetal bovine serum (FBS), containing 100 IU/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at normal culture conditions (37°C in a saturated humidity atmosphere with 95% air and 5% CO 2 ).  (24,48, and 72 μM). After drug intervention, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 30 min. e absorbance was measured at 450 nm using a Synergy ™ H1 instrument (BioTek, American). e cytotoxicity of evodiamine was measured, and rate of cell growth inhibition was calculated in accordance with the instructions. Each experiment was performed at least three times independently under each corresponding experimental condition.

Flow Cytometric Analysis of Cell Apoptosis.
Human AGS and BGC-823 cells were treated with 6.25, 12.5, and 25 μM evodiamine for 24 h. Next, the cells were collected, washed, and resuspended in PBS. e apoptotic cells were counted by Annexin V-FITC and 7-amino-actinomycin (Apoptosis detection kit; BD Biosciences, San Jose, CA, USA) double staining using the Annexin V-FITC Apoptosis Detection kit according to the manufacturer's instructions. e early apoptotic cells, late apoptotic cells, and necrotic cell death were detected by Annexin V positive, Annexin V and 7-AAD positive, and 7-AAD positive, respectively. e cells were washed twice with cold PBS and then resuspended in annexin-V-binding buffer at a concentration of 1 × 10 6 cells/ mL. Transfer 100 μL of the solution to a 5 mL culture tube. e suspension was stained with 5 μL of annexin V-FITC and 5 μL of 7-AAD and incubated for 15 min at room temperature in the dark condition. en, 400 μL of 1 × binding buffer was added to each tube. Cell apoptosis rate was detected using a cytofluorimeter and analyzed by FACScan and CellQuest software (Becton, Dickinson).

High-Content Analysis.
e nuclear morphology and cell proliferation of gastric cancer cells were detected by a High-Content System ( ermo Scientific, MA, USA). e 2 Evidence-Based Complementary and Alternative Medicine localization, cell morphology, and quantitative analysis of AGS and BGC-823 cells were investigated by three fluorescent dyes, including Hoechst 33342 (H3570, Invitrogen), Calcein AM (C3099, Invitrogen), and EthD-1 (L3224, Invitrogen). In HCS system, cell health profiling assay module was chosen and corresponding wavelength channels were set to catch fluorescence images. e parameters and formats of the measurements were similar to our previous study [30]. Finally, an Array Scan XTI was performed to quantify the mean fluorescence intensity of gastric cancer cells.
2.6. Wound-Healing Assay. Wound-healing assay is performed to measure cell migration and repair ability. AGS and BGC-823 cells were seeded into 6-well plates. Cells were scraped off with the end of a 10 μL pipette tip while grew to a certain density. en, the detached cells were removed by washing the plate with PBS; the remaining cells were cultured in the medium containing 0, 12.5, and 25 μM evodiamine solution for another 24 h. Cell migration and repair ability was detected under a 40x magnification phase contrast microscope after 0 and 24 h. AGS and BGC-823 cells migrated to the scratch area in 6 random fields were quantitatively analyzed by computer-assisted microscope.

Transwell Migration Assay.
e migration ability of AGS and BGC-823 cells were evaluated by Transwell assay, which examined the ability of the cells to move under the membrane filter. Cell migration ability was conducted using Transwell polyester membrane filter inserts with 8 μm pores (Corning Inc., Corning, NY, USA). Cells were treated with 0, 12.5, and 25 μM evodiamine solution for 24 h. Cells on the top of the Transwell chambers were removed, and the migrated or invasion cells on the bottoms of the membranes were fixed with 4% paraformaldehyde, followed by staining with 0.1% crystal violet (Beyotime, Haimen, China). Migratory cells were imaged and counted under a light microscope (Olympus, Tokyo, Japan). Finally, three random fields of view for each transmembrane were analyzed and averaged.  e antibody was detected by enhanced chemiluminescence. Data were normalized using β-actin as an endogenous control.

Data and Statistical
Analysis. Data are presented as means ± standard deviation (SD) from at least three separate experiments.
e IC 50 curves were estimated by plotting percentage of viability from the triplicate treatment versus concentration. e IC 50 value was defined and calculated using the 4-full-parameter equation as defined by the GraFit software version 5.0.4 from Erithacus Software (Surrey, UK). Statistical differences of the data were performed using an unpaired Student's t-test and ANOVA. GraphPad Prism Software (version 8.2.0) was used for data analysis. Statistical significance was defined as P < 0.05.

Evodiamine Suppresses Wound-Healing of AGS and BGC-823 Cells.
To explore the antimetastasis potential of evodiamine on gastric cancer cells, wound-healing assay was performed to evaluate the ability of evodiamine on cell migration.
At 0 h, a wound-healing was drawn between AGS and BGC-823 cells with a 10 μL tip, and the effects of different concentrations of evodiamine on wound-healing were observed. e wound-healing migration assay indicated that the wound-healing ability of AGS cells and BGC-823 cells treated with different concentrations of evodiamine (6.25, 12.5, and 25 μM) for 12, 24, and 48 h was significantly decreased in a dose-dependent manner relative to untreated controls ( Figure 2). With the prolongation of time, the cells have different degrees of healing and migration tendency.

Evodiamine Suppresses AGS and BGC-823 Cell Migration.
To explore the antimetastasis potential of evodiamine on gastric cancer cells, cell invasion was performed to evaluate the ability of evodiamine on cell migration. e results showed that the invasion potential of AGS and BGC-823 cells in Transwell migration assay was effectively inhibited by evodiamine treatment (Figure 3(a)). Specifically, compared with the control group, 6.25 μM, 12.5 μM, and 25 μM evodiamine groups could significantly reduce the migration rate of AGS and BGC-823 cells (P < 0.01), especially 12.5 μM and 25 μM groups (Figure 3(b)). e results showed that evodiamine significantly inhibited AGS and BGC-823 cell migration in a dose-dependent manner in the cell invasion assay ( Figure 3). erefore, both the wound-healing and cell invasion assays suggested that evodiamine could inhibit the motility of AGS and BGC-823 cells.

Evodiamine Inhibits Gastric Cancer Cell Proliferation.
In order to intuitively display the effect of evodiamine on the morphology and proliferation of gastric cancer cells, cell proliferation, viability, and morphology of AGS and BGC-823 cells were analyzed by HCA. Cell nucleus (blue fluorescence), living cells (green fluorescence), and dead cells (red fluorescence) were labeled with Hoechst 33342, Calcein AM, and EthD-1, respectively. Representative photomicrographs for HCS image analysis of evodiamine on AGS   ). Evodiamine can significantly reduce the proliferation rate of gastric cancer cells, causing the nucleus fragmentation, cytoplasmic dissolution, and cell deformation of AGS (Figure 4(a)) and BGC-823 cells (Figure 4(b)). ese results indicate that evodiamine could promote the proliferation of gastric cancer cells, destroy the normal morphology of gastric cancer cells, and produce cytotoxicity on gastric cancer cells.

Effects of Evodiamine on the Relative mRNA Expression of PTEN-Mediated EGF/PI3K Signaling Pathways.
PTEN expression levels may be an important molecular issue in the occurrence and development of gastric cancer. Also, it can be regarded as a molecular marker as well as a reliable prognostic indicator of gastric cancer [31,32]. To investigate the potential mechanisms by which evodiamine inhibits the relative mRNA expression of PTEN-mediated EGF/PI3K signaling pathways, AGS and BGC-823 cells were treated with different concentration of evodiamine (6.25, 12.5, and 25 μM) for 24 h. Next, the mRNA expression levels of PTENmediated EGF/PI3K pathway (i.e., PTEN, EGF, EGFR, AKT, mTOR, Beclin-1, Bcl-2, Bax, caspase-3, and caspase-8) were measured by RT-PCR. As shown in Figure 6, evodiamine could significantly increase the relative mRNA expression of PTEN, Beclin-1, Bax, caspase-3, and caspase-8 (P < 0.01), while dramatically decrease the relative mRNA expression of EGF, EGFR, AKT, mTOR, and Bcl-2 (P < 0.01). e results indicated that evodiamine dramatically increased the expression levels of all apoptosis and autophagy-related genes to values found in AGS and BGC-823 cells. us, evodiamine could induce apoptosis of gastric cancer cells, indicating that it might have the potential to treat gastric cancer.

Discussion
e present study aimed to investigate the role of evodiamine, a natural active ingredient of the Traditional Chinese medicine Evodia rutaecarpa, in gastric cancer cells. e results revealed that evodiamine inhibited the proliferation of AGS and BGC-823 cells, destroying the structure and function of gastric cancer cells.
e present study also provided the pharmacodynamics and mechanism evidence that the evodiamine has potential therapeutic effects of antigastric cancer in vitro. Based on the effects of promoting apoptosis and inhibiting migration of gastric cancer cells in vitro, evodiamine might serve as the active component of Evodia rutaecarpa. Furthermore, evodiamine inhibits gastric cancer cells' directional migration by inhibiting PTEN-mediated EGF/PI3K signaling pathways. is study explored the effect of evodiamine on the proliferation and migration of gastric cancer cells, indicating that evodiamine could inhibit the proliferation and metastasis of gastric cancer in vitro.
Numerous studies have shown that evodiamine has anticancer activity in various tumor cells and has inhibitory effect on gastric cancer [16], lung cancer [33], breast cancer cells [34], liver cancer [35], pancreatic cancer cells [36], ovarian cancer [37], chronic myeloid leukemia cells [38], and bladder cancer [5]. In this study, CCK-8 assay was used to assess the gastric cancer cells' viability in vitro. e results indicated that evodiamine could significantly inhibit AGS and BGC-823 cells' viability compared with the control groups. To directly display the effects of evodiamine on cell viability, HCS was used to qualitatively and quantitatively evaluate cell proliferation and morphology of gastric cancer cells. e results showed that evodiamine could significantly decrease the Hoechst and Calcein AM fluorescence intensity but substantially increase the EthD-1 fluorescence intensity of AGS and BGC-823 cells, indicating cell death compared with the control group. Notably, the nucleus and cytoplasm of AGS and BGC-823 cells were broken with incomplete morphology, indicating toxic effect of evodiamine on gastric cancer cells. It has been shown that inducing apoptosis is the key factor responsible for the anticancer role of evodiamine, which can alter the balance of relative gene and protein expression between proapoptotic Bax and antiapoptotic Bcl-2 family members and induce cell apoptosis by activating either effector caspase-3 or initiator caspase-8 [39]. In the present study, it was demonstrated that gastric cancer cells treated with evodiamine exhibited a concentration-dependent increase in the number of apoptotic and necrotic cells. Annexin V staining directly showed that it could induce apoptosis of gastric cancer cells. e results of qRT-PCR confirmed that 25 μM evodiamine could upregulate the expression of proapoptotic Bax and downregulate the antiapoptotic Bcl-2 in gastric cancer cells, resulting in the upregulation of Bax/Bcl-2 ratio. In addition, it activates caspase-3, caspase-8, and caspase-9 genes in AGS and BGC cells, simultaneously. ese results suggested that endogenous and exogenous apoptotic pathways are involved in evodiamine-induced apoptosis of gastric cancer cells.
Previous studies have reported a close association between apoptosis and tumor development [40,41]. erefore, the exploration of the mechanisms underlying drug-induced tumor cell apoptosis is essential for improving drug efficacy and the development of novel anticancer drugs. PTEN is one of the most common tumor-suppressor genes in cancer. PTEN deficiency showed overactivation of PI3K/AKT pathway activation markers and upregulation of mTOR [42]. In addition, previous study investigated the effects of evodiamine on human pancreatic cancer cell, and the results indicated that evodiamine-induced pancreatic cancer cell apoptosis by inhibiting PI3K/AKT and mitogen-activated protein kinase/ ERK [29]. However, whether evodiamine regulates gastric cancer cell apoptosis through regulating PI3K/Akt and its upstream and downstream pathways has not been further explored. To clarify the potential action mechanisms of evodiamine, the mRNA and protein expression levels of PTEN-mediated EGF/PI3K signaling pathways were measured. As downregulation of PTEN decreases anticancer effects in various tumors, we speculated that the PTENmediated EGF/PI3K signaling pathways is responsible for the gastric cancer cell apoptosis and cell migration. Consistent with the previous report, the mRNA and protein expression levels of PTEN were markedly increased in the evodiamine treatment, but the levels of EGF, EGFR, ErbB2, PI3K, AKT, and mTOR were significantly decreased compared to the control group. Furthermore, the expression of PTEN was substantially decreased, while the AGS and BGC-823 cells were pretreated with VO-OHpic trihydrate. e mRNA and protein expression levels of EGF/PI3K signaling pathways increased in various degrees. However, evodiamine could inhibit this increase. ese results suggest that evodiamine may play a role in the treatment of gastric cancer by regulating PTEN-mediated EGF/PI3K signaling pathways.
is study explored the anticancer effects and potential mechanism of evodiamine on gastric cancer cells in vitro. However, this study still has some limitations. Firstly, this study only examined the effects of evodiamine on the cell proliferation, apoptosis, and migration of AGS and BGC-823 gastric cancer cells. However, the effect of evodiamine on other gastric cancer cells, such as MKN-45, MKN-28, MGC-803, and HGC-27, needed further investigated. Secondly, this study only explored the anticancer effect of evodiamine in vitro, but not from the overall animal level and clinical patients in vivo, which led to the validation of conclusion being weaken. Lastly, there are many active compounds in Euodia rutaecarpa. Whether other compounds, such as rutaecarpine and dehydroevodiamine, have antigastric cancer activity remains to be further studied.

Conclusions
e results of the present study indicated that evodiamineinduced AGS and BGC-823 cell apoptosis may be associated with increased expression levels of PTEN and decreased Evidence-Based Complementary and Alternative Medicine expression levels of EGF/PI3K signaling pathway. us, evodiamine may present a potential and novel preventive candidate for gastric cancer in vitro. In addition, further studies are required to investigate the mechanism of other alkaloids in Evodia rutaecarpa for the treatment of gastric cancer in vivo.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
As this study does not involve animal and patient experiments, the ethical approval and consent to participate are not applicable.