Cisplatin resistance in gastric cancer cells is involved with GPR30‐mediated epithelial‐mesenchymal transition

Abstract Cisplatin is the major chemotherapeutic drug in gastric cancer, particularly in treating advanced gastric cancer. Tumour cells often develop resistance to chemotherapeutic drugs, which seriously affects the efficacy of chemotherapy. GPR30 is a novel oestrogen receptor that is involved in the invasion, metastasis and drug resistance of many tumours. Targeting GPR30 has been shown to increase the drug sensitivity of breast cancer cells. However, few studies have investigated the role of GPR30 in gastric cancer. Epithelial‐mesenchymal transition (EMT) has been shown to be associated with the development of chemotherapeutic drug resistance. In this study, we demonstrated that GPR30 is involved in cisplatin resistance by promoting EMT in gastric cancer. GPR30 knockdown resulted in increased sensitivity of different gastric cancer (GC) cells to cisplatin and alterations in the epithelial/mesenchymal markers. Furthermore, G15 significantly enhanced the cisplatin sensitivity of GC cells while G1 inhibited this phenomenon. In addition, EMT occurred when AGS and BGC‐823 were treated with cisplatin. Down‐regulation of GPR30 with G15 inhibited this transformation, while G1 promoted it. Taken together, these results revealed the role of GPR30 in the formation of cisplatin resistance, suggesting that targeting GPR30 signalling may be a potential strategy for improving the efficacy of chemotherapy in gastric cancer.

oestrogen receptor that is involved in the invasion, metastasis and drug resistance of many tumours. Targeting GPR30 has been shown to increase the drug sensitivity of breast cancer cells. However, few studies have investigated the role of GPR30 in gastric cancer. Epithelial-mesenchymal transition (EMT) has been shown to be associated with the development of chemotherapeutic drug resistance. In this study, we demonstrated that GPR30 is involved in cisplatin resistance by promoting EMT in gastric cancer. GPR30 knockdown resulted in increased sensitivity of different gastric cancer (GC) cells to cisplatin and alterations in the epithelial/mesenchymal markers. Furthermore, G15 significantly enhanced the cisplatin sensitivity of GC cells while G1 inhibited this phenomenon. In addition, EMT occurred when AGS and BGC-823 were treated with cisplatin. Down-regulation of GPR30 with G15 inhibited this transformation, while G1 promoted it. Taken together, these results revealed the role of GPR30 in the formation of cisplatin resistance, suggesting that targeting GPR30 signalling may be a potential strategy for improving the efficacy of chemotherapy in gastric cancer.

| INTRODUC TI ON
Gastric cancer (GC) is the fifth most common cancer in the world and the third leading cause of cancer deaths. It accounts for 6.8% of the total cancer incidence and 8.8% of the total mortality rate worldwide, thus posing a serious threat to human health. 1,2 Due to the lack of obvious clinical symptoms in the early stages of gastric cancer, patients with GC are usually diagnosed only at the advanced stage, resulting in a poor prognosis. Although GC treatment includes surgery, chemotherapy and progress has been made in targeted therapy in recent years, the efficacy has not yet reached expectations. 3 Chemotherapy plays a very important role in the treatment of GC, especially advanced GC. Platinum drugs (cisplatin and oxaliplatin) are the first-line treatment in chemotherapy of GC. Cisplatin-based treatment has been proved to effectively improve the survival rate of patients with advanced GC. 4,5 Nevertheless, with the widespread use of chemotherapeutic drugs, tumour cells often develop single or multiple drug resistance (MDR) through various mechanisms, which has become a major obstacle in GC chemotherapy. 6,7 Although some patients are initially sensitive to chemotherapy, almost all patients with GC will develop resistance and recurrence with the continued use of chemotherapeutic drugs. 8 Therefore, investigating the potential mechanism of chemoresistance is of clinical significance.
The mechanism of resistance development is very complex, and it involves multiple protein-coding genes and multiple signalling pathways. Epithelial-mesenchymal transition (EMT) is one of the most widely studied mechanisms not only in GC 9 but also in pancreatic cancer, 10 breast cancer, 11 and lung cancer. 12 EMT is a reversible process in which epithelial cells lose polarity and cell-cell adhesion characteristics and acquire mesenchymal cell characteristics. 13 EMTexpressing cancer cells showed decreased expression of epithelial cell markers such as E-cadherin and ZO-1, while expression of mesenchymal cell markers such as vimentin and N-cadherin increased.
Although there is increasing evidence that EMT is closely related to the development of drug resistance in gastric cancer cells, its underlying mechanisms have not yet been fully elucidated. [14][15][16] To date, many studies have investigated the role of oestrogen receptor (ER) in gastric cancer, and the possible mechanisms of these effects and the clinical relevance of dysregulated ER in GC.
However, the results of these studies are conflicting and controversial. [17][18][19] GPR30 is a seven-transmembrane domain protein that not only structurally differs from classical oestrogen receptors (ERα and ERβ) but also significantly differs in terms of its mechanism and effect. 20,21 Binding of GPR30 to its ligand is known to cause rapid activation of many intracellular signal transduction pathways such as an increase in adenylate cyclase (cAMP), mobilization of intracellular calcium stores, transactivation of EGFR and activation of downstream signal transduction pathways such as PI3K/AKT and ERK1/2. These signalling pathways play an important role in the proliferation, metastasis and formation of drug resistance of human tumours. 22,23 G1 (GPR30 agonist) and G15 (GPR30 inhibitor) are quinoline derivatives that are widely used to activate or block GPR30 signalling and are used to explore the potential of GPR30 as a therapeutic target. 24,25 A recent study showed that targeting GPR30 reduced metastasis and drug resistance in breast cancer. 26 However, few studies have investigated the effect of targeting GPR30 in gastric cancer. Analysis of the KMPLOT database shows that high GPR30 expression leads to poor prognosis in gastric cancer, implicating the potential role of GPR30 in gastric cancer. 27 In this study, we aimed to investigate the role of GPR30 in the formation of cisplatin resistance in GC cells and to explore the potential of GPR30 as a therapeutic target for improving the efficacy of chemotherapy in GC.

| Cell culture and reagents
The GC cell lines AGS, BGC823 were obtained from the Cell Bank of Chinese Academy of Science. All the cells were cultured in RPMI-1640 medium containing 10% foetal bovine serum (FBS) at 37°C with 5% CO 2 . Foetal bovine serum (FBS) and RPMI-1640 medium were purchased from Gibco (Gibco). G1, G15 and cisplatin were obtained from Sigma-Aldrich.

| siRNA transfection
According to the manufacturer's instructions, GC cells were seeded into the wells of six-well plates and cultured until approximately 80% confluence, and they were then transfected with GPR30 siRNA Antisense: 5′-ACGUGACACGUUCGGAGAAdTdT-3′.

| CCK-8 cell viability experiment
Gastric cancer cells were seeded into 96-well plates at a density of 3 × 10 3 cells/well and incubated at 37°C for 24 hours. They were then cultured in complete medium containing serially diluted drugs for 48 hours. Finally, 10 µL of CCK-8 was added to each well (Dojindo), and the plates were placed on a microplate reader (MRX II; Dynex) to evaluate the absorbance at 450 nm after incubation for 2 hours at 37°C. Tween (TBST). The blot was visualized using a chemiluminescence kit (GE Healthcare), and protein expression was determined by a Syngene gel imaging system.

| EdU assay
The inhibition rate of cell proliferation was determined using a Click-iT EdU Imaging Kit (Invitrogen), according to the manufacturer's instructions.

| Immunofluorescence
Gastric cancer cells were fixed with 4% formaldehyde for 15 minutes after being treated with drug for 48 hours, washed with phosphate-buffered saline (PBS) and incubated with 5% BSA for 30 minutes at room temperature. Then, the cells were incubated with primary antibodies against E-cadherin and vimentin (Abcam) overnight at 4°C. After washing with PBS, the cells were incubated with secondary antibodies (Abcam) for 2 hours. The cells were then incubated with 4,6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 2 minutes at room temperature. Finally, the cells were washed with PBS and then observed with an inverted fluorescence microscope (Olympus).

| Statistical analysis
Each of the above experiments was performed in triplicate and repeated thrice. Statistical analysis was performed using SPSS 23.0 software. Data were expressed as the means ± SD of three independent experiments. Student's t test was used for comparison between the two groups. A P value of <.05 was considered to represent a statistically significant difference.

| GPR30 participates in cisplatin resistance in GC cells via EMT
We found that GPR30 expression displayed increasion in GC cells after cisplatin treatment( Figure 1A). However, When the cells were cotransfected with Twist plasmid and GPR30 siRNA, the effect of GPR30 knockdown on cisplatin sensitivity disappeared ( Figure S1). These data preliminarily demonstrated that GPR30 is involved in the cisplatin resistance of GC cells by promoting EMT.

| G15 increases the sensitivity of GC cells to cisplatin
Gastric cancer cells were treated with different concentrations of G15 (0-20 µmol/L) and their cell viability was measured using the CCK-8 assay. We selected the highest G15 concentration (2.5 µmol/L) that did not affect cell viability for the next experiment ( Figure 2A,B). To investigate the toxic effects of cisplatin and G15 on GC cells, we used the CCK-8 and EdU staining assays to determine the viability and proliferation of GC cells. G15 was found to significantly increase the cisplatin sensitivity of AGS and BGC-823 ( Figure 2C,D) and inhibit their DNA copies ( Figure 2E,F). Western blot analysis was carried out to determine whether G15 effectively inhibited the GPR30 expression. The results indicated that GPR30 expression was significantly inhibited by G15 ( Figure 2G,H), suggesting that G15 can increase the sensitivity of GC cells to cisplatin by inhibiting GPR30.

| G15 reverses cisplatin-induced EMT by inhibiting GPR30
To verify that G15 functions via GPR30, we knocked out GPR30 using siRNA and used CCK-8 to measure the viability of siRNAtransfected GC cells treated with cisplatin alone or in combination with G15. The results showed that GPR30 knockdown did not significantly affect the G15-induced cisplatin sensitivity of gastric cancer cells (Figure 4A,B). Results of the EdU incorporation assay also showed consistent results ( Figure 4C,D). Western blot analysis showed that GPR30 expression was significantly inhibited when the cells were transfected with GPR30 siRNA (Figure 4E,F). The above results indicate that G15 increases cisplatin sensitivity by inhibiting GPR30, which in turn reverses EMT.

| G1 activates GPR30, promoting EMT and leading to cisplatin resistance
To further clarify the role of GPR30 in cisplatin-induced EMT, we used the GPR30 agonist G1 to activate GPR30 to amplify this effect. First, we used the CCK-8 assay to determine the effect of different concentrations (0-200 nmol/L) of G1 on the viability of GC cells. Then, we selected the highest concentration (50 nmol/L) of G1 that did not affect cell viability for the subsequent experiment ( Figure 5A,B).  Figure 5G). Immunofluorescence staining also showed consistent results ( Figure 5H-J). These data indicate that G1 promotes EMT by activating GPR30, leading to cisplatin resistance in GC cells.
In addition, based on the KMPLOT database, we found that a high expression of GPR30 in GC can lead to poor prognosis ( Figure 6A,B), suggesting that GPR30 may play a role in tumorigenesis in GC.

| D ISCUSS I ON
Cisplatin has been clinically used as a first-line chemotherapeutic drug for more than 30 years. Cisplatin alone or in combination with other drugs has been shown to be effective in relieving symptoms, improving the quality of life and improving survival in patients with F I G U R E 3 G15 reversed EMT in epithelial GC cells. A-C, Western blot analysis of E-cadherin and vimentin in the AGS and BGC-823 cell lines treated with cisplatin alone or in combination with G15 for 48 h compared with the control. *P < .05, **P < .01 and ***P < .001. D, Immunofluorescence analysis of E-cadherin and vimentin in the AGS and BGC-823 cells treated with cisplatin alone or in combination with G15 for 48 h compared with the control GC. 28 Tumour cells develop resistance to chemotherapeutic drugs including cisplatin via a variety of mechanisms, thereby greatly limiting their therapeutic potential. Therefore, it is extremely important to study the mechanisms leading to chemoresistance. Studies have reported that cisplatin is capable of inducing cell apoptosis, which is caused by cross-linking of intracellular DNA and DNA damage leading to regression of tumours; however, apoptosis can also lead to increased expression of EMT-inducible factors (such as Twist and Snail) and treatment failure. 29 These findings indicate that the formation of cisplatin resistance is associated with EMT. Numerous studies have shown that EMT plays an important role in the progression, invasion, metastasis and drug resistance of a variety of cancers including GC. [9][10][11][12] In this study, E-cadherin expression was down-reg- Furthermore, our data show that G15 reverses cisplatin-induced EMT in AGS and BGC-823 cells, while G1 promotes it. These findings reveal that GPR30 is involved in cisplatin resistance in GC by promoting EMT.
In conclusion, the results of this study show that GPR30 plays a non-negligible role in EMT and cisplatin resistance in GC. Therefore, treatment with targeted GPR30 is a potential strategy to improve the efficacy of chemotherapy in GC. In the future, animal experiments and clinical analyses should be conducted to validate the conclusions of this study.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

AUTH O R S ' CO NTR I B UTI O N S
CXD and CW conceived the research idea; XZY, SJC and LH performed the experiments; WYP, NYX, CSQ, HC and WLJ analysed the data; WXF wrote the manuscript. All authors read and approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.