Sulfasalazine, a potent suppressor of gastric cancer proliferation and metastasis by inhibition of xCT: Conventional drug in new use

Abstract The aim of this study was to explore the role of sulfasalazine on proliferation and metastasis in gastric cancer by inhibition of xCT. The relationships between clinical characteristics and xCT expression were analysed. An immunohistochemical staining assay and Western blot were performed among gastric cancers and normal gastric tissues. qPCR and Western blot were also used to evaluate the mRNA and protein expression in the normal gastric cell and eight gastric cancer cells, respectively. CCK‐8 and colony formation assays were used to evaluate the effect of sulfasalazine on the proliferation and colony formation ability of three gastric cancers. The effect of sulfasalazine on the migration and invasion abilities of three cancer cells was assessed by the Transwell assay. xCT protein is up‐regulated in gastric cancer specimens and cells. Three gastric cancer cells with high, medium and low expression of xCT were selected for the following analyses. CCK‐8 assays revealed that sulfasalazine could attenuate the proliferation of HGC‐27 and AGS. Also, the colony formation assay revealed that sulfasalazine might attenuate the colony formation ability in HGC‐27 and AGS cells. Plus, the Transwell assays demonstrated that sulfasalazine might attenuate the migration and invasion abilities in HGC‐27 and AGS cells. In conclusion, higher expression of xCT is associated with advanced tumour stage and poor overall survival of gastric cancer. Sulfasalazine can attenuate the proliferation, colony formation, metastasis and invasion of gastric cancer in vitro. Further study is required to validate our findings.

standardized D2 lymphadenectomy. 5 However, advanced gastric cancers with distant metastasis are usually incurable. 6 Therefore, alternative therapeutic options are usually and urgently needed. System x c − has been reported to play an essential role in capturing cysteine from the extracellular environment. The cysteine is the basic raw material for intercellular glutathione synthesis. xCT plays a predominant role in the System x c − ; therefore, the intracellular glutathione levels are determined by the expression and function of xCT. Accumulated evidence has demonstrated that xCT plays a crucial role in many cancers, like bladder cancer, 7 thyroid cancer, 8 triple-negative breast cancer 9 and prostate cancer. 10 However, the role of the xCT in gastric cancer has not been well elucidated yet.
Sulfasalazine, a conventional drug, which is widely utilized in treating inflammatory diseases and rheumatoid arthritis, can also inhibit the expression and function of xCT. 10 Kim et al 11  In this study, the mRNA expression and protein expression of xCT in human gastric cancer tissues and normal gastric tissues were evaluated. Also, the mRNA and protein expression of xCT in eight different gastric cancer cell lines was evaluated. Then, we utilized a conventional drug, sulfasalazine, to attenuate the expression of xCT to evaluate the effect of sulfasalazine on proliferation, colony formation, metastasis and invasion of gastric cancer.

| Clinical specimens
All gastric tissue specimens were obtained from the First Affiliated Hospital of Fujian Medical University. Informed consents were conducted, and written consents were obtained before the operation.

| Immunohistochemistry assay
A total of 136 patients with gastric adenocarcinoma who underwent surgery and pathologically confirmed at the First Affiliated Hospital of Fujian Medical University were included in this study.
Immunohistochemistry assay was performed to validate the expression of xCT between gastric cancer and normal gastric tissues.
Briefly, formalin-fixed and paraffin-embedded gastric cancer tumour specimens and normal gastric tissues were prepared and sectioned.

| Cell proliferation assay
CCK-8 (Dojindo) was utilized to evaluate cell proliferation. Cells were seeded in a 96-well plate. The density is 1000 cells/well. Cells were then incubated at 37°C, 5% CO 2 . 100 μL of CCK-8 serum-free medium was added at 24, 48, 72 and 96 hours after discarding the culture solution. We estimated cell proliferation by using a microplate reader (BioTek) after 1 hour of incubation.

| Colony formation assay
We seeded the cells (500/well) in a 6-cm plate. We utilized a culture medium containing 10% FBS, and cells were cultured for 14 days.
The cells were infiltrated with methanol and stained with crystal violet after discarding the culture solution for 10 minutes. After that, cells were washed with water and then dried and counted.

| Cell migration and invasion assay
Gastric cancer cells were cultured in a serum-free medium, and then, we utilized 4 × 10 4 gastric cancer cells in the migration assay. After that, we discarded the medium and washed the well by utilizing calcium-free PBS. Then, we utilized methanol to fix the cells for 30 minutes and stain the cells with 0.1% crystal violet for 20 minutes.
We gently wiped off the un-migrated cells in the upper chamber with a cotton swab. Finally, these un-migrated cells were counted under the microscope.

| Statistical analysis
We utilized GraphPad Prism 7 software to perform statistical analyses. The data were presented as the means ± standard deviation (SD) and analysed by one-way ANOVA or Student's t test. P < .05 was considered to be statistically significant.

| Clinical characteristics and the differentially expressed xCT in gastric cancer
The clinicopathological features of gastric cancer patients with differently expressed xCT are demonstrated in Table 1. There was no significant difference in age, gender, location, tumour size, the grade of differentiation, lymphatic metastasis, vascular invasion and nerve invasion between the low xCT expression and high xCT expression groups. Nevertheless, in terms of invasion depth, the high xCT expression group has more T3 and T4 patients (P = .024). Plus, when it comes to the pTNM stage, the high xCT expression group has more stage III and stage IV patients than the low xCT expression group (P = .027).

| The immunohistochemical staining confirms the expression of xCT
The haematoxylin-eosin (HE) staining demonstrates the morphological differences between gastric cancers and normal gastric tissues.
An immunohistochemical staining assay was performed among gastric cancers and normal gastric tissues. The results demonstrated that xCT is differentially expressed between gastric cancers and normal gastric tissues. The immunohistochemical staining results demonstrated that the expression of xCT was up-regulated in gastric cancer compared with normal gastric tissues ( Figure 1A).

| xCT protein is up-regulated in gastric cancer specimen
A total of eight paired gastric cancer tissues and corresponding nor-

| The effect of sulfasalazine on the proliferation of three cancer cells by CCK-8 assay
We

| The effect of sulfasalazine on colony formation ability of three cancer cells by colony formation assay
We utilized colony formation assays to evaluate the impact of the sulfasalazine on the proliferation of three cancer cells. The con-

| The effect of sulfasalazine on migration and invasion abilities of three cancer cells by Transwell assay
We utilized the Transwell assay to evaluate the impact of the sul-

| D ISCUSS I ON
Gastric cancer remains the fourth most prevalent malignant cancer all over the world and the second leading cause of cancer-specific death worldwide survival. 16,17 The incidence of gastric cancer is high in China, with approximately 400 000 newly diagnosed cases every year. 18 Nowadays, surgical resection remains the mainstay for gastric cancer. Nevertheless, when distant metastasis appears, chemotherapy is required. However, chemotherapy's side effects usually make its administration difficult in some patients, especially those geriatric patients, with comorbidities or decreased general status. 19 Therefore, alternative therapeutic options are usually and urgently needed.
In this study, we thoroughly compared In this study, we utilized a conventional drug, sulfasalazine, to evaluate the effects of attenuating the proliferation, colony formation, metastasis and invasion of gastric cancer. Three gastric cancer cells with high, medium and low expression of xCT were selected for the following analyses. CCK-8 assays revealed that sulfasalazine could attenuate the proliferation of HGC-27 and AGS.
The colony formation assay also revealed that sulfasalazine might attenuate the colony formation ability in HGC-27 and AGS cells. In conclusion, higher expression of xCT is associated with advanced tumour stage and poor overall survival of gastric cancer.
Sulfasalazine can attenuate the proliferation, colony formation, metastasis and invasion of gastric cancer in vitro. Further study is required to validate our findings.

This study was funded by the Foundation of Fujian Provincial
Department of Finance (Grant number: 2019B032).

CO N FLI C T O F I NTE R E S T
The authors declared that they have no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study