2. Patients and Methods
2.1. Patients
110 patients newly diagnosed with gastric adenocarcinomas were recruited in our study. They were diagnosed and received treatment from January 1, 2008 to December 31, 2016 in the Department of Oncology, the First Affiliated Hospital of Xi’an Jiaotong University. All the GC patients received examination and treatment following the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology. Follow-up was performed 3 times per year for the first 5 years, and annually thereafter. The project was terminated on December 31, 2016.
2.2. Instruments
During treatment and follow-up, assessment of the mood status among these patients was performed by physicians in the Department of Psychology, the First Affiliated Hospital of Xi’an Jiaotong University. The psychology assessment was performed every 6 months for 5 years and annually thereafter, and terminated when the patient died or when the study was terminated, which ever came earlier. We measured psychiatric symptoms with the method described in our previous work [23].
2.3. Ethical Standards
This study was approved by the First Affiliated Hospital of Xi’an Jiaotong University Ethics and Scientific Committee and met international standards for informed consent. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent to be included in the study, or the equivalent, was obtained from all patients. All animal experiments were approved by the review board of Zhongnan Hospital of Wuhan University, and all institutional and national guidelines for the care and use of laboratory animals were followed.
2.4. Cell Culture
GC cell lines SGC-7901 and AGS were cultured in RPMI-1640 (HyClone) medium containing 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin (HyClone) in a 37°C incubator with a constant temperature and high humidity under 5% CO2 atmosphere.
2.5. MTT Assay
The MTT assay was used for evaluating cell proliferation. Briefly, cells in log phase were trypsinized into a single cell. Then, the GC cell lines were seeded into a 96-well plate ( cells/well) and cultured in 200 μl of culture medium. 72 h later, the MTT solution (Sigma-Aldrich) (1 : 10 to complete culture medium) was added into each well. After incubation for 4 h, the supernatant of each well was aspirated carefully, and the crystals were dissolved with 100 μl of DMSO (Sigma-Aldrich) by shaking for 5 min. The absorbance of each well was read by a microplate reader. The study was performed in triplicate.
2.6. Migration Assay
A wound-healing assay was used to detect cell migration. Briefly, the proper density of cells was passaged into a 6-well plate to ensure the 90%–100% monolayer cell confluence on the next day. Then, a 200 μl pipette tip was used to form a wound. 48 hours later, the number of cells that migrated into the wound were recorded.
2.7. Drug Treatment
GC cell lines SGC-7901 and AGS were seeded into 96-well plates and treated with a serial concentration of Imatinib and Nilotinib (a gift from Novartis) to determine the half-maximal inhibitory concentration (IC50). Then, GC cells were treated with the two drugs at a dose of IC50 to explore the effect of ABL1 on inflammation signaling.
2.8. Flow Cytometric Analysis
Flow cytometry was accomplished with a FACS flow cytometer (BD Biosciences). SGC-7901 cells were stained with anti-E-cadherin (rabbit, Cell Signaling Technology) and anti-N-cadherin (mouse, Abcam) for 45 min, then stained with Alexa Fluor 488- and Alexa Fluor 594-labeled secondary antibody for 30 min after washing with PBS. All procedures were performed on ice. The analysis for the data was done with the FlowJo software (TreeStar Inc., Ashland, OR, USA).
2.9. PDX Model
The generation of the PDX mice model was established as described before [24]. Briefly, fresh surgical PDX tissue fragments (from GC patients of the Zhongnan Hospital of Wuhan University) (approximately 15 mm3) were implanted subcutaneously directly into male nude (8-week-old) mice (Hubei Provincial Laboratory Animal Public Service Center, Wuhan, China) and serial transplantation was conducted. The animal experiments in this study were performed according to the policies approved by IACUC. Tumor size was monitored twice per week, and tumor volumes () were calculated with the following formula: .
2.10. CMS
Mice were randomly exposed to several stressors in the morning and in the afternoon. The stressors included dampened sawdust, removal of sawdust, replacement of sawdust with water at 21°C, repeated alteration of sawdust, inclining the cage with 45°C, restraint stress, change of the dark and light cycle, and predator voice [25].
2.11. Behavioral Tests
2.11.1. Open-Field Test (OFT)
OFT was adopted for measuring the spontaneous locomotor activities of mice. Briefly, mice were carefully placed in an open-field apparatus ()(Chengdu Techman Software Co. Ltd., Chengdu, China). The movement traces (5 min) of the mice were monitored by infrared cameras and analyzed with corresponding software [26].
2.11.2. Sucrose Preference Test (SPT)
As described before, both water and food were denuded 24 h before SPT. During the test, two bottles with the same size were supplied to each animal: one with clean water and the other one with 1% sucrose solution. After 1 h, fluid consumptions were checked. Sucrose preference proportion was calculated with the following formula: [27].
2.11.3. Tail Suspension Test (TST)
TST was performed for 6 minutes by suspending a mouse 30 cm from the floor. The immobile duration was monitored for a 6 min period. Mice were defined immobile only when they hung passively and were totally motionless [28].
2.12. Enzyme-Linked Immunosorbent Assay (ELISA)
The levels of 8-OHdG in patients’ or mice’ blood samples were quantitatively assayed with a DNA-damage ELISA kit (Ann Arbor, MI, USA). A hydrogen peroxide assay kit and a catalase assay kit were used to detect the levels of hydrogen peroxide and catalase in patients’ blood samples (Nanjing Jiancheng Biotechnology Co., Nanjing, China). The concentrations of IL-6, IL-1β, COX2, and 5-HT of mice’ blood samples were determined with a commercial ELISA kit for mouse IL-6, IL-1β, COX2, and 5-HT (Elabscience Biotechnology Co. Ltd., China). Procedures were performed based on the manufacturers’ protocols, and the absorbance values were read at the recommended wave lengths.
2.13. Western Blot Analysis
Proteins from both the hippocampus and tumor tissue samples and cell lines were extracted. The concentration of proteins was detected with the BCA protein assay reagent (Shenzhen Wolsen Technology Co. Ltd., China). A total 15 μg of proteins from each sample was loaded on 12% gel, then the proteins were transferred from the gel to the polyvinylidene difluoride membranes (EMD Millipore Corporation, Billerica, MA, USA), followed by blocking the membrane with 5% nonfat milk at room temperature for 2 h. The membrane was then incubated with a primary antibody specific for ABL1/p-ABL1(y412), IL-6, IL-1β, and COX2 (Abcam, Shanghai, China); NF-κB1/p-NF-κB1 (ser933) (Cell Signaling Technology, Danvers, MA, USA); STAT3/p-STAT3 (Tyr 705) (Santa Cruz Biotechnology (Shanghai) Co. Ltd., Shanghai, China); and β-actin antibody (Proteintech Group, Wuhan, China) at 4°C for 12 h. The membranes were washed 5 times with TBST (6 min each time), followed by 1 h incubation with the secondary antibody at room temperature. Image Lab software was used for the quantification of protein levels. Western blots were repeated three times for each sample.
2.14. Measurement of Serum Levels of ROS
The sera of mice were harvested, and serum levels of ROS were determined by the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs Inc., California, USA) in accordance with the protocol provided. The fluorescence was read with a fluorescence plate reader at excitation 480 nm and emission 530 nm.
2.15. Statistical Analyses
Statistical analysis was performed by GraphPad Prism. Data are presented as . Significant difference at each comparison point is shown as and .