Glutathione peroxidase 2 knockdown suppresses gastric cancer progression and metastasis via regulation of kynurenine metabolism

Cell lines and reagents

Human GC cell lines (NUGC-4, MKN-74, AZ-521, MKN-1, NUGC-3, AGS, HGC-27, MKN-45) were obtained from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). The human gastric epithelial cell line GES-1 was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). All cell lines were recently authenticated by short tandem repeat authentication and tested for mycoplasma contamination. NUGC-4, MKN-74, AZ-521, MKN-1, NUGC-3, HGC-27, MKN-45, and GES-1 cells were cultured in RPMI-1640 (BasalMedia, Shanghai, China), and AGS cells were cultured in Ham’s F12 (Cienry, Hu Zhou, China) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, USA) and 1% penicillin/streptomycin (Kino Co., Ltd., Hangzhou, China) at 37 °C under 5% CO2 in a cell culture incubator [35]. NAC, a ROS scavenger, was purchased from Sigma–Aldrich (St. Louis, MO, USA). L-kyn was also purchased from Sigma‒Aldrich.

Patients and clinicopathological characteristics

In this study, 269 patients who underwent gastrectomy were enrolled at Zhejiang Cancer Hospital from 2007 to 2017. Meanwhile, we collected demographic information and clinicopathological characteristics to analyze the relationship between those characteristics and GPx2 expression, including age, sex, Lauren type (a histo-clinical classification, which divides GC into intestinal GC, diffuse GC and mixed GC), tumor size, T stage (which reflects the depth of tumor infiltration), N stage (which reflects tumor lymph node metastasis), TNM stage (which refers to the eighth edition of the AJCC staging standard), CEA level, CA199 level, and KI67 expression.

Tissue microarray (TMA) construction and immunohistochemistry (IHC) analysis

Primary GC tissues and adjacent noncancerous tissues from 269 patients who underwent gastrectomy were collected at Zhejiang Cancer Hospital from 2007 to 2017. Informed consent was obtained from all subjects. TMAs were constructed, including 248 GC tissues and 213 noncancerous tissues. Then, IHC staining and analysis were performed. IHC staining with antibodies against GPx2 (#ab140130, Abcam), KYNU (#11796-1-AP, Proteintech), AhR (#67785-1-Ig, Proteintech), E-cadherin (#3195 S, Cell Signaling Technology), N-cadherin (#13116 S, Cell Signaling Technology), and Vimentin (#5741 S, Cell Signaling Technology) was performed to measure protein expression levels using standard procedures. Protein expression was assessed using the H-score system. The formula for the H‐score was as follows: H‐score = ∑ (IS × AP), where IS represents the staining intensity (0, no staining; 1, weak staining; 2, intermediate staining; 3, strong staining) and AP represents the percentage of positively stained tumor cells (0, <5% of the total cells; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%), producing an H-score ranging between 0 and 12. To assess the average degree of staining within a tumor sample, multiple regions were analyzed, and at least 100 tumor cells were assessed. Two experienced pathologists who were blinded to the clinical outcomes performed the scoring independently. The protocol was approved by the Committee on the Ethics of Zhejiang Cancer Hospital (IRB-2020-109).

Generation of concentrated lentiviral vectors and infection

Lentivirus containing shRNA targeting GPx2 or GPx2 overexpression constructs or a negative control shRNA was synthesized by GeneChem Biotechnology Company (Shanghai, China). The sequences of 21 nucleotide shRNAs targeting GPx2 were CCGATCCCAAGCTCATCATTT and GCGCCTCCTTAAAGTTGCCAT. GC cells were transfected with lentivirus according to the manufacturer’s instructions. After 72 h, stable cell lines were screened using 1 µg/ml puromycin. Transfection efficiency was determined by Western blotting.

RNA isolation and quantitative RT‒PCR

Total RNA was extracted using the RNA-Quick Purification Kit (Yishan Biotech, Shanghai, China) and reverse transcribed by using a ReverTra Ace qPCR RT kit (Toyobo). Subsequently, qPCR was performed using SYBR Green reagent (CWBIO) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The relative gene expression was calculated using the 2−ΔΔCq method. The primers used in the present study were as follows: GAPDH forward, AACGGATTTGGTCGTATTG and reverse, GGAAGATGGTGATGGGATT; GPx2 forward, CCCTCATGACCGATCCCAAG and reverse, TCCGGCCCTATGAGGAACTT.

Quantitative proteome analysis

Stable control and shGPx2 cells were collected and sent to GeneChem Biotechnology Company (Shanghai, China) for quantitative proteome library construction and sequencing. Then, protein extraction, protein quantification, protein enzymatic hydrolysis and mass spectrometry detection were performed. The MS data were analyzed using MaxQuant software (version 1.6.17.0). MS data were searched against the database. The cutoff of the global false discovery rate (FDR) for peptide and protein identification was set to 0.01. Protein abundance was calculated on the basis of the normalized spectral protein intensity (LFQ intensity). Proteins with a fold change > 1.5 and P-value (Student’s t-test) <0.05 were considered to be differentially expressed [36]. The differentially expressed proteins were selected for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

Cell viability assay

CCK-8 (GLPBIO, United States) assays were conducted to measure cellular viability. The transfected NUGC-4, MKN-45 and NUGC-3 cell lines were seeded into 96-well culture plates and incubated with CCK-8 reagent for 3 h. Thereafter, a microplate reader (Thermo Varioskan LUX, MA, United States) was used to measure the absorbance (OD) at 450 nm.

EdU incorporation assay

EdU incorporation was measured with the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555. Transfected GC cells were seeded into 12-well plates. After 24 h of incubation, the cells were incubated with 10 µM EdU (Beyotime, Shanghai, China) for 2 h. Then, the cells were harvested and fixed with 4% paraformaldehyde. After washing and permeabilizing, the cells were incubated with Click-iT® EdU reaction cocktail for 30 min. After washing, the EdU-positive cells were measured by flow cytometry (Agilent).

Transwell migration and invasion (Matrigel) experiments

For migration assays, 1 × 105 cells in 200 µL of serum-free media were seeded in the upper chamber of an insert (8 µm pore size, Corning, USA). For invasion assays, 1 × 105 cells in 200 µL of serum-free media were seeded in the upper chamber of an insert coated with Matrigel (BD Biosciences, San Diego, CA). Then, 600 µL of medium containing 20% FBS was added to the lower chamber. After incubation for 72 h, the cells attached onto the upper side of the transwell were mechanically removed with a cotton stick. Next, the cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde for 10 min and then stained with 0.4% crystal violet solution for 10 min [37]. Images of the migrated and invaded cells were captured with a Nikon Digital Sight DS-L1 camera.

Detection of ROS levels

Intracellular ROS levels were measured using the ROS Assay Kit (Beyotime, Shanghai, China). Cells were incubated with 10 µM DCFH-DA for 1 h and then treated with the indicated concentrations of H2O2. The DCF fluorescence intensities were then monitored by flow cytometry [38].

Kyn level assessment

Kyn levels in the supernatant and cell lysates were measured by ELISA (#ab287800, Abcam) according to the supplier’s instructions [39].

Cell treatment conditions

NUGC-4 and MKN-45 cells were exposed to the following conditions: control, transfected with empty lentivirus for 48 h; control+kyn, transfected with GPx2 empty lentivirus for 48 h, followed by treatment with 150 µM kyn for 24 h; shGPx2, transfected with GPx2-specific shRNA lentivirus for 48 h; shGPx2+kyn transfected with GPx2-specific shRNA lentivirus for 48 h, followed by treatment with 150 µM kyn for 24 h; H2O2, transfected with the empty/GPx2-specific shRNA lentivirus for 48 h or treated with H2O2 (0.01, 0.05, 0.1, 0.5 and 1.0 mM) for 30 min; and NAC, transfected with empty/GPx2-specific shRNA lentivirus or treated with NAC (20 mM) for 1 h.

Western blotting analysis

Cells were lysed with 1X sodium dodecyl sulfate lysis buffer. Total protein was quantified, separated by SDS‒PAGE, and transferred onto PVDF membranes (Millipore, MA, USA). The target proteins were probed with antibodies against GPx2 (#ab140130, Abcam), KYNU (#11796-1-AP, Proteintech), AhR (#67785-1-Ig, Proteintech), E-cadherin (#3195 S, Cell Signaling Technology), N-cadherin (#13116 S, Cell Signaling Technology), Vimentin (#5741 S, Cell Signaling Technology) and GAPDH (#60004-1-Ig, ProteinTech). Anti-mouse IgG (926-6807, Invitrogen) and anti-rabbit IgG (926-68070, Invitrogen) were used as secondary antibodies. Finally, the protein bands were visualized with enhanced chemiluminescence (ECL; Fdbio Science, Hangzhou, China). Intensity was measured by ImageJ software.

Subcutaneous xenograft model

To establish the GC xenograft tumor model, 1 × 107 NUGC-4 GC cells in 100 µL of PBS mixed with 100 µL of Matrigel (BD Biosciences) were injected subcutaneously into the right flanks of four-week-old male nude mice. We randomly divided the mice into the control, shGPx2#1 and shGPx2#2 groups (n = 5/each group). Mice were monitored for body weight and tumor size (length × width2 × 0.5) once a week according to the animal protocol. After 4 weeks, the nude mice were sacrificed, and the tissues were collected for detection [40]. The number of macroscopic nodules was then recorded. Blinding was maintained during the experiments. The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhejiang Chinese Medical University.

Peritoneal metastasis models

A total of 5 × 106 MKN-45 cells suspended in 200 µL of saline were intraperitoneally injected into five- to six-week-old male nude mice. We randomly and blindly divided the mice into the control, shGPx2#1 and shGPx2#2 groups (n = 6/each group). The body weight, living status, and tumor size of the nude mice were recorded. Mice were sacrificed 22 days after GC cell injection, and a small shallow section was cut to expose the abdominal cavity [41]. The number of macroscopic nodules was then recorded. Blinding was maintained during the experiments. The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhejiang Chinese Medical University.

In vivo luminescence imaging

Mice were anesthetized, and luminescence was measured 5 min after IP injection of D-luciferin sodium salt (150 mg/kg) by using the in vivo imaging system (IVIS) Lumina LT (Caliper Life Sciences, USA). Luciferase activity, which represents the volume of peritoneal metastasis, was measured using the IVIS. Living Image Ver. 4.3 (Caliper Life Sciences, USA) software was used to access the images and acquire the data.

Statistical analysis

All statistical analyses were performed using GraphPad Prism software version 8.0. Parametric tests (Student’s t-test or one-way ANOVA) or nonparametric tests were used, depending on the type of data distribution and homogeneity of variance. Survival curves were generated using the Kaplan‒Meier method. Count data are presented as the rate or composition ratio using the chi-square test. Data are presented as the mean ± SEM, and P < 0.05 was considered to indicate statistical significance.

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