Helicobacter pylori-induced cystathionine-γ-lyase drives redox imbalance and metabolic remodeling in gastric cancer

Introduction

Gastric cancer (GC) is the fifth most common cancer and the third most prevalent cause of cancer-related death globally (1). Its major risk factors include Helicobacter pylori (H. pylori) infection, genetic predisposition, age, smoking, high-salt diet, and alcohol consumption (2). Over half of the global population is infected with H. pylori, which accounts for ~75% of GC cases, and is classified as a class I carcinogen by World Health Organization (WHO) (3,4). Persistent H. pylori infection has been directly linked to ulcer disease,non-ulcer dyspepsia and GC risk (5,6). Persistent infection alters the gastric microbiome, induces chronic inflammation, and promotes gastric carcinogenesis (7,8).

H. pylori survives in the gastric mucosa by utilizing host-derived nutrients, particularly amino acids rather than sugars (9-11), making amino acid metabolism a potential therapeutic target (12). Among these, cysteine is essential for redox balance, mitochondrial function, and protein synthesis (13), and is supplied by the cystine/glutamate antiporter or synthesized through the transsulfuration (TSS) pathway. Key enzymes, cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE/CTH), 3-mercaptopyruvate sulfurtransferase (3-MST) generate cysteine, hydrogen sulfide (H₂S), and other reactive sulfur species (RSS), which regulate redox homeostasis and cellular signaling (14,15). Cysteine metabolism is crucial in regulating oxidative stress and inflammation, which are two major factors in GC development. However, the role of cysteine/TSS metabolism in H. pylori colonization and GC remains unclear.

Sulfur-containing dietary compounds, particularly garlic-derived metabolites such as allicin, S-allylmercaptocysteine (SAMC), diallyl disulfide (DADS), and diallyl trisulfide (DATS), exhibit anticancer activities by disrupting mitochondrial function, elevating reactive oxygen species (ROS), and inducing apoptosis (16-18). Clinical trials in Shandong and elsewhere showed that garlic supplementation significantly reduced GC incidence and mortality (19). Yet, whether garlic-derived sulfur compounds act through modulation of host TSS metabolism in the context of H. pylori infection has not been addressed.

Here, we investigated the expression of TSS enzymes (CBS, CSE), and their downstream sulfur-containing products, including RSS, H₂S, and metabolic changes in gastric epithelial and cancer cells exposed to H. pylori and DATS. Our findings indicate that H. pylori infection increases CSE expression and ROS levels, potentially linking infection-driven inflammation with altered cysteine metabolism and GC progression. We further explored how garlic-derived sulfur compounds may modulate this pathway, aiming to identify novel metabolic vulnerabilities in H. pylori-associated GC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0397/rc).

Methods

Materials

RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Western blotting kits were purchased from BOSTER (Wuhan, China; AR0138). CBS and CSE was obtained from Proteintech (Wuhan, China; 14787-1-AP, 1:2,000; 60234-1-Ig, 1:5,000). Anti-caspase 3 and caspase 9 were acquired from Abcam (Cambridge, MA, USA; ab32351 and ab32539). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and secondary anti-rabbit or anti-mouse IgG antibodies were obtained from Absin (Shanghai, China; abs830030, 1:5,000). The transfection reagent was purchased from Polyplus 2000 (Illkirch, France; 114-15). The RNA extraction kit was obtained from SparkJade (Shandong, China; AC1001). If not specifically mentioned, the other kits and reagents were all purchased from Solarbio (Beijing, China).

Bacterial culture

H. pylori strain 26695 and SS1 were obtained from our laboratory microbes bank, and cultured on Columbia blood agar or Mueller-Hinton agar (Oxoid, Basingstoke, England) containing 5% sterile sheep blood and antibiotics mixture (vancomycin, amphotericin B, polymyxin B, and trimethoprim; Hopebio, Qingdao, China) in a micro-aerobic environment (5% O₂, 10% CO₂, and 85% N₂) at 37 ℃ for 3–5 days.

Cell culture and H. pylori infection

Human GES1, MKN 45, HGC 27, and MGC 803 cell lines were obtained from our laboratory cell bank, and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin placed in an incubator at 37 ℃ with 5% CO₂. All cell lines used in this study (GES1, MKN 45, MGC 803, and HGC 27) were routinely tested for mycoplasma contamination using a polymerase chain reaction (PCR)-based Mycoplasma Testing Kit (Sigma-Aldrich, St. Louis, MO, USA; MP0050-25TST) before the experiments performed, and all results were negative. The H. pylori strain was harvested during exponential amplification periods via centrifugation at 6,000 ×g for 10 min at 25 ℃ and added to cultured gastric cell lines (80% density) at different multiplicity of infection (MOI) values (MOI =50, 100, and 150) and four time points (3, 6, 9, and 12 h) for co-culture. After that, cells were harvested for total protein and RNA extraction or other functional experiments.

Quantitative real-time PCR (qRT-PCR)

The total RNA was isolated using a SPARK easy cell RNA kit according to the manufacturer’s instructions. The RNA purity and concentration were determined using NanoDrop ND-2000 (Thermo Fisher Scientific, Inc.). The RNA was reversely transcribed into complementary DNA (cDNA) according to the following procedure: denaturation for 30 s at 95 ℃, 40 amplification cycles of 15 s at 95 ℃, 60 s at 60 ℃, and 15 s at 95 ℃, and an extension for 34 s at 60 ℃. Thus, the relative RNA expression were analyzed using the 2^−ΔΔCt method and normalized to the expression of GAPDH. The primers used for qRT-PCR are provided in Table S1.

Western blotting

The total protein from the cell lines and human GC tissues was extracted using RIPA lysis buffer (Solarbio; R0010) on ice, and the protein concentrations were determined using NanoDrop ND-2000. The proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF; Millipore, Billerica, MA, USA) membranes, which were blocked with 5% non-fat milk for 2 h at 25 ℃ and incubated with the primary antibodies at 4 ℃ overnight. Then, the membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody for 45 min after washing thrice with tris-buffered saline with Tween (TBST) buffer at room temperature. The membranes were developed using the enhanced chemiluminescence method (Millipore). The band intensities were quantified using ImageJ software.

Cell treatment and small interfering RNA (siRNA) transfection

The CSE and negative control (NC) siRNAs, listed in Table S1, were designed by Sigma Company. Polyplus 2000/siRNA complexes were prepared in OPTI-MEM (Invitrogen, Waltham, MA, USA) and supplemented with 2% FBS according to the manufacturer’s instructions (1 μL siRNA per 50 μL jetPRIME buffer). After the cells reached 70–80% confluence on the six-well plates, the MKN45, MGC803, and HGC27 cells were transfected with 50 nM of CSE siRNA or NC siRNA using Polyplus 2000/siRNA complexes for 6 h. The medium was then replaced with fresh complete RPMI-1640 with 10% FBS for another 24 h.

Cell proliferation assay

After 48 h of siRNA transfection, 3×10³ cells were seeded on 96-well plates with 100 μL per well and incubated at 37 ℃ overnight. A volume of 10 μL of Cell Counting Kit-8 (CCK-8) reagent (Apex Bio, Houston, TX, USA) was added to each well. After incubation for 2 h, the absorbance of the wells was measured at 450 nm every 24 h for 5 days.

5-ethynyl-2'-deoxyuridine (EdU) staining was performed to evaluate cell proliferation using an EdU kit according to the manufacturer’s protocol (Cell-Light EdU Apollo 567 kit, RIBOBIO, Guangzhou, China). A total of 1.2×10⁴ cells were seeded on 96-well plates with a volume of 100 μL of medium per well and incubated with 50 μM EdU for 2 h at 37 ℃. After being fixed with 4% paraformaldehyde, the cells were exposed to 100 μL of 1× reaction cocktail and then incubated with 5 μg/mL Hoechst 33342 to stain the cell nucleus. Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan). The percentage of EdU-positive cells was used to define the proliferation rate. All the experiments were performed in triplicate.

Cell migration assay

The migration abilities of cells were assayed using Transwell inserts. The cells were collected after siRNA transfection for 48 h. To minimize the influence of cell proliferation on migration assays, cells were pre-treated with 10 μg/mL mitomycin C (MCE, Monmouth Junction, NJ, USA; HY-13316) for 2 h. After treatment, cells were washed three times with phosphate-buffered saline (PBS) and incubated in fresh medium before the migration experiments was performed. Then, the MKN 45, HGC 27, and MGC 803 cells were seeded on the upper chamber of 24-well plates at a density of 5×10⁵ cells per 130 μL of serum-free medium. RPMI-1640 medium (600 μL) containing 20% of serum was added to the lower chamber. After incubation for 48 h, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet for 15 min. Then the cells were washed three times with PBS, and the upper layer of cells were removed. The number of migrating cells was analyzed using a microscope in three independent experiments.

Detection of ROS fluorescence

An intracellular oxidative stress ROS kit (MCE; Cat. No. HY-126793) was used for intracellular ROS level detection. Briefly, after the cells were grown to 70% confluence on 24-well plates, NaHS, H₂O₂, or DATS was added to cells, respectively, and incubated for 30 min. A GENMED working solution (500 μL) with 2',7'-dichlorofluorescein diacetate (DCFH2-DA) was added to each well after the culture medium was discarded. Then, after incubating the plates for 20 min, the upper layer of the GENMED working solution was discarded. In addition, 500 μL GENMED preservation solution was added to the cells, which were photographed using a fluorescence microscope.

Analysis of the intracellular sulfane sulfur species

To perform a sulfane sulfur probe (SSP4) test, SSP4 (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) and cetrimonium bromide (CTAB; Sangon Biotech, Shanghai, China) were dissolved in dimethyl sulfoxide (DMSO) and ddH₂O, respectively, to make 10 and 100 mM stock solutions. A total of 1×10⁴ cells per well were seeded on a 96-well plate and incubated overnight. After the culture medium was discarded, the cells were washed with serum-free 1640 medium Then 100 μL of 800 μM NaHS, 400 μM H₂O₂, and 40 μg/mL DATS were added to each well, respectively, followed by incubation with a culture medium for 30 min. Cells were washed with serum-free medium twice after the supernatant was discarded. A volume of 100 µL of 20 µM SSP4 working solution was added to the cells, followed by incubation for 15 min in the dark with gentle shaking (125 ×g) and the cells were washed with PBS twice again. PBS was added to each well, and the cells were analyzed using a fluorescence microscope.

Measurement of H₂S level in the cell culture supernatants

HSip-1 DA (Dojindo Molecular Technologies, Inc.) was dissolved in DMSO to prepare 10 and 100 mM stock solutions according to the manufacturer’s protocol. The cells were harvested and washed twice with serum-free DMEM after growing to a confluence of 70% on a 24-well plate. A HSip-1 DA working solution (200 µL, 5 μM) was added to each well. After 30 min of incubation, the supernatant was removed, and the cells were washed twice with Hanks’ balanced salt solution (HBSS). NaHS (800 μM), H₂O₂ (400 μM), and DATS (40 μg/mL) were used to treat cells in a culture medium for 30 min. After washing twice with HBSS, 200 μL of HBSS was added to the cells, and these were observed a fluorescence microscope (20). DATS and H₂S both are the more volatile and oxidizable components. In vitro systems demonstrate that the amount of free H₂S in solution varies between 6% and 18% of the original donor concentration. DATS and H₂S donor concentration used in our experiments (10, 20, and 40 μg/mL of DATS, 400 and 800 μM of H₂S donor) are reasonably represent the amount of H₂S reaching the respiratory physiological concentration of cells in vivo.

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

TMA from human GC tissues were purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Shanghai Outdo Biotech Co., Ltd. (ethical code No. HStm-Ade060CS-01). The enrolled patients provided written informed consent for the publication, and all the personal data of patients were deleted (table available at https://cdn.amegroups.cn/static/public/jgo-2026-0397-1.xlsx). TMA included 28 pairs of GC tumors and matched adjacent tissues. The expression and cellular localization of the CSE protein in gastric tissues were detected using IHC analysis. The GC tissues were stained with anti-CSE antibodies.

Immunohistochemical staining was performed on paraffin-embedded sections. Briefly, sections were baked at 63 ℃ for 1 h, followed by deparaffinization using an automated staining system. Antigen retrieval was conducted in a retrieval apparatus, after which sections were naturally cooled in distilled water for >10 min. Endogenous peroxidase activity was blocked, and sections were incubated with primary antibody at 4 ℃ overnight. After washing with 1× PBS, the sections were processed on a DAKO Autostainer Link 48 for secondary antibody incubation and 3,3'-diaminobenzidine (DAB) chromogenic detection according to the manufacturer’s instructions. Sections were then counterstained with hematoxylin for 1 min, differentiated in 0.25% hydrochloric acid (HCl) alcohol (70% ethanol with concentrated HCl) for approximately 10 s, and rinsed under running tap water for 5 min. Finally, sections were dehydrated, cleared, and cover-slipped with neutral resin.

Untargeted metabolomics analysis

The MKN45 GC cell samples were placed in Eppendorf tubes after the treatment with DATS or H. pylori for 24 h and resuspended in prechilled 80% methanol using a well vortex. The no-treated cells were also harvest for control. Then, the samples were melted on ice and whirled for 30 s. After sonification for 6 min, they were centrifuged at 5,000 rpm, 4 ℃, for 1 min. The supernatant was freeze-dried and dissolved with 10% methanol. Finally, the solution was injected into the liquid chromatography with a tandem mass spectrometry (LC-MS/MS) system for analysis.

Ultra-high performance liquid chromatography (UHPLC)-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive TM HF mass spectrometer (Thermo Fisher) in Novogene Co., Ltd. (Beijing, China). The raw data files generated from UHPLC-MS/MS were processed using the Compound Discoverer 3.1 (CD3.1; Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. These metabolites were also annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/pathway.html), Human Metabolome Database (HMDB) (https://hmdb.ca/metabolites), and LIPID Maps database (http://www.lipidmaps.org/) by Novogene Co., Ltd. (Beijing, China).

Animal model and H. pylori infection

C57BL/6J CSE knockout (Cse⁻/⁻) male mice and the wild-type (WT) were used for in vivo infection experiments. The WT mice were purchased from Beijing HFK Bioscience Company, while the CSE knockout (Cse⁻/⁻) were obtained from our laboratory animal bank. All mice were maintained under specific pathogen-free (SPF) conditions and used at 6–8 weeks of age. For H. pylori infection, the SS1 strain was cultured on columbia blood agar plates supplemented with 5% sheep blood under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) at 37 ℃ for 48–72 hours. Bacteria were harvested and resuspended in sterile PBS at a concentration of approximately 1×10⁹ colony-forming unit (CFU)/mL. All animal experiments were performed under a project license (No. KYLL2025693) granted by the Research Ethics Committee of The Second Qilu Hospital of Shandong University, in compliance with institutional guidelines for the care and use of animals.

Amoxicillin (Sigma-Aldrich) was freshly prepared in sterile distilled water to a final concentration of 1 g/L and administered ad libitum in drinking water for 5 consecutive days. The antibiotic-containing water was replaced daily to maintain potency. Control mice received sterile water without antibiotics.

After the final antibiotic dose, mice were maintained on regular sterile water for 12 hours prior to bacterial gavage. Mice were orally gavaged with 200 µL of the bacterial suspension (2×10⁸ CFU) every other day for a total of seven administrations. Infected mice were monitored and euthanized after 2 weeks for fetal and sample collection and analysis.

Gastric tissues were collected for histopathological examination, quantitative PCR, and hematoxylin and eosin (H&E) staining to evaluate inflammation, and the expression of relevant host response genes. Two weeks after H. pylori infection, fecal samples of mice were collected. Bacterial DNA was extracted from stool samples and the hypervariable region V3–V4 of the 16S ribosomal RNA (rRNA) gene was amplified for sequencing analysis by Novogene Company (Tianjin, China). Gastric tissues were harvested and subjected to western blot and H&E staining for histological evaluation.

Statistical analysis

All experiments were repeated at least thrice, and three parallel groups were set up at each instance. Statistical analysis was performed using SPSS 22.0 software (PASW, Chicago, IL, USA). Data are expressed as the mean ± standard deviation (SD). We used a group t-test for two independent samples. Statistical significance was set at P<0.05.

Results

CSE is up-regulated in GC cells and tissues

To investigate the functional role of CBS and CSE in GC, we analyzed the protein and messenger RNA (mRNA) expression in cell lines (GES1, MGC 803, MKN 45, and HGC 27). Results showed that the protein and mRNA expression levels of CSE were higher in GC cell lines than in the normal GES1 gastric epithelial cell line (Figure 1 and Figure S1). These results indicate that CSE expression was significantly higher in GC cell lines than in the normal gastric epithelial cell line. Unlike CSE, the protein expression levels of CBS in the tumor cell lines was no statistical difference in protein expression (Figure 1B). Then we mainly focused on CSE enzyme function in the follow-up studies. To verify the results from these cell lines, the protein extracted from three pairs of GC and adjacent tissues were collected and the CSE protein expression was detected by western blot. We found that CSE protein was obvious increase in GC tissues than that of the adjacent tissues (Figure 1C,1D). To further validate these findings, we analyzed 28 paired human gastric tissue samples using a tissue microarray, wherein each sample pair consisted of a GC tissue sample and a paired adjacent non-tumor gastric tissue sample from the same patient. The CSE protein levels were similar with cell lines, being higher in the tumor tissues than in para-cancer (Figure 1E,1F). Our data showed that the level of CSE protein in cancer cell lines and GC patient tissues significantly increased, which indicated that CSE of TSS pathway might be a potential function protein related with human GC.

Figure 1 Expression of H₂S-producing enzymes (CSE and CBS) in different cell lines and GC tissues. (A,B) Western blot analysis and densitometric analysis of the CSE and CBS protein expression in normal gastric mucosal epithelial cells (GES1) and in GC cells (MKN 45, HGC 27, and MGC 803). Tissues protein expression (C,D) and tissues microarray and IHC analyses of CSE (immunohistochemical staining; 20×) (E,F). The tissues were collected from gastric tumor tissues and adjacent non-tumor gastric tissues of GC patients (28 sample pairs). The relative quantification of western blot. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. CBS, cystathionine-β-synthase; CSE, cystathionine-γ-lyase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC, gastric cancer; H₂S, hydrogen sulfide; IHC, immunohistochemistry.

CSE expression was significantly increased after H. pylori stimulation

Previous studies have confirmed that H. pylori infection is the main etiological agent of GC (2,5). To explore the effects of H. pylori on normal epithelial and GC cells, we co-cultured H. pylori 26695 (standard strain) with different cell lines. The different H. pylori infection times was based on the specific biological processes being investigated in the experiment. Short-term infection periods (e.g., 6–24 h) were used to evaluate early cellular responses, including oxidative stress, inflammatory signaling, and alterations in sulfur metabolism. Western blot analysis of CSE protein expression was performed following H. pylori infection at MOIs of 50, 100, and 150 (Figure 2A). Among these conditions, an MOI of 100 induced the most consistent and robust increase in CSE protein expression and was therefore selected for subsequent experiments. After the strain was added into cell lines for 0, 3, 6, 9, and 12 h, the mRNA and protein were extracted separately to analyze the CSE expression levels, respectively. Reverse transcription quantitative PCR (RT-qPCR) analysis demonstrated that CSE mRNA expression increased following H. pylori infection in both GES1 cells and GC cell lines. Significant upregulation of CSE mRNA was observed at 6 and 9 h post-infection in MGC 803 and HGC 27 cells. These findings indicate that H. pylori infection induces CSE expression at the transcriptional level, although the magnitude and duration of the response vary among cell lines (Figure 2B). At the protein level, H. pylori stimulation led to an increase in CSE expression in GES1 cells, and similar, albeit moderate, increases were seen in MKN 45, MGC 803, and HGC 27 cells at 9 and 12 h (Figure 2C). Taken together, these results demonstrate that H. pylori infection induces CSE protein expression in gastric epithelial and GC cells, potentially pointing to a role for CSE in the host response.

Figure 2 JenyH. pylori upregulated the CSE mRNA and protein expression. (A) The CSE protein expression in normal gastric mucosa epithelial (GES1) and GC cells (MKN 45, HGC 27, MGC 803) co-cultured with different concentrations of H. pylori 26695 (MOIs =50, 100, and 150) using western blot and densitometric analysis. (B) qRT-PCR assay results showed the CSE expression after H. pylori co-cultured with GES1 and GC cells (MKN 45, HGC 27, MGC 803) for 0, 3, 6, 9, and 12 h. (C) Western blot analysis and densitometric analysis of the expression of CSE levels after H. pylori was co-cultured with normal gastric mucosal epithelial (GES1) and GC cells (MKN 45, HGC 27, and MGC 803) for 0, 3, 6, 9, and 12 h. The H. pylori strain used was 26695, MOI =100. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. CSE, cystathionine-γ-lyase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GC, gastric cancer; H. pylori, Helicobacter pylori; MOI, multiplicity of infection; mRNA, messenger RNA; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction.

SiRNA-mediated inhibition of CSE expression reduced the tumor cells proliferation and migration

The above results demonstrated that H. pylori infection may be associated with increased CSE protein expression in gastric epithelial and GC cells. To further elucidate the biological significance of this finding, we sought to determine whether the elevated expression of CSE have a functional relationship with tumor processes. Three different CSE-specific siRNA sequences (Table S1) were designed to inhibit the expression of CSE in cells. Meanwhile, NC sequences were established. SiRNA1 and siRNA3 were chosen for further study because the CSE mRNA and protein levels expression were dramatically suppressed after adding the siRNA based on our comprehensive evaluation (Figure S2).

Moreover, functional experimental studies were performed after the siRNA-mediated inhibition of CSE expression in GC tumor cells. Compared to untreated cells, CSE silencing significantly affected GC cell proliferation (of MKN 45, HGC 27, and MGC 803 cells) (Figure 3A). We further verified that the proliferation ability of cells was significantly reduced after CSE silencing using EdU staning (Figure 3B and Figure S3A,S3B). Furthermore, the transwell experiment results showed that the migration ability of the three cell lines decreased significantly after the CSE protein expression was decreased in GC cells (Figure 3C). Collectively, these results indicate that CSE functionally contributes to the malignant phenotype of GC cells. Based on this observation, we next explored whether pharmacological modulation of CSE-related sulfur metabolism could regulate H. pylori-associated cellular responses.

Figure 3 CSE affects the proliferation and migration of GC cells. (A) A CCK-8 assay was used to test GC cell (MKN 45, HGC 27, and MGC 803) proliferation after the transfection of si-CSE. (B) An EdU assay was used to test GC cell (MKN 45) proliferation after the transfection of si-CSE (20×). (C) Transwell migration assays of GC cells. GC cells migrate to the lower chamber containing RPMI-1640 medium with 20% FBS within 48 h (crystal violet staining; 20×). NC sequences (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). CCK-8, Cell Counting Kit-8; CSE, cystathionine-γ-lyase; EdU, 5-ethynyl-2'-deoxyuridine; FBS, fetal bovine serum; GC, gastric cancer; NC, negative control; si, small interfering RNA.

CSE expression decreased after DATS and NaHS treatment

Our previous studies have demonstrated an association between garlic consumption and a reduced risk of GC, and we hypothesized that the active compounds in garlic may exert protective effects through multiple signaling pathways (21). Given that CSE is implicated in promoting the malignant phenotype of GC cells, we investigated whether DATS or H₂S could modulate CSE expression. As shown in Figure S4A-S4D, normal gastric epithelial cells (GES1) and various GC cell lines were treated with DATS at concentrations of 10, 20, and 40 μg/mL, based on previous studies (22). Cell viability was not significantly affected at any of these concentrations. However, qRT-PCR and western blot analyses revealed that DATS significantly downregulated CSE expression at 20 and 40 μg/mL in the examined cell lines (Figure S4A-S4D). Since DATS is a RSS, we speculated whether DATS inhibits CSE by releasing product H₂S or other sulfur-containing compounds in the cells.

In order to verify the inference, we used NaHS, an exogenous H₂S donor, to treat the cells. After treatment with different concentrations (400 and 800 μM) of NaHS refered to previous studies (23), the growth of GC cells was not affected significantly. Western blot analysis showed that NaHS inhibited CSE expression in GES1 and GC cells. Among these, the CSE protein expression of GES1 and MGC 803 cells decreased significantly at 800 μM, whereas that of HGC 27 cells decreased most significantly at 400 and 800 μM (Figure S4E). Collectively, these results demonstrated that DATS and exogenous H₂S suppress CSE expression without affecting cell viability, suggesting a feedback regulatory relationship between sulfur metabolites and CSE. These findings prompted us to further investigate whether DATS modulates H. pylori-induced CSE upregulation and related cellular responses.

DATS inhibits the H. pylori-induced CSE over-expression

Building on our observation that H. pylori infection upregulated CSE expression in gastric epithelial and cancer cells, we next examined whether DATS could modulate this infection-induced response. Different infection durations were selected according to the biological endpoints analyzed. To further characterize the cellular phenotype during the acute infection phase, we chose to assess physiological indicators at 24 h post-infection in subsequent experiments (24). Cells were first co-cultured with H. pylori strain 26695 (MOI =100) for 24 h, followed by treatment with DATS (20 μg/mL). H. pylori exposure markedly increased CSE protein expression in GES1, HGC27, and MGC803 cells, whereas subsequent DATS treatment significantly attenuated this upregulation (Figure S5). To further determine whether DATS exerts a preventive effect, cells were pretreated with DATS for 24 h prior to H. pylori infection. Notably, under these conditions, H. pylori failed to induce a significant increase in CSE expression (Figure S6). Collectively, these findings indicate that DATS not only suppresses H. pylori-induced CSE upregulation but also confers resistance to infection-associated CSE activation, suggesting that DATS may modulate downstream redox and metabolic responses associated with H. pylori infection, which we next investigated.

DATS orchestrates intracellular redox signaling via ROS and RSS in GC cells

H. pylori infection is known to trigger chronic inflammatory responses in gastric epithelial cells and impair the cellular antioxidant defense mechanisms (5). DATS, a sulfur-containing compound derived from garlic, possesses well-documented anti-inflammatory and antimicrobial activities (17). To evaluate whether DATS mitigates the oxidative stress by modulating intracellular levels of ROS, H₂S, and RSS, we examined oxidative stress markers in cells subjected to different compound treatments. To gain insights into the action mechanisms of CSE protein and TSS pathway on gastric epithelial cells and GC cells, we used fluorescence probes to evaluate the endogenous ROS, H₂S, and RSS levels. Notably, compared with that of control group, NaHS and DATS significantly inhibited ROS production, whereas H₂O₂ significantly increased the production of ROS (Figure 4A). Interestingly, ROS levels in tumor cells were significantly lower than the control group GES1. Additionally, H₂S and RSS production was slightly increased in the GC cell lines compared to that in GES1 cells (Figure 4B,4C). This observation suggests that more H₂S and RSS might be generated and stored in GC cells and involved in GC development and progression. After treating the GC cells with NaHS, H₂O₂, or DATS, respectively, we observed that they could significantly increase the production of RSS and H₂S compared with that of the NC group (DMSO treated). We speculate that the produced RSS and H₂S contribute to the antioxidant effect and react with ROS. Those results might imply that the DATS function is also related to the levels of RSS and H₂S, and that DATS exerts an antioxidant effect through RSS. The ability to produce H₂S and RSS in GC cells are better than normal controls, which might be related to the CSE high expression. The ROS detection results in HGC 27 cells are not shown because these cells were intolerant to the HSip-1 DA fluorescence probe and show a weak adhesion property. As an undifferentiated gastric carcinoma cell line, HGC 27 cells are more prone to detachment during washing or under physical stress, which likely contributed to the observed experimental limitation.

Figure 4 DATS inhibits the ROS and promotes the H₂S and RSS production. (A) ROS production in different cell lines (20×). (B) H₂S production in different cell lines (20×). (C) Sulfane sulfur production in different cell lines (20×). The cells were treated with NaHS (400 µM), H₂O₂ (200 µM) and DATS (20 µg/mL) before the immunofluorescence detection, respectively. DATS, diallyl trisulfide; H₂S, hydrogen sulfide; NC, negative control; ROS, reactive oxygen species; RSS, reactive sulfur species.

Metabolomic profiling reveals metabolic reprogramming induced by H. pylori and its modulation by DATS

We performed untargeted metabolomics analysis on MKN45 GC cells treated with DATS or infected with H. pylori, compared with untreated cells, and identified differential metabolites using tandem mass spectrometry (MS/MS) data. In Figure 5A, the venn diagrams showed the overlap and unique differential metabolites among different groups, demonstrating the relationships among multiple groups of differential metabolites. The overlapping region between the two treatment groups contained two significantly altered metabolites, Glu-Gln and ureidoisobutyric acid. Volcanic map and hierarchical clustering analysis (HCA) was conducted on all the differential metabolites among the obtained comparison pairs. In H. pylori-treated cells compared with the NC group, 18 metabolites were downregulated, while 22 metabolites were upregulated (Figure 5B), In DATS-treated cells compared with the NC group, 5 metabolites were downregulated, while 33 metabolites were upregulated (Figure 5C). Among the metabolite altered between the two treatment groups, several metabolites showed the pronounced alterations. Compared with the NC group, the H. pylori group exhibited significant alterations in amino acids, peptides, and analogues, indicating that the H. pylori-infection significantly affected cellular amino acid metabolism, particularly cystine, L-cysteine-glutathione gisulfide, and homoarginine (Figure 5D and table available at https://cdn.amegroups.cn/static/public/jgo-2026-0397-2.xlsx). Compared with the NC group, DATS treatment significantly altered metabolites including glutamate-glutamine (Glu-Gln), serine, and γ-glutamylcysteine (Figure 5E and table available at https://cdn.amegroups.cn/static/public/jgo-2026-0397-2.xlsx).

Figure 5 Untargeted metabolomics analysis of DATS-treatment or H. pylori-infected MKN 45 GC cells compare with no-treated cells (A) Numbers of overlap and unique differential metabolites between different groups. (B,C) Volcanic diagram of metabolites difference between DATS-treated group and H. pylori infection group. Each point represents a metabolite, up-regulated metabolites are represented by red dots, down-regulated metabolites are represented by green dots, and the size of the dots represents the VIP value. (D,E) Differential metabolites clustering heat map. DATS, H. pylori groups and NC groups are on the bottom, with six repetitions for each group. (F,G) KEGG enrichment bubble plot. The size of the dot represents the number of differential metabolites in the corresponding pathway, and the larger the dot, the more differential metabolites in the pathway. DATS, diallyl trisulfide; DE_Meta, differentially expressed metabolites; GC, gastric cancer; H. pylori, Helicobacter pylori; HP, Helicobacter pylori; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; VIP, variable importance in projection.

Differential metabolite clustering and pathway cluster analyses were conducted according to the characteristics of the screened differential metabolites. The enrichment results were determined via KEGG pathway analysis, and a hypergeometric test was applied, as shown in Figure 5F,5G (only show the top 20 results). The significantly enriched pathways after H. pylori infection were metabolic pathways, carbon and riboflavin metabolism. Notably, it has a substantial impact on vitamin digestion and absorption, particularly on the absorption of vitamin B6. The analysis after DATS treatment revealed that the most significantly affected pathways included metabolic pathways, purine and carbon metabolism and biosynthesis of amino acids. Our metabolomics analysis highlights that H. pylori infection primarily perturbs amino acid metabolism, with a notable impact on cysteine-related metabolites and vitamin B6 absorption, which are closely linked to redox balance and host immune responses. In the TSS pathway, cysteine is a direct product of CSE, which converts cystathionine to cysteine. Thus, cysteine levels may reflect CSE enzymatic activity in generating cysteine from its upstream precursor. Furthermore, CSE also utilizes homocysteine as a substrate to produce H₂S (15). Vitamin B6, in its active form pyridoxal 5'-phosphate (PLP), is an essential cofactor for CSE (as well as CBS). Disrupted vitamin B6 absorption therefore further compromises CSE catalytic efficiency at cysteine synthesis and H₂S generation. Together, the concurrent alterations in cysteine and vitamin B6 metabolism reveal a link to CSE/H₂S axis dysregulation in H. pylori infection. In contrast, DATS treatment primarily altered purine, carbon, and amino acid metabolism, indicating a broad impact on cellular energy and biosynthetic pathways. Notably, several of these metabolic changes opposed those induced by H. pylori infection, suggesting that DATS may partially counteract infection-associated metabolic reprogramming. Together, these data indicated an association between H. pylori infection, sulfur-and vitamin-related metabolic alterations, and the metabolic effects of DATS, supporting a potential role for organosulfur compounds in modulating infection-related metabolic changes.

CSE deficiency exacerbates H. pylori-induced gastric inflammation in vivo

To evaluate the in vivo functional role of CSE during H. pylori infection, WT and Cse^−/− mice were subjected to acute H. pylori infection (Figure 6). No significant differences in body weight or general condition were observed between groups, indicating comparable systemic tolerance to infection.

Figure 6 Acute H. pylori infection models in WT (n=5) and Cse^−/− mice (n=6). (A) Experimental workflow of the acute H. pylori infection model. (B) Western blot analysis of IL-6 expression in mice gastric tissues. (C) H&E staining of gastric tissues. (D-F) 16S rRNA gene sequencing evaluating the effect of CSE on the intestinal microbiome in H. pylori-infected mice. Differences in microbial composition between WT and Cse^−/− mice were analyzed using Spearman’s correlation. The codes KO044–WT058 correspond to different mice. *, P<0.05. CFU, colony-forming unit; CSE, cystathionine-γ-lyase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H. pylori, Helicobacter pylori; H&E, hematoxylin and eosin; KO, knockout; rRNA, ribosomal RNA; WT, wild-type.

Histopathological analysis revealed that Cse^−/− mice exhibited more severe gastric mucosal injury following H. pylori infection, characterized by epithelial disruption, glandular disorganization, and inflammatory cell infiltration, whereas WT mice displayed only mild inflammatory changes (Figure 6B,6C). Consistently, elevated interleukin-6 (IL-6) expression was detected in a subset of infected Cse^−/− mice but not in WT controls, supporting an enhanced inflammatory response in the absence of CSE.

To explore whether CSE deficiency influences host-microbiota interactions during infection, 16S rRNA sequencing was performed. Cse^−/− mice exhibited reduced microbial diversity compared with WT mice after H. pylori challenge, indicating altered microbial community structure associated with CSE loss (Figure 6D-6F). The different intestinal microbiomes between the WT and Cse^−/− mice were analyzed with spearman’s correlation, as shown in Figure 6D,6E. 16S rRNA sequencing revealed significant alterations in microbial community composition following H. pylori infection. Compared with WT mice, Cse^−/− mice exhibited reduced microbial diversity, indicating an association between CSE deficiency and gut microbiota dysbiosis (Figure 6D-6F). Differential abundance analysis identified significant increases in Alloprevotella, Lactococcus, Methanobrevibacter, and DNF00809, whereas Quinella, Candidatus Saccharimonas, Allobaculum, and Anaerovorax were decreased in the Cse^−/− group (Figure 6F). These findings demonstrate that CSE deficiency is associated with distinct microbial community shifts during H. pylori infection. However, the functional consequences of these microbial alterations and their contribution to host inflammatory responses remain to be further investigated. Together, these observations suggest that CSE deficiency is associated with altered host-microbiota interactions during H. pylori infection.

These findings suggest that CSE deficiency is associated with both enhanced gastric inflammation and altered microbial community structure following H. pylori infection. Whether these microbiota changes contribute directly to the inflammatory phenotype requires further investigation.

Discussion

The TSS pathway contributes to de novo cysteine synthesis in cancer cells, and most cells express CSE at high levels (14). The CBS and CSE (or CTH) are functional enzymes involved in the TSS pathway, a biochemical mechanism that links the methionine metabolism to the biosynthesis of cellular redox-controlling molecules, such as cysteine, glutathione, and H₂S (25), regulating various physiological and pathological processes in humans (26). In this study, we demonstrated that the CSE mRNA and protein levels were higher in GC cells than in GES1 normal mucosal epithelial cells. This notion is further supported by the findings that the tumor tissues have an overall higher CSE protein production than para-cancer. H. pylori infection increased the CSE protein expression in vitro (Figure 1). Moreover, H₂S and DATS can inhibit CSE overexpression and prevent further H. pylori infections. The in vitro functional experiments showed that down-regulation of CSE expression could significantly reduce the proliferation and migration of tumor cells (Figure 3). Taken together, these data are consistent with a model in which DATS contributes to anti-tumor activity in vivo, possibly through coordinated regulation of CSE and RSS.

In our study, the silencing of the H₂S-synthesizing enzyme CSE inhibits cell proliferation and migration, which suggests that a high expression of CSE plays a critical role in cells and might be closely related to tumor development. Several studies have reported an increased expression of CSE and/or increased levels of H₂S in several types of cancer (27,28). CSE is over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of the H₂S/CSE pathway decreases the proliferation of hepatoma cells and enhances ROS production and mitochondrial disruption, as well as DNA damage and apoptosis (29). The inhibition of CSE activity or its knockdown could decrease colon cancer cell proliferation, in vitro migration, and xenograft tumor growth in vivo, providing robust evidence that CSE accelerates colon cancer growth (30). In this study, we found that CSE over-expression was related to high ROS levels in GC cells and could be part of negative feedback regulation by DATS and H₂S. We speculated that the intracellular ROS production was suppressed by the production of H₂S and RSS. Therefore, we measured the intracellular ROS, RSS, and H₂S concentration in different cell lines and under different conditions. The results were consistent with our hypotheses. The ROS levels were higher in tumor cells than in normal epithelial cells. When H₂O₂ was added, cancer cells were more resistant to ROS stimulation (Figure 4). The results demonstrated the antioxidative function of CSE over-expression in cancer cells to produce more H₂S and RSS to maintain the physiological ROS levels. These findings suggest a complex interaction between CSE expression, H₂S production, and redox homeostasis in GC cells, which warrants further investigation.

DATS, a major bioactive organosulfur compound derived from garlic, has been widely reported to exert anticancer effects through modulation of oxidative stress pathways (22,31). In the present study, DATS altered intracellular ROS/RSS levels and modulated CSE expression in H. pylori-infected GC cells, supporting the involvement of the CSE-H₂S-redox axis in the cellular response to infection (Figure 4 and Figure S4). These findings suggest that sulfur-metabolism-related pathways may contribute to the biological effects of DATS in GC. Our findings provide preliminary evidence that DATS may modulate H. pylori-associated GC-related pathways through regulation of sulfur metabolism and redox balance. Further studies are required to evaluate its therapeutic potential in vivo.

Metabolic reprogramming is closely associated with inflammation and tumor progression in GC. Our metabolomics analysis analysis showed that H. pylori infection altered carbon, purine, vitamin B6 metabolism, and amino acids biosynthesis, which might be related to the inflammation, tumor migration and ROS response. Cysteine is both a product and a substrate of CSE and is then used by CSE to produce H₂S. Thus, decreased cysteine levels indicate impaired CSE synthetic function while also limiting substrate availability for H₂S production. Vitamin B6 (as PLP), serves as a coenzyme in numerous amino acid metabolic and antioxidant reactions, and its demand increases under inflammatory conditions, leading to an apparent deficiency (32). Together, these alterations provide metabolomic evidence for the dysregulation of CSE/H₂S axis in H. pylori infection. However, the inference of transcriptome analysis needs to be verified by subsequent experiments.

To further evaluate the role of CSE in vivo, we established an acute H. pylori -infection model in WT and Cse^−/− mice. CSE deficiency aggravated gastric inflammatory injury following H. pylori infection in vivo (Figure 6). Together, these findings support a context-dependent role of CSE in H. pylori-associated gastric pathogenesis, with distinct effects in tumor cells and host tissues. Furthermore, DATS modulated CSE expression and sulfur metabolism-related responses, suggesting that the CSE-associated sulfur metabolic pathway may warrant further investigation as a potential therapeutic target in H. pylori-associated gastric disorder.

Taken together, our findings support a context-dependent role of CSE in H. pylori-associated gastric pathogenesis. While CSE promotes proliferative and migratory phenotypes in GC cells, it also appears to protect against infection-associated inflammatory injury in vivo. These results highlight the CSE-associated sulfur metabolic pathway as a potential mediator of host-pathogen interactions and gastric disease progression.

Conclusions

Our findings suggest that CSE is a context-dependent regulator of sulfur metabolism and redox homeostasis during H. pylori infection. CSE appears to support host tissue protection during gastric inflammation and deficiency exacerbates gastric injury while being associated with metabolic adaptations linked to gastric carcinogenesis. Conversely, Importantly, we show that the garlic-derived compound DATS can modulate CSE-related sulfur metabolism and mitigate oxidative stress, suggesting that sulfur metabolic pathways are amenable to pharmacological or dietary intervention. These findings provide a basis for further investigation of CSE-mediated sulfur metabolism in H. pylori-associated GC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0397/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0397/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0397/prf

Funding: This research was financially supported by grants from the Taishan Scholars Program of Shandong Province (No. tsqn202306361), the National Natural Science Foundation of China (No. 32000098), the Shandong Provincial Natural Science Foundation (Nos. ZR2025MS1511, ZR2023QC251, and ZR2023QH214), and The Second Qilu Hospital of Shandong University Key Interdisciplinary Cultivation Fund (No. 2023JX02).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0397/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Shanghai Outdo Biotech Co., Ltd. (ethical code No. HStm-Ade060CS-01). The enrolled patients provided written informed consent for the publication. All animal experiments were performed under a project license (No. KYLL2025693) granted by the Research Ethics Committee of The Second Qilu Hospital of Shandong University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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