Introduction

NAD(P)H quinone oxidoreductase 1 (NQO1) is an oxidation-sensitive enzyme that protects cells against redox imbalance. Involvement of NQO1 in gastrointestinal (GI) health and disease has been poorly studied. Hydrogen sulfide (H₂S) is an endogenous and anti-oxidative molecule that exerts anti-oxidative activity due to the posttranslational persulfidation of proteins. Bioactivity of H₂S is related e.g. to the regulation of antioxidant response elements (AREs) in the nucleus that subsequently affect many molecular factors. We assume that this phenomenon could also include NQO1. Importantly, triggerable H₂S-prodrugs, activated by reactive oxygen species (ROS) are a new class and promising agents in therapeutic applications but remain poorly explored in GI pharmacology. Novel, click-and-release carbonyl sulfide/H₂S donor (Peroxy-TCM), in the presence of ROS is capable to rapidly release cytoprotective H₂S. Of note, oxidative stress and ROS generation have been linked to the development of Barrett's esophagus (BE) or acute gastric mucosal injuries.

Aims & Methods

Therefore, we verified the contribution of the interplay between NQO1 and H₂S-signaling in the pathogenesis of oxidation-dependent GI diseases. We assessed the therapeutic capacity of the novel “click-chemistry”-based pharmacology, implementing this approach in experimental gastroenterology.
Clinically, proteome profile and pathway-specific PSSH shifts were evaluated using recent chemoproteomic approach in 3 sets of endoscopic esophageal samples: BE, squamous segments exposed to gastroesophageal reflux (GERD), and squamous segments from patients w/o GERD. Experimentally, male Wistar rats were administered intragastrically (i.g.) with i) vehicle, ii) peroxy-TCM (4 mg/kg) as a novel ROS-activated COS/H₂S donor or iii) peroxy-CM, as a structural control w/o H₂S-releasing ability. Peroxy-TCM was also combined with pharmacological NQO1 inhibitor (dicumarol, applied i.g.). Next, animals underwent microsurgical ischemia/reperfusion (I/R), selectively targeting gastric mucosa. Gastric mucosal damage was assessed micro- and macroscopically. Bioavailability of H₂S and oxidative RNA damage reflected by 8-hydroxyguanosine (8-OHG) levels were assessed in gastric mucosa. Pathway-specific proteome changes in the gastric mucosa were evaluated using mass spectrometry-based proteomics.

Results

Clinically, NQO1 protein and its persulfidation was progressively increased at subsequent phases of BE pathogenesis. Experimentally, peroxy-TCM in contrast to its negative control, reduced gastric damage and RNA oxidation induced by I/R. Gastric mucosal I/R activated peroxy-TCM and evoked H₂S-release in gastric mucosa. Proteomic screening showed that gastroprotective effect of peroxy-TCM was accompanied by the increased mucosal level of NQO1. Pharmacological inhibition of NQO1 attenuated beneficial effects of H₂S-prodrug.

Conclusion

NQO1 emerged as an important molecular target that contributes in anti-oxidative response of GI epithelium to pathology-driven redox imbalance. NQO1 and its persulfidation could be also considered as an early biomarker of BE development. NQO1 activity in gastric mucosa depends on the H₂S-bioavailability. Click-chemistry-based H₂S-therapeutics appeared here to selectively release this gaseous molecule in controllable-manner (only in the presence of ROS) when applied i.g. This therapeutic approach mechanistically activates gastric mucosal NQO1, increasing the ability of gastric epithelium to cope with oxidative stress. [Funding: National Science Centre (Poland)- 2019/33/B/NZ4/00616, 2023/07/X/NZ4/01052]

References

1. Steiger, Andrea K et al. “Inhibition of Mitochondrial Bioenergetics by Esterase-Triggered COS/H2S Donors.” ACS chemical biology vol. 12,8 (2017): 2117-2123. doi:10.1021/acschembio.7b00279
2. Levinn, Carolyn M et al. “N-Methylation of Self-Immolative Thiocarbamates Provides Insights into the Mechanism of Carbonyl Sulfide Release.” The Journal of organic chemistry vol. 86,8 (2021): 5443-5451. Doi:10.1021/acs.joc.0c02778
3. Zhao, Yu, and Michael D Pluth. “Hydrogen Sulfide Donors Activated by Reactive Oxygen Species.” Angewandte Chemie (International ed. in English) vol. 55,47 (2016): 14638-14642. doi:10.1002/anie.201608052
4. Zhao, Yu et al. “Kinetic Insights into Hydrogen Sulfide Delivery from Caged-Carbonyl Sulfide Isomeric Donor Platforms.” Journal of the American Chemical Society vol. 139,45 (2017): 16365-16376. Doi:10.1021/jacs.7b09527
5. Gilbert, Annie K, and Michael D Pluth. “Subcellular Delivery of Hydrogen Sulfide Using Small Molecule Donors Impacts Organelle Stress.” Journal of the American Chemical Society vol. 144,38 (2022): 17651-17660. doi:10.1021/jacs.2c07225
6. Zhao, Yu et al. “Fluorogenic hydrogen sulfide (H2S) donors based on sulfenyl thiocarbonates enable H2S tracking and quantification.” Chemical science vol. 10,6 1873-1878. 10 Dec. 2018, doi:10.1039/c8sc05200j
7. Zivanovic, Jasmina et al. “Selective Persulfide Detection Reveals Evolutionarily Conserved Antiaging Effects of S-Sulfhydration.” Cell metabolism vol. 30,6 (2019): 1152-1170.e13. doi:10.1016/j.cmet.2019.10.007

Disclosure

All authors declare that they have no conflicts of interest.

Introduction

Endogenous hydrogen sulfide (H₂S) and H₂S-prodrugs contribute in the maintenance of gastrointestinal (GI) integrity and redox balance. Biological effects of any H₂S-donor was predominantly mitochondrial. However, even non-targeted H₂S-releasing compounds were shown to decrease the non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrotoxicity but when applied in relatively high doses. Importantly, new derivatives of NSAIDs, such as H₂S-naproxen (ATB-346, Antibe Therapeutics Inc., Canada) or H₂S-ketoprofen (ATB-352) exerted GI safety compared to mother compounds in preclinical or clinical studies. However, mitochondria were not considered as a direct molecular targets for H₂S-prodrugs in terms of attenuation of NSAIDs gastrotoxicity. Targeted delivery of H₂S to gastric mitochondria to improve NSAIDs-pharmacology remains overlooked.

Aims & Methods

Thus, we aimed here to evaluate for the first time if i.g. treatment with mitochondria-targeted and H₂S-releasing AP39 counteracts development of NSAIDs-induced gastric mucosal injuries. Wistar rats were treated i.g. with vehicle, AP39 (0.004-0.5 mg/kg), AP-219 (0.02 mg/kg) as structural control without H₂S-releasing ability, or AP39 + SnPP (10 mg/kg) as a heme oxygenase (HMOX) inhibitor. Next, animals were administered i.g. with acetylsalicylic acid (ASA, 125 mg/kg) as NSAIDs representative to induce translational hemorrhagic gastric lesions. NSAIDs-gastrotoxicity was assessed micro- and macroscopically. Gastric mucosal activity of mitochondrial complexes IV and V, as well as DNA oxidation were assessed biochemically. Gastric mucosal and/or serum content of IL-1b, IL-10, TNF-a, TGF-b1/2/3, ARG1, GST-a, or phosphorylation of NF-κB, ERK, Akt, JNK, STAT3/5 were evaluated by microbeads-fluorescent xMAP®-assay. Gastric mucosal mRNA level of HMOX-1/2, COX-1/2, SOD-1/2 were determined by real-time PCR.

Results

AP39 (but not AP219) dose-dependently (0.02 and 0.1 mg/kg) diminished the development of NSAID-induced gastric lesions and DNA oxidation, restoring mitochondrial complexes activity, ARG1, GST-a protein levels and increasing anti-oxidative HMOX-1 and SOD-2 expression. AP-39 decreased proteins levels or phosphorylation of state-of-the-art gastric mucosal inflammation/oxidation-sensitive markers. Pharmacological inhibition of HMOX-1 attenuated AP39-gastroprotection.

Conclusion

We showed that mitochondria-targeted H₂S, released from very low i.g. doses of AP-39 improved gastric mucosal capacity to cope with NSAIDs-induced mitochondrial dysfunction and redox imbalance, mechanistically requiring the activity of anti-oxidative HMOX-1 and mitochondrial SOD-2. We assume that mitochondria-targeted H₂S moieties could significantly increase the GI-safety of NSAIDs-pharmacology.

Introduction

Persulfidation of proteins (PSSH) appears functionally significant, e.g. in aging, preventing cellular oxidation, but its role in GI diseases remains clinically elusive. This posttranslational proteins modification relies on endogenous hydrogen sulfide (H₂S) signaling. GERD induces oxidation in esophageal squamous cells. However, the contribution of persulfidation and H₂S bioavailability in etiology, diagnosis or treatment of Barrett’s esophagus (BE) is unknown. We hypothesized that GERD-induced pathway-specific PSSH impairments underlie BE pathogenesis. We explored whether restoration/depletion of esophageal H₂S impacts persulfidation-sensitive, BE-associated targets and BE development.

Aims & Methods

We clinically performed a wide-scale analysis of persulfidated proteins (normalized to total proteins levels) in three-sets of endoscopic biopsies representing BE or squamous segments +/- GERD (n=4-5 each), using the recent chemoproteomic method. We used the microsurgical rat model of GERD/BE (esophago-gastroduodenal anastomosis) +/- i.g. pharmacological treatments to up-/downregulate esophageal H₂S levels over 10 weeks. In parallel, we employed in vitro model of chronic GERD +/- H₂S donors using human esophageal squamous cell lines with (NES-G2T) or without (EPC2) clinical GERD and a Barrett’s cells (BAR-T). CRISPR-Cas9 was used to knockout H2S-producing enzymes.

Results

Global persulfidation level (reflecting H₂S bioavailability) was decreased in clinical BE vs GERD-exposed squamous biopsies. GERD induced statistically significant pathway-specific PSSH changes of ≈90 proteins in squamous segments compared to patients w/o GERD. These persulfidomic alterations further increased in BE where >900 targets were deregulated. IL-1b, prostaglandin (PG)E2 pathways or mTOR-associated proteins, but not exclusively, showed persulfidation shift (e.g. PSSH fall of IL-1b-receptor antagonist (IL-1RA)). H2S-decrease was noted in vitro or in vivo after long-term GERD in rats with chronic esophageal lesions and in human squamous, but not BAR-T cells. In cells and/or rats, treatment with NaSH or GYY4137 to restore H2S induced IL-1RA and inhibited IL-1b/PGE2/mTOR signaling, RNA oxidation, limiting molecular or morphological BE development. H₂S-enzymes inactivation promoted BE-related events in both models.

Conclusion

BE is preceded by GERD-induced, pathway-specific persulfidation shifts, initiated early in squamous epithelium and enhanced in metaplasia. Decreased but stable biosynthesis of H2S reflects the adaptative resistance of the metaplastic cells to GERD. Restored H2S signaling counteracts BE development by interfering in oxidative balance and BE-associated IL-1β, PGE2 and mTOR pathways activation. These findings suggest that BE is a mechanistic outcome of reduced esophageal H₂S caused by GERD. [Funding: National Science Centre (Poland): 2016/23/D/NZ4/01913, 2019/33/B/NZ4/00616, 2023/07/X/NZ4/01052]

Introduction

Barrett’s esophagus (BE), the major precursor of esophageal adenocarcinoma (EAC), is a condition where the normal stratified squamous esophageal epithelium undergoes transdifferentiation into the specialized, intestinal-like epithelium. This reprogramming, also termed ‘paligenosis’, is predominantly caused by gastroesophageal reflux disease (GERD)¹. On the other hand, hydrogen sulfide (H₂S) next to carbon monixide (CO) is an important endogenous gaseous mediator. H₂S is biosynthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Both, H₂S and CO have been shown to maintain gastrointestinal (GI) integrity^2,3,4. However, the contribution of gaseous mediators signaling to esophageal transdifferentiation process is overlooked.

Aims & Methods

Thus, in this study we aimed to explore the role of H₂S bioavailability in mTOR-dependent paligenosis induced by chronic epithelial injury. Furthermore, we evaluated the relationship between GERD, the barrier function of cultured cells, and the pattern of selected components involved in the formation of the tight junctions (TJs) and responsible for epithelial barrier integrity.
We implemented an animal model of GERD, based on Male Wistar rats with esophagogastroduodenal anastomosis (EGDA) treated i.g. for 8 weeks with vehicle or with H₂S- releasing chemicals: GYY4137 (5.0 mg/kg)/NaHS (1.0 mg/kg) or CTH/CBS inhibitors: PAG (15 mg/kg)/AOAA (11 mg/kg). In parallel, we employed optimized in-vitro GERD model based on human-derived esophageal epithelial (EPC2) cells +/- CRISPR-Cas9-induced knockout of H₂S-producing enzymes that were treated with acidified bile mixture (BM) or with GYY4137/NaHS (100 µM). We evaluated activation of AKT/mTOR pathway by multiplex microbeads fluorescent assay and an expression of TJ-associated protein (ZO-1) and occludin by capillary immunoblotting. The barrier function of cultured cells was evaluated by measurement of transepithelial electrical resistance (TER).

Results

We observed that either chronic reflux in EGDA-rats and/or treatment of human esophageal cells with BM led to a slight or a significant activation of AKT/mTOR signaling pathway and its down-stream effector - p70S6K. Additionally, we observed that pharmacologically-derived H₂S reversed this effect, while inhibition of endogenous H₂S biosynthesis (pharmacological or induced by knockout of H₂S-producing enzymes) had either no effect or increased GERD-induced phosphorylation of AKT/mTOR pathway components. Moreover, we showed that ZO-1 is highly expressed in BM treated EPC2 cells and this was reflected by the increase in TER measurment. Level of occludin remained unchanged. We did not observe any changes in expression of ZO-1 and occludin after GYY4137 treatment; however, TER measurment decreased in comparison to BM treated cells. Selective knockout of H₂S-producing enzymes led to a decrease in ZO-1 protein content in comparison to wild-type cells.

Conclusion

We conclude that interventional enhancing or decreasing of H₂S bioavailability in esophageal epithelium respectively delay or accelerate the GERD-induced paligenosis process via regulation of the mTOR pathway. H₂S-prodrugs may provide a protective effect on the esophageal epithelium by substituting the phisiological defensive response of the tissue against GERD, which is reflected by an increase in the expression of ZO-1 and tightening of the cellular barrier.

References

1. Souza RF, Spechler SJ. Mechanisms and pathophysiology of Barrett oesophagus. Nat Rev Gastroenterol Hepatol 2022.
2. Fiorucci S, Distrutti E, Cirino G, Wallace JL. The emerging roles of hydrogen sulfide in the gastrointestinal tract and liver. Gastroenterology 2006.
3. Magierowska K, Korbut E, Wójcik-Grzybek D, Bakalarz D, Sliwowski Z, Cieszkowski J, Szetela M, Torregrossa R, Whiteman M, Magierowski M. Mitochondria-targeted hydrogen sulfide donors versus acute oxidative gastric mucosal injury. J Control Release 2022.
4. Magierowska K, Brzozowski T , Magierowski M. Emerging role of carbon monoxide in regulation of cellular pathways and in the maintenance of gastric mucosal integrity. Pharmacol Res 2018.

[Funding: National Science Centre, Poland (UMO-2016/23/D/NZ4/01913, UMO-2019/33/B/NZ4/00616) and Statutory Grant (N41/DBS/000874), Jagiellonian University Medical College].