Hypoxia-mediated upregulation of xanthine oxidoreductase causes DNA damage of colonic epithelial cells in colitis

doi:10.21203/rs.3.rs-3362379/v1

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Hypoxia-mediated upregulation of xanthine oxidoreductase causes DNA damage of colonic epithelial cells in colitis

https://doi.org/10.21203/rs.3.rs-3362379/v1

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published 11 Jan, 2024

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Xanthine oxidoreductase (XOR) is the primary source of hydrogen peroxide and superoxide anions in the intestinal mucosa. However, its specific contribution to the colonic disease progression remains unclear. In this study, we investigated the role of XOR in ulcerative colitis (UC) and attempted to identify the underlying mechanisms. We used the dextran sulfate sodium (DSS)-induced mouse model to mimic UC and found that the XOR inhibitors (allopurinol and diphenyleneiodonium sulfate (DPI) significantly alleviated UC in mice. Also, cobalt chloride (CoCl₂) and 1% O₂ treatment increases the expression of XOR and caused DNA oxidative damage in colonic epithelial cells. Furthermore, we found that XOR accumulated in the nucleus may directly cause DNA oxidative damage and regulates HIF1α protein levels. In addition, allopurinol effectively protected colon epithelial cells from CoCl₂-induced DNA damage. Altogether, our data provide new evidence that XOR could induce DNA damage under hypoxic conditions indicating a significant role of XOR in the initiation and early development of colitis-associated colorectal cancer (CAC).

Xanthine oxidoreductase

Oxidative stress

Ulcerative colitis

Hypoxia

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of the mucosa that gradually develops from the distal to the proximal end of the colon. UC symptoms include abdominal pain, diarrhea, and rectal bleeding[1]. As a result of frequent rectal bleeding associated with UC, patients with UC experience a substantial impact on their quality of life as well as a higher risk of colon cancer compared to patients with Crohn's disease[2]. According to previous studies, UC is caused by an abnormal immune response of genetically susceptible individuals in response to specific triggers. However, the underlying pathogenic mechanism is unclear. Several factors contribute to the UC initiation, including genetics, environmental factors, abnormal innate and adaptive immune responses, and the gut microbiota[3]. Oxidative stress significantly contributes to UC progression, which is also regulated by various factors. Producing reactive oxygen free radicals (ROS) by intracellular enzymes in colonic epithelial cells may directly damage liposomes, proteins, and DNA [4, 5]. During mucosal inflammation, innate cells such as neutrophils and macrophages are recruited to lamina propria; the production of neutrophils and macrophages releases ROS, which results in further damage to the mucosal epithelial cells and aggravates the inflammatory response[6].

XOR is mainly distributed in the liver and intestine, with the small intestine being the most abundant source[7, 8]. This enzyme is also found in the intestinal mucosa, and its activity increases gradually from the base of the villi to the tip[9], and from the ileum mucosa to the duodenal mucosa[10]. As a target for treating gouty arthritis, XOR catalyzes uric acid production during purine metabolism. It has been demonstrated that XOR is involved in the oxidation of aldehydes[11]. XOR-governed catalytic reactions can directly produce superoxide, hydrogen peroxide, or nitric oxide (NO), which react with superoxide anions[12]. Thus, XOR represents the primary source of oxidants in the intestine and is considered a key source[13]. A study has indicated that the reduced form of XOR can reversibly or irreversibly transform into an oxidized form under hypoxic and inflammatory conditions, which is why XOR promotes cardiovascular disease progression[14]. Our previous studies found that the inflammatory factor TNFα can induce XOR expression and cause DNA damage in colonic epithelial cells [15].Meanwhile, in the basal state, the component epithelial cells that line the mucosa exist in an oxygen-deprived environment, also known as physiological hypoxia[16]. Large metabolic shifts often occur at sites of active mucosal inflammation, rapidly depleting nutrients and rapidly depleting local oxygen, causing hypoxia or even anaerobiosis[17]. Also, XOR has been associated with the pathogenesis of ischemia-reperfusion injuries to the intestine[18, 19]as well as other tissues[20, 21]. Allopurinol, a competitive inhibitor of XOR, can provide ischemic injury protection comparable to that induced by oxygen-free radical scavengers[19–21]. Furthermore, XOR inhibitors have been shown to attenuate experimental UC[22, 23] and to delay the progression of colitis-associated colorectal cancer (CAC)[15]. It is worth noting that ischemia-reperfusion can also cause hypoxia. Therefore, these studies suggest a potentially significant role for hypoxia- or inflammation-related XOR in colonic mucosal tissue and colitis development.

This study aimed to explore XOR's function and underlying mechanisms in the inflammatory stage of CAC development. We found that inhibitors of XOR can effectively alleviate inflammation in DSS-induced UC in mice. We observed elevated expression and activity of XOR at the site of the colon of mice with UC. Furthermore, we discovered that hypoxia caused by intestinal inflammation could induce epithelial cells to express XOR and lead to DNA damage. These findings indicate that XOR may promote the development of UC and act as an oncogene or preventive target in CAC.

2.1 Materials, chemicals, and reagents

Primary antibodies against xanthine oxidase (#ab109235), phospho-H2A histone family member X (γH2AX; Ser139; #ab131382), hypoxia-inducible factor 1-alpha (HIF1α; #ab179483), and nuclear factor erythroid 2-related factor 2 (NRF2; #ab62352), were purchased from Abcam (Cambridge, U.K.). Antibodies to detect p53 (#9282), phospho-p53 (Ser15, #9284) and β-actin (#3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). Tumor necrosis factor-α (TNFα) and Interleukin 6 (IL-6) ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). XOR activity, uric acid (UA) content, and hydrogen peroxide assay kits were purchased from Solarbio Science & Technology (Beijing, China). Evaluating DNA damage using comet, reduced glutathione (GSH), and myeloperoxidase (MPO) assay kits were purchased from Jiancheng Technology (Nanjing, China). Allopurinol was purchased from Sigma-Aldrich (St. Louis, MO, USA), while diphenyleneiodonium sulfate (DPI) was purchased from Toronto Research Chemicals (Toronto, ON, Canada). The siRNA transfection reagent (sc-29528) used to transfect siRNA into cells was purchased from Santa Cruz Biotechnology. Dextran sulfate sodium (DSS) was purchased from MP Biomedicals. Azoxymethane (AOM), CoCl₂, and other chemicals were provided by Sigma-Aldrich (St. Louis, MO, USA).

2.2 Mouse models

All animal studies were performed according to guidelines approved by the Institutional Animal Ethics Committee of Jiangnan University (JN. No. 20170324-0423) and were carried out in the animal facility of Jiangnan University. As reported previously, a mouse model of acute colitis was generated[24]. Male C57BL/6 mice (5-week-old) were administered 3% dextran sulfate sodium (DSS) in drinking water for 7 days. Allopurinol (10 mg/kg) and DPI (1.5 mg/kg) were administered by gavage every day. The disease activity index (DAI) was calculated as reported previously[25] as [body weight decrease + stool consistency + rectal bleeding]/3. Mice were euthanized on day 7. The distal colons were washed three times with cold PBS, fixed in formalin (pH 7.4), embedded in paraffin, sectioned at 5 µm, stained with hematoxylin and eosin and the inflammatory cell infiltration areas in the intestinal mucosa were quantitatively analyzed by using image J.

2.3 Cell culture, plasmid construction, and lentivirus infection

All cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in a monolayer in accordance with the supplier's instructions. Human colorectal carcinoma cell lines (HCT116 and SW620) were cultured with Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C in a 5% CO₂ incubator. Cells were tested cytogenetically and authenticated before being frozen. Subsequently, each vial of frozen cells was thawed and maintained for approximately 2 months. According to the supplier's instructions, approximately 60% of the cells were seeded into a 6-well plate, cultured overnight, and then transfected with siRNA (50 pmol/well). The sequences of the XDH-siRNA are listed in Table S1.

The XOR was amplified from pEnCMV-XOR by the polymerase chain reaction (PCR). The conditions of amplification were 95°C for 3 min, followed by 34 cycles of 95°C for 30 s, 51°C for 30 s, 72°C for 4 min and 30 s, and then 72°C for 5 min. The PCR product was inserted into the pCDH1 plasmid vector using restriction enzyme XbaI (NEB) at 5′ terminal and NotI-HF (NEB) at 3′ terminals of the PCR fragment. For XOR gene ablation, three single guide RNA (sgRNA) targeting different XOR gene sequences (Supplementary Table 1) were cloned into a lenti-CRISPR-v2 vector.

HEK-293T cells were seeded at the density of 6 × 10⁶ cells per 10 cm dish 1 day prior to transfection. Transfection was performed using PEI (Polyethylenimine, Beyotime), and the medium was refreshed 12 h later. Virus-containing supernatant was collected 48 h post-transfection. Next, the virus was filtered through a 0.45 µm membrane, snap-frozen and stored at -80°C. HCT116 was infected and selected with Puromycin 48 h after transduction for at least 4 days until no surviving cells remained in an uninfected control plate.

2.4 RT-qPCR analysis

Total RNA was extracted with Trizol following the manufacturer's protocol. The concentration and purity of the isolated RNA were then estimated using an ND-1000 micro spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA purity was evaluated using the A260/A280 ratio. Then, 2 µg of RNA was used to synthesize cDNA with a RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR procedure was performed as follows: initial denaturation at 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 s; and an annealing extension at 60°C for 30 s. Absorbance values were measured at the extension stage. Data are presented in arbitrary units and were calculated by the 2^−ΔΔCT method.

2.5 Comet assay

HCT116 cells were seeded on a 6-well plate and cultured overnight. Following adherent cell growth, cells were pretreated with 100 µmol/L of allopurinol for 1 h and then stimulated with 150 µmol/L of CoCl₂ for 6 h at 37°C. After trypsin digestion, prepare single cell suspension using cold PBS resuspension. Combing the cell suspension with 1% low melting point agarose at a ratio 1:10 (v/v), mix gently by pipetting up and down, and immediately pipette 30 µL onto the slide with a transparent agarose film. Gently remove the coverslip and immerse in lysis buffer for 1 h at 4°C. After rinsing with PBS, place the slide in a horizontal electrophoresis tank containing alkaline electrophoresis buffer, allowing DNA unwinding at room temperature for 1 hour. Setting the power supply voltage to 20V and run for 30 min. Then, gently immerse slides three times in 0.4 mmol/L Tris-HCl (pH7.5) solution for 10 min each at room temperature. Discard Tris-HCl buffer and add 20 µL of PI solution to stain for 10 min. Fluorescent signals were detected using a confocal fluorescence microscope at 560 nm and take photos.

2.6 Immunofluorescence staining

Immunofluorescence staining was performed as described previously[26]. HCT116 cells were seeded on a 24-well plate with coverslips and cultured overnight. Following adherent cell growth, cells were pretreated with 100 µmol/L of allopurinol for 1 h and then stimulated with 150 µmol/L of CoCl₂ for 6 h at 37°C. Next, we discarded the culture medium and fixed the cells with 4% paraformaldehyde for 10 min at room temperature. After washing three times with phosphate-buffered saline (PBS), cells were permeabilized with 0.1% saponin for 15 min. After blocking with 10% FBS for 30 min, cells were incubated overnight at 4°C with primary antibodies against γH2AX (Ser139, dilution 1:100) and xanthine oxidase (dilution 1:50). The next day, the cells were incubated with cyanine3 (Cy3)-goat anti-rabbit IgG (H + L) and coralite488-conjugated affinipure goat anti-mouse secondary antibody (dilution 1:200) at room temperature for 1 h. Nuclei were stained with 4,6-diamidino-2-phenylindole (0.2 µg/ml in PBS; Sigma Chemicals) for 1 min. Fluorescent signals were detected using a confocal fluorescence microscope (Nikon EZ-C1, Nikon, Tokyo, Japan).

2.7 Western blotting

After treatment, cells were harvested in radio immunoprecipitation assay (RIPA) buffer. A standard Bradford assay determined protein concentration; then, cell lysates were subject to western blotting. Proteins (20 µg) were resolved by SDS-PAGE and transferred onto Hybond C nitrocellulose membranes (Amersham Corporation, Arlington Heights, IL). After blocking with non-fat milk, the membranes were hybridized with primary antibody (1:1000) overnight at 4°C. Finally, the protein bands were hybridized with a horseradish peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence (GE Healthcare, Pittsburgh, PA) with FluorChem FC3 (Protein Simple, CA, USA). Image J software was then used to quantify the gray values produced by the western blotting procedure.

2.8 Statistical analysis

Statistical analysis was performed using the GraphPad Prism 5.0 statistical package. Statistical significance was determined using a two-tailed Student's t-test to compare data between two groups and one-way ANOVA for multiple groups. Data are shown as the means ± standard deviation (SD), and differences were considered statistically significant if p ≤ 0.05.

3.1 The inhibition of XOR alleviated ulcerative colitis in a mouse model

XOR is a well-characterized gene involved in purine metabolism and is a target for treating gouty arthritis. In our previous studies, we observed the up-regulated expression of XOR in the colonic mucosa of patients with UC and found that XOR inhibitors reduced the number of tumors at the site of the colon in mice induced by AOM/DSS[15]. However, its functional role in cancer initiation and development is mainly unknown. Thus, it is attractive to investigate whether XOR exerts an essential role during the inflammation stages of CAC. Therefore, we evaluated the effect of XOR inhibitors (allopurinol and DPI) on mice body weight of the acute colitis model induced by DSS. Interestingly, we found that two inhibitors of XOR significantly reversed weight loss caused by DSS in mice, which indicated that XOR is a potential candidate for DSS-induced acute colitis (Fig. 1A). Additionally, the inhibitors reduced the DAI index compared with the 3% DSS treated group, indicating attenuated colitis response (Fig. 1B). In line with the results of DAI index, we also noticed that the colon length dramatically decreased during colitis caused by DSS, and XOR activity inhibition significantly rescued this phenomenon, indicating reduced UC symptoms upon XOR inhibition (Fig. 1C-1D). These results suggest that XOR activity can regulate the DSS-induced acute colitis process.

To further assess whether XOR affects the severity of inflammation during DSS-induced colitis in mice, we then detected the levels of pro-inflammatory factors (such as TNFα and IL-6) in the colon and serum of our mouse model. As expected, TNFα and IL-6 levels were up-regulated in the colon and serum during DSS-induced colitis. Surprisingly, our results showed that the XOR inhibitors were sufficient to compromise the upregulation of TNFα and IL-6 levels induced by DSS (Fig. 1E-F and Fig. S1A-B). This data indicates that activated XOR is responsible for developing inflammation during colitis. We also confirmed this conclusion by our histological analysis of the distal colon tissue, showing that XOR inhibitors greatly reduced the area of lymphocyte infiltration induced by DSS in the colon mucosa (Fig. 1G-H). These results indicated that the inhibition of XOR effectively alleviated the development of DSS-induced acute colitis in mice.

3.2 XOR-mediated oxidative stress linked to the progression of acute colitis in mice

The main pathogenic factor underlying UC is an intestinal mucosal injury caused by oxidative stress, while the specific mechanisms involved have yet to be identified. We evaluated whether XOR inhibitors could alleviate oxidative stress caused by free radicals during DSS-induced UC. It was found that mice with acute UC had significantly reduced glutathione (GSH) levels and increased myeloperoxidase (MPO) activity. In our DSS mouse model, allopurinol, and DPI significantly increased GSH levels and inhibited MPO activity (Fig. 2A-B). These results revealed that XOR inhibitors could directly alleviate oxidative stress in UC. Therefore, we next tested XOR expression and activity in the colons of mice. According to our findings, XOR expression and activity were significantly increased in the mucosa of DSS-induced mice. Interestingly, XOR expression and activity were decreased by allopurinol and DPI treatment (Fig. 2C-E). Uric acid levels were significantly increased in mice with acute colitis. In line with the XOR expression and activity results, uric acid levels were decreased by allopurinol and DPI treatment (Fig. 2F). These results indicated that the upregulation of XOR expression mediated the effects of oxidative stress and contributed to the development of acute inflammation in the colons of mice.

3.3 Hypoxia upregulated XOR expression and induced DNA damage in colonic epithelial cells

Interestingly, we found that HIF1α mRNA levels significantly increased in DSS (colitis) and AOM/DSS (CAC)-induced mouse colon tissues (Fig. 3A-B). This result showed that during the inflammatory phase of CAC development, the continuation of inflammation of the intestinal mucosa exposes intestinal epithelial cells to a severe hypoxic state, causing activation of the HIF1α pathway. Therefore, we evaluated XOR activity in colonic epithelial cells under CoCl₂-induced hypoxia. We found that uric acid and hydrogen peroxide levels increased in the supernatant of colonic epithelial cells (Fig. 3C-D). In line with the results of upregulation of hydrogen peroxide and uric acid levels, we also observed the increased expression levels of XOR, which exhibited a time and dose-dependent response (Fig. 3E-G). To confirm the results, we cultured HCT116 cells in a 1% O₂ condition, not surprisingly, the results also showed elevated levels of XOR (Fig. 3H).

Our previous results showed that the induction of XOR by TNFα could cause DNA damage in colonic epithelial cells. Similarly, we found that levels of γH2AX is up-regulated in colon tumor epithelial cells induced by CoCl₂ or 1% O₂ condition (Fig. 3F-H). The results also showed increased levels of pp53 and NRF2 (Fig. 3F). This indicated that XOR-mediated oxidative stress might activate an NRF2-regulated antioxidant enzyme-mediated response against oxidative stress and DNA damage repair mechanism regulated by pp53. Additionally, hypoxia induces elevated levels of XOR in intestinal epithelial cells while increasing levels of HIF1α (Fig. 3F-H), suggesting an underlying regulatory mechanism.

3.4 XOR regulates HIF1α protein levels and DNA damage in intestinal epithelial cells

To confirm whether XOR expression is related to HIF1α protein stability, our data showed that the protein levels of HIF1α were significantly blocked by the targeted inhibition of XOR expression in colon tumor epithelial cells under the induction of CoCl₂ (Fig. 4A). To validate our results, we identified three sgRNAs to target XOR for CRISPR/Cas9 gene knockout, two non-targeting sgRNAs were used as control. The results showed that the efficiency of the third sgRNA knockout XOR gene was higher in HCT116 cells (Fig. 4B). It was further confirmed that, unlike control cell lines, XOR gene knockout cell lines were insensitive to the increased HIF1α protein levels induced by CoCl₂ (Fig. 4C) and the increased γH2AX protein levels induced by 1% O₂ condition (Fig. 4D). To further confirm whether XOR can directly regulate HIF1α protein levels and cause DNA damage, we found elevated levels of γH2AX, HIF1α, and pp53 proteins in XOR overexpressed HCT116 cell lines, and immunofluorescence results showed that γH2AX and XOR increased aggregation in the nucleus region in XOR overexpressed cell lines (Fig. 4E-4F). The results indicated that the accumulation of hydrogen peroxide and superoxide anions generated by XOR in the nucleus might directly damage DNA oxidation damage and may increase the stability of the HIF1α protein.

3.5 The inhibition of XOR alleviated DNA damage caused by hypoxia

We further evaluated the protective effect of XOR inhibitor against hypoxia-induced DNA oxidative damage. The results showed that allopurinol was sufficient to decrease the hydrogen peroxide and uric acid levels in the supernatant of colonic epithelial cells induced by CoCl₂ (Fig. 5A-B). Meanwhile, XOR, HIF1α, NRF2, and γH2AX levels decreased after allopurinol treatment (Fig. 5C-D). We also evaluated the protective effect of allopurinol against hypoxia-induced DNA damage through classic comet assay. The results showed that comet tail of COCl₂-treated HCT116 cells was longer and had higher DNA intensity compared with the control group, suggesting a substantial accumulation of fragmented DNA. The length of the comet's tail was reduced after allopurinol treatment (Fig. 5E), These results indicated that allopurinol could protect DNA from damage caused by hypoxia-induced upregulation of XOR. In addition, we confirmed the location of XOR through immunofluorescence; surprisingly, our data showed that XOR accumulated in the nucleus under the induction of CoCl₂, allopurinol could effectively reduce their accumulation in the nuclear region (Fig. 5F). The results indicated that the accumulation of hydrogen peroxide and superoxide anions generated by XOR in the nucleus might directly damage DNA oxidation damage and increase the stability of the HIF1α protein.

A combination of hypoxia and inflammation-induced XOR contributes to the toxic effects of ROS on endothelial cells and the development of cardiovascular diseases[27]. The rapid oxygen consumption at the site of inflammation will also exert extreme stress on the intestinal mucosa via physiological hypoxia and anaerobiosis. Colitis is believed to be caused by inflammation and hypoxia[28]. In this study, allopurinol and DPI could alleviate acute colitis associated with DSS by inhibiting XOR activity. It was important to note that DPI directly affected the FAD center, catalyzing oxygen-free radicals' production[29]. Therefore, when DPI inhibits XOR in the colonic mucosa, it also inhibits other enzymes containing FAD centers, such as non-phagocytic cell oxidases (NOXs)[30]. Moreover, studies have shown that oxidative stress mediated by the NOXs family aggravates the development of acute colitis[31]. In our study, the results showed that DPI is better than allopurinol regarding alleviating UC. Specifically, the TNFα level, MPO activity and the area of lymphocyte infiltration in the intestinal mucosa of the DPI group were lower than those in the allopurinol group. These results highlighted that oxidative stress, as a key factor, contributes to the progression of UC. Studies have also shown that allopurinol can inhibit XDH mRNA transcription in the adipocytes of alloxan-induced diabetes rats[32]. Our results showed that DPI and allopurinol could inhibit the XOR upregulation in colonic mucosa and that allopurinol can also inhibit CoCl₂-induced XOR upregulation in colon epithelial cells. Therefore, a positive feedback regulatory mechanism may exist between XOR and its catalytic products, although these specific products need to be verified.

Several studies show that XOR is the primary source of oxygen-free radicals during intestinal ischemia and reperfusion[18, 19]. Under the physiological hypoxia conditions in the intestinal mucosa, ischemia may exacerbate hypoxia stress. Furthermore, XOR activity increases during the process of intestinal ischemia-reperfusion[33, 34]. These studies provided evidence for a link between hypoxia and XOR. As a result of CoCl₂-induced hypoxia, colonic epithelial cells showed an increase in XOR expression and activity. Consequently, ischemia-reperfusion may increase XOR activity by mediating hypoxia in the intestinal mucosa. Therefore, whether ischemia-reperfusion increases XOR activity by meditating hypoxia in other tissues remains to be determined.

Interestingly, XOR accumulation in the nucleus may directly damage DNA due to hypoxia. In contrast to previous results that XOR is primarily located in the cytoplasm, new data indicated that it was mainly found in the nucleoplasm and centriolar satellites (https://www.proteinatlas.org/ENSG00000158125-XDH/antibody). In addition to the possibility that XOR may directly catalyze the purine groups of DNA or RNA in the nucleoplasm, its biological significance may be important. In addition to the functions described above, XOR may also be involved in centriolar satellite-mediated mitosis and autophagy. Therefore, the exact role of XOR accumulation in the nucleoplasm and centriolar satellites must be determined to understand its functions.

In our study, we also demonstrated that both inflammation and hypoxia could regulate XOR to cause DNA damage in the epithelial cells in colon cancer cells. This result may suggest that the upregulation of XOR expression is an early event at the beginning of CAC carcinogenesis. Hypoxia may always be accompanied by inflammation and the initiation and development of CAC. The present study found that hypoxia induces elevated levels of XOR in intestinal epithelial cells while increasing levels of HIF1α and the targeted inhibition of XOR expression significantly blocked HIF1α protein levels. Studies have also shown that CoCl₂ inhibits the binding of HIF1α to von Hippel-Lindau protein (pVHL), the recognition component of the E3 ubiquitin-protein ligase, thus protecting HIF1α from proteasome degradation via multiple mechanisms[35, 36]. In normoxic conditions, the O₂-dependent hydroxylation of proline (P) residues 402 and 564 in HIF1α by the enzyme PHD (prolyl hydroxylase-domain protein) 1–3 is required for the binding of pVHL[37]. Studies have shown that oxidative stress can inhibit the hydroxylation of the substrate HIF1α by PHD via multiple mechanisms, including the direct inhibition of the PHD enzyme activity[38], thus inducing the decarboxylation of α-ketoglutarate (α-KG)[39], and by oxidizing iron (Fe²⁺) to Fe³⁺ [40]; both Fe²⁺ and α-KG are necessary PHD cofactors. Therefore, we suspect that XOR-mediated oxidative stress inhibits the HIF1α hydroxylation by the PHD enzyme via the above-mentioned pathways and stabilizes the HIF1α protein. However, the exact mechanism involved has yet to be identified.

On the other hand, researchers have shown that HIF1α regulates the polarization of macrophages and exacerbates inflammation[41]. In addition, there was evidence that HIF1α-mediated signaling protects the overall integrity of the intestinal barrier in colitis[42]. However, barrier integrity may also be associated with the survival of intestinal epithelial cells against hypoxic stress since HIF1α can regulate high oxygen consumption-oxidative phosphorylation metabolic shifts towards glycolytic to resist hypoxic stress; this form of metabolic switch is known as the Warburg effect in cancer cells[43]. Therefore, HIF1α-mediated metabolic reprogramming may occur in colonic epithelial cells during the inflammatory phase of tumorigenesis. In addition, hypoxic stress and inflammation may jointly promote the initiation and development of CAC. In summary, this study provides new evidence that XOR may mediate DNA oxidative damage during the inflammatory stages of CAC under hypoxic conditions.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81773064, 31972973 and 31530056), National Youth 1000 Talents Plan, the Jiangsu Specially-Appointed Professor Program, Jiangsu Province Recruitment Plan for High-level, Innovative and Entrepreneurial Talents (Innovative Research Team), Jiangsu Funding Program for Excellent Postdoctoral Talent and Collaborative innovation center of food safety and quality control in Jiangsu Province.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Hongling Li, Qi Zhang, and Haitao Li designed and supervised the experiments. Hongling Li prepared the manuscript. Hongling Li, Weiyu Han, and Yupeng Wang performed experiments.

Ibrahim CB, Aroniadis OC, Brandt LJ (2010) On the role of ischemia in the pathogenesis of IBD: a review. Inflamm Bowel Dis 16: 696-702. https://doi: 10.1002/ibd.21061.
Baumgart DC, Carding SR (2007) Inflammatory bowel disease: cause and immunobiology. Lancet 369: 1627-1640. https://doi: 10.1016/S0140-6736(07)60750-8.
Porter RJ, Kalla R, Ho GT (2020) Ulcerative colitis: Recent advances in the understanding of disease pathogenesis. F1000Res 9. https://doi: 10.12688/f1000research.20805.1.
Colgan SP, Taylor CT (2010) Hypoxia: an alarm signal during intestinal inflammation. Nat Rev Gastroenterol Hepatol 7: 281-287. https://doi: 10.1038/nrgastro.2010.39.
Iborra M, Moret I, Rausell F, Bastida G, Aguas M, Cerrillo E, Nos P, Beltrán B (2011) Role of oxidative stress and antioxidant enzymes in Crohn's disease. Biochem Soc Trans 39: 1102-1106.https://doi: 10.1042/BST0391102.
Roessner A, Kuester D, Malfertheiner P, Schneider-Stock R (2008) Oxidative stress in ulcerative colitis-associated carcinogenesis. Pathol Res Pract 204: 511-524. https://doi: 10.1016/j.prp.2008.04.011.
Battelli MG, Corte ED, Stirpe F (1972) Xanthine oxidase type D (dehydrogenase) in the intestine and other organs of the rat. Biochem J 126: 747-749. https://doi: 10.1042/bj1260747.
Krenitsky TA, Tuttle JV, Cattau EL, Jr., Wang P (1974) A comparison of the distribution and electron acceptor specificities of xanthine oxidase and aldehyde oxidase. Comp Biochem Physiol B 49: 687-703. https://doi: 10.1016/0305-0491(74)90256-9.
Auscher C, Amory N, Pasquier C, Delbarre F (1977) Localization of xanthine oxidase acitivty in hepatic tissue. A new histochemical method. Adv Exp Med Biol 76a: 605-609.PMID:855733.
Roy RS (1985) THE ROLE OF XANTHINE OXIDASE IN SUPEROXIDE-MEDIATED ISCHEMIC INJURY (XANTHINE DEHYDROGENASE) [M]. University of South Alabama.
Morpeth FF (1983) Studies on the specificity toward aldehyde substrates and steady-state kinetics of xanthine oxidase. Biochim Biophys Acta 744: 328-334. https://doi: 10.1016/0167-4838(83)90207-8.
Bortolotti M, Polito L, Battelli MG, Bolognesi A (2021) Xanthine oxidoreductase: One enzyme for multiple physiological tasks. Redox Biol 41: 101882. https://doi: 10.1016/j.redox.2021.101882.
Martin HM, Hancock JT, Salisbury V, Harrison R (2004) Role of xanthine oxidoreductase as an antimicrobial agent. Infect Immun 72: 4933-4939. https://doi: 10.1128/IAI.72.9.4933-4939.2004.
Cantu-Medellin N, Kelley EE (2013) Xanthine oxidoreductase-catalyzed reactive species generation: A process in critical need of reevaluation. Redox Biol 1: 353-358. https://doi: 10.1016/j.redox.2013.05.002.
Li H, Zhang C, Zhang H, Li H (2021) Xanthine oxidoreductase promotes the progression of colitis-associated colorectal cancer by causing DNA damage and mediating macrophage M1 polarization. Eur J Pharmacol 906: 174270. https://doi: 10.1016/j.ejphar.2021.174270.
Shepherd AP (1982) Metabolic control of intestinal oxygenation and blood flow. Fed Proc 41: 2084-2089. PMID:7075783.
Taylor CT, Colgan SP (2007) Hypoxia and gastrointestinal disease. J Mol Med (Berl) 85: 1295-1300. https://doi: 10.1007/s00109-007-0277-z.
Granger DN, Rutili G, McCord JM (1981) Superoxide radicals in feline intestinal ischemia. Gastroenterology 81: 22-29. PMID:6263743.
Parks DA, Bulkley GB, Granger DN, Hamilton SR, McCord JM (1982) Ischemic injury in the cat small intestine: role of superoxide radicals. Gastroenterology 82: 9-15. PMID:6273253.
Itoh M, Guth PH (1985) Role of oxygen-derived free radicals in hemorrhagic shock-induced gastric lesions in the rat. Gastroenterology 88: 1162-1167. https://doi: 10.1016/s0016-5085(85)80075-5.
Adkison D, Höllwarth ME, Benoit JN, Parks DA, McCord JM, Granger DN (1986) Role of free radicals in ischemia-reperfusion injury to the liver. Acta Physiol Scand Suppl 548: 101-107. PMID:3463123.
Amirshahrokhi K (2019) Febuxostat attenuates ulcerative colitis by the inhibition of NF-κB, proinflammatory cytokines, and oxidative stress in mice. Int Immunopharmacol 76: 105884. https://doi: 10.1016/j.intimp.2019.105884.
El-Mahdy NA, Saleh DA, Amer MS, Abu-Risha SE (2020) Role of allopurinol and febuxostat in the amelioration of dextran-induced colitis in rats. Eur J Pharm Sci 141: 105116. https://doi: 10.1016/j.ejps.2019.105116.
Neufert C, Becker C, Neurath MF (2007) An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression. Nat Protoc 2: 1998-2004. https://doi: 10.1038/nprot.2007.279.
Cooper HS, Murthy SN, Shah RS, Sedergran DJ (1993) Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238-249. PMID:8350599.
Dmitrieva NI, Cui K, Kitchaev DA, Zhao K, Burg MB (2011) DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts. Proc Natl Acad Sci U S A 108: 20796-20801. https://doi: 10.1073/pnas.1114677108.
Houston M, Estevez A, Chumley P, Aslan M, Marklund S, Parks DA, Freeman BA (1999) Binding of xanthine oxidase to vascular endothelium. Kinetic characterization and oxidative impairment of nitric oxide-dependent signaling. J Biol Chem 274: 4985-4994. https://doi: 10.1074/jbc.274.8.4985.
Xue X, Ramakrishnan S, Anderson E, Taylor M, Zimmermann EM, Spence JR, Huang S, Greenson JK, Shah YM (2013) Endothelial PAS domain protein 1 activates the inflammatory response in the intestinal epithelium to promote colitis in mice. Gastroenterology 145: 831-841. https://doi: 10.1053/j.gastro.2013.07.010.
Kundu TK, Velayutham M, Zweier JL (2012) Aldehyde oxidase functions as a superoxide generating NADH oxidase: an important redox regulated pathway of cellular oxygen radical formation. Biochemistry 51: 2930-2939. https://doi: 10.1021/bi3000879.
Kono H, Rusyn I, Uesugi T, Yamashina S, Connor HD, Dikalova A, Mason RP, Thurman RG (2001) Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat. Am J Physiol Gastrointest Liver Physiol 280: G1005-1012. https://doi: 10.1152/ajpgi.2001.280.5. G1005.
Lam G, Apostolopoulos V, Zulli A, Nurgali K (2015) NADPH oxidases and inflammatory bowel disease. Curr Med Chem 22: 2100-2109. https://doi: 10.2174/0929867322666150416095114.
Ivanov V, Shakhristova E, Stepovaya E, Litvjakov N, Perekucha N, Nosareva O, Fedorova T, Novitsky V (2018) Oxidative stress in the pathogenesis of type 1 diabetes: the role of adipocyte xanthine oxidase. Bulletin of Siberian Medicine 16: 134-143. https://doi.org/10.20538/1682-0363-2017-4-134-143
Poles MZ, Bódi N, Bagyánszki M, Fekete É, Mészáros AT, Varga G, Szűcs S, Nászai A, Kiss L, Kozlov AV et al (2018) Reduction of nitrosative stress by methane: Neuroprotection through xanthine oxidoreductase inhibition in a rat model of mesenteric ischemia-reperfusion. Free Radic Biol Med 120: 160-169. https://doi: 10.1016/j.freeradbiomed.2018.03.024.
Caty MG, Schmeling DJ, Friedl HP, Oldham KT, Guice KS, Till GO (1990) Histamine: a promoter of xanthine oxidase activity in intestinal ischemia/reperfusion. J Pediatr Surg 25: 218-222; discussion 222-213. https://doi: 10.1016/0022-3468(90)90406-y.
Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A et al (2001) C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 43-54 https://doi: 10.1016/s0092-8674(01)00507-4.
Yuan Y, Hilliard G, Ferguson T, Millhorn DE (2003) Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 278: 15911-15916. https://doi: 10.1074/jbc.M300463200.
Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721-732. https://doi: 10.1038/nrc1187.
Lee G, Won HS, Lee YM, Choi JW, Oh TI, Jang JH, Choi DK, Lim BO, Kim YJ, Park JW et al (2016) Oxidative Dimerization of PHD2 is Responsible for its Inactivation and Contributes to Metabolic Reprogramming via HIF-1α Activation. Sci Rep 6: 18928. https://doi: 10.1038/srep18928.
Guzy RD, Sharma B, Bell E, Chandel NS, Schumacker PT (2008) Loss of the SdhB, but Not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Mol Cell Biol 28: 718-731. https://doi: 10.1128/MCB.01338-07.
Hagen T (2012) Oxygen versus Reactive Oxygen in the Regulation of HIF-1alpha: The Balance Tips. Biochem Res Int 2012: 436981. https://doi: 10.1155/2012/436981.
Galván-Peña S, O'Neill LA (2014) Metabolic reprograming in macrophage polarization. Front Immunol 5: 420. https://10.3389/fimmu.2014.00420.
Louis NA, Hamilton KE, Canny G, Shekels LL, Ho SB, Colgan SP (2006) Selective induction of mucin-3 by hypoxia in intestinal epithelia. J Cell Biochem 99: 1616-1627. https://doi: 10.1002/jcb.20947.
Corcoran SE, O'Neill LA (2016) HIF1α and metabolic reprogramming in inflammation. J Clin Invest 126: 3699-3707. https://doi: 10.1172/JCI84431.

No competing interests reported.

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Hypoxia-mediated upregulation of xanthine oxidoreductase causes DNA damage of colonic epithelial cells in colitis

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Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Materials, chemicals, and reagents

2.2 Mouse models

2.3 Cell culture, plasmid construction, and lentivirus infection

2.4 RT-qPCR analysis

2.5 Comet assay

2.6 Immunofluorescence staining

2.7 Western blotting

2.8 Statistical analysis

3. Results

3.1 The inhibition of XOR alleviated ulcerative colitis in a mouse model

3.2 XOR-mediated oxidative stress linked to the progression of acute colitis in mice

3.3 Hypoxia upregulated XOR expression and induced DNA damage in colonic epithelial cells

3.4 XOR regulates HIF1α protein levels and DNA damage in intestinal epithelial cells

3.5 The inhibition of XOR alleviated DNA damage caused by hypoxia

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1