Curcumin Resuscitate Gliadin Induced Oxidative Damage And Altered Cellular Responses In Human Intestinal Cells Via Cross-Talk Between The Transcription Factor Nrf-2 And Multifunctional Protein APE1

An imbalance between the production of oxygen and nitrogen free radicals and their degradation by the antioxidant system are the major causative factors for the wheat intolerance diseases. In the present study, we have examined the wheat gliadin protein-induced oxidative and nitrosative stress and downstream responses in the human intestinal cell lines viz. HCT-116 and HT-29. The role of phytochemical curcumin was investigated to alleviate the gliadin associated cellular damages. The focus of the study was to identify the role of key DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) in gliadin protein-induced toxicity in the intestine, which may be crucial for establishing the gut-associated diseases. Reactive oxygen species (ROS); reactive nitrogen species (RNS); mitochondrial ROS; mitochondrial trans-membrane potential; protein carbonylation; lipid peroxidation; and the oxidized DNA base damage was estimated in HCT-116 and HT-29 cells after 24 h treatment of 160 µg/ml of gliadin, 10 µM of curcumin and its combination. In addition, the transcriptional expression and enzymatic activities of antioxidants (SOD; Catalase; and GSH) were measured in the in these cells. Furthermore, the cross-talk between the nuclear factor erythroid 2-related factor-2 (Nrf-2) and the multifunctional enzyme APE1 was analyzed by the immunouorescent based imaging and co-immunoprecipitation assays. The endonuclease activity of APE1 and the DNA-protein interaction of NRF-2 with ARE was analyzed by using electrophoretic mobility shift assay (EMSA) with the nuclear lysates of HCT-116 and HT-29 cells. Results suggest that 3 h pre-treatment of curcumin followed by the treatment of gliadin protein for 24 h time protect the HCT-116 and HT-29 cells via (1) decreasing the ROS, RNS, oxidative stress, mitochondrial ROS, recuperate mitochondrial trans-membrane potential; (2) reestablishing the cellular antioxidant defence systems; (3) enhancing the DNA-repair via APE1 and which further activates the ARE elements via activation of Nrf-2. In conclusion, wheat gliadin induces the oxidative/nitrosative stress, mitochondrial damage and damages the cellular biomolecules; hence is associated with the disease pathogenesis and tissue damage in wheat intolerance diseases. The gliadin induced stress and its consequences are signicantly reduced by the pre-treatment of curcumin via DNA repair pathways and oxidative stress which is evident through the interaction between two essential proteins of these pathways APE1 and Nrf2 hence suggesting the role of curcumin based management of wheat intolerance diseases like celiac disease. µg) were mixed with 15% TCA, 0.375% TBA and 0.25 M hydrochloric acid (HCl) followed by heating at 95°C for 45 min and cooling on ice for 30 min. The samples were then centrifuged at 1000 rpm for 10 min at 4°C, the absorption was recorded at 532 nm using microplate reader (BioTek® Synergy H1), and the TBARS content was calculated by using 1.56X10 5 M − 1 cm − 1 molar extinction coecient, and results were expressed as nM/mg of protein (Gill et al. 2017). most common marker for oxidative stress-induced DNA damage is 8-oxo deoxyguanosine (8-oxo-dG) is formed by the oxidation of guanine base. The oxidized DNA damage was detected with 8-oxo-dG antibody (Trevigen®), the cells were cultured on coverslips and treated with gliadin protein, curcumin and its combination. The cells were with chilled methanol and acetone (1:1 for 15 min and air-dried. The RNA was digested with RNase enzyme followed by in-situ DNA denaturation (0.15 N NaOH in 70% ethanol for 5 min). The cells were washed with 70% ethanol containing 4% formaldehyde (v/v), 50 % ethanol, 35 % ethanol and nally PBS for 2 min each and treated with protease K (TE buffer, pH 7.5) to digest the remaining proteins. The coverslips were blocked with 5% FBS for 1 h at 37°C, then incubated with primary antibody of 8-oxo-dG (Trivegen®, 1:250 dilution) in a humidied chamber. Cells were then incubated with Alexa Fluor 488 tagged secondary antibody (Invitrogen, 1/500 dilution) and nucleus was counterstained with Hoechst 33342 (Invitrogen). The images were captured using the OLYMPUS FV1200 Confocal Laser Scanning Microscope (Upadhyay et al. 2020). signicantly the of celiac The content of protein carbonylation and MDA signicantly reduced in the curcumin pre-treated HCT-116 and HT-29 cells as compared to only gliadin treated cells. These results suggest the role of phytochemical curcumin in lowering of gliadin induced oxidative stress responses in celiac patients. in HCT-116 cells, and (ii) in HT-29 cells were examined. (D) The mRNA expression prole of different antioxidant genes viz. SOD-1, SOD-2, Catalase, GPx and Nrf-2 were evaluated through semi-quantitative RT-PCR method by keeping β-actin as an internal control; (i) representative agarose gel images, and densitometric analysis of gel images after the normalization with the expression of β-actin. The protein expression proling of Nrf-2 protein was carried out through Western blot using β-actin as an internal control in both the cell lines; representative immunoblot images and densitometric analysis of these images after normalization with the expression of β-actin. Results mean spectrophotometrically. Immunouorescence based detection of oxidative stress-induced base damage (8-oxo-dG site) was performed using confocal microscopy. The representative images are shown for: (E) HCT-116, and (F) HT-29 cells. The mRNA expression prole of APE1 gene (a key enzyme of DNA base damage repair) was performed by semi-quantitative RT-PCR using GAPDH as an internal control, (G) representative agarose gel image for APE1 gene expression, and (H) densitometric analysis of gel images after the normalization with the expression of GAPDH in both the cell lines. (I) The protein expression prole of APE1 protein was performed by Western blot using β-actin as an internal control, and (J) densitometric analysis of after normalization with the expression of β-actin in both the cell lines. The ability to abasic (AP) site repair of different nuclear extracts prepared after different treatments of both the cell lines was estimated using AP-endonuclease activity assay: (K) the representative gel images, and L) densitometric data of cleaved product for HCT-116 cells, and (M) The representative gel images, and (N) densitometric data of the cleaved product for HT-29 cells. The results were expressed as mean ± standard deviation (n=3). ***P ≤ 0.005, *P ≤ 0.05 (Control vs. Gliadin/Curcumin/H2O2 in both the cell lines), #P ≤ 0.05 (Gliadin Vs. Gliadin+Curcumin in both the cell lines).


Introduction
Wheat is a staple food of many countries, including India and provides about 20% of calories intake from the total food consumption. It is an important source of dietary proteins, carbohydrates and other essential nutritional components. Despite their plenty of bene ts, wheat proteins are the main cause of many diet-induced health issues like wheat protein-induced allergies, Celiac disease (CD), Non-celiac gluten sensitivity (NCGS), wheat intolerance etc. Wheat grain proteins are of two types, either metabolic proteins or storage proteins. Metabolic proteins are albumin and globulin; whereas storage protein is gluten which includes gliadin and glutenin. These storage proteins are responsible for the unique viscoelastic characteristics of wheat dough and provide rheological properties to the wheat our, which is essential for food processing and bakery (Liu et al. 2010). The stored proteins are the main trigger for different allergic reactions and can induce autoimmune responses in genetically susceptible individuals.
In the recent years, the prevalence of wheat-related allergies have drastically increased in the general population, where the prevalence of CD is about 1% worldwide (Singh et al. 2018), NCGS is about 0.5-13% (Molina-Infante et al. 2015), and other wheat allergies/hypersensitivity is about 0.1% − 3.6% (Scherf 2019). The key players of these diseases are the excessive production of ROS/RNS, oxidative stress, and imbalance in the antioxidant defence system (Monguzzi et al. 2019; Moretti et al. 2018). Oxidative stress due to de ciencies in cellular repair processes or change in mitochondrial redox potentials can result in persistently high levels of oxidative base lesions in the DNA. APE1, a primary enzyme responsible for recognition and incision of apurinic/apyrimidinic/abasic (AP) sites in the DNA; and is also responsible for the redox activation of multiple cellular transcription factors (TFs) like nuclear factor-kappa B (NF-κB), activator protein 1 (AP-1), hypoxia-inducible factor 1-alpha (HIF-1α), Nrf-2 etc. (Thakur et al. 2015). Nrf-2, a redox-sensitive transcription factor and the master regulator of phase-II antioxidant enzymes; controls the expression of a range of antioxidant response element (ARE) dependent genes to regulate the physiological and pathological consequences (Sun et al. 2007). There are many phytochemicals with antioxidant properties out of which one of the most common is curcumin; a most studied and primary active curcuminoid of turmeric which is derived from the rhizome of Curcuma longa (Amalraj et al. 2017). Curcumin scavenges the endogenous ROS and RNS by modulating the Nrf-2, NF-κB, and AP-1 proteins (González-Reyes et al. 2013; Pinkus et al. 1996). In the present study, we have examined the gliadin (a main storage protein of the wheat grain) induced oxidative and nitrosative stress and its consequences in the human colon cancer cell lines HCT-116 and HT-29. The role of phytochemical curcumin to modulate the harmful effects of gliadin via functional interaction between APE1 and Nrf-2 are studied, advocating for the functional role, and the cross-talk between these two critical cellular bio-molecules is essential for therapeutic interventions for the human wheat allergies.

Materials And Methods
Cell culture Human colon cancer cell lines HCT-116 and HT-29 were purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Both the cell lines were cultured in high glucose Dulbecco's Modi ed Eagle's Minimal Essential Medium (DMEM; Gibco/ HyClone) supplemented with 10% heat-inactivated FBS (Gibco/ Sigma-Aldrich), 1X antibiotic solution (penicillin-streptomycin; HiMedia) in tissue culture asks (Corning) at 37°C in a humidi ed atmosphere of 5% CO 2 incubator (New Brunswick, Galaxy® 170S). The 70 to 80% con uent cells were trypsinized using 1X trypsin-EDTA and sub-cultured for further experiments (Gupta et al. 2018).
Preparation of whole-cell lysates, cytoplasmic extracts and nuclear extracts The whole-cell lysates were prepared using the RIPA buffer [20 mM  1 h at 37°C in the dark. The unbound dye was removed, and cells were washed with PBS. The uorescence intensity was recorded at an excitation/emission 485/535 nm using a microplate reader (BioTek® Synergy H1). Percent change in the level of ROS was calculated considering control as absolute. (b). Nitroblue tetrazolium (NBT) based assay: As a second measure for ROS NBT assay was accomplished as HCT-116, and HT-29 cells were cultured in 96 well plates at a cell density 1×10 4 cells per well, and overnight cultured cells were treated for 24 h. After completion of treatment, cells were incubated with freshly prepared NBT solution (0.1% w/v) for 3 h at 37°C (Sim Choi et al. 2006). The cells were washed twice with PBS and once with chilled methanol, the NBT deposited inside the cells were then dissolved in 2 M KOH and DMSO. The absorbance was read at 570 nm using a microplate reader (BioTek® Synergy H1) (Sarkar et al. 2017). (c). DAF-FM assay: The intercellular RNS level in the HCT-116 and HT-29 cells treated with gliadin, curcumin and their combinations and untreated as control cells were measured using cell-permeable uorescent probe-based DAF-FM dye. Brie y, both HCT-116 and HT-29 cells were cultured in 96 well plates at a cell density 1×10 4 per well, and after the treatments, these cells were incubated with 20 µM DAF-FM (Molecular Probes™ Invitrogen) in the dark at 37°C for 1 h, the unbound dye was removed, and cells were washed with PBS. Fresh PBS was added, and uorescence intensity was recorded at an excitation/emission wavelength at 478/515 nm using a microplate reader (BioTek® Synergy H1) again the plate was incubated in the dark at 37°C for 30 min, and another uorescence intensity was recorded and the data represented as percentage change concerning control (Dhiman, Garg 2014;Gupta et al. 2018). (d). Nitric Oxide (NO) Level: The secretory/extracellular nitric oxide (NO) levels were measured in the culture supernatants using Griess assay. Brie y, equal volumes of culture supernatant and Griess reagent (1% sulfanilamide and 0.1% of NEDD) were mixed. The absorbance of formed chromophore was recorded at 543 nm in a microplate reader (BioTek® Synergy H1). The nitrite content of each sample was evaluated from a standard curve obtained after linear regression made with the known concentration of sodium nitrate (0-100µM) and represented as the percent change in NO levels considering control as absolute (Gupta et al. 2018;Dhiman et al. 2013).
Assays to Examine the Mitochondrial Health washed thrice with PBS and resuspended in 100 µl of PBS. The JC-1 monomers (green) and JC-1 aggregates (red) were detected by FL1 and FL2 channels, respectively using BD Accuri C6 ow cytometer. MTP is calculated as a change in the green/red uorescence intensity ratio (Gupta et al. 2009).

Antioxidant Enzymes Status
(a) SOD activity: Superoxide dismutase (SOD) activity was measured using pyrogallol auto-oxidation. Brie y, the reaction was started in a mixture having 0.1 mM sodium phosphate buffer (pH 8), 3 mM EDTA, and 8.1 mM pyrogallol with cell lysate. The change in absorbance was recorded at 0 min and 3 min at 420 nm wavelength using Shimadzu double-beam spectrophotometer against buffer as blank. SOD activity is expressed as Unit/mg of protein where one unit of SOD activity is de ned as the amount of enzyme that causes half-maximal inhibition of auto-oxidation of pyrogallol. The nal graph was plotted by converting it in percentage concerning untreated control cells (Gill et al. 2017). (b) Catalase activity: The activity of catalase enzyme was measured using the method as described by Aebi (1984) with slight modi cations. Brie y, the reaction was started by adding the cell lysate into the reaction mixture having 10 mM H 2 O 2 (substrate for Catalase) and 25 mM potassium phosphate buffer (pH 7.0). The activity was recorded for the degradation of H 2 O 2 at 240 nm over 1 min using spectrophotometer (Aebi 1984). The activity of the enzyme was expressed as µM/min/mg protein using 39.4 mM − 1 cm − 1 molar extinction coe cient (de Sousa et al. 2013). (c) Total glutathione content: Amount of total glutathione in the treated and untreated cells were measured using DTNB [(5,5′-Dithiobis-(2-nitrobenzoic acid)] also known as Ellman's reagent. DTNB reacts with the glutathione and forms a yellow coloured 5-thionitrobenzoic acid (TNB) product (Rahman et al. 2006). Brie y, 100 µg of total cell lysates were precipitated using 25% trichloroacetic acid (TCA), after centrifugation supernatant was mixed with 0.6 mM DTMB and incubated in the dark for 15 min at room temperature. Absorbance was recorded at 412 nm using a microplate reader (BioTek® Synergy H1). The total content of glutathione was estimated using linear regression mode with the known concentration of glutathione (0-50 µM) (Gill et al. 2017). . Brie y, cell lysates (100 µg) were mixed with 15% TCA, 0.375% TBA and 0.25 M hydrochloric acid (HCl) followed by heating at 95°C for 45 min and cooling on ice for 30 min. The samples were then centrifuged at 1000 rpm for 10 min at 4°C, the absorption was recorded at 532 nm using microplate reader (BioTek® Synergy H1), and the TBARS content was calculated by using 1.56X10 5 M − 1 cm − 1 molar extinction coe cient, and results were expressed as nM/mg of protein (Gill et al. 2017). (c) DNA damage detection: The most common marker for oxidative stress-induced DNA damage is 8-oxo deoxyguanosine (8-oxo-dG) which is formed by the oxidation of guanine base. The oxidized DNA damage was detected with 8-oxo-dG antibody (Trevigen®), the cells were cultured on coverslips and treated with gliadin protein, curcumin and its combination. The cells were then xed with chilled methanol and acetone (1:1 ratio) for 15 min and airdried. The RNA was digested with RNase enzyme followed by in-situ DNA denaturation (0.15 N NaOH in 70% ethanol for 5 min). The cells were washed with 70% ethanol containing 4% formaldehyde (v/v), 50 % ethanol, 35 % ethanol and nally PBS for 2 min each and treated with protease K (TE buffer, pH 7.5) to digest the remaining proteins. The coverslips were blocked with 5% FBS for 1 h at 37°C, then incubated with primary antibody of 8-oxo-dG (Trivegen®, 1:250 dilution) in a humidi ed chamber. Cells were then incubated with Alexa Fluor 488 tagged secondary antibody (Invitrogen, 1/500 dilution) and nucleus was counterstained with Hoechst 33342 (Invitrogen). The images were captured using the OLYMPUS FV1200 Confocal Laser Scanning Microscope (Upadhyay et al. 2020).

Determination of Oxidative Stress
Apurinic/Apyrimidinic Endonuclease (APE) activity assay A 5' FAM labelled, 52 mer oligonucleotide having tetrahydrofuran (THF; analogue of AP site) at 31st nt (5 -GATCTGATTCCCCATCTCCTCAGTTTCACTTHFAGTGAAGGCATGCACCCTTCT-3') and its complementary strand with 'A' opposite THF was procured from Imperial Life Sciences (ILS), India. These oligonucleotides are annealed in annealing buffer (10 mM Tris base, 50 mM NaCl, 0.1 mM EDTA). 4 pM duplex probe was incubated with freshly prepared nuclear lysates in a reaction buffer [50 mM Tris base (pH 8.5), 8 mM MgCl 2 , 1 mM DTT, 1 mg/ml BSA, 4% Glycerol] at 37°C for 3 min. The reaction was stopped by adding 10 µl of stop buffer [10 mM EDTA, 3-4% Formaldehyde, 0.01% BPB, 30% Glycerol] followed by heating at 95°C for 5 min and kept it on ice till resolution of denaturing gel. The substrate and cleaved product was resolve on 12% polyacrylamide gel having 7 M urea in 1X TAE buffer. The uorescence image was captured using the Bio-Rad Chemi Doc™ MP system, and densitometric analysis was done using Image Lab™ software of Bio-Rad version 5.

Isolation of total RNA and Semi-quantitative RT-PCR analysis
Total RNA from the treated and untreated cells of HCT-116 and HT-29 was isolated using TRIzol® reagent (Invitrogen, USA) as per manufacturer's protocol. The quality of isolated RNA was quanti ed on NanoDrop 2000 (Thermo Scienti c) and on denaturing agarose gel. Genomic DNA contamination was removed by using the TURBO DNA-free™ Kit (Invitrogen, USA), followed by synthesis of cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad). The cDNA was used as a template for the ampli cation of the various genes by using gene-speci c primer pairs (SOD-1, SOD-2, Catalase, GPx, NOS-2, NOS-3, Nrf-2, APE1) along with housekeeping genes (β-actin and GAPDH) as an endogenous control ( Table 1). The PCR product was separated on 1.5% agarose gel contains ethidium bromide (EtBr) along with 100bp DNA ladder (Tracklt™100 bp DNA ladder, Invitrogen). The gel images were captured using the Bio-Rad Gel Doc™XR system, and densitometric analysis was done using Image Lab™ software of Bio-Rad version 5.  The whole-cell lysates were resolve on 10% denaturing SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Ponceau S staining was performed to con rm equal protein loading and transfer. The membrane was blocked with 5% nonfat dry milk (NFDM) in PBS having 0.1% Tween-20 (PSBT) for 2 h. Membranes were then incubated with primary antibody for anti-Nrf-2 (Santa Cruz Biotechnology, 1:1000 dilution), anti-APE1 (Biogenuix, 1:1000 dilution), anti-LaminB1 (Invitrogen, 1:750 dilution) and anti-β-actin (Invitrogen, 1:5000 dilution) in 5% NFDM-PSBT for overnight at 4°C. After three washes with PBST membrane was further incubated with respective horseradish peroxidase (HRP) conjugated antimouse/rabbit IgG secondary antibody (GeNei™, 1:5000) in 5% NFDM-PSBT. Peroxidase activity was captured using enhanced chemiluminescence reagent of Bio-Rad, and images were visualized using Bio-Rad Chemi Doc™ MP system and densitometric analysis was done using Image Lab™ software of Bio-Rad version 5.2.
Immuno uorescence Based Subcellular Distribution and Co-localization Studies of APE1 and Nrf-2 Subcellular distribution and co-localization of APE1, as well as Nrf-2, were examined using immuno uorescence based confocal laser scanning microscopy (CLSM). The HCT-116 and HT-29 cells were grown on sterile coverslips and treated as described in the previous section. After treatment, the cells were xed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature followed by three times wash with PBS. Cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washing with PBS, cells were blocked with 10% FBS for 1 h at 37°C followed by overnight incubation with primary antibodies against anti-APE1 (Biogenuix, 1:100 dilution) and anti-Nrf-2 (Santa Cruz Biotechnology, 1:100) in a humidi ed chamber at 4°C. The glass coverslips were washed with PBS three times and incubated at room temperature for 1 h with respective secondary antibodies, Alexa Fluor 488-conjugated anti-mouse IgG or Alexa Fluor 647-conjugated anti-rabbit IgG (Invitrogen, 1:100 dilution). After washing thrice, the coverslips were incubated in 0.1% solution of Hoechst 33342 (Invitrogen) at 37°C for 10-15 min. The glass coverslips were washed with PBS and mounted with a mounting solution (Sarkar et al. 2017). The uorescence images were captured using a Laser Scanning Confocal Microscope (Olympus FV1200).
Co-Immunoprecipitation for con rming the interaction between APE1 and Nrf-2 In vitro interaction of APE1 and Nrf-2 was stabilized by a co-immunoprecipitation reaction of APE1 and Nrf-2 using magnetic beads coated with protein A/G from Bio-Rad (SureBeads™). Brie y, 100 µl of SureBeads™ were taken in 1.5 ml tube after thorough resuspension, magnetized beads and discard the supernatant and wash with PBST thrice. The beads were incubated with anti-APE1 antibody (10 µg) for 2 h at 4°C on rotation. After incubation beads were again magnetized and the supernatant was discarded followed by 3 times washing with PBST and again incubation with whole-cell lysates (about 300 µg) and rotate for overnight at 4°C. Beads were magnetized and discard the supernatant and wash thrice with PBST. Now, elute it in fresh Laemmli buffer having β-mercaptoethanol by heating at 70°C for 10 min followed by magnetization and collection of supernatant in a fresh tube. Again heat it at 95°C for 5 min and performed Western blotting as described above (in western blotting section) against primary antibody anti-Nrf-2 (Santa Cruz Biotechnology at 1:1000 dilution). After taking image again, the blot was In-silico studies for understanding the interactions between APE1 and Nrf-2 The possible interacting partners of APE1 protein was predicted in-silico by using the STRING database (version 11.0). The database shows the direct and indirect association of proteins in over 2000 organisms. This database critically assesses the known interactions (from experimentally proven, curated database), and predict/possible interactions (from text mining, protein homology study, gene neighboring, co-occurrence and co-expression analysis (Sarkar et al. 2017).

Statistical Analysis
Experiments were performed in triplicates unless it speci ed, and data is represented as the mean value ± standard deviation (S.D), and statistical analysis of obtained data was done using student's t-test. The values of p ≤ 0.05 were considered statistically signi cant.

Assessment of Mitochondrial ROS and Trans-membrane Potential
The ROS produced within the mitochondria upon treatment with gliadin in the HCT-116, and HT-29 cell lines were estimated via MitoSox™ dye (green), and mitochondria were tracked by MitoTracker™ dye (red) using uorescence microscopy. Green uorescence intensity is directly proportional to the ROS production within the mitochondria. The microphotographs obtained using confocal uorescence microscope showed that the uorescence intensity of green dye is high in gliadin treated HCT-116 and HT-29 cells, curcumin, and H 2 O 2 treated cells also show an increase in green uorescence intensity when compared with untreated control cells. In the cells pre-treated with curcumin followed by treatment with gliadin show relatively low green uorescence signal as compared to only gliadin treated cells ( Figure: 1.E. i & ii). Mitochondrial Transmembrane Potential (MTP) (ΔΨm), is a thoughtful parameter to sense the mitochondrial health which can be estimated using JC-1 dye which forms orange-red aggregates in healthy cells and remain green coloured monomers in unhealthy cells. The ratio of red to green uorescence indicates mitochondrial health. The results show that red to green ratio decreases by 70-80% in HCT-116 and HT-29 cells when treated with gliadin protein and H 2 O 2 as compared with untreated control cells; whereas the cells pre-treatment with curcumin followed by treatment with gliadin show about 2-4 fold increase in red to green uorescence ratio as compared to only gliadin treated cells ( Figure: 1.E. iii & iv).

Assessments of Cellular Antioxidant Enzyme Status
Superoxide dismutase Levels: The SOD enzyme activity was measured spectrophotometrically and was Interaction study for APE1 and Nrf-2 The interaction between the APE1 and Nrf-2 protein was retrieved in-silico by using STRING database and found that the interaction of APE1 (APEX1) with Nrf-2 (NFE2L2) protein was mediated through some intermediate proteins like FEN1, RBX1, MCM2, KEAP1, CDKN1A and others ( Figure: 4.I). For further con rmation of the physical interaction of APE1 and Nrf-2 protein co-immunoprecipitation (Co-IP) reaction was performed in both the cell lines which show that APE1 and Nrf-2 are potentially interacting partners and they physically interact with each other. This interaction gets stronger by gliadin stress, and again the pre-treatment of curcumin try to normalize this interaction in both the cell lines studied ( Figure: 4.J).

Discussion
Gluten is the main seed storage protein of wheat, barley and rye, which are consumed worldwide. It has complex protein networks and composed of gliadin and glutenin parts (Wieser 2007  Currently, the only effective treatment for these diseases is a strict lifelong gluten-free diet, i.e. excluding all wheat, rye and barley containing food products from the diet. The pathophysiology of wheat intolerance is not fully elucidated, and the bene cial role of phytochemical curcumin (herbal products) which is having a wide array of bene cial effects in human health are also not studied so for in these diseases.
The present study attempts to explore the in vitro molecular pathways of harmful effects of gliadin protein and also the role of curcumin (one of the most used spices) in maintaining the cellular In the present study, it was found that ROS level was signi cantly reduced near to the level of untreated control HCT-116 and HT-29 cells when these cells were pre-treated with the curcumin (10 µM) for 3 h followed by the exposure/treatment of gliadin. The pretreatment of curcumin also signi cantly diminished the production of NO against gliadin exposure.
Gliadin protein is rich in proline and glutamine amino acids (a member of prolamin family) and the de ciency of prolyl endopeptidases enzyme; its digestion is not completed (Marti et al. 2005  Abasic sites, the DNA damage which is created by oxidative stress is mainly repaired via BER pathway by using APE1 enzyme along with other BER enzymes. APE1 is a multifunctional enzyme, along with its role in DNA base repair, cleaving of AP sites, it also helps in maintaining the cellular redox homeostasis by interacting and activating many key transcription factors like Nrf-2, AP-1, NF-κB, p53, HIF1α and others From the literature and STRING database analysis, it was predicted that these two critical proteins are directly interacting with each other. Further, the physical interaction was con rmed in vitro conditions using co-immunoprecipitation (Co-IP) experiment. Previous reports have also identi ed an interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumour cells and suggested to be playing a crucial role in overcoming the resistance against experimental drugs targeting Ref -1 activity. The present study and other studies from our group (Sarkar et al. 2017;Thakur et al. 2020) advocate the physical interaction between APE1 and Nrf-2 which is associated with regulation of many cell survival pathways leading to the re-establishment of cellular homeostasis against oxidative stress.
However, the speci c transcription factors and their downstream translational implications need to be elucidated further in the human pathophysiology diseases including wheat protein-induced allergies.

Summary And Conclusion
The experimental data presented suggests that pure gliadin protein induces oxidative stress by increasing ROS/RNS and disturbs the antioxidant system which damages the cellular macromolecules in both the human colon cancer HCT-116 and HT-29 cell lines. Additionally, the pivotal role of APE1 and Nrf-2 proteins along with their subcellular localization during the oxidative stress being demonstrated. This study also establishes the interaction of these two proteins and their key role in maintaining the cellular homeostasis against gliadin induced oxidative stress (Figure: 5). Further, the role of curcumin was investigated which signi cantly revitalizes the colon cancer cells against gliadin induced stress and modulated its adverse effects via DNA repair pathways and oxidative stress which is evident through the cross-talk between two crucial proteins of these pathways APE1 and Nrf-2 hence suggesting the role of curcumin based management of wheat intolerance diseases like celiac disease.