Evaluation of the antioxidant properties of carexanes in AGS cells transfected with the Helicobacter pylori's protein HspB

Microbial Pathogenesis 108 (2017) 71e77

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Evaluation of the antioxidant properties of carexanes in AGS cells transfected with the Helicobacter pylori's protein HspB Elisabetta Buommino a, *, 1, Brigida D'Abrosca b, 1, Giovanna Donnarumma c, Annamaria Parisi c, Monica Scognamiglio b, Antonio Fiorentino b, 1, Antonio De Luca d, 1 a

Department of Pharmacy, University of Naples Federico II, 80131, Italy
degli Studi della Campania Luigi Vanvitelli, 81100 Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Universita Caserta, Italy c
degli Studi della Campania Luigi Vanvitelli, Naples, Department of Experimental Medicine, Section of Microbiology and Clinical Microbiology, Universita 80138, Italy d
degli Studi della Campania Luigi Vanvitelli, Department of Mental and Physical Health and Preventive Medicine, Section of Human Anatomy, Universita Naples, 80138, Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 December 2016 Received in revised form 26 April 2017 Accepted 2 May 2017 Available online 4 May 2017

Naturally derived compounds represent a potential source of pharmacologically active drugs able to contrast different diseases, including gastric cancer, a multifactorial disease, in which the important role played by H. pylori infection has been demonstrated. Carexanes, stilbene derivatives, isolated from plants of the Carex distachya Desf., are unusual secondary metabolites with a tetracyclic skeleton arising from a cyclization of prenylstilbenoid precursors. In this study we firstly showed the ability of three purified carexanes CxB, CxG, and CxI to enhance the antioxidant response of AGS cells and to contrast the effect of the H. pylori's protein HspB. Among them CxI was the molecule that best modified the expression of genes involved in the antioxidant response. In particular, CxI was able to reduce Keap-1 gene expression and induce NQO1 gene expression, both at 4 and 24 h in AGS cells, as showed by real time PCR. Nrf2 induction was evident only at 24 h. Interestingly, the effect of CxI was stronger in HspB-transfected AGS cells, where Keap-1 gene expression was nearly abrogated. Finally, we demonstrated that CxI was able to reduce also COX-2 gene expression in HspBtransfected AGS cells, compared with untreated HspB-transfected cells, both at 4 and 24 h. This study first report that carexanes might represent candidate molecules able to contrast the deleterious effect of HspB protein but also to reduce the inflammatory process induced by H. pylori infection. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Helicobacter pylori HspB Nrf2/keap-1 pathway Phase II enzyme Carexanes

1. Introduction Gastric cancer is one of the most common malignancies in the world, although the incidence and mortality rate have been decreasing in recent decades. The association between H. pylori and gastric cancer has attracted great interest worldwide. The International Agency for Research on Cancer (IARC) identified H. pylori as a “group 1 (definite carcinogen)” in 1994 on the basis of the

* Corresponding author. Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano, 49, 80131, Italy. E-mail address: [email protected] (E. Buommino). 1 The first two authors, E.B. and D.B., and the last two (A.F and A.DL) equally contributed to this work. http://dx.doi.org/10.1016/j.micpath.2017.05.007 0882-4010/© 2017 Elsevier Ltd. All rights reserved.

results of epidemiologic studies [1]. Gastric mucosal infection with H. pylori is accompanied by infiltration of neutrophils, and activated inflammatory cells are known to produce oxygen radicals. Davies et al. have reported an increase of oxygen radical production in both the duodenal and gastric pyloric mucosa after infection with H. pylori [2]. Reactive oxygen species (ROS) activate signaling pathways involved in cell proliferation and migration, as well as damage macromolecules like DNA and proteins, leading to mutations [3]. Antioxidants counteract oxidative stress, reducing ROS levels in the intracellular environment; thus the beneficial impact of antioxidant molecules in reducing cancer risk is well appreciated, since they may confer a survival advantage [4,5]. Nrf2 (nuclear factorerythroid 2-related factor 2) is an important regulator of the

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cellular response against oxidative stress [6]. It coordinates induction of genes encoding numerous antioxidants, phase II detoxifying enzymes and related proteins such as catalase, superoxide dismutase (SOD), NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1 (HO-1), glutathione S-transferase (GST), glutathione peroxidase, and others. When oxidative stress occurs, Nrf2 is released from its repressor molecule, Keap1 (Kelch-like ECHassociated protein 1), and translocates to the nucleus to promote the transcription of the antioxidant and detoxifying enzyme [7]. During the past decades, a widespread search has been launched to identify new anti-cancer therapies from natural sources. Herbs, medicinal plants, spices, vegetables have showed to represent a potential source to combat various diseases, including gastric cancer [8e10]. It is now accepted that Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic-ulcer disease and gastric carcinoma [1,11]. We recently reported evidences that the HspB protein of H. pylori interferes in the Nrf2/Keap-1 pathway [12]. The increasing therapeutic failures against H. pylori infection has determined the need to develop new drugs. 3,5,4-trihydroxy-transstilbene known as trans-resveratrol, a phenolic compound present in grapes, wine, peanuts, and other food products, has antioxidant properties, exerts a potent anti carcinogenic activity and protective effects against atherogenesis and cardiovascular diseases [13]. Moreover it has been reported to possess anti-H. pylori activity in vitro [14]. Recently, trans-resveratrol has also been tested on H. pylori CagA þ strains from patients with gastric carcinoma, displaying a strong antibacterial activity [15]. Plants of the Carex genus produce oligostilbenes comprising from two to four monomers of resveratrol, most of them showing antimicrobial activity [16,17]. Carexanes are secondary metabolites isolated only from Carex distachya Desf., so far characterized by an unusual tetracyclic skeleton arising from a cyclization of prenylstilbenoid precursors [18]. Few articles report the isolation of prenylated stilbenes from natural sources. These compounds have been identified as cytotoxic against ovarian cancer cell lines [19]. Here we demonstrated that carexanes enhance the antioxidant response of HspB-transfected human gastric epithelial (AGS) cells reducing the risk of gastric cell transformation. 2. Materials and methods 2.1. General experimental procedures Optical rotations were measured on a PerkineElmer 141 in MeOH solution. UV spectra were performed on UV-1700 Shimadzu spectrophotometer in MeOH. NMR spectra were recorded at 300.03 MHz for 1H and 75.45 MHz for 13C on a Varian Mercury300 spectrometer Fourier transform NMR in CD3OD or CDCl3 solutions at 25  C. Analytical TLC was performed on Merck Kiesel gel 60 F254 plates with 0.2 mm layer thickness. Spots were visualized by UV light or by spraying with H2SO4/AcOH/H2O (1:20:4). The plates were then heated for 5 min at 110  C. Preparative TLC was performed on Merck Kiesel gel 60 F254 plates, with 0.5 or 1.0 mm film thickness. Column chromatography (CC) was performed on Merck Kieselgel 60 (70e240 mm), Merck Kieselgel 60 (40e63 mm), Sephadex LH-20®. 2.2. Plant material Plants of Carex distachya Desf. (Cyperaceae) were collected in June 2012, in the vegetative state, in the Nature Reserve of Castelvolturno, near Caserta (Italy), and identified by Dr Assunta
degli Studi della Campania Luigi Vanvitelli. A Esposito of Universita

voucher specimen (CE278) has been deposited at the Herbarium of the Dept. of Environmental, Biological and Pharmaceutical Sciences
degli Studi della Campania Luigi and Technologies of Universita Vanvitelli. 2.3. Extraction and isolation of the metabolites Fresh roots of Carex distachya (3.0 kg) were extracted with hexane for 5 days at 4  C in the dark. The organic solutions were distilled under reduced pressure by a Rotavapor® to obtain a crude extract (54.0 g), which was chromatographed on CC SiO2 eluting with hexane/EtOAc increasing polarity solutions and collecting three fractions (AeC). Fraction A, eluted with hexane-EtOAc(9:1) was chromatographed on Sephadex LH-20® eluting with hexane-CHCl3-MeOH (3:1:1) to obtain a fraction which was purified by SiO2flash-CC eluting with hexane-EtOAc (7:3) to obtain pure carexane I (CxI, 25.0 mg). Fraction B, eluted with hexane-EtOAc(9:1) re-chromatographed on Sephadex LH-20® eluting with hexane-CHCl3-MeOH (3:1:1) to obtain a fraction which was purified by SiO2TLC eluting with hexane-EtOAc (4:1) to obtain pure carexaneB (CxB, 10.2 mg). Fraction C, eluted with hexane-EtOAc(9:1) and rechromatographed on Sephadex LH-20® eluting with hexaneCHCl3-MeOH (1:1:1) gave pure carexane G (CxG, 23.6 mg). 2.4. Cell cultures, stable DNA transfection and treatments AGS human gastric epithelial cells (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 50 mg/mL penicillinestreptomycin in an atmosphere of 5% CO2 at 37  C. Stable transfected AGS cells were generated with mammalian expression vector for pcDNA3T7Tag-HspB (Invitrogen - Carlsbad, California USA) and pcDNA3 empty vector as control using Lipofectamine (Invitrogen) according to the manufacturer's instruction as previously described [12,20]. Afterwards, the cells were plated in 60 mm dishes (1  106 cells/dish) 24 h before transfection. Stably transfected cells were selected with 800 mg/ml G418. Experiments were performed on AGS cells stably transfected with HspB and cultured with complete DMEM supplemented with 800 mg/mL G418 for at least 1 month. Semi confluent AGS and stable transfected AGS cells were treated with different concentrations of CxB, CxG, CxI (50-75-100 mg/mL) for 4 and 24 h, to better analyze the effect of carexanes on gene expression after a short and longer treatment. Any differences was observed between control cell and pcDNA3 empty vector, in terms of gene expression modification, as also previously demonstrated [12]. Thus, in this study control cells refers to untreated AGS cells. 2.5. Morphological analysis The morphological features of AGS cells treated with CxB, CxG and CxI were defined by phase-contrast microscopy (Olympus CDK40) at 20 magnification. 2.6. MTT cell proliferation assay AGS cells (4  103) treated or not with CxB, CxG, CxI(50-75100 mg/mL) were grown in microplates (tissue culture grade, 96 wells, flat bottom) in a final volume of 100 ml DMEM, at 37  C and 5% CO2. After 24 h, 10 ml of the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) labelling reagent (Roche Diagnostics, Basel, Switzerland; final concentration 0.5 mg/ml) were added to each well. After 4 h, 100 ml of the solubilisation solution

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2.5 U of Taq DNA polymerase (Roche Diagnostics) in a final volume of 50 mL. PCR was carried out in the presence of 0.5 mM sense and antisense HspB primers. Conditions and size of the product are shown in Table 1. The reaction was carried out in a DNA thermal cycler (Mastercycler gradient; Eppendorf, Milan, Italy). b-actin RTPCR was performed on mRNA extracted to confirm that the mRNA was suitable for RT-PCR analysis. PCR products were analyzed by electrophoresis on 1.8%agarose gel in TBE.

(10% SDS in 0.01 M HCl) were added and the cultures were incubated overnight. The spectrophotometric absorbance was measured using a microplate ELISA reader (Biorad) at 570 nm wavelength. 2.7. Real-time polymerase chain reaction (real-time PCR) Total RNA was isolated with the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany). One hundred nanograms of total cellular RNA was reverse-transcribed (Expand Reverse Transcriptase, Roche Diagnostics) into complementary DNA (cDNA) using random hexamer primers (random hexamers, Roche Diagnostics), at 42  C for 45 min, according to the manufacturer's instructions. The resulting cDNA was subjected to realtime PCR analysis by rapid cycling in glass capillaries with a thermocycler (Light-Cycler; Roche Molecular Biochemicals Germany). The reaction was performed in a final volume of 20 mL with LightCycler-DNA Master SYBR Green I (Roche Diagnostics), which contains nucleotides, Taq DNA polymerase, buffer, SYBR Green I dye, and 10 mmol/L MgCl2; cDNA(2 mL), primers, and sterile H2O were added. For primer sequences, PCR conditions, and size of products, see Table 1. Each real-time PCR was performed three times for each sample. SYBR Green I fluorescence was monitored at the end of each cycle to assess the amount of PCR product formed. After completion of the amplification protocol, a melting curve analysis was performed to confirm the specificity of amplification by cooling the sample to 65  C at a rate of 20  C/second, maintaining a temperature of 65  C for 10 s and then heating at a rate of 0.2  C/second to 95  C, with continuous measurement of fluorescence. Cycle-tocycle fluorescence emission readings were monitored and analysed by Light-Cycler Data Software (Roche Molecular Biochemicals Germany). The specificity of the amplification products was verified by electrophoresis on a 1.4% agarose gel stained with ethidium bromide (1 mg/mL). All quantifications were normalized to the housekeeping gene b-actin. The percentage of gene expression increase was calculated using the following formula: [(gene expression in unstimulated conditions-target gene expression)/gene expression in unstimulated conditions] x 100.

2.9. Statistical analysis Each experiment was performed at least five times. The results are expressed as mean ± standard deviation (SD). Student's t-test was used to determine statistical differences between the means, and P < 0.05 was considered a significant difference. 3. Results 3.1. Carexanes characterization Spectroscopic data of the carexanes B, G and I (reported in the text as CxB, CxG and CxI, respectively) were identical to those already reported in our previously phytochemical investigations [21e23]. Carexanes B and G (Fig. 1), have a tetracyclic skeleton and are characterized, respectively, by two and three oxygenated functions in the ring A. On the contrary, carexane I is a tricyclic derivative bearing two methoxyls functions on the ring A and 2propenyl group on the saturated ring (Fig. 1). 3.2. Effect of carexanes treatment on the antioxidant response of AGS cells We have previously demonstrated that efficient co-expression of CagA, VacA and HspB is able to influence human gastric epithelial (AGS) cell growth [24]. Furthermore, it was demonstrated that in AGS cells stably transfected with HspB protein of H. pylori the defense mechanisms of oxidative stress are altered [12]. On the basis of the experimental evidences, we were prompted to analyze the effect of CxB, CxG and CxI on the antioxidant response of AGS cells transfected or not with HspB of H. pylori. Firstly, AGS cells were treated with different concentrations of CxB, CxG and CxI (50, 75 and 100 mg/ml) for 24 h. Since the antioxidant and anticancer properties of many compounds is largely explained by their pro-apoptotic effect it was important to verify the possible induction of apoptotic phenomena in AGS cells. As showed in Fig. 2 MTT assay demonstrated that the three compounds induced cell proliferation reduction at all used concentrations. The effect was stronger at the highest concentration (100 mg/ ml) at which cell death was also observed by phase-contrast microscopy, with increased cell detachment from the substratum, modified morphology and cell rounding (data not shown). Since at

2.8. RT-PCR (reverse transcription polymerase chain reaction) analysis Total cell RNA was isolated using the High Pure RNAIsolation Kit (Roche Diagnostics, Milan, Italy) fromAGS cells stably transfected or not with pcDNA3 empty vector and pcDNA3-HspB. One hundred nanograms of total cell RNA was reverse-transcribed (Expand Reverse Transcriptase; Roche Diagnostics) into cDNA using random hexamer primers (Random hexamers; Roche Diagnostics) at 42  C for 45 min, according to the manufacturer's instructions. Two microliters of cDNA was amplified in a reaction mixture containing 10 mMTriseHCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 200 mM dNTP, and Table 1 Human sense and antisense primers sequences. Gene Nrf2 Keap 1 NQO1 HspB COX-2

sense and antisense sequences 0

Conditions 0

5 - ACCACCCACAACTTACTgCC -3 5- gCCATAggAgTATgggggAT -30 50 CACAgCAATgAACACCATCC-30 50 TgTgACCATCATAgCCTCCA -30 50 ggCTgAACAAAAgAAgCTgg -30 5 AATgACATTCATGTCCCCgT-30 50 ATgACTTCCAAGCTggCCgTg30 5 TgAATTCTCAgCCCTCTTCAAAAA CTT CTC -30 50 gCCTTCTCTAACCTCTCC -30 5 CTgATgCgTgAAgTgC-30

45 60 45 60 40 60 40 56 50 56

cycles at 95  C for 5 s  C for 3 s, 72  C for 5 s cycles at 95  C for 5 s  C for 5 s, 72  C for 9 s cycles at 94  C for 5 s  C for 5 s, 72  C for 10 s cycles at 94  C for 5 s  C for 6 s, 72  C for 12 s cycles at 94  C for 5 s  C for 4 s, 72  C for 8 s

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Fig. 1. Chemical structures of Carexane B (CxB), G (CxG) and I (CxI).

shown). On contrary, NQO1 gene expression was modified, at both time tested, reaching a significant increase mainly at 4 h (21%). CxB slightly modified NQO1 gene expression (14%) (Fig. 4a and b, black bars) while we didn't observe NQO1 gene expression modulation in presence of CxG. Finally, Keap-1 gene expression was reduced by CxI treatment at 4 and 24 h with a decrease of 18 and 20%, respectively (Fig. 5a and b, black bars). On contrary, CxB and CxG were not able to significantly modify Keap-1 gene expression. 3.3. Effect of carexanes treatment on the antioxidant response of HspB-transfected-AGS cells

Fig. 2. Effect of carexanes on AGS cell proliferation. AGS cells were treated with various concentrations of carexanes CxG, CxB, and CxI for 24 h. The data are presented as the means ± SD of the results for five independent experiments.

At this point, to verify the effect of carexanes in an in vitro experimental model of infection, in particular on HspB protein, the latter was stably transfected in AGS cells, as previously reported [12]. As shown in Fig. 6 the levels of HspB mRNA expression were

up to 75 mg/ml we observed only an effect on cell proliferation, without evident signs of cell suffering, carexanes concentration of 75 mg/ml was used for further investigations. Next, to analyze the effect of CxB, CxG and CxI on the antioxidant response of AGS cells, gene expression of Nrf2, NQO1 (a representative downstream target of Nrf2), and keap-1 was investigated [7]. AGS cells were treated with carexanes at the concentration of 75 mg/mL for 4 and 24 h Nrf2 gene expression was modified at 24 h only by CxI compound with an increase of 32%, compared with untreated cells, while CxB and CxG did not (Fig. 3, black bars). At 4 h we didn't observe Nrf2 gene expression modulation (data not

Fig. 3. Real time PCR analysis at 4 (panel a) and 24 h (panel b) using specific primers for Nrf2. Black bars: AGS cells treated with 75 mg/ml CxB, CxG, and CxI. Grey bars: HSPB-transfected AGS cells treated with 75 mg/ml CxB, CxG, and CxI. The data shown are representative of five different experiments ±SD.*P < 0.05.

Fig. 4. Real time PCR analysis at 4 (panel a) and 24 h (panel b) using specific primers for NQO1. Black bars: AGS cells treated with 75 mg/ml CxB, CxG, and CxI. Grey bars: HSPB-transfected AGS cells treated with 75 mg/ml CxB, CxG, and CxI. The data shown are representative of five different experiments ±SD.*P < 0.05.

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significantly expressed in AGS cells stably transfected with the HspB-pcDNA3 vector (abbreviated with HspB-AGS cells), compared with pcDNA3 empty vector, demonstrating that AGS had acquired HspB plasmid. Successively, we went to analyze the effect of carexanes on HspB-AGS cells, comparing the results with untreated-HspBtransfected cells. The results obtained were of great interest, since we observed the ability of carexanes, in particular CxI, to strongly contrast the effect of HspB protein. HspB induced the downregulation of Nrf2, NQO1 and induction of Keap-1 gene expression in HspB-AGS cells, compared with non-transfected control cells (Figs. 3e5, grey bars). These results were in line with our previous study [12]. However, the treatment of HspB-AGS cells with 75 mg/ml CxB and CxI strongly induced Nrf2 gene expression; in particular, CxI increment exceeded 100% if compared with untreated-HspB-AGS cells (Fig. 3, grey bar). Similarly, CxI was the compound that best induced NQO1 gene expression in HspB-AGS cells at 4 and 24 h, compared with untreated-HspB-AGS cells (49 and 41% at 4 and 24 h, respectively) (Fig. 4a and b, grey bar). Finally, CxI nearly abrogated Keap-1 gene expression in HspB-AGS cells at 24 h (91,5%), if comparing the result with untreated-HspB-AGS cells where Keap-1 gene induction was observed (Fig. 5a and b, grey bar).

3.4. Effect of carexanes treatment on COX-2 gene expression Finally, we evaluated the effect of carexanes on COX-2 gene

Fig. 5. Real time PCR analysis at 4 (panel a) and 24 h (panel b) using specific primers for Keap-1. Black bars: AGS cells treated with 75 mg/ml CxB, CxG, and CxI. Grey bars: HSPB-transfected AGS cells treated with 75 mg/ml CxB, CxG, and CxI. The data shown are representative of five different experiments ±SD.*P < 0.05.

Fig. 6. RT-PCR analysis using specific primers for HspB. Lane 1: AGS cells stably transfected with pcDNA3 empty vector. Lane 2: AGS cells stably transfected with HspBpcDNA3. M:100 bp ladder MW-marker (Roche Diagnostics).

Fig. 7. Real time PCR analysis at 4 (panel a) and 24 h (panel b) using specific primers for COX-2. Black bars: AGS cells treated with 75 mg/ml CxB, CxG, and CxI. Grey bars: HSPB-transfected AGS cells treated with 75 mg/ml CxB, CxG, and CxI. The data shown are representative of five different experiments ±SD.*P < 0.05.

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expression, a gene involved in inflammation and reported to be induced in HspB-AGS cells [12]. The results in Fig. 7 showed that while HspB induced a strong increase of COX-2 in transfected AGS cells (34 and 45% at 4 and 24 h, respectively), only CxI reduced COX2 gene expression in HspB-AGS cells, compared with untreatedHspB-AGS cells; the down regulation of COX2 was evident both at 4 and 24 h and the levels of gene expression returned similar to control AGS cells.

glutamyltranspeptidase, which is known to trigger oxidative stress [35,36]. In addition, it represents also an important biomarker of inflammation, in part under the control of Nrf2 [6]. Our results demonstrated that CxI was able to reduce COX-2 gene expression in HspB-AGS cells, compared with untreated-HspB-AGS cells, both at 4 and 24 h.

4. Discussion

The results of the present study show that carexanes might represent candidate molecules able to contrast the deleterious effect of HspB protein but also to reduce the inflammatory process induced by H. pylori infection.

Gastrointestinal cancers are amongst the leading causes of cancer mortality. These forms of cancer are complex and multifactorial diseases, emerging from the combined influence of environmental factors such as diet, lifestyle, H. pylori infection, and the individual genetic background [25]. The inflammatory processes induced play an important role in the development and progression of several gastrointestinal cancers [26]. H. pylori is one of the main causes of the abundant inflammatory response in the gastric mucosa associated to stimulated oxidative burst from neutrophils. The induced oxidative stress on gastric mucosa is responsible of mucosal damage retard in the repair [27]. Mucosal damage quantitatively correlates with bacterial load and ROS production. However, cells can respond to chronic oxidative stress by enhancing the activities of anti-oxidant enzymes, which scavenge free radicals, thus protecting cell from damage. We have previously demonstrated that HspB protein of H. pylori interferes in the Nrf2/Keap-1 pathway [12]. In addition, HspB induced a strong increase of IL-8, COX2, MMP3 and MMP7 both in AGS cells transiently and stably HspB-transfected. Consequently, from our previous results we hypothesized that HspB might create the conditions enhancing the risk of gastric adenocarcinoma. A lot of natural product-derived compounds have been established as inducers of the Nrf2/ARE pathway, including polyphenols, quinones, organosulfur compounds, polyenes, flavonoids, chalcones, and terpenoids. These agents have been successfully tested in experimental prevention studies with inflammatory bowel and neurodegenerative diseases, diabetes, and metabolic disorders, as well as lung, heart, gastric and kidney diseases [28e31]. Carexanes, showed different biological activity, some of them showed antioxidative properties, comparable to ascorbic acid [32]. Here we firstly showed that carexanes could effectively protect AGS cells from oxidative injury inducing the antioxidant response. CxI reduced Keap-1 gene expression was accompanied by the induced NQO1 gene expression, both at 4 and 24 h; on contrary, CxB and CxG showed a reduced ability in modifying Keap-1 and NQO1 gene expression. At 4 h Nrf2 gene expression was not modulated, even though the regulation of NQO1 was already observed at this time. This result is not surprising, since the induced expression of NQO1 may be a consequence of the reduced rate of degradation of Nrf2 at 4 h, and not by a modification of its expression, as reported [33]. Additional results are, however, needed to examine this possibility. Interestingly, the effect of carexanes was stronger when AGStransfected cells were used. The antioxidant response of AGS cells stably transfected with HspB was, effectively, impaired, as demonstrated by our results, but recovered by treatment with carexanes. Also in this case CxI was the molecule that showed the best ability in contrasting the effect of HspB protein. Finally, the result on COX-2 was of great interest. COX-2 overexpression has been reported in several common human malignancies, predominantly of the gastrointestinal tract, including gastric adenocarcinoma also as in their associated precancerous lesions [34]. The increased expression of COX-2 following H. pylori infection has also been demonstrated in MKN28 cells and was at least in part attributed to up-regulation of H. pylori gamma-

4.1. Conclusion

Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Alessandra Fusco, Anna De Filippis, and Lucrezia Manente for technical assistance. References [1] A. De Luca, E. Buommino, L. Manente, M.A. Tufano, Relation between H. pylori infection and gastroduodenal diseases, Curr. Cancer Ther. Rev. 3 (2007) 109e115. [2] G.R. Davies, N. Banatvala, C.E. Collins, M.T. Sheaff, Y. Abdi, L. Clements, D.S. Rampton, Relationship between infective load of Helicobacter pylori and reactive oxygen metabolite production in antral mucosal, Scand. J. Gastroenterol. 29 (1994) 419e424. [3] J.E. Klaunig, L.M. Kamendulis, B.A. Hocevar, Oxidative stress and oxidative damage in carcinogenesis, Toxicol. Pathol. 38 (2010) 96e109. [4] A. Lau, N.F. Villeneuve, Z. Sun, P.K. Wong, D.D. Zhang, Dual roles of Nrf2 in cancer, Pharmacol. Res. 258 (2008) 262e270. [5] J.M. Lu, P.H. Lin, Q. Yao, C. Chen, Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems, J. Cell. Mol. Med. 14 (2010) 840e860. [6] W. Li, T.O. Khor, C. Xu, G. Shen, W.S. Jeong, S. Yu, A.N. Kong, Activation of Nrf2antioxidant signaling attenuates NF-kB inflammatory response and elicits apoptosis, Biochem. Pharmacol. 76 (2008) 1485e1489. [7] T. Nguyen, P. Nioi, C.B. Pickett, The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress, J. Biol. Chem. 284 (2009) 13291e13295. [8] C. Chen, D. Pung, V. Leong, V. Hebbar, G. Shen, S. Nair, et al., Induction of detoxifying enzymes by garlic organosulfur compounds through transcription factor Nrf2: effect of chemical structure and stress signals, Free Radic. Biol. Med. 37 (2004) 1578e1590. [9] A. Yanaka, J.W. Fahey, A. Fukumoto, M. Nakayama, S. Inoue, S. Zhang, et al., Dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in Helicobacter pylori-infected mice and humans, Cancer Prev. Res. (Phila) 2 (2009) 353e360. n, M. Brenes, [10] M. Castro, C. Romero, A. de Castro, J. Vargas, E. Medina, R. Milla Assessment of Helicobacter pylori eradication by virgin olive oil, Helicobacter 17 (2012) 305e311. [11] A. De Luca, G. Iaquinto, Helicobacter pylori and gastric diseases: a dangerous association, Cancer Lett. 213 (2004) 1e10. [12] E. Buommino, G. Donnarumma, L. Manente, A. De Filippis, F. Silvestri, S. Iaquinto, et al., The Helicobacter pylori protein HspB interferes with Nrf2/ Keap1 pathway altering the antioxidant response of Ags cells, Helicobacter 17 (2012) 417e425. [13] M.H. Aziz, R. Kumar, N. Ahmad, Cancer chemoprevention by resveratrol: in vitro and in vivo studies and the underlying mechanisms, Int. J. Oncol. 23 (2003) 17e28. [14] K. Yahiro, D. Shirasaka, M. Tagashira, A. Wada, N. Morinaga, F. Kuroda, et al., Inhibitory effects of polyphenols on gastric injury by Helicobacter pylori VacA toxin, Helicobacter 10 (2005) 231e239. [15] S. Martini, C. Bonechi, C. Rossi, N. Figura, Increased susceptibility to resveratrol of Helicobacter pylori strains isolated from patients with gastric carcinoma, J. Nat. Prod. 74 (2011) 2257e2260. [16] J. Kawabata, M. Mishima, H. Kurihara, J. Mizutani, Stereochemistry of two tetrastilbenes from Carex species, Phytochemistry 40 (1995) 1507e1510.  L. Dinan, Identification and ecdyste[17] Y. Meng, P.C. Bourne, P. Whiting, V. Sik, roid antagonist activity of three oligostilbenes from the seeds of Carex pendula (Cyperaceae), Phytochemistry 57 (2001) 393e400. [18] B. D'Abrosca, A. Fiorentino, A. Golino, P. Monaco, P. Oriano, S. Pacifico,

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