Increased oxidative DNA damage, inducible nitric oxide synthase, nuclear factor κB expression and enhanced antiapoptosis-related proteins in Helicobacter pylori-infected non-cardiac gastric adenocarcinoma

Abstract

AIM: Several epidemiological studies have demonstrated a close association between Helicobacter pylori (H Pylori) infection and non-cardiac carcinoma of the stomach. H pylori infection induces active inflammation with neutrophilic infiltrations as well as production of oxygen free radicals that can cause DNA damage. The DNA damage induced by oxygen free radicals could have very harmful consequences, leading to gene modifications that are potentially mutagenic and/or carcinogenic. The aims of the present study were to assess the effect of H pylori infection on the expression of inducible nitric oxidative synthase (iNOS) and the production of 8 -hydroxy-deoxyguanosine (8-OHdG), a sensitive marker of oxidative DNA injury in human gastric mucosa with and without tumor lesions, and to assess the possible factors affecting cell death signaling due to oxidative DNA damage.

METHODS: In this study, 40 gastric carcinoma specimens and adjacent specimens were obtained from surgical resection. We determined the level of 8-OHdG formation by HPLC-ECD, and the expression of iNOS and mechanism of cell death signaling [including nuclear factor-κB(NFκB), MEKK-1, Caspase 3, B Cell lymphomal leukemia-2 (Bcl-2), inhibitor of apoptosis protein (IAP ) and myeloid cell leukemia-1 (Mcl-1)] by Western-blot assay.

RESULTS: The concentrations of 8-OHdG, iNOS, NFκB, Mcl-1 and IAP were significantly higher in cancer tissues than in adjacent non-cancer tissues. In addition, significantly higher concentrations of 8-OHdG, iNOS, NFκB, Mcl-1 and IAP were detected in patients infected with H pylori compared with patients who were not infected with H pylori. Furthermore, 8-OHdG, iNOS, NFκB, Mcl-1 and IAP concentrations were significantly higher in stage 3 and 4 patients than in stage 1 and 2 patients.

CONCLUSION: Chronic H pylori infection induces iNOS expression and subsequent DNA damage as well as enhances anti-apoptosis signal transduction. This sequence of events supports the hypothesis that oxygen-free radical-mediated damage due to H pylori plays a pivotal role in the development of gastric carcinoma in patients with chronic gastritis.

Key Words: $[Keywords]

INTRODUCTION

Helicobacter pylori ( H pylori) infection is associated with an increased risk of both peptic ulcer and gastric cancer[1] Chronic gastritis induced by H pylori significantly increases the risk for non-cardiac gastric cancer[2,3], and host responses that may affect the threshold for carcinogenesis include alteration of epithelial cell proliferation and apoptosis. Although the linkage between H pylori infection and gastric cancer is convincing, the molecular mechanism or mechanisms responsible are unclear, and both bacterial and host factors are implicated. One possible factor is that increased inflammation generates reactive oxygen and nitrogen intermediates that have been shown to damage DNA directly[4].

Mucosal hyperproliferation has been reproducibly demonstrated in H pylori-infected human[5-7] and rodent gastric tissue[8-10], and proliferation scores normalize following successful eradication in humans[5-7]. However, maintenance of tissue integrity requires that enhanced cell production is accompanied with increased rates of cell loss consequently, studies have also examined the effect of H pylori on apoptosis. In contrast to hyperproliferation, H pylori has been associated with increased[11-14], unchanged[15], or even decreased[16] levels of apoptosis in vivo, and within a particular population, substantial heterogeneity exists among apoptosis scores[11-14] . These observations suggest that increases in proliferation that are not balanced by concordant increases in apoptosis over years of colonization may heighten the retention of mutated cells, ultimately enhancing the risk for gastric malignancy in certain populations. Alteration of epithelial cell proliferation and apoptosis is a manifestation of H pylori-induced gastritis. However, the precise mechanisms underlying these effects remain incompletely clarified. In this study, we aimed to assess the effect H pylori infection on the expression of iNOS and the production of 8 -OHdG, a sensitive marker of oxidative DNA injury in human gastric mucosa with and without tumor lesions, as well as the possible factors affecting cell death signaling due to oxidative DNA damage.

MATERIALS AND METHODS

Patients

In this study, 40 patients (aged 68.3 ± 11.4 years, range 39-84 years, M/F: 26/14) with non-cardiac gastric adenocarcinoma were included. Specimens from tumor site and adjacent non-tumor site were obtained from surgical resection. By TNM staging according to the criteria described by the International Union Against Cancer [17], there were 2 cases in stage 1, 6 cases in stage 2, 8 cases in stage 3 and 24 cases in stage 4 (Table 1). H pylori infection was assessed by the following three methods: histology (haematoxylin and eosin staining or Giemsa staining, rapid urease test (CLO test, Delta West Pty Ltd, Perth, Australia) and serum H pylori ELISA IgG assays (Trinity Biotech USA, Jamestown, NY). A patient was considered H pylori positive if one or more of the diagnostic methods applied were positive, and H pylori negative if all methods were negative. There were 29 patients (72.5%) with H pylori infection and 11 patients (27.5%) without H pylori infection. We determined the level of 8-OHdG formation by HPLC-ECD, and the expression of iNOS and cell death signaling, including NFκB, MEKK1, Caspase 3, Bcl-2, Mcl-1 and IAP by Western-blot assay.

Table 1 Characteristics of all patients.

Case No	Stage	H pylori infection	Case No	Stage	H pylori infection
1	II	+	21	III	+
2	IV	+	22	IV	+
3	IV	+	23	IV	+
4	III	+	24	IV	+
5	IV	+	25	IV	-
6	IV	+	26	IV	+
7	IV	+	27	I	+
8	III	-	28	I	-
9	III	+	29	IV	-
10	III	+	30	II	+
11	IV	+	31	IV	+
12	IV	-	32	IV	+
13	IV	-	33	IV	+
14	IV	+	34	III	+
15	IV	+	35	II	+
16	II	+	36	III	+
17	III	-	37	IV	-
18	IV	+	38	IV	+
19	IV	+	39	IV	+
20	II	-	40	II	-

DNA extraction and digestion

DNA was extracted with Dneasy tissue kit (QIAGEN) according to the protocol of Dahlhaus and Apple[18] with minor modifications as described previously[19]. Briefly, one volume of nuclear fraction obtained from surgical tissue was mixed with ten volumes of extraction buffer (1 mol/L NaCl, 10 mmol/L Tris–HCl, 1 mmol/L EDTA, 5 g/L SDS, pH 7.4) and one volume of chloroform:isoamylalcohol (12:1 v/v). After vigorous shaking, the aqueous phase was separated by centrifugation. This step was repeated three times before DNA was precipitated with absolute ethanol (-70 °C). DNA pellet was dissolved in TE buffer (10 mmol/L Tris–HCl, 1 mmol/L EDTA, pH 7.4), and then incubated with RNase A to remove RNA. After incubation, DNA was extracted with chloroform:isoamylalcohol, and precipitated with absolute ethanol again. The DNA pellet was dissolved in 10 mmol/L Tris–HCl (pH 7.0) and hydrolyzed according to the previous procedure[20]. Briefly, 200 µg DNA in 200 µL 10 mmol/L Tris-HCl (pH 7.0) was first denatured at 95 °C for 3 min. The digestion was carried out at 37 °C for 1 h with 100 units DNase I (from bovine pancreas, Sigma) in a buffer containing 10 mmol/L MgCl₂, followed by incubation with 5 units nuclease P₁ (from penicillium citrinum, Sigma) in 100 µmol/L ZnCl ₂ for an additional hour. The mixture was incubated finally with 3 units alkaline phosphatase (from Escherichia coli, Sigoma) at 37 °C for 1 h. The incubation was terminated with acetone (HPLC grade, Merck, Germany) to precipitate proteins. After 12 000 g centrifugation for 15 min, the supernatant was dried under vacuum in a rotary evaporator (Eyela, Tokyo, Japan) and DNA was dissolved in distilled H₂O.

8-OHdG assay

8-OHdG levels were determined by HPLC/ECD consisting of a BAS PM-80 pump (Bioanalytical Systems, West Lafayette, IN, USA), a CMA-200 microautosampler (CAM/Microdialysis, Stockholm, Sweden), a BAS-4C amperometric detector (Bioanalytical Systems, West Lafayette, IN, USA), a microbore reversed-phase column (Inertsil-2, 5-µm ODS, 1.0 Kanai 150 mm I.D. G.L. Sciences, Tokyo, Japan) Beckman I/O 406 interface, and Beckman system gold data analysis software (Beckman Instruments). A potential of +0.6 V was selected for the glassy carbon working electrode which corresponded to a silver/silver chloride reference electrode. The mobile phase consisted of 12.5 mmol/L citric acid, 30 mmol/L NaOH, 25 mmol/L sodium acetate, 10 mmol/L acetic acid glacial and 50 mL/L methanol (pH 5.3). The flow rate was set at 1 mL/min. Under these conditions, the retention time of 8-OHdG was 8.49 min with a significantly higher signal than background noise.

Immunoblot analysis

Cancerous and non- cancerous tissue samples were homogenized with ten volumes of homogenization buffer (50 mmol/L Tris, 5 mmol/L EDTA, 150 mmol/L sodium chloride, 10 g/L Nonidet P-401 g/L SDS, 5 g/L deoxycholic acid, 1 mmol/L sodium orthovanadate, 170 µg/mL leupeptin, 100 µg/mL phenylsulfonyl fluoride; pH 7.5). After 30 min of mixing at 4 °C, the mixture was centrifuged at 800 r/min for 10 min, and the supernatant was collected. The protein content of the samples was determined with the Bio-Rad protein assay reagent using BSA as a standard. For Western blotting analysis, 50 µg protein from both cancerous and non-cancerous tissue samples was resolved on 100 g/L SDS-PAGE gels along with prestained protein molecular weight standards (Bio-Rad). The separated proteins were then blotted onto NC membrane (0.45 µm, Bio-Rad) and reacted with primary antibodies (against iNOS, NFκB, MEKK1, Caspase 3, Bcl-2, Mcl-1 and IAP). After washed, the blots were incubated with peroxidase-conjugated goat anti-mouse antibody. Immunodetection was carried out using the enhanced chemiluminescence (ECL) Western blotting detection kit (Amersham Corp., UK). Relative protein expression levels were quantified by densitometric measurement of ECL reaction bands and normalized with values of tubulin. Finally, enhanced chemiluminescence (ECL) was used to detect the membrane, and then the membrane was exposed to X-ray film to observe results. All the immunoblot results were determined by Alpha Imager 2000 documentation & analysis system (Alpha Innotech Corporation).

Statistical analysis

Each parameter expressed by fold of increase of the data from cancer tissue was compared to the adjacent non-cancer tissue. The results were reported as mean ± SE from individual magnitudes. Data were analyzed with Student’s t-test. Statistical differences were considered significant when P < 0.05.

RESULTS

To assess the effect of H pylori infection on each determined parameter, the results were expressed by fold of increase comparing the data of cancer tissue with those of the adjacent non-cancer tissue. The concentrations of 8-OHdG were analyzed by HPLC/ECD. There were significantly higher concentrations of 8-OHdG in cancer tissues than in adjacent non-cancer tissues (Figure 1). In addition, significantly higher concentrations of 8-OHdG were detected in patients infected with H pylori than in those without H pylori infection. 8- OHdG levels were also significantly higher in stage 3 and 4 patients than in stage 1 and 2 patients.

Figure 1 Concentrations of 8-OHdG. A: Higher concentration of 8-OhdG in cancer tissues compared with adjacent non- cancer tissues. B: Higher concentration of 8-OhdG in cancer tissues of patients with and without H pylori infection. C: Significantly higher F8-OHdG concentrations in stage 3 and 4 patients ^aP < 0.05, N: non-cancer tissue, C: cancer tissue, Hp(-): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4.

Immunoblot analysis of several factors affecting cell death signaling related to oxidative DNA damage showed increased concentrations of iNOS and NFκB in patients infected with H pylori compared to patients who were tested negative for H pylori (Figure 2A-B). Furthermore, iNOS and NFκB concentrations were significantly higher in stage 3 and 4 patients than in stage 1 and 2 patients.

Figure 2 Immunoblot analysis of factors affecting cell death. A: Immunoblot analysis of iNOS. 1: Significantly higher concentra-tions of iNOS in cancer tissues compared with adjacent non-cancer tissues. 2: Significantly higher concentrations of iNOS in cancer tissues of patients with and without H pylori infection. 3: Significantly higher iNOS concentrations in stage 3 and 4 patients. #=H pylori infected, ^aP < 0.05, ^bP < 0.01, N: non-cancer tissue, C: cancer tissue, Hp(-): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4. B: Immunoblot analysis of NFκB. 1: Significantly higher concentrations of NFκB in cancer tissues in comparison with adjacent non-cancer tissues. 2: Significantly higher concentrations of NFκB in cancer tissues of patients with and without H pylori infection. 3: Significantly higher NFκB concentrations in stage 3 and 4 patients #=H pylori infected, P < 0.05, N: non-cancer tissue, C: cancer tissue, Hp( -): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4. C: Immunoblot analysis of MEKK1. 1: Concentra-tions of MEKK1 in cancer tissues and adjacent non-cancer tissues. 2: Significantly higher concentrations of MEKK1 in cancer tissues of patients without H pylori infection. 3. No differences in MEKK1 concentrations in cancer tissues with different stages. #= H pylori infected, ^aP < 0.05, N: non-cancer tissue, C: cancer tissue, Hp(-): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4. D: Immunoblot analysis of Caspase 3. 1: Similar concentrations of Caspase 3 in cancer tissues and adjacent non-cancer tissues. 2: Similar concentrations of Caspase 3 in cancer tissues of patients with and without H pylori infection. 3: Significantly lower Caspase 3 concentrations in stage 1 and 2 patients. #=H pylori infected, ^bP < 0.01, N: non- cancer tissue, C: cancer tissue, Hp(-): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4. E: Immunoblot analysis of Bcl-2. 1: Similar concentrations of Bcl-2 in cancer tissues and adjacent non-caner tissues. 2: No differences in concentrations of Bcl-2 in cancer tissues of patients with and without H pylori infection. 3: Significantly higher Bcl-2 concentrations in stage 1 and 2 patients. #= H pylori infected, ^bP < 0.01, N: non-cancer tissue, C: cancer tissue, Hp( -): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4. F: Immunoblot analysis of Mcl-1. 1: Significantly higher concentrations of Mcl-1 in cancer tissues in comparison with adjacent non-cancer tissues. 2: Significantly higher concentration of Mcl-1 in cancer tissues of patients with and without H pylori infection. 3: Significant higher Mcl -1 concentrations in stage 3 and 4 patients than #= H pylori infected, ^aP < 0.05, N: non -cancer tissue, C: cancer tissue, Hp(-): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4. G: Immunoblot analysis of IAP. 1: Significantly higher concentrations of IAP in cancer tissues compared with adjacent non- cancer tissues, 2: Significantly higher concentrations of IAP in cancer tissues with H pylori infection, 3: Significantly higher IAP concentrations in stage 3 and 4 patients #=H pylori infected, ^aP < 0.05, N: non -cancer tissue, C: cancer tissue, Hp(-): without H pylori infection, Hp(+): with H pylori infection, 1&2: gastric cancer stages 1 and 2, 3&4: gastric cancer stages 3 and 4.

Although there were some increases in the concentrations of MEKK1 and Caspase-3 in the cancer tissue (Figure 2C-D), these changes were not statistically significant. In addition, no significant increases of MEKK1 and Caspase-3 in patients infected with H pylori were noted. The concentrations of Caspase-3 in stage 1 and 2 patients were significantly lower than those in stage 3 and 4 patients.

Among the anti-apoptosis-related proteins, there were some increases in the concentrations of Bcl-2 in the cancer tissue (Figure 2E). However, the changes were not statistically significant. In addition, no significant increase of Bcl-2 in patients infected with H pylori was noted. The concentrations of Bcl-2 in stage 1 and 2 patients were significantly lower than those in stage 3 and 4 patients. Mcl-1 and IAP concentrations were significantly higher in the cancer tissues than in the adjacent non-cancer tissues (Figure 2F-G). In addition, Mcl-1 and IAP concentrations were significantly higher in H pylori-positive patients than in H pylori-negative patients. Stage 3 and 4 patients had significantly higher concentrations of Mcl-1 and IAP than stage 1 and 2 patients.

In summary, 8- OHdG, iNOS, NF κB, Mcl-1 and IAP concentrations were significantly higher in cancer tissues than in adjacent non-caner tissues (Table 2). In addition, significantly higher concentrations of 8-OHdG, iNOS, NFκB, Mcl-1 and IAP were detected in patients infected with H pylori compared to patients who were not infected with H pylori.

Table 2 8-oHdG, iNOS, NFκB, Mel-1 and IAP Concentrations in cancer and adjacent non-cancer tissues.

	Cancertissue	H pylori (+)	Stages I & II	Stages III & IV
8-OHdG	↑a	↑a	-	↑a
iNOS	↑b	↑b	-	↑b
NF-κB	↑a	↑a	-	↑a
MEKK1	-	-	-	-
Caspase 3	-	-	↓b	-
Bcl-2	-	-	↓b	-
Mcl-1	↑a	↑a	-	↑a
IAP	↑a	↑a	-	↑a

↑: increase, ↓: decrease, -: no significant change, ^aP < 0.05, ^bP < 0.01 compared with control, by one sample t-test and independent t-test, respectively.

Furthermore, 8-OHdG, iNOS, NFκB, Mcl-1 and IAP concentrations were significantly higher in stage 3 and 4 patients than in stage 1 and 2 patients.

DISCUSSION

H pylori infection may influence the progression from chronic gastritis to adenocarcinoma of the distal stomach by a multifactorial, multistep process, eas which chronic inflammation plays a major role. Free radical mediated oxidative DNA damage was involved in this process[21-23]. The DNA damage provoked by oxygen free radicals can have very harmful consequences, leading to gene modifications that are potentially mutagenic and/or carcinogenic. Recently, 8-OHdG has been accepted as a sensitive marker for oxidative DNA damage in affected organs. Chronic gastritis due to H pylori infection is characterized by the accumulation of oxidative DNA damage measured by concentrations of 8-OHdG, indicating mutagenic and carcinogenic potential[24]. In this study, we demonstrated an increase in the concentrations of 8-OHdG in cancer tissues. Furthermore, the increase was significantly higher in patients infected with H pylori as well as in patients with advanced gastric cancer. The increased level of oxidative DNA damage suggested a mechanistic link between H pylori infection and gastric carcinoma[25,26].

H pylori-associated inflammation related to DNA damage is indicated by increased levels of oxidative DNA damage, increased occurrence of apoptosis, as well as increased expression of iNOS, which seemed to provide the mechanistic links between H pylori infection and gastric carcinogenesis[27,28]. Upregulation of iNOS expression might contribute to the oxidative DNA damage observed during H pylori infection[25]. Recent studies have shown that nitric oxide (production of iNOS) reacts rapidly with superoxide anions produced by H pylori-infected gastric tissues to form peroxynitrate, which could induce oxidative damage[29]. This may increase the mutation rate in infected hyperproliferative gastric mucosa. In addition, the expressions of iNOS are closely related to tumor angiogenesis, and involved in progression of the disease as well as lymph node metastasis. Thus the expressions of iNOS might serve as indexes for evaluating staging of gastric carcinoma. In this study, we observed significantly higher increases in the concentrations of iNOS in patients infected with H pylori and in patients with advanced gastric cancer, compared with H pylori-negative patients and low stage gastric cancer patients, respectively.

NFκB has been found to be a critical regulator of genes involved in inflammation, cell proliferation, and apoptosis[30]. Recent studies suggested that NFκB might play a critical role in protecting cells against apoptosis[31,32]. The magnitude of the stimulus and the cell type involved may determine whether

NFκB leads to cell survival or cell death. Maeda et al[33] reported cag PAI positive H pylori was capable of inducing apoptotic effects mainly through the mitochondrial pathway. Antiapoptotic effects mediated by NFκB activation were also observed by Maeda et al[33]. In this study, we revealed an increase in the concentrations of NFκB in cancer tissues. In addition, the increase was significantly higher in patients infected with H pylori as well as in patients with advanced gastric cancer compared with H pylori-negative patients and low stage gastric cancer patients, respectively.

Recent investigations implicated mitogen-activated protein kinases (MAPK) as additional mediators of H pylori-dependent NFκB activation and IL-8 expression. Studies have demonstrated the presence of cross-talk between the MAPK and NFκB pathways[21-34]. MAPK cascades are signal transduction networks that target transcription factors and thus participate in a diverse array of cellular functions including cytokine production. In this study, there was an increase in the concentrations of MEKK1, a member of the MAPK signaling cascade in cancer tissues. However, the increase was not significant in all the studied patient groups.

Kanai et al noted that transforming growth factor-α played an anti-apoptotic role in gastric mucosal cells by enhancing the expression of Bcl-2 family proteins via an NFκB-dependent pathway[35] NFκB is now known to upregulate transcription of several anti-apoptotic genes, including a cellular inhibitor of apoptosis, a Bcl-2 homolog, and cyclooxygenase-2, as well as pro-apoptotic genes, such as bax and p53. Tumor cells can have abnormal expression levels of factors such as Bcl-2 family proteins that slow the progression of apoptosis and elevate NFκB-regulated transcription, which can inhibit TNF-α -induced apoptosis. However, in this study, no significant changes in the concentrations of Bcl-2 in cancer tissues or patients infected with H pylori were noted. Furthermore, upregulation of anti-apoptotic gene mcl-1 might play a role in interleukin-6-mediated protection of gastric cancer cells from the apoptosis induced by hydrogen peroxide[36]. The anti-apoptotic role played by NFκB also involves the ability of this transcriptional factor to induce expression of genes that promote cell survival, such as the genes coding for TRAF1, TRAF2, and the cellular inhibitors of apoptosis 1 and 2 (c-IAP1, c- IAP2)[37]. In this study, we demonstrated an increase in the concentrations of IAP and Mcl-1, anti- apoptosis related proteins in cancer tissues. Furthermore, the increase was significantly higher in patients infected with H pylori as well as in patients with advanced gastric cancer, compared with H pylori-negative and non-advanced gastric patients, respectively. Therefore, enhanced antiapoptosis-related protein expression may contribute to disease progression.

Caspase3 is a member of the cell death effector (CED)-3 family, which is involved in the induction of apoptosis. Hoshi et al[38] reported Caspase 3 was involved in the development or regulation of apoptotic cell death in cell turnover of normal and neoplastic mucosa of the human stomach. These results indicate that gastric adenocarcinoma is associated with an inhibition of apoptosis and the augmentation of proliferative activity of tumor cells when compared to non-neoplastic gastric mucosa. However, in this study, there was no difference of increase in Caspase 3 between patients with or without H pylori infection or patients with or without advanced gastric cancer.

In conclusion, chronic H pylori-induced iNOS expression and subsequent DNA damage as well as signal transduction appear to make an important contribution to H pylori-related gastric carcinogenesis. This sequence of events found in this study supports the hypothesis that oxygen- free radical-mediated damage induced by H pylori plays a vital role in the development of gastric carcinoma in patients with chronic gastritis.

ACKNOWLEDGEMENT

The authors would like to thanks the Biostatistics Task Force of Taichung Veterans General Hospital for their help with the statistical analyses performed in this study.