Abstract
OBJECTIVE To explore the expression of inducible nitric oxide synthase (iNOS), p53 and bcl-2 in gastric precancerous and Cancerous lesions and to examine the expression of these proteins In relation to clinical features.
METHODS The expressions of iNOS, p53 and bcl-2 proteins in gastric precancerous and cancerous lesions and their correlations with the clinical features were determined using immunohistochemical assays (Power VisionTM two-step method) on 84 gastric carcinomas and 54 gastric atypical hyperplastic tissues. Apoptotlc cells were evaluated by terminal deoxynucléotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL).
RESULTS Expression of iNOS, p53 and bcl-2 was significantly higher in gastric carcinoma (GC) tissues than in gastric atypical hyperplastic tissues. Among the 84 carcinomas, the expression of p53 was observed in 50 (59.52%), bcl-2 in 43 (51.19%), and iNOS In 65 (77.58%). Overexpression of iNOS and bcl-2 In gastric carcinoma was related to tumor size and iNOS was related to the presence of lymph node metastasis (P< 0.05). The expression of proteins did not correlate with age, sex, stage of disease, or differentiation. Expression of iNOS In gastric carcinoma tissues was positively correlated with bcl-2 expression (χ2=8.926, P=0.003), and also with p53 expression (χ2= 5.2430, P= 0.022). The mean apoptotic indexes (AI) were 1.29%±0.50 in low-grade atypical hyperplasia (LG), 0.96%±0.36 In high-grade atypical hyperplasia (HG) and 0.70%±0.43 in GC, with the difference being significant between LG, HG and GC (P< 0.05). There was a significant positive correlation between iNOS expression and the AI in GC (t=3.0815, P=0.0028).
CONCLUSION iNOS was expressed in the majority of gastric carcinoma tissues and correlated with cellular apoptosis associated with p53 and bel-2 expression. iNOS overexpression is closely associated with p53 and bcl-2 accumulation status. iNOS may play a synergistic role in the pathogenesis of GC.
keywords
ABBREVIATIONS- LG
- low grade atypical hyperplasia
- HG
- high grade atypical hyperplasia
- GC
- gastric adenocarcinoma
- iNOS
- inducible nitric oxide synthase
- AI
- apoptotic index.
Gastric carcinoma (GC) is one of the most common malignant diseases in China. Although the mortality rate from GC has declined, it remains the leading cause of cancer-related deaths in this country. GC is considered to be a multistage progressive process. The early indicator for a predisposition to GC is abnormal hyperproliferation of gastro-epithelial cells, such as chronic atrophic gastritis, dysplasia and intestinal metaplasia. These conditions have been considered to be precancerous lesions for GC. However, what is known concerning the process of gastric carcinogenesis is very limited.
A new class of proto-oncogenes has been defined that contributes to malignancy by inhibiting programmed cell death or apoptosis. The understanding of apoptosis has provided the basis for carcinogenesis and novel targeted therapies that can induce death in cancer cells or sensitize them to established cytotoxic agents and radiation therapy. Many pathways and proteins control the apoptosis machinery, including p53, the bcl-2 family, and other pathways.[1]
Inducible nitric oxide synthase (iNOS) is one of several isoenzymes that catalyze the synthesis of nitric oxide (NO). These enzymes are important in that they mediate the inflammatory processes. In recent years, it has been demonstrated that iNOS plays an important role in various tumors.[2-4] NO functions in many physiological and pathological conditions, in serving as an interand intra-cellular messenger.[5] NO also has been demonstrated to induce in vitro apoptosis and necrosis in a variety of cell types,[6] including myoblasts [7] and T cells.[8] But its role in vivo as a messenger is much more complex, as both the cytotoxic and an anti-apoptotic effects have been recognized in recent years.[9] NO generated by iNOS is capable of inducing mutations in the p53 tumor suppressor (also known as TP53) gene and contributes to human colon carcino-genesis. [10] And at the same time, NO may also play a role in tumor cell survival via induction of HO-1, HSP70 and bcl-2 expression.[11]
The p53 tumor suppressor protein plays a central role in the cellular response to DNA damage, mediating a cell-cycle checkpoint that leads to growth arrest or apoptosis. Loss of p53 in many cancers leads to genomic instability, impaired cell cycle regulation, and inhibition of apoptosis. Previous studies have indicated that the p53 protein, detected by immunohistochemistry, seems to represent mostly mutant p53. Accumulation of p53 therefore is an indicator for a loss of p53 tumor suppressor function. Both mutations and protein-protein interactions can cause a p53 accumulation.[12]
The bcl-2 protein is one of the most important regulators of the apoptotic pathway as the bcl-2 protein acts as repressor of apoptosis. Increased expression of bcl-2 in cells counteracts pro-apoptotic protein function, thereby hypothetically enhanceing cell survival, and also causes resistance to chemotherapeutic drugs and radiation therapy. Conversely decreasing bcl-2 expression may promote apoptotic responses to anticancer drugs.[13]
The correlation of expression of p53, iNOS and bcl-2 with clinical features in GC, however, has not been well characterized. We hypothesized that alterations of p53, iNOS and bcl-2 expression may contribute to GC and influence the clinical behavior. In the present study using immunohistochemisty, we have examined the expression of iNOS, p53 and bcl-2 in 54 gastric atypical hyperplastic tissues and in 84 GC with and without lymph node metastasis from surgically resected human GC and from gastric biopsies excised from symptom-free subjects. Furthermore we assessed the number of apoptotic cells using the terminal deoxynucléotidyl transferase-mediated dUTP-biotin nick end-labeling assay.
Materials and Methods
Patients and specimens
The present investigation included a study of tissues from 84 GC and 54 gastric atypical hyperplastic tissues. These patients were received GC surgery and gastric biopsy at the Affiliated Hospital of Xuzhou Medical college from 2001 to 2004. Before surgery, the patients had received no chemical or radiation therapy. The median age of the patients was 53.5 years (31~77 years). The gastric cancers were classified based on the depth of invasion, as Stage T1 (within the mucosa, 9 patients, 10.71%), T2 (within the submucosa, 39 patients, 46.43%), T3 (muscle layer, 26 patients, 30.95% ) and T4 (serous layer, 10 patients, 11.90%). The tumors were histologically graded as well-differentiated (7 patients, 8.33%), moderately-differentiated (27 patients, 32.1%) and poorly-differentiated (50 patients, 59.52%). Of the 84 patients, 56 had local lymph node metastasis (66.67%). The 54 gastric atypical hyperplasitic tissues included 33 LG and 21 HG.
Tissue processing
All the surgically resected specimens and biopsy samples were fixed with 10% neutral formalin, embedded with paraffin, and serially sectioned at 5 p.m. The sections were mounted onto the histostick-coated slides. Four or 5 adjacent strips were collected for histopathological analysis (H&E), TUNNEL assay and immunohistochemical staining.
Histopathological analysis
Histopathological diagnosis for gastric epithelia was made according to cellular morphological changes and tissue architecture using previously established criteria. In brief, DYS: characterized by nuclear atypia with or without architectural abnormalities in the gastric epithelium, but without invasion; adenocarcinoma (AC): invasion of neoplastic gastric cells through the basement membrane.
Terminal deoxynucleotidyl tmnsferasemediated dUTP nick-end labeling ffUNNEL)assay
Sections(5m)cut from formalin.fixed,paraffin-embedded specimens were deparaffinized in xylene and rehydrated in graded alcoh01.The tissues on the slides were then pretreated with freshly diluted proteinase K (Proteinase K,Dako)for 1 5 min at room temperature, and the endogenous peroxidase quenched by treatment with 3%hydrogen peroxide in phosphate.buffered saline for 1 0 min.Equilibration buffer was then applied to the slides,followed by incubating with terminal deoxynucleotidyl transferase for 1 h at 37°C The slides were then placed in the anti-digoxigenin peroxidase coniugate for 30 min at room temperature.Di-aminobenzidine was used as the chromogen and the sections then counterstained with hematoxylin.The numbers of apoptotic cells in a total of 5 high-power fields were counted and the results expressed as a percentage ofpositive cells.
Immunohistochemical staining
To detect the p53, iNOS and bcl-2 protein, rabbit polyclonal antibodies from Santa Cruz Biotechnology, Inc were used (sc-6243, dilution 1:50; sc-649, dilution 1: 50; sc-492, dilution 1:75). The Powervision TM two-step method was used for the immunostaining of p53, iNOS and bcl-2. In brief, 5 urn sections cut from formalin-fixed, paraffin-embedded specimens were deparaffinized in xylene and rehydrated in graded alcohol. For antigen retrieval, the deparaffinized slides were high-pressure treated in 0.01 mol/L sodium citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was then blocked by incubating the sections with 3% hydrogen peroxide in phosphate-buffered saline for 10 min. The sections were then incubated with the primary antibodies diluted as described above. After incubation, these sections were washed with 0.02 mol/L sodium phosphate buffer. The slides were then incubated with a secondary antibody for 30 min at room temperature. Diaminobenzidine was used as the chromogen and the sections were counterstained with hematoxylin. Negative controls were established by replacing the primary antibody with PBS, and known immunostaining-positive slides were used as positive controls.
Specific staining for each protein was categorized as either positive or negative based on the presence of brown-color staining. More than 10% cells positively stained were graded as positive. Clear staining for nuclei (p53), cytoplasm (iNOS and bcl-2 ) were the criteria for a positive reaction. All immunostained slides were observed by two pathologists. The slides with different diagnosis by two pathologists were read again, until the agreed diagnosis was made.
Statistical analysis
The degree of apoptosis is presented as the mean±SD and comparisons made based on one-way ANOVA using STATA 8.0. The χ2 test was used for the percentage of samples with positive stain and for the correlations between iNOS and bcl-2 as well as iNOS and p53. The t test was used for the correlation between iNOS and AI in GC (P<0.05 was considered significant).
RESULTS
Histopathologically, all 84 surgically-resected gastric specimens were confirmed as adenocarcinoma. Of the 84 GC patients, 10 cases displayed invasion within the mucosa, 26 within the submucosa, 39 with the muscle layer and 9 with the serous layer. Of the 54 biopsy samples examined, 33 were diagnosed as LG and 21 as HG.
Fig.l shows the positive apoptotic cells in GC. From LG, HG to GC, the Als were 1.29%±0.50, 0.96%±0.36 and 0.70% ±0.43 respectively, i.e. they gradually diminished, significantly showing a difference between LG, HG and GC (P<0.05). The correlation coefficient showed a significant positive correlation between iNOS expression and AI in GC (t=3.0815, P=0.0028, Table 1).
Coincidental expression of iNOS protein and AI in the gastric carcinomas
Apoptotic cells in gastric carcinomafTUNELx 400)
Positive immunostaining for p53, iNOS and bcl-2 was observed in the gastric epithelial cells and carcinoma cells with different rates in LG, HG and GC lesions. Figs.2, 3 and 4 show typical p53, iNOS and bcl-2 immunostaining in GC. p53 immunoreactivity was located in the nuclei whereas iNOS and bcl-2 immunoreactivity was located mainly in the cytoplasm. The positive immunostaining rate for p53 was very low in LG, and slightly increased in HG and significantly increased in GC (Table 2). Similar results were observed with iNOS and bcl-2, i.e. there was a significant difference between LG and GC (P<0.05, Table 2). Also the positive rate of bcl-2 staining (51.19%) in GC was significantly higher than that in HG (P<0.05, Table 2). Overexpression of iNOS and bcl-2 in GC was related to tumor size and the iNOS immunoreactivity was related to lymph node metastasis (P<0.05, Table 3).
Immunoreactivity of p53, iNOS and bel-2 in gastric epithelia with different severities of lesions
Immunoreactivity of iNOS, p53 and bcl-2 in GC and its correlation with clinical features
Coincidental expression of iNOS and bcl-2 protein in gastric carcinomas
Coincidental expression of iNOS and p53 protein in gastric carcinomas
Expression of p53 in gastric carcinoma(Power Vision™ × 400).
Expression of iNOS in gastic carcinoma(Power Vision™ × 400).
Expression of bcl-2 in gastric carcinoma(Power Vision™ × 400).
DISCUSSION
There are two major mechanisms of cell death, namely necrosis and apoptosis. Cells that are damaged by external injury undergo necrosis, while cells induced to commit programmed suicide because of internal or external stimuli undergo apoptosis.m Apoptosis, or programmed cell death, is a mechanism by which cells undergo death to control cellular proliferation or in response to DNA damage. The decrease of apoptosis in tumors is an important process in carcinogenesis. In our study, we found the AI from LG, HG to GC was 1.29%±0.50, 0.96%±0.36 and 0.70%±0.43 respectively, i.e., gradually diminished and there was a significant difference between LG, HG and GC (P<0.05).
Although an understanding of the detailed signaling pathways that trigger apoptosis is incomplete, this process is controlled by a number of complex proteins, which are activated by various triggers and arranged in sequential signaling modules, such as p53, the bcl-2 family, and other pathways.1'1
An interesting observation in our study was that the immunoreactivity of p53, iNOS and bcl-2 occurred in the early stage of GC. With a progression of the lesions from LG, HG to GC, the positive immunostaining rates for p53, iNOS and bcl-2 increased significantly and the increasing tendency for p53, iNOS and bcl-2 expression had a good linear correlation with lesion progression. Atypical hyperplasia has been strongly associated with stomach carcinoma. The present results demonstrated that from LG to HG, the accumulation of the iNOS and bcl-2 and p53 proteins became more significant, so these are early active molecular changes in GC.
Another interesting observation was that expression of iNOS in GC tissues was positively correlated with bcl-2 expression (χ2=8.926, P=0.003), as well as with p53 expression (χ2=5.2430, P=0.022). These results were consistent with the findings in human lung colon carcinomas and in UVA-induced apoptosis.[10, 11] The correlation coefficient showed a significant positive correlation between iNOS and AI in GC (t=3.0815, P=0.0028, Table 5).correlation between iNOS and AI in GC (t=3.0815, P=0.0028, Table 5).
Increased iNOS expression has been demonstrated in many tumors, such as gastric cancer,™ brain tumors [3] and colon cancer.[2] The molecular regulation of iNOS expression is complex and occurs at multiple levels in the gene-expression pathway. iNOS can produce high, persistent levels of NO upon induction with cytokines in many cell types and is expressed in the resting state in other cells, potentially resulting in cytotoxicity, tissue damage, or DNA damage. Tumor associated NO production might modify DNA directly, or inhibit DNA repair activities.[l4] The study of primary human colon tumors and hepatitis C virus infection established a strong positive relationship between the presence of iNOS in the tumors and the frequency of G:C to A:T transitions at CpG dinucleotides in the p53 gene.[l0,15] p53 is known to play an important role in safeguarding the genomic integrity of mammalian cells in response to DNA damage. Because NO production also induces accumulation of wild-type p53, [16, 17] the resulting growth inhibition can provide an additional strong selection pressure for nonfunctional, mutant p53.[18] And also, NO can promote tumor cell survival via induction of bcl-2 expression.[11] NO may, therefore, act as both an endogenous initiator and a promoter in human tumor carcinogenesis.
Finally, the present study demonstrated that the positive immunostaining rates of iNOS were correlated well with GC lymph node metastasis, increased expressions of bcl-2, and iNOS protein accumulation was related to the tumor size. These findings indicate that p53, iNOS and bcl-2 may be useful biomarkers for GC aggressiveness.
Considering that p53, iNOS and bcl-2 protein accumulation was correlated with GC clinical features, we conclude that iNOS protein accumulation may lead tö DNA damage, p53 mutants and upregulation of the expression levels of bcl-2 in GC, which may lead to the decrease of apoptosis. Thus, the high coincidental expression of iNOS, p53 and bcl-2 protein accumulation may be important events that enhance gastric carcinogenesis and may be useful biomarkers to assess risk for the development of GC. The molecular basis for the observed expression of iNOS, bcl-2 and p53 protein accumulation and their roles in the progression of gastric pre-cancerous lesions suggests the need for further careful laboratory investigations and follow-up studies.
Footnotes
This work was supported by the Project of NaturaI Science Foundation of the Education Department of Jiangsu Province of China (No.05KJD320234 and 01KJB320011).
- Received June 27, 2006.
- Accepted September 12, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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