Japanese Journal of Clinical Oncology 32:162-166 (2002)
© 2002 Foundation for Promotion of Cancer Research
Nitric Oxide Levels and Lipid Peroxidation in Plasma of Patients with Gastric Cancer
1 Department of Biochemistry, School of Medicine, 2 Biotechnology Application and Research Center and 3 General Surgery, Atatürk University, Medical School, Erzurum, Turkey
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Background: The aim was to investigate the levels of malondialdehyde and total NO2– plus NO3– marker for NO• generation in gastric carcinoma and to correlate their levels with the cancer stage.
Method: The pretreatment plasma samples were obtained from 38 patients with gastric cancer (seven patients at stage II, 19 at stage III and 12 at stage IV). Nitrite (NO2–) and nitrate (NO3–) levels, the end products of nitric oxide (NO•), were determined in these samples. NO2– was measured by using the Griess reaction and after enzymatic conversion of NO3– into NO2– by nitrate reductase, the resultant NO2– was also measured by the same method. Malondialdehyde (MDA), a lipid peroxidation marker, was measured by the thiobarbituric acid method.
Results: The levels of plasma MDA, NO• and NO3– were significantly higher in patients with gastric cancer compared with the healthy control group. Higher levels of MDA, NO• and NO3– were observed as the stage of the disease increased.
Conclusion: We found that increased NO• production and MDA levels were present in plasma of patients with gastric cancer. These increases can be associated with the oxidant–antioxidant status in these patients.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Gastric cancer is the second most common fatal malignancy in the world and is the cause of more than 750 000 deaths annually. In 1990, it was the fourteenth most frequent cause of death globally and, despite a general decline in its incidence, projections indicate that the annual number of new cases will increase significantly in the developing world during the next few decades as a result of adult population growth. Most gastric cancer is diagnosed at an advanced stage and survival is uniformly poor, usually no more than 15% at 5 years (1). Gastric cancer formation is a multifactorial process and possible mechanisms leading to gastric carcinogenesis have not yet been clarified. Therefore, this subject needs further molecular studies (2). Growing evidence indicates that reactive oxygen species (ROS) are associated with the different steps of carcinogenesis, either through structural DNA damage, interaction with oncogenes or tumor suppressor genes or immunological mechanisms (3).
For a variety of human cancers, chronic infection and inflammation have long been recognized as risk factors. It has been suggested that active oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical generated in inflamed tissues can damage the target cells, resulting in DNA damage and being able to contribute to tumor development (4). There is growing evidence that nitric oxide (NO•), an unpaired electron and its derivatives produced by activated phagocytes may also play a role in the multistage carcinogenesis process (4–6). NO• is known, together with other ROS, to induce cytotoxicity and cytostasis. Several studies on NO•- and H2O2-induced oxidative damage have cited similarities between the two chemicals in their enzymatic generation, chemical interaction with macromolecules and resulting cytotoxicity (7).
NO• is an inorganic free radical gas produced from L-arginine by a family of isoenzymes called NO synthases (8,9). Two of them are constitutively expressed and a third is inducible by immunological stimuli. It is the NO• released by the constitutive enzymes that acts as an important signaling molecule in the cardiovascular and nervous systems and NO• released by the inducible NO synthase (iNOS) is generated for long periods by cells of the immune system among others and has been shown to be cytostatic/cytotoxic for tumor cells and a variety of microorganisms (9).
The role of NO• in tumor biology is still poorly understood (9,10). Interactions of endothelial cells of the tumor vasculature, tumor-infiltrating immune cells such as T lymphocytes and macrophages and the tumor cells themselves regulate the growth of solid tumors. Most of these cellular components have been shown to generate NO• in vitro (9). Thomsen and co-workers (11,12) showed that NOS was present in fresh human tumor tissue, where it was localized to tumor cells in gynecological cancers and to stroma of breast cancers. Subsequently, increased activity of NOS was reported in patients with human gastric cancer (13,14).
It is well known that NO• possesses either antioxidant or pro-oxidant properties. It has been found that the concentrations of NO•, under non-pathological conditions, are at nanomolar levels and, under conditions of oxidant injury, at micromolar levels (10,13,15).
Endogenous NO• plays a dual role in specialized tissues and cells, where it is an essential physiological signaling molecule mediating various cell functions but also induces cytotoxic and mutagenic effects when present in excess. NO• reacts rapidly with superoxide anion to form peroxynitrite, which may be cytotoxic by itself or easily decompose to the highly reactive and toxic hydroxyl radical and nitrogen dioxide (NO2) (4):
NO• + O2• 
ONOO–
ONOO– + H+ ONOOH
ONOOH HO– + NO2–
It is well known that peroxynitrite is much more reactive than NO• or superoxide, which will cause diverse chemical reactions in biological systems including nitration of tyrosine residues of proteins, triggering of lipid peroxidation, inactivation of aconitases, inhibition of mitochondrial electron transport and oxidation of biological thiol compounds (16). NO• could also be directly oxidized to NO2–, which induces DNA damage. In addition, the reaction of NO• with H2O2 has recently been shown to produce potentially cytotoxic singlet oxygen (4).
The process of lipid peroxidation is one of oxidative conversion of polyunsaturated fatty acids to products known as malondialdehyde (MDA) or lipid peroxides, which is the most studied, biologically relevant, free radical reaction (17). MDA itself, owing to its high cytotoxicity and inhibitory action on protective enzymes, is suggested to act as a tumor promoter and a co-carcinogenic agent (18). On the other hand, it is reported that lipid hydroperoxides decompose to yield reactive aldehydes, such as MDA and 4-hydroxynoneal. MDA is a well-characterized mutagen that reacts with deoxyguanosine to form a major endogenous adduct found in the DNA of human liver (19).
The aim of this study was to investigate the plasma nitrite (NO2–), nitrate (NO3–), total NO• and MDA levels in the plasma of patients with gastric cancer and to correlate their levels with the disease stage.
|
PATIENTS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Patients
Thirty-eight patients (25 males, 13 females) with gastric cancer comprised the patient group and 24 healthy subjects (16 males, 8 females) were taken as control group, with the age range being 39–72 years (mean ± SD, 54.5 ± 11.2 years) for the patients and 38–69 years (mean ± SD, 51.9 ± 10.7 years) for the controls. Patients’ characteristics are shown in Table 1. Tumors were classified according to UICC criteria (20). Seven patients were in stage II, 19 in stage III and 12 in stage IV. Seven of the stage IV patients had distant organ–tissue metastases. Seven patients had peritoneal dissemination. Distant organ and lymph node involvement was documented by chest radiography, abdominal ultrasonography and computed tomography.
|
Blood Sampling
Gastric cancer patients and healthy control subjects were recruited into the study after obtaining their informed consent. Venous blood (10 ml) was taken from controls and patients. The blood samples were centrifuged and the plasma samples obtained were stored at –80°C until the analysis date.
Biochemical Measurements
The measurement of plasma NO• is difficult because this radical is poorly soluble in water and has a short half-life in tissue (10–60 s), but its half-life may be as long as 4 min in the presence of oxygen. For these reasons, the NO• itself can be determined only with difficulty and requires the handling of radioisotopes. In spite of this, the end products of the phenomena, nitrate and nitrite, are preferentially used in clinical biochemistry (21,22). Plasma total nitrite + nitrate levels were measured with use of the Griess reagent as described previously (22,23). The Griess reagent consists of sulfanilamide and N-(1-naphthyl)ethylenediamine. The method is based on a two-step process. The first step is the conversion of nitrate to nitrite using nitrate reductase. The second step is the addition of Griess reagent, which converts nitrite into a deep-purple azo compound; photometric measurement of the absorbance at 540 nm due to this azochromophore accurately determines the nitrite concentration (sodium nitrate is used as a standard). Protein interference was eliminated by treatment of the reacted samples with zinc sulfate and centrifugation for 5 min at 10 000 g. MDA, an end product of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a colored substance. Measurement of MDA by TBA reactivity is the most widely used method for assessing lipid peroxidation. MDA was estimated according to the modified method of Satoh (24). To 0.5 ml plasma was added 0.5 ml of 35% TCA. After vortex mixing, 0.5 ml of Tris–HCl buffer (50 mM; pH 7.4) was added, followed by further mixing and incubation at room temperature for 10 min. A 1 ml volume of 0.75% TBA in 2 M Na2SO4 was added and then the mixture was heated at 100 °C for 45 min. After cooling, 1 ml of 70% TCA was added and the mixture was vortex mixed and then centrifuged at 950 g for 10 min. The absorbance of the supernatant was measured at 532 nm. Total TBA-reactive materials were expressed as MDA, using a molar absorptivity for MDA of 1.56x105 cm–1 M–1. The results were expressed as µmol/l.
Statistical Analysis
The findings were expressed as the mean ± SD. Statistical and correlation analyses were undertaken using the Mann–Whitney U-test and Pearson’s rank correlation test, respectively. A P value <0.05 was accepted as statistically significant. SPSS (for Windows, Version 10.0) was used for statistical analyses.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our findings on the assessed parameters and correlations between parameters in gastric cancer and healthy control subjects are shown in Tables 2 and 3, respectively (Fig. 1). Mean plasma NO•, NO3– and MDA levels were found to be lowest in the control group and highest in the stage IV patients. As seen in Table 2, as the stage of the disease increased, higher levels of plasma NO•, NO3– and MDA were determined. Plasma NO•, NO3– and MDA levels in the patients of each stage were compared with those of control groups. For MDA, the difference between the control group and stage II group was at the P < 0.01 level and that between the controls and the stage III and IV groups at the P < 0.001 level. For NO• and NO3–, the difference between control group and stage II, III and IV groups was at the P < 0.001 level. The difference in plasma NO3– between stage II and stage IV groups was at the P < 0.001 level. The difference in plasma NO•, NO3– and MDA between total patient group and control group was at the P < 0.001 level. Plasma NO2– levels were of no significance in all comparisons. There was no statistically significant difference in the parameters when the patients were grouped with respect to metastasis. Table 3 summarizes the correlations between the parameters.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
NO• is a multifunctional species that is implicated in a wide variety of physiological and pathological processes (3,9). NO2– and NO3– in plasma reflect the level of NO• formation. Various studies showed that NOS activity and NO• synthesis were high in tumor tissue and in plasma (13–16,25–29). The progression of a tumor might be related to the oxidant–antioxidant status (21,30,31). To our knowledge, plasma NO• and MDA levels are reported together in gastric cancer for the first time in this study. Our results showed that nitrite, nitrate, NO• and MDA levels were higher in gastric cancer than in the control group.
It has been claimed that MDA acts as a tumor promoter and co-carcinogenic agent because of its high cytotoxicity and inhibitory action on protective enzymes (18). There are controversial results on this subject in the literature. For example, decreased plasma MDA (21,31), thiobarbituric acid reactive substances (TBARS) (32) and tissue MDA (33) levels have been reported in patients with breast cancer. Gerber et al. (27) reported that plasma MDA levels decreased with increasing tumor size and progression in breast cancer. However, Huang et al. (34) reported significantly increased lipid peroxidation, measured as MDA, in the serum of breast cancer patients. On the other hand, Seven et al. (18) and Samir and el Kohly (17), in their studies on patients with laryngeal carcinoma, reported that plasma MDA levels were significantly increased compared with healthy controls. Nagini et al. (35) found that MDA levels were lower in tissue with oral squamous cell carcinoma than in the control group. Increased serum MDA levels were reported in patients with gastric cancer (36). Haklar et al. (25) reported significantly increased NO• in tissues in patients with colon tumors. Our results for MDA and NO• levels in plasma of gastric cancer patients are in agreement with those obtained by Choi et al. (36), Alagöl et al. (21) and Haklar et al. (25).
In conclusion, we found that increased NO• production and MDA levels were present in plasma of patients with gastric cancer, which can be related to an alteration in oxidant–antioxidant status. Since NO• seems to have a dual role in tumor progression, high concentrations of NO• for long periods could result in damage to DNA, leading to mutations and cancer. One should take into consideration that the levels of NO2– and NO3– are also influenced by dietary factors in addition to endogenous synthesis and that further studies to clarify this issue are necessary. Moreover, the role of NO• and nitrous compounds in carcinogenesis is still under discussion. Further studies are needed.
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Ebubekir Bakan, Ataturk University Medical School, Department of Biochemistry, 25240 Erzurum, Turkey. E-mail: ebubekirbakan@usa.net
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 Forman D. Helicobacter pylori: the gastric cancer problem. Gut 1998;43 Suppl 1:S33–4.
2 Durak I, Cetin R, Canbolat O, Cetin D, Yurtarslani Z, Unal A. Adenosine deaminase, 5'-nucleotidase, guanase and cytidine deaminase activities in gastric tissues from patients with gastric cancer. Cancer Lett 1994;84:199–202.[ISI][Medline]
3 Carla B, Patrizia F, Sergio AT, Carla V, Francesco R, Antonia F. Vitamin E serum levels and gastric cancer: results from a cohort of patients in Tuscany, Italy. CancerLett 2000;151:15–8.[ISI][Medline]
4 Ohshima H, Bartsch H. Chronic infections and inflammatory process as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994;305:253–64.[ISI][Medline]
5 Tamir S, Tannenbaum ST. The role of nitric oxide (NO•) in the carcinogenic process. Biochim Biophys Acta 1996;1288:F31–6.[Medline]
6 Kanner J, Harel S, Granit R. Nitric oxide as an antioxidant. Arch Biochem Biophys 1991;289:130–6.[ISI][Medline]
7 Abu-Shakra A, McQueen ET, Cunnigham ML. Rapid analysis of base-pair substitutions induced by mutagenic drugs through their oxygen radical or epoxide derivatives. Mutat Res 2000;470:11–8.[ISI][Medline]
8 Ignarro LJ, Buga GM, Wei LH, Bauer PM, Wu G, Soldato PD. Role of arginine–nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation. Proc Natl Acad Sci USA 2001;98:4202–8.
9 Jenkins DC, Charles IG, Thomsen LL, Moss DW, Holmes LS, Baylis SA, et al. Role of nitric oxide in tumor growth. Proc Natl Acad Sci USA 1995;92:4392–6.
10 Mahesh SJ, Julie LP and Jack RLJR. Cellular antioxidant and pro-oxidant actions of nitric oxide. Free Rad Biol Med 1999;27:1357–66.[ISI][Medline]
11 Thomsen LL, Lawton FG, Knowles RG, Beesley JE, Riveros-Moreno V, Moncada S. Nitric oxide synthase activity in human gynecological cancer. Cancer Res 1994;54:1352–4.
12 Thomsen LL, Miles DW, Happerfield L, Bobrow LG, Knowles RG, Moncada S. Nitric oxide synthase activity in human breast cancer. Br J Cancer 1995;72:41–4.[ISI][Medline]
13 Koh E, Noh SH, Lee YD, Lee HY, Han JW, Lee HW, et al. Differential expression of nitric oxide synthase in human stomach cancer. Cancer Lett 1999;146:173–80.[ISI][Medline]
14 Goto T, Haruma K, Kitadai Y, Ito M, Yoshihara M, Sumii K, et al. Enhanced expression of inducible nitric oxide synthase and nitrotyrosine in gastric mucosa of gastric cancer patients. Clin Cancer Res 1999;5: 1411–5.
15 Vakkala M, Kahlos K, Lakari E, Paakko P, Kinnula V, Soini Y. Inducible nitric oxide synthase expression, apoptosis and angiogenesis in in situ and invasive breast carcinomas. Clin Cancer Res 2000;6:2408–16.
16 Maeda H, Akaike T. Nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry (USSR) 1998;63:854–65.[Medline]
17 Samir M, el Kholy NM. Thiobarbituric acid reactive substances in patients with laryngeal cancer. Clin Otolaryngol 1999;24:232–4.[ISI][Medline]
18 Seven A, Civelek S, Inci E, Korkut N, Burçak G. Evaluation of oxidative stress parameters in blood of patients with laryngeal carcinoma. Clin Biochem 1999;32:369–73.[ISI][Medline]
19 Stone WL, Papas AM. Tocopherols and the etiology of colon cancer. J Natl Cancer Inst 1997;89:1006–14.
20 Sobin LH, Wittekind CH. In: Wittekind CH, editor. TNM Classification of Malignant Tumours, 5th ed. International Union Against Cancer. New York: Wiley-Liss 1997;59–62.
21 Alagöl H, Erdem E, Sancak B, Turkmen G, Camlibel M, Bugdayci G. Nitric oxide biosynthesis and malondialdehyde levels in advanced breast cancer. Aust N Z J Surg 1999;69:647–50.[Medline]
22 Moshage H, Kok B, Huizenga JR, Jansen PLM. Nitrite and nitrate determinations in plasma: a critical evaluation. Clin Chem 1995;41:892–6.
23 Bories PN, Bories C. Nitrate determination in biological fluids by an enzymatic one step assay with nitrate reductase. Clin Chem 1995;41:904–7.
24 Hunter MIS, Nlemadim BC, Davidson DLW. Lipid peroxidation products and antioxidant proteins in plasma and cerebrospinal fluid from multiple sclerosis patients. Neurochem Res 1985;10:1645–52.[ISI][Medline]
25 Haklar G, Sayin-Özveri E, Yüksel M, Aktan AÖ, Yalçin AS. Different kinds of reactive oxygen and nitrogen species were detected in colon and breast tumors. Cancer Lett 2001;165:219–24.[ISI][Medline]
26 Doi C, Noguchi Y, Marat D, Saito A, Fukuzawa K, Yoshikawa T, et al. Expression of nitric oxide synthase in gastric cancer. Cancer Lett 1999;144:161–7.[ISI][Medline]
27 Gerber M, Astre C, Segala C, Saintot M, Scali J, Simony-Lafontaine J, et al. Tumor progression and oxidant–antioxidant status. Cancer Lett 1997;114:211–4.[ISI][Medline]
28 Mariyama A, Tabaru A, Unoki H, Abe S, Masumota A, Otsuki M. Plasma nitrite/nitrate concentrations as a tumor marker for hepatocellular carcinoma. Clin Chim Acta 2000;296:181–91.[ISI][Medline]
29 Szaleczky E, Pronai L, Nakazawa H, Tulassay Z. Evidence of in vivo peroxynitrite formation in patients with colorectal carcinoma, higher plasma nitrite/nitrate levels, and lower protection against oxygen free radicals. J Clin Gastroenterol 2000;30:47–51.[ISI][Medline]
30 Saintot M, Astre C, Pujol H, Gerber M. Tumor progression and oxidant–antioxidant status. Carcinogenesis 1996;17:1267–71.
31 Gerber M, Astre C, Segala C, Saintot M, Scali J, Simony-Lafontaine J, et al. Oxidant–antioxidant status alterations in cancer patients: relationship to tumor progression. J Nutr 1996;126(4 Suppl):1201S–7S.
32 Seven A, Erbil Y, Seven R, Inci F, Gulyasar T, Barutcu B, Candan G, et al. Breast cancer and benign breast disease patients evaluated in relation to oxidative stress. Cancer Biochem Biophys 1998;16:333–45.[Medline]
33 Portakal O, Ozkaya O, Erden M, Bozan B, Kosan M, Sayek I. Coenzyme Q10 concentrations and antioxidant status in tissues of breast cancer patients. Clin Biochem 2000;33:279–84.[ISI][Medline]
34 Huang YL, Sheu JY, Lin TH. Association between oxidative stress and changes of trace elements in patients with breast cancer. Clin Biochem 1999;32:131–6.[ISI][Medline]
35 Nagini S, Manoharan S, Ramachandran CR. Lipid peroxidations and antioxidants in oral squamous cell carcinoma. Clin Chim Acta 1998;273:95–8.[ISI][Medline]
36 Choi MA, Kim BS, Yu R. Serum antioxidative vitamin levels and lipid peroxidation in gastric carcinoma patients. Cancer Lett 1999;136:89–93.[ISI][Medline]
Received November 2, 2001; accepted February 20, 2002
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. P. Patel, U. M. Rawal, T. K. Dave, R. M. Rawal, S. N. Shukla, P. M. Shah, and P. S. Patel Lipid Peroxidation, Total Antioxidant Status, and Total Thiol Levels Predict Overall Survival in Patients With Oral Squamous Cell Carcinoma Integr Cancer Ther, December 1, 2007; 6(4): 365 - 372. [Abstract] [PDF]
|
|
|
|
 |
|
 T. A. Reiter, B. Pang, P. Dedon, and B. Demple Resistance to Nitric Oxide-induced Necrosis in Heme Oxygenase-1 Overexpressing Pulmonary Epithelial Cells Associated with Decreased Lipid Peroxidation J. Biol. Chem., December 1, 2006; 281(48): 36603 - 36612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y-J Yang, J-L Zhao, S-J You, Y-J Wu, Z-C Jing, W-X Yang, L Meng, Y-W Wang, and R-L Gao Different effects of tirofiban and aspirin plus clopidogrel on myocardial no-reflow in a mini-swine model of acute myocardial infarction and reperfusion Heart, August 1, 2006; 92(8): 1131 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H Ucuncu, S Taysi, B Aktan, M E Buyukokuroglu, and M Elmastas Effect of dantrolene on lipid peroxidation, glutathione and glutathione-dependent enzyme activities in experimental otitis media with effusion in guinea pigs Human and Experimental Toxicology, November 1, 2005; 24(11): 567 - 571. [Abstract] [PDF]
|
|
|
|
 |
|
 S. S. S. Beevi, A. M. H. Rasheed, and A. Geetha Evaluation of Oxidative Stress and Nitric Oxide Levels in Patients with Oral Cavity Cancer Jpn. J. Clin. Oncol., July 1, 2004; 34(7): 379 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Aktan, S. Taysi, K. Gumustekin, N. Bakan, and Y. Sutbeyaz Evaluation of Oxidative Stress in Erythrocytes of Guinea Pigs with Experimental Otitis Media and Effusion Ann. Clin. Lab. Sci., April 1, 2003; 33(2): 232 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Uslu, S. Taysi, and N. Bakan Lipid Peroxidation and Antioxidant Enzyme Activities in Experimental Maxillary Sinusitis Ann. Clin. Lab. Sci., January 1, 2003; 33(1): 18 - 22. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||