Supportive Care In Cancer

Support Care Cancer (2007) 15:1399–1405 DOI 10.1007/s00520-007-0266-3

ORIGINAL ARTICLE

Oxidative stress in lymphocytes, neutrophils, and serum of oral cavity cancer patients: modulatory array of L-glutamine Subhasis Das & Santanu Kar Mahapatra & N. Gautam & Amrita Das & Somenath Roy

Received: 22 January 2007 / Accepted: 2 May 2007 / Published online: 26 June 2007 # Springer-Verlag 2007

Abstract Objectives Our aim was to assess the oxidative stress and ameliorative effect of L-glutamine in serum, neutrophils, and lymphocytes of oral cancer patients by measuring the levels of malondialdehyde (MDA) and antioxidants. Materials and methods This study has been conducted on serum and specific blood cells in adult, male oral cancer patients (stage III-6, stage IV-42) and normal subjects of an equal number of age and sex-matched disease-free healthy subjects. The levels of lipid peroxidation and antioxidant enzymes were assayed using spectrophotometric methods. Results MDA levels were elevated, and antioxidant enzyme status was decreased significantly in all groups of cancer patients simultaneously, but after supplementation of “glutammune” (66.66% L-glutamine), oxidative stress has been alleviated to some extent; especially, it has repaired the glutathione cascade system. Conclusion We conclude that oxidative stress is due to the enhanced lipid peroxidation and decrease in antioxidant enzymes, and it can be restored with dietary supplementation of L-glutamine related drug. Keywords Oral cavity cancer . Lipid peroxidaton . Antioxidant enzymes . Oxidative equilibrium . L-Glutamine

S. Das : S. Kar Mahapatra : N. Gautam : A. Das : S. Roy (*) Immunology and Microbiology Laboratory, Department of Human Physiology with Community Health, Vidyasagar University, Midnapore 721 102 West Bengal, India e-mail: [email protected]

Introduction Oral cancer is the sixth most frequent cancer in the world; some of the highest rates are in developing countries where up to 25% of all malignancies are found in the oral cavity [37]. Tobacco is the predominant cause of this disease [18, 33, 48]. Alcohol use is a risk factor that acts synergistically with tobacco. In India, the use of chewing tobacco and betel quid, usually mixed with other toxins such as slaked lime, is a common custom that causes oral cancer, which is responsible for 50–90% of cases worldwide [12, 39, 41]. Smoking is the main etiology of oral cancer and generates oxygen free radicals in the oral cavity. Free radicals have been implicated in apoptosis and in DNA damage, inducing alteration of the cell cycle [35]. Alkaline saliva generated by chewing betel quid plays an important role in cigarette-related nicotineinduced DNA damage, and reactive oxygen species (ROS) may be involved in generating this DNA damage [49]. ROS can cause DNA base alterations, strand breaks, damage to tumor suppressor genes, and enhanced expression of proto-oncogenes [8]. ROS-induced mutation could also arise from protein damage and attack on lipids, which then initiate lipid peroxidation. It should be noted, however, that the development of human cancer is multifactorial, depending on several factors including the extent of DNA damage, effectiveness of antioxidant defense, DNA repair system, and growth-promoting effect of ROS [6]. The burst of ROS has been implicated in the development of oral cavity cancer in tobacco chewers and smokers [45]. Tobacco consumption in any form has been demonstrated to have carcinogenic, teratogenic, and genotoxic effects and is positively correlated with accumulation of DNA damage. Tobacco is therefore believed to directly induce cellular DNA damages in the human oral cavity [43].

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The successful control of oral cavity cancer will depend on its prevention. Considerable evidence exists, suggesting that the role of enzymatic and non-enzymatic antioxidant defense systems protect cell against ROS produced during normal metabolism and after an oxidative insult. Antioxidant defense systems work cooperatively to alleviate the oxidative stress caused by enhanced free radical production. Any changes in one of these systems may break this equilibrium and cause cellular damages and, ultimately, malignant transformation [13, 47]. It has been reported that ROS production is pathologically high in advance-stage cancer patients [27, 28]. Glutathione peroxidase (GPx), one of the major antioxidant enzymes in glutathione antioxidant defense system, showed significantly lower value in cancer patients as compared to control [27, 28, 38]. Moreover, these anti-oxidants, both natural and synthetic, neutralize metabolic products (including reactive oxygen species), interfere with activation of procarcinogens, prevent binding of carcinogens to DNA, inhibit chromosome aberrations, restrain replication of the transformed cell, suppress actions of cancer promoters, and may even induce regression of precancerous oral lesions [12]. A number of nutrients zinc, epigallcatechin galatee, omega-3 polyunsaturated fatty acids, probiotics all act differently to modulate the immune response, but all seem to have the potential to protect the host from the development of cancer and its progression [5]. Glutamine is a precursor for nucleotide synthesis, a substrate for liver gluconeogenesis and an important fuel source for cells (such as gastrointestinal epithelia, lymphocytes, fibroblasts, and reticulocytes) that have rapid turnover [42]. Visceral organs, enterocytes, T lymphocytes, and macrophages utilize glutamine mobilized from muscle and plasma as a source of fuel [8]. In critically ill patients, glutamine supplementation may reduce morbidity and mortality [32]. Studies in animal models and emerging clinical trials suggest the benefits of enteral or parenteral glutamine supplementation. Although some studies show no apparent benefit, glutamine supplementation of parenteral nutrition, enteral diets, or drinking water improves animal survival, decreases infectious morbidity, and enhances gut mucosal repair in models of chemotherapy and irradiation, sepsis, and inflammation [51]. Glutamine-enriched nutrition attenuates depletion of the key antioxidant reduced glutathione (GSH) in plasma, liver, and gut after chemotherapy, sepsis, and gut ischemia/reperfusion and upregulates systemic and tissue immune function in animals [9, 44, 51, 52]. Considering the fact that glutamine is vital for the rapidly dividing cells like lymphocytes, it is not surprising that its depletion contributes to the development of immunosuppression ill patients [25]. Our aim of this study was to assess the degree of oxidative stress and alleviative role of “glutammune” (66.66% L-glutamine) in serum and immunocompetent cells of oral cavity cancer patients.

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Materials and methods Selection of human subjects Control group (group I) of volunteers were only those who were almost in the same age group (40–70 years), unlike the experimental groups, and proved to be in normal state of health and free from any signs of chronic disease by the careful clinical examinations with similar socioeconomic backgrounds. They were non-smokers and not consuming alcohol. Control subjects were not receiving any drug on a long-term basis. The subjects (age group 40–70 years) were suffering from oral cavity cancer, divided into three groups, ie., group II (just 1 day after operation), group III (14 days after operation), and group IV (supplemented group, supplement was given for 14 days just after the operation). After experimental period, heparinized blood and serum were collected from Royd Nursing Home, 5B Royd Street, Kolkata, India and analyzed in Immunology and Microbiology Laboratory, Vidyasagar University, Midnapur, West Bengal, India. All the carcinomas were graded as well-differentiated squamous cell carcinoma. The ethical committee of the Vidyasagar University approved the study, and informed consent of all the subjects was obtained. None of the patients and control subjects had concomitant diseases such as diabetes mellitus, liver disease, and rheumatoid arthritis. The clinical characteristics of oral cavity cancer patients were shown in Table 1. Experimental design Group I—control (normal healthy subjects) Group II—cancer patients (just 1 day after operation)

Table 1 Clinical characteristics of oral cavity cancer patients Characteristics

Patients Male/female Age (years): mean±SEM Stage III IV Tumor location Buccal mucosa Anterior two thirds of the tongue Retromolar area Body weight: mean±SEM

Number of patients Group II

Group III

Group IV

16 14/2 55.3±7.2

16 15/1 55.7±9.1

16 15/1 58.2±10.4

2 14

3 13

1 15

9 4

10 5

12 3

3 65.89± 11.49

1 63.28± 8.7

1 68.27± 11.92

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Group III—cancer patients (14 days after operation) Group IV—cancer patients (supplemented after operation for 14 days): The dose of “glutammune” per day per patient was 30 g orally, which contains 20 g L-glutamine. The dose and duration was selected as per the previous report by many researchers [1, 2, 10, 14] Chemicals and reagents All fine chemicals were obtained from Sigma Chemical, USA. Other chemicals used were analytical grade and obtained locally. L-Glutamine was obtained as a brand name of “glutammune” from Claris Lifesciences Limited, Corporate Towers, Ellisbridge, Ahmedabad #380 006, India.

transferase was assayed as described by Habig et al. [17]. Total protein content was estimated by the method of Lowry et al. [24]. Statistical analysis The data were expressed as mean±standard error. Comparisons of the means of group I, group II, group III, and group IV were made by model I analysis of variance test with (using a statistical package, Origin 6.1, Northamption, MA 01060, USA) multiple comparison t test, P<0.05 as a limit of significance.

Results Collection of blood samples and separation of serum, neutrophil, and lymphocytes Fasting blood samples were collected from all groups of individuals. Serum was obtained by centrifugation at 1500×g for 15 min of blood samples taken without anticoagulant. Serum was kept at −86°C for further analysis. Heparinized blood samples were used for the separation of neutrophils and lymphocytes. Neutrophils and lymphocytes were isolated from heparinized blood using standard isolation techniques [21]. The pellets of neutrophils and lymphocytes were lysed in a hypotonic solution (kept for 45 min at 37°C) and stored at −86°C until biochemical estimations [40]. Biochemical assays Malondialdehyde level was measured according to the method of Ohkawa et al. [36]. Reduced glutathione content was measured as described by modified procedure of Grifith [15]. Glutathione disulfide content was estimated according to the method described by Grifith [15]. The catalase activity was measured according to the methodology of Beers and Sizer [4]. The activity of superoxide dismutase was measured according to the method of Marklund and Marklund [29]. The activity of glutathione-s-

The findings of the study are presented in Table 2 of control (normal healthy subjects), different groups of cancer patients (groups II and III), and cancer patients with supplementation of “glutammune” (L-glutamine). Result shows that the extent of lipid peroxidation, as evidenced by lymphocytes, neutrophils, and serum malondialdehyde (MDA), was significantly increased in the both group II and group III oral cancer patients (P<0.05) compared to control subjects, whereas supplemented group revealed the significantly decreased MDA level (P<0.05) as compared to group III patients, but it did not show the reversible effect. The enzymatic antioxidants profile in the circulation of oral cancer patients and control subjects was measured. The levels of GSH and oxidized glutathione (GSSG) in lymphocytes, neutrophils, and serum were significantly lower and simultaneously decreased in both group II and group III oral cancer patients (P<0.05) when compared to the control subjects. On the other hand, supplemented group shows significant (P<0.05) increase in both GSH and GSSG levels. More interestingly, glutamine increases the GSH levels rather than GSSG. A significant decrease (P<0.05) in the activities of superoxide dismutase (SOD), glutathione-S-transferase (GST), and catalase (CAT) in the lymphocytes, neutrophils, and serum were seen in the two

Table 2 Lipid peroxidation and antioxidant enzyme status of lymphocytes Groups

Group Group Group Group

I II III IV

MDA (nmol/mg protein)

GSH (μg/mg protein)

GSSG (μg/mg protein)

CAT (nmol min−1 mg−1 protein)

SOD (U min−1 mg−1 protein)

GST (nmol min−1 mg−1 protein)

0.83±0.035 1.48±0.081* 1.99±0.035* 1.31±0.069**

28.17±1.68 15.52±0.94* 6.31±0.61* 17.95±0.88**

36.96±1.64 28.27±0.82* 22.79±1.58* 25.98±1.45**

28.16±0.21 17.22±0.15* 14.59±0.18* 15.76±0.22

46.51±0.31 32.89±0.33* 29.51±0.29* 31.55±0.24

365.26±13.35 268.74±6.37* 177.87±9.29* 292.19±10.31**

Values are expressed as mean±SEM for n=16. *P≤0.05 compared to control (group I) **P≤0.05 compared to patients 14 days after surgery (group III)

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groups of oral cancer patients as compared to control subjects. Maximum significant decrement has been seen in the case of catalase activity. “Glutammune”-supplemented group shows maximum effect on GST activity, which is near to control level, but SOD and CAT did not response significantly on the “glutammune”-supplemented lymphocytes, neutrophils, and serum.

Discussion Cancer is essentially an event occurring at the gene level, and the ultimate step resulting in carcinogenesis is DNA damage. Multiple factors such as viruses, chemicals, irradiations, and the genetic makeup of the individual play a role in carcinogenesis. ROS are found to be involved in both initiation and promotion of multi-step carcinogenesis. They can cause DNA damage, activate pro-carcinogenesis, initiate lipid peroxidation, inactivate enzyme systems, and alter the cellular antioxidants defense system [46]. High levels of oxidative stress result in peroxidation of membrane lipids, with the generation of peroxides that can decompose to multiple mutagenic carbonyl products. They are considered to be mutagenic and carcinogenic [50]. They can also modulate the expression of genes related to tumor promotion [7]. In our study (Tables 2 and 3), we observed significantly simultaneous increase in MDA levels in serum, neutrophil, and lymphocytes of both the two groups (II and III) as compared to control. On the other hand, supplemented group showed significantly decreased level of MDA in comparison to group III, except neutrophil, but it did not show the reversible effect. Elevated levels of lipid peroxidation product support the hypothesis that the cancer cells produce large amount of free radicals [8] and that there exist a relationship between free radical activity and malignancy [11]. So, specifically oxidative stress provides evidence of the relationship between lipid peroxidation and oral cavity cancer. On the contrary, supplemented group showed reduced levels of MDA, which may be due to few increased antioxidant activity in the system out of the four antioxidants (GSH, CAT, SOD, and GST).

Antioxidants have been shown to inhibit both initiation and promotion in carcinogenesis and counteract cell immunization and transformation [34]. Cellular antioxidant enzymes and free radical scavengers protect a cell against toxic oxygen radicals. GSH, an important non-protein thiol, in conjugation with GST and glutathione peroxidase (GPx), plays a significant role in protecting cells by scavenging ROS [31]. A wide variety of oxidizing molecules such as ROS and/or depleting agents can alter the glutathione redox state, a key compound in the regulation of body redox homeostasis. The glutathione redox state is normally maintained by the activity of GSH-depleting (GPx) and -replenishing enzymes (GR) [3, 22]. A significant depletion of lymphocytes, neutrophils, and plasma GSH observed in our study reflects enhanced pro-oxidant milieu of the system and correlates with the increased lipid peroxides in the circulation of oral cavity cancer patients, whereas supplemented group showed significantly increased level of GSH, GSSG, and activity of GST when compared to groups II and III of cancer patients. This increased level of enzymes in glutathione system, especially GSH, may help to minimize the MDA levels in the supplemented group. Antioxidant enzymes such as SOD and CAT provide the first line of cellular defense against toxic-free radicals. These enzymes react directly with oxygen-free radicals to yield non-radical products. These enzymes prevent H2O2mediated intracellular DNA damage, which is thought to be a prerequisite for carcinogenesis [30]. SOD metabolizes
free radicals and dismutates superoxide anions O2 to
H2O2 and protects the cells against O2 -mediated lipid peroxidation. CAT acts on H2O2 by decomposing it, thereby neutralizing its toxicity. It has been reported that superoxide radicals inhibit catalase activity and H2O2 suppresses SOD activity in the cell [19]. Gupta et al. [16] demonstrated that reduction in several antioxidant defense mechanisms correlates with the emergence of the malignant phenotype. The low activities of these antioxidant enzymes observed in our study during the group II and group III of cancer patients might be due to the depletion of the antioxidant defense system. This could occur as a consequence of overwhelming free radicals, as evidenced

Table 3 Lipid peroxidation and antioxidant enzyme status of neutrophils Groups

MDA (nmol/mg protein)

GSH μg/mg protein)

GSSG (μg/mg protein)

CAT (nmol min−1 mg−1 protein)

SOD (U min−1 mg−1 protein)

GST (nmol min−1 mg−1 protein)

Group Group Group Group

1.4±0.021 2.14±0.063* 2.43±0.061* 2.25±0.057**

25.83±1.09 14.12±0.64* 5.16±0.33* 16.91±0.71**

31.55±1.31 24.36±0.54* 16.59±0.83* 20.66±0.98**

22.16±0.29 13.62±0.18* 10.09±0.12* 12.76±0.12

38.92±0.36 27.29±0.35* 23.51±0.39* 24.97±0.29

311.99±11.38 225.57±10.09* 150.9±9.27* 248.18±11.36**

I II III IV

Values are expressed as mean±SEM for n=16. *P≤0.05 compared to control (group I) **P≤0.05 compared to patients 14 days after surgery (group III)

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Table 4 Lipid peroxidation and antioxidant enzyme status of serum Groups

Group Group Group Group

I II III IV

MDA (nmol/mg protein)

GSH (μg/mg protein)

GSSG (μg/mg protein)

CAT (mmol min−1 mg−1 protein)

SOD (U min−1 mg−1 protein)

GST (μmol min−1 mg−1 protein)

9.49±0.939 21.31±1.987* 45.46±4.167* 23.60±1.886**

0.63±0.047 0.51±0.054* 0.26±0.047* 0.64±0.054**

0.23±0.077 0.88±0.076* 0.57±0.047* 0.96±0.119**

0.55±0.054 0.24±0.036* 0.19±0.018* 0.29±0.032

1.22±0.065 0.82±0.034* 0.57±0.049* 0.76±0.044

571.95±24.28 420.38±21.029* 318.70±13.459* 477.99±19.95**

Values are expressed as mean±SEM for n=16. *P≤0.05 compared to control (group I) **P≤0.05 compared to patients 14 days after surgery (group III)

oxidants) in patients with oral cavity cancer. A weak antioxidant defense system makes the macromolecules of immunocompetent cells and serum more vulnerable to the genotoxic effect of ROS. This creates an intracellular environment more favorable for DNA damage and disease progression. “Glutammune”, a rich source of Lglutamine (66.66%), ameliorate the oxidative stress in some extent (Fig. 1). However, the degree of effectiveness with which the redox balance can be restored with dietary supplements including “glutammune” and “glutammune”related drugs so that cancer patients can be benefited. Finally, more research can be continued in this field to know the actual mechanism and development of a suitable drug. Studies on these lines are presently in progress.

by the elevated levels of lipid peroxides in the circulation of oral cavity cancer patients. Reports on CAT activity in cancer are contradictory. Both increase [20] and decrease [23, 26, 38] in CAT activity have been reported previously. Reduction in CAT and SOD activity as observed in this study might be due to increased endogenous production of the superoxide anion, as evidenced by increased MDA. But in the supplemented group, it has been revealed that significantly increased CAT and SOD activity minimizes the MDA level near to the group II subjects, but not to the normal level (Table 4). From this study, it could be concluded that oxidative stress is increased (as evidenced by elevated levels of lipid peroxidation products), and antioxidant defenses are depleted (as evidenced by depletion of enzymatic anti-

Fig. 1 Oxidative stress during the oral cavity cancer and the role of L-glutamine to reduce the potential cancer progression

Cigarette smoke, Tobacco products and other factors

DECREASE THE CANCER PROGRASSION

Initiation of lipid peroxidation and weak antioxidant enzyme

Improved immune functions

OXIDATIVE STRESS

Decreased genotoxic effect of ROS leads to carcinogenic deactivation

Genotoxic effect of ROS leads to carcinogenic activation

Boost up the antioxidant defense system especially glutathione system L-glutamine supplementation

ORAL CAVITY CANCER

1404 Acknowledgment The authors are very much grateful to Dr. Sk. Saidul Islam, maxillofacial onco and microvascular surgeon, Royd Nursing Home, Kolkata for kind cooperation and Claris Lifesciences Limited, Corporate Towers, Ellisbridge, Ahmedabad, 380 006, India for providing the “glutammune” free of cost.

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