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Measurement of serum paraoxonase activity and MDA concentrations in patients suffering with oral squamous cell carcinoma
Clinica Chimica Acta 430 (2014) 38–42
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Measurement of serum paraoxonase activity and MDA concentrations in patients suffering with oral squamous cell carcinoma Uzma Urooj Malik a, Imtiaz Ather Siddiqui b, Zehra Hashim a, Shamshad Zarina a,⁎ a b
National Center for Proteomics, University of Karachi, Karachi, Pakistan Department of ENT, Jinnah Postgraduate Medical Center, Karachi, Pakistan
a r t i c l e
i n f o
Article history: Received 27 May 2013 Received in revised form 12 November 2013 Accepted 23 December 2013 Available online 2 January 2014 Keywords: Oral squamous cell carcinoma Paraoxonase activity Arylesterase activity Oxidative stress Lipid peroxidation Malondialdehyde
a b s t r a c t Background: Oxidative stress is associated with many diseases including cancer. Oral squamous cell carcinoma (OSCC) is a prevalent cancer involving oral cavity. We evaluate the activity of paraoxonase 1 (PON1) in serum samples of subjects suffering from OSCC along with malondialdehyde (MDA) levels, a marker for oxidative stress. Antioxidant status in OSCC may reflect the role of oxidative imbalance in the disease. Methods: Forty-five patients suffering with OSCC and 30 healthy controls were selected for the study. Serum paraoxonase (PON) and arylesterase (ARE) activities were measured in subjects suffering from OSCC and their healthy counterparts. To examine the status of lipid peroxidation, MDA concentrations were estimated and a correlation was determined between PON activities and MDA concentrations. MDA expression in cancer and normal adjacent tissue was studied through immunohistochemical (IHC) analysis. Total reactive oxygen species (ROS) level was determined in serum from normal and diseased subjects. Our results revealed that both PON and ARE activities of PON1 were significantly decreased in OSCC patients. Serum MDA concentrations were inversely correlated to PON activity. Immunohistochemical analysis showed a higher expression of MDA in cancerous tissue. Total ROS levels were found to be significantly elevated in cancer subjects. Conclusions: Along with other antioxidants, PON levels may act as an indicator of oxidative stress in cancer. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Oral cancer is among the most prevalent malignancies constituting a significant component of global cancer burden [1]. Oral squamous cell carcinoma (OSCC) accounts for 90% of malignant oral lesions and is widely recognized as a most frequently occurring malignant tumor of oral cavity [2]. The incidence rate is very high especially in Europe, South East Asia and Brazil [3]. Despite of the advances in surgical techniques and treatment methods, five year survival rate has remained low [4]. In Pakistan, oral cancer is among the 10 most commonly occurring cancers [5], out of which it is second and third most common in women and men, respectively. The survival rates of oral cancer patients are unsatisfactory due to the poor prognosis and late diagnosis of the disease [6]. Chronic irritating factors which play critical part in cancer development are tobacco, alcohol, and betel quid. Studies reveal that cigarette smoking and alcohol drinking are the major risk factors in Western
Abbreviations: OSCC, oral squamous cell carcinoma; PON, paraoxonase; ARE, arylesterase; MDA, malondialdehyde; SOD, superoxide dismutase; ROS, reactive oxygen species; XO, xylenol. ⁎ Corresponding author at: National Center for Proteomics, University of Karachi, Karachi-75270, Pakistan. Tel.: +92 21 34656511; fax: +92 21 34650726. E-mail address: [email protected] (S. Zarina). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.033
countries, whereas, Paan, betel quid and tobacco are major contributing factors for oral cancer in South East Asia [7]. The world practice of tobacco and betel chewing is most prevalent in south Asian countries and thus offers a major risk for oral cancer. Betel quid has been strongly associated with the development of mucosal diseases such as leukoplakia, oral submucosa fibrosis, oral precancer lesions and oral cancer [7,8]. Reactive oxygen species (ROS) are generated in cellular response to various physiological processes and their adverse effects are minimized by cell antioxidant defense system. This balance is critical for maintaining normal cellular activities and prevention of cell damage. The imbalance between ROS generation and removal results in damage to DNA, lipids and proteins [9]. Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is initiated due to the formation of ROS resulting in reactive carbonyl compounds production. Malondialdehyde (MDA) is among secondary products of lipid peroxidation and is extensively studied as a potential biomarker for oxidative stress [10]. MDA concentration has been measured to examine oxidative stress and as an indicator of oxidative imbalance during the onset of many diseases [11]. It is stated that ROS are responsible for pathogenesis of different diseases including cancer [12] and the process of carcinogenesis can be induced by redox imbalance [13]. Limited literature is available regarding the relationship of oxidative stress markers in OSCC. Higher rates of lipid peroxidation and low antioxidants levels have been reported in serum of patients suffering from OSCC [14]. Another report highlighted oxidant/antioxidant status in blood and tumor tissue of
U.U. Malik et al. / Clinica Chimica Acta 430 (2014) 38–42 Table 1 Distribution of subjects with respect to ethnicity, tumor location, habit history and TNM staging. n and % represent number and relative percentage of patients in each category, respectively. n
Percentage (%)
Ethnicity Urdu speaking Pathan Sindhi Punjabi Balochi
19 12 4 4 6
42.22 26.66 8.89 8.89 13.33
Location of tumor Right cheek Left cheek Tongue Lip More than one site
19 12 5 3 6
42.22 26.67 11.11 6.67 13.33
Habit history Paan (Betel quid with tobacco & betel nut) Naswar Gutka Manpuri Betel nut Cigarette & Paan Cigarette & Naswar Cigarette & Manpuri None
9 14 5 4 1 3 1 2 6
20 31.11 11.11 8.89 2.22 6.67 2.22 4.44 13.33
Stage of cancer (TNM staging) Stage I Stage II Stage III
27 15 3
60 33.33 6.67
OSCC patients with specific reference to superoxide dismutase (SOD) and catalase activities [15]. Human serum paraoxonase (PON1) is a 43–45 kDa calcium dependent esterase that functions as a detoxifier of organophosphates [16]. The paraoxonase gene family has 3 members including PON1, PON2 and PON3, which are homologous with each other [17]. Among these, Paraoxonase 1 is the most extensively studied member [18]. Human PON1 is synthesized in liver and released into the blood where it circulates in association with high-density lipoprotein (HDL). PON1 prevents oxidative stress induced by oxidation in HDL, LDL, homocysteine, thiolactone, and platelet aggregating factor [19]. Decreased PON1
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activity has been reported in various disorders including cardiovascular diseases [20], chronic renal failure [21], senile and diabetic cataract [22], hyperthyroidism [23], age-related macular degeneration [24], lung, colorectal and breast cancer [25], esophageal cancer [26], laryngeal cancer [27], and many other diseases [28]. Studies have indicated that apart from paraoxonase activity, PON1 serves as a contributing factor for serum arylesterase (ARE) activity which in turn has shown a strong correlation with oxidative stress [29]. 2. Materials and methods All chemicals used in this study were of reagent grade and purchased by Sigma–Aldrich (St. Louis, USA), Merck, (Germany), and Scharlau (Spain). 2.1. Patients and samples The study was carried out after the ethical approval of Institutional Review board. The study included patients and controls of either sex without any discrimination. Out of 45 patients, 31 were males (mean age ± S.D: 45.7 ± 11.72 y) while 14 were females (mean age ± S.D: 43.5 ± 6.16 y). In case of controls, 24 were males and 6 were females (mean age ± S.D: 38.54 ± 12.19 and 39.66 ± 10.28 y respectively). For serum analysis, 45 OSCC patients were selected who were admitted to ENT department of Jinnah Postgraduate Medical Center (JPMC) Karachi and diagnosed with OSCC. Tumor node metastasis (TNM) staging system was used to diagnose the stage of cancer. Thirty age and sex matched subjects were selected as controls who had no smoking or chewing habits. The subjects and controls were excluded from the study if they had any history of diabetes, cataract, cardiovascular diseases, hypertension, hepatic function disorder, renal dysfunction, anaemia, arthritis, hyperthyroidism, hypothyroidism, breast cancer, colorectal cancer, lung cancer, laryngeal cancer and esophageal cancer. After getting an informed consent, 3 ml venous blood was collected from each patient and control. The blood was allowed to coagulate for 15 min and then centrifuged at 3500 rpm for 10 min. Serum was collected and stored at − 70 °C till further analysis. For IHC study, cancerous tissue and normal mucosa that is at least 2 cm apart from tumor was collected during surgery. 2.2. Paraoxonase activity measurement Serum paraoxonase activity was measured by the method as described previously [30]. The assay mixture contained 1.0 mmol/l CaCl2 and 1.0 mmol/l paraoxon in 50 mmol/l glycine/NaOH buffer of pH 10.5. Paraoxon was used as a substrate and 50 μl serum was added. The change in absorbance due to the formation of 4-nitrophenol was measured at 412 nm using UV mini 1240, Shimadzu UV Spectrophotometer. The activity of paraoxonase was calculated using molar extinction coefficient 18,290 M−1 cm−1. 2.3. Arylesterase activity measurement Arylesterase activity was measured by estimating the rate of phenyl acetate hydrolysis. 50 μl serum was added in 20 mmol/l Tris–HCl buffer of pH 8.0 containing 1.0 mmol/l CaCl2 and 1 mmol/l phenyl acetate as a substrate. The rate of hydrolysis was measured at 270 nm for 2 min and results were articulated as change in absorbance per ml per minute. Molar extinction coefficient used for calculation was 1310 M −1cm−1 [22,30]. 2.4. MDA measurement
Fig. 1. Paraoxonase activity in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. Asterisks indicate statistical significance.
Serum MDA concentrations were determined by estimating the products of the reaction between MDA and thiobarbituric acid (TBA) as described previously [31]. These products were extracted by treating
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U.U. Malik et al. / Clinica Chimica Acta 430 (2014) 38–42
Fig. 2. Arylesterase (ARE) activity in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. *p b 0.001.
50 μl serum with 20% trichloroacetic acid solution containing 8.1% SDS and 0.8% aqueous solution of TBA. Then the mixture was heated at boiling water bath for 1 h followed by cooling on ice. N-butanol was then added and mixture was centrifuged at 4000 rpm for 10 min. The reaction products were separated by taking organic layer and read at 532 nm. 1,1,3,3-Tetraethoxy propane was used to prepare standards. 2.5. Total ROS assay Fifty microliters of serum was added with 3 ml of reagent solution containing 25 mmol/l H2 SO 4 , 100 μmol/l xylenol orange (XO), 100 μmol/l sorbitol, and 200 μmol/l ferrous ammonium sulphate. The pH of the mixture was 1.8 which is optimum for the reaction. The mixture was then incubated for 1 h in the dark and absorbance was read at 560 nm using XO as blank [32]. Hydrogen peroxide (H2O2) was used as standard.
Fig. 4. Correlation between serum PON activity and MDA concentrations in OSCC patients and controls. P b 0.001 was considered statistically significant.
secondary antibody at a dilution of 1:200. Negative controls were also prepared following the same procedure, the only difference was that the primary antibody was not added in the incubation mixture.
2.7. Western blotting Western blot analysis for the presence of PON1 in serum of controls and OSCC patients was performed as described earlier [34]. Briefly, equal amounts of serum proteins from controls and OSCC patients were subjected to 12% SDS–polyacrylamide gel electrophoresis followed by transfer to PVDF membrane. The membrane was blocked with 3% BSA solution for 1 h and incubated overnight with human PON1 monoclonal antibody (Abcam) diluted (1:500) in blocking solution followed by incubation with horseradish peroxidase- conjugated goat anti-mouse IgG (1:2500 in TBS). The reactive PON1 band was visualized using ECL Western blotting detection kit (Millipore).
2.6. Immunohistochemical analysis 2.8. Statistical analysis Immunohistochemical analysis was performed as described elsewhere [33]. Briefly, fresh normal and cancerous tissues were fixed in phosphate-buffered formalin and cryosections (2–3 μm thickness) were collected. Specific human MDA polyclonal antibody (Abcam) was used for immunostaining of each section at a dilution of 1:100 which was then detected by Alexa Fluor-conjugated goat anti-rabbit
Fig. 3. MDA concentrations in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. *P b 0.001.
SPSS software (IBM SPSS Statistics ver 20) was used to analyze data. Mean values with standard deviation were calculated using t-test for both PON and ARE activities. MDA and ROS levels were also tested and in each case, patients and controls were analyzed separately and then compared using one-way ANOVA. To analyze the relationship of PON activity with MDA concentrations, Pearson's correlation coefficient was used. A p b 0.001 was considered statistically significant.
Fig. 5. Total ROS levels in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. *P b 0.001.
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Fig. 6. Immunohistochemical analysis of human oral cavity normal (A) and cancerous (B) tissue stained for MDA.
3. Results
4. Discussion
Demographic details of OSCC subjects including ethnicity, location of tumor, chewing habits, and stage of cancer are given in Table 1. The subjects included in this study belonged to various ethnicities thus representing people from different castes who visited the local hospital.
Reactive oxygen species (ROS) are produced in response to normal cellular processes. Low concentrations of ROS are required for certain sub-cellular events while higher concentrations might have deleterious effects. ROS that are released in excessive amounts not only affect redox balance of the cell but also damage other cellular machinery including DNA, cellular proteins and lipids. Oxidative stress reflects disturbance in ROS homeostasis either due to elevation of ROS or decline of ROS scavenging capacity. This imbalance may originate from a variety of reasons such as exposure to carcinogens, environmental factors or genetic alterations etc. and is found to be increased during pathogenesis of many diseases including cancer. Cancer is a multi-stage process mainly involving initiation, promotion and progression and all of these stages can be triggered by ROS thus promoting tumor growth [35,36]. Being an important etiological factor in carcinogenesis, oxidative stress has been studied in patients suffering from various cancers including OSCC. However, PON1, an endogenous free radical scavenger, has not been studied in OSCC so far. In current study, we have measured serum paraoxonase and arylesterase activities of PON1 in subjects suffering with OSCC. To examine the extent of lipid peroxidation, concentration of MDA, a biomarker for oxidative stress, was also measured. Maintaining normal ROS level is crucial for normal cellular growth and survival [37]. To meet the increasing demand of growing tumor, host tumor cells sequester all essential nutrients from circulation resulting in decreased antioxidant levels due to their utilization. Earlier studies have reported significantly decreased levels of ROS scavenging antioxidants including SOD, catalase and glutathione in OSCC patients indicating oxidative stress [38,39]. Our results demonstrated significantly elevated total ROS in OSCC samples as compared to their respective controls. This result was substantiated by significantly lower paraoxonase and arylesterase activities in the serum of OSCC subjects as compared to age and sex matched healthy controls. Similar trends have been reported in lung [25], pancreatic [40], and ovarian cancer [41]. In gastroesophageal cancer, authors noted a correlation between decreased PON1 activity and lymph node metastasis but due to moderate predictive power, they were reluctant to suggest use of PON1 activity as a biomarker in clinical applications [42]. Our findings propose that along with other antioxidants, PON1 can serve as a potential candidate for studying the process of carcinogenesis implicated due to oxidative stress. Although oxidative imbalance has been reported in various types of cancers, the concentration and implication of lipid peroxidation products in cancer are still controversial. Few studies demonstrate low levels of lipid peroxidation end products in cancer subjects as compared to controls [43] while others highlight an involvement of oxidative stress induced lipid peroxidation products during carcinogenesis [44]. Nevertheless, lipid peroxidation products have always been subject of interest. The protective role of PON1 against LDL oxidation is suggested to contribute towards elimination of carcinogenic lipid soluble radical [25]. Thus, decreased PON levels are expected to increase lipid
3.1. Enzyme activity of PON and lipid peroxidation Both PON (Fig. 1) and ARE (Fig. 2) activities were found to be decreased significantly (p b 0.001) in serum from OSCC patients when compared with respective controls. Serum MDA concentrations were significantly elevated (Fig. 3) in subjects suffering from OSCC compared to their healthy counterparts (p b 0.001). PON activity was found to be negatively correlated to MDA concentrations (r = − 0.534; p b 0.01; Fig. 4) thus reflecting an increased oxidative stress. 3.2. Total ROS levels Total ROS level was found to be significantly increased (p b 0.001) in subjects compared to controls (Fig. 5). This clearly indicates that an oxidative imbalance has occurred in OSCC subjects. 3.3. Expression of MDA in normal and cancerous tissues Immunohistochemical analysis was performed using human MDA antibody. Results of IHC are depicted in Fig. 6 for both normal (A) and cancerous (B) tissue. Cancerous tissues showed an over expression of MDA as compared to Normal tissue. 3.4. Western blot analysis Western blot analysis of PON1 from serum samples revealed a single band at 42 kDa (Fig. 7). The intensity of PON band from healthy controls was found to be higher than from patients (data not shown).
Fig. 7. Western blot analysis for the presence of PON1 in serum from OSCC subjects (right panel) and healthy controls (left panel).
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peroxidation. Indeed, the levels of MDA, a biomarker of lipid peroxidation, were found to be negatively correlated with PON activity depicting increased lipid peroxidation in the body. IHC analysis of cancerous and adjacent normal tissue revealed over expression of MDA in diseased tissue reflecting involvement of lipid peroxidation. Our findings are consistent with earlier studies which highlighted significant increase in lipid peroxidation in OSCC patients [38,39]. OSCC is a multi-factorial disorder and many other factors apart from oxidative stress may be at play; yet finding out the preventive role of antioxidants in pathogenesis of carcinogenesis might be beneficial. ROS act as regulatory factors in pathways associated with tumor development hence they might have potential therapeutic intervention points [45]. Considering the implications of oxidative imbalance in tumor development, clinical trials testing anti-carcinogenic effects of antioxidant supplements have been proposed [25]. A recent study has reported PON1 as potential biomarker of survival in patients with breast cancer recurrence [46]. Although decreased PON activity is likely to be a consequence of cancer rather than an underlying cause [25], understanding the role of PON as an antioxidant might contribute in elimination of carcinogenic radicals. Furthermore, agents that enhance PON1 activity in high risk population and subsequent alleviation of malignancy will help in further confirmation of the protective role of PON1 [28]. Significant decrease of PON1 and ARE activities and concomitant increase in MDA concentrations indicate the contribution of oxidative stress in developing cancer related complications. Current study provides preliminary data that needs to be further verified in other populations in order to understand the exact role of PON in carcinogenesis, the mechanism leading towards reduced activity and its implication in OSCC. Acknowledgment This work was supported by a research project (project no: 2010) funded by Higher Education Commission, Islamabad, Pakistan. References [1] Thun MJ, DeLancey JO, Center MM, Jemal A, Ward EM. The global burden of cancer: priorities for prevention. Carcinogenesis 2010;31:100–10. [2] Lai CH, Chang NW, Lin CF, et al. Proteomics-based identification of haptoglobin as a novel plasma biomarker in oral squamous cell carcinoma. Clin Chim Acta 2010;411:984–91. [3] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74–108. [4] Kademani D. Oral Cancer. Mayo Clin Proc 2007;82:878–87. [5] WHO study group report on control of oral cancer in developing countries. Bull WHO 1984;62:817–30. [6] Merchant A, Husain SS, Hosain M, et al. Paan without tobacco: an independent risk factor for oral cancer. Int J Cancer 2000;86:128–31. [7] Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24:450–3. [8] Chen YK, Huang HC, Lin LM, Lin CC. Primary oral squamous cell carcinoma: an analysis of 703 cases in southern Taiwan. Oral Oncol 1999;35:173–9. [9] Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem-Biol Interact 2006;160:1–40. [10] Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR, Grandjean P. Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. Clin Chem 1997;43:1209–14. [11] Niki E. Lipid peroxidation products as oxidative stress biomarkers. Biofactors 2008;34:171–80. [12] Abdi S, Ali A. Role of ROS modified human DNA in the pathogenesis and etiology of cancer. Cancer Lett 1999;142:1–9. [13] Pala FS, Gürkan H. The role of free radicals in ethiopathogenesis of diseases. Adv Molec Biol 2008;1:1–9. [14] Khanna R, Thapa PB, Khanna HD, Khanna S, Khanna AK, Shukla HS. Lipid peroxidation and antioxidant enzyme status in oral carcinoma patients. Kath Univ Med J 2005;3:334–9.
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Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Measurement of serum paraoxonase activity and MDA concentrations in patients suffering with oral squamous cell carcinoma Uzma Urooj Malik a, Imtiaz Ather Siddiqui b, Zehra Hashim a, Shamshad Zarina a,⁎ a b
National Center for Proteomics, University of Karachi, Karachi, Pakistan Department of ENT, Jinnah Postgraduate Medical Center, Karachi, Pakistan
a r t i c l e
i n f o
Article history: Received 27 May 2013 Received in revised form 12 November 2013 Accepted 23 December 2013 Available online 2 January 2014 Keywords: Oral squamous cell carcinoma Paraoxonase activity Arylesterase activity Oxidative stress Lipid peroxidation Malondialdehyde
a b s t r a c t Background: Oxidative stress is associated with many diseases including cancer. Oral squamous cell carcinoma (OSCC) is a prevalent cancer involving oral cavity. We evaluate the activity of paraoxonase 1 (PON1) in serum samples of subjects suffering from OSCC along with malondialdehyde (MDA) levels, a marker for oxidative stress. Antioxidant status in OSCC may reflect the role of oxidative imbalance in the disease. Methods: Forty-five patients suffering with OSCC and 30 healthy controls were selected for the study. Serum paraoxonase (PON) and arylesterase (ARE) activities were measured in subjects suffering from OSCC and their healthy counterparts. To examine the status of lipid peroxidation, MDA concentrations were estimated and a correlation was determined between PON activities and MDA concentrations. MDA expression in cancer and normal adjacent tissue was studied through immunohistochemical (IHC) analysis. Total reactive oxygen species (ROS) level was determined in serum from normal and diseased subjects. Our results revealed that both PON and ARE activities of PON1 were significantly decreased in OSCC patients. Serum MDA concentrations were inversely correlated to PON activity. Immunohistochemical analysis showed a higher expression of MDA in cancerous tissue. Total ROS levels were found to be significantly elevated in cancer subjects. Conclusions: Along with other antioxidants, PON levels may act as an indicator of oxidative stress in cancer. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Oral cancer is among the most prevalent malignancies constituting a significant component of global cancer burden [1]. Oral squamous cell carcinoma (OSCC) accounts for 90% of malignant oral lesions and is widely recognized as a most frequently occurring malignant tumor of oral cavity [2]. The incidence rate is very high especially in Europe, South East Asia and Brazil [3]. Despite of the advances in surgical techniques and treatment methods, five year survival rate has remained low [4]. In Pakistan, oral cancer is among the 10 most commonly occurring cancers [5], out of which it is second and third most common in women and men, respectively. The survival rates of oral cancer patients are unsatisfactory due to the poor prognosis and late diagnosis of the disease [6]. Chronic irritating factors which play critical part in cancer development are tobacco, alcohol, and betel quid. Studies reveal that cigarette smoking and alcohol drinking are the major risk factors in Western
Abbreviations: OSCC, oral squamous cell carcinoma; PON, paraoxonase; ARE, arylesterase; MDA, malondialdehyde; SOD, superoxide dismutase; ROS, reactive oxygen species; XO, xylenol. ⁎ Corresponding author at: National Center for Proteomics, University of Karachi, Karachi-75270, Pakistan. Tel.: +92 21 34656511; fax: +92 21 34650726. E-mail address: [email protected] (S. Zarina). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.12.033
countries, whereas, Paan, betel quid and tobacco are major contributing factors for oral cancer in South East Asia [7]. The world practice of tobacco and betel chewing is most prevalent in south Asian countries and thus offers a major risk for oral cancer. Betel quid has been strongly associated with the development of mucosal diseases such as leukoplakia, oral submucosa fibrosis, oral precancer lesions and oral cancer [7,8]. Reactive oxygen species (ROS) are generated in cellular response to various physiological processes and their adverse effects are minimized by cell antioxidant defense system. This balance is critical for maintaining normal cellular activities and prevention of cell damage. The imbalance between ROS generation and removal results in damage to DNA, lipids and proteins [9]. Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is initiated due to the formation of ROS resulting in reactive carbonyl compounds production. Malondialdehyde (MDA) is among secondary products of lipid peroxidation and is extensively studied as a potential biomarker for oxidative stress [10]. MDA concentration has been measured to examine oxidative stress and as an indicator of oxidative imbalance during the onset of many diseases [11]. It is stated that ROS are responsible for pathogenesis of different diseases including cancer [12] and the process of carcinogenesis can be induced by redox imbalance [13]. Limited literature is available regarding the relationship of oxidative stress markers in OSCC. Higher rates of lipid peroxidation and low antioxidants levels have been reported in serum of patients suffering from OSCC [14]. Another report highlighted oxidant/antioxidant status in blood and tumor tissue of
U.U. Malik et al. / Clinica Chimica Acta 430 (2014) 38–42 Table 1 Distribution of subjects with respect to ethnicity, tumor location, habit history and TNM staging. n and % represent number and relative percentage of patients in each category, respectively. n
Percentage (%)
Ethnicity Urdu speaking Pathan Sindhi Punjabi Balochi
19 12 4 4 6
42.22 26.66 8.89 8.89 13.33
Location of tumor Right cheek Left cheek Tongue Lip More than one site
19 12 5 3 6
42.22 26.67 11.11 6.67 13.33
Habit history Paan (Betel quid with tobacco & betel nut) Naswar Gutka Manpuri Betel nut Cigarette & Paan Cigarette & Naswar Cigarette & Manpuri None
9 14 5 4 1 3 1 2 6
20 31.11 11.11 8.89 2.22 6.67 2.22 4.44 13.33
Stage of cancer (TNM staging) Stage I Stage II Stage III
27 15 3
60 33.33 6.67
OSCC patients with specific reference to superoxide dismutase (SOD) and catalase activities [15]. Human serum paraoxonase (PON1) is a 43–45 kDa calcium dependent esterase that functions as a detoxifier of organophosphates [16]. The paraoxonase gene family has 3 members including PON1, PON2 and PON3, which are homologous with each other [17]. Among these, Paraoxonase 1 is the most extensively studied member [18]. Human PON1 is synthesized in liver and released into the blood where it circulates in association with high-density lipoprotein (HDL). PON1 prevents oxidative stress induced by oxidation in HDL, LDL, homocysteine, thiolactone, and platelet aggregating factor [19]. Decreased PON1
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activity has been reported in various disorders including cardiovascular diseases [20], chronic renal failure [21], senile and diabetic cataract [22], hyperthyroidism [23], age-related macular degeneration [24], lung, colorectal and breast cancer [25], esophageal cancer [26], laryngeal cancer [27], and many other diseases [28]. Studies have indicated that apart from paraoxonase activity, PON1 serves as a contributing factor for serum arylesterase (ARE) activity which in turn has shown a strong correlation with oxidative stress [29]. 2. Materials and methods All chemicals used in this study were of reagent grade and purchased by Sigma–Aldrich (St. Louis, USA), Merck, (Germany), and Scharlau (Spain). 2.1. Patients and samples The study was carried out after the ethical approval of Institutional Review board. The study included patients and controls of either sex without any discrimination. Out of 45 patients, 31 were males (mean age ± S.D: 45.7 ± 11.72 y) while 14 were females (mean age ± S.D: 43.5 ± 6.16 y). In case of controls, 24 were males and 6 were females (mean age ± S.D: 38.54 ± 12.19 and 39.66 ± 10.28 y respectively). For serum analysis, 45 OSCC patients were selected who were admitted to ENT department of Jinnah Postgraduate Medical Center (JPMC) Karachi and diagnosed with OSCC. Tumor node metastasis (TNM) staging system was used to diagnose the stage of cancer. Thirty age and sex matched subjects were selected as controls who had no smoking or chewing habits. The subjects and controls were excluded from the study if they had any history of diabetes, cataract, cardiovascular diseases, hypertension, hepatic function disorder, renal dysfunction, anaemia, arthritis, hyperthyroidism, hypothyroidism, breast cancer, colorectal cancer, lung cancer, laryngeal cancer and esophageal cancer. After getting an informed consent, 3 ml venous blood was collected from each patient and control. The blood was allowed to coagulate for 15 min and then centrifuged at 3500 rpm for 10 min. Serum was collected and stored at − 70 °C till further analysis. For IHC study, cancerous tissue and normal mucosa that is at least 2 cm apart from tumor was collected during surgery. 2.2. Paraoxonase activity measurement Serum paraoxonase activity was measured by the method as described previously [30]. The assay mixture contained 1.0 mmol/l CaCl2 and 1.0 mmol/l paraoxon in 50 mmol/l glycine/NaOH buffer of pH 10.5. Paraoxon was used as a substrate and 50 μl serum was added. The change in absorbance due to the formation of 4-nitrophenol was measured at 412 nm using UV mini 1240, Shimadzu UV Spectrophotometer. The activity of paraoxonase was calculated using molar extinction coefficient 18,290 M−1 cm−1. 2.3. Arylesterase activity measurement Arylesterase activity was measured by estimating the rate of phenyl acetate hydrolysis. 50 μl serum was added in 20 mmol/l Tris–HCl buffer of pH 8.0 containing 1.0 mmol/l CaCl2 and 1 mmol/l phenyl acetate as a substrate. The rate of hydrolysis was measured at 270 nm for 2 min and results were articulated as change in absorbance per ml per minute. Molar extinction coefficient used for calculation was 1310 M −1cm−1 [22,30]. 2.4. MDA measurement
Fig. 1. Paraoxonase activity in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. Asterisks indicate statistical significance.
Serum MDA concentrations were determined by estimating the products of the reaction between MDA and thiobarbituric acid (TBA) as described previously [31]. These products were extracted by treating
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Fig. 2. Arylesterase (ARE) activity in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. *p b 0.001.
50 μl serum with 20% trichloroacetic acid solution containing 8.1% SDS and 0.8% aqueous solution of TBA. Then the mixture was heated at boiling water bath for 1 h followed by cooling on ice. N-butanol was then added and mixture was centrifuged at 4000 rpm for 10 min. The reaction products were separated by taking organic layer and read at 532 nm. 1,1,3,3-Tetraethoxy propane was used to prepare standards. 2.5. Total ROS assay Fifty microliters of serum was added with 3 ml of reagent solution containing 25 mmol/l H2 SO 4 , 100 μmol/l xylenol orange (XO), 100 μmol/l sorbitol, and 200 μmol/l ferrous ammonium sulphate. The pH of the mixture was 1.8 which is optimum for the reaction. The mixture was then incubated for 1 h in the dark and absorbance was read at 560 nm using XO as blank [32]. Hydrogen peroxide (H2O2) was used as standard.
Fig. 4. Correlation between serum PON activity and MDA concentrations in OSCC patients and controls. P b 0.001 was considered statistically significant.
secondary antibody at a dilution of 1:200. Negative controls were also prepared following the same procedure, the only difference was that the primary antibody was not added in the incubation mixture.
2.7. Western blotting Western blot analysis for the presence of PON1 in serum of controls and OSCC patients was performed as described earlier [34]. Briefly, equal amounts of serum proteins from controls and OSCC patients were subjected to 12% SDS–polyacrylamide gel electrophoresis followed by transfer to PVDF membrane. The membrane was blocked with 3% BSA solution for 1 h and incubated overnight with human PON1 monoclonal antibody (Abcam) diluted (1:500) in blocking solution followed by incubation with horseradish peroxidase- conjugated goat anti-mouse IgG (1:2500 in TBS). The reactive PON1 band was visualized using ECL Western blotting detection kit (Millipore).
2.6. Immunohistochemical analysis 2.8. Statistical analysis Immunohistochemical analysis was performed as described elsewhere [33]. Briefly, fresh normal and cancerous tissues were fixed in phosphate-buffered formalin and cryosections (2–3 μm thickness) were collected. Specific human MDA polyclonal antibody (Abcam) was used for immunostaining of each section at a dilution of 1:100 which was then detected by Alexa Fluor-conjugated goat anti-rabbit
Fig. 3. MDA concentrations in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. *P b 0.001.
SPSS software (IBM SPSS Statistics ver 20) was used to analyze data. Mean values with standard deviation were calculated using t-test for both PON and ARE activities. MDA and ROS levels were also tested and in each case, patients and controls were analyzed separately and then compared using one-way ANOVA. To analyze the relationship of PON activity with MDA concentrations, Pearson's correlation coefficient was used. A p b 0.001 was considered statistically significant.
Fig. 5. Total ROS levels in serum from OSCC subjects and healthy controls. Each bar represents mean ± S.D (n = 45) for OSCC subjects and (n = 30) for controls. *P b 0.001.
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Fig. 6. Immunohistochemical analysis of human oral cavity normal (A) and cancerous (B) tissue stained for MDA.
3. Results
4. Discussion
Demographic details of OSCC subjects including ethnicity, location of tumor, chewing habits, and stage of cancer are given in Table 1. The subjects included in this study belonged to various ethnicities thus representing people from different castes who visited the local hospital.
Reactive oxygen species (ROS) are produced in response to normal cellular processes. Low concentrations of ROS are required for certain sub-cellular events while higher concentrations might have deleterious effects. ROS that are released in excessive amounts not only affect redox balance of the cell but also damage other cellular machinery including DNA, cellular proteins and lipids. Oxidative stress reflects disturbance in ROS homeostasis either due to elevation of ROS or decline of ROS scavenging capacity. This imbalance may originate from a variety of reasons such as exposure to carcinogens, environmental factors or genetic alterations etc. and is found to be increased during pathogenesis of many diseases including cancer. Cancer is a multi-stage process mainly involving initiation, promotion and progression and all of these stages can be triggered by ROS thus promoting tumor growth [35,36]. Being an important etiological factor in carcinogenesis, oxidative stress has been studied in patients suffering from various cancers including OSCC. However, PON1, an endogenous free radical scavenger, has not been studied in OSCC so far. In current study, we have measured serum paraoxonase and arylesterase activities of PON1 in subjects suffering with OSCC. To examine the extent of lipid peroxidation, concentration of MDA, a biomarker for oxidative stress, was also measured. Maintaining normal ROS level is crucial for normal cellular growth and survival [37]. To meet the increasing demand of growing tumor, host tumor cells sequester all essential nutrients from circulation resulting in decreased antioxidant levels due to their utilization. Earlier studies have reported significantly decreased levels of ROS scavenging antioxidants including SOD, catalase and glutathione in OSCC patients indicating oxidative stress [38,39]. Our results demonstrated significantly elevated total ROS in OSCC samples as compared to their respective controls. This result was substantiated by significantly lower paraoxonase and arylesterase activities in the serum of OSCC subjects as compared to age and sex matched healthy controls. Similar trends have been reported in lung [25], pancreatic [40], and ovarian cancer [41]. In gastroesophageal cancer, authors noted a correlation between decreased PON1 activity and lymph node metastasis but due to moderate predictive power, they were reluctant to suggest use of PON1 activity as a biomarker in clinical applications [42]. Our findings propose that along with other antioxidants, PON1 can serve as a potential candidate for studying the process of carcinogenesis implicated due to oxidative stress. Although oxidative imbalance has been reported in various types of cancers, the concentration and implication of lipid peroxidation products in cancer are still controversial. Few studies demonstrate low levels of lipid peroxidation end products in cancer subjects as compared to controls [43] while others highlight an involvement of oxidative stress induced lipid peroxidation products during carcinogenesis [44]. Nevertheless, lipid peroxidation products have always been subject of interest. The protective role of PON1 against LDL oxidation is suggested to contribute towards elimination of carcinogenic lipid soluble radical [25]. Thus, decreased PON levels are expected to increase lipid
3.1. Enzyme activity of PON and lipid peroxidation Both PON (Fig. 1) and ARE (Fig. 2) activities were found to be decreased significantly (p b 0.001) in serum from OSCC patients when compared with respective controls. Serum MDA concentrations were significantly elevated (Fig. 3) in subjects suffering from OSCC compared to their healthy counterparts (p b 0.001). PON activity was found to be negatively correlated to MDA concentrations (r = − 0.534; p b 0.01; Fig. 4) thus reflecting an increased oxidative stress. 3.2. Total ROS levels Total ROS level was found to be significantly increased (p b 0.001) in subjects compared to controls (Fig. 5). This clearly indicates that an oxidative imbalance has occurred in OSCC subjects. 3.3. Expression of MDA in normal and cancerous tissues Immunohistochemical analysis was performed using human MDA antibody. Results of IHC are depicted in Fig. 6 for both normal (A) and cancerous (B) tissue. Cancerous tissues showed an over expression of MDA as compared to Normal tissue. 3.4. Western blot analysis Western blot analysis of PON1 from serum samples revealed a single band at 42 kDa (Fig. 7). The intensity of PON band from healthy controls was found to be higher than from patients (data not shown).
Fig. 7. Western blot analysis for the presence of PON1 in serum from OSCC subjects (right panel) and healthy controls (left panel).
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peroxidation. Indeed, the levels of MDA, a biomarker of lipid peroxidation, were found to be negatively correlated with PON activity depicting increased lipid peroxidation in the body. IHC analysis of cancerous and adjacent normal tissue revealed over expression of MDA in diseased tissue reflecting involvement of lipid peroxidation. Our findings are consistent with earlier studies which highlighted significant increase in lipid peroxidation in OSCC patients [38,39]. OSCC is a multi-factorial disorder and many other factors apart from oxidative stress may be at play; yet finding out the preventive role of antioxidants in pathogenesis of carcinogenesis might be beneficial. ROS act as regulatory factors in pathways associated with tumor development hence they might have potential therapeutic intervention points [45]. Considering the implications of oxidative imbalance in tumor development, clinical trials testing anti-carcinogenic effects of antioxidant supplements have been proposed [25]. A recent study has reported PON1 as potential biomarker of survival in patients with breast cancer recurrence [46]. Although decreased PON activity is likely to be a consequence of cancer rather than an underlying cause [25], understanding the role of PON as an antioxidant might contribute in elimination of carcinogenic radicals. Furthermore, agents that enhance PON1 activity in high risk population and subsequent alleviation of malignancy will help in further confirmation of the protective role of PON1 [28]. Significant decrease of PON1 and ARE activities and concomitant increase in MDA concentrations indicate the contribution of oxidative stress in developing cancer related complications. Current study provides preliminary data that needs to be further verified in other populations in order to understand the exact role of PON in carcinogenesis, the mechanism leading towards reduced activity and its implication in OSCC. Acknowledgment This work was supported by a research project (project no: 2010) funded by Higher Education Commission, Islamabad, Pakistan. References [1] Thun MJ, DeLancey JO, Center MM, Jemal A, Ward EM. The global burden of cancer: priorities for prevention. Carcinogenesis 2010;31:100–10. [2] Lai CH, Chang NW, Lin CF, et al. Proteomics-based identification of haptoglobin as a novel plasma biomarker in oral squamous cell carcinoma. Clin Chim Acta 2010;411:984–91. [3] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74–108. [4] Kademani D. Oral Cancer. Mayo Clin Proc 2007;82:878–87. [5] WHO study group report on control of oral cancer in developing countries. Bull WHO 1984;62:817–30. [6] Merchant A, Husain SS, Hosain M, et al. Paan without tobacco: an independent risk factor for oral cancer. Int J Cancer 2000;86:128–31. [7] Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24:450–3. [8] Chen YK, Huang HC, Lin LM, Lin CC. Primary oral squamous cell carcinoma: an analysis of 703 cases in southern Taiwan. Oral Oncol 1999;35:173–9. [9] Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem-Biol Interact 2006;160:1–40. [10] Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR, Grandjean P. Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. Clin Chem 1997;43:1209–14. [11] Niki E. Lipid peroxidation products as oxidative stress biomarkers. Biofactors 2008;34:171–80. [12] Abdi S, Ali A. Role of ROS modified human DNA in the pathogenesis and etiology of cancer. Cancer Lett 1999;142:1–9. [13] Pala FS, Gürkan H. The role of free radicals in ethiopathogenesis of diseases. Adv Molec Biol 2008;1:1–9. [14] Khanna R, Thapa PB, Khanna HD, Khanna S, Khanna AK, Shukla HS. Lipid peroxidation and antioxidant enzyme status in oral carcinoma patients. Kath Univ Med J 2005;3:334–9.
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