© 2005 Foundation for Promotion of Cancer Research
Induced Oxidative Stress and Decreased Expression of Inducible Heat Shock Protein 70 (ihsp 70) in Patients with Colorectal Adenocarcinomas
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1 Department of Biochemistry and 2 Department of General Surgery, 
stanbul Medical Faculty, University of stanbul, Çapa, stanbul, Turkey
For reprints and all correspondence: G. Özdemirler Erata, Department of Biochemistry, stanbul Medical Faculty, University of stanbul, Çapa, TR-34093, stanbul, Turkey. E-mail: gul_erata{at}yahoo.com
Received September 29, 2004; accepted December 11, 2004
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Background: Colorectal cancer is one of the most common carcinomas observed in humans. Recently we have reported that increased oxidative stress is associated with human colorectal cancer. There are few and controversial studies on the clinical relevance of the expression of heat shock protein 70 (HSP 70), a member of the HSP family, in colorectal cancer. In this study, we assayed lipid peroxide levels, glutathione peroxidase (GPx) activity and the expression of inducible heat shock protein 70 (ihsp 70) in 20 paired samples of colorectal tumor and adjacent normal tissues in order to determine the relationship between oxidative stress and ihsp 70 expression. Histopathological results were also considered to estabish the clinical relevance of ihsp 70 in colorectal cancer.
Methods: Malondialdehyde (MDA) levels as an indicator of lipid peroxidation and GPx activity were assayed by spectrophotometric methods. The Western blotting procedure was used for the determination of ihsp 70 expression.
Results: Significant increases were observed in MDA levels (111%) and GPx activities (50%) of malignant tissues as compared with normal tissues of the patients with colorectal cancer. The expression of ihsp 70 was found to be decreased in malignant tissues as compared with normal tissues of the patients. Significant negative correlations were detected between MDA levels and ihsp expression (r = –0.516; P < 0.05 ) and GPx activity and ihsp 70 expression (r = –0.471; P < 0.05) in malignant tissues of patients. When the patients were grouped according to histopathological characteristics, no difference was found in MDA levels, GPx activity and ihsp 70 expression.
Conclusion: Our results indicate that ihsp70 expression is suppressed under induced oxidative stress conditions in malignant tissues of patients with colorectal cancer. Further research is needed to clarify the mechanisms responsible for this decrease and the definitive role of ihsp 70 in colorectal cancer.
Key Words: ihsp 70 • oxidative stress • colorectal cancer
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INTRODUCTION |
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Colorectal cancer is a heterogeneous neoplasm made up of cancer cells with various growth rates and metastatic potentials. Since colorectal tissue is constantly exposed to a variety of hazardous chemicals, free radicals formed during the metabolic activation of these compounds have been thought to play an important role in carcinogenesis (1,2). Although oxidant–antioxidant balance within the tissue is widely accepted to contribute to the development and progression of cancer in both humans (3,4) and experimental animals (5,6), the molecular pathogenesis of colorectal cancer still remains to be elucidated.
In our previous studies, lipid peroxide and glutathione (GSH) levels, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were found to be increased in post-mitochondrial fractions (7); MnSOD (manganase superoxide dismutase), GPx and glutathione S-transferase (GST) activities but not lipid peroxide levels were observed to be increased in the mitochondrial fractions (8) of tumors compared with normal tissues of patients with colorectal cancer.
In response to injury during various stress conditions, all mammalian cells tend to increase the synthesis of a class of stress proteins called heat shock proteins (HSPs). They are also produced constitutively in the normal state. Constitutively expressed HSPs act as molecular chaperones which function by assisting in folding newly synthesized proteins and their translocation into organelles. Under stress conditions, stress-inducible HSPs (ihsps) are induced to be expressed to cope with the increased concentrations of damaged/unfolded proteins in the cell. HSPs are generally designated according to their molecular weights. The HSP 70 family with a molecular mass of 70 kDa is the best studied class of HSPs. Human cells have several HSP 70 family members, but the most thorougly studied of them are ihsp 70 and constitutively expressed HSP 70 (hsc 70). The role of HSP 70 (both ihsp 70 and hsc 70) expression has been investigated in various type of cancers such as breast (10), lung (12), kidney (13), ovary (14) and osteosarcoma (15). Although investigations on the expression of HSP 70 in colorectal cancer in human are available, there is only little and controversial knowledge about its clinical relevance.
In the present study, we assayed lipid peroxide levels, GPx activity and the expression of ihsp 70 to determine the association of ihsp 70 expression with the oxidative conditions in colorectal tissues. In order to establish the clinical relevance of ihsp 70 in colorectal cancer, the relationship between the histopathological results with ihsp 70, lipid peroxidation and GPx were also considered.
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MATERIALS AND METHODS |
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Samples of colorectal tissue were obtained from 20 cancer patients (7 women and 13 men, aged between 33 and 85) who had undergone an operation at the Department of General Surgery of Istanbul Medical Faculty.
Malignant and normal tissue specimens were immediately transported to the laboratory in ice and washed with ice-cold 0.9% NaCl. Tumor-free tissues were taken from areas at least 10 cm away from the tumor site and used as normal colorectal tissue. Twenty paired samples were analyzed and histopathologically confirmed as adenocarcinoma at the Department of Pathology of Istanbul Medical Faculty.
Tissue samples were homogenized in a buffer containing 10 mM Tris, 1 mM MgCl2, 150 mM KCl, 1 mM EDTA, 1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100 and 1.0 µg/ml aprotonin (pH 7.2) by an ultratorax at 4°C. The homogenates (20% w/v) were centrifuged at 600 g for 10 min to remove crude fractions, and the resultant supernatant was used for the determination of lipid peroxide levels, GPx activity and ihsp 70 expression.
LIPID PEROXIDE LEVELS
Malondialdehyde (MDA) levels, the end-product of lipid peroxidation, were measured spectrophotometrically by using thiobarbituric acid (16).
Results were calculated by using 1,1,3,3-tetraethoxypropane, the breakdown product of lipid peroxidation, as the standard, and expressed as pmol MDA/mg protein.
GPX ACTIVITY
In this method, GPx catalyzes the oxidation of GSH by cumene hydroperoxide. In the presence of glutathione reductase (GR) and NADPH, the oxidized glutathione (GSSG) is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance at 340 nm is measured (17). Results were calculated by using the extinction coefficient of NADPH (6.22 x 103/M/cm) and expressed as mU/mg protein.
WESTERN BLOT ANALYSIS
Gel electrophoresis was performed using the Bio Rad mini gel system according to Laemmli's method (18). The samples of colorectal tissue homogenates containing 20 µg of protein per lane were electrophoresed on a 7.5% SDS–polyacrylamide gel in Tris-glycine buffer. After electrophoresis, separated proteins were transferred onto Immobilon-PVDF membrane (Millipore), at 20 V, 45 mA for 2 h (19).The proteins on the membrane were blocked with 3% low-fat dried milk. After washing in phosphate-buffered saline (PBS) containing 1% Tween-20 three times, blots were treated with a 1:1000 dilution of monoclonal antibody specific for ihsp 70 (SPA-810 Stressgen) and then washed with PBS containing 0.1% Tween-20 three times. The resulting immunocomplexes were exposed to 1:10 000 horseradish peroxidase-conjugated goat anti-mouse IgG (SAB-100, Stressgen) for 30 min. After washing with PBS containing 1% Tween-20, labeled antigen bands were visualized by an ECL Western blotting detection reagent (Amersham). The intensity of bands was semi-quantified densitometrically by using the Vilber Lourmatt gel documentation system and Bio-1D V.97 software, an image analyzing program.
The protein content of colorectal tissue was measured by using bicinchoninic acid (20).
STATISTICAL ANALYSIS
Data were expressed as the means ± SEM and the statistical analyses were performed by using the Wilcoxon matched pairs signed ranks and Mann–Whitney U–Wilcoxon rank sum W. Correlation coefficients were determined by Spearman's method.
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RESULTS |
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The physical properties of the patients such as age and sex, and the histolopathological evaluation of the specimens (stage of TNM classification and the extent of necrotic areas and lymphocyte infiltration within the specimens) are shown in Table 1.
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MDA levels and GPx activity were found to be significantly increased in the range of 111 and 50%, respectively, in malignant colorectal tissues as compared with adjacent normal tissues (Table 2). According to the results of western blot analysis of ihsp 70 in normal and tumor tissues, an unexpected decrease was detected in ihsp 70 expression in malignant tissue compared with normal tissue (Fig. 1). In addition, considerable interindividual variations were observed for all parameters (Fig. 2).
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When the measured parameters of malignant tissues were expressed as a percentage of normal tissues, significant negative correlations were found between both GPx activity and ihsp 70 expression (r = –0.471; P < 0.05) and between MDA levels and ihsp 70 expression (r = –0.516; P < 0.05 ) (Fig. 3).
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When the patients were grouped according to some histopathological evaluation such as stage, necrosis and lymphocyte infiltration, we did not find any difference in MDA levels, GPx activity and ihsp 70 expression in malignant tissues (Table 3).
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DISCUSSION |
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The etiology of colorectal cancer, which is one of the most common carcinomas observed in humans, is still being investigated. Colorectal cancer is generally assumed to be initiated by environmental genotoxic agents causing cellular overproduction of reactive oxygen species (ROS). As a consequence, extensive oxyradical damage can cause genetic alterations required for neoplastic progression and lead to a cycle of cell death and regeneration (21). A growing body of evidence has suggested that oxidative stress plays an important role in the molecular mechanism of colorectal cancer (1,3,6,21). Related studies carried out in both humans (3,4) and experimental animals (5,6) have shown lipid peroxide levels in malignant colorectal tissues to be higher than those in the normal tissues of the same patients. In our previous studies, we had revealed that lipid peroxide levels are increased in total tissue homogenates of patients with colorectal cancer (7). In accordance with these results, we found in the present study that MDA levels are increased in the range of 111% in malignant tissues compared with normal tissues of the same patients.
In addition, GPx is one of the important GSH-dependent enzymes which provides protection against oxidative stress by detoxifying oxygen species. Investigators have reported overexpressed (22) or activated (23,24) GPx activity by various environmental stresses including cancer. We had demonstrated GPx activity to be increased in both total tissue homogenates (7) and mitochondrial fractions (8) of the patients with colorectal cancer in our previous studies. In the present study, significant increases in GPx activity (50%) were found in malignant colorectal tissues as compared with normal tissues of the same patients. This increase seen in GPx activity is probably due to the upregulation of the enzyme as an adaptation process against the increased oxidative stress.
On the other hand, HSPs have also been considered to play a general role in protection from cellular injury (9–11). Recently, the expression of some HSPs such as HSP 27, HSP 60, HSP 90 and HSP 70 in tumors and their role in tumor progression have been explored in several studies (10,15,25,26). HSP 70, one of the best studied of the HSPs, has been reported to be overexpressed in various cancers such as breast (10), lung (12), stomach (25) and melanoma (26). However, there are controversial results regarding the clinical implications of increased HSP 70 expression. For example, in breast cancer, ihsp 70 expression has been shown to be correlated with the poor differentiation of the tumor (10). On the other hand, it has been shown that HSP 70 is not associated with advanced characteristics of tumors in gastric cancers (25).
There is little information available on the clinical significance of HSP 70 expression in human colorectal cancer. In one recent study, no significant relationship of HSP 70 has been reported to the stage and differentiation of colorectal tumors (27). In contrast, Hwang et al. have shown ihsp 70 expression to be correlated with advanced clinical stage in colorectal cancer (28).
In our study, unexpectedly, we have found decreased ihsp 70 expression in malignant tissues of patients with colorectal cancer. Furthermore, the expression of ihsp 70 correlated negatively with MDA levels and GPx levels of malignant tissues. According to our results, it seems that ihsp 70 expression has been suppressed in increased oxidative stress conditions. This decrease seen in colorectal tumor tissues may arise from the genetic alterations of heat shock genes which regulate the expression of the HSPs. The transcription of the heat shock genes is triggered via the binding of an activated transcription factor named heat shock factor (HSF) to DNA. Since extensive oxidative stress can cause some genetic alterations, HSF may not have been able to bind to the damaged DNA region involved in HSPs. Based on the recent knowledge that overexpression of ihsp 70 makes the cells resistant to apoptosis induced by anticancer drugs (10,28), the suppression of ihsp 70 expression might be an advantage for the efficacy of cancer therapy.
In summary, our preliminary results show that ihsp 70 expression is suppressed under induced oxidative stress conditions in malignant tissues of patients with colorectal cancer. The exact mechanisms responsible for decreased ihsp 70 and its definitive role in colorectal cancer should be investigated in larger scale studies.
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Acknowledgments |
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This work was supported by the Research Fund of the University of Istanbul. Project number: 1377/081299.
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References |
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