Abstract: Superoxide dismutase and catalase activities were measured in tissue extracts obtained from cancerous and corresponding non-cancerous tissues of patients with renal adenocarcinoma. The activities of cytosol superoxide dismutase (CuZnSOD) and mitochondrial superoxide dismutase (MnSOD) were decreased significantly (P≤0.02) in cancerous tissues in comparison to non-cancerous tissues, while the activity of catalase (CAT) was increased significantly (P<0.02) in cancerous tissue at stage III-IV. These results suggest that human kidney cancer progression is accompanied by altered antioxidant status of both superoxide dismutase (SOD) and catalase activities.
Introduction
An increasing amount of experimental and epidemiological evidence implicates the involvement of oxygen-derived radicals in the pathogenesis of cancer development (Mates et al., 1999). Oxygen-derived radicals are able to cause damage to membranes, mitochondria and macromolecules including proteins, lipids and DNA. Accumulation of DNA damages has been suggested to contribute to carcinogenesis. It would, therefore, be advantageous to pinpoint the effects of oxygen-derived radicals in cancer development (Portakal et al., 2000). Reactive oxygen species (ROS), represented by superoxide, hydrogen peroxide (H2O2) and hydroxyl radicals (·OH), have been implicated in many diseases including cancer. ROS have been known to play an important role in the initiation and promotion of multistep carcinogenesis. The cellular antioxidants play a crucial role in protection against neoplastic disease (Ray et al., 2000; Bostwick et al., 2000).
In our previous study, investigated the correlation between chromosome aberrations, micronuclei and activity of superoxide dismutases (SOD) in human lymphocytes after irradiation in vitro. The indirect role of intracellular SODs on the yield of radiation-induced DNA damage was observed (Joksic et al., 2000). Related to that, further defined the correlation between SODs activities and intrinsic radiosensitivity induced by radiotherapy in breast cancer patients (Pajovic et al., 1998; Pajovic et al., 2000).
Homma-Takeda et al. (2000) examined the enzyme activity and protein content of superoxide dismutase isozymes in human renal cell carcinoma. Immunoblot analysis showed that the loss in enzyme activity in cancerous tissue was greater than the decrease in protein content for either isozyme. The selective decrease in CuZnSOD activities in cancerous tissue observed in that study suggests that the cytoplasmic defense system against free radical damage appears to be reduced in renal cell carcinoma. Since the oxidant–antioxidant balance within tissues is thought to contribute to the development and progression of cancer.
The aim of the study was to evaluate whether the kidney cancer progression is accompanied by changes in the activity of antioxidant enzymes such as manganese superoxide dismutase, copper-zinc superoxide dismutase and catalase.
Materials and Methods
This study was performed on 7 patients with renal adenocarcinoma (mean age 59 years), hospitalized at Department of Urology, Clinical Center of Serbia, during January to December 2001. After nephrectomy and histopathological evaluation, tissues with III-IV stage of malignancy were selected as well as non-malignant tissues of the same kidneys were used as control.
Cancerous and non-cancerous kidney tissue homogenates were prepared according to slightly modified methods of Rossi et al. (1983) and DeWaziers and Albrecht (1987). Tissues were homogenized in 0.05 M KH2PO4, 0.1 mM EDTA, pH 7.8. Homogenates were vortexed 30 sec several times with intermittent cooling on ice and left frozen at -70°C for 20 h. The homogenates were than defrosted and centrifuged at 35000 rpm for 100 min. Cytosols were kept at -20°C until use. Protein concentration in the cytosol was determined by the method of Lowry et al. (1951).
Enzyme activity of SOD was determined by the method of Misra and Fridovich (1972) before and after the inhibition of CuZnSOD with KCN (Geller and Winge, 1983). This method is based on the ability of SOD to inhibit the auto-oxidation of adrenaline to adrenochrome in 0.05 M Na2CO3, 0.1 mM EDTA, pH 10.2, which was monitored spectrophotometrically at 480 nm. The enzyme activity was expressed in units per mg protein. One unit of SOD was defined as the amount of protein, which caused 50% inhibition of conversion rate of adrenaline to adrenochrome between the third and the fourth minute of auto-oxidation.
CAT activity was assayed as suggested by Beutler (1982) and expressed as μmol H2O2 min-1 mg-1 protein. The method is based on the rate of H2O2 degradation by the action of CAT contained in the samples, monitored spectrophotometrically at 230 nm, in 5 mM EDTA, 1 M Tris-HCl, pH 8.0.
The significance of differences between groups was determined by Student’s t-test for dependent variables.
Results
The activity of mitochondrial MnSOD (C: 4.01 ± 1.29; NC: 9.17 ± 1.49), P=0.02) as well as the activity of cytosol CuZnSOD (C: 19.06 ± 4.83; NC: 34.95 ± 5.56, P<0.02) was significantly reduced in cancerous ( C) kidney tissue in comparison to non-cancerous (NC) tissue (Fig. 1). On the contrary, the activity of CAT was significantly (P < 0.02) increased in neoplastic tissue in comparison to corresponding controls (C: 20.41 ± 4.25; NC: 14.55 ± 2.47) (Fig. 2).
| Fig. 1: | The activities of MnSOD and CuZnSOD in non-cancerous (NC)
and cancerous© kidney tissues of 7 subjects. Data are means±SE
**: P ≤ 0.02 |
| Fig. 2: | The activity of catalase (CAT) in non-cancerous (NC) and cancerous
( C) kidney tissues of 6 subjects. Data are means±SE **: P < 0.02 |
Discussion
Results suggest that antioxidant enzyme activities are significantly altered the human renal adenocarcinoma at stage III-IV. Segura-Aguilar et al. (1994), Durak et al. (1997) and Homma-Takeda et al. (2000) showed that human renal cell carcinomas have significantly reduced the activities of CuZnSOD and glutathione peroxidase (GSH-Px) and non significant variation of MnSOD activity when compared to corresponding non-cancerous renal tissue. Reduced activity of CuZnSOD, in this study (Fig. 1), is in accordance with these findings. The differences with respect to MnSOD activity and increment of CAT activity are in agreement with Oberley et al. (1994) and Oberley and Oberley (1997). They reported that human renal cell carcinomas show greatly altered MnSOD level (either elevated or depressed), which depends on heterogeneity of cell types and number of mitochondria, and the high level of CAT. Also shown that relevant variations of GSH-antioxidant enzyme system exist in the different stages of renal cell carcinoma (Lusini et al., 2001), which together support the hypothesis that oxidative stress plays an important role in renal carcinoma growth and progression. Namely, growing evidence suggest that O2·G and H2O2 mediate the mitogenic signaling of protein kinase activity in transformed cells, as well as the growth factor induction of proto-oncogene expression (Finkel, 1998). Also, it can not be excluded that increment of ROS in malignant cells may be caused by accumulation of DNA damages related to antioxidative defense system.
Free radicals and antioxidant enzymes may play a critical role in cell proliferation and in the resistance of malignant cells against cytotoxic drugs and radiation (Kahlos et al., 2001). Data suggest that enhanced ability in scavenging free radicals by antioxidant enzymes contribute to the resistance of cancer during chemo- and radiotherapy (Ahmed et al., 1999; Suzuki et al., 2000). Therefore, examination of antioxidant status in cancer tissues seems to be necessary for enhancing the efficacy of cancer therapy.
This study indicates that the development of neoplasia in the human kidney is accompanied by significant changes in the activity of superoxide dismutases and catalase. In conclusion, antioxidative disbalance might have a functional role in human kidney carcinogenesis. These findings are used to propose new cancer therapies based on modulation of cellular oxido-redox state.
Acknowledgments
The authors wish to thank Prof. Stevan Matijaševic and Dr. Nevena Ribarac-Stepic for help in organization of the experiments.