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Glutathione peroxidase and glutathione reductase activities in cancerous and non-cancerous human kidney tissues
CAWCER LETTERS I- L S E V I E R
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Cancer Letters 91 (1995) 19-23
Glutathione peroxidase and glutathione reductase activities in cancerous and non-cancerous human kidney tissues Carmine Di Ilio*“,
Paolo Sacchetta”, Stefania Angelucci”, Raffaele Tenagliab, Antonio Aceto”
Andrea Zezza”
“Istiiuto di Scienze Biochimiche. Facoltci di Medicina, Universitb ‘G. D’Annunzio’, 66100 Chieti, It& bIstituto di Uroloqia, Facoltri di Medic&a, Universifci ‘G. D’Annunzio‘. 66100 Chieti. Itu!,
Received 12 January 1995;accepted 19 January 1995
Abstract Selenium-dependent(Se-GSH-Px), selenium-independent (non-Se-GSH-Px) glutathione peroxidase and glutathione reductase (GSSG-Rx) activities have heen determined in cancerous and non-cancerous human adult kidney. Large inter-individual variation in the activities of all enzymestested were found in both turnour and non-tumour specimens. In general a significant decreasein the activities of the three enzymeswas found in turnours. When a comparison was made between cancerous and non-cancerous tissuesof the sameindividual, Se-GSH-Px activity was found to he iower in tumour in 17 casesout of 29, and the non-Se-GSH-Px activity in 20. In 20 casesout of 29 GSSG-Rx was found to he lower in tumour. It was concluded that changesin the factors involved in the anti-oxidative protection actually occur in human kidney tumour. Keywords:
Glutathione peroxidase; Glutathione reductase; Human kidney
1. Introduction Oxygen radicals and lipid peroxidation have been implicated in the initiation and production of malignant processes [ 11. Glutathione peroxidase (GSH-Px) seemsto play a role in the prevention of carcinogenesis [2]. Cytosolic GSH-Px is represented by at least two proteins [I+]. The first is the ‘classical’ GSH-Px, which is a tetrameric selenoenzyme dependent on one selenocysteine residue *Corresponding author, Tel.: +39 871 355274, Fax: +39 871 355356;E-Mail [email protected].
per subunit for its activity (Se-GSH-Px) [2,4]. SeGSH-Px catalyses the reduction of both organic and inorganic peroxides with a specific requirement for GSH [4]. The second is a seleniumindependent enzyme (non-Se-GSH-Px), which has
ken characterized in many tissues and belongs to certain isoenzymes of glutathione transferases [5,6]. Non-Se-GSH-Px, however, reduces only organic peroxides [5,6]. As a result, it may be distinguished from the Se-GSH-Px measuring enzyme activity with both cumene hydroperoxide and hydrogen peroxide, and subtracting the former from the latter results 171. Oxidized
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C. Di llio et al. /Cancer
glutathione produced by peroxidase reactions is reconverted to reduced glutathione by glutathione reductase(GSSG-Rx) in the presenceof NADPH. Hence, working in concert, the peroxidase/reductase couple counteracts oxidative stress in vivo. The majority of experimental studies carried out on human cell lines suggestthat glutathione peroxidase levels are low in tumours [8- 121. GSH-Px activity was found to be lower in tumour than in non-tumour tissues also in human liver and oropharynx [ 13-151. On the other hand, in other human tissues, such as colon and stomach, the opposite was observed [ 16,171. It has also been shown that the activity of GSH redox cycle enzymesalters the responseof cells to chemotherapy [18]. Thus, it may be relevant to evaluate glutathione peroxidase and glutathione reductase activities in many human tumours in comparison with control tissues.To addressthis issue,matched normal and tumour samples from human kidney were specifically tested. 2. Materials and methods Tumour and normal adjacent tissues were obtained from the sameindividuals at the time of surgery. The sampleswere immediately transferred to cold isotonic saline solution, exhaustively washed and frozen at -80°C until analysis. No measurable loss of enzyme activity was noted after at least 6 months of storage under these conditions. The 29 tumour samples were obtained from 23 patients with primary renal cell carcinoma, two patients with a tumour of renal pelvis, one patient with hemangioma, one patient with angiomyolipoma, one patient with oncocytoma and one patient with a carcinoma of collector ductii. None of these patients received antiblastic chemotherapy in the 6 weeks prior to surgical excision. The frozen samples were rapidly warmed at 0°C cut into small pieces and homogenized in 4 ~01s. of Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 2 mM 2-mercaptoethanol. The homogenate was centrifuged at 105 900 x g for 60 min and the enzyme and protein analyseswere promptly performed on the resulting cytosol. Glutathione peroxidase activity was measured by the method of Paglia and Valentine [19]. The final concentrations were 0.25
Letters YI (I99Sj
19-23
mM for hydrogen peroxide and 1.2 mM for cumene hydroperoxide. The assay solution contained 50 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1.5 mM NaN3, 1 mM GSH, 0.16 mM NADPH, 4 pg of glutathione reductase and a suitable sample of enzyme solution. After a 5-min pre incubation, the reaction was started with the addition of peroxides. The value for a blank reaction with the enzyme source replaced by buffer was subtracted for each assay.The rate of reaction was recorded at 24°C by following the decreasein absorbanceat 340 nm. Specific activity is given as nmoles GSH oxidized/min/mg protein. Glutathione reductase activity was measured as previously described [20]. The assay mixture contained 0.1 mM potassium phosphate buffer, pH 7.4, 1 mM EDTA, 1 mM GSSG, 0.16 mM NADPH and an appropriate amount of enzyme source. The blank did not contain GSSG. Enzyme activity was determined at 24°C by measuring the disappearanceof NADPH at 340 nm and expressed as nmoles NADPH/min/mg protein. Protein concentrations were measured by the method of Bradford [21], using y-globulin as standard. The paired t-test was used to compare changes in enzyme levels in tumour versus non-tumour cytosol. 3. Results and discussion Table 1 reports single value ranges, means and standard errors for the activities of Se-GSH-Px, non-Se-GSH-Px, GSSG-Rx. Available clinical data are also presented. In control non- tumour tissues both Se-GSH-Px and non-Se-GSH-Px activities varied greatly from one patient to another, ranging from 20 to 690 nmoles/min/mg protein and from 0 to 205 nmoles/min/mg protein, respectively. A significant inter-individual variation in the distribution of the two GSH-Px activities was also found among the non-tumour kidney samples.Indeed, non-Se-GSH-Px ranged from 7% (patient no. 33) to 77% (patient 21) of total activity. In 17 casesfor Se-GSH-Px activity and in 20 casesfor non-Se-GSH-Px, the activity values were lower in tumour than in non-tumour tissues.In one casefor Se-GSH-Pxand in sevencasesfor non-Se-GSH-Px the activities were undetectable in neoplastic tis-
C. Di Ilio et al. /Cancer Letters 91 (1995) 19-23
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Table I Glutathione peroxidase and glutathione reductase activities in tumour and non-tumour human kidney tissue Patient
Sex
I F M 9 F 10 M II M 12 M 13 F 14 I5 F F 16 I7 M M 18 M 19 F 20 F 21 M 22 M 23 M 24 2s M M 26 M 27 M 28 F 29 M 30 F 31 M 32 F 33 F 34 M 35 F 36 Mean f SE. P-values
Age
68 42 49 54 68 68 41 53 37 13 68 55 NA 49 NA 36 61 NA NA 58 59 NA NA NA 50 50 39 51 59
Grade
III I I NA I III I I I 11 I II I I NA III NA I 11 II II NA I NA I NA I II III
Se-GSH-Px (nmoles/min/mg)
Non-Se-GSH-Px (nmoles/min/mg)
Tumour
Non-tumour
Tumor
130 51 180 30 23 120 260 40 40 65 12 56 IO 0 47 36 22 42 110 54 86 48 40 93 140 540 60 46 170 87 f 19.5 <0.05
I60 82 70 100 80 120 150 45 14 62 99 54 28 49 90 67 20 34 100 80 56 66 130 95 440 690 90 21 360 I21 f 26
I40 0 43 0 70 0 70 7 20 0 150 0 0 35 40 0 IO 22 18 0 31 4 $4 21 I60 I2 I70 26 10 8 123 8 II 16 68 30 30 3 30 24 84 17 112 2 60 205 107 20 120 70 so 40 30 54 73 IO 40 20 f 4.9 70.5 f IO
Non-tumor
GSSG-Px (nmoleslminimg) Tumor
Non-tumor -I__15 I8 II IO IO 10 40 6 IO 9 I’* I2
9 I >I I 2 >I 20 1 5 3 I 10 >l _z .I ‘; >I I ‘i 2 8 I h 20 36 IO 40 21 :i: 20 01( 23 .74 40 ii 12 (0 I3 I’I 13 14 10 10 8.0 f 1.8 I I 8 f 3.6
All samples were from primary renal cell carcinoma with the exception of samples 22 and 24 (tumour of renal pelvis), sample I1 (hemangioma), sample 29 (oncocytoma), sample 31 (collector ducti carcinoma), sample 33 (angiomyolipoma). Se-GSH-Px, seleniumdependent glutathione peroxidase; Non-Se-GSH-Px, selenium-independentglutathione peroxidase; GSSG-Px, glutathione reductase: NA, not available.
sue. Large inter-individual variation in the activities of GSSG-Rx in non-tumour sample of kidney was also observed, ranging from 1 to 60 nmoles/min/mg protein. GSSG-Rx was lower in tumour than in non-tumour tissues, in 20 patients. The present study has evidenced a significant decreaseof Se GSH-Px (P < 0.05), non-SeGSHPx (P c 0.001) and GSSG-Rx (P < 0.001) activities in tumour tiSsuesas compared with controls. The decreaseof GSSG-Rx may be a consequence
of decreasedGSH-Px activity. These results are in agreementwith those reported by Howie et al. f 151 who studied glutathione peroxidase activity in sevensamplesof matched normal and tumour kidney tissues.These results obtained with kidney tissue are different from those observed in other human tissues, such as breast [15,211, lung [E-22,23], colon [15,16], stomach [I&17] and bladder [24], for which a significantly higher GSHPx activity was measured in tumour specimensas
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C. Di Ilio et al. / Cancer Letters 91 (1995) 19-23
compared to control. On the other hand, a significantly lower GSH-Px activity in tumour as compared with non-tumour tissues has been reported in human liver [ 13,151and oropharynx [14]. Thus, changesin the level of GSH-Px activity in cancer cells appear to be tissue dependent. It is noteworthy that in human breast [ 15,221, lung [23,24], colon [22], stomach [22] and bladder [25] only the selenium-dependent enzyme is present, whereas in liver and kidney both the seleniumdependent and independent forms are present. Thus, it seemsthat the decreaseof GSH-Px activity in tumour occurs in those tissues that express both the Se-GSH-Px and the non-Se-GSH-Px isoenzymes,whereas an opposite trend is manifest in tissues expressing only the Se-GSH-Px. The decrease of non-Se-GSH-Px activity found in human tumour kidney well correlates with the decreasedalpha class GST expression shown in this tumour [26,27], since non-Se-GSH-Px activity is maximally associated with GST isoenzyme of class alpha 1281.Data on glutathione peroxidase and glutathione reductase in human tumour tissuesmay be of relevance in cancer chemotherapy. Many of the anticancer drugs seemto destroy target cells by producing reactive oxygen species,including hydrogen peroxide [29]. Thus, it may be postulated that turnouts with lower level of GSHPx and GSSG-Rx are poorly capable of resisting to drug-mediated oxidative injury. Notably, the cytotoxicity of daunomicyn in tumour cells is inversely related with the level of glutathione dependent hydrogen peroxide removing enzymes [30]. Furthermore, tumour cells with acquired resistance to cisplatin and doxorubicin showed elevated expression of glutathione-linked detoxification enzymes, including GSH-Px and GSSGRx [31]. In conclusion, our study presents evidence that changes in the factors involved in anti-oxidative protection actually occur in human kidney tumour. References [l] Shamberger,R.J. (1972) Increase of peroxidation in carcinogenesis.J. Natl. Cancer Inst., 48, 1491-1497. [2] FlohC, L. (1988) The seknoprotein glutathione peroxi-
dase. In: Glutathione - Chemical, Biochemical and Medical Aspects, Part A, pp. 643-731. Editors: D. Dolphin, R. Paulsen and 0. Avramovic. Academic Press, New York. [31 Lawrence, R.A. and Burk, R.F. (1976)Glutathione peroxidase activity in seleniumdeticient rat liver. B&hem. Biophys. Res. Commun., 71, 951-958. 141 Floht, L. (1982) Glutathione peroxidase brought into focus. In: Free Radical in Biology, Vol. V, pp. 233-254. Editor: W.A. Pryor. Academic Press, New York. 151Prohaska, J.R. and Ganther, H.E. (1977)Glutathine peroxidase activity of giutathione S-transferasesfrom rat liver. B&hem. Biophys. Res. Commun., 76, 437-445. I61 Lawrence, R.A., Parkhill, L.K. and Burk, R.F. (1978) Hepatic cytosolic non-selenium-dependent glutathione peroxidase activity: its nature and the effect of the selenium deficiency. J. Nutr., 108, 981-987. [71 Wendel, A. (1980) Glutathione peroxidase. In: Enzymatic Basis of Detoxication, Vol. 1, pp. 333-348. Editor: W.B. Jakoby. Academic Press, New York. 181Bozzi, A., Mavelli, I., Finazzi Agrb, A., Strom, R., Wolf, A.M., Mondovi, B. and Rotilio, G. (1976) Enzyme defence against reactive oxygen derivatives. II. Erytrocytes and tumor cells. Mol. Cell. B&hem., 10, 11-16. [91 Marklund, S.L., Gunnar Westman, N., Lundgren, E. and Ros, G. (1982) Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase and glutathione peroxidase in normal and neoplastic human cell lines and normal tissues. Cancer Res., 42, 1955-1961. WI Peskin, A.V., Koen, Y.M., Zbarski, I.B.I. and Kostantinov, A.A. (1977)Superoxide dismutaseand glutathione peroxidase in tumors. FEBS Lett., 78, 41-45. [I 11 Castro, V.M., Soderstrom, M., Carlberg, I,, Widersten, M., Platz, A. and Mannervik, B. (1990) Differences among human tumor cell lines in the expression of glutathione transferaseand other glutathione-linked enzymes. Carcinogenesis, 11, 1569- 1576. I121Sharma, R., Singhal, S.S., Srivastava, S.K., Bajpai, K.K., Frenkel, E.P. and Awasthi, S. (1993) Glutathione and ghttathione linked enzymes in human small lung cancer cell lines. Cancer Lett., 75, I1 1-I 19. 1131 Casaril, M., Gabrielli, G.B., Dusi, S., Nicoh, N., Bellisola, G. and Corrocher, R. (1985)Decreasedactivity of liver ghttathione peroxidase in human hepatocellular carcinoma. Eur. J. Cancer Clin. Oncol., 21, 941-944. 1141 Goodwin, W.J., Lane, H.W., Bradford, K., Marshall, M.V., GriBin,A.C., Goepfert, H. and Jesse,R.H. (1983) Selenium and ghttathione peroxidase levels in patients with epidermoid carcinoma of the oral cavity and oropharynx. Cancer, 51, I IO- 115. Howie, A.F., Fort-ester, L.M., Glancey, M.J., Schlager, J.J., Powis, G., Beckett, G.J., Hayes, J.D. and Wolf, C.R. (1990) Glutathione Stransferase and. ghttathione peroxidase expression in normal and tumour human tissues. Carcinogenesis, 11, 451-458. WI Baur, G. and Wendel, A. (1980) The activity of the per-
C. Di Ilio et al. /Cancer Letters 91 (1995) 19-23 oxuie metabolising systemin human colon carcinoma. J. Cancer Res. Clin. Oncol., 97, 267-273. [17] Siigers, C.-P., Bose-Younes, H., Thies, E., Hop per&s, R. and Younes, M. (1984) Glutathione and GSH-dependent enzymes in the tumours and nontumours mucosa of the human colon and rectum. J. Cancer Res. Clin. Oncol., 107, 238-241. [ 181 Harris, H.L. (1990) Mechanism of anticancer drug resistance. In: Glutathione S-transferases and drug resistance, pp. 283-293. Editors: J.D. Hayes, C.B. Pickett and T.J. Mantle. Taylor and Francis, London. [19] Paglia, D.E. and Valentine, W.N. (1967) Studies on the quantitative and qualitative characterization of erythrocytes glutathione peroxidase. J. Lab. Clin. Med., 70, 158-W. (201 Di Ilio, C., Polidoro, G., Arduini, A., Muccini, A. and
[21]
[22]
[23]
[24]
Federici, G. (1983) Glutathione peroxidase, glutathione reductase, ghnathione Stransferase and T-glutamyl transpepidase activities in the human early pregnancy placenta. Biochem. Med., 29, 143-148. Bradford, M.M. (1976)A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. B&hem., 72, 248-254. Di Ilio, C., Sacchetta, P., Del Boccio, G., La Rovere, G. and Federici, G. (1985) Glutathione peroxidase, glutathione Stransferase and glutathione reductase activities in normal and neoplastic human breast tissue. Cancer Lett., 29, 37-42. Di Bio, C., Del Boccio, G., Casaccia. R., Aceto, A., Di Giacomo, F. and Federici, G. (1987) Selenium level and giutathione-dependent enzyme activities in normal and neoplastic human lung tissues. Carcinogenesis, 8, 281-284. Carmichael, J., Forrester, L.M., Lewis, A.D., Hayes, J.D. and Wolf, C.R. (1988) Glutathione S-transferases
23
isoenzymes and ghnathione peroxidase activity in normal and tumor sampies from human lung. Carcinogenesis,9, 1617-1621. [25] Singh, S.V., Xu, B.H., Tkakevic, G.T., Gupta, V., Roberts, B. and Ruiz, P. (1994) Glutathione-linked detoxification pathway in normal and malignant bladder tissue. Cancer L&t., 77, 15-24. [26] Di Ilio, C., Aceto, A., Bucciardli, T., Angelucci, S., Felaco, M., Grilli, A., Zezza, A., Tenagha, R. and Federici, G. (1991) Giutathione trans&ase isoenzymes in normal and neophtstic human kidney tissues. Carcinogenesis, 12, 1471-1475. [27] Klone, A., Weidner, U., Hubnatter, R.. Harris, J., Meyer, D., Peter, S., Ketterer, B. and Sies, H. (1990) Decreased expression of the glutathioae S-transferases alpha and pi genesin human renal cell carcinoma. Carcinogenesis, 11, 2179-2183. [28] Mannervik, B., Ahn, P., Guthenberg, C., Jeusson, H., Tahir, M.K., Warholm, M. and Jomvall, H. (1985)Identitication of three classes of cytosolic glutathione transferasescommon to several species, correlation between structural dam and enzymatic properties. Proc. Natl. Acad. Sci. USA, 82, 7202-7206. [29] Arrick, B.A. and Nathan, CF. (1984) Glutathione metabolism as determinant of the terapeutic cfBcacy: a review. Cancer Res., 33, 4224-4232. 1301 Bozzi, A., Mavelli, I., Mondovi, B.. Strom, R. and Rotilio, G. (1981) Differential cytotoxicity of daunomycin in tumor cells is related to glutathione dependent hydrogen peroxide metabolism. Biochem. J.. 194, 369-372.
(311 Hao, X-Y., Berg, J., Brodin, O., Hellman, U. and Mannervik, B. (1994)Aquired resistanceto cisplatin and doxorubiiin in a small cell lung cancer cell line is correlated to elevated expression of glutathione-linked detoxification enzymes. Carcinogenesis, 1.5,1167-i 173.
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Cancer Letters 91 (1995) 19-23
Glutathione peroxidase and glutathione reductase activities in cancerous and non-cancerous human kidney tissues Carmine Di Ilio*“,
Paolo Sacchetta”, Stefania Angelucci”, Raffaele Tenagliab, Antonio Aceto”
Andrea Zezza”
“Istiiuto di Scienze Biochimiche. Facoltci di Medicina, Universitb ‘G. D’Annunzio’, 66100 Chieti, It& bIstituto di Uroloqia, Facoltri di Medic&a, Universifci ‘G. D’Annunzio‘. 66100 Chieti. Itu!,
Received 12 January 1995;accepted 19 January 1995
Abstract Selenium-dependent(Se-GSH-Px), selenium-independent (non-Se-GSH-Px) glutathione peroxidase and glutathione reductase (GSSG-Rx) activities have heen determined in cancerous and non-cancerous human adult kidney. Large inter-individual variation in the activities of all enzymestested were found in both turnour and non-tumour specimens. In general a significant decreasein the activities of the three enzymeswas found in turnours. When a comparison was made between cancerous and non-cancerous tissuesof the sameindividual, Se-GSH-Px activity was found to he iower in tumour in 17 casesout of 29, and the non-Se-GSH-Px activity in 20. In 20 casesout of 29 GSSG-Rx was found to he lower in tumour. It was concluded that changesin the factors involved in the anti-oxidative protection actually occur in human kidney tumour. Keywords:
Glutathione peroxidase; Glutathione reductase; Human kidney
1. Introduction Oxygen radicals and lipid peroxidation have been implicated in the initiation and production of malignant processes [ 11. Glutathione peroxidase (GSH-Px) seemsto play a role in the prevention of carcinogenesis [2]. Cytosolic GSH-Px is represented by at least two proteins [I+]. The first is the ‘classical’ GSH-Px, which is a tetrameric selenoenzyme dependent on one selenocysteine residue *Corresponding author, Tel.: +39 871 355274, Fax: +39 871 355356;E-Mail [email protected].
per subunit for its activity (Se-GSH-Px) [2,4]. SeGSH-Px catalyses the reduction of both organic and inorganic peroxides with a specific requirement for GSH [4]. The second is a seleniumindependent enzyme (non-Se-GSH-Px), which has
ken characterized in many tissues and belongs to certain isoenzymes of glutathione transferases [5,6]. Non-Se-GSH-Px, however, reduces only organic peroxides [5,6]. As a result, it may be distinguished from the Se-GSH-Px measuring enzyme activity with both cumene hydroperoxide and hydrogen peroxide, and subtracting the former from the latter results 171. Oxidized
0304-3835/95/$09.50 0 1995 Ekevier Science Ireland Ltd. All rights reserved SSDI 0304-3835(95)03710-E
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C. Di llio et al. /Cancer
glutathione produced by peroxidase reactions is reconverted to reduced glutathione by glutathione reductase(GSSG-Rx) in the presenceof NADPH. Hence, working in concert, the peroxidase/reductase couple counteracts oxidative stress in vivo. The majority of experimental studies carried out on human cell lines suggestthat glutathione peroxidase levels are low in tumours [8- 121. GSH-Px activity was found to be lower in tumour than in non-tumour tissues also in human liver and oropharynx [ 13-151. On the other hand, in other human tissues, such as colon and stomach, the opposite was observed [ 16,171. It has also been shown that the activity of GSH redox cycle enzymesalters the responseof cells to chemotherapy [18]. Thus, it may be relevant to evaluate glutathione peroxidase and glutathione reductase activities in many human tumours in comparison with control tissues.To addressthis issue,matched normal and tumour samples from human kidney were specifically tested. 2. Materials and methods Tumour and normal adjacent tissues were obtained from the sameindividuals at the time of surgery. The sampleswere immediately transferred to cold isotonic saline solution, exhaustively washed and frozen at -80°C until analysis. No measurable loss of enzyme activity was noted after at least 6 months of storage under these conditions. The 29 tumour samples were obtained from 23 patients with primary renal cell carcinoma, two patients with a tumour of renal pelvis, one patient with hemangioma, one patient with angiomyolipoma, one patient with oncocytoma and one patient with a carcinoma of collector ductii. None of these patients received antiblastic chemotherapy in the 6 weeks prior to surgical excision. The frozen samples were rapidly warmed at 0°C cut into small pieces and homogenized in 4 ~01s. of Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and 2 mM 2-mercaptoethanol. The homogenate was centrifuged at 105 900 x g for 60 min and the enzyme and protein analyseswere promptly performed on the resulting cytosol. Glutathione peroxidase activity was measured by the method of Paglia and Valentine [19]. The final concentrations were 0.25
Letters YI (I99Sj
19-23
mM for hydrogen peroxide and 1.2 mM for cumene hydroperoxide. The assay solution contained 50 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1.5 mM NaN3, 1 mM GSH, 0.16 mM NADPH, 4 pg of glutathione reductase and a suitable sample of enzyme solution. After a 5-min pre incubation, the reaction was started with the addition of peroxides. The value for a blank reaction with the enzyme source replaced by buffer was subtracted for each assay.The rate of reaction was recorded at 24°C by following the decreasein absorbanceat 340 nm. Specific activity is given as nmoles GSH oxidized/min/mg protein. Glutathione reductase activity was measured as previously described [20]. The assay mixture contained 0.1 mM potassium phosphate buffer, pH 7.4, 1 mM EDTA, 1 mM GSSG, 0.16 mM NADPH and an appropriate amount of enzyme source. The blank did not contain GSSG. Enzyme activity was determined at 24°C by measuring the disappearanceof NADPH at 340 nm and expressed as nmoles NADPH/min/mg protein. Protein concentrations were measured by the method of Bradford [21], using y-globulin as standard. The paired t-test was used to compare changes in enzyme levels in tumour versus non-tumour cytosol. 3. Results and discussion Table 1 reports single value ranges, means and standard errors for the activities of Se-GSH-Px, non-Se-GSH-Px, GSSG-Rx. Available clinical data are also presented. In control non- tumour tissues both Se-GSH-Px and non-Se-GSH-Px activities varied greatly from one patient to another, ranging from 20 to 690 nmoles/min/mg protein and from 0 to 205 nmoles/min/mg protein, respectively. A significant inter-individual variation in the distribution of the two GSH-Px activities was also found among the non-tumour kidney samples.Indeed, non-Se-GSH-Px ranged from 7% (patient no. 33) to 77% (patient 21) of total activity. In 17 casesfor Se-GSH-Px activity and in 20 casesfor non-Se-GSH-Px, the activity values were lower in tumour than in non-tumour tissues.In one casefor Se-GSH-Pxand in sevencasesfor non-Se-GSH-Px the activities were undetectable in neoplastic tis-
C. Di Ilio et al. /Cancer Letters 91 (1995) 19-23
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Table I Glutathione peroxidase and glutathione reductase activities in tumour and non-tumour human kidney tissue Patient
Sex
I F M 9 F 10 M II M 12 M 13 F 14 I5 F F 16 I7 M M 18 M 19 F 20 F 21 M 22 M 23 M 24 2s M M 26 M 27 M 28 F 29 M 30 F 31 M 32 F 33 F 34 M 35 F 36 Mean f SE. P-values
Age
68 42 49 54 68 68 41 53 37 13 68 55 NA 49 NA 36 61 NA NA 58 59 NA NA NA 50 50 39 51 59
Grade
III I I NA I III I I I 11 I II I I NA III NA I 11 II II NA I NA I NA I II III
Se-GSH-Px (nmoles/min/mg)
Non-Se-GSH-Px (nmoles/min/mg)
Tumour
Non-tumour
Tumor
130 51 180 30 23 120 260 40 40 65 12 56 IO 0 47 36 22 42 110 54 86 48 40 93 140 540 60 46 170 87 f 19.5 <0.05
I60 82 70 100 80 120 150 45 14 62 99 54 28 49 90 67 20 34 100 80 56 66 130 95 440 690 90 21 360 I21 f 26
I40 0 43 0 70 0 70 7 20 0 150 0 0 35 40 0 IO 22 18 0 31 4 $4 21 I60 I2 I70 26 10 8 123 8 II 16 68 30 30 3 30 24 84 17 112 2 60 205 107 20 120 70 so 40 30 54 73 IO 40 20 f 4.9 70.5 f IO
Non-tumor
GSSG-Px (nmoleslminimg) Tumor
Non-tumor -I__15 I8 II IO IO 10 40 6 IO 9 I’* I2
9 I >I I 2 >I 20 1 5 3 I 10 >l _z .I ‘; >I I ‘i 2 8 I h 20 36 IO 40 21 :i: 20 01( 23 .74 40 ii 12 (0 I3 I’I 13 14 10 10 8.0 f 1.8 I I 8 f 3.6
All samples were from primary renal cell carcinoma with the exception of samples 22 and 24 (tumour of renal pelvis), sample I1 (hemangioma), sample 29 (oncocytoma), sample 31 (collector ducti carcinoma), sample 33 (angiomyolipoma). Se-GSH-Px, seleniumdependent glutathione peroxidase; Non-Se-GSH-Px, selenium-independentglutathione peroxidase; GSSG-Px, glutathione reductase: NA, not available.
sue. Large inter-individual variation in the activities of GSSG-Rx in non-tumour sample of kidney was also observed, ranging from 1 to 60 nmoles/min/mg protein. GSSG-Rx was lower in tumour than in non-tumour tissues, in 20 patients. The present study has evidenced a significant decreaseof Se GSH-Px (P < 0.05), non-SeGSHPx (P c 0.001) and GSSG-Rx (P < 0.001) activities in tumour tiSsuesas compared with controls. The decreaseof GSSG-Rx may be a consequence
of decreasedGSH-Px activity. These results are in agreementwith those reported by Howie et al. f 151 who studied glutathione peroxidase activity in sevensamplesof matched normal and tumour kidney tissues.These results obtained with kidney tissue are different from those observed in other human tissues, such as breast [15,211, lung [E-22,23], colon [15,16], stomach [I&17] and bladder [24], for which a significantly higher GSHPx activity was measured in tumour specimensas
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C. Di Ilio et al. / Cancer Letters 91 (1995) 19-23
compared to control. On the other hand, a significantly lower GSH-Px activity in tumour as compared with non-tumour tissues has been reported in human liver [ 13,151and oropharynx [14]. Thus, changesin the level of GSH-Px activity in cancer cells appear to be tissue dependent. It is noteworthy that in human breast [ 15,221, lung [23,24], colon [22], stomach [22] and bladder [25] only the selenium-dependent enzyme is present, whereas in liver and kidney both the seleniumdependent and independent forms are present. Thus, it seemsthat the decreaseof GSH-Px activity in tumour occurs in those tissues that express both the Se-GSH-Px and the non-Se-GSH-Px isoenzymes,whereas an opposite trend is manifest in tissues expressing only the Se-GSH-Px. The decrease of non-Se-GSH-Px activity found in human tumour kidney well correlates with the decreasedalpha class GST expression shown in this tumour [26,27], since non-Se-GSH-Px activity is maximally associated with GST isoenzyme of class alpha 1281.Data on glutathione peroxidase and glutathione reductase in human tumour tissuesmay be of relevance in cancer chemotherapy. Many of the anticancer drugs seemto destroy target cells by producing reactive oxygen species,including hydrogen peroxide [29]. Thus, it may be postulated that turnouts with lower level of GSHPx and GSSG-Rx are poorly capable of resisting to drug-mediated oxidative injury. Notably, the cytotoxicity of daunomicyn in tumour cells is inversely related with the level of glutathione dependent hydrogen peroxide removing enzymes [30]. Furthermore, tumour cells with acquired resistance to cisplatin and doxorubicin showed elevated expression of glutathione-linked detoxification enzymes, including GSH-Px and GSSGRx [31]. In conclusion, our study presents evidence that changes in the factors involved in anti-oxidative protection actually occur in human kidney tumour. References [l] Shamberger,R.J. (1972) Increase of peroxidation in carcinogenesis.J. Natl. Cancer Inst., 48, 1491-1497. [2] FlohC, L. (1988) The seknoprotein glutathione peroxi-
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