Cancer Letters, 5 l(1990)
43
43-48
Elsevier Scientific Publishers Ireland Ltd.
Differential expression of glutathione S-transferase, glutathione peroxidase and glutathione reductase in normal and malignant human breast tissues S.V. Singh”, S.R. Brunnertb, “Departments
of Oncology and
B. Robertsb and A. Krishan”
*Pathology,
University of Miami School of Medicine, Miami, FL 33136 (U.S.A.)
(Received 27 November 1989) (Revision received 10 January 1990) (Accepted 11 January 1990)
Summary In the present study we have compared the levels of glutathione (GSH) S-transferase, GSH peroxidase and GSH reductase in human breast tumors and adjacent normal fissues obtained from the same individuals. We haoe also quantitated GST T[ type antigen in these samples by western blotting. GST T[ actioity towards 1 -chloro-2,4-dinitrobenzene was found to be elevated in tumors from three out of six patients (patient nos. 2, 4 and 5), whereas this activity was suppressed in tumor from patient no. 1. Results of Western blotting using antibodies raised against GST II of human placenta were in agreement with the GST activity data. GSH peroxidase actioity with cumene hydroperoxide as substrate was found to be eleoated in four tumor samples (patients nos. 2, 4, 5, and 6) but suppressed in tumor from patient no. 1. On the other hand, GSH reductase activity was elevated in three samples [patients nos. 2, 4 and 5) and downregulated in the remaining three samples (patients nos. 1, 3 and 6). These results indicate that GSH-related enzymes are dijferentially altered in human breast tumors and GST TI type isoenzyme(s), unlike certain other human carcinomas such 0s colonic, are not uniformly elevated in human breast tumors. Correspondence
to:
S.V. Singh.
0304-3835/90/$03.50 Published and Printed
in
Keywords: glutathione; glutathione S-transferase; glutathione peroxidase; breast carcinoma. Introduction
Glutathione (GSH) S-transferases (GST) are a family of multifunctional enzymes which participate in detoxification of a wide variety of xenobiotics including carcinogens and their metabolites (8,171. Structural, kinetic and immunological characterizations have enabled to group mammalian GST’s into three major classes a, p and II [18]. Several independent studies have suggested that the placental form of rat GST (GST-P) may be used as a marker for chemically induced rat hepatocarcinogenesis [22,24,29]. GST-P expression is very low in normal rat liver but this isoenzyme is elevated several-fold in chemically induced pre-neoplastic and neoplastic lesions [24f. In a recent study, however, uniform elevation of GST-P was not observed in peroxisome proliferator induced lesions [23]. GST-P has considerable structural homology with human placental GST TI [15,19,21,24]. Overexpression of n class GST isoenzyme has been reported in some human tumors such as primary hepatomas [28] and colonic [ 161, esophageal [31] and renal carcinomas [9]. These studies suggest the possibility of II class GST being used as a human tumor marker.
0 1990 Elsevier Scientific Publishers Ireland Ltd Ireland
44
On the other hand, uniform elevation of GST TI was not observed in human lung tumors [7,13,25] and small cell lung cancer cell lines [2]. Taken together, these studies suggest that induction of GST TIin human tumors may be tissue specific. In order to substantiate this hypothesis and to identify human carcinomas where this isoenzyme may serve as a tumor marker, quantitation of GST TImust be carried out in various types of human tumors. We have, therefore, compared GST activity and relative amount of GST T[ in human breast tumors and adjacent normal tissues obtained from same patients. Increased levels of GST, GSH peroxidase and GSH reductase have been reported in cancer cells resistant to chemotherapeutic agents such as doxorubicin (DOX) [4,20,26] and these enzymes have been implicated in cellular drug resistance. Thus, we have also compared GSH peroxidase and GSH reductase activities in normal and tumorous breast tissue samples. Significance of the findings is discussed. Materials and methods
Procurement and processing of tissues Breast tumors and adjacent normal tissues were collected from mastectomy patients through our Cancer Center Tissue Procurement Services. Samples were obtained from white females of age between 34 and 63 years. A small piece of tissue was fixed in 10% formalin for histopathological examination and the remaining sample was used for the enzyme activity determinations and Western blotting. Histopathological examination was used to differentiate normal and malignant tissues. After fixation, tumors and adjacent normal tissues were dehydrated, embedded, sectioned (5 pm), and stained with hematoxylin and eosin. Preparation of tissue supernatants A 10% (w/v) homogenate was prepared in 10 mM potassium phosphate buffer (pH 7.0)) containing 1.4 mM 2-mercaptoethanol. The homogenate was centrifuged at 14,000 x g for 45 min to obtain supernatant .
Enzyme assays GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate was determined by the method of Habig et al. [ 141. GSH peroxidase and GSH reductase and GSH reductase activities were determined by the methods of Awasthi et al. [l] and Beutler [5], respectively. Protein content was determined by the method of Bradford [6]. Western blotting Immunoblotting was performed by the method of Towbin et al. [30] with slight modifications as described previously [2]. Polyclonal antibodies, specific against GST R of human placenta were kindly provided by Dr. Yogesh C. Awasthi, University of Texas Medical Branch, Galveston, TX. Results and discussion
Table 1 presents clinical data on six patients from whom tumors and normal tissues were obtained. GST activity in 14,000 x g supernatant fractions of paired tumor and normal breast tissues was determined towards CDNB as substrate (Fig. 1). Among normal tissues of these patients, GST activity varied significantly; ranging from 7 to 133 nmol/ min/ mg protein. This activity in tumor from patient no. 1 was about 40% of that in the corresponding normal tissue. GST activity in normal and tumor tissues from patients nos. 3 and 6 was comparable (Fig. 1). On the other hand, this activity in tumors from patients 2, 4 and 5 was higher by 1.5-, 6.7- and 2.0-fold, respectively, compared to that of the corresponding normal tissues (Fig. 1). Quantitation of GST n in normal and tumor tissues of patients 1, 3 and 4 was performed by Western blotting using equal amount of cytosolic protein and antibodies raised against GST n of human placenta (Fig. 2). These samples were specifically chosen for Western blotting because in tumor no. 1 there was a decrease in GST activity, in tumor no. 3 it was unchanged and in tumor no. 4 GST activity was significantly higher (Fig. 1). Similar to GST activity data, Western blotting revealed suppression of
45 Table 1.
Clinical data on patients.
Patient
Chemotherapy
Age
Type of breast cancer
1 2 3 4 5 6
None None None None None None
50 34 52 55 63 -
Ductal carcinoma Poorly differentiated carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Cytosarcoma phyllodes Infiltrating ductal carcinoma
GST in tumor tissue of patient no. 1 (Fig. 2, lane 3). GST n content was similar in normal and tumor tissues of patient no. 3 (Fig. 2, lanes 4 and 5, respectively), whereas GST II content in tumor tissue of patient 4 was significantly higher (Fig. 2, lane 7) compared to that of the corresponding normal tissue (Fig. 2, lane 6). Quantitation of a and p class GST was not undertaken because these isoenzymes have been reported to be absent in human breast tissue [lo]. An isoenzyme of GST immunologically similar to GST rr of human placenta is elevated in human colon [16], esophageal [31] and renal carcinomas [9] suggesting that it could be used as a human tumor marker [9,16,28,31]. Even though the present study was conducted on a small number of human breast tumor samples, it suggests that elevation of GST IT is not a common feature in different types of human 2001
Sample number
Fig. 1. Glutathione S-transferax activity in 14,000 X g supernatant fractions of normal (0) and malignant (n )
breast tissues.
tumors. Out of six tumor samples analysed, GST n was elevated only in three samples. In two of the tumor samples GST activity was similar to those of corresponding normal tissues whereas in the remaining tumor sample GST A level was significantly reduced. These results are similar to those of our recent studies with three small cell lung cancer cell lines [2] where GST n was found to be elevated in cell lines NES and NOC-361 and suppressed in the third cell line, NCLH69 [2]. Interestingly, in a different study GST II was found to be absent in six small cell lung tumors and elevated considerably in squamous cell carcinomas [13]. Similarly, uniform elevation of GST n was not observed in human lung tumors during two separate investigations [7,25]. Our results are also in agreement with another study where out of 17 breast tumors analysed, GST activity was significantly high in only 11 samples [ll]. Taken together, these results suggest that elevation of GST T[ in human tumors may be tissue specific; colon, esophageal and renal tumors exhibiting higher frequency of GST I[ overexpression [9,16,31], whereas lung and breast tumors showing relatively lower rate of elevation. These results also suggest that GST II may not represent an ideal marker for human breast carcinomas. However, a large number of paired normal and tumor breast samples must be analyzed for GST II content before validity of this contention can be established beyond any doubt. Significant individual variation was also observed in GSH peroxidase activity in normal tissues of these patients; ranging between 15
46
6
7
Fig. 2. Western blot analysis using antibodies raised against GST n of human placenta. Lane 1, purified GST n of human placenta (3 c(s); lanes 2 and 3, aliquots (25 pg protein) of 14,000 x g supematant fractions from normal and tumor tissues of patient no. 1, respectively; lanes 4 and 5, aliquots (25 pg protein) of supematant fractions from normal and tumor tissues of patient 3, respectively; lanes 6 and 7, aliquots (50 pg protein) of supematant fractions from normal and tumor tissues of patient no. 4, respectively.
and 103 nmol/ min/ mg protein (Table 2). GSH peroxidase activity with cumene hydroperoxide (CUOH) as a substrate in tumor tissue from patient no. 1 was 48.5% of that in adjacent normal tissue, whereas the activity in normal and tumor tissues of patient no. 3 was comparable. On the other hand, GSH peroxidase activity was elevated in tumor tissues of patients nos. 2, 4, 5 and 6 by 1.25, 1.95-, 6.06- and 1.45-fold, respectively (Table 2) when compared to those in corresponding normal tissues. GSH reductase activity ranged between 5 and 23 nmol/ min/ mg protein in normal tissues of these patients (Table 2). This activity in tumors from patients nos. 1, 3 and 6 was 75%, 33% and 22%, respectively, of that in the corresponding normal tissues whereas the activity in normal and tumor tissues from
patient no. 2 was comparable. On the other hand, GSH reductase activity in tumors from patients nos. 4 and 5 was higher by 2.6- and 2.3-fold, respectively, as compared to those in the corresponding normal tissues (Table 2). These data indicate that GSH related enzymes are differentially altered in human breast carcinomas. However, mechanism(s) by which the expression of GST, GSH peroxidase and GSH reductase genes are altered during carcinogenesis remains to be determined. The central role of GSH and related enzymes in detoxification of oxyradicals [32] (which have been implicated in cytotoxic activity of chemotherapeutic agents such as DOX [3]) and electrophiles (including some of the currently used ’ cytotoxic drugs such as melphalan [12] and BCNU [27]) implies that the relative levels of these enzymes may affect
47
Table 2.
GSH peroxidase and GSH reductase activities in normal and malignant breast tissues. Both the enzymes assayed at 37OC. Values represent means of duplicate or triplicate determinations. Patient
Enzyme activity (nmol/min
per mg protein) GSH reductase
GSH peroxidase
1 2 3 4 5 6 Means f SD. (n = 6)
were
Normal
Tumor
Normal
Tumor
103 68 91 21 15 69 61 f 36
50 85 94 41 91 100 77 f 25
8 9 6 5 6 23 9.5 k 7
6 11 2 13 14 5 8.5 + 5
tumor cell response to certain chemotherapeutic agents. In fact, several independent studies have suggested that GST, GSH peroxidase and GSH reductase may contribute to drug resistance [4,12,20,26,27]. More direct evidence for the involvement of these enzymes in cellular drug resistance has also been presented recently. For example, DOX resistance in a MCF-7 human breast cancer cell line has been attributed to the increased GSH peroxidase mediated detoxification of drug induced free radicals [26]. Similarly, GST levels can affect tumor cell sensitivity to melphalan and BCNU because these drugs have recently been shown to be detoxified by GST [12,27]. The usual procedure for breast cancer treatment involves surgery followed by radiation or chemotherapy. It may be speculated that tumors with elevated levels of GSH related enzymes may show relatively low sensitivity to drugs such as DOX, melphalan and BCNU and eventually causing treatment failure. Thus, it may be useful to compare the levels of GSH related enzymes in tumor biopsies and adjacent normal tissue specimens of same individuals prior to the start of chemotherapy. This information may be valuable in selection of chemotherapeutic agents.
Acknowledgements
This investigation was supported in part by the American Cancer Society Institutional, Biomedical Research Support Institutional Grants (S.V.S.) and US PHS Grant CA 44737 (A.K.). References Awasthi, Y.C., Beutler. E. and Srivastava, S.K. (1975) Purification and properties of human erythrocyte glutathione peroxidase. J. Biol. Chem., 250,5144-5149. Awasthi, Y .C., Singh, S.V., Gupta,
V. (1988)
isoenzymes
in human
Carcinogenesis, Bachur,
H., Moller, P.C. and S-transferase
small cell lung cancer
cell lines.
9.89-93.
N.R.,
Anthracycline
Ahmad,
Expression of glutathione
Gordon,
S.L.
and
antibiotic augmentation
Gee,
M.V.
(1977)
of microsomal elec-
tron transport and free radical formation.
Mol. Pharmacol.,
13,901-910. Batist, G., Tulpule, C.E. and Cowan, anionic
A., Sinha, B.K., K.H.
glutathione
(1986)
Katki, A.G.,
Overexpression
transferase
in
Myers,
of a novel
multidrug-resistant
human breast cancer cells. J. Biol. Chem.,
261,
15544-
15549. Beutler,
E. (1975)
Metabolism,
Glutathione
reductase.
A Manual of Biochemical
In: Red Cell
Methods,
pp. 72-
73. Editor: E. Beutler. Grune and Stratton. New York. Bradford,
M.M.
(1976)
A rapid and sensitive method for
the quantitation of microgram quantities of protein utilizing
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18
19
the principle of protein-dye binding. Anal. Biochem., 72, 248-254. Carmichael, J., Forrester, L.M., Lewis, A.L., Hayes, J.D., Hayes, P.C. and Wolf, C.R. (1988) Glutathione Stransferase isoenzymes and glutathione peroxidase activity in normal and tumor samples from human lung. Carcinogenesis, 9, 1617-1621. Chasseaud, L.F. (1979) The role of glutathione and glutathione S-transferase in the metabolism of chemical carcinogens and other electrophilic agent. Adv. Cancer Res., 29, 175-274. Dillio, C., Del Boccio, G., Aceto, A. and Federici, G. (1987) Alteration of glutathione transferase isoenzyme concentrations in human renal carcinoma. Carcinogenesis, 8,861-864. Dillio, C., Del Boccio, G., Massoud, R. and Federici, G. (1986) Glutathione transferase of human breast is closely related to transferase of human placenta and erythrocytes. Biochem. Int., 13,263-269. Dillio, C., Sacchetta, P., Del Boccio, G., La Rovere, G. and Federici, G. (1985) Glutathione peroxidase, glutathione S-transferase and glutathione reductase activities in normal and neoplastic human breast tissue. Cancer Lett., 29,37-42. Dulik, D.M., Fenselau, C. and Hilton, J. (1986) Characterization of melphajan-glutathione adducts whose formation is catalysed by glutathione transferases. Biochem. Pharmacol., 35,3405-3409. Eimoto, H., Tsutsumi, M., Nakajima, A., Yamamoto, K., Takashima, Y., Maruyama, H. and Konishi, Y. (1988) Expression of glutathione S-transferase placental form in human lung carcinomas. Carcinogenesis, 9, 2325-2327. Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Glutathione S-transferases, the first enzmatic step in mercapturic acid formation. J. Biol. Chem., 249,7130-7139. Kano, T., Sakai, M. and Muramatsu, M. (1987) Structure and expression of human class n glutathione S-transferase messenger RNA. Cancer Res., 47,5626-5630. Kodate, C., Fukushi, A., Nartta, T., Kudo, H., Soma, Y. and Sato, K. (1986) Human placental form of glutathione S-transferase (GST-n) as new immunohistochemical marker for human colonic carcinoma. Gann, 77,226-229. Mannervik, B. (1985) The isoensymes of glutathione transferase. Adv. Enzymol. Relat. Areas Mol. Biol., 57, 357-417. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M.K., Warholm, M and Jomvall, H. (1985) Identification of three classes of cytosolic glutathione transferase common to several mammalian species, correlation between structural data and enzymatic properties. Proc. Natl. Acad. Sci. USA, 82,7202-7206. Moscow, J.A., Fairchild, C.R., Madden, M.J., Ransom, D.T., Wieand, H.S., O’Brien, E.E., Poplack, D.G., Cossman, J., Myers, C.E. and Cowan, K.H. (1989) Expression of anionic glutathione S-transferase and P-glycoprotein genes in human tissues and tumors. Cancer Res., 49, 1422-1428.
20
Nair, S., Singh, S.V., Samy, T.S.A. and Krishan, A. (1989) Anthracycllne resistance in murine leukemic P388 cells, role of drug efflux and glutathione related enzymes. Biochem. Pharmacol., in press. 21 Okuda, A., Sakai, M. and Muramatsu, M. (1987) The structure of rat glutathione S-transferase P gene and related pseudogenes. J. Biol. Chem., 262,3858-3862. 22 Pemble, S.E., Taylor, J.B. and Ketterer, B. (1986) Tissue distribution of rat glutathione transferase subunit 7, a hepatoma marker. Btochem. J., 240,885-889. 23 Rao, M.S., Tatematsu, M., Subbarao, V., ho, N. and Reddy, J.K. (1986) Analysis of peroxisome proliferatorinduced preneoplastic and neoplastic lesions of rat liver for placental form of glutathione S-transferase and r-glutamyltranspeptidase. Cancer Res., 46,5287-5290. 24 Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hatayama, I and Sato, K. (1985) Purification, induction, and distribution of placental glutathione transferase, a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA, 82, 39643968. 25 Singh, S.V., Hague, A.K., Ahmad, H., Medh, R.D. and Awasthi, Y.C. (1988) Glutathione S-transferase isoensymes in human lung tumors. Carcinogenesis, 9, 16811685. 26 Sinha, B.K., Katki, A.G., Batist, G., Cowan, K.H. and Myers, C.E. (1987) Differential formation of hydroxyl radicals by Adrtamycin in sensitive and resistant MCF-7 human breast tumor cells, implications for the mechanism of action. Biochemistry, 26,3776-3781. 27 Smith, M.T., Evans, C.G., Doane-Setzer, P., Castro, V., Tahir, M.K. and Mannervik, B. (1989) Denitrosation of 1,3-bis(2-chloroethyI)-l-nitrosourea by class Mu glutathione transferases and its role in cellular resistance in rat brain tumor cells. Cancer Res., 49,2621-2625. 28 Soma, Y., Satoh, K. and Sato, K. (1986) Purification and subunit structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker. Biochim. Biophys. Acta, 869,247-258. 29 Tatematsu, M., Tsuda, H., Shirai, T., Masui, T. and ho, N. (1987) Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis, in vivo short-term screening for hepatocarcinogenesis. Toxicol. Pathol., 15, 60-68. 30 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA, 76.4350-4354. 31 Tsuchida, S., Sekine, Y., Shineha, R., Nishihira, T. and Sato, K. (1989) Elevation of the placental glutathione Stransferase form (GST-n) in tumor tissues and the levels in sera of patients with cancer. Cancer Res., 49, 52255229. 32 Wendel, A. (1980) Glutathione peroxidase. In: Enzymatic Basis of Detoxification, pp. 333-353. Editors: W.B. Jakoby and W.H. Habig. Academic Press, New York.
43
43-48
Elsevier Scientific Publishers Ireland Ltd.
Differential expression of glutathione S-transferase, glutathione peroxidase and glutathione reductase in normal and malignant human breast tissues S.V. Singh”, S.R. Brunnertb, “Departments
of Oncology and
B. Robertsb and A. Krishan”
*Pathology,
University of Miami School of Medicine, Miami, FL 33136 (U.S.A.)
(Received 27 November 1989) (Revision received 10 January 1990) (Accepted 11 January 1990)
Summary In the present study we have compared the levels of glutathione (GSH) S-transferase, GSH peroxidase and GSH reductase in human breast tumors and adjacent normal fissues obtained from the same individuals. We haoe also quantitated GST T[ type antigen in these samples by western blotting. GST T[ actioity towards 1 -chloro-2,4-dinitrobenzene was found to be elevated in tumors from three out of six patients (patient nos. 2, 4 and 5), whereas this activity was suppressed in tumor from patient no. 1. Results of Western blotting using antibodies raised against GST II of human placenta were in agreement with the GST activity data. GSH peroxidase actioity with cumene hydroperoxide as substrate was found to be eleoated in four tumor samples (patients nos. 2, 4, 5, and 6) but suppressed in tumor from patient no. 1. On the other hand, GSH reductase activity was elevated in three samples [patients nos. 2, 4 and 5) and downregulated in the remaining three samples (patients nos. 1, 3 and 6). These results indicate that GSH-related enzymes are dijferentially altered in human breast tumors and GST TI type isoenzyme(s), unlike certain other human carcinomas such 0s colonic, are not uniformly elevated in human breast tumors. Correspondence
to:
S.V. Singh.
0304-3835/90/$03.50 Published and Printed
in
Keywords: glutathione; glutathione S-transferase; glutathione peroxidase; breast carcinoma. Introduction
Glutathione (GSH) S-transferases (GST) are a family of multifunctional enzymes which participate in detoxification of a wide variety of xenobiotics including carcinogens and their metabolites (8,171. Structural, kinetic and immunological characterizations have enabled to group mammalian GST’s into three major classes a, p and II [18]. Several independent studies have suggested that the placental form of rat GST (GST-P) may be used as a marker for chemically induced rat hepatocarcinogenesis [22,24,29]. GST-P expression is very low in normal rat liver but this isoenzyme is elevated several-fold in chemically induced pre-neoplastic and neoplastic lesions [24f. In a recent study, however, uniform elevation of GST-P was not observed in peroxisome proliferator induced lesions [23]. GST-P has considerable structural homology with human placental GST TI [15,19,21,24]. Overexpression of n class GST isoenzyme has been reported in some human tumors such as primary hepatomas [28] and colonic [ 161, esophageal [31] and renal carcinomas [9]. These studies suggest the possibility of II class GST being used as a human tumor marker.
0 1990 Elsevier Scientific Publishers Ireland Ltd Ireland
44
On the other hand, uniform elevation of GST TI was not observed in human lung tumors [7,13,25] and small cell lung cancer cell lines [2]. Taken together, these studies suggest that induction of GST TIin human tumors may be tissue specific. In order to substantiate this hypothesis and to identify human carcinomas where this isoenzyme may serve as a tumor marker, quantitation of GST TImust be carried out in various types of human tumors. We have, therefore, compared GST activity and relative amount of GST T[ in human breast tumors and adjacent normal tissues obtained from same patients. Increased levels of GST, GSH peroxidase and GSH reductase have been reported in cancer cells resistant to chemotherapeutic agents such as doxorubicin (DOX) [4,20,26] and these enzymes have been implicated in cellular drug resistance. Thus, we have also compared GSH peroxidase and GSH reductase activities in normal and tumorous breast tissue samples. Significance of the findings is discussed. Materials and methods
Procurement and processing of tissues Breast tumors and adjacent normal tissues were collected from mastectomy patients through our Cancer Center Tissue Procurement Services. Samples were obtained from white females of age between 34 and 63 years. A small piece of tissue was fixed in 10% formalin for histopathological examination and the remaining sample was used for the enzyme activity determinations and Western blotting. Histopathological examination was used to differentiate normal and malignant tissues. After fixation, tumors and adjacent normal tissues were dehydrated, embedded, sectioned (5 pm), and stained with hematoxylin and eosin. Preparation of tissue supernatants A 10% (w/v) homogenate was prepared in 10 mM potassium phosphate buffer (pH 7.0)) containing 1.4 mM 2-mercaptoethanol. The homogenate was centrifuged at 14,000 x g for 45 min to obtain supernatant .
Enzyme assays GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate was determined by the method of Habig et al. [ 141. GSH peroxidase and GSH reductase and GSH reductase activities were determined by the methods of Awasthi et al. [l] and Beutler [5], respectively. Protein content was determined by the method of Bradford [6]. Western blotting Immunoblotting was performed by the method of Towbin et al. [30] with slight modifications as described previously [2]. Polyclonal antibodies, specific against GST R of human placenta were kindly provided by Dr. Yogesh C. Awasthi, University of Texas Medical Branch, Galveston, TX. Results and discussion
Table 1 presents clinical data on six patients from whom tumors and normal tissues were obtained. GST activity in 14,000 x g supernatant fractions of paired tumor and normal breast tissues was determined towards CDNB as substrate (Fig. 1). Among normal tissues of these patients, GST activity varied significantly; ranging from 7 to 133 nmol/ min/ mg protein. This activity in tumor from patient no. 1 was about 40% of that in the corresponding normal tissue. GST activity in normal and tumor tissues from patients nos. 3 and 6 was comparable (Fig. 1). On the other hand, this activity in tumors from patients 2, 4 and 5 was higher by 1.5-, 6.7- and 2.0-fold, respectively, compared to that of the corresponding normal tissues (Fig. 1). Quantitation of GST n in normal and tumor tissues of patients 1, 3 and 4 was performed by Western blotting using equal amount of cytosolic protein and antibodies raised against GST n of human placenta (Fig. 2). These samples were specifically chosen for Western blotting because in tumor no. 1 there was a decrease in GST activity, in tumor no. 3 it was unchanged and in tumor no. 4 GST activity was significantly higher (Fig. 1). Similar to GST activity data, Western blotting revealed suppression of
45 Table 1.
Clinical data on patients.
Patient
Chemotherapy
Age
Type of breast cancer
1 2 3 4 5 6
None None None None None None
50 34 52 55 63 -
Ductal carcinoma Poorly differentiated carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Cytosarcoma phyllodes Infiltrating ductal carcinoma
GST in tumor tissue of patient no. 1 (Fig. 2, lane 3). GST n content was similar in normal and tumor tissues of patient no. 3 (Fig. 2, lanes 4 and 5, respectively), whereas GST II content in tumor tissue of patient 4 was significantly higher (Fig. 2, lane 7) compared to that of the corresponding normal tissue (Fig. 2, lane 6). Quantitation of a and p class GST was not undertaken because these isoenzymes have been reported to be absent in human breast tissue [lo]. An isoenzyme of GST immunologically similar to GST rr of human placenta is elevated in human colon [16], esophageal [31] and renal carcinomas [9] suggesting that it could be used as a human tumor marker [9,16,28,31]. Even though the present study was conducted on a small number of human breast tumor samples, it suggests that elevation of GST IT is not a common feature in different types of human 2001
Sample number
Fig. 1. Glutathione S-transferax activity in 14,000 X g supernatant fractions of normal (0) and malignant (n )
breast tissues.
tumors. Out of six tumor samples analysed, GST n was elevated only in three samples. In two of the tumor samples GST activity was similar to those of corresponding normal tissues whereas in the remaining tumor sample GST A level was significantly reduced. These results are similar to those of our recent studies with three small cell lung cancer cell lines [2] where GST n was found to be elevated in cell lines NES and NOC-361 and suppressed in the third cell line, NCLH69 [2]. Interestingly, in a different study GST II was found to be absent in six small cell lung tumors and elevated considerably in squamous cell carcinomas [13]. Similarly, uniform elevation of GST n was not observed in human lung tumors during two separate investigations [7,25]. Our results are also in agreement with another study where out of 17 breast tumors analysed, GST activity was significantly high in only 11 samples [ll]. Taken together, these results suggest that elevation of GST T[ in human tumors may be tissue specific; colon, esophageal and renal tumors exhibiting higher frequency of GST I[ overexpression [9,16,31], whereas lung and breast tumors showing relatively lower rate of elevation. These results also suggest that GST II may not represent an ideal marker for human breast carcinomas. However, a large number of paired normal and tumor breast samples must be analyzed for GST II content before validity of this contention can be established beyond any doubt. Significant individual variation was also observed in GSH peroxidase activity in normal tissues of these patients; ranging between 15
46
6
7
Fig. 2. Western blot analysis using antibodies raised against GST n of human placenta. Lane 1, purified GST n of human placenta (3 c(s); lanes 2 and 3, aliquots (25 pg protein) of 14,000 x g supematant fractions from normal and tumor tissues of patient no. 1, respectively; lanes 4 and 5, aliquots (25 pg protein) of supematant fractions from normal and tumor tissues of patient 3, respectively; lanes 6 and 7, aliquots (50 pg protein) of supematant fractions from normal and tumor tissues of patient no. 4, respectively.
and 103 nmol/ min/ mg protein (Table 2). GSH peroxidase activity with cumene hydroperoxide (CUOH) as a substrate in tumor tissue from patient no. 1 was 48.5% of that in adjacent normal tissue, whereas the activity in normal and tumor tissues of patient no. 3 was comparable. On the other hand, GSH peroxidase activity was elevated in tumor tissues of patients nos. 2, 4, 5 and 6 by 1.25, 1.95-, 6.06- and 1.45-fold, respectively (Table 2) when compared to those in corresponding normal tissues. GSH reductase activity ranged between 5 and 23 nmol/ min/ mg protein in normal tissues of these patients (Table 2). This activity in tumors from patients nos. 1, 3 and 6 was 75%, 33% and 22%, respectively, of that in the corresponding normal tissues whereas the activity in normal and tumor tissues from
patient no. 2 was comparable. On the other hand, GSH reductase activity in tumors from patients nos. 4 and 5 was higher by 2.6- and 2.3-fold, respectively, as compared to those in the corresponding normal tissues (Table 2). These data indicate that GSH related enzymes are differentially altered in human breast carcinomas. However, mechanism(s) by which the expression of GST, GSH peroxidase and GSH reductase genes are altered during carcinogenesis remains to be determined. The central role of GSH and related enzymes in detoxification of oxyradicals [32] (which have been implicated in cytotoxic activity of chemotherapeutic agents such as DOX [3]) and electrophiles (including some of the currently used ’ cytotoxic drugs such as melphalan [12] and BCNU [27]) implies that the relative levels of these enzymes may affect
47
Table 2.
GSH peroxidase and GSH reductase activities in normal and malignant breast tissues. Both the enzymes assayed at 37OC. Values represent means of duplicate or triplicate determinations. Patient
Enzyme activity (nmol/min
per mg protein) GSH reductase
GSH peroxidase
1 2 3 4 5 6 Means f SD. (n = 6)
were
Normal
Tumor
Normal
Tumor
103 68 91 21 15 69 61 f 36
50 85 94 41 91 100 77 f 25
8 9 6 5 6 23 9.5 k 7
6 11 2 13 14 5 8.5 + 5
tumor cell response to certain chemotherapeutic agents. In fact, several independent studies have suggested that GST, GSH peroxidase and GSH reductase may contribute to drug resistance [4,12,20,26,27]. More direct evidence for the involvement of these enzymes in cellular drug resistance has also been presented recently. For example, DOX resistance in a MCF-7 human breast cancer cell line has been attributed to the increased GSH peroxidase mediated detoxification of drug induced free radicals [26]. Similarly, GST levels can affect tumor cell sensitivity to melphalan and BCNU because these drugs have recently been shown to be detoxified by GST [12,27]. The usual procedure for breast cancer treatment involves surgery followed by radiation or chemotherapy. It may be speculated that tumors with elevated levels of GSH related enzymes may show relatively low sensitivity to drugs such as DOX, melphalan and BCNU and eventually causing treatment failure. Thus, it may be useful to compare the levels of GSH related enzymes in tumor biopsies and adjacent normal tissue specimens of same individuals prior to the start of chemotherapy. This information may be valuable in selection of chemotherapeutic agents.
Acknowledgements
This investigation was supported in part by the American Cancer Society Institutional, Biomedical Research Support Institutional Grants (S.V.S.) and US PHS Grant CA 44737 (A.K.). References Awasthi, Y.C., Beutler. E. and Srivastava, S.K. (1975) Purification and properties of human erythrocyte glutathione peroxidase. J. Biol. Chem., 250,5144-5149. Awasthi, Y .C., Singh, S.V., Gupta,
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