Biol. Chem. Hoppe-Seyler Vol. 373. pp. 595-604, July 1992
Cystatins and Cathepsins in Breast Carcinoma TAMARA T. LAH, MAJDA KOKALJ-KUNOVAR, MARINA DROBNIC-KOSOROK, JOZA BABNIK, RASTKO GOLOUH, IVAN VRHOVEC AND VITO TURK Department of Biochemistry, J. Stefan Institute, Jamova 39, 61000 Ljubljana, Slovenia Summary The increased expression of proteolytic systems is one of the characteristics of transformed and malignant cells and their evaluations in whole tumor homogenates were considered as possible diagnostic and/or prognostic factors. Abnormal intracellular distribution, increased activities and secretion of cysteine proteinases (CPs) cathepsin B (Cat B) and L (Cat L), were associated with tumor progression. In the present study of matched pairs of breast carcinoma and normal breast tissue, the activities of Cat B and Cat L in breast carcinoma homogenates were found to be 20 and 50 fold higher, respectively, than in normal tissues. In contrast, a decrease in total inhibitory activity of cysteine proteinase inhibitors (CPIs) was observed but an average ratio between tumor and normal tissues was only 0.75. One of the CPIs, stefin A, was also determined immunochemically. The activities of CPs and CPIs were compared to the increased levels of cathepsin D (Cat D) activities in individual patients, but no statistically significant correlations were found. We correlated CPs and CPIs with morphological and receptor data as well as the axilliary lymph node metastases. There was no statistical correlation of CP and CPIs with the number of lymph node metastases. However, highly elevated levels of Cat B and Cat L and lowered CPI activities in tumor cytosols were often associated with poorly differentiated carcinomas and those with negative ER and PR values. We conclude that cysteine-dependent proteolysis may play an important role in breast tumors.
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Introduction Effective breast cancer management utilizes prognostic factors that alone or in combination with others will aid in identifying women at risk for early relapse and metastasis. Two different types of proteinases, urokinase (u-PA), a serine proteinase, and cathepsin D (Cat D), a lysosomal aspartic proteinase have been reported recently to be valuable predictors in this regard (1,2,3). Cat D has been measured in breast carcinoma immunohistochemically, by Western blotting and by ELISA (for review see 4) and its increase was generally associated with shorter survival rate, although the authors reached somewhat different conclusions on its role in malignancy. Having rather broad specificity, a variety of proteins proved to be good substrates for Cat D "in vitro." Among them are also the inhibitors of cysteine proteinases (CPIs) kininogens, cystatins and stefins (5,6). In this study, a possible role of Cat D in the inactivation of CPIs was considered. "In vivo" this may occur in acid microenvironment, and thereby increase the proteolytic activities of Cat B and Cat L. The balance between CP and CPIs seems to be associated with the increase in metastatic potential in some experimental tumors (7,8). Elevated levels of Cat B were found also in human carcinomas (9,10). For example, malignant breast tissue in culture produced and secreted a precursor form of Cat B (11) and elevated Cat B was demonstrated in human breast carcinomas compared to benign and normal breast tissues and cells (12,13). The present work shows relative increases in tumor Cat B and Cat L activities in matched pairs of breast carcinomas. Total CPI activities in the same samples were decreased or increased in 67 % and 33 % of tumors, respectively. Decreased CPIs correlated to highly increased CPs and also to some clinical parameters. However, CPI levels did not correlate to the Cat D, nor did the increased Cat D levels statistically correlate to the elevated Cat B and Cat L. In addition, we have developed a double sandwich ELISA assay to measure the intracellular CPI stefin A. The results indicate an association of cysteine dependent proteolysis, with the biochemical mechanisms of tumor progression (14).
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Cystatins and Cathepsins in Breast Carcinoma
597
Materials and Methods Patient selection and tissue samples preparation
Patients, diagnosed for intraductal breast carcinoma, were treated by partial or total mastectomy and selected randomly for this study. The histologic parameters considered were: tumor grade, based on tubule formation, pleomorphism, hyperchromasia and mitotic activity. Tumors were graded I, II, and ΠΙ (well, moderately and poorly differentiated, respectively). The regional lymph node involvement was determined histologically. A matched pair represented tumor and adjacent uninvolved control tissue from the same breast. Both tumor and normal breast tissues were trimmed of fat and connective tissue, washed with physiologic saline solution and homogenized in 50 mM TRIS buffer, pH 6.0, containing ImM benzamidine and 0.5 mM phenylmethylsulfonyl fluoride with Waring blender. Aliquotes of the supernatant cytosols were taken for assay of the cathepsins. CPI activities were measured in the same cytosols after inactivation of endogenous proteinases by boiling the samples for 10 min as previously described (14). Analytical grade chemicals were purchased from Sigma (Poole, UK). Proteinase substrates and inhibitors were obtained from Serva, Heidelberg, Germany. Activity assays
Cat D activity was measured using hemoglobin as substrate in acetate buffer, pH 3.5, and expressed in Anson units/ mg protein in the cytosol (15). Cat B activity was measured using fluorimetric peptide substrate, benzoylcarbonyl-arginyl-arginyl-amino-4-methylcoumarine (Z-Arg-Arg-AMC), as previously described (14,16). To discriminate between Cat B and Cat L activities, we measured the hydrolysis of non-specific substrate Z-Phe-Arg-AMC in the presence and absence of 0.5 μΜ (final concentration) of Z-Phe-PheCHN2? the latter inhibiting Cat L but not Cat B (14,17). The activities of CPs are expressed in enzyme units/mg protein, one mEU representing the release of 1 nmol of the reaction product, 7-aminomethylcoumarine, per minute at 37 °C. Inhibition activity was tested against plant CP papain and referred to as the total CPI activity. Commercial papain (Sigma, type IV) was further purified as
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described by Buttle et al. (18) and the active papain fraction was determined by titration with L-epoxy succinylleucylamido(4-guanidino) butane (E-64). One inhibitory unit (IU) equals one EU and was determined as suggested by Barrett and Kirschke (16). Specific activities were expressed in IU/mg protein. Protein concentration in the cytosols was determined according to the Bradford assay with bovine serum albumin as standard (19). Enzyme immunosorbent assay (ELISA) Double sandwich ELISA for stefin A was developed using rabbit polyclonal antibodies (Ab) raised against human stefin A, isolated in our laboratory (14). Immunospecific Ab was diluted to 10 >ig/ml in 0.1 M carbonate/bicarbonate buffer, pH 9.6 and 100 ul were pipetted into each well. After overnight incubation at 4°C and subsequent washing, 100 μ\ of the samples or standard antigens diluted with 2 % bovine serum albumin, were added. The plates were incubated for 2 hrs at 37°C, thoroughly washed and peroxidase-labeled antistefin A Ab in 1:500 dilution, was added. To quantitate the antigen, we added peroxidase substrate, 2,2-azino-bis-3-ethylbenzothiazoline sulphonate ( Boehringer -Mannheim GmbH, FRG) measured the optical density of the product at 410 nm on an automated reader (Dynatech Minireader II, USA). Estrogen and progesterone receptors
Estrogen receptors (ER) and progesterone receptors (PR) were measured according to the protocol of BYK-Diagnostica, using dextran-charcoal assay with radiolabeled ligands. Tumors with the receptor concentration > 10 fmol/mg protein were considered receptor positive. Statistical Analysis
For statistical analysis of matched breast tissue pairs (tumor and normal) we used Wilcoxon's rank test. The levels of signficance were determined using Student t-test, where the probability of 0.05 or less was considered signficant. The correlation between various biochemical parameters was calculated by linear regression analysis and the significance of the Pearson correlation
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Cystatins and Cathepsins in Breast Carcinoma
599
coefficient (r) was evaluated by co-variance test. These analyses were carried out with the PARASTAT Statistics Program (Arlington, VA). Results and Discussion Cathepsins activities in breast carcinoma The activities of lysosomal enzymes aspartic proteinase Cat D and cysteine proteinases, Cat B and Cat L, as well as total inhibitory activity of CPIs, were assayed for their activities in breast ductal carcinoma and normal breast tissue cytosol of the same patient, representing a matched pair. Relative increases were calculated as the ratios between the two activities in matched pairs and the means of the ratios were determined in a randomly chosen population of total 70 patients: The results for CPs, CPIs and Cat D are presented in Fig.l. Of the proteinases, Cat L was significantly more elevated than was Cat B, while Cat D activity was signficantly less increased compared to the activities of CPs. These results are slightly different from a study on the immunochemically measured content of CP in breast carcinomas (20), where the average increase of Cat B was higher than that of Cat L, while another CP, cathepsin H was present in the highest concentration. Also, these authors reported about a 9-fold elevated immunoreactive Cat B in breast carcinoma cytosols while we observed about 22 fold increase in the mean activity. The discrepancies between the activity and the protein measurements might be explained by the presence of Cat B pro-forms and their complexes with the CPIs: in both cases, Cat B might not be equally recognized by the antibodies that were raised against the mature enzyme. Measuring enzyme activity, we determined the fraction of the free enzyme form (15) and the enzyme which was not tightly complexed to the endogenous CPIs and dissociated during the assay conditions (21). Therefore, the advantage of the activity over the immunoassays is that it indicates the level of the functional enzyme. For example, Maciewicz et al. (22) reported high levels of pro-cathepsins B and L in all colorectal carcinoma cell lines, but only malignant cell lines had the ability to process them to the mature active forms. It was suggested that the invasive potential of a tumor is rather related to the amount of the active enzyme forms than to the amount of the synthesized precursors. Consequently, the endogenous inhibitors must play a very important role in ultimately regulating their activities.
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Tumor/Normal Activity
•iCYSTATINS
^CATB
CD CAT L
^ CAT D
Patients population: 50 IOC
Fig.l. Relative increase of cathepsins in matched pairs of carcinomas and normal breast tissue counterparts The ratios of measured specific activities of CPIs (mIU/mg), CPs (mEU/g), and Cat D (Anson Units/g) between the tumor and the normal tissue cytosols of the same breast were calculated. The mean values + - standard error (SE), assuming normal distribution statistics were calculated: CPI = 0.79 + -0.02 (n = 44); Cat B = 18.49 +- 10.98 (n = 50); Cat L= 52.5 + 39.6 (n=50); Cat D = 5.8 +- 28 (n=20) and the mean values are presented. Table 1. Breast carcinoma patients: differences in biochemical and histo-pathological parameters GROUP I Decreased CPI (n =29) T/N = 0.49 +-0.05 Cat B (mU/g, mean +- SE) Cat L (mU/g; mean +- SE) Estrogen Receptor Negative Progesteron Receptor Negative Lymph Node Metastatis Positive Tumor Grade III
50 +- 15 901 +-250 65% 72% 41% 58%
GROUP II Increased CPI (n = 15) T/N1 = 1.53 + 0.1 11 +- 5 250+ -170 27% 46% 41% 20%
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Cystatins and Cathepsins in Breast Carcinoma
0
400
800 1200 1600 2000 Cat O. Anaon U/mg
601
2400
Fig.2. Correlation of Cat D and CPI activities Linear regression analysis of Cat D and CPI activities revealed poor positive correlation (Pearson coefficient, r = 0.57) which was statistically significant at probability (p = 0.05 ). Table 2. Stefin A and CPI activities ratios (T/N) in matched pairs Tumor grade III Stefma A 0.57 1.05 0.60 0.14 0.38 0.24 0.64 0.27 0.87 0.50 0.45 0.50 0.45
CPI 4.70 1.25 0.30 0.17 0.22 0.68 0.12 0.32 1.02 0.39 0.21 0.61 0.59
II
Stefin A 11.4 0.43 1.01
I
CPI 1.38 2.05 0.95
Stefin A 7.15 3.77
CPI 3.27 3.68
Linear regression analysis revealed poor correlation (r=0.55) between stefin A and CPI activities in matched pairs of breast carcinoma (n=26, not shown).
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CPI in breast carcinoma A decrease in the mean tumor CPI activity was observed (Fig.l), indicating a loss of the final control of CP activities. However, as the total decrease was only about 25% of the activities in normal tissues, it is less likely that this could cause about 20 to 50 fold increase in CP activities. Other regulatory mechanisms must be responsible for shifting the balance between CPs and CPIs in tumors in favor of highly increased proteolysis. An important observation was also the heterogenous nature of the tumors regarding their CPI content. The decrease in inhibitory potential was observed in only about two thirds of tumors, others having similar or higher CPI activities. The patients were therefore divided into two groups, group I patients having decreased levels of CPIs and group II patients expressing higher inhibitory potential. When the proteinase activities were compared in the two groups, group I was found to express significantly more active Cat B and Cat L. The differences in tumor grade, metastasis number and estrogen receptors between the two groups of patients, are summarized in Table 1. It is evident, that decreased CPIs in breast tumors are associated with higher relative increase in both Cat B and Cat L and are more often observed in patients with ER negative values and with poorly differentiated tumors. No correlation with metastasis was found, both groups comprising similar proportion of lymph node positive patients. Analysis of larger patients population may reveal the significance of CPIs association with clinical and histopathological status of breast carcinoma. The above findings raise two questions: (a) why are the CPI activities decreased and (b) which of the cytstatins are down-regulated in breast tumors? Based on the "in vitro" observation, Lenarcic et al. (6) hypothesized that Cat D might inactivate kininogens, cystatins and stefins "in vivo". However, Cat D was increased in all tumors, what was not necessarily accompanied by lowered inhibitory potential (see Table 1) and no inverse correlation was observed between Cat D and CPI (Fig.2). Cat D activities were also correlated with Cat B and Cat L activities in the small sample population (n=20) by Cox multivariant analysis. No statistically significant correlations were found, indicating an independent regulation of lysosomal proteinases in breast tumors. Stefin A in breast carcinoma Investigating the changes of stefins in various neoplasms by immunostaining, Jarvinen et al. (23) concluded, that stefin A is closely related to
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Cystatins and Cathepsins in Breast Carcinoma
603
the process of dedifferentiation in epidermoid carcinomas. This is similar to our observation on the decrease of stefin A immunoreactivity in most of the poorly differentiated breast carcinomas ( Table 2). Kolar et al. also found by immunohistochemical methods, that cystatins (stefins A and B and cystatin C) were influenced by estrogen in breast adenocarcinoma cell line (MCF-7) (24). However, rather poor correlation was found between Stefin A and the total inhibitory activity, indicating that other cystatins contribute significantly to the total inhibition potential in the homogenates. This conclusion is strongly supported by recent findings on significant changes of kininogen concentration in breast carcinomas (25). In conclusion, we showed that cysteine dependent proteolysis plays an important role in the progression of breast carcinoma, but is not directly related to metastatic spread. Tumors were found to be highly heterogeneous regarding the total CPI activities and with respect to the increase in cathepsins. A follow-up study on the recurrence of the disease is in progress to evaluate CPs and CPIs as possible diagnostic/prognostic factors for breast carcinoma. Acknowledgement
We thank Dr. Joseph Tabachnick, Albert Einstein Medical Center, Phialdelphia, for comments and suggestions. This work was supported by Grants from Ministry of Science and Technology of Slovenia. References
1.
2. 3. 4. 5. 6.
Duffy, M.J., O'Grady, P. and O'soriani, L. (1988) in: Progress in Cancer Research and Therapy: Hormones and Cancer (Bresciani, T., King, R.J.B., Lippman, M.E. and Ranaud, J.P., eds..) Vol., 35, pp. 300-303, Raven Press, New York. Jaenicke, F., Schmitt, M., Hafter, R., Hollrieder, A., Babic, R., Ulm, K., Goessar, W. and Graeff, H. (1990) Fibrinolysis 4, 69 - 78 . Henry, J.A., McCarthy A.L., Angus, B., Westley, B.R., May, .E.B., Nicholson, S., Cairns, J., Harris, A.L. and Hörne, C.W. (1990) Cancer, 65,5-271. Rochefort, H. (1990) Seminars in Cancer Biology. 1,153-160. Barrett, A.J., Rawling, N.D., Davies, M.E., Machleidt, W., Salvesen,G. and Turk V. (1986) in: Proteinase Inhibitors ( Barrett, A and Salvesen, G. eds) pp 515-569, Elsevier, Amsterdam. Lenarcic, B., Kos, J., Dolenc, I., Lucovnik, P., Krizaj, I. and Turk, V. (1988). Biochem.Biophys.Res.Commun., 154,765-722 .
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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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Sloane, B.R, Dünn, J.R. and Honn , K.V. (1981) Science. 212. 1151-1153. Sloane, B.F. (1990) Seminars in Cancer Biology. I, 137-152 . Sheahan, K., Shuja,S. and Murnane, M.J.(1989) Cancer Res.49.38Q9-3814. Krepela, E., Kasafirek, E., Novak , K. and Vicklicky, J. (1990) Neoplasma 37,61-70. Recklies, A.D., Tiltman,K.J, Stoker, A.M. and Poole, A.R. (1980) Cancer Res.. 40,550-556 . Vashista, ., Baker, P.R., Preece,P.E., Wood, R.A.B. and Cushieri, A. (1988) Anticancer Res.. 8, 785-790. Krepela, E., Bartek, J., Skalkova, D., Vicar J., Rasnick, D., TaylorPapadimitru, J., Hallowes, R.C. (1987) J. Cell Science. 87,1450-1454. Lah, T.T., Kokalj-Kunovar, M., Strukelj, B., Pungercar, J., BarlicMaganja, D., Drobnic-Kosorok, M., Kastelic, L., Babnik, J., Golouh, R., Turk, V. (1992) Int. J Cancer. 50,34-46. Turk,V., Lah, T. and Kregar, I.(1984) in: Methods of Enzymatic Analysis (Bergmayer, V., ed.) pp. 211-222, Verlag Chemie, GmbH, Weinheim . Barrett, A.J. and Kirschke, H. (1981). in: Methods in Enzymology Vol. 80, part C, pp. 535-561, Academic Press, New York. Mason, R.W., Green,G.D.J. and Barrett, A.J. (1985) Biochem J.. 226, 233-241. Buttle, D.J., Kembhavi, A.A., Sharp, S.L., Shute, R., Rich, D.H. and Barrett, A.L. (1989) Biochem. J.. 261,469-476. Bradford,M. (1976) Anal. Biochem.. 72, 248-254. Gabrijelcic, D., Annan-Prah, A., Skrk, J., Kramberger, M., Sebek, S. and Turk, V. (1990) Pcriod.Biol. 92, 154-155. Machleidt-Assflag, L, Jochum, M., Klaubert,W.,Inthorn, D. and Machleidt, W. (1988) Biol.Chem.Hoppe-Seyler. 369, Suppl, pp. 263-269. Maciewicz, R.A., Wardale, R.J., Etherington, DJ. and Paraskeva, C. (1989) IntJ.Cancer. 43, 478-486. Jarvinnen, M., Rinne, A. and Hopsu-Havu V.K (1987) Acta histochem.. 82, 5-18. Kolar, Z., Jarvinnen, M., and Negrini, R. (1989). Neoplasma. 36, 185-188. Gabrijelcic, D., Svetic, B., Spaic, D., Skrk, J., Budihna, M. KININ'91 Munich (1991) Abstract Poster Session 10, Symp. 2, Pst2.16.
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Cystatins and Cathepsins in Breast Carcinoma TAMARA T. LAH, MAJDA KOKALJ-KUNOVAR, MARINA DROBNIC-KOSOROK, JOZA BABNIK, RASTKO GOLOUH, IVAN VRHOVEC AND VITO TURK Department of Biochemistry, J. Stefan Institute, Jamova 39, 61000 Ljubljana, Slovenia Summary The increased expression of proteolytic systems is one of the characteristics of transformed and malignant cells and their evaluations in whole tumor homogenates were considered as possible diagnostic and/or prognostic factors. Abnormal intracellular distribution, increased activities and secretion of cysteine proteinases (CPs) cathepsin B (Cat B) and L (Cat L), were associated with tumor progression. In the present study of matched pairs of breast carcinoma and normal breast tissue, the activities of Cat B and Cat L in breast carcinoma homogenates were found to be 20 and 50 fold higher, respectively, than in normal tissues. In contrast, a decrease in total inhibitory activity of cysteine proteinase inhibitors (CPIs) was observed but an average ratio between tumor and normal tissues was only 0.75. One of the CPIs, stefin A, was also determined immunochemically. The activities of CPs and CPIs were compared to the increased levels of cathepsin D (Cat D) activities in individual patients, but no statistically significant correlations were found. We correlated CPs and CPIs with morphological and receptor data as well as the axilliary lymph node metastases. There was no statistical correlation of CP and CPIs with the number of lymph node metastases. However, highly elevated levels of Cat B and Cat L and lowered CPI activities in tumor cytosols were often associated with poorly differentiated carcinomas and those with negative ER and PR values. We conclude that cysteine-dependent proteolysis may play an important role in breast tumors.
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Introduction Effective breast cancer management utilizes prognostic factors that alone or in combination with others will aid in identifying women at risk for early relapse and metastasis. Two different types of proteinases, urokinase (u-PA), a serine proteinase, and cathepsin D (Cat D), a lysosomal aspartic proteinase have been reported recently to be valuable predictors in this regard (1,2,3). Cat D has been measured in breast carcinoma immunohistochemically, by Western blotting and by ELISA (for review see 4) and its increase was generally associated with shorter survival rate, although the authors reached somewhat different conclusions on its role in malignancy. Having rather broad specificity, a variety of proteins proved to be good substrates for Cat D "in vitro." Among them are also the inhibitors of cysteine proteinases (CPIs) kininogens, cystatins and stefins (5,6). In this study, a possible role of Cat D in the inactivation of CPIs was considered. "In vivo" this may occur in acid microenvironment, and thereby increase the proteolytic activities of Cat B and Cat L. The balance between CP and CPIs seems to be associated with the increase in metastatic potential in some experimental tumors (7,8). Elevated levels of Cat B were found also in human carcinomas (9,10). For example, malignant breast tissue in culture produced and secreted a precursor form of Cat B (11) and elevated Cat B was demonstrated in human breast carcinomas compared to benign and normal breast tissues and cells (12,13). The present work shows relative increases in tumor Cat B and Cat L activities in matched pairs of breast carcinomas. Total CPI activities in the same samples were decreased or increased in 67 % and 33 % of tumors, respectively. Decreased CPIs correlated to highly increased CPs and also to some clinical parameters. However, CPI levels did not correlate to the Cat D, nor did the increased Cat D levels statistically correlate to the elevated Cat B and Cat L. In addition, we have developed a double sandwich ELISA assay to measure the intracellular CPI stefin A. The results indicate an association of cysteine dependent proteolysis, with the biochemical mechanisms of tumor progression (14).
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Cystatins and Cathepsins in Breast Carcinoma
597
Materials and Methods Patient selection and tissue samples preparation
Patients, diagnosed for intraductal breast carcinoma, were treated by partial or total mastectomy and selected randomly for this study. The histologic parameters considered were: tumor grade, based on tubule formation, pleomorphism, hyperchromasia and mitotic activity. Tumors were graded I, II, and ΠΙ (well, moderately and poorly differentiated, respectively). The regional lymph node involvement was determined histologically. A matched pair represented tumor and adjacent uninvolved control tissue from the same breast. Both tumor and normal breast tissues were trimmed of fat and connective tissue, washed with physiologic saline solution and homogenized in 50 mM TRIS buffer, pH 6.0, containing ImM benzamidine and 0.5 mM phenylmethylsulfonyl fluoride with Waring blender. Aliquotes of the supernatant cytosols were taken for assay of the cathepsins. CPI activities were measured in the same cytosols after inactivation of endogenous proteinases by boiling the samples for 10 min as previously described (14). Analytical grade chemicals were purchased from Sigma (Poole, UK). Proteinase substrates and inhibitors were obtained from Serva, Heidelberg, Germany. Activity assays
Cat D activity was measured using hemoglobin as substrate in acetate buffer, pH 3.5, and expressed in Anson units/ mg protein in the cytosol (15). Cat B activity was measured using fluorimetric peptide substrate, benzoylcarbonyl-arginyl-arginyl-amino-4-methylcoumarine (Z-Arg-Arg-AMC), as previously described (14,16). To discriminate between Cat B and Cat L activities, we measured the hydrolysis of non-specific substrate Z-Phe-Arg-AMC in the presence and absence of 0.5 μΜ (final concentration) of Z-Phe-PheCHN2? the latter inhibiting Cat L but not Cat B (14,17). The activities of CPs are expressed in enzyme units/mg protein, one mEU representing the release of 1 nmol of the reaction product, 7-aminomethylcoumarine, per minute at 37 °C. Inhibition activity was tested against plant CP papain and referred to as the total CPI activity. Commercial papain (Sigma, type IV) was further purified as
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described by Buttle et al. (18) and the active papain fraction was determined by titration with L-epoxy succinylleucylamido(4-guanidino) butane (E-64). One inhibitory unit (IU) equals one EU and was determined as suggested by Barrett and Kirschke (16). Specific activities were expressed in IU/mg protein. Protein concentration in the cytosols was determined according to the Bradford assay with bovine serum albumin as standard (19). Enzyme immunosorbent assay (ELISA) Double sandwich ELISA for stefin A was developed using rabbit polyclonal antibodies (Ab) raised against human stefin A, isolated in our laboratory (14). Immunospecific Ab was diluted to 10 >ig/ml in 0.1 M carbonate/bicarbonate buffer, pH 9.6 and 100 ul were pipetted into each well. After overnight incubation at 4°C and subsequent washing, 100 μ\ of the samples or standard antigens diluted with 2 % bovine serum albumin, were added. The plates were incubated for 2 hrs at 37°C, thoroughly washed and peroxidase-labeled antistefin A Ab in 1:500 dilution, was added. To quantitate the antigen, we added peroxidase substrate, 2,2-azino-bis-3-ethylbenzothiazoline sulphonate ( Boehringer -Mannheim GmbH, FRG) measured the optical density of the product at 410 nm on an automated reader (Dynatech Minireader II, USA). Estrogen and progesterone receptors
Estrogen receptors (ER) and progesterone receptors (PR) were measured according to the protocol of BYK-Diagnostica, using dextran-charcoal assay with radiolabeled ligands. Tumors with the receptor concentration > 10 fmol/mg protein were considered receptor positive. Statistical Analysis
For statistical analysis of matched breast tissue pairs (tumor and normal) we used Wilcoxon's rank test. The levels of signficance were determined using Student t-test, where the probability of 0.05 or less was considered signficant. The correlation between various biochemical parameters was calculated by linear regression analysis and the significance of the Pearson correlation
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Cystatins and Cathepsins in Breast Carcinoma
599
coefficient (r) was evaluated by co-variance test. These analyses were carried out with the PARASTAT Statistics Program (Arlington, VA). Results and Discussion Cathepsins activities in breast carcinoma The activities of lysosomal enzymes aspartic proteinase Cat D and cysteine proteinases, Cat B and Cat L, as well as total inhibitory activity of CPIs, were assayed for their activities in breast ductal carcinoma and normal breast tissue cytosol of the same patient, representing a matched pair. Relative increases were calculated as the ratios between the two activities in matched pairs and the means of the ratios were determined in a randomly chosen population of total 70 patients: The results for CPs, CPIs and Cat D are presented in Fig.l. Of the proteinases, Cat L was significantly more elevated than was Cat B, while Cat D activity was signficantly less increased compared to the activities of CPs. These results are slightly different from a study on the immunochemically measured content of CP in breast carcinomas (20), where the average increase of Cat B was higher than that of Cat L, while another CP, cathepsin H was present in the highest concentration. Also, these authors reported about a 9-fold elevated immunoreactive Cat B in breast carcinoma cytosols while we observed about 22 fold increase in the mean activity. The discrepancies between the activity and the protein measurements might be explained by the presence of Cat B pro-forms and their complexes with the CPIs: in both cases, Cat B might not be equally recognized by the antibodies that were raised against the mature enzyme. Measuring enzyme activity, we determined the fraction of the free enzyme form (15) and the enzyme which was not tightly complexed to the endogenous CPIs and dissociated during the assay conditions (21). Therefore, the advantage of the activity over the immunoassays is that it indicates the level of the functional enzyme. For example, Maciewicz et al. (22) reported high levels of pro-cathepsins B and L in all colorectal carcinoma cell lines, but only malignant cell lines had the ability to process them to the mature active forms. It was suggested that the invasive potential of a tumor is rather related to the amount of the active enzyme forms than to the amount of the synthesized precursors. Consequently, the endogenous inhibitors must play a very important role in ultimately regulating their activities.
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Tumor/Normal Activity
•iCYSTATINS
^CATB
CD CAT L
^ CAT D
Patients population: 50 IOC
Fig.l. Relative increase of cathepsins in matched pairs of carcinomas and normal breast tissue counterparts The ratios of measured specific activities of CPIs (mIU/mg), CPs (mEU/g), and Cat D (Anson Units/g) between the tumor and the normal tissue cytosols of the same breast were calculated. The mean values + - standard error (SE), assuming normal distribution statistics were calculated: CPI = 0.79 + -0.02 (n = 44); Cat B = 18.49 +- 10.98 (n = 50); Cat L= 52.5 + 39.6 (n=50); Cat D = 5.8 +- 28 (n=20) and the mean values are presented. Table 1. Breast carcinoma patients: differences in biochemical and histo-pathological parameters GROUP I Decreased CPI (n =29) T/N = 0.49 +-0.05 Cat B (mU/g, mean +- SE) Cat L (mU/g; mean +- SE) Estrogen Receptor Negative Progesteron Receptor Negative Lymph Node Metastatis Positive Tumor Grade III
50 +- 15 901 +-250 65% 72% 41% 58%
GROUP II Increased CPI (n = 15) T/N1 = 1.53 + 0.1 11 +- 5 250+ -170 27% 46% 41% 20%
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400
800 1200 1600 2000 Cat O. Anaon U/mg
601
2400
Fig.2. Correlation of Cat D and CPI activities Linear regression analysis of Cat D and CPI activities revealed poor positive correlation (Pearson coefficient, r = 0.57) which was statistically significant at probability (p = 0.05 ). Table 2. Stefin A and CPI activities ratios (T/N) in matched pairs Tumor grade III Stefma A 0.57 1.05 0.60 0.14 0.38 0.24 0.64 0.27 0.87 0.50 0.45 0.50 0.45
CPI 4.70 1.25 0.30 0.17 0.22 0.68 0.12 0.32 1.02 0.39 0.21 0.61 0.59
II
Stefin A 11.4 0.43 1.01
I
CPI 1.38 2.05 0.95
Stefin A 7.15 3.77
CPI 3.27 3.68
Linear regression analysis revealed poor correlation (r=0.55) between stefin A and CPI activities in matched pairs of breast carcinoma (n=26, not shown).
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CPI in breast carcinoma A decrease in the mean tumor CPI activity was observed (Fig.l), indicating a loss of the final control of CP activities. However, as the total decrease was only about 25% of the activities in normal tissues, it is less likely that this could cause about 20 to 50 fold increase in CP activities. Other regulatory mechanisms must be responsible for shifting the balance between CPs and CPIs in tumors in favor of highly increased proteolysis. An important observation was also the heterogenous nature of the tumors regarding their CPI content. The decrease in inhibitory potential was observed in only about two thirds of tumors, others having similar or higher CPI activities. The patients were therefore divided into two groups, group I patients having decreased levels of CPIs and group II patients expressing higher inhibitory potential. When the proteinase activities were compared in the two groups, group I was found to express significantly more active Cat B and Cat L. The differences in tumor grade, metastasis number and estrogen receptors between the two groups of patients, are summarized in Table 1. It is evident, that decreased CPIs in breast tumors are associated with higher relative increase in both Cat B and Cat L and are more often observed in patients with ER negative values and with poorly differentiated tumors. No correlation with metastasis was found, both groups comprising similar proportion of lymph node positive patients. Analysis of larger patients population may reveal the significance of CPIs association with clinical and histopathological status of breast carcinoma. The above findings raise two questions: (a) why are the CPI activities decreased and (b) which of the cytstatins are down-regulated in breast tumors? Based on the "in vitro" observation, Lenarcic et al. (6) hypothesized that Cat D might inactivate kininogens, cystatins and stefins "in vivo". However, Cat D was increased in all tumors, what was not necessarily accompanied by lowered inhibitory potential (see Table 1) and no inverse correlation was observed between Cat D and CPI (Fig.2). Cat D activities were also correlated with Cat B and Cat L activities in the small sample population (n=20) by Cox multivariant analysis. No statistically significant correlations were found, indicating an independent regulation of lysosomal proteinases in breast tumors. Stefin A in breast carcinoma Investigating the changes of stefins in various neoplasms by immunostaining, Jarvinen et al. (23) concluded, that stefin A is closely related to
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the process of dedifferentiation in epidermoid carcinomas. This is similar to our observation on the decrease of stefin A immunoreactivity in most of the poorly differentiated breast carcinomas ( Table 2). Kolar et al. also found by immunohistochemical methods, that cystatins (stefins A and B and cystatin C) were influenced by estrogen in breast adenocarcinoma cell line (MCF-7) (24). However, rather poor correlation was found between Stefin A and the total inhibitory activity, indicating that other cystatins contribute significantly to the total inhibition potential in the homogenates. This conclusion is strongly supported by recent findings on significant changes of kininogen concentration in breast carcinomas (25). In conclusion, we showed that cysteine dependent proteolysis plays an important role in the progression of breast carcinoma, but is not directly related to metastatic spread. Tumors were found to be highly heterogeneous regarding the total CPI activities and with respect to the increase in cathepsins. A follow-up study on the recurrence of the disease is in progress to evaluate CPs and CPIs as possible diagnostic/prognostic factors for breast carcinoma. Acknowledgement
We thank Dr. Joseph Tabachnick, Albert Einstein Medical Center, Phialdelphia, for comments and suggestions. This work was supported by Grants from Ministry of Science and Technology of Slovenia. References
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