Cathepsin B and Cysteine Protease Inhibitors in Human Tongue Cancer: Correlation with Tumor Staging and In Vitro Inhibition of Cathepsin B by Chicken Cystatin

Yousif Saleh¹, Jan Wnukiewicz, Ryszard Andrzejak, Tadeusz Trziszka,

Maciej Siewinski, Piotr Ziolkowski, and Wieslaw Kopec

Department of Forensic Medicine, Molecular Technical Unit [Y. Saleh], Department of Oral and Maxillo-Facial Surgery [J. Wnukiewicz], Department of Internal Medicine, Occupational Diseases and Hypertension [R. Andrzejak], Faculty of Public Health [M. Siewinski], and Department of Pathology [P. Ziolkowski], Wroclaw Medical University, Wroclaw, Poland; Department of Animal Products, Wroclaw Agriculture University, Wroclaw, Poland [T. Trziszka, W. Kopec]

AIM: The study is aimed to investigate if the sera and the tumor tissues of tongue cancer patients demonstrate elevated cathepsin B activity during cancer progression and in vitro inhibition of the activity of cathepsin B by chicken cystatin. METHODS: Pro-cathepsin B and cathepsin B activities and cystatins as antipapain activity were measured using a fluorogenic substrate in 47 sera from patients and compared with 40 control subjects.　 Peroxidase method was used for immunohistochemical staining. RESULTS: The activities of pro-cathepsin B and cathepsin B were found to be significantly increased and their endogenous inhibitor cystatin C decreased in the sera of patients with tongue cancer.　 Patients with advanced stages had higher serum activities of pro-cathepsin B, cathepsin B and cysteine protease inhibitors if compared with the patients with stage I disease.　 However, the levels of cysteine protease inhibitors were not comparable to those of cathepsin B.　 The serum activity of cathepsin B from tongue cancer patients could be decreased by 2.4-fold after treatment with 5 nM purified egg white cystatin.　 Immunohistochemical staining showed significantly increased expression of cystatin C and cathepsin B in tongue tumor tissues, while negative staining was observed with the non-tumor parts. CONCLUSION: Elevated cathepsin B level was closely correlated with the invasion and progression of tongue cancer.　 The incomparable levels between cathepsin B and its natural inhibitors could contribute to the uncontrolled proteolysis and thus the malignant progression of tongue cancer.　 Chicken cystatin was able to effectively inhibit patients’ cathepsin B activities in sera. Journal of Cancer Molecules 2(2): 67-72, 2006.

Keywords: cathepsin B chicken cystatin tongue cancer cysteine protease

Received 1/3/06; Revised 1/30/06; Accepted 2/8/06.

¹Correspondence: Dr. Yousif Saleh, Department of Forensic Medicine, Molecular Technical Unit, Wroclaw Medical University, Sklodowskiej-Curie-52, 50-369 Wroclaw, Poland. Phone: 48-717841588. E-mail: biolcancer@op.pl

Introduction

Patients with tongue carcinoma treated by intraoral tumor excision should be followed up carefully for cervical lymph node metastasis and receive re-surgery immediately if tumor is recurrent, because some patients present a more aggressive clinical course.　 Elevated levels of lysosomal proteases, such as cathepsins B, H, L, or D, have been reported in many cancer types [1,2].　 These proteases produced by the tumor cells are thought to play a major role in degrading the protein components of the basement membrane.　 Cathepsin B has been shown to participate in the dissolution and remodeling　 of connective　 tissue　 and　 basement membrane　 in the processes of tumor growth, invasion, and metastasis [3,4].　 Increased levels of cathepsins B and L in tumors or in some extracellular fluids are associated with the disease-free and overall survival periods and may therefore serve as prognostic factors for cancer patients [5,6].　 In addition, cathepsins are useful markers for identifying patients who are suffering from breast cancer [7] or colorectal cancer [8]. 　Kawasaki et al. [9] have reported that the expression of cathepsins D and B was closely correlated with cancer invasion and progression of oral squamous cell carcinoma.　 Cathepsins B and L are more frequently overexpressed in chronic atrophic gastritis with dysplasia.　 Cathepsin B protein is also frequently overexpressed in laryngeal carcinoma [10].　 However, little is known about the prognostic value of cathepsins in tongue carcinoma.

Cathepsin B can be regulated by the endogenous cysteine protease inhibitor, named cystatin, in normal tissues and cells [11,12].　 It has been suggested that cysteine protease inhibitors play a role in several diseases including cancer, which are associated with alterations of the proteolytic system [12].　 Cystatins are a group of reversible, tight-binding competitive inhibitors for cysteine peptidases such as cathepsins B, H, and L [13].　 Cystatin C inhibits motility and in vitro invasiveness of cancer cells, and could associate with the maintenance of cell differentiation [14].　 In inflammatory conditions or conditions with tissue breakdown, cystatin C free in the blood or other body fluid inhibits cysteine peptidases and thereby prevents tissue damage. 　The activity and concentration of cystatin seem varied in different cancer tissues, but its interactions with cathepsin B has been widely investigated [15].　 In this study, we investigated the expression status of cathepsin B in the sera and tumor tissues of tongue cancer patients and study its susceptibility to exogenous chicken cystatin.

Materials and Methods

Patients

The study comprised of 47 tongue cancer patients (27 men and 20 women) who visited the First Department of Oral and Maxillo-Facial Surgery Hospital, Wroclaw Medical University.　 Clinical data such as patients’ age, gender, and stage of disease were listed in Table 1.　 All cancer specimens were histologically proved to be squamous cell carcinoma.　 Patients’ age ranged from 34 to 75 years at diagnosis.　 Tumor staging was performed using the International Union Against Cancer (UICC) TNM classification. 　Forty healthy blood donors were enrolled as controls.　 Five ml of blood samples was collected before operation from patients scheduled for tongue cancer surgery.　 Blood samples were clotted at a temperature between 4°C and 8°C and thereafter centrifuged at 3,000 rpm.　 The samples were stored at -80°C until analysis.　 The study protocol was drawn up in accordance with the guidelines of the Helsinki Declaration and approved by our Institutional Review Board and the informed consent was obtained from each patient.

Purification of cystatin from egg white

Cystatin from egg white was purified according to the method described by Saleh et al. and Trziszka et al. [16,17].　 The homogenate of egg white was diluted with an equal volume of 0.25% NaCl, and was brought to pH 3.0 with 3 M HCl and left for 1 h at 4°C.　 Then, the homogenate was brought to pH 6.0 with 3 M NaOH and left overnight.　 The precipitated ovomucin was removed by centrifugation at 14,000 rpm for 1 h.　 The supernatant was subjected to affinity chromatography papain-Sepharose 4B column (7.5 ´ 5 cm; Sigma, Germany) equilibrated with 10 mM Tris-HCl buffer (pH 7.5).　 The proteins bound to papain-Sepharose 4B were eluted　 with 10 mM NaOH (pH 11.0).　　 The　 fractions　 were　 pooled,

Table 1: Clinical data of the 47 patients with tongue cancer

adjusted to pH 7.5 with 3 M HCl, and concentrated.　 The concentrated solution of cysteine protease inhibitor was dialyzed against 10 mM Tris-HCl buffer (pH 7.5) containing 0.1 M sodium chloride, and was applied to Sephadex G-100 column (128 ´ 2.5 cm; Pharmacia Fine Chemicals, Sweden) equilibrated with the same buffer.　 The fractions were separated as two peaks corresponding to molecular weight values of proteins in the range of 13-41 kDa.　 The collected fractions of 13 kDa protein fractions were dialyzed against 10 mM Tris-HCl buffer (pH 8.0) and subsequently applied to DEAE-Sephacel column (Pharmacia) equilibrated with the same buffer.　 Elution of the column with linear gradient of sodium chloride (0.05 -1.0 M) in the same buffer resulted in a single symmetrical peak of protein.　 The solution obtained from the peak fractions was concentrated for additional purification.　 Using reversed phase HPLC on a Nucleosil-100 C₁₈ column (Knauer, Germany) and Waters HPLC system according to Butzow et al. [18], concentrated solution of 200 mg of protein was dissolved in 0.1% trifluroacetic acid and injected onto C₁₈ (8 ´ 100 mm) HPLC column.　 A linear gradient of acetonitrile (0-60%) containing 0.1% trifluroacetic acid was used to elute protein.　 The purity of the protein was checked by SDS-PAGE.　 Samples of the preparations were stored in lyophilized form at -20°C.　 Protein concentrations were determined according to Bradford method [19] using bovine serum albumin as a standard.

Determination of cathepsin B activity

Cathepsin B activity was measured according to the method described by Barrett et al. [20].　 Fluorescence was measured by the luminescence spectrometer (Perkin Elmer LS 50B) at 370 nm excitation and 440 nm emission wavelengths using fluorescent substrate Z-Arg-Arg-AMC (Sigma).　 Fluorescence readings of the samples were standardized with the reaction product 7-AMC (7-amino-4-methylcoumarin).　 The activity unit 1 mEU was defined as the quantity releasing 1 nM of 7-AMC.

Pro-cathepsin B assay

To determine the pro-cathepsin B level, an activation step was carried out according to procedure described elsewhere [21].　 Samples (50 ml) were incubated at 37°C with pepsin (150 ml, 0.7 mg/ml) in 0.1 M acetate buffer (pH 3.0) for 60 min.　 Thereafter the generated active enzyme was assayed as described above.　 Total cathepsin B levels were calculated from the sum of cathepsin B and pro-cathepsin B levels.

Inhibitory activity against papain and cathepsin B

This inhibitory activity was referred to as total cysteine protease inhibitors.　 The method was described by Heidtman et al. [22].　 One inhibitory unit against papain or cathepsins B and L represents the amount of the inhibitor that totally inhibits one activity unit of papain or cathepsin B in the assay.　 This amount is determined by extrapolation of the titration curve to zero papain and cathepsin B activity.

Immunohistochemical staining

Immunohistochemistry was performed as previously described [23] to determine the expression status of cystatin C and cathepsin B proteins.　 Briefly, 5-mm sections were cut and mounted on adherent glass slides.　 Sections were deparaffinized in xylene and rehydrated in graded ethanol.　 Preliminary experiments on control tissues showed that no antigen enhancing methods were needed on similarly processed oral tissues.　 Endogenous peroxidase activity was blocked by immersion in 0.3% aqueous peroxide for 15 min followed by two washes in PBS for 5 min each.　 Endogenous proteins were blocked by incubating in a 2 % solution of bovine serum albumin in PBS for 20 min.　 The sections were then incubated for 1 h at room temperature with primary

antibodies against cystatin C and cathepsin B (Acris Antibodies GmbH, Germany) diluted 1:100 in PBS.　 This was followed by two washes in PBS and then incubation 30 min with peroxidase-conjugated rabbit anti-mouse IgG secondary antibody diluted 1: 50 (DAKO, CA, USA).　 The bound complexes were visualized by adding with 0.03% 3,3-diaminobenzidine tetrahydrochloride containing 0.005% H₂O₂ for 8 min.　 Following incubation, the sections were washed and then lightly counterstained with hematoxylin and coverslipped.　 In negative controls the primary antibody was omitted.

Statistical analysis

Values are expressed as mean ± SD or 25-75 percentile.　 The significance of the mean differences between groups was assessed by the Student’s two-tailed unpaired t test.　 Walloon’s rank test was used to calculate the relation between the level of cathepsin B and its inhibitors during the progression of cancer in comparison with controls.　 The coefficient of correlation was determined by linear regression analysis.　 Differences were considered significant at P < 0.05.　 For each parameter, we also calculated the sensitivity, specificity, productivity (of a positive test) and diagnostic accuracy.

Results

Cathepsin B, pro-cathepsin B and cysteine protease inhibitors in the sera of tongue cancer patients

Table 2 shows the activities of pro-cathepsin and cathepsin B and their cysteine protease inhibitor activities in the sera from patients with tongue carcinoma as well as in healthy individuals.　 The median activities of pro-cathepsin B, free cathepsin B and total cathepsin B were much higher in the sera from patients with tongue carcinoma in comparison with those in the control group (P < 0.001), while the activity of cysteine protease inhibitors was much lower in the patients’ sera compared to the controls.　 Total cathepsin B activity with a median value of 31.5 mEU/ml in the sera of patients with tongue cancer was higher in comparison with 7.4 mEU/ml in controls (P < 0.001).　 Free cathepsin B activity with a median value 18.5 mEU/ml in the sera of patients with tongue cancer was also higher as compared to 3.8 mEU/ml in controls.　 Likewise, for pro-cathepsin B activity a median value was 13.0 mEU/ml in patients’ sera compared to that in the control group (3.6 mEU/ml).　 Tumor cathepsin B activities were elevated 4.3-fold compared to the control.　 Protein median value was 20.2 mg in the sera of patients with tongue cancer while 4.3 mg was measured in controls.　 Table 2

Table 2: Serum activities of cathepsin B, pro-cathepsin B and cysteine protease inhibitors in the sera of patients with tongue cancer

Table 3: Serum activities of cathepsin B, procathepsin B, and cysteine protease inhibitors in different stages of patients with tongue cancer

shows that inhibitory activity of cysteine protease inhibitors was significantly decreased from 2.3 mEU/ml in control to 1.3 mEU/ml in the sera of patients with tongue cancer (P < 0.001).　 The median activity of inhibitors was decreased by ~43%.　 The data suggests that cysteine protease inhibitors were down regulated in the sera of tongue cancer patients as compared to control sera.

Relationships of the serum activities of cathepsin B, pro-cathepsin B, and cysteine protease inhibitors with tumor staging

Table 3 presents the relationships of the serum enzyme and inhibitor activities with tumor stages in tongue cancer patients.　 Both cathepsin B and pro-cathepsin B in the sera of patients were increased with the pathological stage (P < 0.001).　 The activity of their inhibitors also increased with an increase in the stage of disease (P < 0.001).　 Activities of cathepsin B and its pro-cathepsin B in stage I patients were 3.8 ± 1.0 and 3.4 ± 1.0 mEU/ml respectively, while the inhibitor activity was 0.06 ± 0.00 mEU/ml.　 The activities of cathepsin B and its pro-cathepsin B in stage II patients were 5.6 ± 2.5 and 5.6 ± 1.1 mEU/ml respectively, while the inhibitor activity was 0.09 ± 0.02 mEU/ml.　 In the serum from patients with tongue carcinoma in stage III, cathepsin B and its pro-cathepsin B activities were 15.5 ± 5.1 and 10.3 ± 4.1 mEU/ml respectively, while the inhibitor activity in the same stage was 0.50 ± 0.10 mEU/ml.　 The activity of cysteine protease inhibitors increased from 0.06 ± 0.00 in stage I to 0.50 ± 0.10 mEU/ml in stage III.

In vitro inhibition of the activity of cathepsin B by chicken cystatin

The serum cathepsin B activity decreased from 15.8 ± 5.4 mEU/ml to 6.7 ± 0.8 mEU/ml after inhibition with egg white cystatin.　 As shown in Figure 1, highly significant differences were observed between the activities of serum cathepsin B of tongue carcinoma patient before and after treatment with egg white cystatin (P < 0.001).　 The activity of serum cathepsin B decreased about 2.4-fold after inhibition by chicken cystatin.

Elevated expression of cystatin C and cathepsin B in tumor tissues correlated with tongue cancer staging

Diffuse and granular cytoplasmic labelling of cystatin C and cathepsin B was seen in tongue tumor tissues.　 The result from Figure 2A shows that the tumor cells expressed an elevated cystatin C level, while the non-cancerous tissue showed no staining with anti-chicken cystatin C antibody (Figure 2B).　 The immunohistochemical staining showed a granular pattern, indicating a lysosomal　 localization of cath-

Figure 1: Serum cathepsin B activities of 47 tongue cancer patients before and after inhibition with 5 nM of purified chicken egg white cystatin.　 BF, before inhibition; AF, after inhibition.

epsin inhibitors.　 Positive immunohistochemical staining of cystatin C was significantly increased in tongue cancer tissues when compared to the non-cancerous tissues (P < 0.001, Table 4).　 Moreover, elevation of cystatin C expression was correlated with tumor staging.　 The elevated expression of cystatin C was observed in 3 out of 5 tumor cases in stage I (60%), 27 out of 30 tumors in stage II (90 %), and 12 out of 12 tumors in stage III (100 %).　 In addition, a differential expression of cathepsin B was also observed to correlate with tongue cancer staging.　 The positive staining was seen in two out of five tumors in stage I (40%), 22 out of 30 tumors in stage II (73%), and 10 out of 12 tumors in stage III (83%).　 In all positive cases, no difference was seen between the central and the periphery of the tumors.　 The immunohistochemical data are summarised in Table 4.

Discussion

The results of this study showed that patients with tongue cancer had higher expression of cathepsin B than control.　 One of the most important factors in the prognosis of tongue cancer patients is the invasion level of cancer cells in the extracellular matrix of oral mucosa.　 The steps of tumor cell invasion involve attachment of tumor cells to the underlying basement membrane, local proteolysis, and migration of tumor cells through the proteolytically modified region.　 Local proteolysis can be achieved by proteases outside the tumor cells, perhaps bound to the cell surface and/or secreted　 from　 the　 tumor　 cells [24].　　 The recent data suggest that

A B
Figure 2: Representative data of the immunohistochemical staining of cystatin C in the tongue tumor tissue (A) but not in control normal tissue (B) using anti–chicken cystatin antibody.　 Positive staining was observed only with tumor cells, and was diffusely localized in the cytoplasm.

Table 4: Expression status of cathepsin B and cystatin in the patients with different stages of tongue cancer
	^aExpression status is presented by immunohistochemical grading: -, background; +, low expression; ++, intermediate expression; +++, high expression.

proteases from phagocytes also participate in local proteolysis by digesting extracellular matrix [25-27].　 Therefore, proteases are believed to be required for cancer cell metastasis [28].　 Evidence is now emerging indicating that proteases are involved in tumor growth at both primary and metastatic sites [29,30].　 The activities of cathepsin B and pro-cathepsin B were found to be significantly increased in the sera of tongue cancer patients, in accompanying with decreasing endogenous inhibitor activity, if compared with those in control sera (P < 0.001).　 Several studies have shown that there was an inverse correlation between cathepsin B expression and basement membrane staining in bladder [31], gastric [32], lung [33], and colon carcinoma [34].　 The association between pro-cathepsin B and cathepsin B activities as well as the relationship of their inhibitor activity and histopathological grading of the cancer were observed and suggested that cathepsin B was involved in the invasion of tumor cells.　 It is probable that abnormal cathepsin B expression, activation, or/and secretion occurred with the development of malignant stages of the disease [35].　 Although a sufficiently acidic condition (pH < 5.5) is required to activate cathepsin D, cathepsin B can work at neutral pH, which may suggest that cathepsin B mainly degrades the basement membrane at the local sites.　 Wickramasinghe et al. [36] suggested a direct role for cathepsin B in promoting oral cancer spread and invasion, and indicated the possibility of controlling oral carcinoma malignancy and metastasis by targeting cathepsin B with RNA inhibitor strategies.

We showed that cathepsin B activity was decreased significantly in the homogenates of tongue cancer tissues after addition with 5 nM cystatin isolated from chicken egg white in comparison with control groups.　 The level of cathepsin B activity was decreased by 2.4-fold in tumor samples after inhibition with chicken cystatin.　 Our study demonstrated that cathepsin B might have a role in cancer invasion, and our results also provided evidence that the cystatin isolated from egg could stop or retain back the level of cathepsin B to normal values.　 The similar results were obtained with gastric cancer [37] and colorectal cancer [38].　 The result from Figure 2 shows that high levels of cystatin C distributed diffusely in the cytoplasm of tongue tumor cells but not in non-cancerous tissue cells, suggesting that a significant interaction between cystatin C and cathepsin B might exist in tongue tumor cells.　 In addition, we have also noted that the serum levels of cathepsin B and cystatin C activities were both increased in tongue cancer patients (Tables 2 and 3).　 It is possible that cystatin C was released in a complex form with cathepsin B via a translocation pathway of lysosomal vesicles from the perinuclear region to the plasma membrane, possible fusion and subsequent release at the cell surface [39].　 However, the level of cystatin C was not comparable to that of cathepsin B.　 The resultant uncontrolled proteolysis can be the outcome of an imbalance between catalytically active proteases and their natural inhibitors.　 This can be observed in inflammation and tumor growth, although these processes are very complicate.　 The knowledge about the balance between endogenous cysteine protease inhibitors and different papain-type cysteine proteases is needed for better understanding of the regulation of cysteine-dependent proteolysis in tumor progression.　

In conclusion, we observed that the levels of cathepsin B and cystatin C were both elevated in the sera and tumor tissues and correlated with tumor staging in tongue cancer patients.　 The exogenous addition of chicken egg cystatin to the patients’ sera could efficiently reduce the activities of cathepsin B to those like normal control.　 An imbalance between the levels of proteases and their natural inhibitors was suggested to partly account for the malignant progression of tongue cancer.

Acknowledgment

This study was supported by the Project Number 3 T098B 13628 2005 from the State Committee for Scientific Research (KBN) and granted from the Foundation for Polish Science (Techno/Techne project).

　

References

1. Fujise N, Nanashima A, Taniguchi Y, Matsuo S, Hatano K, Matsumoto Y, Tagawas Y, Ayabe H. Prognostic impact of cathepsin B and matrix metalloproteinase-9 in pulmonary adenocarcinoma by immunohistochemical study. Lung Cancer 27: 19-26, 2000.

2. Tumminello FM, Leto G, Pizzolanti G, Candiloro V, Crescimanno M, Crosta L, Flandina C, Montalto G, Soresi M, Carroccio A, Bascone F, Ruggeri L, Ippolito S, Gebbia N. Cathepsin D, B and L circulating levels as prognostic markers of malignant progression. Anticancer Res 16: 2315-20, 1996.

3. Schmitt M, Jänicke F, Graeff H. Protease matrix degradation and tumor cell spread. Fibrinolysis 6: 1-17, 1992.

4. Sloane BF, Moin K, Lah TT. Lysosomal enzymes and their endogenous inhibitors in neoplasia. In: Pretlow TG, Pretlow TP, ed. Biochemical and molecular aspects of selected cancers. New York: Academic Press 1994: 411-466.

5. Kos J, Lah TT. Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer. Oncol Rep 5: 1349-1361, 1998.

6. Duffy MJ. Proteases as prognostic markers in cancer. Clin Cancer Res 2: 613-618, 1996.

7. Kandalaft P, Chang KL, Ahn CW, Traweek ST, Mehta P, Battifora H. Prognostic significance of immunohistochemical analysis of cathepsin D in low-stage breast cancer. Cancer 71: 2756-63, 1993

8. Hirai K, Yokoyama M, Asano G, Tanaka S. Expression of cathepsin B and cystatin C in human colorectal cancer. Hum Pathol 30: 680-686, 1999.

9. Kawasaki G, Kato Y, Mizuno A. Cathepsin expression in oral squamous cell carcinoma: relationship with clinicopathologic factors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 93: 446-454, 2002.

10. Macabeo-Ong M, Shiboski CH, Silverman S, Ginzinger DG, Dekker N, Wong DTW, Jordan RCK. Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression. Oral Oncology 39: 638-647, 2003.

11. Turk V, Bode W. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett 285: 213-219, 1991.　

12. Turk B, Turk V, Turk D. Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol Chem 378: 141-150, 1997.

13. Henskens YMC, Veerman ECJ, Amerongen AVN. Cystatins in health and disease. Biol Chem 377: 71-86, 1996.　　　　　　　

14. Troy AM, Sheahan K, Mulcahy HE, Duffy MJ, Hyland JMP, O’Donoghue DP: Expression of cathepsin B and L antigen and activity is associated with early colorectal cancer progression. Eur J Cancer 40: 1610-1616, 2004.

15. Sokal JP, Schlemann WP. Cystatin C antagonises transforming growth factor　 b signaling in normal and cancer cells. Mol Cancer Res 2: 183-189, 2004.

16. Saleh Y, Siewinski M, Sebzda T, Jelen M, Ziolkowski P, Gutowicz J, Grybos M, Pawelec M. Inhibition of cathepsin B activity in human breast cancer tissue by cysteine peptidase inhibitor isolated from human placenta: Immunohistochemical and biochemical studies. Folia Histochem Cytobiol 41: 161-167, 2003.

17. Trziszka T, Saleh Y, Kopec W, Siewinski M, Wesierska E. Effect of Hen’s Age on the Level of Cystatin in the Chicken Egg White. Intl J Poultry Sci 3: 471-477, 2004.

18. Butzow R, Huhtala ML, Bohn H, Virtanen I, Seppala M. Purification and charactrization of placenta protein 5. Biochem Biophys Res Commun 150: 483-490, 1987.

19. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.

20. Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, cathepsin L. Methods Enzymol 80: 535-561, 1981.

21. Pagano M, Capony F, Rochefort H. Pro-cathepsin D can activate in vitro pro-cathepsin B secreted by ovarian cancers. CR Acad Sci III 309: 7-12, 1989.

22. Heidtman HH, Salge U, Abrahamson M, Bencina M, Kasteliv L, Kopitar N, Turk V, Lah TT. Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines. Clin Exp Metastas 15: 368-381, 1997.

23. Bar JK, Harozinska A, Sobanska E, Cislo M. Relation between carcinoma-associated antigens in tumor tissue and detached cyst fluid cell of patients with ovarian neoplasms. Tumori 80: 50-55, 1994.

24. Turk B, Turk D, Turk V. Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 147: 98-111, 2000.

25. Strojan P, Budihna M, Smid L, Vrhovec I, Skrk J. Cathepsin D in tissue and serum of patients with squamous cell carcinoma of the head and neck. Cancer Lett 130: 49-56,1988.

26. Sánchez-Martín MM, Cabezas JA, Ortega S, García J, García-Criando FJ, Pina J, Go'mez-Alonso A. Levels of serum cathepsin L and several glycosidases in patients operated for colorectal cancer. Cancer Lett 141: 73-77, 1999.

27. Thomas GT, Lewis MP, Speight PM. Matrix metalloproteinases and oral cancer. Oral Oncol 35: 227-233, 1999.

28. Hong SD, Hong SP, Lee JI, Lim CY. Expression of matrix metalloproteinase-2 and -9 in oral squamous cell carcinomas with regard to the metastatic potential. Oral Oncol 36: 207-213, 2000.

29. Koblinski JE, Abram M, Sloane BF. Unraveling the role of proteases in cancer. Clin Chim Acta 291: 113-135, 2000.

30. Koop S, Khokha R, Schmidt EE, MacDonald IC, Morris VL, Chambers AF, Groom AC. Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasations but reduces tumor growth. Cancer Res 54: 4791-4797, 1994.

31. Visscher DW, Sloane BF, Sameni M, Babiarz JW, Jacobson J, Crissman JD. Clinicopathologic significance of cathepsin B immunostaining in transitional neoplasia. Mod Pathol 7: 76-81, 1994.

32. Khan A, Krishna M, Baker SP, Malhothra R, Banner BF. Cathepsin B expression and its correlation with tumor-associated laminin and tumor progression in gastric cancer. Arch Pathol Lab Med 122: 172-177, 1998.

33. Sukoh N, Abe S, Ogura S, Isobe H, Takekawa H, Inoue K, Kawakami Y. Immunohistochemical study of Cathepsin B. Prognostic significance in human lung cancer. Cancer 74: 46-51, 1994.

34. Jessep JM. Cathepsin B and other proteases in human colorectal carcinoma. Am J Pathol 145: 253-262, 1994.

35. Sheahan K, Shuja S, Murnane MJ. Cysteine protease activities and tumor development in human colorectal carcinoma. Cancer Res 49: 3809–3814, 1989.

36. Wickramasinghe NS, Nagaraj NS, Vigneswaran N, Zacharias W. Cathepsin B promotes both motility and invasiveness of oral carcinoma cells. Arch Biochem Biophys 436: 187-195, 2005.

37. Saleh Y, Sebzda T, Warwas M, Kopec W, Ziolkowska J, Siewinski M. Expression of cystatin C in clinical human colorectal cancer tissues. J Exp Ther Oncol 5: 49-53, 2005.

38. Saleh Y, Siewinski M, Kielan W, Ziolkowski P, Grybos M. Regulation of Cathepsin B and L expression in vitro in gastric cancer tissues by egg cystatin. J Exp Therp Oncol 3: 319-324, 2003.　　　

39. Hanewinkel H, Glossl J, Kresse H. Biosynthesis of Cathepsin B in cultured normal and I-cell fibroblasts.　 J Biol Chem 262: 12351-12355, 1987.