J Cancer Res Clin Oncol (1991) 117:249-253 017152169100046I

C~ncer Research Clinical 9 9 Springer-Verlag1991

Dipeptidyl peptidase IV, prolyl endopeptidase and cathepsin B activities in primary human lung tumours and lung parenchyma A. ~edo l, E. K~epela ~, and E. Kasafirek 2 1 Departmentof Biochemistry,Research Institute of Tuberculosis and RespiratoryDiseases, and 2 Laboratoryfor Peptide Synthesis,Research Institute of Pharmacyand Biochemistry,Prague, Czechoslovakia Received 2 November 1990/Accepted7 January 1991

Summary. The activities of dipeptidyl peptidase IV (DPP-IV), prolyl endopeptidase (PE) and cathepsin B (CB) were investigated in primary human lung tumours and matched lung parenchyma, using continuous-rate fluorometric assays of the enzymes. Squamous-cell lung carcinomas showed significantly higher specific activities of all three enzymes studied. In lung adenocarcinomas, only activities of PE and CB were increased significantly. In a limited number of primary human lung tumours of other histological types the activities of DPP-IV, PE and CB were also elevated. Mixing the matched homogenates of lung tumours and lung parenchyma gave additive activities for each enzyme studied. A significant correlation between tumour/lung ratios of specific activities of DPPIV and CB was observed. Key words: Dipeptidyl peptidase IV - Prolyl endopeptidase - Cathepsin B - Primary human lung tumour

Introduction Proteolytic enzymes are hypothesized to play a significant role in the pathogenesis oftumour development and progression (Gottesman 1990). In human lung cancer there is some information on proteinases associated with tumour tissue. Immunohistochemical studies demonstrated the occurrence of cathepsin D (Reid et al. 1986), renin (Taylor et al. 1988), and several blood coagulation factors (Zacharski et al. 1987) in various histological

types of lung cancer. Biochemical studies revealed elevation of activities of plasminogen activator (Markus et al. 1980), collagenases cleaving type I and IV collagens (Kubochi et al. 1986), and cathepsin B (K~epela et al. 1990) in lung tumour tissue as compared with the lung parenchyma. On the cellular level, there has been reported in vitro secretion of urokinase-type plasminogen activators (Keski-Oja et al. 1988; Trefz et al. 1990), type IV collagenase (Collier et al. 1988; Ura et at. 1989; Momiki et al. 1990) and latent cathepsin B (Trefz et al. 1990) from oncogene-transformed human bronchial epithelial cells and several cell lines derived from human non-small-cell lung carcinomas. Secretion of type IV collagenase has been shown to correlate with invasive and metastatic potential of some lung tumour cell lines (Ura et al. 1989; Momiki et al. 1990). Moreover, evidence of plasma-membrane association of some proteinases including collagen- and gelatin-degrading activities (Zucker et al. 1987) and cathepsin B (Erdel et al. 1990) in human lung cancer cell lines was reported. In this communication we have investigated three proteolytic enzymes, two proline-specific peptidases (Walter et al. 1980), dipeptidyl peptidase IV (DPP-IV; EC 3.4.14.5) and prolyl endopeptidase (PE; EC 3.4.21.26), and the cysteine proteinase cathepsin B (CB; EC 3.4.22.1) in matched pairs of primary human lung tumour and lung parenchyma. Both DPP-IV (K~epela et al. 1985) und PE (Zolfaghari et al. 1986) have been identified in normal lung, the former being membrane-bound and the latter soluble. The lysosomal proteinase CB, previously demonstrated immunologically in normal human lung (Burnett et al. 1983; Howie et al. 1985) and recently reported as occurring in human lung tumours at an activity higher than in lung parenchyma (K~epela et al. 1990) was also examined in this study in order to evaluate any correlation with DPP-IV and PE.

Abbreviations: DPP-IV, dipeptidyl peptidase IV; PE, prolyl endopeptidase; CB, cathepsin B; Ac-Leu-Arg-Arg-NHMec,7-(N-acetylL-leucyl-L-arginyl-L-arginylamido)-4-methylcoumarin; Gly-ProNHMec, 7-(glycyl-L-prolylamido)-4-methylcoumarin;Suc-GlyPro-NHMec, 7-(N-succinylglycyl-L-prolylamido)-4-methylcoumarin; NH2Mec, 7-amino-4-methylcoumarin;r, Pearson's correlation coefficient Materials and methods Offprint requests to: A. ~edo, Department of Biochemistry, Research Institute of Tuberculosis and Respiratory Diseases, BuReagents. Ac-Leu-Arg-Arg-NHMec(K~epelaet al. 1990) and Glydinova 67, CS-18071 Prague 8, Czechoslovakia Pro-NHMec (Kato et al. 1978) were synthesizedas described pre-

250 viously. Suc-Gly-Pro-NHMec was obtained from Bachem, Bubendoff, Switzerland. DNA, type III from salmon sperm, and NH2Mec were supplied by Sigma, Poole, Dorset, UK. Bisbenzimide H 33258 (Hoechst 33258) was purchased from Serva, Heidelberg, FRG. Other common reagents were of analytical grade and were obtained from various commercial sources. Highly purified water with specific resistance greater than 16 MF2/cm was used for preparation of solutions. Patients. The patients included in this study were those with newly recognized lung tumour who were subjected to lung surgery (lobectomy, bilobectomy or pneumonectomy). None of the patients had received radiotherapy or chemotherapy before surgery and most of patients were smokers. The age of patients ranged from 36 to 70 years. No evidence for distant extrathoracic metastases was found in the patients at the time of entry to the study. The cell type of lung tumours was classified according to the rules of WHO (World Health Organization 1981). The tumuor disease stage was classified according to the international staging system (Mountain 1987).

9 51.4 030.8 9 015.1

12 2

9

Determination of DNA. DNA was determined in tissue homogenates, treated with heparin at a final concentration of 40 gg/ml (Downs and Wilfinger 1983), using the fluorescence binding assay with bisbenzimide H 33258 (Brunk et al. 1979). DNA assays were carried out with duplicate homogenate aliquots. Highly polymerized DNA from salmon sperm was used as an internal standard. Calculations. DNA concentrations in tissue homogenates were calculated according to a computer program described previously (gedo and Adamec 1990), statistical calculations were done with the EPISTAT program package.

Results

D e t e r m i n a t i o n o f specific activities o f D P P - I V , P E a n d CB in h o m o g e n a t e s o f p r i m a r y h u m a n lung t u m o u r s o f six different h i s t o l o g i c a l types (Fig. 1, T a b l e s 1-3) a n d m a t c h e d lung p a r e n c h y m a r e v e a l e d g r e a t i n t e r i n d i v i d u a l v a r i a t i o n s . I n l u n g t u m o u r s , the r a n g e s o f D P P - I V , P E

0

10-

OB-

9

9

9

A

0 0

9

6-

O

0

Tissues. Specimens of tumour tissue and matched lung parenchyma were obtained and processed as described previously (K~epala et al. 1990). Enzyme assays. Activities of DPP-IV, PE and CB in tissue homogenates were measured by fluorometric continuous-rate assays incubated at 37~ C. The total volume of the reaction mixture was 2 ml in each case and the stirred enzyme reactions were recorded for the initial 2~4 rain. The activity of DPP-IV was assayed with 0.2 mM Gly-Pro-NHMec as the substrate in 75 mM TRIS/HC1 buffer, pH 8.0 (Sedo et al. 1989). The activity of PE was measured with 0.2 mM Suc-Gly-Pro-NHMec as the substrate (Kato et al. 1980) in 100mM TRIS/HC1 buffer, pH 7.4, containing 0.25mM Na 2 ETDA and 0.25 mM dithiothreitol. The activity of CB was determined with 0. l mM Ac-Leu-Arg-Arg-NHMec as the substrate in 100 mM NaH2PO4/Na2HPO4 buffer, pH 6.0, containing 1 mM Na2EDTA and I mM dithiothreitol (K~epela et al. 1990). Enzyme assays were carried out with duplicate homogenate aliquots. It has been proved in preliminary experiments that the activity of the enzymes followed a linear relationship with respect to the reaction time and amount of the homogenate in the reaction. The enzymically liberated NHzMec was monitored at excitation and emission wavelengths of 380 nm and 460 nm, respectively, using a Shimadzu spectrofluorophotometer RF-540. The fluorometer was calibrated with NHzMec in the respective reaction buffer. The activity of the enzymes was calculated from the slope of progress curves and was expressed as nkat/mg DNA, i.e. as nmol liberated NH2Mec s mg tissue D N A - ~.

0

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oo 9 ~176 e e oOO 9 ooO I

D

9 A

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,I

Fig. 1. Ratio of specific activities of dipeptidyl peptidase IV (DPP-

IV), prolyl endopeptidase (PE) and cathepsin B (CB) in primary human lung tumours and matched lung parenchyma. , the ratio 1.5 used as a cut-off line for assessment of enzyme activity levels. Squamous cell carcinoma, o; adenocarcinoma, e; small-cell carcinoma, zx; bronchioloalveolar carcinoma, []; sarcoma, A; carcinoid, 9

a n d CB activities were 0.11-8.73, 0.06~1.42 a n d 8 . 8 124.9 n k a t / m g D N A , respectively. In l u n g p a r e n c h y m a , the r a n g e s o f D P P - I V , P E a n d CB activities were 0 . 0 9 1.99, 0.04 0.63 a n d 3 . 1 - 6 5 . 5 n k a t / m g D N A , respectively. T h e i n t r a a s s a y v a r i a t i o n coefficients o f D P P - I V , PE, CB a n d D N A m e a s u r e m e n t s in lung t u r n o u t a n d lung tissue h o m o g e n a t e s d i d n o t exceed 5 % . T h e v a r i a t i o n o f specific activities o f D P P - I V , P E a n d CB a m o n g several specimens o f the s a m e lung t u m o u r a n d m a t c h e d l u n g p a r e n c h y m a was n o t higher t h a n 2 0 % . Thus, the specific activity o f D P P - I V , P E a n d CB was c o n s i d e r e d to be h i g h e r in lung t u m o u r (T) t h a n in m a t c h e d lung p a r e n c h y m a (L) if the r a t i o T / L w a s . e q u a l to o r higher t h a n the value 1.5. T h e r a t i o s T / L for D P P - I V , P E a n d CB specific activities are i n d i c a t e d in Fig. 1. I n c r e a s e d activities o f D P P - I V , P E a n d CB in lung t u m o u r s over t h o s e in m a t c h e d lung p a r e n c h y m a were o b s e r v e d in 64.3%, 68.3% a n d 67.4% respectively, o f all cases e x a m i n e d . I n lung t u m o u r there was an increase o f activity o f all three, two, one a n d n o n e o f the s t u d i e d e n z y m e s in 33.3%, 4 1 . 7 % , 16.7% a n d 8.3% o f p a t i e n t s ( n = 36), respectively, where all the enz y m e s were a s s a y e d in parallel. In m i x i n g e x p e r i m e n t s using m a t c h e d lung t u m o u r a n d lung p a r e n c h y m a h o m o g enates m i x e d in e q u a l p r o p o r t i o n s we h a v e f o u n d a d d i tive activities o f D P P - I V , P E a n d CB.

251 Table 1. Dipeptidyl peptidase IV activity in primary human lung tumours and matched lung parenchyma Tumour histology

Disease stage

Number Dipeptidylpeptidase IV activity" of (nkat/mg DNA) patients Tumour (Y) Lung (L)

Statistical comparison (*'): T vs L b

Squamous-cell carcinoma

I II III Total

13 9 5 27

1.39_+1.06 0.52_+0.41 0.88_+0.81 1.01 -+0.91

0,64_+0.65 0.35_+0.10 0.30_+0.12 0.48_+ 0.47

0.031 0.225 0.191 0.005

Adenocarcinoma

I II

5 3

III Total

1 9

1.29_+0.95 1.79 2.11 8.73 1.42 1.99_+2.63

0.26_+ 0.21 0.22 0.29 0.17 0.55 0.28_+0.18

0.150 0.089

Small-cell carcinoma

I

1

0.37

0.09

-

Bronchioloalveolar carcinoma Sarcoma

I

1

0.61

0.54

-

II III

1 2

0.66 2.28 1.06

1.60 0.14 0.29

-

Carcinoid

-

1

1.48

0.13

-

" Grouped data indicated as mean + SD b Statistical comparison of tumour versus lung enzyme activity was performed using the two-tailed paired t-test Table 2. Prolyl endopeptidase activity in primary human lung tumours and matched lung parenchyma Tumour histology

Disease stage

Number Prolyl endopeptidase activity" of (nkat/mg DNA) patients Ynmour (T) Lung (L)

Statistical comparison (P): T vs L b

Squamous-cell carcinoma

I II III Total

12 9 5 26

0.50_+0.42 0.30_+ 0.22 0.32+_0.17 0.40_+0.33

0.27_+0.20 0.18_+ 0.09 0.13_+0.04 0.21 ___0.16

0.058 0.141 0.028 0.003

Adenocarcinoma

I II

5 3

III Total

1 9

0.17_+0.03 0.28 0.12 0.37 0.13 0.19_+0.08

0.11 _+0.08 0.13 0.06 0.10 0.18 0.11_+0.06

0.161 0.004

Small-cell carcinoma

I

1

0.63

0.09

-

Bronchioloalveolar carcinoma Sarcoma

I

1

0.55

0.18

-

II III

1 2

0.55 0.24 0.27

0.07 0.04 0.07

-

-

1

0.54

0.13

-

Carcinoid

a Grouped data indicated as mean • SD b Statistical comparison of tumour versus lung enzyme activity was performed using the two-tailed paired t-test

T a k i n g i n t o a c c o u n t the disease stage, s q u a m o u s - c e l l l u n g c a r c i n o m a s o f disease stage I h a d significantly increased m e a n activities o f D P P - I V a n d CB as c o m p a r e d to m e a n e n z y m e activities in m a t c h e d l u n g p a r e n c h y m a (Tables 1 a n d 3). I n the case o f PE, the m e a n activity o f the enzyme was significantly higher in s q u a m o u s - c e l l carc i n o m a s o f disease stage III (Table 2). D i s r e g a r d i n g the disease stage, s q u a m o u s - c e l l c a r c i n o m a s showed signifi-

c a n t l y increased m e a n activities o f D P P - I V , P E a n d CB (Tables 1-3). I n l u n g a d e n o c a r c i n o m a s , o n l y P E a n d CB displayed significantly increased m e a n activity (Tables 2 a n d 3). There was n o significant difference o f m e a n activity o f each e n z y m e studied b e t w e e n s q u a m o u s - c e l l l u n g c a r c i n o m a a n d l u n g a d e n o c a r c i n o m a (Tables 1-3). Activities o f D P P - I V , PE a n d CB were increased also in a limited n u m b e r o f other p r i m a r y h u m a n l u n g t u m o u r s ,

252 Table 3. Cathepsin B activity in primary human lung tumours and matched lung parenchyma

Tumour histology

Disease Number Cathepsin B activitya stage of (nkat/mg DNA) patients Tumour (T) Lung (L)

Statistical comparison (P): T vs L b

Squamous-cell carcinoma

I II II[ Total I II III Total I I

0.021 0.244 0.297 0.004 0.058 0.039

Adenocarcinoma

Small-cell carcinoma Bronchioloalveolar carcinoma Sarcoma

Carcinoid

16 9 6 31 4 4 1 9 1 1

II III

1 2

-

1

34.1 +-31.1 26.0 • 20.1 40.2+_23.2 32.9 • 26.7 29.8+_12.5 57.4+32.5 90.2 48.8___29.8 11.0 32.6

14.9+_ 14.7 16.0 +_11.5 25.8+_18.9 17.3 • 14.9 15.4+ 5.3 17.6+-10.9 26.7 14.6+- 8.2 5.4 6.4

16.4 63.0 20.9 10.9

38.3 5.6 5.3 8.1

0.004

a Grouped data indicated as mean +-SD b Statistical comparison of tumour versus lung enzyme activity was performed using the two-tailed paired t-test

including small-cell carcinoma, bronchioloalveolar carcinoma, sarcoma and carcinoid (Tables 1-3). Patients (n = 36) whose matched pairs of lung tumour and lung parenchyma were examined for all three enzymes were included in the correlation analysis. There was significant intratissue correlation between DPP-IV and PE (P=0.041; r=0.34), DPP-IV and CB ( P = 2.2 x 10-4; r=0.58), PE and CB (P=0.006; r=0.453) in lung parenchyma. In lung tumours, however, only activities of DPP-IV and CB showed significant correlation (P = 0.019; r-- 0.39). Significant intertissue correlation ( P < 0 . 0 5 ) was found only for PE activity in matched pairs of lung tumours and lung parenchyma (P = 0.005; r--0.46). When evaluating the specific activity ratios of enzymes, T/L (see above), only ratios obtained for DPPIV and CB displayed significant correlation (P=0.007; r=0.44).

Discussion

In the present study we have demonstrated for the first time an increased activity of two proline-specific peptidases, DPP-IV and PE in a high number of primary human lung tumours as compared with matched lung parenchyma. The finding of significantly higher CB activity in primary lung tumours, including sarcomas, confirmed and extended previous studies (Trefz et al. 1989; K~epela et al. 1990). In contrast to the report ofTrefz et al. (1989) we have not found CB activitiy to be significantly different in human squamous-cell lung carcinomas and lung adenocarcinomas. The higher activities of DPP-IV, PE and CB in lung tumours of various histological types were probably not caused by a decreased concentration of active endogenous inhibitors because the activity of each enzyme stud-

ied was additive in mixed homogenates of matched lung tumour and lung parenchyma. To date there is no information about the occurrence of a potent endogenous inhibitor of DPP-IV in mammalian tissues and tumours. The contribution of endogenous inhibitors of PE (Yoshimoto et al. 1982) and of cysteine proteinases including CB (Kyll6nen et al. 1984; Chapman et al. 1990) to regulation of PE and CB in lung tumours and lung parenchyma may be of lesser importance or limited to specific cellular and tissue compartments. CB in lung tumour tissue may be derived from different cellular sources including cancer cells (Erdel et al. 1990; Trefz et al. 1990), macrophages (Burnett et al. 1983) and ciliated or glandular epithelia of the bronchus (Howie et al. 1985). The cellular origin of DPP-IV and PE in primary lung tumours is not known yet. The finding of significant correlation between tumour/lung ratios of both DPP-IV and CB activities suggests that the alterations affecting the expression or regulation of these active enzyme molecules may involve the same cell population(s) in primary lung tumours. The concordant increase of both DPP-IV and CB activities in human lung tumours contrasts with the situation in gastric carcinoma, where increased CB (Chung and Kawai 1990) and decreased DPP-IV (Yoshii et al. 1981) activities were reported. The lack of correlation of PE activitiy with DPPIV and CB activities in lung tumours may reflect not only differences in the expression and regulation of activie PE molecules but also a distinct cellular source of the enzyme. Further studies are needed to elucidate the cellular expression, regulation and the role of DPP-IV, PE and CB in lung cancer.

Acknowledgements. We thank the staff of the Department of Surgery and Dr. J. Viklick2~,head of the Department of Cytology and

253 Histology, Research Institute of Tuberculosis and Respiratory Diseases, Prague, Czechoslovakia, for excellent cooperation during this study and for histopathological determinations, respectively. The skilful technical assistance of Mrs. A. Velechovskfi and Mrs. J. Hronovfi is gratefully acknowledged.

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