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Adenosine deaminase, 5′nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human bladder tissues
Free Radical Biology & Medicine, Vol. 16, No. 6, pp. 825-831, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All fights reserved 0891-5849/94 $6.00 + .00
Pergamon
0891-5849(93)E0055-A
Brief Communication ADENOSINE DEAMINASE, 5'NUCLEOTIDASE, XANTHINE OXIDASE, SUPEROXIDE DISMUTASE, AND CATALASE ACTIVITIES IN CANCEROUS AND NONCANCEROUS HUMAN BLADDER TISSUES ILKER DURAK,* HAKKI PERK, t MUSTAFA KAVUT~U,* ORHAN CANBOLAT,* {~MER AKYOL,* and YA~AR BEDOK t
*Ankara University,Faculty of Medicine, Departmentof Biochemistry,Ankara, Turkey; and *AnkaraUniversity, Faculty of Medicine, Departmentof Urology, Ankara, Turkey (Received 24 June 1993; Revised 16 December 1993; Accepted 21 December 1993) Abstract--Activities of adenosine deaminase (ADA), 5'nucleotidase (5NT), xanthine oxidase (XO), superoxide dismutase (SOD), and catalase (CAT) enzymes were measured in cancerous and cancer-free adjacent bladder tissues from 36 patients with bladder cancer and in control bladder tissues from 9 noncancer patients. Increased ADA and decreased XO, SOD, and CAT activities were found in cancerous bladder tissues compared with those of cancer-free adjacent tissues and of control bladder tissues. Differences were also found between enzyme activities in the bladder of different disease stages and grades. In the cancerous tissues, only positive intracorrelations were found, but in the cancer-free adjacent tissues and control tissues, both positive and negative correlations were established between enzyme activities. Results suggested that purine metabolism and salvage pathway activity of purine nucleotides were accelerated in the cancerous human bladder tissues via increased ADA and decreased XO activities, probably together with changes in some other related enzyme activities and, free radical metabolisingenzyme activities were depressed in cancerous bladder tissues, which indicated exposure of cancerous tissues to more radicalic stress. Keywords---Cancer, Bladder tissue, Adenosine deaminase, 5'nucleotidase, Xanthine oxidase, Superoxide dismutase, Catalase,
Free radicals
INTRODUCTION
decomposition of toxic reactive intermediates because nucleic acids, proteins, and some enzymes in the cell are found to be attacked by FRs. 5 An increase of activated forms of molecular oxygen such as superoxide (02"-), hydroperoxide (OH), singlet oxygen (O'), and hydrogen peroxide ( H 2 0 2 ) due to overproduction and/ or to inability to destroy them, may lead to severe damage in cellular structures, the consequences of which are mutations, chromosomal aberrations, and carcinogenesis. 6 Active free radicals may also damage specific genes that control the growth and differentiation during promotion phase and stimulate a more rapid growth and malignancy of cells. 7'8 Among the free radical metabolising enzymes, superoxide dismutase (SOD) and catalase (CAT) are of particular importance. SOD (EC 1.15.1.1) catalyses the removal reaction of O2"-, thereby eliminating its toxic effects with production of H202. CAT (EC 1.11.1.6), which is localized mainly in peroxisomes, catalyses dismutation of n 2 0 2 produced to molecular oxygen and water. Although there was accumulating evidence that SOD and CAT activities were suppressed in can-
Cancer development follows a series of biochemical changes, and susceptibility to cancer can be modulated by several factors such as radiation, diet, hormonal status, genetic factors, environmental conditions, etc. However, little is yet known of the mechanisms of cancer induction, promotion and development processes. 1 In recent years, free radicals (FR) have been implicated in the cancer process and some cancer-causing factors have been thought to involve a series of stages generating FRs, particularly those of molecular oxygen. 2 The use of free radical-generating substances as promoters) and scavengers of free radicals as antipromoters 4 seems in accordance with these suggestions. Protection of cellular structures from damage by free radicals can be accomplished through enzymatic and nonenzymatic defense mechanisms. Intracellular distribution of these systems is quite important in the Address correspondence to: Ilker Durak, Ankara Oniversitesi Tip
Faktlltesi, Biyokimya Anabilim Dah, (Dekanhk Binas0, 06100 Slhhlye/Ankara, Tttrkiye. 825
826
i. DURAK et al.
cerous tissues, 9'~° some researchers found unchanged or increased activity in some tumor tissues. 1~ Adenosine deaminase (ADA) (EC.3.5.4.4) catalyses the hydrolytic deamination of either adenosine or deoxyadenosine. ADA is an important enzyme in the degradation of adenine nucleotides. Because of the irreversibility of the reaction catalyzed by ADA, this enzyme reaction seems to be one of the rate-limiting steps in adenosine degradation. 12 In several studies, ADA activity was found increased in cancerous tissue and cells compared to noncancerous ones] 3-~6 Some researchers, however, found low lymphocyte ADA activities in cancer patients.~7'~8 Detoxification of its substrates, namely adenosine and deoxyadenosine, is very important for normal cells because their high concentrations are toxic. Several mechanisms have been proposed for the toxicities of adenosine and deoxyadenosine. It has been suggested that adenosine and deoxyadenosine cause dATP accumulation, which is a strong inhibitor of ribonucleotide reductase and causes inhibition of DNA synthesis,19 and that deoxyadenosine inactivates S-adenosyl homocystein hydrolase, inhibition of which causes interference with critical methylationdependent processes such as synthesis, maturation, or function of DNA. 2° 5'nucleotidase (5NT) (EC 3.1.3.5) is another enzyme functioning in nucleotide metabolism. It generates adenosine from 5'AMP. In several studies, 5NT activity was found decreased in cancer tissue and cell] 5'21'22 It has been suggested that decreased 5NT activity is an attempt to preserve the mononucleotide pool in tumor tissues] 5 Another enzyme playing a part in purine metabolism is xanthine oxidase (XO) (EC 1.2.3.2). This enzyme is widely distributed among species (from bacteria to man) within the various tissues of mammals. Although the enzyme participates in the oxidation of a wide variety of endogenous and exogenous substrates, it is most recognized for its role as the ratelimiting enzyme in nucleic acid degradation, through which all purines are channeled for terminal oxidation. Enzyme catalyses the conversion reaction of xanthine and hypoxanthine to uric acid with production of toxic 02"- radical. In this regard, it is a key enzyme between purine and free radical metabolisms. There is growing evidence that Oz'- radicals generated by XO are primarily responsible for the cellular deterioration associated with several conditions. While enzyme activity was found increased in some publications, 23 it is mostly found decreased in tissues. 24-26 In a previous work, we carried out a similar study by using larynx tissue as a model, and established that ADA and 5NT activities were lower and XO, SOD, and CAT activities were higher in cancerous larynx tissues compared with noncancerous ones. 27 The results of the study mentioned above prompted us to
investigate the subject in detail by using different cancerous tissues such as bladder, kidney, liver tissues, etc. Our aims in these studies are: 1. To establish the activities of some key enzymes participating in purine metabolism in cancerous tissues; 2. To establish the activities of some key enzymes participating in free radical metabolism in cancerous tissues; and 3. To elucidate possible relations between purine and free radical-metabolising enzyme activities, namely interaction between cancer and free radical metabolisms.
MATERIALS AND METHODS
Bladder tissue samples were taken by cystectomy in the Urology Department of Ankara University. We assayed enzyme activities in both cancerous tissues and cancer-free adjacent tissues from 36 patients with bladder cancer ranging in ages from 49 to 72 years, (mean ___ SD 60 _+ 18), and in control bladder tissues from 9 noncancer patients ranging in ages from 45 to 67 years (mean _+ SD 56 ___ 12). All the cancerous tissues were of transitional cell cancer type and most of them were primary cancer. Cancer-free adjacent tissues were obtained a from pathologically noncancer region near to tumor area. Seventeen cancerous tissues were of Grade I - I I and 19 were of Grade III-IV. Five cancerous tissues were at Stage T1, 7 tissues at Stage T2, 8 tissues at T3a, 9 tissues at T3b, and 7 tissues at T4a (TNM stage grouping). Duration of having complaints from bladder cancer ranged from 3 months to 2 years (mean ___SD 9.2 ___3.6 months). Twenty-seven patients underwent only surgical treatment and 9 patients with advanced stage and grade were under classical MVEC (Methotraxate, vinblastin, epirubicin, and cis-platinium) management before surgical treatment. After samples were obtained, they were immediately prepared for enzyme activity assays as given below. Tissue samples (200-500 mg), washed out from contaminated blood with cold water, were homogenised in equal amounts of cold destilled water by using a Potter Elvehzem homogenizer with a Teflon pastle. The homogenate was centrifuged at 15,000 × g for 60 min to remove debris. Clear upper supernatant fluid was taken and the assays were carried out in this part. All the procedures mentioned above were performed at +4°C. Protein assay was made by Lowry's method. 28 XO, SOD, CAT, ADA, and 5NT activities were measured as described. 25'29-32 Results were expressed in international units per mg protein (IU/mg protein) for ADA and 5NT and, mili international unit per mg protein
Human bladder tissues
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Table 1. Mean ±_ SD Values of ADA, 5'NT, XO, SOD, and CAT Activities in Normal, Cancerous and Cancer-Free Adjacent Bladder Tissues from Noncancer and Cancer Patients Grouped According to Tumor Stage. M a n n - W h i t n e y - U Test (#) and Student's t-Test Results Between Control and Other Groups (a, b, c, d) and Between x and y Groups (e, f, g) A (n = 9)
ADA
5' NT
0.98 ± 0.48
0.014 _+ 0.007
0.234 XO
SOD
CAT
± 0.112
C (n = 5)
D (n = 7)
E (n = 8)
F (n = 9)
G (n = 7)
15.9IY ± 9.16
3.15 b ± 2.52
14.0# +__ 5.6
34.12 ~ ± 22.30
22.34 a ± 13.40
12.97 b ___ 5.76
#
#
6.29 c ± 2.60 0.022 +- 0.012
2.32 ± 2.02 0.017 +_ 0.010
2.31 ___ 1.14 0.025 ± 0.009
8.20 a ± 4.72 0.012 ± 0.005
8.17# ± 5.25 0.002# --- 0.020
9.72 ¢ _ 2.41 0.010 ± 0.008
0.044# ± 0.023 0.045# ± 0.020
0.032 b ± 0.004 0.027 b ± 0.010
0.015 --- 0.008 0.110 --- 0.056
0.015 + 0.008 0.041 b ± 0.025
0.010 _+ 0.002 0.068# --- 0.038
0.147 ___ 0.112 2.76 b ± 1.33
0.044 ~ ± 0.023 1.064 c +__0.581
0.154 ± 0.084 1.824 ~ ± 0.627
0.100 +- 0.056 0.998 d -+ 0.370
0.034 a ± 0.009 1.402 c --- 0.593
1.23 d ± 0.45 33.7 b ± 11.5
7.44 a --- 3.98 13.3# ± 6.7
0.801 c ± 0.394 29.0# ___ 4.98
3.752 c _ 1.697 31.9# ±7.1
0.689 d ± 0.254 32.9# ± 16.9
1.583 c ± 0.672 52.3 ± 21.7
32.7 a ± 16.9
14.9# --- 6.7
x a
y
x
f
# y
0.024 ± 0.012 0.064 a _+ 0.000
x y
16.5
x
+- 4.5
y
60.2 +-. 26.9
B (n = 36)
x y
e
0.064 c +-. 0.035 1.49 d + 0.61
#
#
35.5# ___ 13.3
50.7 + 17.3
#
30.8# --- 4.98
50.0 + 16.9
A: Control group; B: General cancer group; C: Cancer group with stage TI; D: Cancer group with stage T2; E: Cancer group with stage T3a; F: Cancer group with stage T3b; G: Cancer group with stage T4~; x: Cancerous tissue; y: Cancer-free-adjacent-tissue. a'~p < 0.05. b.fp < 0.01. C.gp < 0.005. d p < 0.0005. # p < 0.05 ( M a n n - W h i t n e y U-test).
(mlU/mg protein) for XO activities. One unit of SOD activity was defined as the enzyme protein amount causing 50% inhibition in NBT reduction rate, and results of SOD were also given in U/mg protein. However, CAT activity was expressed in k/g protein as described. 3°
RESULTS
Results are given in the tables. As shown in Table 1, ADA activities of cancerous tissues were higher than those of cancer-free adjacent tissues and, those of adjacent tissues were higher than that of control group. There were, however, no statistically meaningful differences in 5NT activities of control group and of cancer groups. Regarding the XO, SOD, and CAT enzymes, activities were found decreased in cancerous tissues compared with control values. In the cancerous tissues, we could not find meaningful relations between stage and enzyme activities except CAT, in which activity increased with stage. In the adjacent tissues, however, ADA activity increased and 5NT activity de-
creased as disease stage increased. There were no relations between stage and XO, SOD, and CAT activities. In Table 2, enzyme activities in bladder tissues of different grades are given. As shown, ADA activity of cancerous tissues increased and SOD activity decreased with grade. There were no such relations between grade and other enzyme activities. In the adjacent tissues, however, there were no differences between ADA activities of grade groups. In this respect, XO, SOD, and CAT activities were found decreased with grade. Furthermore, ADA activities of cancerous tissues were higher than those of adjacent tissues in both grade groups. XO, SOD, and CAT activities, however, were lower in cancerous tissues with Grade I - I I relative to adjacent tissue activities. In the cancerous tissues with Grade III-IV, only XO activity was lower than cancer-free adjacent tissue activity. In Table 3, we compare enzyme activity values in bladder tissues from cancer patients having advanced clinical stage and grade with and/or without chemotheraphy management. In the cancerous tissues, ADA, 5NT, and CAT activities were higher, and XO and SOD activities were lower in the chemotheraphy man-
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i. DURAR et al.
Table 2. Mean ± SD Values of ADA, 5'NT, XO, SOD, and CAT Activities in Normal, Cancerous, and Cancer-Free Adjacent Bladder Tissues from Noncancer and Cancer Patients Grouped According to Tumor Grade and Mann-Whitney U-Test (#) and Student's t-Test Results Between Control and Other Groups (a, b, c, d), Between x and y Groups (e, f) and Between B and C Groups (g, h) A (n = 9) ADA
0.99 ± 0.48
5' NT
0.014 ± 0.007
XO
0.234 ± 0.112
SOD
16.5 ± 4.5
CAT
60.2 ± 26.8
B (n = 17) x y x y x y x y x y
C (n = 19)
10.14 ___4.91# 6.36 + 8.18# 0.019 ___0.008 0.022 _+ 0.010 0.067 - 0.028 b 0.079 e ± 0.031 b 2.189 ± 1.081 d 4.189 ± 2.770 d 23.6 ___ 10.6 b 41.4 _+ 20.8
g h
h h
20.61 ___7.50~ 6.22 ± 4.13# 0.021 _ 0.009 0.012 ~ ± 0.006 0.061 -+ 0.017 c 0.091" ± 0.034 b 0.796 ± 0.394 d 0.839 ± 0.313 d 30.8 ± 17.4# 32.8 ± 11.7#
A: Control group; B: Grade I-H; C: Grade III-IV; x: Cancerous tissue; y: Cancer-free adjacent tissue. ....g p < 0.05. b,f,hp < 0.01. °p < 0.005. d p < 0.0005. # p < 0.05 (Mann-Whitney U-test).
agement group. In the adjacent tissues, however, no significant differences existed. As seen from the table, ADA and 5NT activities of the groups were higher, XO and SOD activities were lower in cancerous tissues r e l a t i v e to a d j a c e n t t i s s u e v a l u e s . I n T a b l e s 4 a n d 5, i n t r a - a n d i n t e r c o r r e l a t i o n a n a l y sis r e s u l t s a r e g i v e n . A s s e e n i n T a b l e 4, all t h e a c t i v i t y r e l a t i o n s w e r e p o s i t i v e in c o n t r o l g r o u p s . I n t h e c a n c e r ous and adjacent tissues, however, intrarelations bet w e e n e n z y m e a c t i v i t i e s w e r e d i s o r d e r e d . I n t h i s respect, there were negative correlations between ADA and SOD, ADA and CAT, and ADA and XO activities, and there were positive correlations between 5NT and SOD, and 5NT and CAT activities in cancerous tissues.
In the cancer-free adjacent tissues, correlations between ADA and SOD, and ADA and XO were negative and the ones between 5NT and XO, and 5NT and SOD were positive. No meaningful correlations were present, however, between ADA and CAT, and 5NT and CAT activities. ADA and 5NT relations were posit i v e in c a n c e r o u s t i s s u e s a n d w e r e n e g a t i v e in a d j a c e n t tissues. Intrarelations between XO, SOD, and CAT a c t i v i t i e s w e r e also d i s o r d e r e d . I n t h i s r e g a r d , t h e r e l a tions between XO and SOD, and XO and CAT were p o s i t i v e in b o t h t i s s u e s w h i l e t h e r e l a t i o n b e t w e e n S O D and CAT was negative in cancerous tissue and positive in adjacent tissues. Intercorrelation analysis results carded out between
Table 3. Mean _ SD Values of ADA, 5'NT, XO, SOD, and CAT Activities in Normal, Cancerous, and Cancer-Free Adjacent Tissues from Noncancer and Cancer Patients Grouped According to Chemotherapy Management and Mann-Whitney U-test (#) and Student's t-test Results Between Control and Other Groups (a, b, c, d) and Between x and y Groups (e, f) A (n = 9) ADA
0.99 ± 0.46
5' NT
0.014 z 0.006
XO
0.234 ___0.112
SOD
16.5 ± 4.5
CAT
60.2 ___26.7
B (n = 9) x y x y x y x y x y
24.94 ___ 12.46 b 8.48 e ± 5.34# 0.035 --- 0.022# 0.010 ± 0.006 0.050 --- 0.017 b 0.079 ± 0.034" 0.462 ± 0.225 d 1.024 f _ 0.249 c 42.9 ___ 17.1 40.0 _ 17.5
C (n = 10) 17.00 6.22 0.020 0.012 0.061 0.091 0.796 0.839 30.8 32.9
± 8.57 b ± 3.83# ___0.016 ___0.007 ± 0.017 b ± 0.016 ± 0.414 d ___0.310 d ___ 16.4# ± 9.7#
A: Control group; B: Advanced clinical stage and grade group with chemotherapy; C: Advanced clinical stage and grade group without chemotherapy; x: Cancerous tissue; y: Cancer-free adjacent tissue. a.Op < 0.05. b,fp < 0.01. Cp < 0.005. d p < 0.00005. # p < 0.05 (Mann-Whitney U-test).
Human bladder tissues Table 4. Intracorrelation Coefficients Between Enzyme Activities in Normal, Cancerous, and Cancer-free Adjacent Tissues from Noncancer and Cancer Patients A (n=9) ADA-5' NT
0.87
ADA-XO
0.59
ADA-SOD
0.91
ADA-CAT
0.25 (n.s.) 0.31 (n.s.) 0.90
5'NT-XO 5'NT-SOD 5'NT-CAT XO-SOD XO-CAT SOD-CAT i
0.33 (n.s.) 0.43 (n.s.) 0.79 0.75
B (n=36) x y x y x y x y x y x y x y x y x y x y
+0.25 -0.21 -0.40 -0.20 -0.33 -0.31 -0.30 +0.10 +0.10 +0.32 +0.17 +0.70 +0.47 +0.05 +0.72 +0.85 +0.22 +0.21 -0.17 +0.13
(n.s.) (n.s.) (n.s.)
(n.s.) (n.s.) (n.s.)
(n.s.)
(n.s.) (n.s.) (n.s.) (n.s.)
A: Control group; B: General cancer group; x: Cancerous bladder tissue; y: Noncancerous bladder tissue; n.s.: nonsignificant (p <
0.05).
enzyme activities of cancerous and adjacent tissues exhibited only positive relations.
DISCUSSION
The results of the study presented here exhibited contrast with the results of our previous study in which human cancerous larynx tissue was used for this purpose. The controversy found between two studies indicated that enzyme metabolism in cancer might show great differences depending on cancerous tissues studied, and that mechanisms put forward to explain enzyme changes in carcinogenesis might be specific only for the material analyzed. These different findings also display difficulties encountered in the explanation of enzymatic mechanisms in cancer processes. As to the results of this study, the following conclusions can be made. In this study, we have established that ADA activity increased in cancerous tissues and cancer-free adjacent tissue. Although in several studies, ADA activities were found increased in cancerous tissue ~3-15 and cells, ~6 some researchers measured low activity values in lymphocytes from cancer patients. 17"18 However, Sufrin et al. 16 found that ADA levels of iymphocytes from patients with bladder cancer were also elevated with transitional cell carcinoma and corraleted with stage, activity, clinical course, and tumor resection, but not tumor grade. These researchers found higher erythrocyte ADA activities as well in
829
the same patients and suggested that lymphocyte ADA levels might be a sensitive indicator of bladder carcinoma. However, Dasmahapatra et al. 17 and Kojima et al) s found low lymphocyte ADA activities in head and neck cancer patients and gastric cancer patients, respectively. These researchers suggested that low lymphocyte ADA activities might be a more sensitive indicator of suppressed cellular immunity) 7"18 High ADA activity determined in cancerous bladder tissue reflected accelerated purine turnover and high salvage pathway activity. In fact, it has been established that activities of purine salvage enzymes including ADA were elevated in neoplastic human tissues. 15'33 Although we have no detailed information of the physiological significance of increased ADA activities in cancerous and adjacent tissues at present, it seems to be a secondary phenomenon, which reflects rapid salvage pathway activity of nucleic acid metabolism associated with tissue hyperproliferation. Although some researchers found lower 5NT activity in cancerous tissue, 15'2a we could not find statistically meaningful differences between 5NT activities of cancerous and noncancerous tissues. This difference might arise from the fact that analyses were carded out in different organs and tissues. 5NT activities in cancerous tissues were to some extent higher compared to noncancerous ones in some of the groups of Tables 1 and 2. However, increases in enzyme activity were generally not statistically meaningful. This finding showed that 5NT activity was not a limiting factor in accelerated purine metabolism of cancerous bladder tissue. Regarding the XO activity, Ktko~lu et al.23 found higher enzyme activity in tumoral brain tissues and suggested that the levels of XO in brain tissues could be used as a biochemical marker for the differentiation of tumoral tissues from normal ones. However, several researchers found lower XO activities in cancerous tissues. 15'25"34Natsumeda et al. 33 established decreased XO activities in all hepatomas irrespective of growth rate or differentiation and Weber et al. 26 established decreased XO activities in slowly and rapidly growing
Table 5. Intercorrelation Coefficients Between Enzyme Activities of Cancerous and Cancer-Free Adjacent Tissues from Cancer Patients B
(n = 36) ADA-ADA 5'NT-5'NT XO-XO SOD-SOD CAT-CAT B: Generfl cancer group
0.80 0.39 0.61 0.81 0.21
830
i. DLrRAKet aL
human colon carcinoma xerografts. Our results are in a good agreement with those of Prajda and Weber. 25 Decreased XO activities in human tissues might contribute to accelerated salvage pathway synthesis of purine nucleotides needed by tumor cells. On the one hand, increased ADA activity, and on the other, decreased XO activity, might give selective advantage to cancer cells to grow and develop more rapidly. In this study, we found lower SOD and CAT activities in cancerous tissue and adjacent bladder tissue compared with the activity values of tissues from noncancer patients. In fact, several researchers found decreased SOD and CAT activities in various types of cancer tissues. For example, Vo et al.9 determined low SOD and CAT activities in rat hepatocytes exposed to carcinogenic treatment, and suggested that changes in the level of endogenous defense mechanisms might modify cellular homeostasis and hence might contribute to the promotion of neoplasia. Similar observations were made after treatment of mouse skin with a tumor promoter. 35 Corrocher et al. 36 established decreased CAT activity in human hepatoma and suggested that antioxidant defense system of hepatocellular carcinoma cells was severely impaired. Balgoy and Robe r t s 37 also determined lower SOD levels in transplantable mouse and rat tumors. Similar results were obtained by Bize et al. 38 in Morris Hepatomas, Hoffman et al. 1° in human colorectal cancer, and Reiners et al. 39 in chemically induced skin cancer. However, Nakada et al. 11 found no changes in SOD activities between renal carcinoma cells and tumor-uninvolved renal tissues, and they added that grade and stage of tumors had no apparent effect on SOD activities. Accordingly, Howie et al. 4° established that some enzyme activities participating in free radical metabolism exhibit differences depending on type of organ used. For example, they found higher glutathione peroxidase activities in tumoral human lung, colon, stomach, and breast tissues, and low activities in tumoral kidney and liver tissues from the same subjects. Low SOD and CAT activity results obtained in this study supported general previous observations that enzymatic antioxidant defense mechanisms were impaired in tumor cells and tissues, although this was not a universal characteristic of neoplastic cells. At this stage, two general questions appear regarding the free radical enzyme metabolism in cancer. First, may changes in antioxidant enzyme activities arise from cancer disease itself? Second, may changes in free radical metabolism play a part in the carcinogenic process? It seems quite possible that disordered metabolism in cancer cells and tissues may also lead to inhibition or repression of synthesis of free radical-metabolising enzymes. For example, decreased XO activity, as established in this study, may cause decreased production
of 02"- radical. Steadily decreased 02"- radical production in the cancer cells may lead to suppression of free radical enzymes using 02"- and other oxygen radicals as substrate. Another hypothesis is that impairment of free radical metabolism due to several factors may also accelerate carcinogenic process in the cell. For example, inhibition of SOD, CAT or other free radical enzyme activities with treatment of some chemicals such as 12-O-tetradecanoyl-phorbol-13-acetate as made by Reiners et al.39 or with some toxic elements such as nickel, chromium, etc., may cause inhibition of activities of some free radical-metabolising enzymes, thereby resulting in increased production of free radicals in the cell. Radicalic stress in the cell may then lead to tumor promotion and development process by known mechanisms. Furthermore, it is also possible that some cancer-causing factors may affect free radical-metabolising systems as well. For example, treatment of mouse skin with a tumor promoter may independently lead to both tumor promotion and inhibition, and/or repression of free radical enzymes in skin cells. Neverthless, a vicious cycle can be mentioned about the subject. On the one hand, destroyed free radical metabolism can impaire purine and DNA metabolisms in cancer cells, and on the other, changed metabolism due to cancer process in cancer cells may deteriorate the situation of antioxidant defense system. Negative relations established between ADA and SOD, and ADA and CAT, reveal this negative interaction between free radical and purine metabolisms. From the results of this study, we obtained the following conclusions: 1. In cancerous human bladder cells, purine metabolism and salvage pathway activity (high ADA and low XO activities) were accelerated. 2. Enzymatic antioxidant defense mechanism of bladder cancer cells was impaired (low SOD and CAT activities). 3. There were negative relations between ADA and free radical-metabolising enzyme activities, namely purine metabolism and free radical metabolism in cancerous bladder tissues. 4. Enzyme activity pattern of cancerous and cancerfree adjacent tissues presented partial similarity. 5. In cancerous bladder tissue, no important relations existed between stage and activities of the enzymes mentioned. However, there were positive relations between grade and ADA activity and negative relations between grade and SOD and CAT activities. REFERENCES
1. Welsch, C.W. Host factors affecting growth of carcinogen-inducedrat mammarycarcinomas:A reviewand tributeto Charles Brenton-Huggins. Cancer Res. 45:3415-3443; 1985.
Human bladder tissues 2. Ames, B. N. Dietary carcinogens and anticarcinogens: Oxygen radicals and degenerative diseases. Science 221:1256-1264; 1983. 3. Slaga, T. J.; Klein-Szantu, A. J. P.; Triplett, L. L.; Yotti, L. P.; Trosko, J. E. Skin tumor promoting activity of benzoyl peroxide, a widely used free radical generating compound. Science 213:1023-1025; 1981. 4. Kensler, T. W.; Bush, D. M.; Kozumbo, W. J. Inhibition of tumor promotion by biomimetic superoxide dismutase. Science 221:75-77; 1983. 5. HalliweU, B.; Gutteridge, J,M.C. Oxygen toxicity, oxygen radicals, transition metal and disease. Biochem. J. 219:1-14; 1984. 6. Player, T. J.; Mills, P. L.; Horton, A. A. Age-dependent changes in rat liver microsomal and mitocontrial NADPH-dependent lipid peroxidation. Biochem. Biophys. Res. Commun. 78:397402; 1977. 7. Freeman, B. A.; Crapo, J. D. Biology of disease, free radicals and tissue injury. Lab. Invest. 47:412-426; 1982. 8. Heckler, E.; Fusenig, N.; Marx, W.; Thielman, ; Carcinogenesis: A comprehensive survey. Vol. 7. New York: Raven Press; 1982. 9. Vo, T. K. O.; Druez, C.; Delzenne, N,; Taper, H. S.; Roberfroid, M. Analysis of antioxidant defense systems during rat hepatocarcinogenesis. Carcinogenesis 9:2009-2013; 1988. 10. Hoffman, C. E. J.; Webster, N. R.; Wiggins, P. A.; Chisholm, E. M.; Giles, G. R.; Leveson, S. H. Free radical detoxifying systems in human colorectal cancer. Br. J. Cancer 51:127-129; 1985. 11. Nakada, T.; Akiya, T.; Koike, H.; Katayama, T. Superoxide dismutase activity in renal cell carcinoma. Eur. Urol. 14:5055; 1988. 12. Iiznka, H.; Koizumi, H.; Kamigaki, K.; Aoyagi, T.; Miura, Y. Two forms of adenosine deaminase in pig epidermis. J. Dermatol 8:91-95; 1981. 13. Koizumi, H.; Iizuka, H.; Aoyagi, T.; Miura, Y. Adenosine deaminase In human epidermis from healthy and psoriatic subjects. Arch. Dermatol. Res 275:310-314; 1983. 14. Koizumi, H.; Iizuka, H.; Aoyagi, T.; Miura, Y. Characterization of adenosine deaminase from normal human epidermis and squamous cell carcinoma of the skin. The Journal oflnvestigarive Dermatology 84:199-202; 1985. 15. Camici, M.; Tozzi, M. G.; Allegrini, S.; Corso, A. D.; Sanfilippo, O.; Daidone, M. G.; Marco, C. D.; Ipata, P. L. Purine salvage enzyme activity in normal and neoplastic human tissues. Cancer Biochem. Biophys 11:201-209; 1990. 16. Sufrin, G.; Tritsch, G. L.; Mittelman, A.; Murphy, G. P. Adenosine deaminase activity in patients with carcinoma of the bladder. The Journal of Urology 119:343-346; 1978. 17. Dasmahapatra, K. S.; Facs, H. Z. H.; Dasmahapatra, A.; Suarez, S. Evaluation of adenosine deaminase activity in patients with head and neck cancer. Journal of Surgical Research 40:363373; 1986, 18. Kojima, O.; Majima, T.; Uehara, Y.; Yamane, T.; Fujita, Y.; Takahashi, T.; Majima, S. Alteration of adenosine deaminase levels in peripheral blood lymphocytes of patients with gastric cancer. Japanese Journal of Surgery 15:130-133; 1985. 19. Donofrio, J.; Coleman, M. S.; Hutton, J. J.; Daoud, A.; Lampkin, B.; Dyminski, J. Overproduction of adenosine deoxynucleosides and deoxynucleotides in adenosine deaminase deficiency with severe combined immunodeficiency disease. J. Clin. Invest 62:884-887; 1978. 20. Hershfield, M. S.; Kredich, N, M. Resistance of an adenosine kinase-deficient human lyphoblastoid cell line to effects of deoxyadenosine on growth. S-adenosylhomocysteine hydrolase inactivation, and dATP accumulation, Proc. Nat. Acad. Sci. USA 771:4292-4296; 1980. 21. Lamirande, G. D.; Allard, C.; Cantero, A. Purine-metabolizing
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enzymes in normal rat liver nandovikoff hepatoma. Cancer Res. 18:952-958; 1958. Domand, J.; Bonnafous, J. C.; Favero, J.; Mani, J. C. Ecto5'nucleotidase and adenosine deaminase activities of lymphoid cells. Biochemical Medicine 28:144-156; 1982. Ktko~lu, E.; Belce, A.; Ozyurt, E.; Tepeler, Z. Xanthine oxidase levels in human brain tumors. Cancer Letters 50:179-181; 1990. Prajda, N.; Donohue, J. P.; Weber, G. Increased amidophosphoribosyl-transferase and decreased xanthine oxidase activity in human and rat renal cell carcinoma. Life Sci. 29:853-860; 1981. Prajda, N.; Weber, G. Malignant transformation-linked imbalance: Decreased xanthine oxidase activity in hepatomas. Febs Letts. 59:245-249; 1975. Weber, G.; Hager, J. C.; Lui, S. M.; Prajda, N.; Tzeng, D. Y.; Jackson, R. C.; Takeda, E.; Eble, J. N. Biochemical programs of slowly and rapidly growing human colon carcinoma xenografts. Cancer Res. 41:854-859; 1981. Durak, |.; I§ik, A. U., Canbolat, O.; Akyol, 04 Kavutcu, M. Adenosine deaminase 5'nucleotidase, xanthine oxidase, superoxide dismutase and catalase activities in cancerous and noncancerous human laryngeal tissues. Free Radic. Biol. Med. 15:681 684; 1993. Lowry, O.; Rosenbraugh, N.; Farr, L.; Rondall, R. Protein measurement with the theofilin phenol reagent. J. Biol. Chem. 183:265-275; 1951. Sun, Y.; Oberly, L. W.; Li, Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34/3:497-500; 1988. Aebi, H. Catalase. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis. New York and London: Academic Press, Inc.; 1974:1-2:673-677. Guisti, G. Enzyme activities. In: Bergmeyer, H. U., ed. Methods of enzymatic analysis. Gmblt Weinheim, Bergest: Verlag Chemie; 1974:1092-1098. Donald, W. M.; et al. Enzymes. In: Tietz, N. W., ed. Textbook of clinical chemistry. Philadelphia: W. B. Saunders Company; 1986:718-720. Natsumeda, Y.; Prajda, N.; Donohue, J. P.; Glover, J. L.; Weber, G. Enzymic capacities of purine de novo and salvage pathways for nucleotide synthesis in normal and neoplastic tissues. Cancer Res. 44:2475-2479; 1984. Prajda, N.; Morris, H. P.; Weber, G. Imbalance of purine metabolism in hepatomas of different growth rates as expressed in behavior of xanthine oxidase (EC 1.2.3.2), Cancer Res. 36:4639-4646; 1976. Solanki, V.; Rana, R. S.; Slage, T. J. Diminution of mouse epidermal superoxide dismutase and catalase activities by tumor promoters. Carcinogenesis 2" 1141 - 1146; 1981. Corrocher, R.; Casafil, M.; Bellisola, G.; GabrieUi, G. B.; Nicoli, N,; Guidi, G. C.; De Sandre, G. Severe impairment of antioxidant system in human hepatoma. Cancer 58:1658-1662; 1986. Balgoy, J. N. A. V.; Roberts, E. Superoxide dismutase in normal and malignant tissues in different species. Comp. Biochem. Physiol. 6/1:263-268; 1979. Bize, I. B.; Oberley, L. W.; Morris, H. P. Superoxide dismutase and superoxide radical in Morris Hepatomas. Cancer Res. 40:3686-3693; 1980. Reiners, J. J.; Thai, G.; Rupp, T.; Cantu, A. D. Assessment of the antioxidant/prooxidant status of murine skin following topical treatment with 12-O-tetradecanoylphorbol-13-acetate and throughot the ontogeny of skin cancer. Part I: Quantitation of superoxide dismutase, catalase, glutathione peroxidase and xanthine oxidase. Carcinogenesis 12:2337-2343; 1991, Howie, A. F.; Forrester, L. M.; Glancey, M. J.; Schlager, J. J.; Powis, G.; Beckett, G, J.; Hayes, J. D.; Wolf, C. R. Glutathione S-transferase and glutathione peroxidase expression in normal and turnour human tissues. Carcinogenesis 11:451-458; 1990.
Pergamon
0891-5849(93)E0055-A
Brief Communication ADENOSINE DEAMINASE, 5'NUCLEOTIDASE, XANTHINE OXIDASE, SUPEROXIDE DISMUTASE, AND CATALASE ACTIVITIES IN CANCEROUS AND NONCANCEROUS HUMAN BLADDER TISSUES ILKER DURAK,* HAKKI PERK, t MUSTAFA KAVUT~U,* ORHAN CANBOLAT,* {~MER AKYOL,* and YA~AR BEDOK t
*Ankara University,Faculty of Medicine, Departmentof Biochemistry,Ankara, Turkey; and *AnkaraUniversity, Faculty of Medicine, Departmentof Urology, Ankara, Turkey (Received 24 June 1993; Revised 16 December 1993; Accepted 21 December 1993) Abstract--Activities of adenosine deaminase (ADA), 5'nucleotidase (5NT), xanthine oxidase (XO), superoxide dismutase (SOD), and catalase (CAT) enzymes were measured in cancerous and cancer-free adjacent bladder tissues from 36 patients with bladder cancer and in control bladder tissues from 9 noncancer patients. Increased ADA and decreased XO, SOD, and CAT activities were found in cancerous bladder tissues compared with those of cancer-free adjacent tissues and of control bladder tissues. Differences were also found between enzyme activities in the bladder of different disease stages and grades. In the cancerous tissues, only positive intracorrelations were found, but in the cancer-free adjacent tissues and control tissues, both positive and negative correlations were established between enzyme activities. Results suggested that purine metabolism and salvage pathway activity of purine nucleotides were accelerated in the cancerous human bladder tissues via increased ADA and decreased XO activities, probably together with changes in some other related enzyme activities and, free radical metabolisingenzyme activities were depressed in cancerous bladder tissues, which indicated exposure of cancerous tissues to more radicalic stress. Keywords---Cancer, Bladder tissue, Adenosine deaminase, 5'nucleotidase, Xanthine oxidase, Superoxide dismutase, Catalase,
Free radicals
INTRODUCTION
decomposition of toxic reactive intermediates because nucleic acids, proteins, and some enzymes in the cell are found to be attacked by FRs. 5 An increase of activated forms of molecular oxygen such as superoxide (02"-), hydroperoxide (OH), singlet oxygen (O'), and hydrogen peroxide ( H 2 0 2 ) due to overproduction and/ or to inability to destroy them, may lead to severe damage in cellular structures, the consequences of which are mutations, chromosomal aberrations, and carcinogenesis. 6 Active free radicals may also damage specific genes that control the growth and differentiation during promotion phase and stimulate a more rapid growth and malignancy of cells. 7'8 Among the free radical metabolising enzymes, superoxide dismutase (SOD) and catalase (CAT) are of particular importance. SOD (EC 1.15.1.1) catalyses the removal reaction of O2"-, thereby eliminating its toxic effects with production of H202. CAT (EC 1.11.1.6), which is localized mainly in peroxisomes, catalyses dismutation of n 2 0 2 produced to molecular oxygen and water. Although there was accumulating evidence that SOD and CAT activities were suppressed in can-
Cancer development follows a series of biochemical changes, and susceptibility to cancer can be modulated by several factors such as radiation, diet, hormonal status, genetic factors, environmental conditions, etc. However, little is yet known of the mechanisms of cancer induction, promotion and development processes. 1 In recent years, free radicals (FR) have been implicated in the cancer process and some cancer-causing factors have been thought to involve a series of stages generating FRs, particularly those of molecular oxygen. 2 The use of free radical-generating substances as promoters) and scavengers of free radicals as antipromoters 4 seems in accordance with these suggestions. Protection of cellular structures from damage by free radicals can be accomplished through enzymatic and nonenzymatic defense mechanisms. Intracellular distribution of these systems is quite important in the Address correspondence to: Ilker Durak, Ankara Oniversitesi Tip
Faktlltesi, Biyokimya Anabilim Dah, (Dekanhk Binas0, 06100 Slhhlye/Ankara, Tttrkiye. 825
826
i. DURAK et al.
cerous tissues, 9'~° some researchers found unchanged or increased activity in some tumor tissues. 1~ Adenosine deaminase (ADA) (EC.3.5.4.4) catalyses the hydrolytic deamination of either adenosine or deoxyadenosine. ADA is an important enzyme in the degradation of adenine nucleotides. Because of the irreversibility of the reaction catalyzed by ADA, this enzyme reaction seems to be one of the rate-limiting steps in adenosine degradation. 12 In several studies, ADA activity was found increased in cancerous tissue and cells compared to noncancerous ones] 3-~6 Some researchers, however, found low lymphocyte ADA activities in cancer patients.~7'~8 Detoxification of its substrates, namely adenosine and deoxyadenosine, is very important for normal cells because their high concentrations are toxic. Several mechanisms have been proposed for the toxicities of adenosine and deoxyadenosine. It has been suggested that adenosine and deoxyadenosine cause dATP accumulation, which is a strong inhibitor of ribonucleotide reductase and causes inhibition of DNA synthesis,19 and that deoxyadenosine inactivates S-adenosyl homocystein hydrolase, inhibition of which causes interference with critical methylationdependent processes such as synthesis, maturation, or function of DNA. 2° 5'nucleotidase (5NT) (EC 3.1.3.5) is another enzyme functioning in nucleotide metabolism. It generates adenosine from 5'AMP. In several studies, 5NT activity was found decreased in cancer tissue and cell] 5'21'22 It has been suggested that decreased 5NT activity is an attempt to preserve the mononucleotide pool in tumor tissues] 5 Another enzyme playing a part in purine metabolism is xanthine oxidase (XO) (EC 1.2.3.2). This enzyme is widely distributed among species (from bacteria to man) within the various tissues of mammals. Although the enzyme participates in the oxidation of a wide variety of endogenous and exogenous substrates, it is most recognized for its role as the ratelimiting enzyme in nucleic acid degradation, through which all purines are channeled for terminal oxidation. Enzyme catalyses the conversion reaction of xanthine and hypoxanthine to uric acid with production of toxic 02"- radical. In this regard, it is a key enzyme between purine and free radical metabolisms. There is growing evidence that Oz'- radicals generated by XO are primarily responsible for the cellular deterioration associated with several conditions. While enzyme activity was found increased in some publications, 23 it is mostly found decreased in tissues. 24-26 In a previous work, we carried out a similar study by using larynx tissue as a model, and established that ADA and 5NT activities were lower and XO, SOD, and CAT activities were higher in cancerous larynx tissues compared with noncancerous ones. 27 The results of the study mentioned above prompted us to
investigate the subject in detail by using different cancerous tissues such as bladder, kidney, liver tissues, etc. Our aims in these studies are: 1. To establish the activities of some key enzymes participating in purine metabolism in cancerous tissues; 2. To establish the activities of some key enzymes participating in free radical metabolism in cancerous tissues; and 3. To elucidate possible relations between purine and free radical-metabolising enzyme activities, namely interaction between cancer and free radical metabolisms.
MATERIALS AND METHODS
Bladder tissue samples were taken by cystectomy in the Urology Department of Ankara University. We assayed enzyme activities in both cancerous tissues and cancer-free adjacent tissues from 36 patients with bladder cancer ranging in ages from 49 to 72 years, (mean ___ SD 60 _+ 18), and in control bladder tissues from 9 noncancer patients ranging in ages from 45 to 67 years (mean _+ SD 56 ___ 12). All the cancerous tissues were of transitional cell cancer type and most of them were primary cancer. Cancer-free adjacent tissues were obtained a from pathologically noncancer region near to tumor area. Seventeen cancerous tissues were of Grade I - I I and 19 were of Grade III-IV. Five cancerous tissues were at Stage T1, 7 tissues at Stage T2, 8 tissues at T3a, 9 tissues at T3b, and 7 tissues at T4a (TNM stage grouping). Duration of having complaints from bladder cancer ranged from 3 months to 2 years (mean ___SD 9.2 ___3.6 months). Twenty-seven patients underwent only surgical treatment and 9 patients with advanced stage and grade were under classical MVEC (Methotraxate, vinblastin, epirubicin, and cis-platinium) management before surgical treatment. After samples were obtained, they were immediately prepared for enzyme activity assays as given below. Tissue samples (200-500 mg), washed out from contaminated blood with cold water, were homogenised in equal amounts of cold destilled water by using a Potter Elvehzem homogenizer with a Teflon pastle. The homogenate was centrifuged at 15,000 × g for 60 min to remove debris. Clear upper supernatant fluid was taken and the assays were carried out in this part. All the procedures mentioned above were performed at +4°C. Protein assay was made by Lowry's method. 28 XO, SOD, CAT, ADA, and 5NT activities were measured as described. 25'29-32 Results were expressed in international units per mg protein (IU/mg protein) for ADA and 5NT and, mili international unit per mg protein
Human bladder tissues
827
Table 1. Mean ±_ SD Values of ADA, 5'NT, XO, SOD, and CAT Activities in Normal, Cancerous and Cancer-Free Adjacent Bladder Tissues from Noncancer and Cancer Patients Grouped According to Tumor Stage. M a n n - W h i t n e y - U Test (#) and Student's t-Test Results Between Control and Other Groups (a, b, c, d) and Between x and y Groups (e, f, g) A (n = 9)
ADA
5' NT
0.98 ± 0.48
0.014 _+ 0.007
0.234 XO
SOD
CAT
± 0.112
C (n = 5)
D (n = 7)
E (n = 8)
F (n = 9)
G (n = 7)
15.9IY ± 9.16
3.15 b ± 2.52
14.0# +__ 5.6
34.12 ~ ± 22.30
22.34 a ± 13.40
12.97 b ___ 5.76
#
#
6.29 c ± 2.60 0.022 +- 0.012
2.32 ± 2.02 0.017 +_ 0.010
2.31 ___ 1.14 0.025 ± 0.009
8.20 a ± 4.72 0.012 ± 0.005
8.17# ± 5.25 0.002# --- 0.020
9.72 ¢ _ 2.41 0.010 ± 0.008
0.044# ± 0.023 0.045# ± 0.020
0.032 b ± 0.004 0.027 b ± 0.010
0.015 --- 0.008 0.110 --- 0.056
0.015 + 0.008 0.041 b ± 0.025
0.010 _+ 0.002 0.068# --- 0.038
0.147 ___ 0.112 2.76 b ± 1.33
0.044 ~ ± 0.023 1.064 c +__0.581
0.154 ± 0.084 1.824 ~ ± 0.627
0.100 +- 0.056 0.998 d -+ 0.370
0.034 a ± 0.009 1.402 c --- 0.593
1.23 d ± 0.45 33.7 b ± 11.5
7.44 a --- 3.98 13.3# ± 6.7
0.801 c ± 0.394 29.0# ___ 4.98
3.752 c _ 1.697 31.9# ±7.1
0.689 d ± 0.254 32.9# ± 16.9
1.583 c ± 0.672 52.3 ± 21.7
32.7 a ± 16.9
14.9# --- 6.7
x a
y
x
f
# y
0.024 ± 0.012 0.064 a _+ 0.000
x y
16.5
x
+- 4.5
y
60.2 +-. 26.9
B (n = 36)
x y
e
0.064 c +-. 0.035 1.49 d + 0.61
#
#
35.5# ___ 13.3
50.7 + 17.3
#
30.8# --- 4.98
50.0 + 16.9
A: Control group; B: General cancer group; C: Cancer group with stage TI; D: Cancer group with stage T2; E: Cancer group with stage T3a; F: Cancer group with stage T3b; G: Cancer group with stage T4~; x: Cancerous tissue; y: Cancer-free-adjacent-tissue. a'~p < 0.05. b.fp < 0.01. C.gp < 0.005. d p < 0.0005. # p < 0.05 ( M a n n - W h i t n e y U-test).
(mlU/mg protein) for XO activities. One unit of SOD activity was defined as the enzyme protein amount causing 50% inhibition in NBT reduction rate, and results of SOD were also given in U/mg protein. However, CAT activity was expressed in k/g protein as described. 3°
RESULTS
Results are given in the tables. As shown in Table 1, ADA activities of cancerous tissues were higher than those of cancer-free adjacent tissues and, those of adjacent tissues were higher than that of control group. There were, however, no statistically meaningful differences in 5NT activities of control group and of cancer groups. Regarding the XO, SOD, and CAT enzymes, activities were found decreased in cancerous tissues compared with control values. In the cancerous tissues, we could not find meaningful relations between stage and enzyme activities except CAT, in which activity increased with stage. In the adjacent tissues, however, ADA activity increased and 5NT activity de-
creased as disease stage increased. There were no relations between stage and XO, SOD, and CAT activities. In Table 2, enzyme activities in bladder tissues of different grades are given. As shown, ADA activity of cancerous tissues increased and SOD activity decreased with grade. There were no such relations between grade and other enzyme activities. In the adjacent tissues, however, there were no differences between ADA activities of grade groups. In this respect, XO, SOD, and CAT activities were found decreased with grade. Furthermore, ADA activities of cancerous tissues were higher than those of adjacent tissues in both grade groups. XO, SOD, and CAT activities, however, were lower in cancerous tissues with Grade I - I I relative to adjacent tissue activities. In the cancerous tissues with Grade III-IV, only XO activity was lower than cancer-free adjacent tissue activity. In Table 3, we compare enzyme activity values in bladder tissues from cancer patients having advanced clinical stage and grade with and/or without chemotheraphy management. In the cancerous tissues, ADA, 5NT, and CAT activities were higher, and XO and SOD activities were lower in the chemotheraphy man-
828
i. DURAR et al.
Table 2. Mean ± SD Values of ADA, 5'NT, XO, SOD, and CAT Activities in Normal, Cancerous, and Cancer-Free Adjacent Bladder Tissues from Noncancer and Cancer Patients Grouped According to Tumor Grade and Mann-Whitney U-Test (#) and Student's t-Test Results Between Control and Other Groups (a, b, c, d), Between x and y Groups (e, f) and Between B and C Groups (g, h) A (n = 9) ADA
0.99 ± 0.48
5' NT
0.014 ± 0.007
XO
0.234 ± 0.112
SOD
16.5 ± 4.5
CAT
60.2 ± 26.8
B (n = 17) x y x y x y x y x y
C (n = 19)
10.14 ___4.91# 6.36 + 8.18# 0.019 ___0.008 0.022 _+ 0.010 0.067 - 0.028 b 0.079 e ± 0.031 b 2.189 ± 1.081 d 4.189 ± 2.770 d 23.6 ___ 10.6 b 41.4 _+ 20.8
g h
h h
20.61 ___7.50~ 6.22 ± 4.13# 0.021 _ 0.009 0.012 ~ ± 0.006 0.061 -+ 0.017 c 0.091" ± 0.034 b 0.796 ± 0.394 d 0.839 ± 0.313 d 30.8 ± 17.4# 32.8 ± 11.7#
A: Control group; B: Grade I-H; C: Grade III-IV; x: Cancerous tissue; y: Cancer-free adjacent tissue. ....g p < 0.05. b,f,hp < 0.01. °p < 0.005. d p < 0.0005. # p < 0.05 (Mann-Whitney U-test).
agement group. In the adjacent tissues, however, no significant differences existed. As seen from the table, ADA and 5NT activities of the groups were higher, XO and SOD activities were lower in cancerous tissues r e l a t i v e to a d j a c e n t t i s s u e v a l u e s . I n T a b l e s 4 a n d 5, i n t r a - a n d i n t e r c o r r e l a t i o n a n a l y sis r e s u l t s a r e g i v e n . A s s e e n i n T a b l e 4, all t h e a c t i v i t y r e l a t i o n s w e r e p o s i t i v e in c o n t r o l g r o u p s . I n t h e c a n c e r ous and adjacent tissues, however, intrarelations bet w e e n e n z y m e a c t i v i t i e s w e r e d i s o r d e r e d . I n t h i s respect, there were negative correlations between ADA and SOD, ADA and CAT, and ADA and XO activities, and there were positive correlations between 5NT and SOD, and 5NT and CAT activities in cancerous tissues.
In the cancer-free adjacent tissues, correlations between ADA and SOD, and ADA and XO were negative and the ones between 5NT and XO, and 5NT and SOD were positive. No meaningful correlations were present, however, between ADA and CAT, and 5NT and CAT activities. ADA and 5NT relations were posit i v e in c a n c e r o u s t i s s u e s a n d w e r e n e g a t i v e in a d j a c e n t tissues. Intrarelations between XO, SOD, and CAT a c t i v i t i e s w e r e also d i s o r d e r e d . I n t h i s r e g a r d , t h e r e l a tions between XO and SOD, and XO and CAT were p o s i t i v e in b o t h t i s s u e s w h i l e t h e r e l a t i o n b e t w e e n S O D and CAT was negative in cancerous tissue and positive in adjacent tissues. Intercorrelation analysis results carded out between
Table 3. Mean _ SD Values of ADA, 5'NT, XO, SOD, and CAT Activities in Normal, Cancerous, and Cancer-Free Adjacent Tissues from Noncancer and Cancer Patients Grouped According to Chemotherapy Management and Mann-Whitney U-test (#) and Student's t-test Results Between Control and Other Groups (a, b, c, d) and Between x and y Groups (e, f) A (n = 9) ADA
0.99 ± 0.46
5' NT
0.014 z 0.006
XO
0.234 ___0.112
SOD
16.5 ± 4.5
CAT
60.2 ___26.7
B (n = 9) x y x y x y x y x y
24.94 ___ 12.46 b 8.48 e ± 5.34# 0.035 --- 0.022# 0.010 ± 0.006 0.050 --- 0.017 b 0.079 ± 0.034" 0.462 ± 0.225 d 1.024 f _ 0.249 c 42.9 ___ 17.1 40.0 _ 17.5
C (n = 10) 17.00 6.22 0.020 0.012 0.061 0.091 0.796 0.839 30.8 32.9
± 8.57 b ± 3.83# ___0.016 ___0.007 ± 0.017 b ± 0.016 ± 0.414 d ___0.310 d ___ 16.4# ± 9.7#
A: Control group; B: Advanced clinical stage and grade group with chemotherapy; C: Advanced clinical stage and grade group without chemotherapy; x: Cancerous tissue; y: Cancer-free adjacent tissue. a.Op < 0.05. b,fp < 0.01. Cp < 0.005. d p < 0.00005. # p < 0.05 (Mann-Whitney U-test).
Human bladder tissues Table 4. Intracorrelation Coefficients Between Enzyme Activities in Normal, Cancerous, and Cancer-free Adjacent Tissues from Noncancer and Cancer Patients A (n=9) ADA-5' NT
0.87
ADA-XO
0.59
ADA-SOD
0.91
ADA-CAT
0.25 (n.s.) 0.31 (n.s.) 0.90
5'NT-XO 5'NT-SOD 5'NT-CAT XO-SOD XO-CAT SOD-CAT i
0.33 (n.s.) 0.43 (n.s.) 0.79 0.75
B (n=36) x y x y x y x y x y x y x y x y x y x y
+0.25 -0.21 -0.40 -0.20 -0.33 -0.31 -0.30 +0.10 +0.10 +0.32 +0.17 +0.70 +0.47 +0.05 +0.72 +0.85 +0.22 +0.21 -0.17 +0.13
(n.s.) (n.s.) (n.s.)
(n.s.) (n.s.) (n.s.)
(n.s.)
(n.s.) (n.s.) (n.s.) (n.s.)
A: Control group; B: General cancer group; x: Cancerous bladder tissue; y: Noncancerous bladder tissue; n.s.: nonsignificant (p <
0.05).
enzyme activities of cancerous and adjacent tissues exhibited only positive relations.
DISCUSSION
The results of the study presented here exhibited contrast with the results of our previous study in which human cancerous larynx tissue was used for this purpose. The controversy found between two studies indicated that enzyme metabolism in cancer might show great differences depending on cancerous tissues studied, and that mechanisms put forward to explain enzyme changes in carcinogenesis might be specific only for the material analyzed. These different findings also display difficulties encountered in the explanation of enzymatic mechanisms in cancer processes. As to the results of this study, the following conclusions can be made. In this study, we have established that ADA activity increased in cancerous tissues and cancer-free adjacent tissue. Although in several studies, ADA activities were found increased in cancerous tissue ~3-15 and cells, ~6 some researchers measured low activity values in lymphocytes from cancer patients. 17"18 However, Sufrin et al. 16 found that ADA levels of iymphocytes from patients with bladder cancer were also elevated with transitional cell carcinoma and corraleted with stage, activity, clinical course, and tumor resection, but not tumor grade. These researchers found higher erythrocyte ADA activities as well in
829
the same patients and suggested that lymphocyte ADA levels might be a sensitive indicator of bladder carcinoma. However, Dasmahapatra et al. 17 and Kojima et al) s found low lymphocyte ADA activities in head and neck cancer patients and gastric cancer patients, respectively. These researchers suggested that low lymphocyte ADA activities might be a more sensitive indicator of suppressed cellular immunity) 7"18 High ADA activity determined in cancerous bladder tissue reflected accelerated purine turnover and high salvage pathway activity. In fact, it has been established that activities of purine salvage enzymes including ADA were elevated in neoplastic human tissues. 15'33 Although we have no detailed information of the physiological significance of increased ADA activities in cancerous and adjacent tissues at present, it seems to be a secondary phenomenon, which reflects rapid salvage pathway activity of nucleic acid metabolism associated with tissue hyperproliferation. Although some researchers found lower 5NT activity in cancerous tissue, 15'2a we could not find statistically meaningful differences between 5NT activities of cancerous and noncancerous tissues. This difference might arise from the fact that analyses were carded out in different organs and tissues. 5NT activities in cancerous tissues were to some extent higher compared to noncancerous ones in some of the groups of Tables 1 and 2. However, increases in enzyme activity were generally not statistically meaningful. This finding showed that 5NT activity was not a limiting factor in accelerated purine metabolism of cancerous bladder tissue. Regarding the XO activity, Ktko~lu et al.23 found higher enzyme activity in tumoral brain tissues and suggested that the levels of XO in brain tissues could be used as a biochemical marker for the differentiation of tumoral tissues from normal ones. However, several researchers found lower XO activities in cancerous tissues. 15'25"34Natsumeda et al. 33 established decreased XO activities in all hepatomas irrespective of growth rate or differentiation and Weber et al. 26 established decreased XO activities in slowly and rapidly growing
Table 5. Intercorrelation Coefficients Between Enzyme Activities of Cancerous and Cancer-Free Adjacent Tissues from Cancer Patients B
(n = 36) ADA-ADA 5'NT-5'NT XO-XO SOD-SOD CAT-CAT B: Generfl cancer group
0.80 0.39 0.61 0.81 0.21
830
i. DLrRAKet aL
human colon carcinoma xerografts. Our results are in a good agreement with those of Prajda and Weber. 25 Decreased XO activities in human tissues might contribute to accelerated salvage pathway synthesis of purine nucleotides needed by tumor cells. On the one hand, increased ADA activity, and on the other, decreased XO activity, might give selective advantage to cancer cells to grow and develop more rapidly. In this study, we found lower SOD and CAT activities in cancerous tissue and adjacent bladder tissue compared with the activity values of tissues from noncancer patients. In fact, several researchers found decreased SOD and CAT activities in various types of cancer tissues. For example, Vo et al.9 determined low SOD and CAT activities in rat hepatocytes exposed to carcinogenic treatment, and suggested that changes in the level of endogenous defense mechanisms might modify cellular homeostasis and hence might contribute to the promotion of neoplasia. Similar observations were made after treatment of mouse skin with a tumor promoter. 35 Corrocher et al. 36 established decreased CAT activity in human hepatoma and suggested that antioxidant defense system of hepatocellular carcinoma cells was severely impaired. Balgoy and Robe r t s 37 also determined lower SOD levels in transplantable mouse and rat tumors. Similar results were obtained by Bize et al. 38 in Morris Hepatomas, Hoffman et al. 1° in human colorectal cancer, and Reiners et al. 39 in chemically induced skin cancer. However, Nakada et al. 11 found no changes in SOD activities between renal carcinoma cells and tumor-uninvolved renal tissues, and they added that grade and stage of tumors had no apparent effect on SOD activities. Accordingly, Howie et al. 4° established that some enzyme activities participating in free radical metabolism exhibit differences depending on type of organ used. For example, they found higher glutathione peroxidase activities in tumoral human lung, colon, stomach, and breast tissues, and low activities in tumoral kidney and liver tissues from the same subjects. Low SOD and CAT activity results obtained in this study supported general previous observations that enzymatic antioxidant defense mechanisms were impaired in tumor cells and tissues, although this was not a universal characteristic of neoplastic cells. At this stage, two general questions appear regarding the free radical enzyme metabolism in cancer. First, may changes in antioxidant enzyme activities arise from cancer disease itself? Second, may changes in free radical metabolism play a part in the carcinogenic process? It seems quite possible that disordered metabolism in cancer cells and tissues may also lead to inhibition or repression of synthesis of free radical-metabolising enzymes. For example, decreased XO activity, as established in this study, may cause decreased production
of 02"- radical. Steadily decreased 02"- radical production in the cancer cells may lead to suppression of free radical enzymes using 02"- and other oxygen radicals as substrate. Another hypothesis is that impairment of free radical metabolism due to several factors may also accelerate carcinogenic process in the cell. For example, inhibition of SOD, CAT or other free radical enzyme activities with treatment of some chemicals such as 12-O-tetradecanoyl-phorbol-13-acetate as made by Reiners et al.39 or with some toxic elements such as nickel, chromium, etc., may cause inhibition of activities of some free radical-metabolising enzymes, thereby resulting in increased production of free radicals in the cell. Radicalic stress in the cell may then lead to tumor promotion and development process by known mechanisms. Furthermore, it is also possible that some cancer-causing factors may affect free radical-metabolising systems as well. For example, treatment of mouse skin with a tumor promoter may independently lead to both tumor promotion and inhibition, and/or repression of free radical enzymes in skin cells. Neverthless, a vicious cycle can be mentioned about the subject. On the one hand, destroyed free radical metabolism can impaire purine and DNA metabolisms in cancer cells, and on the other, changed metabolism due to cancer process in cancer cells may deteriorate the situation of antioxidant defense system. Negative relations established between ADA and SOD, and ADA and CAT, reveal this negative interaction between free radical and purine metabolisms. From the results of this study, we obtained the following conclusions: 1. In cancerous human bladder cells, purine metabolism and salvage pathway activity (high ADA and low XO activities) were accelerated. 2. Enzymatic antioxidant defense mechanism of bladder cancer cells was impaired (low SOD and CAT activities). 3. There were negative relations between ADA and free radical-metabolising enzyme activities, namely purine metabolism and free radical metabolism in cancerous bladder tissues. 4. Enzyme activity pattern of cancerous and cancerfree adjacent tissues presented partial similarity. 5. In cancerous bladder tissue, no important relations existed between stage and activities of the enzymes mentioned. However, there were positive relations between grade and ADA activity and negative relations between grade and SOD and CAT activities. REFERENCES
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