Experimental cancer prophylaxis

Exp. Path. 24, 83-89 (1983) Institute for Cancer Research, University of Vienna, Austria

Experimental cancer prophylaxis By H.

WRBA

With 3 figures (Received January 28, 1983)

Address for correspondence: Prof. Dr. Dr. HEINRICH WRBA, Institut fiir Krebsforschung der Universitat Wien, Borschkegasse 8a, A - 1090 Wien, Austria Key wo r ds: cancer prophylaxis, experimental; ENU-induced leukemia; MCA-induced sarcoma; Vitamin A; proteolytic enzymes; ethylnitrosourea (ENU); methylcholanthrene (MCA); mouse

Summary In A-Jax mice, the appearance of MCA-induced sarcomas in delayed by Vitamin A application. Continuous uptake of drinking water containing 0.05 % of a retinol palmitate emulsion leads to a significant inhibition. Administration of ethylnitrosourea (ENU) to pregnant Swiss mice on the 17th day of gravidity results in the formation of lymphoblastoid leukemias from the beginning of the 12th week after birth, 90 % of the animals dying within 25 weeks. In this case, continuous addition of 0.1 % retinol palmitate emulsion to drinking water leads to a dramatic reduction of the tumor risk to about 50 %. By additional administration of proteolytic enzymes of animal and plant origin this risk can be further reduced. The lung adenomas developing to 100 % in the animals are not influenced by this treatment. Vitamin A, added to drinking water, exerts a strong inhibitory effect on transplacentally induced leukemia. Introduction

Based upon an analysis performed by a panel of experts, not later than in 1964 it was published by WHO that at least 3/4of malignant diseases in man are either caused or enhanced by extrinsic factors [20]. As a logical consequence, the majority of human cancer can - at least in theory - be prevented. There is a considerable number of substances and mechanisms known for their potentials to induce transformation, i.e. to transform a normal cell into a potentially malignant one. This primary process takes place in the chromatin of the cell - certainly much more frequently than the actual appearance of malignant tumors. The biological behavior of the cell is determined by subsequent mechanisms of repair and regulation. Manifold factors - partly identified, partly quite unspecific ones - either lead to an increase of the number of malignant cells or prevent promotion. Acting over long periods, these socalled cofactors are able to positively or negatively influence the balance between potentially malignant cells and defence mechanisms of the organism, as is clearly evidenced by epidemiological investigations. These factors are, in many cases, entirely unspec}fic ones such as use of tobacco and alcohol, nutritional habits, occupational situation, etc. For these reasons, the panel of experts mentioned above proposed three different approaches to cancer prophylaxis: 1) This paper is to Professor BOLCK in friendship on the occasion of his 65th birthday_

83

1. Identification and elimination of noxious substances from the environment before they start acting - primary prophylaxis. Molecular biologically viewed, this means prevention of transformation. 2. Prevention of cancer formation - secondary prophylaxis - by keeping transformed cells from multiplication (prevention of promotion). 3. Detection and removal of the disease in an early stage - tertiary prophylaxis (early diagnosis of the malignant disease). Actually, a more or less intense, though continuous and low influence of initiator substances on the organism has to be accepted. On the other hand, there is no possibility of identifying and, subsequently, eliminating all these noxious substances and factors. Measures taken to inhibit induced carcinogenesis seem to be a practical approach for testing effective preventive agents. Experiments of this kind had already been performed in the past; the effect obtained was designated "anticarcinogenesis" by BERENBLUM in 1931 (2). Meanwhile, it has been demonstrated by both experimental and epidemiological investigations that cofactors effective in tumor development during the promotion phase are able to not only exert a positin, i.e. enhancing, effect but also an inhibiting one (11, 17). In recent years, many rather hE'terogeneous substances have been tested; among them, a considerable number has been proven to be chemo-immunoprotective in carcinogenesis. As already stated 30 yrars ago, many very heterogeneous substances exert a certain anti carcinogenic effeet, some of them definitely inhibiting and inactivating the carcinogen on a molecular level (1 ). Classification of the presently known effective substances is not feasible from a chemical point of view. Moreover, the modes of action are very different and partly unclear either. Presently, the following substances are under discussion: Biological response modifiers (a heterogeneous group of substances, comprising small synthetic moleeules ai' well as preparations from microorganisms and undefined large molecules) antioxydant substances antiinflammatory steroids protease inhibitors proteolytic enzymes retinoids (Vitamin A) Vitamin E and, possibly,

Vitamin C. We have been dealing with the effect of retinoids and proteolytic enzymes in cancer prophylaxis for more than 10 years. In the present paper several aspects of the application of both these groups of substances are discussed.

Materials and Methods Experiment 1: Sixty male A-Jax mice, 10 weeks old, were used. At the start of experiment all animals were given 0.25 mg methylcholanthrene (MGA) dissolved in 0.05 ml Trioctanoin by injection into the left calf muscle. 30 animals received a suspension of 0.05 % Vitamin A-Palmitate added to their drinking water for the entire experiment. For this purpose, a commercially available, stable oily suspension was used whose resorption by the intestinal tract can be quantitatively controlled (A-Mulsin Hochkonzentrat®; 300,000 IUJml). For both experimental groups, the averagp weights of the animals were recorded from day 0 to day 65. The anima.ls were kept in cages at 15 mice each and fed standardized pellet food and water ad libitum. The appearance of tumors at the injection site was controlled regularly by palpation and recorded. Dead animals were prepared 'and histologically examined. Experiment 2 In 9 female Swiss miet' of the outbred stock of the Institute for Cancer Research, after mating presence of the vaginal plug was determined. On the 17th day of gravidity the animals were given i.p. 120 mg/kg of ethylnitrosourea (ENU) dissolved in water. 30 male mice of the offspring of these mothers w('re included in thp experiment ,titer weaning on day 30 of their life. 10 animals (group A)

84

served as controls, 10 animals (group B) received Vitamin A-Palmitate 0.1 % in drinking water until the 3rd month (for suspension, see experiment 1). 10 animals (group C) were given Vitamin A as described for group B and, additionally, 1.0 mg of proteolytic enzymes once weekly injected s.c. into the neck. The preparation applied is commercially available, consisting of proteolytic enzymes of both animal and plant origin (Wobe-Mugos®; courtesy of Mucos Emulsionsges. mbH, FRG). For the entire duration of the experiment the animals were housed in cages at 10 mice each and given standard food and water ad libitum. The Swiss strain of the Institute for Cancer Research used in these experiments is very sensitive; without treatment, life expectance is approx. 2 to 3 years, about 30 % of the animals dying of spontaneous lymphoblastoid leukemia, 7 % of adenoma or carcinoma of the lung. After death, all animals were examined both macroscopically and histologically.

Results In male A-Jax animals tumor formation starts after the 10th week from application of 0.25 mg MeA (fig. 1), the first tumors appearing approx. at the same time in both groups, i.e. also in those animals treated with Vitamin A-Palmitate. For the whole time of the experiment, the average weights of the animals of both groups is comparable, the weight of Vitamin A-treated animals even being higher over long periods (fig. 2). Therefore, a feigned effect

fJ ~

.-- ------------ -- -- --,

, ,--,

______ 1

. - - __ 1

i--- B --~ , .e,

~ ~ ~

f==l

, . - - - A _ _.....

,-----'

!

, ,--,, ,--,

60

,

1-------

~-,

.... -_1

I

a::

o

1:

,,--'

::> ....

,-

u..

,i-I ,--,

o

o

WEEKS AFTER MCA APPLICATION

Fig. 1. 60 male A-Jax mice, 10 weeks old. 0.25 mg MCA injected on day O. ---AI - 3o untreated controls, - - - Bl - 3U Vitamin A (0.05 %; A-Mulsin) suspension in drinking water (approx. 33,000 lUI kg/day) from day of MCA applieation to end of experiment .

............../

LL.I

t.:l

«

a::

10

A

2

~

WEEKS AFTER MeA APPLICATION

LL.I

> «

Fig. 2. Comparison of mean body weights of the 2 groups of animals during the whole experiment.

85

~--A--........Jo~

,

.• I

G

-THERAPY----t

,-----I

...---..+----------..!

:

,.~ I B------~ I

;-.-.-., C .-.-.j

______ 1 :-.-._._._._._._.1~ 1

,...-.-.1 1

o

WEEKS

AFTER BIRTH

Fig. 3. Male 8wiss mice. Offspring of mothers treated with 120 mg/kg END on day 17 of pregnancy. In the surviving animals killed after 37 weeks, lung adenomas had developed to 100 %. A: control group, B: 0.1 % Vitamin A ('" 170,000 IU/kg daily) in drinking water, starting three weeks after birth, continued for 12 weeks, C: see B, additionally 1 mg proteolytic enzymes twice;weekly for 12 weeks.

caused by either toxic influence of the substance or by reduced uptake of food due to this influence can be ruled out. Such effects may well lead to an inhibition of tumor growth. From the 30th to the 60th week of the experiment, tumor appearance is significantly delayed in Vitamin A-treated animals, the effect of Vitamin A in drinking water being significant for the entire experiment. In the second experiment, there is a very high rate of lymphoblastoid leukemia in the offspring of mothers having been treated with END on day 17 of gravidity. In mice of group A (fig. 3), leukemia formation starts between the 10th and 15th week, reaching its final stage of 90 %) leukemias after 25 weeks. The leukemia - frequently appearing spontaneously in old animals of this strain - is strongly enhanced by administration of END, being present to a very high percentage even in 12 weeks old animals. There are two different ways of manifestation of the disease: in part of the animals leukosis starts in the thymus which is considerably enlarged, a change made obvious by respiration difficulties of the animals. The second way is a beginning of the disease in the mesenterial and abdominal lymph nodes, leading to an enlargement of lymph nodes in general, with both inguinal and axillary lymph nodes being palpable. The disease - an immature lymphoblastoid leukemia - leads to death within approx. 2 weeks after the appearanee of the first comprehensible symptoms. In this experiment, the addition of Vitamin A to drinking water produced a marked preventive effect even at a very low concentration. After 30 weeks generally no further leukemias arose. When the animals were killed after 37 weeks, leukemias were found in 90 % of the untreated animals. Animals receiving Vitamin A developed leukemias to only 50 %, with a significant delay of appearance. In the group treated additionally with proteolytic enzymes 60 % of the mire were found tumor-free, the leukemia risk thus being reduced for more than one half. Similar to results obtained by therapy experiments, efficiency for prophylactic purposes was optimal when Vitamin A was administered together with proteolytic enzymes. It is worth mentioning that the animals of all groups had adenomas of the lung at the end of the experiment.

86

Discussion To date, a great number of different substances and factors have been described with regard to their efficiency in preventing induced carcinogenesis. The group of biological response modifiers consists of very heterogeneous substances which are characterized by the influence on tumor-host-relations they have in common. These factors are, by far, iiot extensively investigated, the number of substances with potentially identical qualities increasing constantly. We have already succeeded in giving evidence to appropriate activities in various agents. In the maternal share of the bovine placenta - which can be separated easily in its two parts - there is a high molecular factor of specific efficiency (8, 21). Corynebacterium parvum as a preparation suited for injections for immunotherapeutical purposes exerts an identical prophylactic effect against tumor formation (22). In this context, BCG is under discussion as well. Tests conducted with antioxydants in the preventing of chemical carcinogenesis have by now reached a stage allowing considering an active prophylaxis (12, 18), the more, as some of these substances are added occasionally even to food. In experiments, antiinflammatory steroids display a variety of important effects which, especially in combination with other agents, might lead to a prevention of promotion (16). origins has produced a rich literature with clearcut results applying for certain systems (14). There is an increasing number of indications that the rise of Vitamin E level is of importance for cancer prophylaxis (7). In connection with carcinogenesis by at least certain substances, Vitamin C has been proposed to reduce the formation of nitroso-compounds (10). Early clinical results gave rise to great interest in a possible use of proteolytic enzymes and anticoagulants in tumor prophylaxis. In cancer patients who had been given such drugs after primary treatment the rate of metastases was reduced for 50% in comparison to untreated patients under comparable conditions (9, 13). The combination of proteolytic enzymes of animal and plant origin in our own experiments has been effective in the prophylaxis of metastases also when applied clinically (19). The rationale for this application were experimental findings demonstrating a remarkable reduction of frequency of metastases in animals after heparin or fibrinolysin administration (5, 15). The use of the enzyme in the clinical situation suggests an uncovering of the tumor cell as well as a modulation of the cell membrane, inducing not only a reduction of the frequency of metastases but also, presumably, an increase of the efficieney of oncolytic mechanisms. The authors quoted propose that the reduction of the stiekiness of the tumor cells might be responsible for this effect in their experiments (5). Based upon these results we decided to use this preparation in prophylaxis experiments. We consider the increased efficiency of retinoids in cancer prevention obtained by addition of proteolytic enzymes ~ an affirmation. Proteolytic enzymes ~ factors modulating the tumor cell membrane -- exert a positive effect on transformed cells in recognizing and eliminating, at least when combined with retinoids. Not later than at the heginning of the 1960es HOEFER-JANKER pointed to the oncolytic capacity of Vitamin A when applied elinically (6). In 1970, BOLLAG (4) provided the experimental basis. Since then, a large number of substituted retinoids has been clinically tested. Though, no compound showing lower toxicity with better clinical efficacy was found. A preventive effect of Vitamin A in human lung cancer was first reported by BJELKE in 1975 (3) who based his findings on a field study. The relations between the biology of cancer and retinoids has been investigated under many aspects. Immunomodulating effect, enhancement of both differentiation and RNA-synthesis as well as many other actions have been described. The very stable emulsion of Vitamin A-Palmitate we have used in our study offers the decisive advantage of being quantitatively resorbed after 120 minutes, normal conditions given. The effective dose in the serum is indeed corresponding to the dosis applied. Toxic side effects of this preparation have been thoroughly investigated and defined by our group (23).

87

In the present investigation Vitamin A has been applied in a very simple way - by addition to drinking water -, acting continuously as a tumor inhibitor. It seems important that the applieation modality chosen was effective against cells transformed with purpose, inhibiting the promotion with only little inconveniences and continuous action. After having been tested in various systems, the substance must be considered to be of special importance not only because of the lack of toxicity at the doses applied but also because of its mode of application. The lack of toxicity at the effective dose is evidenced by the weight curves. Therefore, no serious reserves were possible even in cases of long-term application. The efficiency of Vitamin A in the prophylaxis of hematological disorders is still controversial and unclear. From this point of view, there are some remarkable aspects in this experiment. First, there is a significant prevention of the developing leukemia. Especially in male animals the inhibition of leukemogenesis is highly significant, reaching more than 50 %. From this, it can be concluded that even transplacentally induced malignant processes can be influenced by subsequent prophylaxis. With special regard to the situation in man, this results is certainly of special interest, since the effect was achieved in a very simple manner (addition to drinking water) in this case, too. Another important aspect is the suppression of leukemogenesis without influencing the formation of lung adenomas. There is no explanation for possible modes of action, though a regulating or differentiating influence of Vitamin A on bone marrow cells might be assumed (23). The third important aspect is eertainly the possibility of combining prophylactically effective agents with different modes of action. It is obvious that proteolytic enzymes - usually ineffective in experiments of this sort - increase the effect in a kind of synprophylaxis when combined with retinoids. Any practical development of prophylactic mechanisms against cancer will certainly require exact analyses of possible combined effects. Similar to other analyses of combination effects of substances, a eonsiderable problem will be presented by the great number of agents with biological response-modifying potential and the variety of possible combinations resulting from it. For these reasons, it is our conviction that chemo-immunoprophylaxis of cancer will be the most promising approach in oncology for the near future.

Lit?rature 1. BAUER, K. H., Das Krebsproblem. Antiblastogenese. Springer, Berlin-Heidelberg 1963, pp. 509-

518.

2. BERENBLUM, 1., The antiearcinogenie aetion of DL-chlorodiethylsulphide (mustard gas). J. Path. Bact. 34, 731-764 (1931). 3. BJELKE, E., Dietary Vitamin A and human lung caneer. Int. J. Cancer 10, 561-565 (1975). 4. BOLLAG, W., Vitamin A and Vitamin A acid in the prophylaxis and therapy of epithelial tumours. Int. Z. Vitaminforsch. 40, 299-314 (1970). 5. CLIFFTON, E., and D. AGOSTINO: Factors affecting the development of metastatic cancer. Effect of alterations in clotting mechanism. Cancer 11>, 276-283 (1962). 6. HOEFER-JANKER, H., F. KHAZNE, and W. SCHEEF: Erste kJinische Erfahrungen mit subtoxischen Vjta,min-A-Dosen im Rahmen der radiologisehen und zytostatischen Tumortherapie. Krebsarzt 24, 203-207 (1969).

7. Ip, C., Dietary Vitamin E·intake and mammary carcinogenesiS in rats. Carcinogenesis 3, 14531456 (1982).

8. LETNANSKY, K., Inhibition of thymidine incorporation into the DNA of normal and neoplastic cells by a factor from bovine maternal placenta: interaction of the inhibitor with cell membranes. Bioscience Rep. 2, 39-45 (1982). 9. MICHAELIS, L., Cancer incidl'nn' and mortality in patients having anticoagulant therapy. Lancet I, 832-835 (1964). 10. MIRVISH, S. S., L. WALLCAVE, :\1. EAGEX and P. SHUBIK, Ascorbate-nitrite-reaction: Possible means of blocking the formation of carcinogenic N-nitroso compounds. Science 177, 65-68 (1972).

88

11. PETO, P., Carcinogenesis as a multistage process - evidence from human studies. In: Host Factors in Human Carcinogenesis, R. BARTSCH & B. ARMSTRONG, eds. IACR Scientific Publication No. 39, Lyon 1982. . 12. RAO, A. R., Inhibitory action of BRA on carcinogenesis in Fl and Ft descendants of mice exposed to DMBA during pregnancy. Int. J. Cancer 30,121-124 (1982). 13. RlEs, J., H. LUDWIG and W. ApPEL, Antikoagulantien bei der Strahlenbehandlung weiblicher Genitalkarzinome. Med. Welt 38, 2042-2046 (1968) . . 14. ROSSMANN, T. G., and W. TROLL: Protease inhibitors in carcinogenesis: Possible site of action. In: Modifiers of Chemical Carcinogenesis: An Approach to Biochemical Mechanisms of Cancer Prevention. Raven Press, N.Y. 1980, pp. 127-143. 15. SCHMAHL, D., Experimentelle Grundlagen der Tumormetastasierung. Krebsforschung und Krebsbekiimpfung VI; 176-188 (1967). 16. SLAGA, T. J., Antiinflammatory steroids: Potent inhibitors of tumor promotion. In: Modifiers of Chemical Carcinogenesis: An Approaeh to the Biochemical Mechanism of Cancer Prevention. Raven Press, N. Y. 1980, pp. 111-12G. 17. SPORN, M. B., and D. L. NEWTON, Chemoprcvention of raneer with retinoids. Fed. Proe. 38, 2528-2534 (1979). 18. WATTENBERG, L. W., Inhibition of chemical carcinogenesis. J. Nat!. Cancer Inst. 60, 11-18 (1978). 19. WOLF, M., and K. RANSBERGER, Enzymtherapy. Invantage Press Corp. N.Y. 1972. 20. World Health Organization: Prevention of Cancer. Technical Report series No. 216, 1964. 21. WRBA, H., Comparative effects of various strains of Corynebacterium parvum and other prophylactic agents on tumor development in animals. In: Corynebacterium Parvum, B. HALPERN, ed., Plenum Publish. Compo N.Y. 1975, pp. 314-318. 22. - Immune prophylaxis of rxperimental cancer by Corynebacterium parvum. Exp. Path. 16, 154-156 (1978). 23. - and A. RIEDER, Retinoids. In: Fortschritte der Krebsforschung. Akademie Verlag Berlin (in press).

7 Exp. Path. 24, H. 2/a

89