Journal of Surgical Oncology 22:273-277 (1983)

The Effects and ADR-132

of Cholestyramine, Colestipol, on the Rat Prostate and Dunning -

R-3327 Adenocarcinoma

WILLIAM J. BROWN, MS, JAMES P. KARR, PhD, MICHAEL McGARRY, PhD,

PHYLLIS D. WILLIAMS, BS, LARRY M. HAGERMAN, PhD, AND GERALD P. MURPHY, MD

From the Department of Experimental Surgery, Roswell Park Memorial Institute, Buffalo, New York, and Adria Laboratories, Inc, Plain City, Ohio

The effects of three compounds known to have hypocholesterolemic activ- ity in several species were investigated on the rat prostate and the hor- mone-dependent R-3327 rat prostatic adenocarcinoma. Cholestyramine, colestipol, and ADR- 132 are bile acid-sequestering anion exchange resins which were fed to separate groups of adult male Copenhagen x Fischer (F1) hybrid rats in doses of 0.25%, 1.00%, and 2.00% of diet. The results indicate that serum cholesterol levels in tumor-bearing rats and controls fed these compounds for 29 days were not reduced. The body and organ weights as well as the histological features of the prostate gland, seminal vesicles, and the R-3327 tumor were unaffected by these agents.

KEY WORDS: hypocholesterolemia, rat prostate, R-3327 adenocarcinoma, cholestyramine, colestipol

INTRODUCTION Several reports have indicated that the size of the

prostate gland can be reduced in laboratory animals with benign prostatic hypertrophy by compounds which re- duce the level of cholesterol in the blood [ 1-31. Since the development and function of the prostate gland is depen- dent upon the level of serum testosterone, and since cholesterol is an intermediate metabolite in testosterone biosynthesis, the possibility that these compounds may reduce the levels of testosterone available to the prostate was postulated [2]. Cholestyramine, colestipol, and ADR- 132 are bile acid-sequestering anion exchange resins [4] which reduce serum cholesterol levels by binding bile acid salts in the intestinal tract [5-81. The metabolism of serum cholesterol by the liver is commensurately in- creased to replace the lost bile acid salts [9] .

Gordon and Schaffner first reported a study to test the toxicity of orally administered candicidin, a polyene an- tifungal antibiotic, in 1968 [ 11. Aged dogs with histolog- ically confirmed benign prostatic hyperplasia were fed daily doses varying from 5-20 mglkg body weight. Oral treatment with candicidin for 30 days resulted in diar- rhea, emesis, and loss of appetite. It also resulted in

@ 1983 Alan R. Lm, InC.

marked reduction in granularity, papillations, and con- gestion of the prostate gland, suggesting that candicidin may be an effective oral treatment for benign prostatic hyperplasia. These investigators subsequently reported that candicidin reduced the level of serum cholesterol in groups of male and female beagle dogs aged 10-12 months [2]. These studies established a basis for their hypothesis that cholesterol levels have a direct relation- ship to steroid hormone levels.

Wang and Schaffner subsequently tested the hypothesis that hypocholesterolemic compounds can reduce the size and weight of testosterone-dependent organs [3]. Their studies involved measurement of the effects of candicidin and colestipol on the testes and prostate of bio 87.20 hamsters, animals which develop cystic prostatic hyper- trophy spontaneously [ 101. Administered orally, both candicidin and colestipol resulted in a significant reduc- tion in prostate size and weight at low and high dose levels of 75 mglkg body weightlday and 400 mglkg body weight/day, respectively.

Accepted for publication September 9, 1982. Address reprint requests to Phyllis D. Williams, BS, Department of Experimental Surgery, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263.

274 Brownetal

One widely quoted case of three [3] prostate cancer patients reported in 1972 aroused interest in the potential clinical usefulness of hypocholesterolemic agents in treat- ment of disease of the human prostate. In that report, cholestyramine was claimed to have brought sympto- matic relief by lowering the level of serum cholesterol [ll]. In an effort to test these subjective observations, other investigators examined the effects of cholestyr- amine on androgenic activity in the prostate glands of Wistar rats and concluded that cholestyramine exhibited no ability to inhibit prostatic growth in this species and displayed minimal antiandrogenic effects as judged by the lack of reduction in arginase concentration in the dorsolateral lobes [ 121.

In another clinical setting, the effects of candicidin were measured on patients with benign prostatic hypertrophy scheduled for prostatectomy [ 131. Each patient had been recommended for surgery after rectal examination and excretion urography . The presence of bladder outlet ob- struction was confirmed by a pressure/flow analysis. Other symptoms included diurnal frequency, nocturia, deterioration in stream, hesitancy, and urgency. The pa- tients took 100 mg of candicidin orally each day for six months. Prostatic size was assessed by rectal examina- tion. Urodynamic investigations were performed rou- tinely in order to objectively evaluate the effects of candicidin. The results of this trial failed to provide any evidence to support the claim that candicidin is an effec- tive drug for the treatment of benign prostatic hyper- trophy.

Recently, Resnick and Boyce studied the effects of the hypocholesterolemic drug probucol on the symptoms and objective signs of bladder obstruction in men with benign prostatic hypertrophy [ 141. Seventy-five patients were given probucol in a double-blind manner over a one-year period. Both groups showed a reduction in the mean residual urine volume during the first six months. The improvement disappeared in the treatment group toward the end of the year but persisted in the placebo group to the last visit. No reduction in prostatic size was noted in either group as measured by prostatic ultrasonography . Although there was a dramatic placebo effect, probucol proved ineffective in relieving the symptoms of benign prostatic hypertrophy.

In spite of these mainly negative results, the search continues for useful application of this group of agents in the non-surgical treatment of prostatic disease. For ex- ample, Yamanaka et a1 studied the effects of simfibrate- induced hypolipidemia on testosterone-stimulated pros- tatic growth in castrated rats [15]. Simfibrate induces hypolipidemia by reducing levels of triglycerides and cholesterol in the serum. Rats fed simfibrate were com- pared to controls after 21 days. While the level of serum cholesterol was reduced by simfibrate, no significant

differences were found in the weights of the adrenal, testis, prostate, and seminal vesicles between those ani- mals with induced hypolipidemia and control animals.

The object of the present study was to determine whether bile acid sequesterants (cholestyramine, colesti- pol, and ADR-132) affected the rat R-3327 prostatic adenocarcinoma. We used the slow-growing hormone- dependent rat R-3327 tumor which was first observed as a spontaneous lesion in 1961 by Dr. W.F. Dunning [16]. The tumor, discovered in the dorsolateral lobe of an aged Copenhagen rat, is now in the 17th transfer generation and was provided for this study by the Papanicolaou Cancer Research Center in Miami through the National Prostatic Cancer Project.

MATERIALS AND METHODS Male Copenhagen X Fischer (F1) hybrid rats were

implanted subcutaneously in each flank with 2.0 mm3 of R-3327 tumor. The animals were fed standard pelleted Ralston Purina Rat Chow and water ad libitum. Body weight and tumor volume (L X W X H X 0.5236) measurements were taken weekly [ 171.

The rats were randomly assigned to 12 groups of six animals each and were housed two to a cage. Day 0 of the ex eriment was marked when the tumors had reached 1 cm in volume (tumor implanted for 105 days), at which point one group was sacrificed as baseline con- trols. The rest of the groups were then placed on pow- dered Ralston Purina Certified Rodent Chow for one week. The chemical composition of this diet meets or exceeds the nutrient requirements of the laboratory rat set by the National Academy of Sciences [18]. Another group of six animals was sacrificed as diet controls after seven days on this diet; the remaining rats were allocated to the nine experimental and one control group outlined in Table I.

After 29 days on experimental diets, the rats were anesthesized with ether, and blood samples were taken by cardiac puncture. Serum samples were obtained by centrifugation of the whole blood at 2,OOOg and frozen until analyses for cholesterol were performed at Adria Laboratories using standard autoanalyzer methodology. Tissues were trimmed free of fat coverings, blotted dry,

P

TABLE I. Exwrimental Diet Grouos

Control diet Cholestyramine Certified rodent chow 0.25% of diet

ADR-132 0.25% of diet 1.00% of diet 2.00% of diet

1.00% of diet 2.00% of diet

0.25% of diet 1.00% of diet 2.00% of diet

Colestipol

Hypocholesterolemic Compounds and Rat Prostate 275

influence the level of serum cholesterol in the Copen- hagen X Fischer (F,) hybrid rat. After 29 days on exper- imental diets the mean serum cholesterol level in the control group was 65 f 5 mg/100 ml. This value did not differ significantly from any of the experimental group mean values (Fig. 1).

The ratios of the weights of each accessory sex organ to the animal’s body weight were compared, and again there was no significant difference between the control group and experimental groups. For example, the mean ratio of the ventral prostate weight to body weight for the controls was 0.9724 f 0.121 X This value did not differ significantly from other mean values recorded for the experimental diet groups (Fig. 2). Although the variability within the experimental diet groups was greater in terms of the relative size of their dorsolateral prostate than within the control diet group, their means did not differ significantly from the mean observed in the control group, 0.5750 f 0.160 X (Fig. 3). The control group’s mean ratio of seminal vesicle weight to body weight was 3 f 0.3 x a value which was not significantly different from any of the means of the ex- perimental diet groups (Fig. 4). There was wide variabil- ity in total tumor volume per animal within each experimental group. The mean for the control group total tumor volume was 18 7, the range of which encom- passed all the means of the other experimental diet groups (Fig. 5) .

and weighed. The weights of the body, tumors, ventral prostate, dorsolateral prostate, and seminal vesicles were recorded, and selected tissue specimens were preserved in 10% buffered formalin, imbedded in paraffin, sec- tioned at 6 pm, and stained with hematoxylin and eosin for light microscopy.

Correlation analysis was performed on the levels of serum cholesterol and the weights of the ventral prostate, dorsolateral prostate, seminal vesicles, and tumor vol- ume. Analysis for statistically significant differences in these parameters between control and experimental groups was carried out according to Student’s t-test [ 191.

Accessory sex organ weights were each standardized as a ratio to body weight (tumor weight not included), such that the larger the ratio the greater the relative size of the androgen-dependent organ [20].

RESULTS Diets which contained three concentrations of choles-

tyramine, colestipol, and ADR-132 did not significantly

Serum Cholesterol Levels

80.00

75.00 [T T

T

% L A0

J i rtyromir s

0.4500 0.4400 0.4 300 0.4 200

Ventral Prostate Wt. ’ Of Body Wt.-Tumor Wt.

T

Fig. 1. Serum cholesterol levels in Copenhagen X Fischer (F1) hybrid rats fed varying amounts of cholestyramine, colestipol, or ADR-132. The hypocholesterolemic activity of these compounds does not manifest itself in the rat at the concentrations fed in their diets (see text). Note that only one animal survived the full treatment period in the 2.00% cholestyramine group.

Fig. 2. Index of the ratio of ventral prostate weight to body weight minus tumor weight. Organ weights were normalized in this way to provide a means to compare the activity of testosterone between groups. No significant differences were observed in the ratio of ventral prostate weight to body weight according to Student’s t-test. Note that only one animal survived the full treatment period in the 2.00% cholestyramine group.

276 Brownetal

Seminal Vesicle Wt. Body Wt.-Tumor Wt. X Ratio of Dorsolateral Prostate Wt.

Body Wt.-Tumor Wt. X Ratio of

0.(000 0.9000 0.8OOO 0.7000

(do3) 0.6000 0.5OoO 0.4000 0.3000 0.2000 0.4000 o.oooo 0 0 - N 0 - 0 6 p

$ i u O O i u O o o ~ o g ~ o g 5 3 a p a p a p a p a e a e ~ a p 9 u - u

3 ADR-432 Chobctyomine Colestipol 111111111 f ADR-432 Cholrrtyraminr Cokstipd

Fig. 3. No significant difference was observed in the ratio of dor- solateral prostate weight to body weight minus tumor weight. Note that only one animal survived the full treatment period in the 2.00% cholestyramine group.

Fig. 5 . The growth of the R-3327H tumor was used as a measure of the effects of cholestyramine, colestipol, and ADR-132 on hormone levels in the Copenhagen X Fischer (F,) hybrid rat. There were no significant differences between the means of the group’s total tumor volume. Note that only one animal survived the full treatment period in the 2.00% cholestyramine group.

X of Total Tumor Volume per Animal

trials studied the induction of hypocholesterolemia in relation to heart disease, the dose levels and durations used were similar to those of the current study, which revealed that the three bile acid-sequestering anion ex- change resins used were ineffective in significantly re- ducing the level of serum cholesterol in adult male Copenhagen X Fischer (F1) hybrid rats over a period of 29 days at doses of 0.25%, 1.00%, and 2.00% of diet. It is further unlikely that these compounds could exert a significant influence on the level of serum cholesterol if, as reported, they affect only the low-density lipoprotein levels [9], which represent < 10% of the lipoproteins in the sera of normal rats [21-231. Moreover, increased de novo synthesis of the steroid nucleus by the liver could offset the increased hepatic catabolism of cholesterol to bile acids in rats fed bile acid-sequestering anion ex- change resins [24]. Others have reported that cholestyr-

slight to moderate effect on fat-soluble vitamin utilization

Since the compounds studied were unable to influence the level of serum cholesterol, it is not surprising that they displayed no apparent ability to affect the androgen- dependent sex organs in this species. No statistically significant differences were measured in the weights of these organs or in the volume of the androgen-dependent R-3327 tumor. However, the effects of bile acid seques- terants on serum cholesterol and the effects of hypocho-

O O & p Q - N O - N $ g o g g g O i u o o o w 0 0

Z f ~ a p a p ~ a p a P a p a e g u u u g ADR-132 Cholestymrnine Colrstipol

Fig. 4. The ratios of seminal vesicle weight to body weight minus tumor weight showed no statistically significant differences between groups. Note that only one animal survived the full treatment period in the 2.00% cholestyramine group.

amine had a minimal effect on fecal fat excretion and a

[25-261.

DISCUSSION Bile acid-sequestering anion exchange resins such as

cholestyramine and colestipol have proven to be effective in lowering levels of serum cholesterol in some species including man, cockerels, and dogs [5-81. Although these

Hypocholesterolemic Compounds and Rat Prostate 277

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13. Abrams PH: A double blind trial of the effects of candicidin on patients with benign prostatic hypertrophy. Br J Urol 49:67-71, 1977.

14. Resnick M, Boyce W: Clinical study of the efficacy of probucol in the treatment of benign prostatic hypertrophy. Presented at the 93rd Annual Meeting of the American Association of Genitouri- nary Surgeons, Palm Springs, California, March, 1982.

15. Yamanaka H, Shimazaki J, Koya A, Mayuzumi T, Imai K, Ito Y, Shida K: Effect of hypolipidemia on testosterone stimulated growth in castrated rats. Endocrinol Jpn 24:213-217, 1977.

16. Voight W, Dunning WF: In vivo metabolism of te~tosterone-~H in R3327, an androgen sensitive rat prostatic adenocarcinoma. Cancer Res 34: 1447-1450, 1974.

17. Janik P, Briand P, Hartman N: The effects of estrone-progester- one treatment on cell proliferation kinetics of hormone dependent GR mouse mammary tumors. Cancer Res 35:3698-3704, 1975.

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lesterolemia-inducing compounds on the prostate and its tumors may provide alternative information in species other than the rat tumor model used in this report.

ACKNOWLEDGMENTS This investigation was supported in part by the Frater-

nal Order of the Eagles Cancer Fund, and by the United States Public Health Service grant RR 05648.

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