Enzyme activity and distribution in rat prostatic adenocarcinoma.

The activity and distribution of acid phosphatase, alkaline phosphatase, nonspecific esterases, beta-glucuronidase, and aminopeptidase were examined in the transplantable R-3327 rat prostatic adenocarcinoma and compared with those in the four prostatic lobes of the rat. Both the well and poorly differentiated tumor cells in R-3327 carcinoma were characterized by high activities of beta-glucuronidase and aminopeptidase. The poorly differentiated cells had a high alkaline phosphatase activity. The enzymatic profile of the R-3327 adenocarcinoma closely resembled that of the anterior and dorsal prostate. The origin of the tumor in one of these prostatic lobes is probable, but not certain.

Inhibitory effects of Estracyt on R-3327 rat prostatic carcinoma.

Estracyt (estramustine phosphate) injected intraperitoneally, 100 mg, per Kg. three days a week for four weeks, retarded growth of the R-3327 tumor in intact rats and in orchiectomized rats given androgen. The growth inhibition was accomplished by reduction of tumor deoxyribonucleic acid concentration and of the activities of acid phosphatase, leucine aminopeptidase, and other hydrolases. Histologic examination revealed cellular necrosis particularly prominent in the orchiectomized, androgen-treated rats. Estracyt did not affect the uptake of 65-Zn in the tumors but markedly reduced the high uptake in the dorsolateral prostate. There was no accumulation of 3H or 14C in the tumors after intravenous administration of 3H, 14C-labeled Estracyt, but the isotope concentrations decreased much in the same way as they decreased in the dorsolateral prostate. The isotopes were retained in the ventral prostate, where their concentrations were approximately twenty times higher than those in the muscle four hours after injection. The results demonstrate the value of the R-3327 tumor in the evaluation of drugs of potential clinical use for the treatment of prostatic cancer. The results also show that Estracyt has an antitumor effect which is not dependent on the antigonadotropic action of the drug.

Uptake in vivo of 45Ca in male accessory sex organs of the rat: effect of estramustine phosphate and diethylstilbestrol diphosphate.

The radioactivity following a single i.v. injection of 45Ca into male rats was found to be significantly higher in seminal vesicles, dorsolateral prostate, coagulating glands and ventral prostate than in muscle. The acute effect of a single dose given intraperitoneally of estramustine phosphate (Estracyt) and diethylstilbestrol diphosphate (Honvan) were examined on 45Ca uptake. Generally, the 45Ca concentration in the accessory sex organs with the exception of the dorsolateral prostate increased after administration of estramustine phosphate. Diethylstilbestrol phosphate treatment also significantly increased 45Ca uptake in the ventral prostate when measured on the basis of tissue wet weight but not on the basis of protein. The implications of these findings are discussed in relation to the therapeutic action of Estracyt in prostatic carcinoma.

Regulation of prostatic growth. Growth-limiting mechanisms in the rat ventral prostate.

The possibility that a prostatic chalone is a factor regulating growth of the gland was investigated in rats submitted to hemiprostatectomy to reduce the level of a hypothetical circulating chalone. No evidence of regeneration was found 4 weeks postoperatively. Orchiectomized rats with intrasplenic, intramuscular or intraprostatic implants of ventral prostatic tissue from other orchiectomized rats, were given androgen. The cell divisions in the prostatic tissue did not end until the total cell number was 150-170% of that in control rats. These results indicate that other factors than a prostatic chalone have a dominant role in the mechanism limiting prostatic growth. As introduction of an artificial stroma led to increased prostatic growth, it appears likely that the fibromuscular stroma is one of the factors controlling growth of the prostatic epithelial cells.

Androgen and collagen as growth regulators of the rat ventral prostate.

With the aim of studying a possible role of the fibromuscular stroma as growth regulator for the rat ventral prostate the weight, DNA content, and hydroxyproline content were analyzed in the ventral prostate of rats of varying age. Exogenous androgen increased the age-dependent weight and contents only in young and old rats. Orchiectomy reduced prostatic weight, DNA content, and hydroxyproline content although to different extent, and exogenous androgen restored the weight and contents. Exogenous estrogen to intact animals had effects similar to orchiectomy. Estrogen combined with androgen increased DNA content over that found in animals given androgen only, while weight and hydroxyproline content were not changed. It is concluded that the endogenous androgen determines the size of the prostate in young and old animals. Androgen also determines prostatic collagen content. The good correlation between prostatic DNA and hydroxyproline indicates a more or less fixed number of epithelial cells per amount of collagen. A hypothesis suggesting a crucial role for prostatic collagen in the growth-limiting mechanism in the prostate is presented.

The prognostic value of acid phosphatase and beta-glucuronidase activity in biopsy specimens from patients with reactivated prostatic cancer.

The quotient between the activities of acid phosphatase and beta-glucuronidase in biopsy specimens of malignant prostatic tissue varied among 11 patients with reactivated, estrogen-resistant prostatic carcinoma. No correlation was found between the quotient and the therapeutic response to estramustine phosphate. In biopsy specimens obtained after 2 months' treatment, the quotient was lower in those patients who responded to the treatment. Thus, the quotient before treatment is of no prognostic use but diminution of the quotient found after treatment for 2 months is a sign of good clinical effect of the therapy.

Chemotherapy principles in the treatment of prostatic cancer.

The efficacy of chemotherapy for prostatic cancer is difficult to evaluate owing to the low incidence of measurable indicator lesions and the resulting need for indirect response criteria. Although complete regressions remain exceptional, a number of agents, eg, doxorubicin and cisplatin have been shown to be effective in the treatment of this disease. So far, combinations of effective agents with or without concomitant hormone therapy have not proven to be more effective than single agents. Androgen priming has considerable theoretical appeal and deserves further consideration. A higher effectiveness of chemotherapeutic agents might be obtained by linkage to various carriers. Estramustine phosphate is an example of such a complex that has a cytotoxic effect in test systems in which estrogen has no effect and in patients with hormone-refractory prostatic cancer. The use of hormonal and other carriers that could increase the specificity of chemotherapeutic agents deserves extensive exploration.

Creatine phosphokinase in prostatic tissue.

A histochemical study of creatine phosphokinase (CPK) in the rat ventral prostate revealed that the epithelial cells had a high cytoplasmic activity which was retained or even increased when the cells were atrophic as a consequence of castration. Both the fibromuscular stroma, which is prominent in the ventral prostate of castrated rats, and the secretion had a high level of activity. In human benign prostatic tissue where CPK was present in the epithelium, secretion and stroma, pronounced differences in activity could sometimes be observed between the epithelium of adjacent acini and also between cells in the same acinus. There was no obvious correlation between CPK activity and degree of epithelial hyperplasia. The activity in carcinomatous prostatic epithelial cells varied considerably between specimen from different patients but was uniform within a given specimen in contrast to the variation of epithelial CPK activity within non-carcinomatous tissue. Thus, a difference in the pattern of CPK activity was observed between malignant and non-malignant epithelial cells.

Pilot study on the growth inhibition by estramustine phosphate (Estracyt) of rat mammary tumours sensitive and insensitive of oestrogen.

Rat mammary tumours induced by 7,12-dimethylbenz(a)anthracene had a higher concentration of 3H and 14C than muscle after injection of estramustine phosphate labelled with 3H in the oestrogen moiety and 14C in the alkylating moiety. Thin-layer chromatography showed that dephosphorylated estramustine phosphate was present in the tumours but no free oestradiol-17beta. The uptake of the drug in the tumours was parallelled by a dose dependant retardation of tumour growth and a prevention of tumour number increase. Estramustine phosphate also retarded growth of mammary tumours resistant to treatment with oestradiol-17 beta. It is concluded that estramustine phosphate has a greater effect on tumour growth than oestrogen.

Treatment of prostatitis in the rat.

A spontaneous, nonacute, age-dependent prostatitis was found in a high incidence in the lateral prostatic lobes of Lewis rats. Such rats were treated with methylprednisolone, indometacin , testosterone, hexyloxyphenylproprionate , polyestradiol phosphate, various antibiotics, or were caged together with female rats. The effect of the different treatment modalities was evaluated microscopically by blind observation of the degree of inflammatory reaction in the lateral prostate. Methylprednisolone and the testosterone ester caused a reduction of the inflammatory reactions and so did caging with female rats. The similarity of the rat prostatitis to the human condition may suggest the possibility of using corticosteroids or androgens for the treatment of patients with nonacute prostatitis.

The growth characteristics of metastases from experimental renal tumors.

Wistar-Furth rats and BALB/c mice were implanted with tumor cells from metastases or primaries of an experimental Wilms' tumor or an experimental renal cell adenocarcinoma. The survival time, metastasis formation and growth of the primary tumor after implantation of metastatic tumor cells did not exceed those found after implantation of tumor cells from the tumor primaries. It is concluded that the higher growth rate usually found in metastases is not necessarily due to a selection of metastatic cells with a short cell growth cycle but that there are other environmental factors enhancing the growth of metastases.

Chalones and stroma as possible growth-limiting factors in the rat ventral prostate.

In an attempt to determine the mechanism that limits the growth of the rat ventral prostate, atrophied ventral prostates from orchiectomized inbred rats were implanted into the ventral prostate of other inbred rats. Under the influence of endogenous or exogenous androgen the DNA content of the ventral prostates was increased above normal. This indicates that there is no soluble growth inhibitor diffused in the normal prostate in a concentration sufficient to block cell divisions of the epithelial cells. In other experiments pieces of plastic sponge were implanted into the rat ventral prostate. Orchiectomy and subsequent androgen treatment led to an increased cell number in the prostate. Histologic examination revealed an ingrowth of loose connective tissue and also of normal-looking prostatic tissue in the sponge. The results suggest that implantation of pieces of sponge induced increased growth of the fibromuscular stroma and a secondary proliferation of the epithelial cells. A growth control of the prostatic epithelial cells by the fibromuscular stroma is suggested.

Prostatitis in the rat.

A high incidence of spontaneous, non-acute, age-dependent prostatitis was observed in the lateral prostate of Copenhagen rats and Wistar rats. The lumen of infected acini was filled with polymorphonuclear leucocytes, shed epithelial cells and cell residues. Epithelial cells lining such acini showed degenerative changes. Lymphocytes and macrophages were seen in the stroma. A histochemically observed increase in acid phosphatase and beta-glucuronidase activity in affected epithelial cells may indicate an increased lysosomal activity. Some bacteriological cultures of infected lateral prostates were positive for Proteus vulgaris and diphtheroids. It is suggested that this spontaneous rat prostatitis may be a useful model for the study of the pathogenesis and treatment of human non-acute prostatitis.

Mammary tumour inhibition and subacute toxicity in rats of prednimustine and of its molecular components chlorambucil and prednisolone.

Prednimustine, a chlorambucil ester of pregnisolone, retarded growth of DMBA-induced mammary tumours in rats and reduced the number of tumours. A combination of chlorambucil and prednisolone (C + P) in the same proportion as in prednimustine, had similar effects, 8 and 26 mg per kg of the C + P combination being equipotnet to 16 and 64 mg per kg of prednimustine, respectively. The mortality figures suggested that prednimustine was considerably less toxic than equipotent doses of C + P. This toxicity difference was confirmed in a parallel investigation of the subacute toxicity in rats of prednimustine and C + P. This study showed that the mortality, reduction of lymphocytes and platelets, and bone marrow depression was much lower after pregnimustine than after equimolar amounts of the C + P combination. The results suggest that the low toxicity of prednimustine makes this drug a better cytostatic agent than the C + P combination treatment.

Direct and indirect effects of docosanol (IK.2), the active principle in Tadenan, on the rat prostate.

To better understand the mode of action of Tadenan, a drug used in the treatment of benign prostatic hyperplasia, the effect of its active principle docosanol, IK.2, was investigated in rats. IK.2 had no effects on the weight and histologic appearance of the prostate in intact rats but increased the RNA/DNA quotient in the ventral prostate. The plasma concentrations of luteinizing hormone and testosterone were reduced. In orchiectomized animals IK.2 increased the weight of the prostate and the adrenals. In adrenalectomized, orchiectomized animals IK.2 did not increase prostatic weight but on the contrary caused a further weight reduction. IK.2 had a thymolytic effect in intact rats but not in adrenalectomized rats in which the thymus weight was increased. The results indicate that IK.2 increases adrenal steroid secretion. The supposedly higher concentration of adrenal androgens causes a stimulation of the prostate most easily discernible in orchiectomized animals. The further weight reduction of the ventral prostate in orchiectomized, adrenalectomized animals, and the increased thymus weight in adrenalectomized animals after IK.2 administration may suggest that IK.2 has effects other than the stimulatory effect on the adrenals.

An immunohistochemical technique for localization and semiquantitation of estramustine-binding protein (EMBP) in rat and human prostate.

An immunohistochemical technique for determination of "estramustine-binding protein" (EMBP) in rat prostate is described. The localization and staining intensity of this protein were correlated to prostatic morphological structures in intact animals and at different time intervals after androgen deprivation by castration. EMBP was found almost exclusively in epithelial cells, while the fibromuscular stroma seemed to be negative. Intracellularly, immunostaining was confined to the cytoplasm, but was absent in nuclei. In intact rats, acinar lumina demonstrated heavy immunostaining, indicating secretion of EMBP. Orchiectomy caused a diminution of EMBP expression as well as secretion, suggesting that EMBP synthesis is under androgenic regulation. Human benign hyperplastic and cancerous prostatic specimens were also examined. All human specimens examined so far exhibited positive epithelial staining although of varying intensity. Therefore, this immunohistochemical technique may be used for studying the correlation of EMBP with tumor malignancy grade and for clinical investigations of how various treatments affect EMBP expression in prostatic carcinomas.