[CANCER RESEARCH 49, 3045-3049, June 1. 1989] Effect of Coumarin on the Normal Rat Prostate and on the R-3327H Prostatic Adenocarcinoma1 Bashar Omarbasha, William R. Fair, and Warren D. W. Heston2 Urologie Oncology Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 ABSTRACT Coumarin, the parent compound of warfarin, has been observed to stimulate macrophages, increase phagocytosis, and induce changes in lymphocyte-mitogen responsiveness in cancer patients. Coumarin has been reported to have antitumor activity in human melanomas and renal cancer when used in conjunction with the 11-2 antagonist, cimetidine. We have observed that coumarin has antiprostatic activity in rats. When coumarin was given to mature rats at a dose of 40 mg/kg, a significant decrease in the size of the prostate, seminal vesicles, and testes was observed. Testosterone levels were unchanged or slightly elevated, consistent with an antiandrogenic-like activity. Similarly, cou marin significantly inhibited the androgen-induced increase in prostatic size when administered to castrated rats receiving testosterone. Coumarin given to rats bearing the R-3327H androgen-sensitive, prostate-derived tumor decreased the size of the primary tumor. The effect was greater than that produced by castration. Coumarin is worthy of further consid eration as an agent for use in controlling the normal and abnormal growth of the prostate. INTRODUCTION Coumarin (2//-l-benzopyran-2-one, also 1,2-benzopyrone), the parent compound of warfarin, has been widely used in the past as a flavoring agent in foods and drugs (1, 2) (Fig. 1). Originally obtained as a natural product from tonka beans, commercial coumarin is now prepared synthetically. Coumarin, unlike warfarin, has no antiprothrombin activity. Multiple stud ies on the physiology, pharmacology, and toxicology of cou marin have been reported in the early literature, but it was not until recently that significant immunological and antitumor effects have been reported (2-8). Coumarin has been observed to induce changes in lymphocyte-mitogen responsiveness in cancer patients, and to increase the number and activation state of macrophages (3, 4). Such activities are thought to be of importance in the control of metastasis and destruction of tumor cells (9). The mechanism of the above findings is un known. Coumarin as a single agent or in combination with other immune modulators has demonstrated antitumor activity against human malignant melanoma and metastatic renal cell carcinoma (6-8). Our interest in coumarin originated following multiple experiments which showed that coumarin had signifi cant antiprostatic activity. In preliminary observations pub lished in abstract forms, we reported the results of coumarin treatment in the Mat-LyLu metastatic, anaplastic, androgen- independent, prostate cancer rat animal model (10, 11). Cou- marin-treated groups showed statistically significant decreases in the number and size of metastatic lesions in their lungs. During these studies we observed that coumarin produced a remarkable decrease in size and weight of the male rat testes and sex accessory organs (11). This antiprostatic activity of Received 10/20/88; revised 3/2/89; accepted 3/8/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported in part by Grant PDT-364 from the American Cancer Society. 2To whom requests for reprints should be addressed, at Box 334, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York. NY 10021. coumarin has not been previously described nor characterized. This report further characterizes the effect of coumarin in non- tumor-bearing male rats and rats bearing the Dunning R- 3327H3 prostatic adenocarcinoma. MATERIALS AND METHODS Animals Sprague-Dawley rats were obtained from Sprague-Dawley (Indian apolis, IN), and Copenhagen rats were obtained from the National Cancer Institute. All rats were maintained in the animal facility with a 12-h cycle of light and dark. Rat chow and water were given ad libitum. Chemicals Coumarin was purchased from Sigma Chemical Co. (St. Louis, MO). Coumarin was dissolved in a vehicle containing 10% ethanol, 10% propylene glycol, and 80% normal saline. The control animals received injections of vehicle only, 2.0 ml/animal/day. All injections were given i.p. between 10:00 and 11:00 a.m. Testosterone as the propionate (4- androstene-17/3-ol-3-one propionate) was purchased from Steraloids, Inc. (Manchester, NH), dissolved in sesame oil (Fisher Chemical Co., Fairlawn, NJ), and given 1.0 ml/kg/s.c. in the flank. Serum testosterone concentrations were measured using a '"I-testosterone radioimmuno- assay kit from Radioassay Systems Laboratories, Inc. (Carson, CA) (12). Tumors The R-3327H tumor was provided by Dr. John Isaacs of the Brady Urological Research Institute, John Hopkins Hospital, Baltimore, MD. A single cell suspension containing 1.5 x 10* of the R-3327H tumor cells was injected in the flank, each tumor was measured weekly with microcalipers, and tumor volumes were calculated by the formula (/ x w x H x 0.524) as previously described (13). Treatment Regimens Non-Tumoring-bearing Animals. The initial experiments were de signed to study the dose effect of coumarin on sexually mature, non- tumor-bearing male Sprague-Dawley rats. The animals were randomly assigned to four groups of six rats per group. All rats were of the same age and weighed between 350 g and 370 g. The first group received coumarin, 80 mg/kg/day, i.p.; the second group received 40 mg/kg/ day; the third group received 20 mg/kg/day; the fourth group, 10 mg/ kg/day; and the fifth group received i.p. injections of vehicle only, 20% ethanol and 80% saline, 2.0 ml/animal/day, i.p., and was considered as the control group. The animals were treated for 21 days. On Day 21 all animals were euthanized. None of the rats in any of the treatment groups expired during the experiment. In other protocols 10% of the ethanol was replaced with 10% of propylene glycol. Animal weight, ventral prostate, and testes weight were recorded. A blood sample was obtained via cardiac puncture for determination of serum testosterone levels. Tissue specimens were preserved in formalin for histopatholog- ical studies. The second treatment protocol was performed on castrated rats which received androgen replacement therapy. All rats were castrated 3The abbreviations used are: R-3327H. the androgen-sensitive, well-differen tiated Copenhagen rat transplantable prostate-derived tumor; TP, testosterone propionate; VC, vehicle for dissolving coumarin, 10% ethanol, 10% propylene glycol, and 80% normal saline; VTP, vehicle for dissolving testosterone propio nate and sesame oil. 3045 Research. on October 14, 2021. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from