Antitumor Effect of Two Oral Steroids, Mepitiostane and Fluoxymesterone, on a Pregnancy-Dependent Mouse Mammary Tumor (TPDMT-4)1

[CANCER RESEARCH 37, 4408-4415, December 1977]

Antitumor Effect of Two Oral Steroids, Mepitiostane and Fluoxymesterone, on a Pregnancy-dependent Mouse Mammary Tumor (TPDMT-4)1

Akio Matsuzawa and Tadashi Yamamoto

Laboratory Animal Research Center Â¡A.M.Â¡and Department of Oncology [T. Y.], Institute ot Medical Science, University of Tokyo, P. O. Takanawa, Tokyo 108, Japan

SUMMARY (9). Pituitary glands released from hypothalamic control secreted primarily prolactin, with both luteotropic and Pregnancy-dependent TPDMT-4 mammary tumors, char mammotropic effects (17). Continuous tumor growth also acterized by requiring estrogen, progesterone, and pituitary occurred in virgins given implants of a pellet containing hormones for growth, grew continuously in female ODD 17/3-estradiol and progesterone (9). These experimental mice carrying pituitary isografts. The experimental model systems have provided useful models for studies on endo- was used to investigate the antitumor effects of two p.o. crinotherapy of hormone-dependent mammary tumors. In steroids, mepitiostane and fluoxymesterone. When tumors hosts with ectopie pituitary isografts, the tumors ceased to implanted with pituitary Â¡sograftsinto the fat-pad reached grow and began to regress immediately after treatment palpable size, animals received 6 doses/week of 0.1, 0.3, with epitiostanol [2a,3Â«-epitho-5Â«-androstan-17/3-ol(10275-, 1.0, and 3.0 mg of either steroid intragastrically. Mepitio S)] (10). The steroid also suppressed mammary gland devel stane significantly suppressed tumor growth with regression opment in the hosts. The steroid has been used with some in 25 and 29% of animals at 1.0 and 3.0 mg, respectively, efficacy for treatment of advanced breast cancer (3). A but had no inhibitory effects at other doses. Fluoxymester derivative of the steroid that exerts an antiestrogenic effect one retarded tumor growth during the first week of treat after p.o. administration was synthesized (5); the derivative ment at 3.0 mg but finally had no inhibitory effects at any is mepitiostane [2a,3a-epitho-5a-androstan-170-yl 1-meth- doses. Under similar conditions ovariectomy caused tumor oxycyclopentyl ether (10364-S)]. Mepitiostane has been tried regression immediately, and epitiostanol, the parent steroid with some efficacy in advanced human breast carcinomas of mepitiostane, significantly suppressed tumor growth (6). Fluoxymesterone or 9a-fluoro-11/3, 17/3-dihydroxyan- when given in 3 injections/week of 0.5 mg s.c. Tumors had drost-4-en-3-one is another p.o. steroid that has been ex papillary structures and almost lacked secretory activity. A tensively used for treating human breast carcinomas (4, 7, comparison of these findings to those obtained with earlier 20). Both mepitiostane and fluoxymesterone antagonized generations suggests that TPDMT-4 tumors became less the uterine weight gain and vaginal cornification produced sensitive to the antitumor effect of epitiostanol and were by exogenous estrogen in immature or ovariectomized able to grow at lower hormone levels with succeeding mice, but only mepitiostane inhibited ductal growth of the generations. Morphologically, progression to more cancer mouse mammary gland and growth of hormone-dependent and less secretory activity was noticed. rat mammary tumors (15). This study was conducted to investigate the antitumor effects of mepitiostane and fluoxymesterone on TPDMT-4 INTRODUCTION mammary tumors in pituitary Â¡sograft-bearing hosts. Treat ments with epitiostanol and ovariectomy were included to A very stable, transplantable pregnancy-dependent mam examine possible changes in TPDMT-4 tumors in response mary tumor line designated TPDMT-4 was first successfully to hormones during the course of serial transplantations. isolated in ODD mice, a strain of European origin (8). TPDMT-4 tumors are characterized by growth during preg nancy, rapid regression after parturition in breeders, and MATERIALS AND METHODS virtually no growth in virgins. These mammary tumors are very similar to the normal mammary gland in their response Animals. ODD mice, bred and maintained in the mouse to a variety of hormones. Estrogen and progesterone colony in the Laboratory Animal Research Center, were secreted from the ovary (9) and pituitary hormones includ used in all experiments. Female mice, 6 to 7 weeks old, ing prolactin (11) are essential for TPDMT-4 tumor growth. served as recipients. Pituitary isografts were obtained from The tumors have both cytoplasmic and nuclear estrogen syngeneic male and female mice 2 to 4 months old. The receptors. TPDMT-4 tumors grew without regression in origin and some characteristics of the strain have been syngeneic female mice carrying ectopie pituitary isografts described in detail elsewhere (12). ODD mice originated in Europe, carry a virulent strain of murine mammary tumor 1 Supported by a grant-in-aid for cancer research from the Ministry of virus (MTV-P), and are characterized by rare and late Education, Science, and Culture. Japan. Received May 3, 1977; accepted September 15, 1977. development of mammary tumors. Under the present exper-

4408 CANCER RESEARCH VOL. 37

Â¡mentalconditions, spontaneous mammary tumors did not 80, 0.5% (w/v) carboxymethylcellulose, and 0.9% (v/v) ben affect the results. Fourteen, 15, and 7 animals were allo zyl alcohol and was injected s.c. into the neck region of cated to each group in Experiments 1, 2, and 3. respec the back 3 times weekly. Control and ovariectomized ani tively. Animals were given free access to F1 laboratory mals received s.c. injections of the vehicle alone at the chow (Funabashi Nojo Co. Ltd., Funabashi, Japan) and tap same frequency during the same period. In all experiments water throughout the experimental period. the first treatment day was designated as Day 0. At the Tumors. TPDMT-4 is a transplantable pregnancy-depend termination of treatments, all animals were killed to obtain ent mammary tumor line established from a spontaneous the weights of the body, ovaries, uterus, adrenal glands, mammary tumor of a ODD mouse. The response to hor and thymus. mones and other properties of this particular tumor line Morphology. In Experiments 1 and 2 the tumors and have been described in detail (8, 9, 11). A tumor at trans ovaries were fixed in 10% formalin, processed routinely, plant generation 23 or 24 was obtained from a female, and stained with hematoxylin and eosin for histological close to termination of her second pregnancy, and was cut study. The whole skins from all control and experimental into fragments measuring approximately 2x2x2 mm. A animals were fixed in 10% formalin, and whole mounts of tumor fragment and 3 pituitary isografts were implanted the third mammary glands were prepared as described into the right inguinal fat-pad of each recipient. These elsewhere (13, 14). The extent of lobuloalveolar develop animals were inspected, and the 2 diameters, bisecting ment was classified into 5 grades numbering 0 to 4, accord palpable tumors at a right angle to each other, were deter ing to the criteria described in 'Results." mined twice weekly. The arithmetic mean of the 2 diameters Statistics. The significance of difference in tumor diame was designated as the tumor diameter. Growth curves were ter and tumor, body, and organ weights between each obtained by charting the mean diameters versus time for treated group and the corresponding control was examined each group. by Student's t test. The differences were evaluated as Treatments. In Experiment 1 the antitumor effect of significant at p < 0.05. mepitiostane and fluoxymesterone was examined at daily doses of 0.1, 0.3, and 1.0 mg. The experiment was carried out at transplant generation 23. When tumor sizes had RESULTS reached 4x4 mm to 11 x 11 mm 27 to 30 days after implantation, the animals were randomly divided into 7 Experiment 1. Antitumor Effect of Mepitiostane and Fluox groups of 14 animals each; no significant difference in ymesterone at Small Doses tumor diameters, pretreatment periods (intervals between Each group included 14 animals with palpable tumors at tumor implantation and the start of treatments), and latent the commencement of treatment. However, 1 animal in periods (intervals between tumor implantation and the ap each group was discarded due to unsuccessful intubation pearance of palpable tumors) was noticeable among them. in all groups except for those treated with 0.1 and 0.3 mg Each test dose of steroids was dissolved or suspended in fluoxymesterone in which groups all animals were success 0.1 ml sesame oil and was p.o. administered with a stomach fully treated throughout the experimental period. As indi tube 6 times weekly over a treatment period of 36 days, cated in Table 1, mepitiostane at doses of 0.1 and 0.3 mg starting on the day of animal grouping. Control animals received i.g.2 doses of sesame oil alone. and fluoxymesterone at all doses had no influence on tumor weights and diameters. Although it is not illustrated Experiment 2 was carried out at transplant generation 24 in the charts, the growth curve of the tumor followed to examine antitumor effect of both steroids at a higher virtually the same course in these groups as in the control. daily dose of 3.0 mg. Treatment with 1.0 mg of either Mepitiostane significantly suppressed tumor growth as re steroid was included for reference because the growth of flected by weight and diameter at a dose of 1.0 mg (Table tumors varied to some degree with the tumors that served 1). Tumor regression occurred in 2 animals of this group as sources of tumor grafts (10). When tumor sizes reached (Chart 3). The degree of tumor growth retardation resulting 3x3 mm to 12 x 12 mm after 27 to 32 days, animals were from this dose was similar to that obtained at the same randomly divided into 5 groups of 15 animals each as dose in Experiment 2 and is presented in Chart 1. described in Experiment 1. Treatments were then per formed for 35 days in the same manner and at the same frequency as in Experiment 1. Experiment 2. Antitumor Effect of Mepitiostane and Fluox In Experiment 3 the antitumor effect of epitiostanol and ymesterone at Larger Doses ovariectomy on TPDMT-4 tumors was investigated at trans Each group included 15 animals with palpable tumors at plant generation 24. When tumor sizes reached 3x3 mm the start of treatment. However, 1 animal in each group to 6 x 6 mm after 25 days, animals were randomly divided was discarded due to failure in intubation during the treat into 5 groups of 7 animals each; no significant difference ment period in all groups except in the control group and in tumor diameters and latent periods was noted. A group the group treated with 1.0 mg mepitiostane. At both doses of animals was ovariectomized immediately. Another group was treated with epitiostanol during a 41-day period com mepitiostane gave rise to the same significant suppression of tumor growth (Table 1; Chart 1). Tumor regression mencing on the day of animal grouping. Epitiostanol (0.5 occurred in 5 and 4 animals receiving 1.0 and 3.0 mg mg) was suspended in 0.1 ml of an aqueous solution mepitiostane, respectively (Chart 3). It also occurred in 1 containing 0.9% (w/v) NaCI solution, 0.4% (v/v) polysorbate animal of the group treated with 1.0 mg fluoxymesterone. ! The abbreviation used is: i.g., intragastric. However, there was no significant difference in mean tumor

DECEMBER 1977 4409

diameter and weight between this group and the control as COT-CNI CO CO CMr^ COT^CO co >~ T-:b01-ti Wti ^ti O 0 ^ evidenced in Experiment 1 (Table 1; Chart 1). At a higher ti ti titiâ€¢Â«J- ti00 ti ti titiCO dose, fluoxymesterone inhibited tumor growth especially T-TJ- IV LO CO LOCO Y-COCO O C3) during the first week of treatment, although in the end the CMCMCM N. 'Â«t LO1â€” LOCMLO LO co T-CMâ€¢O- Tâ€”CNI T-CO T- inhibitory effect did not reach a significant level.

-Ã•0 CM CNJ CNJ ThO CO0 LO CO CO Experiment 3. Antitumor Effect of Epitiostanol and Ovar- CO"coC0)1coWcu5cu'C5oi 0ti OOO Oti Oti OOO iectomy tiO) ti ti ti tiÂ»- ti ti titiLO CO|vLO â€¢>Â»â€¢â€¢Â»IV CO 1O) O) 00 Â»~ tvCOCO CO IO COCJ) COIv CO co Iv Experiment 3 was conducted to investigate the possible variation in response of the TPDMT-4 tumor to both treat ments during serial transplantations. At the eighth trans O1â€”tiO CO CO LOti O0)T-. CNJ CO T-^â€”ti CNJti plant generation the tumors ceased to grow and began to ti ti titiO tiCM ti ti titiCO ti IVCMCO CM CO COcoCO T- LO O ,~ regress immediately after ovariectomy or after the start of -Â«a-Tâ€”^ ^ co CM*~ co*-o co â€¢* CDCO^1Â¿tiCOÃ¶S*"coCNÃŒ+1COcoo4-1CMCJ>y- T-.00 T- T- T- *~co T-co CM CNJ T- epitiostanol treatment at daily doses higher than 0.1 mg (10). In the present observation carried out at the 24th generation, ovariectomy also gave rise to immediate tumor Iv00000tiO) fv LO co0 cooco co TJ. 0+1 o o ooti regression in all animals (Chart 2). In contrast, 3 injections/ ti ti titiIO tiCM ti ti titiCNJ week of 0.5 mg epitiostanol s.c. did not cause tumor >-eneoM- O i- IVCO IVCOCO i- CO regression in any animals, although it suppressed tumor cofv M- >- CDT_ CNJLOO O CO growth to a significant degree (Table 1; Chart 2). These facts show that the tumor had become less sensitive to the

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4410 CANCER RESEARCH VOL. 37

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. p.o. Steroids and Hormone-dependent Mammary Tumor antitumor effect of epitiostanol between transplant genera groups were collectively indicated. Pituitary isografts al tions 8 and 24. lowed the attainment of mammary gland pregnant levels in 27 of 28 host animals (Figs. 3 to 5) and the late-pregnant state of Grade 4+ in one-half of these animals (Fig. 5). All Effect of Mepitiostane and Fluoxymesterone on Body and these animals showed significant tumor growth. Neither Organ Weights, Mammary Gland Development, and Ovary 0.1 to 0.3 mg mepitiostane nor 0.1 to 1.0 mg fluoxymester and Tumor Morphology one had a significant effect on mammary gland develop ment and tumor weight. Mammary gland development was Body and Organ Weights (Table 1). Body weight was suppressed by 1 or more grades in about 60 and 50% of ani significantly increased in groups given 1.0 and 3.0 mg mals treated with 1.0 and 3.0 mg mepitiostane, respectively, mepitiostane and 3.0 mg fluoxymesterone. Uterine weight and in about 40% of animals treated with 3.0 mg fluoxy gain was significant in the groups treated with 0.3 to 3.0 mesterone. Significantly, lower tumor weight was recorded mg mepitiostane and 3.0 mg fluoxymesterone, although only in the first 2 groups (Table 1). Tumor regression was the reason for this is unclear. Both steroids gave rise to a mostly observed in animals with virgin mammary glands significant decrease in ovarian weight at all of the doses (Figs. 1 and 2), although it noticeably occurred in a few tested. The decrease was especially remarkable in the animals with mid- to late-pregnant glands. In general the groups receiving 1.0 and 3.0 mg mepitiostane, in which extent of mammary gland development was positively cor significantly suppressed tumor growth and conspicuous related with tumor weight as previously reported (9, 10). degeneration of luteal elements of the ovary were observed. Ovary Morphology. In the pituitary isograft-bearing con Although significant weight loss of the thymus occurred in trols, the ovaries had many apparently active corpora lutea all treated groups, this phenomenon was far more striking and follicles of various developmental stages (Fig. 6). The in the mepitiostane group. The particular action spectrum ovaries, although significantly lower in weight, showed of mepitiostane on these parameters is very similar to that similar morphology in the groups treated with 0.1 mg observed in its parent steroid, epitiostanol (10). mepitiostane and 0.1 or 0.3 mg fluoxymesterone. Stronger Mammary Gland Development. After the observation of all whole-mount preparations, the following criteria were degeneration of luteal components and more decrease in the number of solid, active corpora lutea occurred at higher established to classify the extent of lobuloalveolar develop doses in both steroids (Fig. 7). In particular no apparently ment into 5 classes: 0, the presence of end buds and the active corpora lutea were found in 3 animals receiving 3.0 absence of alveoli; 1, the presence of a few alveoli and few mg fluoxymesterone and in 19 and 12 animals administered lobules (Fig. 1); 2, the presence of many alveoli and a few 1.0 and 3.0 mg mepitiostane, respectively (Fig. 8). Follicu- small lobules (Fig. 2); 3, the presence of a considerable logenesis was not markedly disturbed in any of these number of small and developed lobules (Fig. 3); 4, the animals except for 1 receiving 3.0 mg mepitiostane. The presence of highly developed lobules or complete lobuloal significant decrease in ovary weight observed at all test veolar development (Fig. 4). The gland of Grade 4 is comparable to the mid- to late-pregnant state, and that of doses of both antiestrogens (Table 1) is attributable to their Grade 3 is comparable to the 7- to 10-day-pregnant state. selective effect on luteal components. Epitiostanol, the The mammary glands composed of well-formed, thicker parent compound of mepitiostane, also had a primary effect on these components of the ovary (10). lobules of enlarged alveoli and less empty spaces were Tumor Morphology. Tumors from the pituitary isograft- specificially expressed as Grade 4+ (Fig. 5). The glands of bearing controls were composed of small cuboidal epithe Grades 0 to 2 remained at virgin levels. lial cells. The cells were arranged in 2 or more layers and The state of mammary gland development was graded in lined angular spaces or clefts in most areas (Fig. 9). These relation to tumor weight in all animals (Chart 3). As there epithelial strands were separated by a small amount of was no difference in lobuloalveolar development between 0.1- and 0.3-mg groups in either steroid, the results in both connective tissue and occasionally had papillary projec tions into the spaces (Fig. 10). In minor areas tubular structures were formed by cells of the same type arranged in single rows (Fig. 11). However, tumors had no obvious s'SÃ®Ã¯'5 0 mg(39) iÂ«)O9(22)Omg(64) glandular and acinar structures, and most lumina and OO(IS)(25) 8136) spaces formed were devoid of secretion or fluid (Figs. 9 to â€¢ cPâ€¢0 11). In this respect they presented a sharp contrast to the Â° tumors at earlier generations, which grew under the same 0â„¢â€¢Ã®02) oÃ‡oo* hormonal condition and were characterized by predomi 21 jeO Â°'aÂ«a Ã‡CF0 a(4)00 "(U) rf>00^8 nant glandular and acinar structures with the presence of â€¢lilt30mg(430 0o oO 5>O secreted material in lumina (9. 10). No especially noticeable ,Mfpitiostan*S-.(541(34)cPaP o f1rfi <Ã^ g o1 cPÂ¿ morphological changes occurred in any of the treated 0Controlmâ€¢;3k:'S.' aiFluoxvmvÃ¯tvrontoÃ¬"1Â«o1 â€¢0 1 Ã® 3 &3.0mg(Â¿6)Oo01234 animals. Thus, the peculiar cystadenomatous structure with ORADE OF MAMMARY Gt-ANO DEVELOPMENT much secreted material induced by either epitiostanol or Chart 3. Relationship between final tumor weight and mammary gland development. â€¢.mammary gland development of Grade 4+; A, regressed testosterone at earlier generations (10) was not observed tumor; number in parentheses, percentage of animals classified in the after treatment with epitiostanol, mepitiostane, or fluoxy corresponding grade. In the control. 1.0-mg-mepitiostane. and 1.0-mg-fluox- mesterone at generations 23 and 24 in the present study. ymesterone groups, animals of Experiments 1 and 2 are presented together. Animals in groups treated with 0.1 or 0.3 mg are presented together in both These findings suggest that the tumor cells may have steroids. altered their response to hormones and secretory activity

DECEMBER 1977 4411

during serial transplantations. Tumors were classified as closely related to the antitumor effect in the experimental type B according to criteria of Dunn (1) in all animals. model. However, as evidenced in epitiostanol (10), mepi tiostane might inhibit tumor growth through partial sup DISCUSSION pression of ovary hormone production and direct action on tumor cells, phenomena that are ascribable to its antiestro Epitiostanol is a sulfur-containing steroid exhibiting anti- genic and androgenic activities. With regard to its direct tumor activity on hormone-dependent rat and mouse mam action, epitiostanol, believed to be an active form of mepi mary tumors (10, 22) and on some advanced human breast tiostane in vivo, competitively inhibited the binding of 17/3- cancers (3). Mepitiostane, a derivative of epitiostanol, was estradiol to cytoplasmic estrogen receptors from the tu synthesized to obtain the antitumor effect p.o. (5). The new mors, but fluoxymesterone did not (T. Hori and A. Matsu P.O. steroid also manifested an antitumor effect on hor zawa, unpublished data). mone-dependent rat mammary tumors (15) and gave rise to Ovariectomy gave rise to immediate regression of clinical remission in about one-fourth of the advanced TPDMT-4 tumors in the present experiment (Chart 2) as in breast carcinomas (6). Fluoxymesterone is an p.o. effective, the previous one (10). As for epitiostanol, however, the highly potential androgen and has been used with some result was inconsistent in both experiments. The steroid efficacy for treatment of advanced breast cancer (4, 7, 20). caused tumor regression at the 7th and 8th transplant This study has focused on antitumor effect of both p.o. generation (10), but it only suppressed tumor growth at steroids on the TPDMT-4 tumor. Emphasis also was placed transplant generation 24 (Chart 2). This discrepancy can be on progression of the tumor. TPDMT-4 is a transplantable explained by progression of the tumor cells toward de pregnancy-dependent mammary tumor line characterized creased sensitivity to the antitumor effect in the course of by its requirement for estrogen, progesterone, and pitui successive transplants. The morphological evidence also tary hormones for growth and by growth during pregnancy supports this explanation. Tumor morphology varied with and rapid regression after parturition (8, 9, 11). It can grow generations, although individual cells have remained of the without regression in pseudopregnant mice bearing pitui same cuboidal epithelial type. Tumors were primarily com tary isografts. Pituitary grafts free from hypothalamic con posed of glandular and acinar structures with strong secre trol gave rise to tumor growth and lobuloalveolar develop tory activity at earlier generations (9, 10). In contrast they ment of host mammary glands by producing primarily consisted mainly of papillary structures with little secreted prolactin, with a luteotropic effect on the ovary and a material and cells arranged in multiple layers resulting in mammotropic effect on the mammary gland (10, 17). In this lack of glandular and acinar formation at later generations experimental model, mepitiostane suppressed the tumor (Figs. 9 to 12) and appeared morphologically more malig growth to a significant degree at daily doses of 1.0 and 3.0 nant. However, the content of cytoplasmic estrogen recep mg (Table 1; Chart 1), with tumor regression in some tors determined by the dextran-coated charcoal assay has animals (Chart 3). In contrast, fluoxymesterone caused not changed during this period of time (A. Matsuzawa, T. moderate but insignificant retardation of tumor growth at a Hori, and T. Suzuki, unpublished data). daily dose of 3.0 mg (Table 1; Chart 1). As in the previous The TPDMT-4 tumor line since its isolation has been observations (9, 10), there was positive parallelism between maintained on the criterion of pregnancy-dependent growth tumor growth and mammary gland development; tumor in breeders and no appreciable growth in virgins. This weight tended to be larger in animals with more highly condition had been met at least up to generation 23 (11) at developed mammary glands (Chart 3). At a dose of 1.0 mg, which these experiments were carried out. At generation mepitiostane inhibited both tumor growth and mammary 27 tumor cells remained dormant and did not produce gland development, but fluoxymesterone exhibited no influ significant outgrowths during a period of 3 months in the 6 ence on either of them. In addition, the former suppressed virgins observed. However, tumors grew sporadically in estrogen-stimulated mammary duct growth, whereas the some virgins at the transplant generation 28 and thereafter. latter did not exhibit this effect (15). These findings are In fact, significant tumor growth occurred in 3 of 7 virgins very interesting because this particular tumor line was at generation 29; their sizes reached 4x7 mm, 4x7 mm, thought to be closely related with ductal cells in its origin and 7x8 mm, respectively, 2 months after implantation. (11). The action spectrum of mepitiostane on body and More significantly, these tumors grew irregularly although organ weights (Table 1) and on ovary morphology (Figs. 7 not linearly and regressed occasionally. In breeders, tumors and 8) is very similar to that of epitiostanol (10). Especially even have grown during pregnancy and regressed after marked atrophy of the thymus and degeneration of luteal parturition. Even at earlier generations the tumor was able elements of the ovary are specific to both steroids, support to grow to a small although appreciable size in ODD x C3H ing the hypothesis that mepitiostane may manifest its activ virgins (A. Matsuzawa, unpublished data), which are prob ity after it has reverted to epitiostanol in the body. Recently, ably different from ODD virgins in the levels of hormones this reaction has been proven to occur in the body after that regulate tumor growth. These observations suggest p.o. administration (H. Yoshida, unpublished data). that the TPDMT-4 tumor has gradually progressed so that it Mepitiostane has been demonstrated to have androgenic, is able to grow at lower hormone levels and that it will be antiestrogenic, anabolic, and myotropic activities (15, 16). autonomous finally. Response of tumors grown in virgins These activities were reflected in body weight gain, thymic to ovariectomy and hormones is under investigation. atrophy, uterine weight gain, adrenal involution, and sup Many investigators have reached the consensus that pressed mammary tumor development (Table 1; Chart 3). It hormone-dependent tumors show progression to autonomy is difficult to determine which of the activities was most (2, 18, 19, 21). Generally, the progression occurs rapidly in

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. P.O. Steroids and Hormone-dependent Mammary Tumor murine tumor virus-induced mouse mammary tumors. Many 8. Matsuzawa, A., and Yamamoto, T. A Transplantable Pregnancy-depend ent Mammary Tumor Line (TPDMT-4) in Strain ODD Mice. Gann, 65. hormone-dependent mouse mammary tumors have become 307-315. 1974. autonomous in situ or during several successive transplants 9. Matsuzawa. A., and Yamamoto. T. Response of a Pregnancy-dependent (2, 21). In this respect TPDTM-4 is a very exceptional mouse Mouse Mammary Tumor to Hormones. J. Nati. Cancer Inst., 55: 447- 453, 1975. mammary tumor. Such a slow progression of hormone- 10. Matsuzawa, A., and Yamamoto, T. Inhibited Growth In Vivo of a Mouse dependent tumors to autonomy was reported in rats (18, Pregnancy-dependent Mammary Tumor (TPDMT-4) by an Antiestrogen, 19). A series of studies on TPDMT-4 tumors (9-11) including 2a,3a-Epithio-5a-androstan-17/3-ol (10275-S). Cancer Res., 36. 1598- 1606, 1976. this one indicate the difficulty of predicting what type of 11. Matsuzawa, A., and Yamamoto, T. Lack of Growth of a Pregnancy- endocrine therapy will be most effective against a hormone- dependent Mouse Mammary Tumor (TPDMT-4) in the Absence of Pitui tary Hormones. J. Nati. Cancer Inst., 58: 1087-1091, 1977. dependent breast cancer. 12. Matsuzawa, A., Yamamoto, T., and Suzuki, K. Studies on ODD Mouse with Special Reference to Mammary Gland Tumors. Japan. J. Exptl. Med. ,40: 159-181. 1970. 13. Matsuzawa. A., Yamamoto, T., and Suzuki, K. Pregnancy Dependence ACKNOWLEDGMENTS of Mammary Tumors in Strain ODD Mice. J. Nati. Cancer Inst., 52: 449- 456, 1974. We express our gratitude to Dr. T. Hori of the Shionogi Research 14. Medina, D. Neoplastic Lesions in Mouse Mammary Tumorigenesis. Laboratory for the assistance and advice he extended throughout the Methods Cancer Res., 7: 3-53, 1973. present work. 15. MiyakÃ©,T., Hori, T., Kato, G., Ide, M., Uchida, N., and Yamaguchi, K. 2a,3a-Epithio-5o-androstan-17/3-yl 1-Methoxycyclopentyl Ether (10364-S), a New Orally Active Antiestrogenic Steroid. Steroids, 23: 929-937, 1974. REFERENCES 16. MiyakÃ©,T.,Uchida, K., Kakushi, H., Nomura, Y., Kadowaki, M.. Miyata, K., Hanafusa, T., and Muranaka, R. 2a.3a-Epithio-5a-androstan-170-yl 1. Dunn, T. B. Morphology of Mammary Tumors in Mice. In: F. Homberg 1-Methoxycyclopentyl Ether (10364-S), a New Orally Active Anabolic-An- (ed.), The Physiopathology of Cancer, Ed. 2, pp. 38-84. New York: Paul drogenic Steroid. Japan. J. Pharmacol., 24: 551-558, 1974. B. Hoeber, Inc., 1959. 17. MÃ¼hlbock,O., and Boot, L. M. Induction of Mammary Cancer in Mice 2. Foulds, L. Neoplastic Development. Vol. 1, pp. 41-75. New York: without the Mammary Tumor Agent by Isografts of Hypophyses Cancer Academic Press, Inc., 1969. Res., Ã•9:402-412, 1959. 3. Japanese Cooperative Group of Hormonal Treatment for Breast Cancer 18. Noble, R. L. Hormonal Control of Growth and Progression in Tumors of (Chairman: M. Fujimori). 2a,3a-Epithio-5a-androstan-17/3-ol in the Treat Nb Rats and a Theory of Action. Cancer Res.. 37: 82-94, 1977. ment of Advanced Breast Cancer. Cancer, 31: 789-792. 1973. 19. Noble, R. L., and Hoover, L. A Classification of Transplantable Tumors 4. Kennedy, B. Fluoxymesterone in Treatment of Advanced Breast Cancer. in Nb Rats Controlled by Estrogen from Dormancy to Autonomy. Cancer Cancer, 10: 813-818, 1957. Res.,35: 2935-2941, 1975. 5. Komeno, T., Hayashi, S., Ishihara, S., Itani, H., Iwakura. H., and Takeda, 20. Segaroff, A.. Bowers. C. Y.. Roncone. E. L., Murison, P. J., and K. Studies on 2a,3a-Epithio-5a-androstan-17/3-ol and Related Com SchlÃ¶ssen,J. V. Hormonal Therapy in Cancer of Breast. XIII. The Effect pounds. Shionogi Kenkyusho Nempo, )9: 3-19, 1961. of 9a Fluoro-17a-methyl-A4-androsten-3-one-110,17/j-diol (Fluoxymes 6. Kumaoka, S., Takatani, O., Yoshida, M., Miura, S.. Takao, T., Haman terone) Therapy on Clinical Course and Hormonal Excretion. Cancer, aka. Y., Izuo, M., and Okada, T. 2a,3o-Epithio-5a-androstan-17/3-yl 1- 11: 1187-1189. 1958. Methoxycyclopentyl Ether in the Treatment of Advanced Breast Cancer. 21. Sluyser, M., Evers, S. G., and DeGoeij, C. C. J. Sex Hormone Receptors A Preliminary Clinical Trial. Japan. J. Clin. Oncol., 6: 65-68, 1974. in Mammary Tumors of GR Mice. Nature. 263: 386-389, 1976. 7. Lowe. R., De Lorimier, A. A., Gordan. G. S.. and Goldman, L. Antitumor 22. Yamaguchi, K., Uchida. N.. and Kasai. H. Studies on 2Â«,3a-Epithio-5a- Efficacy of Fluoxymesterone. Use in Advanced Breast Cancer. Arch. androstan-17/3-ol on Mammary Tumors of Rats and Mice. Shionogi Internal Med., 707: 241-244, 1961. Kenkyusho Nempo. 20: 96-100. 1970.

Fig. 1. Mammary gland from pituitary isograft-bearing mouse receiving doses of 1.0 mg mepitiostane i.g. Note the presence of some alveoli and a few small lobules (Grade 1). Hematoxylin, x 3. Fig. 2. Mammary gland from pituitary isograft-bearing mouse receiving doses of 1.0 mepitiostane i.g. Note the presence of many alveoli and a few lobules (Grade 2). Hematoxylin. x 3. Fig. 3. Mammary gland from pituitary isograft-bearing control mouse. Note the presence of many small and developed lobules (Grade 3). Hematoxylin. x 3. Fig. 4. Mammary gland from pituitary isograft-bearing control mouse. Note the presence of highly developed lobules (Grade 4). Hematoxylin. x 3. Fig. 5. Mammary gland from pituitary isograft-bearing control mouse. Note the presence of well-formed, thicker lobules and little empty space (Grade 4+). Hematoxylin, x 3. Fig. 6. Ovary from pituitary isograft-bearing control mouse. Note many large, active corpora lutea and follicles of various developmental stages. H & E. x 40. Fig. 7. Ovary from pituitary isograft-bearing mouse treated with 0.3 mg mepitiostane. Note degenerative changes of luteal components and decrease in number of active corpora lutea. H & E, x 40. Fig. 8. Ovary from pituitary isograft-bearing mouse treated with 1.0 mg mepitiostane. Note the absence of apparently active corpora lutea and the far smaller size than that in Fig. 6. H & E, x 40. Fig. 9. Tumor from pituitary isograft-bearing control mouse Note the presence of many angular spaces or clefts of various sizes and the absence of secreted material in the lumina. H & E, x 40. Fig. 10. Higher magnification of Fig. 9. Note double layers of cuboidal epithelial cells lining angular spaces and papillary projections. H & E. x 200. Fig. 11. Tumor from pituitary isograft-bearing control mouse. Note tubular structures formed by cuboidal epithelial cells in single rows and the absence of secretion in lumina. H & E, x 200. Fig. 12. Tumor from pituitary isograft-bearing control mouse. Note cuboidal epithelial cells arranged randomly in multiple layers without forming glandular and acinar structures. H & E, x 200.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. Antitumor Effect of Two Oral Steroids, Mepitiostane and Fluoxymesterone, on a Pregnancy-dependent Mouse Mammary Tumor (TPDMT-4)

Akio Matsuzawa and Tadashi Yamamoto

Cancer Res 1977;37:4408-4415.

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