H. Michna

Antitumor activity of the antiprogestins ZK 98.299 and RU 38.486 in hormone dependent rat and mouse mammary tumors: Mechanistic studies

SummaryIn the transplantable MXT mammary tumor model of the mouse and in the DMBA- and MNU-induced mammary tumor models of the rat, the progesterone antagonists ZK 98.299 and RU 38.468 were shown to have potent antitumor activity. The weight and/or morphology of the ovaries, uterus, and vagina, as well as the effects on serum hormone levels, indicate that the antitumor activity of both antiprogesterones in these models does not depend on a blockade of the ovarian and pituitary functions and does not depend on a non receptor-mediated cytotoxic effect.On the other hand, the morpholoy of the MXT and the DMBA-induced mammary tumors after treatment with the progesterone antagonists is completely different from that observed after ovariectomy. Treatment with the antiprogesterones seems to trigger differentiation of the mitotically active polygonal tumor cells towards glandular structures and acini with a massive sequestering of secretory products, as well as towards spindle-shaped necrobiotic subpopulations. By contrast, the induction of tumor cell degeneration and cytolysis is the predominant feature of the mammary tumors after ovariectomy.In conclusion, our results indicate that the main mechanism of the antitumor action of antiprogesterones in these models is a direct progesterone receptor-mediated antiproliferative effect at the level of the mammary tumor cells, most probably via the induction of terminal differentiation associated with terminal cell death. This antiproliferative effect seems to be dissociated from the antihormone (antiprogestational) activity of these progesterone antagonists.

The antitumor mechanism of progesterone antagonists is a receptor mediated antiproliferative effect by induction of terminal cell death

The antiprogesterones Onapristone, ZK 112.993 (Schering AG), and Mifepristone (Roussel-Uclaf) proved to possess progesterone receptor-mediated antiproliferative effects in experimental mammary carcinomas. In this study, the potency and mechanism of the antitumor action of Onapristone and ZK 112.993 is characterized by ovariectomized, progestagen and/or estradiol substituted mice bearing hormone-dependent MXT(+) mammary tumours. Medroxyprogesterone acetate (MPA, 0.8 mg/mouse, 3 times weekly, s.c.) could only induce a poor stimulation of tumour growth (% T/C = 40; intact control % T/C = 100), which was only marginally inhibited (% T/C = 21) by Onapristone (0.2 mg/mouse, 6 times weekly, s.c.) during a 6-week therapy. Therefore, the antitumor mechanism of antiprogesterones cannot preferably depend on a classical progesterone antagonism. In contrary, the pronounced stimulation of tumor growth (% T/C = 152) by estradiol benzoate (EB, 0.33 microgram/mouse, 3 times weekly, s.c.) was completely inhibited (% T/C = 7) by the antiprogesterones. An even more stimulated tumour growth was achieved by a combination of EB and MPA (% T/C = 365 using 0.17 mg; % T/C = 225 using 0.8 mg MPA). Onapristone dramatically blocked tumor growth (% T/C = 7) at the lower dose of MPA; no inhibition (% T/C = 203), however, was detected at the higher dose of MPA. These data and a morphological analysis indicate that the potent antitumor activity of the progesterone antagonists depends on the binding to a number of available progesterone receptors high enough to trigger an antiproliferative effect via the induction of terminal differentiation associated with terminal cell death.

Progesterone antagonists: Tumor-inhibiting potential and mechanism of action☆

A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical anti-hormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the G0G1 phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G1 arrest has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.

Differentiation Therapy with Progesterone Antagonists

It is widely and readily accepted that a receptor antagonist may antagonize the functions of the specific receptor; therefore, it would be surprizing if a pure receptor antagonist possessed the ability to induce some of the physiological functions of this receptor: we will demonstrate that progesterone antagonists may indeed succeed in inducing the physiological function of the progesterone receptor. In females this function is accepted as being differentiation in steroid hormone-dependent tissue; in males the function has been neglected so far, although progesterone receptors have been localized in the male genital tract of different species.’-3

The antiovulatory effect of the antiprogestin onapristone could be related to down-regulation of intraovarian progesterone (receptors).

The present study was undertaken to investigate intraovarian mechanism(s) for the antiovulatory effect of Onapristone (ON), because antiprogestins possessing the same antiprogestational activity and inhibiting the preovulatory LH surge to the same extent differ in their antiovulatory potency. Ovulation was induced by treating immature female rats with pregnant mare serum gonadotropin (PMSG) for folliculogenesis and hCG for the induction of ovulation. The animals were treated twice with ON (200 mg/kg 42 h and 48 h after PMSG) and killed at different times. The ovulation rate was assessed by counting the number of ova in the fallopian tubes and uteri. Blood and ovaries were collected for radioimmunoassay (RIA) of steroid hormones and histological analysis for 3beta-hydroxysteroid dehydrogenase (3beta-HSDH), 17beta-hydroxysteroid dehydrogenase (17beta-HSDH), progesterone (PR), estrogen (ER) and androgen (AR) receptors. Treatment with ON totally blocked ovulation and the progesterone (P4) surge was significantly diminished in comparison to the control (6-8 h post-hCG), whereas androgen levels remained unaffected. The decreased P4 concentrations correlated well with a reduced staining intensity of 3beta-HSDH in granulosa cells of tertiary follicles. Moreover, we observed a down-regulation of PR in granulosa cells of tertiary follicles. Additionally, in secondary and tertiary follicles the expression of AR between 0 and 6 h after hCG was reduced. These results suggest that the antiovulatory effect of the antiprogestin ON is related to down-regulation of intraovarian progesterone, caused by attenuated 3beta-HSDH activity and PR expression. One can thus assume that intraovarian P4 is an important factor for the induction of ovulation. An effect of ON on the staining intensity of 17beta-HSDH in theca and granulosa cells could not be observed at any time. In conclusion, the inhibition of ovulation induced by the antiprogestin, ON, could be related to decreased intraovarian progesterone production through reduced 3beta-HSDH activity and the down-regulation of PR.

The antitumor potency of progesterone antagonists is due to their differentiation potential

A new therapy for the progesterone receptor positive mammary carcinoma may be the treatment with progesterone antagonists. This new class of antihormones causes a strong inhibition of tumor growth comparable to the potency of ovariectomy in a panel of experimental mammary carcinomas. The mechanisms of the strong tumor-inhibiting action of progesterone antagonists on experimental mammary carcinomas mainly depends on a progesterone receptor mediated process leading to induction of terminal differentiation and a blockade of the cell cycle. To further characterize the antitumor mechanism of progesterone antagonists we analyzed the effects of Onapristone and ZK 112.993 on DMBA- and MNU-mammary tumors of the rat and MXT-tumors of the mouse after different therapy intervals. These hormone-dependent mammary tumors normally display intraductal growth in papillary, cribiform or solid formation, whereas after treatment periods of 2-6 weeks with progesterone antagonists they displayed dysplastic ductal and acinous formations, usually filled with secretory material. Whereas tumor size, mitotic index, and the grade of tumor malignancy decreased distinctly, the volume fraction of glandular structures in the tumors as well as the appearance of apoptosis increased 3-fold compared to the controls. In addition, the mammary glands of progesterone antagonist treated animals showed the morphological features of differentiation with the appearance of secretory activity. Interestingly, the staining pattern of some of the lectins used, especially UEA 1 binding pattern, fits to the concept of differentiation since recent studies revealed a higher degree of fucosylation only in benign lesions of human breast cancers. Therefore, these data underline the concept of a differentiation potential of progesterone antagonists on progesterone receptor positive mammary carcinomas.

A bioassay for the evaluation of antiproliferative potencies of progesterone antagonists

A bioassay which allows quantification of the antiproliferative potency of progesterone antagonists on the mammary gland was developed. For this purpose, ovariectomized rats were substituted with oestrone and progesterone and a further group simultaneously treated with the progesterone antagonists Mifepristone (= RU 38.468), Onapristone (= ZK 98.299), or ZK 112.993 (Schering AG, Berlin). A morphometric analysis of the tubulo-alveolar buds in the inguinal mammary glands revealed a dramatic antiproliferative effect of the progesterone antagonists after as little as 3 days of treatment. Several less specific mammary gland growth parameters (weight, DNA- and RNA-content) proved to be less sensitive. This bioassay measures the potency of progesterone antagonists to competitively antagonize the specific effects of progesterone on the target organ mammary gland. Further advantages of this bioassay are the use of a hormonally standardized biological system, the quantitative results, the small amount of test compound necessary, as well as the substitution with progesterone and oestrone since the antiproliferative potency of progesterone antagonists on experimental hormone dependent mammary carcinomas is most potently displayed in ovariectomized animals substituted with both sex hormones.

Morphology of the rat uterus after long-term treatment with progesterone antagonists

The effects of long-term treatment with the progesterone antagonists ZK 98.299 and ZK 112.993 on the uterus of intact mature rats were investigated with light and electron microscopy. After 3-4 weeks treatment with both progesterone antagonists, the uterine luminal epithelium showed ongoing mitotic activity, increased apoptosis and invasion by granulocytes. Many uteri showed metaplastic areas with stratified squamous epithelium. Basically, the same changes occurred, but to a lesser extent, in the glandular epithelium. At the ultrastructural level, the epithelial cells displayed the morphological features of a certain degree of differentiation. The dissociation of collagen fibres, infiltration by granulocytes and dilatation of small vessels were observed in the subepithelial connective tissue. The myometrium increased in thickness and electron microscopic examination revealed hypertrophic myocytes with a well developed granular endoplasmic reticulum. Most of the morphological reactions may be regarded as due to the direct inhibitory action of progesterone antagonists at the level of the different uterine tissues and the resulting unopposed action of estrogen. The metaplastic changes and the suppression of the anti-proliferative action of progesterone on uterine epithelial cells should be taken into account when treating women in their reproductive years with these drugs for long periods of time, as may be necessary for the endocrine treatment of mammary cancer and endometriosis.

The tumour-inhibiting potential of the progesterone antagonist Onapristone in the human mammary carcinoma T61 in nude mice

SummaryThe progesterone antagonist Onapristone proved to possess strong tumour-inhibiting activity in a panel of experimental mammary carcinomas. Its underlying mechanism of action is due to a progesterone-receptor-mediated induction of terminal differentiation and a specific blockade of the cell cycle and is also present in the absence of progesterone as was shown in the MXT mammary tumour. To prove this further, the tumour-inhibiting activity of Onapristone was investigated in the human postmenopausal T61 mammary tumour implanted in castrated male nude mice. Whereas Onapristone given alone had no effect on growth of established tumours, after stimulation of the relatively low progesterone receptor content of this tumour line with an oestrogen, Onapristone significantly inhibited tumour growth. Thus, we suggest that Onapristone exerts its antitumour action via progesterone receptors. As there is no endogenous progesterone in these mice, the tumour-inhibiting activity of Onapristone is not primarily due to a classical antihormonal effect.

Antitumor effect of a specific aromatase inhibitor, 1 -methyl-androsta-1,4-diene-3,17-dione (Atamestane), in female rats bearing DMBA-induced mammary tumors

Atamestane is a potent competitive inhibitor of estrogen biosynthesis (aromatase) in several species, in vitro and in vivo, and has no endocrine side effects. In this study, the efficacy of atamestane in suppressing tumor growth was evaluated in comparing with that of a non-steroidal aromatase inhibitor (CGS 16949A, Ciba-Geigy) and ovariectomy. Female Sprague-Dawley rats bearing DMBA-tumors were treated s.c. once daily either with 30 or 150 mg/kg atamestane or with 0.1 or 0.5 mg/kg CGS 16949A for 4 weeks. At these biologically equivalent doses both aromatase inhibitors effectively inhibited tumor growth: at the end of treatment they caused a marked reduction in tumor size (up to 70%), while ovariectomy led to a complete remission of tumor growth. The histo-morphological pictures of the mammary tumors from treated animals were qualitatively almost similar to those of the control. In hosts, neither compound exerted any influence on the weight of genital organs (ovary, uterus and vagina), although the peripheral LH levels were significantly elevated by the higher dose of the aromatase inhibitors. This effect on LH levels is probably due to the elimination of the negative feed-back effect of estrogens on gonadotropin secretion (counter regulation). The serum prolactin levels were decreased by the aromatase inhibitors, indicating a diminution of estrogen levels in the treated animals. The present results clearly demonstrate that, in spite of the counter regulation, a pure aromatase inhibitor such as atamestane in sufficiently high doses is able to inhibit the growth of DMBA-induced mammary tumors in intact female rats.