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Androgen ablation therapy induces apoptosis only in androgen-sensitive prostate cancer cells; therefore, other cytotoxic drugs are being used to induce apoptosis in androgen-refractory cells. Mifepristone, an antiprogestin used individually or together with the antiestrogen Tamoxifen, has been recommended for induction of cell death and treatment of several hormonal cancers. However, little is known about the mechanism of action of these drugs in prostate cancer. Therefore, we investigated the effect of Mifepristone on the tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) pathway, a newly identified and very effective member of tumor necrosis factor-alpha family. Mifepristone and Tamoxifen induced significant expression of death receptors in prostate cancer cells in vitro and in xenografts. However, Mifepristone in combination with Tamoxifen did not increase prostate cancer cell death compared with their individual values. The involvement of the TRAIL pathway was further confirmed by the activation of caspase-8 in Mifepristone-treated cells. This was followed by truncation of Bid, confirming that Mifepristone activates the TRAIL pathway. This knowledge is being used to design a combination treatment of TRAIL and Mifepristone to induce significant apoptosis in prostate cancer cells.

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BACKGROUND: Published data indicate that antiprogestins and antiestrogens could inhibit prostate cancer cell growth in vitro and in vivo. The main objective of the present studies was to explore the role of bcl(2) and TGFbeta(1) for induction of apoptosis in LNCaP prostate cancer cells growing in culture as a treatment response to the antiprogestin, mifepristone, and the antiestrogen, 4-hydroxytamoxifen. METHODS: In vitro cell viability (cytotoxicity), DNA fragmentation, and changes in the expression of bcl(2) and TGFbeta(1) proteins were assessed using the sulforhodamine B protein dye-binding assay, specific ELISA, and competitive inhibition assays. RESULTS: Both steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of viable cells was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl(2), and induction of TGFbeta(1) protein. Abrogation of the mifepristone- and 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta(1)-neutralizing antibody and by the addition of mannose-6-phosphate confirmed the correlation between induction of active TGFbeta(1) and subsequent prostate cancer cell death. The effect of mifepristone was not significantly reduced or prevented by occupying the progesterone or glucocorticoid receptors by their corresponding high-affinity native ligands. On the contrary, the effect of a combination of mifepristone with progesterone or hydrocortisone on the increase in DNA fragmentation, bcl(2) downregulation, and induction of TGFbeta(1) protein was additive and significantly different (P < 0.05) from the effect of mifepristone monotherapy. CONCLUSIONS: Our data suggest that mifepristone and tamoxifen are effective inducers of apoptosis and may represent nonandrogen-ablation, novel therapeutic approaches to overcome a potential intrinsic apoptosis resistance of androgen-independent prostate cancer cells.

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BACKGROUND: Antiprogestins are a promising new class of mammary tumor inhibitors with a unique mechanism of action. Previously published results also suggest a tumor-inhibitory effect of antiprogestins in prostate cancer models. The objective of the present studies was to determine whether androgen-sensitive and androgen-insensitive variants of the well-characterized LNCaP human prostate cancer cell line exhibit stable differences in their sensitivity to in vivo antitumor activity of the antiprogestin, mifepristone. METHODS: Exponentially growing LNCaP, LNCaP-C4, and LNCaP-C4-2 prostate cancer cells in culture were mixed with Matrigel and injected subcutaneously (s.c.) into the flank of 6-8-week-old male nude mice. The tumors were permitted to grow until they reached a volume of 270-300 mm(3). The animals were then randomly assigned to two groups. One group received mifepristone (50 mg/kg/day s.c.). Control animals were treated with vehicle. Tumor volume was determined every 4 days. After 28 days of treatment, the tumors were harvested and wet weights were determined. RESULTS: The inoculated tumor cells produced progressively growing tumors in male nude mice. However, the androgen-insensitive LNCaP-C4-2 cells showed the most aggressive and most rapid growth rate and shortest time to tumor progression. The tumors derived from the LNCaP-C4 cells exhibited a higher rate of tumor growth as compared with those derived from the parental androgen-sensitive LNCaP cells. In all three models, mifepristone treatment caused a significant retardation of tumor progression: after 28 days of treatment, about 50% inhibition of tumor weight was observed in the mifepristone treatment groups (P < 0.05) compared with the corresponding control groups. CONCLUSIONS: This is the first report demonstrating significant antitumor activity of mifepristone in both androgen-sensitive and androgen-insensitive variants of the LNCaP human prostate cancer model in nude mice. These results suggest a potential clinical benefit of the use of antiprogestins as a novel nonandrogen ablation therapeutic approach in the management of prostate cancer.

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This is the first report demonstrating an in vivo antitumor activity of antiprogestins (mifepristone, onapristone) alone and in combination with tamoxifen in the MCF-7 human breast cancer model. The MCF-7 cells produced progressive growing tumors in female nude mice supplemented with 17beta-estradiol. Tumor regression was observed following either estrogen ablation alone or estrogen ablation in combination with tamoxifen. Monotherapy with tamoxifen or antiprogestins caused a retardation of estrogen-induced tumor progression. Complete inhibition or prevention of tumor growth occurred as a result of simultaneous administration of mifepristone and tamoxifen. The addition of mifepristone in this combination treatment was also effective in delaying or preventing tumor escape (relapse) from the antiestrogenic (antitumor) effect of tamoxifen. These results suggest a potential clinical benefit of adding an antiprogestin to antiestrogen therapy of breast cancer patients.

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MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.

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In 1993, medical therapy for BPH is a reality. Androgen deprivation therapy (LHRH agonists, antiandrogens, and 5 alpha-reductase inhibitors) has been shown to be effective by reducing the static component of BPH. Of these agents, the 5 alpha-reductase inhibitors have the greatest promise because of their low toxicity profile. Aromatase inhibitors, which function via a different mechanism, however, have not been demonstrated, as monotherapy, to be effective in the treatment of symptomatic BPH. It is still theoretically possible that aromatase inhibitors could have a role in the management of prostatism if they are utilized in conjunction with an antiandrogen or 5 alpha-reductase inhibitor. Although the early results for this endocrine therapies are encouraging, several issues relating to medical treatment remain unanswered. Not everyone with significant prostatism will respond to androgen deprivation therapy. How does the physician identify pre-treatment the patient most likely to achieve a significant improvement in voiding function with 5 alpha-reductase inhibitor therapy? At the present time, there is no method--based on symptoms, DRE findings, serum hormone and PSA levels, or histopathologic criteria--for recognizing the ideal patient for androgen deprivation therapy. Without question, it is a subjective decision. As a result, the benefits and risks of these medical approaches as well as those of the minimally invasive and surgical therapies must be discussed with the patient so that he can participate in the management decision. In this manner, the expectations and needs of the patient will be best served.

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The pathogenesis of human benign prostatic hyperplasia (BPH) has not been fully elucidated. There is, however, evidence that estrogens--besides other factors--might play an important role for the growth of the prostate. Consequently, estrogen deprivation might be a new, useful principle for a conservative treatment of BPH. Atamestane, a new, highly selective steroidal aromatase inhibitor has been proven to be successful in antagonizing experimentally-induced estrogen-related stromal overgrowth of the prostate in dogs and monkeys. Double-blind placebo controlled studies are now underway in Europe and the U.S.A. It is anticipated that these studies will give us a definite answer of the clinical validity of this concept in BPH patients in the near future. However, it is very important to take into consideration that for an effective treatment of BPH, a reduction of both the glandular and stromal elements has to be achieved. In other words, both androgens and estrogens seem to be involved in the regulation of (over)growth of the prostate. Therefore, a combination of an androgen and estrogen deprivation might be a more promising approach than any single treatment.

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Benign prostatic hyperplasia (BPH) is the most common neoplastic growth in men and is the most frequent cause of urinary flow obstruction at the bladder neck. In addition to the clear evidence in favor of the androgen dependency of BPH, the involvement of the stroma, stromal-epithelial interaction and the role of estrogens have gained much interest in connection with the pathogenesis of this disease. For this reason, specific aromatase inhibitors such as atamestane (1-methyl-1,4-androstadiene-3,17-dione) have recently attracted attention due to their potential use in the treatment of BPH. The pharmacological action of atamestane as a new competitive and irreversible inhibitor of estrogen biosynthesis has been evaluated in mice, rats, rabbits, dogs, monkeys and in man. In all species tested so far, atamestane lacks other intrinsic hormonal or antihormonal activities and shows no inhibition of other cytochrome-P450 dependent enzymes of adrenal steroidogenesis. However, it inhibits the estrogen-related negative feed-back. The extent and consequence of the induced counter-regulation of the pituitary-hypothalamic axis show major sex- and species-specific differences. In BPH animal models, atamestane is highly effective in inhibiting estrogen-induced hyperplastic changes in the fibromuscular stroma of the prostate in androstenedione-treated dogs and monkeys. In male volunteers and BPH patients, atamestane induces an expected dose-dependent reduction of serum estrogen concentrations with slight increases in androgen level. In conclusion, all available results indicate that atamestane is a selective (no inhibition of adrenal function), pure (= specific--no endocrine side-effects) and highly effective steroidal aromatase inhibitor with excellent safety profile. Based on our preliminary results aromatase inhibitors seem to be promising compounds for the treatment of BPH.

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Today, human benign prostatic hyperplasia (BPH) is considered primarily to be a disease of the stroma, in which estrogens are thought to play a considerable causative or permissive role. The growing incidence of BPH with increasing age coincides with a shift in the androgen:estrogen ratio in favor of estrogens, not only in terms of serum hormone values, but also in the prostate itself. Furthermore, evidence has been provided for a preferential accumulation of estrogens in the stroma of human hyperplastic tissue, and the presence of an estrogen receptor satisfying the classical criteria of high affinity and low capacity has been demonstrated. Also, animal studies have emphasized the potential role of estrogens in the pathogenesis of BPH. Experimentally, stimulation of the stroma, particularly of smooth muscle, can be induced by aromatizable substrates, such as androstenedione, in the prostates of beagles and cynomolgus monkeys. These effects can be antagonized by aromatase inhibitors, such as atamestane. In addition, the increase in intraprostatic estrogen concentrations and immunohistochemically detectable estrogen receptor content induced by androstenedione in intact dogs is completely reversed by simultaneous treatment with atamestane. In conclusion, clinical data, as well as that from animal models, emphasize an important role for estrogens in the development of BPH. Estrogen deprivation might, therefore, represent a useful treatment for human BPH.

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Atamestane is a new, competitive, and irreversible inhibitor of estrogen biosynthesis. Its pharmacologic action has been evaluated in mice, rats, rabbits, dogs, monkeys, and humans. In rats, atamestane leads to a decrease of pregnant mare serum gonadotropin-stimulated ovarian estrogen production, and inhibits androstenedione-induced estrogenic effects such as uterine growth and abortion. In all species tested, atamestane lacks other intrinsic hormonal or antihormonal activities, and shows no inhibition of other cytochrome P450-dependent enzymes of adrenal steroidogenesis. However, it inhibits estrogen-related negative feedback. The extent and consequences of the induced counterregulation of the pituitary-hypothalamic axis show major sex- and species-specific differences. Atamestane is highly effective in inhibiting estrogen-induced hyperplastic changes in the fibromuscular stroma of the prostate in androstenedione-treated dogs and monkeys. In male volunteers and patients with benign prostatic hyperplasia (BPH), atamestane induces an expected reduction of serum (and BPH tissue) estrogen concentrations without significant changes in androgen levels. In conclusion, all available results indicate that atamestane is a selective (no inhibition of adrenal function), pure (no endocrine side effects), and highly effective steroidal aromatase inhibitor, with an excellent safety profile. Based on the discussion of its clinical potential, atamestane seems to be a promising compound for the management of BPH.

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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.

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Onapristone and other antiprogestins proved to possess a potent antitumor activity in several hormone-dependent experimental breast cancer models. This activity is as strong or even better than that of tamoxifen or ovariectomy in the MXT-mammary tumor of the mouse and the DMBA-and MNU-induced mammary tumor of the rat. The antitumor activity is evident in these models in spite of elevated serum levels of ovarian and pituitary hormones. The detailed analysis of all our data including the morphological (ultrastructure) studies of the mammary tumors of treated animals and the effects on growth and cell cycle kinetics using DNA flow cytometry indicates that the antitumor action of antiprogestins is mediated via the progesterone receptor and related to the induction of terminal cell differentiation leading to increased cell death. The strong antitumor activity of antiprogestins in our experimental breast cancer models does not primarily depend on a classical antihormonal mechanism. The antiprogestin-related reduction of the number of mammary tumor cells in the S-phase in our experimental tumor models (G0G1 arrest) emphasizes the unique innovative mechanism of action of these new agents in the treatment of human breast cancer.

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UNLABELLED: The ability of the new LHRH antagonist antide to induce a long-term chemical castration in adult male rats and cynomolgus monkeys was investigated. The animals were treated subcutaneously with different doses either once or on 5 consecutive days. The effects on serum concentration of LH (only rat) and testosterone and on the weights of the testes, prostates and seminal vesicles were investigated after different periods of time. Histological evaluation of testes, pituitary and hypothalamus was also performed. In the rat a clear dose-dependent inhibitory effect on the above mentioned parameters was observed whereby long-lasting castration-like effects were achieved at concentrations between 6 (less than or equal to 8 weeks) and 15 mg/kg (greater than 8 weeks). In the cynomolgus monkey a prolonged inhibitory effect was induced only at 15 mg/kg and the duration was only 2-3 weeks. Histologically, no signs indicative of irreversible effects were observed in either species. IN CONCLUSION: although species differences became evident in terms of the duration of a long-lasting inhibitory effect on the male reproductive system, antide exhibited such an effect in the rat and the monkey and was able to induce a chemical castration in both species. In addition, using the rat Dunning R 3327 prostatic carcinoma model, 10 mg/kg antide given subcutaneously every 6 weeks for a total period of 26 weeks, had an inhibitory effect on tumor growth identical to that of castration emphasizing the suitability of this compound for treatment of prostatic cancer.

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The progesterone antagonists Onapristone (ZK 98.299) and Mifepristone (RU 486) proved to be strong inhibitors of various rodent mammary tumors. Therefore, a further potent antiprogestin, ZK 112.993, and 11 beta-(4-acetyl-phenyl)-analog of Mifepristone, with a high progesterone receptor affinity was tested in experimental rodent and human breast cancer models. In the hormone-dependent MXT(+) mammary tumor of the mouse, treatment of tumors immediately after implantation with 5 mg/kg for 6 weeks led to an inhibition of growth by 95%, being significantly superior to that caused by tamoxifen, diethylstilbestrol and Onapristone. Treatment of established MXT(+) tumors by ZK 112.993 at doses of 0.5, 1.0 and 2.0 mg/kg led to a strong inhibition that equalled that of ovariectomy and surpassed that of Onapristone in the lower doses. In the human, receptor positive mammary carcinoma T61 implanted in male, castrated nude mice, ZK 112.993 (10 mg/kg) significantly retarded tumor growth. Its effect was again superior to Onapristone though weaker than that of tamoxifen. The NMU-induced mammary carcinoma of the rat (established tumors) was inhibited by ZK 112.993 (5 and 10 mg/kg) in a dose-dependent manner slightly superior to Onapristone but weaker than after ovariectomy. Due to its strong antitumor activity and because of the innovative mechanism of action of antiprogesterones in tumor treatment, ZK 112.993 could be of great value for the treatment of breast cancer.

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LHRH-antagonists might represent a useful new type of androgen deprivation to treat prostatic cancer. In this context adult intact male rats were treated subcutaneously with different concentrations of the new LHRH-antagonist antide either once (1, 3, 6, 10, 15 mg/kg) or on 5 consecutive days (5 x 3 mg/kg). The effect on serum concentration of LH and testosterone and the effect on the weights of testes, prostate, and seminal vesicles was investigated after different periods of time (24 hours, 1, 2, 3, 5, and 8 weeks). Histological evaluation of the testes was also performed. A clear dose-dependent inhibitory effect on the above-mentioned parameters was observed. The most effective treatment schedule was the single application of 15 mg/kg resulting in castration-like inhibition of prostate weights and marked inhibition of spermatogenesis within 2 weeks, which was maintained 8 weeks after the injection. Serum LH and serum testosterone concentrations were below the detection limit of the assay within 2 weeks and showed first signs of recovery after 8 weeks. Histologically, no signs indicative of irreversible effects (testes) were observed. To summarize, the LHRH-antagonist antide was found to have a profound long-lasting inhibitory but reversible effect on the reproductive system of adult intact male rats. These data emphasize the suitability of this type of compound for the treatment of prostatic cancer.

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In 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 morphology 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 antiproliferation 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.

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Metachromasia has been shown to be a stromal marker for human benign prostatic hyperplasia (BPH). So far, it is not known whether comparable changes can be demonstrated in experimentally induced BPH in the dog or in a subhuman primate species. In the present study, the phenomenon of metachromasia could be demonstrated in the prostate of cynomolgus monkeys, too. The reaction was quantitatively intensified by the treatment with androstenedione--an aromatizable androgen which caused hyperplastic and hypertrophic changes--especially a stimulation of the smooth muscle--in the stroma of the prostate. Simultaneous treatment with the aromatase inhibitor 1-methyl-ADD prevented both the phenomenon of metachromasia and the stimulation of the stroma. In conclusion, it is not only possible to induce hyperplastic/hypertrophic changes in the prostate of a subhuman species by means of an aromatizable androgen but, in addition, this effect is accompanied by a phenomenon which is thought to be typical for human BPH suggesting the suitability of this type of experiment for the study of the factors involved in the pathogenesis of BPH.

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