The anthocyanidins cyanidin and delphinidin are potent inhibitors of the epidermal growth-factor receptor.

The aglycons of the most abundant anthocyanins in food, cyanidin (cy) and delphinidin (del), were found to inhibit the growth of human tumor cells in vitro in the micromolar range, whereas malvidin (mv), a typical anthocyanidin in grapes, was less active. The aglycons preferentially inhibited the growth of the human vulva carcinoma cell line A431, overexpressing the epidermal growth-factor receptor (EGFR). The glycosides cyanidin-3-beta-D-galactoside (cy-3-gal, idaein) and malvidin-3-beta-D-glucoside (mv-3-glc, oenin) did not affect tumor cell growth up to 100 microM. The tyrosine kinase activity of the EGFR, isolated from A431 cells, was potently inhibited by cy and del. Mv and the glycosides cy-3-gal and mv-3-glc were inactive up to 100 microM. In intact cells the influence of anthocyanin treatment on downstream signaling cascades was investigated by measuring the phosphorylation of the transcription factor Elk-1. A431 cells were transiently transfected with a luciferase reporter gene construct whose expression is controlled by MAP kinase pathway dependent phosphorylation of a GAL4-Elk-1 fusion protein. We found that cy and del inhibited the activation of the GAL4-Elk-1 fusion protein in the concentration range where growth inhibition was observed. Thus, the anthocyanidins cy and del are potent inhibitors of the EGFR, shutting off downstream signaling cascades. These effects might contribute substantially to the growth-inhibitory properties of these natural food constituents.

Antiestrogenic activities of 3,8-dihydroxy-6,11-dihydrobenzo[a]carbazoles with sulfur-containing side chains.

The objective of this study was to explore whether the conversion of the 2-phenylindole system into the tetracyclic benzo[a]carbazole changes the endocrine profile when the side chain structure was kept constant. Five different sulfur-containing side chains were linked to the nitrogen of the tetracycle. The biological evaluation revealed that the character of the indole derivatives remained unchanged after the conversion to the respective benzocarbzoles but the potency decreased by one order of magnitude. In vitro, all derivatives acted as pure antiestrogens without any agonist activity. They strongly inhibited the growth of estrogen-sensitive MCF-7 breast cancer cells with IC50-values in the nanomolar range. In the mouse uterine weight test, the derivatives with an aliphatic side chain were devoid of estrogenic activity and antagonized the effect of estradiol. The presence of an aromatic ring in the side chain gave rise to significant agonist activity in vivo independently of the carrier structure. All data revealed the equivalence of both carrier structures in respect to the endocrine profile but showed a decrease in potency upon the conversion of the 2-phenylindole system into the benzocarbazole structure.

Tubulin as a target for anticancer drugs.

Nature provides a large spectrum of agents that either inhibit the polymerization of tubulin or prevent the disassembly of microtubules. Some of them are already used as anticancer drugs, but acquired resistance limits their application. Recently, a variety of new substances that inhibit tubulin aggregation have been synthesized or isolated from natural sources. Examples are vinflunine, the combretastatin analog AC-7700, indanocine, the sulfonamide T-138067, oncocidin A1, natural and modified dolastatins, and cryptophycins. Among the substances which stabilize microtubules, the taxanes play the dominant role. A second generation of synthetically modified derivatives is in development as exemplified by IDN-5109. A number of other natural products, such as the epothilones, eleutherobin, sarcodictyin and discodermolide, share the mode of action with the taxanes, but exceed them in potency and activity in multidrug-resistant cells. However, toxicity and availability might prevent their medical application.

Methoxy-substituted 3-formyl-2-phenylindoles inhibit tubulin polymerization.

The aim of this study was the identification of the essential structural elements in the 12-formyl-5,6-dihydroindolo[2, 1-a]isoquinoline system required for the inhibition of tubulin polymerization which is understood to be the predominant mode of action of this class of cytostatics. Since 2-phenylindole forms the main fragment of this tetracycle, it was used as the basic structure and modified with respect to the number and positions of the oxygen functions in the aromatic rings. Further modifications related to the nitrogen, which was both replaced by oxygen and sulfur and alkylated. All derivatives were tested for cytostatic activity in human breast cancer cells (MDA-MB 231, MCF-7) and inhibition of tubulin polymerization. The spectrum of activity ranged from inactive to IC50 values of 35 nM (cell growth inhibition) and 1.5 microM (tubulin polymerization), respectively, for the most active derivative 3e (3-formyl-6-methoxy-2-(4-methoxyphenyl)indole). Although the correlation between antiproliferative activity and inhibition of tubulin polymerization was not very pronounced, all of the potent cytostatic agents in this study disrupted microtubule assembly completely at the standard concentration of 40 microM. By fluorescence microscopy it was demonstrated that the derivative 3e degrades the cytoskeleton in a similar fashion as colchicine does leading to the condensation of the microtubules around the nucleus after treatment. The comparison between hydroxy and methoxy derivatives revealed a striking difference between the 2-phenylindole derivatives and the indoloisoquinolines. In the 2-phenylindole series, the methoxy compounds were much more effective than the free phenols, whereas in the tetracyclic system the effect of the hydroxy derivatives exceeded that of the methylated compounds by 1 order of magnitude. Preliminary studies on the binding mode showed that both the 2-phenylindole derivatives and the indoloisoquinolines bind to the colchicine site on tubulin.

2-Phenylbenzo[b]thiophene-based antiestrogens with mammary tumor inhibiting activity.

In this study we extended our studies on heterocyclic antiestrogens to 2-phenylbenzo[b]thiophenes which can be considered as isosteric to the 2-phenylindole system. We synthesized a number of 6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophenes with carbamoyl and amino functions in the side chain at carbon-3 and analyzed their biological properties. The binding affinities for the estrogen receptor are mainly influenced by the chain length whereas the hormonal profile depends on the nature of the functional group. From this study 3-[10-(2,2,3,3,4,4,4-heptafluorobutyl-methylcarbamoyl) decyl]-6-hydroxy-2-(4-hydroxyphenyl)benzo-[b]thiophene (6e) emerged as an antiestrogen with all the characteristics of a pure antagonist. It did not stimulate gene expression in HeLa cells cotransfected with the expression vector for the human estrogen receptor HEG0 and the luciferase reporter plasmid EREwtc luc nor did it show any estrogenic activity in the mouse uterus weight test. In the latter assay, it completely abrogated the stimulatory effect of estrone. Due to its antiestrogenic potency it strongly inhibited the growth of estrogen-sensitive human MCF-7 breast cancer cells with an IC50 value of 5 nM. These data suggest that an amide function in combination with the fluorination of the terminal carbon atoms is an appropriate modification to abolish the estrogenic action of the 2-phenylbenzothiophene system.

1-Benzyl-2-phenylindole- and 1,2-diphenylindole-based antiestrogens. Estimation of agonist and antagonist activities in transfection assays.

In the 2-phenylindole system, the side chain at the nitrogen atom dominates the endocrine profile both in respect to the reduction of estrogenic action and the increase of antiestrogenic potency. In previous papers we reported on 2-phenylindoles with aliphatic side chains and various functional groups [Biberger, C. and von Angerer, E., J. Steroid Biochem. Molec. Biol., 1996, 58, 31-43 and references therein]. In this study, we incorporated one or two phenyl rings into the side chain in order to lower the flexibility of the side chain. The sulfone group which was used as a polar function was linked to various positions of a benzyl or a phenyl group attached to the indole moiety. The relative binding affinities (RBA) ranged from 1.5 to 8.4% of estradiol. Agonist and antagonist activities were estimated in transfection assays using transiently transfected HeLa cells (cotransfected with the HEG0 vector) and stably transfected MCF-7/2a human breast cancer cells. The reporter plasmid contained the ERE from the Vitellogenin 2A gene, a viral tk promotor and the luciferase gene. Many of the new derivatives showed no or only very low estrogenic activity except for the compound 4e which contained two benzyl elements in the side chain. The antiestrogenic potency was very variable when concentrations 100-fold higher than that of estradiol were applied. The compound with the para-substituted benzyl fragment (4b) proved to be a pure antagonist in the transfection assays. It antagonized the effect of estradiol (10 nM) with an IC50 value of 10(-7) M. It also inhibited strongly the growth of estrogen-sensitive human MCF-7 mammary carcinoma cells (IC50, 3 nM). Its activity was comparable to the one of the corresponding aliphatic 2-phenylindole derivative ZK 164.015. The data from the transcription and proliferation assays suggest that a phenyl ring can be incorporated into the side chain of pure antiestrogens without reducing their potency, provided the aromatic ring is para-substituted and a methylene group between the indole nitrogen and the phenyl group can act as hinge.

Inhibition of tubulin polymerization by 5,6-dihydroindolo[2,1-alpha]isoquinoline derivatives.

6-Alkyl-12-formyl-5,6-dihydroindolo[2,1-alpha]isoquinolines have been shown to inhibit the growth of human mammary carcinoma cells by an unknown mode of action. One of the possible molecular targets is the tubulin system which is involved in cell division. A number of 5,6-dihydroindolo[2,1-alpha]isoquinolines with methoxy or hydroxy groups in positions 3, 9, and/or 10 and various functional groups such as formyl, acetyl, cyano, alkylimino, and alkylamino in position 12 were synthesized and evaluated for both inhibition of tubulin polymerization and cytostatic activity in MDA-MB 231 and MCF-7 human breast cancer cells. In the tubulin polymerization assay, only hydroxy derivatives were active, whereas both the hydroxy derivatives and some of the methoxy compounds inhibited cell growth. In order to establish a correlation between the inhibition of tubulin polymerization and cytostatic activity in the hydroxy series, two of the most active racemates were separated into the enantiomers. In both assays, the relative potencies of the hydroxy derivatives were in a similar order. Highest activity was found for the (+)-isomers of 6-propyl- (6b) and 6-butyl-12-formyl-5,6-hydro-3,9-dihydroxyindolo[2,1-alpha]isoquino line (6c) with IC50 values of 11 +/- 0.4 and 3.1 +/- 0.4 microM, respectively, for the polymerization of tubulin at 37 degrees C (colchicine: 2.1 +/- 0.1 microM). The active hydroxy derivatives displaced 40-70% of [3H]colchicine from its binding site in the tubulin at concentrations 10-fold higher than that of colchicine. The data suggest that hydroxy-substituted indolo[2,1-alpha]isoquinolines bind to the colchicine-binding site and inhibit the polymerization of tubulin. This action can be assumed to be responsible for the cytostatic activity of the hydroxy derivatives and might also contribute to the antitumor effect of the corresponding methyl ethers.

Effect of growth factors on estrogen receptor mediated gene expression.

The proliferation of mammary carcinoma cells can be stimulated by estrogens and various growth factors such as EGF and IGF-I. Steroid hormones and growth factors are understood to exert their effects via different receptors and signal transduction pathways. Recently, it has been shown that growth factors can utilize the unliganded estrogen receptor (ER) as a transcription factor. This study was aimed at identifying the growth factors that can act via the estrogen receptor, and finding new estrogen antagonists that block this activity. Originally, a transcription assay was used in which HeLa cells had been transiently co-transfected with the expression vector for the human ER and a reporter plasmid EREwtc luc. EGF and, to a lesser extent, insulin stimulated the expression of the reporter gene in the absence of estradiol (E2), whereas IGF-I was inactive. The stimulatory effect of E2 and insulin was suppressed when the ER was blocked by the pure antiestrogen ICI 182,780. In ER-positive MCF-7 cells, transfected transiently with the reporter plasmid, EGF had no stimulatory effect on luciferase expression. IGF-I stimulated the transcription to about 50% of the E2 value. Similar activity was found for insulin. The effect of both growth factors was only partly reversed by the addition of a pure antiestrogen. The combination of E2 and IGF-I or insulin led to a synergistic activation of transcription. Because transiently transfected cells do not allow one to study the influence of chromatin structure on gene expression, an MCF-7 subline (MCF-7/2a) was established, in which the reporter construct had been integrated in the genome. IGF-I stimulated luciferase expression in these cells, but showed no overadditive effect with E2. The effects of both agents were completely suppressed by the pure antiestrogen ICI 182,780. These data suggest the existence of an ER-independent mechanism for the activation of the reporter gene in transiently transfected cells, but not in stable transfectants.

2-Phenylindoles with sulfur containing side chains. Estrogen receptor affinity, antiestrogenic potency, and antitumor activity.

The 2-phenylindole system has been identified as a suitable structure for the design of non-steroidal pure estrogen antagonists [E. von Angerer et al., J. Steroid Biochem. Molec. Biol. 49 (1994) 51-62]. Derivatives with an amide function in the side chain antagonized the stimulatory effect of estrogens both in vitro and in vivo, and showed no agonistic activity when given alone. The findings of other groups who studied steroidal antiestrogens prompted us to replace the amide function by sulfide, sulfoxide, sulfone, sulfonamide and related groups. The compounds with polar sulfur functions retained the high binding affinity for the calf uterine estrogen receptor (RBA: 1-5% of estradiol; ICI 182,780; 6.2%). The estrogenic effect was quantified in a transcription assay using HeLa cells cotransfected with the expression vector HEG0 for the human estrogen receptor and a reporter plasmid that harbored a Vit. A2 ERE and the luciferase gene driven by a thymidine kinase promotor. Pentylsulfide, -sulfinyl, and -sulfonyl groups, linked to the indole nitrogen by a decamethylene spacer, were devoid of any transcriptional activity. These results were confirmed in the mouse uterine weight test. The sulfone (ZK 164,015) completely abolished the effect of a standard dose of estrone at a daily dose of 7 mg/kg. This compound strongly inhibited the growth of hormone-sensitive human MCF-7 breast cancer cells with an IC50-value close to 1 nM. Similar activity was found for the steroidal sulfoxide ICI 182,780. We were also able to demonstrate significant antineoplastic activity in vivo for some of these new 2-phenylindole derivatives.

Studies on heterocycle-based pure estrogen antagonists.

2-Phenylindoles and isosteric structures such as benzo[b]furans and benzo[b]thiophenes were used as estrogen receptor binding moiety for the syntheses of new nonsteroidal antiestrogens. The antiestrogenic potency was considerably enhanced following the introduction of polar functional groups into the side chain in position 1 (indole) or 3 (benzofuran, benzothiophene). The amino compounds could be characterized as mixed agonist/antagonists. Among the derivatives with an amide group compounds without any agonistic activity both in vitro and in vivo were identified. The amide function can be replaced by alkylthio or alkylsulfonyl groups without changing the endocrine profile very much. In this study, the estrogenic activity was determined in a new transcription assay with luciferase as the reporter. The results obtained in this assay were in very good agreement with those from the conventional mouse uterine weight test. Antitumor activity was determined in hormone-sensitive MCF-7 breast cancer cells. There was no difference in activity between partial and pure estrogen antagonists. However, the derivatives with sulfur containing side chains were much more active than the corresponding heterocycles with amino or carbamoyl functions. They reached IC50-values of about 1 nM. 2-Phenylindoles and 2-phenylbenzothiophenes were rather similar in their potencies whereas the benzofuran derivatives were less active probably due to their lower binding affinities for the ER.

Effect of the nonsteroidal antiestrogen ZK 119.010 on growth and metastasis of the EnDA endometrial carcinoma.

For patients with disseminated endometrial cancer the prognosis is poor. Radiotherapy, chemotherapy or high-dose progestins have been of limited value in the clinic, with low response rates and a usually short duration. Because of the role of estrogen in the etiology of this disease, a rationale exists for therapies using estrogen antagonists. In order to test this strategy, we used the EnDA endometrial carcinoma of the rat recently described by us. The nonsteroidal antiestrogen ZK 119.010 inhibited the primary-tumor growth of the s.c. implanted EnDA endometrial carcinoma by 50%, being superior to high-dose progestin and tamoxifen (TAM). Moreover, in intact as well as in castrated estrogen (E2)-substituted rats, ZK 119.010 substantially reduced metastatic-tumor growth in the lymph nodes and lungs. With TAM, however, the number of lung metastases in intact and in castrated E2-substituted rats either rose or remained stable and the weight of lymph nodes in intact rats increased. After TAM treatment, almost no low-salt-extractable (cytosolic) estrogen receptor (ER) was measurable in the tumor, whereas ZK 119.010 did not alter ER concentrations. The stimulation of metastatic tumor growth, as well as the loss of cytosolic ER under TAM therapy, may reflect the well-known agonist activity of this compound in uterine tissues. ZK 119.010, however, not only lacks this agonist activity, but it exerts a strong antagonistic one. In conclusion, pure antiestrogens may help to improve treatment of endometrial cancer.

[3-Substituted 2-phenylindoles: synthesis and biological properties]. / 3-Substituierte 2-Phenylindole: Synthese und biologische Eigenschaften.

Knoevenagel-reaction of indol-3-carbaldehydes 5a,b and 7a,b with nitromethane leads to the nitroethenes 12 and 14, the analogous reaction with malodinitrile to the methylidenemalonic acid dinitriles 16.- Michael-addition of nitromethane at 12 and 14 affords the 1,3-dinitropropanes 13 and 15, reduction of 16 the methylmalonic acid dinitriles 17.- Reaction of indoles 3 with n-BuLi/phenylsulfonylchloride leads to the 3-chloroindoles 18, reaction with NaH/ethyliodide, either cleavage and acylation to derivatives 19.- Compounds 7-11, 14-17, and 19 show affinity to the estrogen receptor. Compounds 7b, 9-11, 17b, and 19b inhibit the growth of MCF-7- and MDA-MB-231-cells. IC50-values are determined and structure-activity relationships are discussed.

1-Carbamoylalkyl-2-phenylindoles: relationship between side chain structure and estrogen antagonism.

The 2-phenylindole system has proved to be a versatile structure for the design of potent antiestrogens, especially when functional groups have been introduced into the alkyl side chain in position 1. In analogy to steroidal structures such as ICI 164,384 a number of 2-phenylindoles with carbamoylalkyl and aminoalkyl side chains were synthesized. They bind to the calf uterine estrogen receptor with relative binding affinities between 2.1 and 21 (estradiol = 100). The antiestrogenic effect of these compounds was demonstrated by the inhibition of transcriptional activity which was measured in a new luciferase assay with the EREwtc luc as reporter plasmid. The derivative with a methyl-n-propyldodecanamide side chain (4h) antagonized the effect of estradiol (10(-9) M) completely at concentrations of 10(-7) M and higher. As a sensitive model for quantification of estrogenic and antiestrogenic effects in vitro we used HeLa-cells cotransfected both with the reporter plasmid and estrogen receptor expression vectors HEG0 and HE0. In cells transfected with these vectors transcriptional activity was strongly dependent on side chain structure. With mutated receptors we were able to show that this activity was mainly due to TAF-1 whereas TAF-2 remained silent. When we studied the effect of some of the new compounds in vivo using the mouse uterine weight assay, we observed a correlation between transcriptional activity in transfected HeLa cells and estrogenic effects in mice. Two of the 1-carbamoylalkyl-2-phenylindoles (4f, 4h) proved to be "pure" antiestrogens both in vitro and in vivo. In estrogen-sensitive MCF-7 breast cancer cells, they strongly inhibit cellular growth. Some of the IC50-values were close to 10(-8) M.

The activity of epitestosterone in hormone dependent prostate tumour models.

Epitestosterone has been shown previously to counteract the testosterone activity in some experimental models. In the present study the activity of epitestosterone in an in vitro model of human LNCaP/FCS prostate cells and in vitro in Dunning R 3327-GH rat prostate carcinoma was tested. In LNCaP/FGC cells cultivated with fetal calf serum (FCS) treated with dextran-coated charcoal epitestosterone displayed rather androgenic than antiandrogenic properties, whereas the cultivation with native FCS resulted in a very weak inhibition of tumour cell growth with epitestosterone in higher concentration. The growth of Dunning R 3327-GH carcinoma of prostate was very weakly enhanced by epitestosterone alone as late as at the end of the 5-week experiment. Epitestosterone did not significantly inhibit the testosterone stimulated tumour growth.

Dibenzo[a,f]quinolizines: syntheses and cytostatic activity in estrogen-sensitive tumor cells.

A number of methoxy-substituted 7,11b,12,13-tetrahydro-6H-dibenzo-[a,f]quinolizines with short alkyl groups in position 6 or 12 were synthesized by the Bischler-Napieralski reaction using the appropriate starting material followed by a second ring closure reaction involving a base-generated benzyne intermediate. The methoxy functions in positions 2 or 3 and 9 were cleaved with BBr3 and the free hydroxy groups converted into the acetates. The enantiomers of two of these derivatives were separated by liquid chromatography on triacetylcellulose. Compounds with alkyl substituents bind strongly to the estrogen receptor except those with a cis-orientation at the central ring connection. The RBA values ranged from 2.2-10.8 (17 beta-estradiol: RBA = 100). There was no major difference in binding between the (+) and (-)-enantiomers. The 3,9-diacetoxy-6-alkyl derivatives also showed binding affinity for the progesterone receptor (RBA: 1.2-3.1). The 2,9-diacetoxydibenzoquinolizines trans-61 and -6m with ethyl and propyl respectively in position 12 strongly inhibited the growth of hormone-sensitive MCF-7 breast cancer cells at concentrations of 10(-6) M and higher but were inactive in hormone-independent MDA-MB 231 breast cancer cells. Preliminary tests with hormone-dependent MXT mouse mammary tumors as model showed that these compounds have also antineoplastic activity in vivo. Derivative trans-61 at a dose of 20 mg/kg body weight, administered 3 times/week, inhibited the growth of these tumors by 78% (tamoxifen: 76% inhibition). Studies on the estrogenic and antiestrogenic properties of these agents in mice revealed that they are mixed agonists/antagonists with strong antiestrogenic activity at low doses but significant estrogenic effects at higher doses.

Antitumour activity of coumarin in prostate and mammary cancer models.

The antineoplastic and antimetastatic activities of coumarin were evaluated in transplanted prostate tumours of the rat. The growth of Noble Nb-R prostate tumours was strongly inhibited by coumarin (40 mg/kg; administered three times per week), whereas the hormonally more sensitive Dunning R3327-G rat prostate carcinoma did not respond. Coumarin was also shown to possess antimetastatic activity in a Dunning R3327-MatLu tumour model. The number of lung metastases was reduced significantly by 40%-50% following the administration of coumarin (40 mg daily). Preliminary data from experiments with rats bearing DMBA-induced mammary carcinomas showed that these tumours are as sensitive to coumarin as Noble-R-prostate tumours.

Antitumour activity of coumarin and 7-hydroxycoumarin against 7,12-dimethylbenz[a]anthracene-induced rat mammary carcinomas.

Female SD rats with established 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumours were treated with coumarin (20 mg/kg body weight; six times per week) or its metabolite 7-hydroxycoumarin (20 mg/kg) for 4 weeks. The anti-oestrogen tamoxifen (8.8 mg/kg) served as the reference drug. The inhibitory effect of coumarin was similar to that of tamoxifen [mean change of tumour area: 428% (coumarin) compared to 528% (tamoxifen); control 822%]. The strongest inhibition was observed with 7-hydroxycoumarin (248%); the difference compared to the control was significant (P < 0.01). Neither coumarin nor 7-hydroxycoumarin reduced the number of tumours appearing during treatment as tamoxifen did. However, the size of the tumours treated with coumarin or its metabolite was generally much smaller than those in the tamoxifen group or in the control group. From the data obtained it appears that coumarin and 7-hydroxycoumarin inhibit the growth of tumours that have reached a certain size but do not prevent the formation of tumours after exposure to the carcinogen.

A rapid luciferase transfection assay for transcription activation effects and stability control of estrogenic drugs in cell cultures.

A rapid assay system for measuring the potential of estrogenic drugs is introduced. Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc. The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug. The assay system was used to measure the stability of the drug diaqua-[1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine] platinum(II) sulfate, containing an estrogenic ligand and reactive platinum. Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active. It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.

Antiproliferative activity of casodex (ICI 176.334) in hormone-dependent tumours.

The nonsteroidal antiandrogen casodex has been described as a peripherally selective drug for the treatment of prostatic cancer. In this study we determined its activity in various models of hormone-dependent malignancies including those of the prostate and the breast. Analysis of endocrine effects in rats after 15 days of treatment revealed a strong reduction of the weights of prostates and seminal vesicles and a significant rise of testosterone serum levels as a result of the interference with central feedback mechanisms. The growth of androgen-sensitive human LNCaP/FGC prostate cancer cells was strongly inhibited by casodex. Unlike hydroxyflutamide, casodex was also active in hormone-depleted medium. The inhibitory effect was overcome by addition of testosterone propionate, which indicates an androgen-receptor-mediated mode of action. In rats bearing Dunning R3327-G prostate carcinomas casodex exerted a strong antitumour effect at the beginning of therapy. However, after 4 weeks of treatment tumours resumed growth whereas diethylstilboestrol-treated tumours remained static. In MXT-M3.2 mouse mammary tumours with significant quantities of androgen receptors casodex was also effective in inhibiting tumour growth. After 6 weeks of treatment, tumour weights were reduced by 69% whereas uterine weights were significantly increased, possibly because of a progestin-like activity of the drug. Csodex is very active in various models of hormone-dependent carcinomas. However, the limited duration of action in prostatic tumours and the incomplete growth inhibition in mammary tumours suggest that it should be used only combination with other endocrine therapies.