Inhibition of the spindle assembly checkpoint kinase TTK enhances the efficacy of docetaxel in a triple-negative breast cancer model.

BACKGROUND: Triple-negative breast cancers (TNBC) are considered the most aggressive type of breast cancer, for which no targeted therapy exists at the moment. These tumors are characterized by having a high degree of chromosome instability and often overexpress the spindle assembly checkpoint kinase TTK. To explore the potential of TTK inhibition as a targeted therapy in TNBC, we developed a highly potent and selective small molecule inhibitor of TTK, NTRC 0066-0. RESULTS AND CONCLUSIONS: The compound is characterized by long residence time on the target and inhibits the proliferation of a wide variety of human cancer cell lines with potency in the same range as marketed cytotoxic agents. In cell lines and in mice, NTRC 0066-0 inhibits the phosphorylation of a TTK substrate and induces chromosome missegregation. NTRC 0066-0 inhibits tumor growth in MDA-MB-231 xenografts as a single agent after oral application. To address the effect of the inhibitor in breast cancer, we used a well-defined mouse model that spontaneously develops breast tumors that share key morphologic and molecular features with human TNBC. Our studies show that combination of NTRC 0066-0 with a therapeutic dose of docetaxel resulted in doubling of mouse survival and extended tumor remission, without toxicity. Furthermore, we observed that treatment efficacy is only achieved upon co-administration of the two compounds, which suggests a synergistic in vivo effect. Therefore, we propose TTK inhibition as a novel therapeutic target for neoadjuvant therapy in TNBC.

Determination of the oestrogenic (uterotrophic) activity of extracts of 'general purpose polystyrene (GPPS)' using immature female rats.

In Japan there is growing concern about the possible adverse effects of consumption of food from styrene containers (mainly those made from polystyrene paper) due to the alleged oestrogenic activity of styrene oligomers (dimers and trimers), which may migrate into the food. To examine the possible oestrogenic activity of styrene dimers and trimers, extracts were made from 'general purpose polystyrene (GPPS)' and administered orally to immature female rats over a 4 day period. Increase of uterus weight (wet and blotted) was used for assessment of possible oestrogenic activity. To establish the sensitivity of the test method, immature rats were treated with diethylstilboestrol (DES), a well-known oestrogenic compound. It was found that treatment of rats with levels of up to 60 microg of styrene dimers and 930 microg of styrene trimers per kilogram body weight per day did not give any statistically significant increase of the uterus weight (wet or blotted), whereas DES caused statistically significant, dose-related increases in uterus weight at levels as low as 0.89 microg kg(-1) body weight day(-1). It was concluded that, compared with the estimated maximum human daily intake of styrene trimers of 1 microg kg(-1) body weight day(-1) from polystyrene food containers, the risk of adverse human health effects with respect to oestrogenicity may be considered negligible.

Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data.

An arachidonic acid-enriched oil (AA-oil), derived from Mortierella alpina was subjected to a programme of studies to establish its preliminary safety for use in infant nutrition. This was addressed at two levels: (1) HPLC analysis of metabolites produced by the production strains at various conditions, and (2) an evaluation of the toxicity of the final product. The following studies were carried out on the AA-oil: gene mutation assays in bacteria and mammalian cells in vitro; chromosome aberration assays both in vitro and in vivo and acute and subacute (4-wk) oral toxicity in the rat. No known mycotoxins were produced by the production strains under the conditions tested. Further, the oil did not show mutagenic or clastogenic activity and the acute oral toxicity, expressed as the LD50 value, exceeded 20 ml/kg body weight, that is, 18.2 g/kg body weight. In the subacute oral toxicity study the AA-oil was tested as such and in combination with a docosahexaenoic-enriched oil (DHA-oil) derived from fish oil at a ratio of 2:1 (AA:DHA). This was done because high dose levels of AA may result in adverse effects; DHA can compensate for these effects. Furthermore, human milk contains both AA and DHA at a ratio of AA:DHA of 2 to 3:1. No obvious signs of toxicity were observed. Levels of phospholipids and triglycerides tended to be decreased in the highest dose groups. The no-observed-adverse-effect level of the AA-oil in the subacute 4-wk toxicity study was placed at the highest levels tested, namely 3000 mg AA-oil/kg body weight/day as such and in the combination of 3000 mg AA-oil and 1500 mg DHA-oil/kg body weight/day. This corresponds to an intake of 1000 mg AA/kg body weight/day, which represents approximately 37 times the infant intake of AA in human milk.

The chicken enucleated eye test (CEET): a practical (pre)screen for the assessment of eye irritation/corrosion potential of test materials.

The enucleated eye test with chicken eyes (CEET) obtained from an abattoir proved to be a valuable and practical alternative for the 'traditional' enucleated eye test with eyes of laboratory rabbits. Since 1992, the CEET has been incorporated in standard contract toxicity testing at the Toxicology Division of the TNO Nutrition and Food Research Institute as a (pre)screen for the Draize eye test with rabbits. The results of the first 44 compounds tested showed excellent correlation with the in vivo results. The CEET identified non-irritating or severely irritating compounds, and predicted (slightly to moderately) irritating compounds. Statistical analysis of the CEET and the rabbit in vivo scores showed high linear correlations between the critical values of both tests and confirmed the relevance of this assay with respect to ocular effects. In general, tiered in vitro/in vivo testing was considered a meaningful approach for further validation of alternative methods and for reducing the use of suffering of laboratory animals to a minimum. Tiered testing of compounds in cases of eye irritation hazard assessment should be incorporated in the legislation of the European Community.

Justification of the enucleated eye test with eyes of slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits.

The enucleated eye test (EET) with the isolated eye of rabbits has been recognized as a valuable alternative to the Draize test, because it represents a test system nearest to the in vivo test, without the need to use live animals. In this ex vivo bioassay, three parameters are measured to detect possible adverse eye effects, namely corneal swelling, corneal opacity and fluorescein retention. The measurement of corneal swelling in this assay guarantees a highly objective and discriminative parameter. In combination with the detailed observation of corneal opacity and fluorescein retention, a reliable evaluation of the eye irritation potential of test materials is achieved. However, laboratory animals are still necessary as eye donors. The use of slaughter animals, such as the cow, the pig and the chicken, as possible as eye donors for the EET was therefore examined. From these candidates, the chicken appeared to be the most practicable. 21 reference compounds, ranging from non-irritant to severe irritant, which had been tested previously in a validation study on alternative test methods for eye irritation testing, sponsored by the Commission of the European Communities, were examined in the Chicken Enucleated Eye Test (CEET). When compared with the in vivo EC classification, the CEET correctly classified each of the compounds that must be labelled in the EC as irritant (R36) or severely irritant (R41). In addition, since the CEET recognizes three levels of irritancy rather than two (as in the case of the EC classification) a small number of the compounds were recognized as slightly irritant, which according to the EC classification need not be labelled. It was concluded that this ex vivo test system is highly accurate in the assessment of eye irritation potential without the use of laboratory animals.

Induction of skin and thyroid tumors in male rats by N-methyl-N-nitrosourea after sequential treatment with cyproterone acetate and testosterone propionate: effects of castration, rat strain and time of carcinogen injection.

The purpose of this study was to determine the carcinogenic effect in male rats of a single i.v. injection of N-methyl-N-nitrosourea (MNU) after sequential treatment with cyproterone acetate (for 21 days) and testosterone propionate (for 3 days). This treatment has previously been shown to induce carcinomas of the prostate and other male accessory sex glands. A wide spectrum of non-melanoma skin tumors was found in 38-48% of Wistar (Cpb:WU) rats given this sequential treatment, but only in 5% of rats that received only MNU. Castration long and, particularly, early after MNU markedly reduced this skin tumor response to a 10-13% incidence. The skin tumorigenic efficacy of MNU was dependent on the time between the start of the testosterone propionate treatment and carcinogen administration: MNU injection after 48-50 or 60-63 h induced skin tumors in 17-21% of Wistar rats, whereas injection after 72-74 h induced a 48% incidence. The Fischer F344 and Sprague-Dawley strains were not very sensitive to induction of skin tumors by this approach. Thyroid follicular cell tumors were also induced by MNU only after the hormonal pretreatment, and their induction was influenced by the time of MNU injection as well. The time of MNU injection and rat strain used did not significantly influence the induction of sebaceous-squamous neoplasms of the ear-duct/Zymbal's glands or other tumors. These data indicate that endogenous androgens are critically involved in the later stages of rat skin tumorigenesis and suggest that androgen-induced cell proliferation influences the initiation stage of this process and, possibly, of thyroid tumorigenesis.

Induction of proliferative lesions of ventral prostate, seminal vesicle, and other accessory sex glands in rats by N-methyl-N-nitrosourea: effect of castration, pretreatment with cyproterone acetate and testosterone propionate and rat strain.

Wistar (Cpb:WU), F344 or Sprague-Dawley rats were sequentially treated with cyproterone acetate (CA) for 21 days, testosterone propionate (TP) for 3 days, followed by a single i.v. injection of N-methyl-N-nitrosourea (MNU). One group of Wistar rats was castrated 4 weeks after MNU injection, and another group 58 weeks after MNU, when the first prostatic carcinoma was detected. Control groups received only CA + TP, CA, MNU, or they remained untreated. Early or late castration inhibited the development of atypical hyperplasia of the ventral prostate in Wistar rats. This lesion was induced by the CA + TP + MNU treatment in F344 rats, but not Sprague-Dawley rats; in Wistar rats, it was induced by CA + TP treatment, irrespective of whether MNU was given. Hypertrophic-hyperplastic lesions of the seminal vesicle were induced by MNU, irrespective of pretreatment, and their development was prevented by early castration and inhibited by late orchiectomy. Dorsolateral prostate carcinomas and preneoplasia occurred only in low incidence in Wistar and Sprague-Dawley rats. These lesions were absent in F344 rats that had received treatment with CA + TP + MNU. No dorsolateral prostate (pre)neoplasia was found in Wistar rats subjected to early orchiectomy, but rats castrated at 58 weeks had an incidence similar to that for the intact group treated with CA + TP + MNU. This finding supports the contention that androgens are required for the development of MNU-induced prostatic cancer in rats but that advanced carcinomas are androgen insensitive. Differences in incidence and localization of prostatic proliferative lesions between F344 and Wistar rats and between dorsolateral and ventral prostate could not be explained by differences in epithelial cell proliferative responses to CA + TP treatment at the time of MNU injection, since they were similar in ventral and dorsolateral prostate and were more prominent in F344 rats than in Wistar rats. DNA damage as estimated by MNU-induced unscheduled DNA synthesis also did not differ between dorsolateral and ventral prostate.

Induction of dorsolateral prostate adenocarcinomas and other accessory sex gland lesions in male Wistar rats by a single administration of N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl after sequential treatment with cyproterone acetate and testosterone propionate.

Groups of 20-25 male Wistar rats (Cpb:WU), nine groups of 4-week-old rats, and nine groups of 8-week-old rats, were given cyproterone acetate (CA) s.c. or by gavage daily for 18 days at a dose of 50 mg/kg/day. Directly following CA treatment, the rats received 3 daily s.c. injections with testosterone propionate (TP) at a dose of 100 mg/kg/day. On the day after the last TP administration, a single dose of one of the following carcinogens was given to 3 groups: N-methyl-N-nitrosourea (MNU), 50 mg/kg i.v.; 7,12-dimethylbenz(a)anthracene, 30 mg/kg i.v.; 3,2'-dimethyl-4-aminobiphenyl, 250 mg/kg s.c. Three other groups received the same carcinogen treatments after 7 days of recovery from the CA administration. The last 3 groups received carcinogen without TP treatment, but immediately after CA pretreatment was stopped. A 25% incidence of invasively growing, metastasizing adenocarcinomas was found in the dorsolateral prostate region of 8-week-old rats that had received MNU after treatment with CA plus TP. In addition, this group had a 5% incidence of carcinoma in situ and a 5% incidence of atypical hyperplasia in the dorsolateral prostate. Lower incidences of adenocarcinoma of the dorsolateral prostate region and of carcinoma in situ and atypical hyperplasia of the dorsolateral prostate were found in other groups that were treated with MNU or 7,12-dimethylbenz(a)anthracene after pretreatment with CA, followed by TP or recovery, but never in rats that had been treated with CA only. In the groups treated with 3,2'-dimethyl-4-aminobiphenyl, which is slowly metabolized, these lesions were also found in groups that were pretreated with only CA. The carcinomas seemed to originate from the dorsolateral prostate and their average latency time was approximately 61 weeks. The 8-week-old rat given a MNU injection after sequential treatment with CA and TP may provide a relevant animal model for human prostatic cancer.

Characterization of adenocarcinomas of the dorsolateral prostate induced in Wistar rats by N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl, following sequential treatment with cyproterone acetate and testosterone propionate.

Carcinomas of the rat prostate induced by a single injection of N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl, after sequential treatment with cyproterone acetate and testosterone propionate, were evaluated as potential animal models for prostatic cancer. All ten carcinomas examined were located in the dorsolateral prostate region and did not involve the distal parts of the seminal vesicles and coagulating glands. The incidence of urinary obstruction leading to the animals' death was 6 of 10 rats, and metastases in the lung, abdominal lymph nodes, and/or liver also occurred in 6 of 10 rats. The tumors were invasive adenocarcinomas, showing frequent perineural invasion and a variable degree of differentiation. There were ultrastructural similarities with human prostatic carcinomas, such as intracellular lumina. Plasma acid phosphatase was increased. Enzyme histochemical analysis revealed similarities with the Dunning R3327H and -HI prostatic carcinomas but was not helpful in determining the site of origin of the tumors. The gross and microscopic appearance of the tumors and the observation of preneoplastic lesions exclusively located in the dorsolateral prostate suggest this lobe as site of origin of the carcinomas. Preneoplastic lesions (n = 9) included atypical hyperplasias (n = 5) and lesions with all histological characteristics of carcinoma except for local invasion and metastases, which were classified as carcinoma in situ (n = 4). Although androgen sensitivity could not be assessed, the observed characteristics of the tumors [their long latency time (46-80 weeks), the presence of preneoplastic lesions, and the short duration of the treatment, leaving the animals intact] all indicate that the present approach is a valid animal model for the study of prostatic carcinogenesis.

Adenocarcinomas of the prostate induced by N-nitroso-N-methylurea in rats pretreated with cyproterone acetate and testosterone.

Prostatic adenocarcinomas were induced in 5 out of 20 Wistar rats upon a single administration of 50 mg/kg N-nitroso-N-methylurea (NMU). The rats were pretreated with a daily dose of 50 mg/kg cyproterone acetate for 3 weeks followed by 3 daily injections of 100 mg/kg testosterone. All tumours developed in the dorsolateral prostate and were invasively growing. In 2 cases distant metastases were found. Three proliferative lesions classified as carcinomas in situ were also found in the dorsolateral prostate. A total of 7/20 animals (35%) carried an adenocarcinoma and/or a carcinoma in situ. In addition, 6 epithelial hyperplasias were observed in the dorsolateral and 1 in the ventral prostate of non-tumour-bearing rats. The method described may provide a good animal model for cancer of the prostate and lead to a better understanding of prostatic carcinogenesis.