Effects of sandostatin and castration on pancreatic carcinogenesis in rats and hamsters.

The effects of treatment with the somatostatin analogue Sandostatin, separately and in combination with surgical castration, on the development of azaserine-induced lesions in rat pancreas and N-nitrosobis(2-oxopropyl)amine (BOP)-induced lesions in hamster pancreas were investigated. The animals were divided in 4 groups and treated as follows: (a) controls, injected s.c. with saline solution (0.9% NaCl); (b) orchiectomy directly after the last treatment with carcinogen; (c) Sandostatin (SMS 201-995) subcutaneously; (d) orchiectomy followed by treatment with Sandostatin. No significant suppressive effects on plasma EGF or IGF-I concentrations were noted after Sandostatin treatment, but plasma gastrin levels decreased slightly in the rats, not in the hamsters. In rats, Sandostatin treatment enhanced rather than inhibited growth of acidophilic atypical acinar cell nodules. In hamster pancreas, by contrast, Sandostatin inhibited the development of putative pre-neoplastic ductular lesions. There was no interaction between treatment with Sandostatin and surgical castration. It was concluded that Sandostatin, when administered prophylactically, has an inhibitory effect on the growth of putative pre-neoplastic ductular, but not acinar, lesions.

Effects of castration, alone and in combination with aminoglutethimide, on growth of (pre)neoplastic lesions in exocrine pancreas of rats and hamsters.

We studied the effects of hormonal manipulation by orchiectomy, alone or in combination with the aromatase inhibitor aminoglutethimide (AGT), and by luteinizing hormone-releasing hormone agonist (LH-RH-A) (goserelin) treatment on the development of early putative (pre)neoplastic lesions induced in the pancreas of rats and hamsters by azaserine and N-nitrosobis(2-oxopropyl)amine respectively. Treatment of the animals started 1 week after the last injection with carcinogen and continued for 4 months. Orchiectomy caused a significant inhibition of growth of acidophilic atypical acinar cell nodules in the rat model, whereas surgical castration did not show an effect in the hamster model. In rats, but not in hamsters, orchiectomy resulted in a significant decrease in body weight and in absolute, but not relative pancreatic weight. Treatment of the animals with AGT or goserelin did not cause a significant effect on the development of either putative preneoplastic acinar lesions in rat pancreas or early ductular lesions in hamster pancreas. Hamsters showed clearly higher plasma epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) concentrations than rats, while plasma testosterone levels were significantly lower. Plasma EGF and IGF-1 levels decreased with increasing age in both control and treatment groups. Compared to controls there were no clear unequivocal effects of treatment on EGF, IGF-1 and gastrin levels. Plasma testosterone levels decreased by orchiectomy and LH-RH-A treatment. In rats hormone-induced effects on food intake and altered nutritional status might be important with respect to the development of carcinogen-induced preneoplastic pancreatic lesions.

Treatment of breast cancer with different antiprogestins: preclinical and clinical studies.

UNLABELLED: Treatment with antiprogestins is a new treatment modality for breast cancer. Previously, in rats with DMBA-induced mammary tumors we observed significant growth inhibitory effects of chronic treatment with the antiprogestin mifepristone (RU486). In addition, in 11 postmenopausal breast cancer patients, we observed one objective response, six instances of short-term stable disease, and four instances of progressive disease. Side-effects appeared mainly due to antiglucocorticoid properties of the drug. Increased plasma estradiol levels were observed which probably resulted from ovarian (rat) and adrenal (patients) steroidogenesis. Combined treatment with an antiestrogen in the rat model caused additive growth inhibitory effects. Tumor inhibition after single treatment with mifepristone or tamoxifen was 90 and 75%, respectively. In contrast, when combined, tumor remission similar to that caused by LHRH-agonist treatment (50%) was observed. Even higher tumor remission was found after combined treatment with mifepristone plus LHRH-agonist (75%). In first studies in the rat model we observed significant tumor growth inhibitory effects with two new antiprogestins of seemingly greater potency which cause less unfavorable endocrine side-effects. IN CONCLUSION: combined treatment (antiprogestin plus antiestrogen or LHRH-agonist) may be of value in endocrine therapy of breast cancer.

Growth factor-receptor pathway interfering treatment by somatostatin analogs and suramin: preclinical and clinical studies.

UNLABELLED: Interference in growth factor mediated pathways is a new strategy in the treatment of cancer. Somatostatin analogs can inhibit hormone and growth factor secretion, while suramin can block the binding of several growth factors to their receptors. In addition, somatostatin analogs can cause direct growth inhibitory effects after binding to tumoral somatostatin receptors. We tested the efficacy and endocrine effects of chronic treatment with three somatostatin analogs (Sandostatin, R RC-160 and CGP 15-425) or suramin in several tumor models and in patients with various types of cancer. Treatment with somatostatin analogs caused growth inhibition of breast cancer cells (MCF-7) in vitro, and of rat transplantable pancreatic (50-70% inhibition) and prostatic Dunning tumors (12% inhibition). No tumor growth inhibition was observed with respect to DMBA-induced rat mammary tumors, a transplantable colon tumor and a rhabdomyosarcoma in rats. In 34 patients with metastatic pancreatic or gastrointestinal adenocarcinomas chronic Sandostatin treatment caused stable disease in 27% of the patients, but no objective remissions. Somatostatin receptors were found in the responding MCF-7 mammary tumor cells, rat pancreatic tumors and in 20-45% of human breast cancer specimens [J. Steroid Biochem. Molec. Biol. 37 (1990) 1073-1077], but not in rat DMBA-mammary tumors or in 10 human pancreatic adenocarcinomas. Suramin caused significant dose-dependent growth inhibition of human breast cancer cells in vitro and of rat pancreatic tumors in vivo in the presence of plasma levels up to 150 micrograms/ml. In a preliminary clinical study concerning 11 patients with various tumor types we observed significant hematological, biochemical, endocrine and clinical side effects, but no objective remissions in spite of relevant peak plasma suramin concentrations of 270-330 micrograms/ml. IN CONCLUSION: somatostatin analogs and suramin can cause growth inhibition of various experimental tumors in vitro and in vivo, but the clinical value has to be established for several types of cancer, especially with respect to suramin and suramin-like compounds.

The somatostatin analog Sandostatin (SMS201-995) in treatment of DMBA-induced rat mammary tumors.

The effects of treatment with a somatostatin analog (Sandostatin, SMS201-995) were investigated in female rats with dimethylbenzanthracene (DMBA)-induced rat mammary tumors. A 3-week treatment was performed using sandostatin, the LHRH-agonist buserelin alone, or buserelin in combination with sandostatin. Twice daily sandostatin treatment was performed with dosages of 0.05 microgram, 0.2 microgram, 1 microgram, 5 micrograms, and 20 micrograms. Buserelin was used in a 2 x 5 micrograms/day dosage. The combined results from six different experiments show that the various dosages of sandostatin caused no tumor growth inhibition. Somatostatin receptors could not be demonstrated in these mammary tumors. Sandostatin treatment by daily injections did not suppress levels of growth hormone, prolactin, or epidermal growth factor-like activities. Estrogen (ER) and progesterone (PgR) receptor contents of the mammary tumors were not changed. In contrast, buserelin treatment caused highly significant tumor remission. The combined treatment with sandostatin and buserelin did not alter the treatment results obtained after treatment with buserelin alone. In conclusion, sandostatin treatment in this tumor model had no direct growth inhibitory effect and did not cause an endocrine inhibition of mammary tumor growth. However, these results do not exclude antitumor effects in human breast cancer in view of the presence of somatostatin receptors in approximately 20-45% of human tumors, besides possible different endocrine effects.

Endocrine and antitumor effects of combined treatment with an antiprogestin and antiestrogen or luteinizing hormone-releasing hormone agonist in female rats bearing mammary tumors.

Rats bearing mammary tumors induced with dimethylbenzanthracene were treated with the antiprogestin mifepristone (RU486; 10 mg/kg.day, sc), the antiestrogen tamoxifen (400 micrograms/kg.day, sc), LHRH agonists administered by either sc injections (buserelin; 40 micrograms/kg.day) or implant (buserelin or zoladex), or combinations of mifepristone and tamoxifen or LHRH agonists. Single treatment with mifepristone or tamoxifen caused a significant inhibition of tumor growth (90% and 75%, respectively), but no tumor remission. In contrast, single treatment with LHRH agonists caused remission of mammary tumor growth by 50% (injection) or 70% (implant), respectively. Combined treatment with mifepristone and tamoxifen caused additive tumor growth inhibitory effects resulting in the same extent of tumor remission as that observed after treatment with LHRH agonist injections alone. Combination of mifepristone with either manner of LHRH agonist administration resulted in the highest tumor remission (approximately 75%). Significant reductions in cytosolic steroid (estrogen and progesterone) receptor contents of mammary tumors were noted after various treatment modalities. The most pronounced decrements were observed after combined treatment with mifepristone and tamoxifen (residual estrogen receptor; 10%; residual progesterone receptor, 0%). On the other hand, suppression of pituitary-ovarian function was most pronounced after treatment with LHRH agonist implants alone or in combination with mifepristone. It is concluded that combination treatment with an antiprogestin and an antiestrogen or an LHRH agonist may be of great value in the endocrine therapy of breast cancer.

Antiprogestins, a new form of endocrine therapy for human breast cancer.

The antitumor, endocrine, hematological, biochemical, and side effects of chronic second-line treatment with the antiprogestin mifepristone (RU) 486) were investigated in 11 postmenopausal patients with metastatic breast cancer. We observed one objective response, 6 instances of short-term stable disease, and 4 instances of progressive disease. Mean plasma concentrations of adrenocorticotropic hormone (P less than 0.05), cortisol (P less than 0.001), androstenedione (P less than 0.01), and estradiol (P less than 0.002) increased significantly during treatment accompanied by a slight decrease of sex hormone binding globulin levels, while basal and stimulated gonadotropin levels did not change significantly. The increased basal cortisol levels could not be further stimulated by synacthen, nor suppressed by 1 mg of dexamethasone. Plasma estradiol concentrations were significantly correlated with both androstenedione (P less than 0.05) and cortisol levels (P less than 0.01). The percentage of eosinophilic white blood cells (P less than 0.02) and mean plasma creatinine concentration (P less than 0.05) increased significantly. Side effects frequently occurred during long-term treatment and appeared to be caused mainly by the antiglucocorticoid properties of the drug. It is concluded that antiprogestins form a new treatment modality in the endocrine treatment of human breast cancer. New antiprogestins with less antiglucocorticoid side effects might be especially of value as an adjunct to antiestrogenic treatment in view of our finding that combined antiestrogenic and antiprogestational treatment caused additive growth-inhibitory effects in rat mammary tumors.

Comparison of the actions of the antiprogestin mifepristone (RU486), the progestin megestrol acetate, the LHRH analog buserelin, and ovariectomy in treatment of rat mammary tumors.

The effects of the synthetic antiprogestin mifepristone (RU486) on growth of dimethylbenzanthracene (DMBA)-induced mammary tumors in female rats were investigated. Prophylactic treatment with mifepristone (10 mg/kg/day) for 3 weeks starting from the day of first DMBA injection resulted in a doubling of the average tumor latency period (81 +/- 16 days, n = 17 treated, versus 39 +/- 5 days, n = 75 controls; P less than 0.005) and was accompanied by a significant growth retardation as shown by lower body weight increments. A 3-week therapeutic treatment of rats bearing mammary tumors was performed by administration of different dosages of mifepristone (2.5, 10, or 40 mg/kg/day) or megestrol acetate (2.5 or 10 mg/kg/day), with the luteinizing hormone-releasing hormone agonist buserelin (40 micrograms/kg/day), buserelin plus mifepristone (10 mg/kg/day), or by ovariectomy. The effects of treatment on tumor load, pituitary, adrenal and reproductive organ weights, steroid receptor contents of mammary tumors, and blood plasma hormone concentrations were investigated. Mifepristone and megestrol acetate treatment gave rise to inhibition of mammary tumor growth with all dosages studied, in which mifepristone was more potent than megestrol acetate (80%-90% vs 40% inhibition, P less than 0.01). In contrast, buserelin treatment and ovariectomy resulted not only in inhibition, but in tumor remission by about 50%. Combined treatment with buserelin and mifepristone gave the same tumor remission as resulted from ovariectomy or single treatment with buserelin. Estradiol-stimulated growth of the human mammary cancer MCF-7 cells in culture was fully abolished by mifepristone (3.6 X 10(-8) M) or tamoxifen (4 X 10(-8) M), whereas growth of MCF-7 cells under control incubation was not affected by either agent. Therefore, a direct inhibition of the growth of rat mammary tumor cells by mifepristone appears likely. Based on the effects of mifepristone on plasma hormone levels (increased: luteinizing hormone, estradiol, progesterone; unchanged: follicle-stimulating hormone, adrenocorticotropic hormone, corticosterone), organ weights (increased: pituitary, ovaries, uterus; unchanged: adrenals) and steroid receptor contents of mammary tumors (decreased: estrogen receptor and progesterone receptor contents), the main mechanism of action is probably a direct antiprogestational effect at the level of the mammary tumor cells through occupancy of the progesterone receptor.

Effects of the progesterone antagonist RU486 on ovarian activity in the rat.

Adult female rats were treated for 2 or 4 weeks with the progesterone antagonist RU486 to study its effect on the regulation of ovarian function. In rats with 5-day ovarian cycles, the vaginal cyclicity disappeared. Uninterrupted vaginal cornification emerged within 4 days after the start of treatment and cornification persisted for the whole period of treatment. It took more than 2 weeks after cessation of 2-4 weeks of treatment before 5-day vaginal cycles reappeared. Ovarian weights increased rapidly resulting from the accumulation of large numbers of corpora lutea. In addition, the ovaries developed occasional follicular cysts which could reach an extremely large size (2 mm or more). Analysis of serial histological sections of ovaries, combined with plasma concentrations of estradiol-17 beta and progesterone, indicated cyclic ovulation and corpus luteum formation together with persistence of functional activity of already existing and newly formed corpora lutea. RU486 seems to have the unique property of dissociating cessation of luteal activity and ovulation in rats. After treatment with RU486, pituitary enlargement and mammary gland alveolar development were observed. It is hypothesized that these effects result from unopposed estrogen action on PRL secretion. The effects of RU486 are reversible: 4 to 5 weeks after the end of treatment ovarian activity seems normal (as evidenced by reduction of ovarian weights and 5-day vaginal cycles) except for the presence of occasional large follicular cysts which may require longer periods for their regression.

Lutropin stimulates de novo synthesis of short-lived proteins required for lutropin-dependent steroid production in tumour Leydig cells.

Continuous protein synthesis is required for the hormonal regulation of cholesterol side chain cleavage activity. A protein with a short half-life (t1/2 = 2-13 min) is believed to play an important role, but the regulation of the synthesis of this putative rapidly-turning-over protein is largely unknown. The steroid production rate in tumour Leydig cells can be increased more than 4-fold after addition of lutropin. However, steroid production by cells preincubated for 60 min with medium containing cycloheximide (89 microM) could not be stimulated when lutropin was added to the medium. Thus, the putative protein with the short half-life is apparently not derived from a stable precursor protein. Moreover, in tumour Leydig cells incubated with low concentrations of cycloheximide (0.2-0.8 microM), inhibition of steroid production was significantly greater in lutropin-stimulated cells than in control cells. These results support the hypothesis that lutropin regulates the de novo synthesis of rapidly-turning-over proteins by increasing the rate of initiation of the translation step of protein synthesis.

LH-dependent steroid production and protein phosphorylation in culture of rat tumour Leydig cells.

The possible cause of the decreased responsiveness of cultured Leydig cells to stimulation with LH was investigated with rat tumour Leydig cells cultured at 32 degrees C for 2 days. Pregnenolone production and phosphorylation of proteins were studied in combination with the activity of cyclic AMP-dependent protein kinase. Pregnenolone production in cultured tumour Leydig cells decreased from 45 +/- 16 on day 0 to 14 +/- 7 on day 2 (ng/60 min/mg protein) under basal conditions and from 226 +/- 108 on day 0 to 30 +/- 24 on day 2 (ng/60 min/ng protein) under LH-stimulated conditions (means +/- SD, n = 4). This decrease may be accounted for by decreased capacity of cholesterol side-chain cleavage which decreased (in the presence of 25-hydroxycholesterol) from 1.68 +/- 0.26 on day 0 to 0.74 +/- 0.48 on day 2 (microgram/60 min/mg protein; mean +/- SD, n = 4) and decreased activity of cyclic AMP-dependent protein kinase (as apparent from a direct assay of protein kinase activity and the extent of phosphorylation of LH-dependent phosphoproteins).

Subcellular localization of LH-dependent phosphoproteins and their possible role in regulation of steroidogenesis in rat tumour Leydig cells.

Stimulation of rat tumour Leydig cells with LH resulted in phosphorylation of 7 proteins of 17, 22, 24, 33, 43, 57 and 76 kDa, and in dephosphorylation of a single protein of 20 kDa. The subcellular localization of these LH-dependent phosphoproteins in combination with effects of inhibitors of microfilament formation and protein synthesis, suggest that phosphoproteins of 20, 43 and 76 kDa present in the cytosol may be involved in the action of microfilaments, whilst phosphoproteins of 24 and 33 kDa present in microsomes may be involved in specific protein synthesis.

The role of phosphoproteins and newly synthesized proteins in the hormonal regulation of steroidogenesis in Leydig cells.

LH activation of steroidogenesis in Leydig cells is accompanied by increased phosphorylation of at least 5 phosphoproteins. The 17000 Da protein is localized in nuclei and appears not to be involved in the acute stimulation of steroidogenesis. The 57,000 Da protein is the regulatory subunit of the cAMP-dependent protein kinase. The 22,000, 24,000 and 33,000 phosphoproteins are present in microsomes and may regulate the synthesis of specific rapidly-turning-over protein(s) which in turn may stimulate the mitochondrial cholesterol side-chain cleavage activity.

Lutropin increases phosphorylation of a 33 000-dalton ribosomal protein in rat tumour Leydig cells.

Addition of lutropin (luteinizing hormone, 'LH') and 3-isobutyl-1-methylxanthine to tumour Leydig cells stimulated phosphorylation of five proteins, of 17 000, 22 000, 24 000, 33 000 and 57 000 Da. Phosphorylation of these proteins coincided with increased pregnenolone production. Phosphorylation of a 33 000-Da protein was lutropin-dependent in Leydig cells isolated from a Leydig-cell tumour, from immature testes or from mature testes. In tumour Leydig cells this protein was present in the small ribosomal subunit. Incubation of tumour Leydig cells with either cycloheximide or puromycin inhibited both basal and lutropin-dependent pregnenolone production, by approx. 90% and 98% respectively. In contrast, basal pregnenolone production in Leydig cells from immature and mature testes was insensitive to cycloheximide or puromycin. Cycloheximide or puromycin increased phosphorylation of the 33 000-Da phosphoprotein by approx. 130% and 80% respectively (effect of lutropin/3-isobutyl-1-methylxanthine on phosphorylation: 100%). The molecular mass, the subcellular localization and the sensitivity to phosphorylation in the presence of inhibitors of protein synthesis indicate that the 33 000-Da protein could be similar to ribosomal protein S6.

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells.

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.