Macrophage role in the anti-prostate cancer response to one class of antiangiogenic agents.

BACKGROUND: Tumor-associated macrophages (TAMs) can either promote angiogenesis (i.e., the formation of new blood vessels) in tumors by secreting tumor necrosis factor-alpha (TNF-alpha) or inhibit angiogenesis by producing granulocyte-macrophage colony-stimulating factor (GM-CSF), which in turn stimulates production of the antiangiogenic protein plasminogen activator inhibitor type 2 (PAI-2). We tested, alone or in combination, the anti-prostate cancer activity of agents that perturb macrophage function. METHODS: By use of enzyme-linked immunosorbent assays, we measured the effects of Linomide (roquinimex), thalidomide, pentoxifylline, and genistein on TNF-alpha and GM-CSF production in vitro by virally transformed RAW 264.7 mouse macrophages and on PAI-2 production in vitro by human macrophages. The antitumor effects of these agents were tested in vivo on transplanted Dunning R-3327 MAT-Lu rat prostate cancers; TAM numbers and blood vessel densities in these cancers were determined by use of immunocytochemistry. RESULTS: Linomide selectively inhibited mouse macrophage secretion of TNF-alpha but not of GM-CSF; however, thalidomide, pentoxifylline, and genistein inhibited the production of both cytokines. Linomide, but not thalidomide or pentoxifylline, increased production of PAI-2 by human macrophages. When administered to rats bearing MAT-Lu tumors, each of the tested agents reduced TAM numbers (Linomide, by 46%; thalidomide, by 94%; pentoxifylline, by 71%; and genistein, by 96%). However, all of the agents reduced tumor blood vessel density and tumor growth, with Linomide being the most effective (44% reduction in blood vessel density and 69% inhibition of tumor growth). None of the other agents potentiated Linomide's antitumor effect. CONCLUSIONS: Linomide is unique among the antiangiogenic agents tested, in that it inhibits the stimulatory effects of TAMs on tumor angiogenesis without eliminating their antiangiogenic effects, and may thus prove to be more effective against prostate cancer.

Anti-angiogenic treatment with linomide as adjuvant to surgical castration in experimental prostate cancer.

PURPOSE: Escape from "castration inhibition," be it surgical or chemically induced, is still the major problem in prostate cancer treatment. New agents that can be given as adjuvant therapy are needed. Linomide has demonstrated both anti-tumor and anti-angiogenic activity with little toxicity in the Dunning R-3327 rat prostate tumor system. Therefore it was deemed essential to study the efficacy of this drug in the adjuvant situation. MATERIALS AND METHODS: Linomide, roquinimex, was administered 3 times a week i.p. alone or in conjunction with castration to rats bearing the Dunning R-3327 PAP rat prostate tumor and its effect on tumor growth analyzed. Similar experiments, in which Linomide 25 mg./kg./day was given in the drinking water were carried out in rats with the Dunning R-3327 G tumor. The effect of treatment on blood vessel density and blood flow in the tumor was also assessed using an image analysis system. RESULTS: Linomide, 2.5 & 40 mg./kg., administered from the day after castration inhibited the regrowth of the Dunning R-3327 PAP tumors In addition, Linomide 40 mg./kg. administered after tumor regrowth occurred following castration(week 10) inhibited further tumor growth. Inhibition of tumor regrowth after castration was also found in the Dunning G tumor. When Linomide treatment was stopped regrowth of the tumors occurred, either in the same animal or on transplantation to new intact hosts, demonstrating that the tumor cells were still viable. Tumor blood vessel density was decreased both after castration and Linomide treatment alone, 40 and 32% respectively. On combination of castration and Linomide a 60% decrease in blood vessel density was found. This was significantly different from either of the two treatments given alone. The enhancement on combining castration and Linomide was confirmed by a further decrease in blood flow, from 19 and 22 to 12 ml. per minute/gm. tissue respectively. CONCLUSIONS: Linomide, an anti-angiogenic drug, inhibits escape from "castration inhibition".

Potentiation of the antiangiogenic ability of linomide by androgen ablation involves down-regulation of vascular endothelial growth factor in human androgen-responsive prostatic cancers.

Linomide is a p.o. active antiangiogenic agent that has been demonstrated to be effective in suppressing the in vivo growth of rat and human prostatic cancer xenografts. The present studies were conducted to determine whether the angiogenic molecules, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and basic fibroblast growth factor (bFGF) are expressed in vitro by DU-145, PC-3, TSU-PR1, and LnCaP human prostate cancer cell lines and whether Linomide inhibits the secretion of these angiogenic molecules. Additionally, two different androgen-responsive human prostatic cancer xenograft models (i.e., PC-82 and A-2) were used to determine whether androgen ablation-induced reduction in tumor growth is associated with a reduction in tumor VEGF and/or bFGF levels. These studies demonstrated that both VEGF and bFGF proteins are expressed to different degrees in the human prostatic cancer cell lines. The secretion of VEGF but not bFGF is up-regulated by hypoxia. Linomide is unable to inhibit either basal or hypoxia-induced secretion of VEGF. Linomide also has no effect on secreted bFGF levels. Castration inhibited tumor VEGF but had no effect on bFGF levels in both the androgen-responsive PC-82 and A-2 human prostatic cancers when grown in severe combined immunodeficient mice. When given in combination, castration potentiated the inhibition of tumor growth induced by Linomide alone. This potentiation is not due to a further inhibition in tumor VEGF levels induced by castration. Although both castration and Linomide inhibit angiogenesis, the former accomplishes it by inhibiting VEGF secretion, whereas the latter has multiple effects at several steps in the angiogenic process other than VEGF secretion. Based on their different but complementary mechanisms of action, simultaneous combination of androgen ablation with Linomide enhances the anti-prostatic cancer efficacy compared to either monotherapies alone and warrants testing in humans.

Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue.

In previous studies, we have demonstrated that androgen ablation-induced growth inhibition of androgen-responsive PC-82 and A-2 human prostate cancer xenografts involves not only direct activation of programmed (apoptotic) death of these cells but also indirect activation of this death process via a decrease in tumor angiogenesis secondary to a reduction in tumor vascular endothelial growth factor (VEGF) levels. To determine whether androgens consistently regulate angiogenesis via control of VEGF levels, an additional human (i.e., LnCaP) and two rodent (i.e., Dunning G and H) androgen-sensitive prostate cancer sublines were tested. Androgen ablation causes a decrease in the subsequent growth rate of each of these three additional prostate cancer sublines, and this growth inhibition is consistently associated with a >60% reduction in tumor VEGF levels. To examine whether androgens regulate VEGF levels not only in malignant but also in normal prostatic tissue, male rats were castrated, and the temporal changes in the VEGF content of ventral prostate tissue were determined. One week after castration, VEGF content decreased to <20% within the ventral prostate. Subsequent replacement with exogenous androgen to long-term castrated rats stimulated an 8-fold rise in ventral prostate VEGF content within 1 week. To evaluate whether androgen regulation of VEGF is due to a direct effect of androgen on prostatic cells, the dose-response ability of androgens to increase VEGF levels in media of LnCaP cells grown in vitro was tested. These studies demonstrate that androgens directly stimulate VEGF secretion in these cells. The presence of 4-5-fold higher levels of VEGF in prostatic fluid versus seminal vesicle fluid obtained from benign prostatic hyperplasia and clinically localized prostate cancer patients suggests that elevated levels of VEGF may contribute to the progression of these prostatic conditions by promoting angiogenesis. In summary, one of the mechanisms for androgen sensitivity for the control of the growth of both normal and malignant prostatic tissue is via its stimulation of VEGF levels.

The antiangiogenic agent linomide inhibits tumor necrosis factor-alpha secretion via inhibition of its synthesis.

We have previously reported that linomide, a quinoline-3-carboxamide, has antitumor effects against prostatic cancers in vivo through its ability to inhibit tumor angiogenesis. Subsequently, we reported that linomide inhibits several steps in the process of angiogenesis, including direct effects on endothelial cell proliferation and their chemotactic migration and invasion. Besides these direct effects, linomide's antiangiogenic activity also involves indirect effects secondary to inhibition of tumor infiltration of macrophages and their ability to secrete the angiogenic factor tumor necrosis factor-alpha (TNF-alpha). The current studies were conducted to gain insight into the mechanism by which linomide inhibits macrophage TNF-alpha secretion. The virally transformed RAW 264.7 mouse macrophage cell line was used as a model system. Chronic in vitro exposure (7 days) to 81-650 microM linomide is cytostatic to RAW cells. Such chronic exposure to linomide significantly decreased (P < 0.05) RAW cells' baseline ability to secrete TNF-alpha and also their ability to up-regulate TNF-alpha secretion in response to lipopolysaccharide (LPS) challenge. Ribonuclease protection assays demonstrated that linomide's ability to inhibit baseline and LPS-challenged TNF-alpha secretion is not functioning at the mRNA level, because steady-state levels of TNF-alpha mRNA do not change in response to linomide. Linomide's ability to inhibit TNF-alpha secretion is not associated with an increase in cell-associated TNF-alpha levels. Immunoprecipitation experiments demonstrated that linomide did not inhibit the normal proteolytic processing of the initial 26 kDa plasma membrane-bound TNF-alpha to the secreted 17 kDa soluble form. These results demonstrate that linomide inhibits TNF-alpha secretion by inhibition of the synthesis of the TNF-alpha protein. Linomide's ability to inhibit TNF-alpha protein synthesis is not due to an inhibition of general protein synthesis or secretion and is not mediated via a change in cyclic adenosine monophosphate levels.

Antiangiogenic treatment with linomide as chemoprevention for prostate, seminal vesicle, and breast carcinogenesis in rodents.

There are two distinct phases during prostatic carcinogenesis with regard to tumor blood vessel development. During the first or prevascular phase, which may persist for years, cells that have undergone some but not all of the transformation steps undergo a limited amount of net growth, producing premalignant prostatic intraepithelial neoplastic (PIN) lesions. Most of these PIN lesions do not continue net growth and do not progress to produce histologically detectable cancer. Even the PIN lesions that do progress to cancer remain of limited virulence unless they undergo conversion to the second or angiogenic phase. Once this angiogenic phase is reached, new blood vessel development is greatly enhanced within the cancer. It is this enhanced tumor angiogenesis which allows these cancers both to grow continuously and to metastasize. Thus, inhibition of angiogenesis should be an effective chemopreventive approach for prostatic carcinogenesis. Linomide is a low molecular weight, water-soluble agent with excellent p.o. absorption and bioavailability. We have previously demonstrated that daily p.o. treatment with Linomide has antiangiogenic abilities against a series of rat and human prostatic cancer xenografts growing in vivo. In the present studies, we have demonstrated using Matrigel in in vivo angiogenesis assays that daily p.o. Linomide at 25 mg/kg/day inhibits angiogenesis induced by tumor necrosis factor alpha, acidic fibroblast growth factor, basic fibroblast growth factor, and vascular endothelial growth factor. Using an N-methylnitrosourea initiation-androgen promotion model, Linomide was given p.o. at a daily dose as high as 25 mg/kg/day for at least 1 year without major toxicity while inhibiting the development of seminal vesicle/prostate cancers in male rats by >50%. Dose-response analysis demonstrated that a Linomide blood level of 50-100 microM is optimal for such chemoprevention. In addition, Linomide treatment at a dose of 25 mg/kg/day was able to inhibit by approximately 60% the incidence of N-methylnitrosourea and approximately 50% of 7,12-dimethyl-benz(a)anthracine-induced mammary carcinogenesis in female rats.

Effects of time after ovariectomy, season and oestradiol on luteinizing hormone and follicle-stimulating hormone secretion in ovariectomized ewes.

During the breeding season, five groups of three ewes were implanted at ovariectomy with 0.36, 0.5, 1.0 and 6.0 cm oestradiol implants or implants containing no steroid. Eleven days after receiving implants, blood samples were taken every 10 min for 6 h; implants were then removed. Treatments were repeated three times during each of two consecutive breeding seasons and four times during the intervening anoestrus. In ovariectomized ewes without steroid treatment, luteinizing hormone (LH) pulse frequency increased from early to mid-breeding season, decreased to a minimum at mid-anoestrus and increased to reach a maximum at the mid-point of the second breeding season, subsequently declining. LH pulse amplitude was inversely related to frequency. Basal serum LH concentrations decreased gradually from the first breeding season to reach a minimum at mid-anoestrus and gradually increased to reach a maximum at the end of the second breeding season. Mean serum LH and follicle-stimulating hormone (FSH) concentrations were higher at the end of the second breeding season compared with the beginning of the first breeding season. All parameters of gonadotrophin secretion were decreased much more by oestradiol during the anoestrus than during the breeding season. LH pulse frequency was decreased during anoestrus and at high oestradiol concentrations during the first breeding season. Apart from LH pulse amplitude, the decreases in all parameters of gonadotrophin secretion were less during the second compared with the first breeding season. The minimum effective dose of oestradiol required to decrease mean and basal serum concentrations of LH during anoestrus was lower than in the breeding season. The minimum effective dose of oestradiol required to decrease mean serum concentrations of FSH was lower in the first compared with the second breeding season. Oestradiol depression of LH pulse amplitude and mean serum concentrations of LH and FSH showed a dose dependency during the breeding season. During anoestrus dose dependency was seen for basal concentrations of LH and mean serum concentrations of LH and FSH. We conclude that significant chronic changes in gonadotrophin secretion occur in the ewe with time after ovariectomy. Sensitivity to oestradiol also changes, and the effects of oestradiol are not always dose dependent. We suggest that the circannual pattern of LH pulse frequency and basal LH secretion are directly linked to the circannual cycle of photoperiod.(ABSTRACT TRUNCATED AT 400 WORDS)

Maturational changes in opioidergic control of luteinizing hormone and follicle-stimulating hormone in ram lambs.

Stimulation by naloxone, an opioid antagonist, of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion was examined in spring-born crossbred ram lambs raised under natural photoperiod. Vehicle (n = 6) or 1 mg naloxone/kg vehicle (n = 6) was injected (i.m.) 3 times at 2-h intervals at 5, 10 and 15 weeks of age and 4 times at 2-h intervals at 20, 25, 30 and 35 weeks of age. Blood samples were taken every 12 min for 6 h at 5, 10 and 15 weeks of age and for 8 h at 20, 25, 30 and 35 weeks of age. Naloxone had no effect on age at sexual maturity (controls 239 +/- 23 days; naloxone 232 +/- 33 days). The only significant (P less than 0.05) effect of naloxone on FSH was a greater pulse amplitude in 10-week-old treated lambs than in control lambs. Naloxone treatment resulted in greater LH pulse amplitude at 5 and 10 weeks of age (P less than 0.05), lower basal serum concentration of LH at 10 weeks of age (P less than 0.05), greater LH pulse frequency at 25 weeks of age (P less than 0.05), and greater mean serum concentrations of LH, basal LH and LH pulse amplitude at 35 weeks of age (P less than 0.01) than in the controls. In both groups of lambs, mean and basal FSH, and LH and FSH pulse amplitude were highest at 5 weeks of age and fell with age. LH pulse amplitude was lowest at 35 weeks of age (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Morphine, naloxone and the gonadotrophin surge in ewes.

Possible endogenous opioid peptide regulation of the preovulatory gonadotrophin surge was examined in ewes during the breeding season. Intact ewes (n = 54) were synchronized by treatment for 12 days with intravaginal sponges releasing medroxyprogesterone acetate. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion prior to and during the gonadotrophin surge were not affected by naloxone (0.33 mg/kg body wt per h) administered from the time of medroxyprogesterone acetate withdrawal until 30 h after the onset of oestrus (n = 6). Morphine was administered in 4 patterns: (i) 0.25 mg morphine/kg body wt per h from medroxy-progesterone acetate withdrawal until 30 h after the onset of oestrus (n = 6), (ii) 0.25 mg morphine/kg body wt per h from 24 to 48 h after medroxyprogesterone acetate withdrawal (n = 6), (iii) 0.50 mg morphine/kg body wt per h from 24 to 36 h after medroxyprogesterone acetate withdrawal (n = 6) and (iv) 0.50 mg morphine/kg body wt per h from 18 to 30 h after medroxyprogesterone acetate withdrawal (n = 6). Oestrus and the gonadotrophin surge were delayed, but not blocked, in all cases of morphine administration (P less than 0.05). Inconsistent effects of morphine on circulating oestradiol and gonadotrophin concentrations prior to the gonadotrophin surge suggest that the delays are not due to reduced gonadotrophic support of ovarian oestradiol output. Morphine may reduce responsiveness of central behavioural and gonadotrophin surge-generating centres to the oestradiol signal. The absence of effects of naloxone on gonadotrophin secretion suggest that suppression of LH secretion by opioid peptide activity is reduced after the end of the luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of progesterone restores the nocturnal surge of prolactin following suppression with a gonadotropin-releasing hormone agonist during early pregnancy in the rat.

Continuous administration of a GnRH agonist (GnRH-Ag) at a dose of 5 micrograms/day, commencing on day 7 of pregnancy resulted in the suppression of daily nocturnal surges of prolactin (PRL) on day 8, and serum progesterone (P4) levels with subsequent termination of pregnancy. Replacement with dydrogesterone, a synthetic analog of P4 at a dose of 4 mg/day s.c. restored the magnitude of nocturnal PRL surges. These data suggest that GnRH-Ag may act either at the level of the brain to suppress the nocturnal PRL surge, resulting in a fall in serum P4 levels or at the level of the corpus luteum itself or at both sites simultaneously to terminate pregnancy.