A novel hypoxia-dependent 2-nitroimidazole KIN-841 inhibits tumour-specific angiogenesis by blocking production of angiogenic factors.

Tumour angiogenesis is initiated by angiogenic factors that are produced in large amounts by hypoxic tumour cells. The inhibition of this step may lead to tumour-specific antiangiogenesis because normal tissues are not usually hypoxic. On the other hand, blocking a biological function of endothelial cells is known to result in angiogenic inhibition. To produce a tumour-specific and powerful antiangiogenesis, we determined whether potent angiogenic inhibition could be achieved by inhibiting the production of angiogenic factors by hypoxic tumour cells and simultaneously blocking certain angiogenic steps in endothelial cells under normoxia. We focused on the 2-nitroimidazole moiety, which is easily incorporated into hypoxic cells and exhibits its cytotoxicity as hypoxic cytotoxin. We designed and synthesised 2-nitroimidazole derivatives designated as KIN compounds, and investigated their antiangiogenic activities under normoxia using a chick embryo chorioallantoic membrane. KIN-841 (2-nitroimidazole 1-acetylhydroxamate) showed a potent angiogenic inhibition in a dose-dependent manner. This compound inhibited the proliferation of bovine pulmonary arterial endothelial (BPAE) cells more strongly than that of tumour cells, such as Lewis lung carcinoma (3LL) cells, under normoxia. The inhibition of cell proliferation by KIN-841 under hypoxia increased about five-fold compared to that under normoxia. Moreover, under hypoxia, KIN-841 significantly decreased the excessive production of vascular endothelial cell growth factors induced by 3LL cells as determined by tritium-labelled thymidine ([(3)H]thymidine) incorporation into BPAE cells and by ELISA. Intraperitoneal administration of KIN-841 suppressed 3LL-cell-induced in vivo angiogenesis in the mouse dorsal air sac system. These results indicate that the regulation of the production of angiogenic factors by hypoxic tumour cells is a useful target for tumour-specific angiogenesis inhibition, and that KIN-841, which causes simultaneous direct inhibition of endothelial cell function and production of angiogenic factors by hypoxic tumour cells, is a very potent inhibitor of tumour-specific angiogenesis. Thus, the potential for clinical use of KIN-841 as an antitumour drug is very high.

Inhibition of angiogenesis by humulone, a bitter acid from beer hop.

On the basis of our previous finding that humulone, a bitter acid from beer hop extract, was a potent inhibitor of bone resorption and inhibited the catalytic activity of cyclooxygenase-2 (COX-2) and more potently the transcription of the COX-2 gene, we examined the effect of humulone on angiogenesis, using chick embryo chorioallantoic membranes (CAMs) and vascular endothelial and tumor cells. Humulone significantly prevented in vivo angiogenesis in CAM in a dose-dependent manner with an ED(50) of 1.5 microg/CAM. Humulone also inhibited in vitro tube formation of vascular endothelial cells. Moreover, it suppressed the proliferation of endothelial cells and the production of vascular endothelial growth factor (VEGF), an angiogenic growth factor, in endothelial and tumor cells. Thus, humulone is a potent angiogenic inhibitor, and may be a novel powerful tool for the therapy of various angiogenic diseases involving solid tumor growth and metastasis.

Three novel synthetic retinoids, Re 80, Am 580 and Am 80, all exhibit anti-angiogenic activity in vivo.

In a previous study, we demonstrated that retinoic acid or a synthetic retinoid, Ch 55 ((E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid), significantly affects in vivo angiogenesis, on the basis of our working hypothesis that a cell differentiation modulator could also exhibit anti-angiogenic activity. In the present study, three novel synthetic retinoids, Re 80 (4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl- 2- naphthalenyl)-1-propenyl]benzoic acid), Am 580 (4-[(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid) and Am 80 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl] benzoic acid), whose cell differentiation-modulating effects are roughly comparable to or more potent than that of Ch 55, which was the most effective angiostatic retinoid identified previously, were examined. Their anti-angiogenic effects were tested in an in vivo assay system involving chorioallantoic membranes of growing chick embryos. They were all found to exert dose-dependent anti-angiogenic effects in the picomolar range. Their rank order for inhibitory potency was Re 80 > Am 580 > Am 80, the ID50 values being 6.3, 23 and 28 pmol/egg, respectively. These results indicate that treatment involving these three novel synthetic retinoids might have potential therapeutic efficacy in various angiogenesis-dependent disorders, including solid tumors, psoriasis, rheumatoid arthritis and diabetic retinopathy.

Radicicol, a microbial cell differentiation modulator, inhibits in vivo angiogenesis.

Angiogenesis plays a significant role in various pathological states, including the progressive growth of solid tumors, rheumatoid arthritis, psoriasis, and diabetic retinopathy, in addition to its crucial role in embryonic development. Recent studies have revealed that an angiogenesis inhibitor is efficacious for these so-called angiogenic diseases. In the previous studies, we found that retinoids and vitamin D3 analogs, which are known to exhibit cell differentiation-modulating activity, effectively inhibit angiogenesis in vivo, thus forming the basis of our working hypothesis that a modulator of cell differentiation is capable of affecting angiogenesis. In this study, to verify this hypothesis further, radicicol (syn. monorden; 5-chloro-6-(7,8-epoxy-10-hydoxy-2-oxo-3,5-undecadienyl)-beta -resorcylic acid mu-lactone), a microbial cell differentiation modulator from a fungus, a strain of Neocosmospora tenuicristata, was examined for its anti-angiogenic activity in a bioassay system involving chorioallantoic membranes of growing chick embryos. The microbial cell differentiation modulator dose dependently inhibited embryonic angiogenesis, the ID50 value being 200 ng/egg. Radicicol also inhibited both the proliferation of and plasminogen activator production by vascular endothelial cells in the nM concentration range in a concentration-dependent manner, suggesting the possible involvement of these inhibitory effects in the anti-angiogenic action of the microbial product. These results indicate that radicicol might be a potential drug for treating different angiogenesis-dependent diseases, such as solid tumors, psoriasis, rheumatoid arthritis, and diabetic retinopathy.

Inhibition of angiogenesis by erbstatin, an inhibitor of tyrosine kinase.

Here we describe the inhibitory effect of erbstatin, a specific tyrosine kinase inhibitor, on in vivo angiogenesis. Inhibition of angiogenesis was determined in a bioassay system involving chorioallantoic membranes of growing chick embryos. Erbstatin produced a dose-dependent inhibitory action on embryonic angiogenesis. This inhibition occurred at as small a dose as 10 ng/egg and the ID50 value was 80 ng/egg. To analyze this inhibition, in vitro experiments involving vascular endothelial cells were also performed. Erbstatin affected the proliferation of vascular endothelial cells, one of angiogenic components. This inhibition was dose-dependent, the IC50 value being 3.6 microM. These data indicate that erbstatin-sensitive tyrosine kinase(s) is involved in angiogenic endothelial cell proliferation, and that experiments involving erbstatin will provide an important due to understand a mechanism of angiogenesis.

Inhibition of angiogenesis by staurosporine, a potent protein kinase inhibitor.

The effect of staurosporine, a potent inhibitor of protein kinases, on embryonic angiogenesis was studied in an in vivo assay system involving chorioallantoic membranes of growing chick embryo. Staurosporine inhibited embryonic angiogenesis in a dose-related manner, the ID50 value being 71 pmol/egg. Staurosporine dose-dependently suppressed the proliferation of vascular endothelial cells, an important event involved in the angiogenesis process. The IC50 value was 0.88 nM. In contrast, staurosporine did not affect the migration of vascular endothelial cells. These results suggest that staurosporine affected embryonic angiogenesis probably by inhibiting endothelial cell proliferation. In addition, these results might support the notion that certain protein kinase(s) could be implicated in induction of angiogenesis and also that staurosporine would be a useful compound for studying a mode of action of angiogenesis occurring in various diseases, including tumor development.

Effect of 15-deoxyspergualin, a microbial angiogenesis inhibitor, on the biological activities of bovine vascular endothelial cells.

We found recently that 15-deoxyspergualin, an analog of spergualin, which is an antibiotic and includes a spermidine moiety in its structure, exhibits anti-angiogenic activity. We have now carried out in vitro experiments with bovine vascular endothelial cells to determine which events occurring during angiogenesis are affected by this microbial angiogenesis inhibitor. 15-Deoxyspergualin did not inhibit the production of urokinase-type plasminogen activator (u-PA) or type IV collagenase by vascular endothelial cells. The direct inhibition of u-PA activity by 15-deoxyspergualin was not observed either. The angiostatic antibiotic neither affected the migration of vascular endothelial cells nor inhibited the endothelial cell proliferation in a two-dimensional culture system. We also examined the effect of 15-deoxyspergualin on the proliferation of endothelial cells in a three-dimensional culture system involving collagen gel, in which cell growth resembles more closely the endothelial cell proliferation during in vivo angiogenesis than that in a two-dimensional culture system without collagen gel. The antibiotic inhibited cell proliferation in a dose-dependent manner, indicating that the three-dimensional culture system is useful for finding a new angiogenesis inhibitor with a different mode of action from those of angiogenesis inhibitors found by using a two-dimensional assay system; however, no cause-effect relationship has yet been established. Taken together, these results suggest the possible involvement of the inhibition of vascular endothelial cell growth by 15-deoxyspergualin in its angiogenesis-inhibitory effect. 15-Deoxyspergualin appears to be a promising candidate as an angiogenesis inhibitor for controlling aberrant angiogenic responses occurring in different states, including tumor development.(ABSTRACT TRUNCATED AT 250 WORDS)

Eponemycin, a novel antibiotic, is a highly powerful angiogenesis inhibitor.

Eponemycin, a novel antibiotic, was examined as to its anti-angiogenic activity in an in vivo assay system involving chorioallantoic membranes (CAMs) of growing chick embryos. Eponemycin powerfully inhibited angiogenesis in the CAMs. This powerful inhibition was dose-dependent, the inhibitory activity becoming detectable at a dose of 7.5 fmol/egg and the ID50 value being 250 fmol/egg, suggesting that eponemycin exhibits more potent anti-angiogenic activity than Ch 55, a synthetic retinoid, which had been the strongest angiogenesis inhibitor identified so far. To determine which event(s) in the angiogenesis process was affected by eponemycin, experiments were conducted using systems involving cultured vascular endothelial cells. Eponemycin effectively inhibited both the proliferation and migration of endothelial cells, indicating that the antibiotic affected these two important events during angiogenesis, resulting in effective inhibition of angiogenesis. These results strongly suggest that eponemycin could be a promising candidate as an angiogenesis inhibitor for the control of aberrant angiogenesis occurring in different diseases such as tumor development and diabetic retinopathy.