o-(chloroacetylcarbamoyl)fumagillol and Breast-Neoplasms

Sustained breast cancer growth and metastasis requires paracrine signals between the tumor cells and the normal surrounding host tissue. One crucial function of these signals is to recruit endothelial cells and thus new blood vessels for the nourishment of the expanding tumor mass. This proliferation and migration of endothelial cells in the vicinity of progressing tumors contrasts with the extremely low turn-over rate of endothelial cells in the healthy adult. A blockade of tumor-induced endothelial cell proliferation should inhibit tumor growth and potentially metastasis with only few adverse effects. Different therapeutic approaches that take advantage of this situation are discussed with respect to their interaction with conventional therapies of breast cancer.

Topics: Animals; Breast Neoplasms; Carrier Proteins; Clinical Trials, Phase I as Topic; Cyclohexanes; Cytokines; Endothelium, Vascular; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mammary Neoplasms, Experimental; Neoplasm Proteins; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Pentosan Sulfuric Polyester; Rats; Sesquiterpenes

Angiogenesis plays an important role in tumor growth and metastasis; therefore, inhibition of angiogenesis is a promising strategy for developing new anticancer drugs. Type 2 methionine aminopeptidase (MetAP2) protein is likely a molecular target of angiogenesis inhibitors.. Nitroxoline, an antibiotic used to treat urinary tract infections, was identified from a high-throughput screen of a library of 175,000 compounds for MetAP2 inhibitors and from a parallel screen using the Johns Hopkins Drug Library to identify currently used clinical drugs that can also inhibit human umbilical vein endothelial cells (HUVEC) proliferation. To investigate the mechanism of action of nitroxoline, inhibition of MetAP2 activity and induction of senescence were assessed in HUVEC. To test the antiangiogenic activity of nitroxoline, endothelial tube formation in Matrigel and microvessel formation in Matrigel plugs in vivo were assessed. Antitumor efficacy of nitroxoline was evaluated in mouse models of human breast cancer xenograft (n = 10) and bladder cancer orthotopic xenograft (n = 11). Furthermore, the mechanism of action of nitroxoline was investigated in vivo.. Nitroxoline inhibited MetAP2 activity in vitro (half maximal inhibitory concentration [IC(50)] = 54.8 nM, 95% confidence interval [CI] = 22.6 to 132.8 nM) and HUVEC proliferation (IC(50) = 1.9 μM, 95% CI = 1.54 to 2.39 μM). Nitroxoline inhibited MetAP2 activity in HUVEC in a dose-dependent manner and induced premature senescence in a biphasic manner. Nitroxoline inhibited endothelial tube formation in Matrigel and reduced microvessel density in vivo. Mice (five per group) treated with nitroxoline showed a 60% reduction in tumor volume in breast cancer xenografts (tumor volume on day 30, vehicle vs nitroxoline, mean = 215.4 vs 86.5 mm(3), difference = 128.9 mm(3), 95% CI = 32.9 to 225.0 mm(3), P = .012) and statistically significantly inhibited growth of bladder cancer in an orthotopic mouse model (tumor bioluminescence intensities of vehicle [n = 5] vs nitroxoline [n = 6], P = .045).. Nitroxoline shows promise as a potential therapeutic antiangiogenic agent.

Topics: Acetylation; Aminopeptidases; Angiogenesis Inhibitors; Animals; Anti-Infective Agents, Urinary; Breast Neoplasms; Cell Proliferation; Collagen; Cyclohexanes; Disease Models, Animal; Drug Combinations; Drug Synergism; Endothelial Cells; Endothelium, Vascular; Female; Glycoproteins; Humans; Immunoblotting; Immunohistochemistry; Laminin; Methionyl Aminopeptidases; Mice; Mice, Nude; Neovascularization, Pathologic; Nitroquinolines; O-(Chloroacetylcarbamoyl)fumagillol; Proteoglycans; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sesquiterpenes; Sirtuin 1; Transfection; Umbilical Veins; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Methionine aminopeptidase (MetAP)-2 has been suggested as a novel target for cancer therapy because the anticancer agent TNP-470 irreversibly inactivates the catalytic activity of this enzyme. However, the importance of MetAP2 in cell growth and tumor progression was uncertain because previous data were based on the chemically reactive TNP-470. Here we show that a rationally designed reversible MetAP2 inhibitor, A-357300, suppresses tumor growth preclinically without the toxicities observed with TNP-470. We have synthesized this bestatin-type MetAP2 inhibitor with the aid of crystal structures of the enzyme-inhibitor complexes and parallel synthesis. A-357300 induces cytostasis by cell cycle arrest at the G(1) phase selectively in endothelial cells and in a subset of tumor cells, but not in most primary cells of nonendothelial type. A-357300 inhibits angiogenesis both in vitro and in vivo and shows potent antitumor efficacy in carcinoma, sarcoma, and neuroblastoma murine models. These data affirm that MetAP2 plays a pivotal role in cell growth and establish that reversible MetAP2 inhibitors are promising novel cancer therapeutic agents.

Topics: Aminopeptidases; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Division; Chlorobenzenes; Cornea; Cyclohexanes; Drug Design; Endothelium, Vascular; Female; Fibrosarcoma; Humans; Metalloendopeptidases; Mice; Mice, SCID; Models, Molecular; Neovascularization, Physiologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Protease Inhibitors; Sesquiterpenes; Xenograft Model Antitumor Assays

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Lewis Lung; Cyclohexanes; Cyclophosphamide; Female; Humans; Maximum Tolerated Dose; Mice; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Sesquiterpenes; Tumor Cells, Cultured; Vinblastine

In our previous study, the growth of KPL-1 human breast cancer cells was found to be stimulated by an antiestrogen, ICI 182, 780, and inhibited by 17 beta-estradiol (E2) in vivo but not in vitro. To investigate the action mechanisms of these paradoxical responses, the effects of E2, ovariectomy (Ovex) and medroxyprogesterone acetate (MPA) on the growth, angiogenesis, apoptosis and expression of vascular endothelial growth factor (VEGF) were investigated. E2 stimulated the growth of KPL-1 cells but MPA inhibited it in vitro. In contrast, E2 propionate inhibited the growth of KPL-1 cells in female nude mice but Ovex and MPA stimulated it. E2 propionate suppressed angiogenesis and increased apoptosis in KPL-1 tumors, but Ovex and MPA promoted angiogenesis and decreased apoptosis. Both mRNA expression and secretion of VEGF were stimulated by MPA in KPL-1 cells, but in E2-dependent ML-20 cells they were both inhibited by MPA. E2 did not significantly influence VEGF expression in either cell line. These findings suggest that the abnormal modulation of VEGF expression by MPA and of the other angiogenic factor by E2 are responsible for the paradoxical growth responses of KPL-1 cells in vivo. To support this hypothesis, an antiangiogenic agent, TNP-470, was administered to mice bearing KPL-1 tumors. TNP-470 significantly inhibited the growth of KPL-1 tumors stimulated by MPA. Antiangiogenic agents may be effective for the treatment of hormone-refractory breast cancer.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Cell Division; Cyclohexanes; Endothelial Growth Factors; Female; Humans; Lymphokines; Medroxyprogesterone Acetate; Mitotic Index; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; RNA, Messenger; Sesquiterpenes; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The effects of eicosapentaenoic acid (EPA) and an angiogenesis inhibitor (TNP-470) on the suppression of breast cancer cell growth were examined in five human breast cancer cell lines (MDA-MB-231, T-47D, MCF-7, KPL-1, and MKL-F). In all five cell lines, EPA and TNP-470 alone both showed tumor growth inhibition in a time- and dose-dependent manner, and in combination, a synergistic effect was seen at high concentrations. EPA plus TNP-470 treatment evoked apoptosis as confirmed by the appearance of sub G1 populations, by DNA fragmentation, and by cell morphology. With the combination, the expression of Bax and Bcl-xS, the apoptosis-enhancing proteins, was more up-regulated and that of Bcl-2 and Bcl-xL, the apoptosis-suppressing proteins, was more down-regulated compared to the use of EPA or TNP-470 alone, suggesting that their synergistic effect was due to an acceleration of apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Cyclohexanes; DNA Fragmentation; Drug Screening Assays, Antitumor; Drug Synergism; Eicosapentaenoic Acid; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; O-(Chloroacetylcarbamoyl)fumagillol; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Signal Transduction; Tumor Cells, Cultured

Angiogenesis inhibitor TNP-470, 6-O-(N-chloroacetyl-carbamoyl)-fumagillol, semisynthetic analogue of fumagillin, has strong inhibitory activities against in vivo tumor growth and metastasis in a wide variety of tumors. However, it is still unknown whether this agent inhibits bone metastasis. We examined the effects of TNP-470 in a bone metastasis model in nude mice in which intracardiac injection of the human breast cancer cell line MDA-MB-231 (MDA-231) produced osteolytic bone metastasis. After inoculation of MDA-231 cells into the left heart ventricle, TNP-470 (30 mg/kg, three times a week) or PBS was s.c. administrated for 4 weeks. After this period, the TNP-470 had reduced not only the number and area of osteolytic bone metastases (approximately 60 and 70%, respectively) but also their radiolucency. Histological examination of the femurs of the untreated group revealed that most of the cancellous bone had been replaced by the metastatic cancer. Numerous active osteoclasts were present along the trabecular bone surface surrounded by the metastatic MDA-231 cancer cells aggressively invading the bone marrow. In contrast, in the bone from TNP-470-treated mice, bone destruction was markedly inhibited, and there were much fewer osteoclasts. In a murine bone marrow culture under 1,25-dihydroxyvitamin D3 in which mature functional osteoclasts formed in vitro, TNP-470 significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells. And also, TNP-470 suppressed the in vivo bone resorption in calvaria treated with interleukin-1beta, an osteoclast stimulator. These data suggested that TNP-470 inhibited bone metastasis through not only antitumor action by its angiogenesis inhibition but also by the inhibition of osteoclastic bone resorption. Our results indicate that TNP-470 should be a potentially beneficial drug to be used in the treatment of osteolytic metastasis.

Topics: Animals; Antibiotics, Antineoplastic; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Cachexia; Calcitriol; Cells, Cultured; Cyclohexanes; Disease Models, Animal; Female; Heart Ventricles; Humans; Injections; Interleukin-1; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplastic Cells, Circulating; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Osteoclasts; Osteolysis; Sesquiterpenes; Tumor Cells, Cultured

Antitumor and antimetastatic activity of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), a semisynthetic analogue of fumagillin, was evaluated in breast cancer cell lines. In an in vitro MTT assay, after 72 hrs continuous exposure to TNP-470, growth inhibition was observed in all seven cell lines of murine (JYG-A, JYG-B, DD-762, and BALB/c-MC) or human (KPL-1, MDA-MB-231, and MKL-F) origin, in which the 50% inhibitory concentrations (IC50) at 72 hrs treatment were 4.6, 4.4, 4.6, 10.1, 35.0, 25.3, and 33.4 micrograms/ml, respectively. In an in vivo assay using JYG-A, JYG-B, KPL-1, and MDA-MB-231 cells by orthotopic (right thoracic mammary fat pad) transplantation in female nude mice, TNP-470 at 30 or 50 mg/kg body weight was injected s.c. every other day from the day of tumor cell inoculation until the end of the experiment. The inhibitory effect on primary tumor growth was obtained in all four cell lines in a dose-dependent manner. In the 50 mg/kg TNP-470-treated group, the reductions in tumor weight of the JYG-A, JYG-B, KPL-1, and MDA-MB-231 cells with respect to the controls were 50%, 30%, 4%, and 49%, respectively. Metastasis was seen in the JYG-A, JYG-B, and KPL-1 cells. The numbers of mice bearing pulmonary metastases of JYG-A and JYG-B cells and regional axillary lymph node metastases of KPL-1 cells were reduced, and TNP-470 at the 50 mg/kg dose to KPL-1 cells significantly reduced lymph node metastases compared with the control. Although the weight gain was retarded in the TNP-470-treated mice, weight loss was not seen. TNP-470 was highly effective in the treatment of breast cancer cells. These results suggest that the clinical use of TNP-470 may be a promising treatment for breast cancer patients.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Division; Cyclohexanes; Female; Humans; Lung Neoplasms; Lymphatic Metastasis; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

Previously, we described FGF-1- or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM-1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM-1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGF- or FGF-4-transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 10(5) times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF-transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Division; Cell Line; Cyclohexanes; Drug Implants; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Pentosan Sulfuric Polyester; Proto-Oncogene Proteins; Recombinant Proteins; Sesquiterpenes; Tamoxifen; Time Factors; Transfection; Tumor Cells, Cultured

The angiogenesis inhibitor O-(chloroacetyl-carbamoyl)fumagillol (TNP-470) showed antitumor activity in three human cancer xenograft systems. TNP-470 potently inhibited the tumor growth of hormone-independent prostate cancer PC-3 cells and breast cancer MDA-MB-231 cells dose dependently at weekly s.c. doses of 50-200 mg/kg with maximum inhibition of 96 and 88% (tumor growth, 4 and 12% of that in the respective control). In experiments of combination therapy with chemotherapeutic agents, the combination of TNP-470 (100 mg/kg) and cisplatin (5 mg/kg) showed an additive antitumor effect (from treated versus control, 38 and 22% to 5%) against PC-3 carcinoma. 5-Fluorouracil and Adriamycin alone did not significantly inhibit MDA-MB-231 tumor growth (treated versus control, 131 and 64%, respectively). TNP-470 also inhibited tumor growth of WiDr colon cancer; although the inhibition was less marked (treated versus control, 39%) than that observed with the hormone-independent cancers used in this study. In an in vitro study, all the cell lines tested were considerably insensitive to TNP-470 in monolayer cultures (50% inhibitory concentration, approximately 5 micrograms/ml), whereas TNP-470 inhibited the anchorage-independent growth of PC-3 and MDA-MB-231 cells (50% inhibitory concentration, 0.05 and 470 ng/ml, respectively). The inhibitory activity of TNP-470 against anchorage-independent growth correlated well with the in vivo antitumor activity among the cell lines tested. Thus, this inhibitory action may partly contribute to the potent antitumor activity of the angiogenesis inhibitor TNP-470, at least in the case of PC-3 and MDA-MB-231. These results suggest that hormone-independent prostate and breast cancers may be appropriate target diseases for TNP-470 clinical trials.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Division; Cell Line; Cell Survival; Cisplatin; Cyclohexanes; Dose-Response Relationship, Drug; Doxorubicin; Drug Synergism; Fluorouracil; Humans; Male; Mice; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Prostatic Neoplasms; Sesquiterpenes; Transplantation, Heterologous; Tumor Cells, Cultured