o-(chloroacetylcarbamoyl)fumagillol and Carcinoma--Squamous-Cell

A Phase I study of the novel angiogenesis inhibitor TNP-470 was performed. Patients with inoperable recurring or metastatic squamous cell cancer of the cervix with evaluable disease, no coagulopathy, and adequate renal, hepatic, and hematological function were eligible. One course of treatment consisted of an i.v. infusion of TNP-470 over 60 min every other day for 28 days, followed by a 14-day rest period. The starting dose was 9.3 mg/m2. Eighteen evaluable patients were treated, with a median age of 48 years (range 27-55) and performance status Zubrod 1 (range 0-2). Grade 3 neurotoxicities consisting of weakness, nystagmus, diplopia, and ataxia were encountered in two patients receiving the 71.2 mg/m2 dose. An intermediate dose level of 60 mg/m2 was evaluated and found to be well tolerated by three patients. Only one patient experienced grade 3 nausea on the 60 mg/m2 dose level. No myelosuppression, retinal hemorrhage, weight loss, or significant alopecia were observed. One patient had a complete response, which continues for 26 months, and three patients with initially progressive disease stage had stable disease for 5, 7.7, and 19+ months. Other Phase I studies, including over 200 patients, were performed concurrently with this study. Based on this experience, the dose of TNP-470 recommended for further studies is 60 mg/m2 as a 60-min i.v. infusion every Monday, Wednesday, and Friday. Neurotoxicity was dose limiting, but appears to be reversible. Otherwise, the treatment was well tolerated. The drug may be active in squamous cell cancer of the cervix. Further studies of TNP-470 in squamous cell cancer of the cervix are warranted.

Topics: Adult; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclohexanes; Dose-Response Relationship, Drug; Female; Humans; Infusions, Intravenous; Middle Aged; Nausea; Neovascularization, Pathologic; Nervous System Diseases; O-(Chloroacetylcarbamoyl)fumagillol; Salvage Therapy; Sesquiterpenes; Treatment Outcome; Uterine Cervical Neoplasms

Blocking angiogenesis may enhance conventional anticancer treatments such as radiation therapy. In this study, we examined the effects of the angiogenesis inhibitor TNP-470 on human OSCC cell lines HSC2 and KB, with combining radiation therapy in the nude mouse. We evaluated cell-induced neovascularization with dorsal air sac assay, and selected two cells (HSC2: low, KB: high) with different level of cell-induced angiogenesis. The angiogenesis inhibitor TNP-470 was given 30 mg/kg s.c. daily on day 1-5, and irradiation, 8 Gy x 1, was administered on day 1 each week for 3 weeks. Significant inhibition of tumor growth relative to untreated controls was achieved in KB cells showing high induced angiogenesis with both TNP-470 (P < 0.01) and radiation (P < 0.01) and combining TNP-470 and radiation (P < 0.01). We saw little effect of TNP-470 either alone or in addition to the effect of radiation on the HSC2 cells showing low induced angiogenesis. These results suggested that TNP-470 significantly enhanced the effect of radiation on the cells with high neovascularization. These findings indicated that individual evaluation of each tumor neovascularization potential will be important before deciding the anti-angiogenesis treatment.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclohexanes; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

Metastatic activity is one parameter indicating the malignancy of tumor cells. Angiogenesis has now been extensively studied to clarify the mechanisms of tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine expressed by many human and animal tumors. We studied the role of VEGF in tumor growth by transfecting the VEGF gene into tumor cells and analyzing the survival period of nude mice implanted with these transfected tumor cells.. Cell line: The tumor cell line, OKK-LN, was established from human maxillary squamous cell carcinoma and used in this study. The tumor cells did not produce VEGF in the culture supernatant. Transfection: OKK-LN cells were stably transfected with sense VEGF165 cDNA or with the vector alone. The full-length VEGF165 cDNA was cloned into an expression vector (pCIneo). The DNA transfection was performed by the lipofection method, and the limiting dilution method was used for cloning. ELISA was used to measure VEGF in the culture supernatant. As a control, OKK-LN cells were transfected with the vector alone without VEGF (OKK-LN/pCIneo). The tumor cells were subcutaneously injected into nude mice (Balb/c nu/nu, 6W), and the survival period and tumor volume were analyzed. Effects of angio-suppressive agent, TNP-470, and anti-VEGF antibody on tumor growth and angiogenesis: TNP-470 (supplied by Takeda Pharmaceutical Co., Ltd.) and monoclonal anti-human VEGF antibodies were intraperitoneally administered to mice implanted with tumor cells once a week and twice a week for 5 weeks, respectively. The effects of TNP-470 and anti-VEGF antibodies were analyzed by examining tumor size and survival rate and immunohistologically using CD31 monoclonal antibody.. Tumor cells transfected with sense VEGF 165 cDNA (referred to as OKK-LN/pCIneo VEGF ) produced VEGF in the supernatant permanently, confirming the establishment of a VEGF-producing human cancer cell line. We observed marked tumor growth and a shortened survival period by nine days in the OKK-LN/pCIneo-VEGF group, compared to the control group. The administration of TNP-470 and anti-VEGF antibody significantly suppressed tumor growth. The immunohistological study showed the significant suppression of a number of tumor vessels in anti-VEGF antibody-administered mice.. Our data strongly suggests that VEGF plays an important role in tumor growth and that treatment by anti-VEGF antibody may be a promising strategy against head and neck cancers.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclohexanes; DNA; Head and Neck Neoplasms; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

To investigate the antitumor apoptotic effect of AGM-1470 by comparing it to that induced by methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) therapy, currently the standard chemotherapy for bladder cancer, in a rat bladder cancer model.. A total of 45 six-week-old female rats were divided into 3 equal groups: those receiving AGM-1470 treatment; those receiving MVAC treatment, and controls. All rats were cystectomized to evaluate the therapeutic effect with regard to macroscopic tumor findings, hematoxylin and eosin pathology, apoptosis detection, and immunohistochemistry (IHC) for bcl-2.. Our experimental protocol succeeded in producing invasive bladder tumors in the majority of rats. Tumor volume was significantly reduced in the AGM-1470 and MVAC treatment groups compared with that in the control group. The apoptotic indices of tumor cells was significantly higher in the AGM-1470 and MVAC treatment groups than in the control group. IHC for bcl-2 demonstrated no statistical difference in expression among the groups. However, the apoptotic indices of high-level bcl-2 expression were significantly lower than the indices of low-level expression in the AGM-1470 group.. AGM-1470 and MVAC appear to exert a prominent mass reduction effect via tumor cell apoptosis in cases of invasive bladder tumor, although these therapies did not demonstrate any obvious modulation of bcl-2 protein expression status. Bcl-2 overexpression might be an obstacle to AGM-1470 therapy because of its significant inhibitory effect on apoptosis.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Cyclohexanes; Doxorubicin; Female; Gene Expression; Genes, bcl-2; Hyperplasia; Methotrexate; Models, Animal; O-(Chloroacetylcarbamoyl)fumagillol; Papilloma; Rats; Rats, Wistar; Sesquiterpenes; Treatment Outcome; Urinary Bladder; Urinary Bladder Neoplasms; Vinblastine

The purpose of this study was to investigate the effect of an angiogenesis inhibitor (TNP-470) on the ultra micro-structural morphological changes of GNM cell line, which was derived from human oral squamous cell carcinomas in vitro.. The GNM cells were cultured and, the effect of TNP-470 on ultra micro-structural morphological changes of GNM cells was observed under the inverted microscope, the scanning electron microscope (SEM) and the transmission electron microscope (TEM).. Numerous round cells, shrinkage of cellular membrane and dead cells were observed 48 hours after 2 micrograms/ml of TNP-470 was added into the GNM cellular suspension. After 72 hours, GNM cells became shortened and, the number of microvilli of the cellular surface was observed under the SEM and TEM. A large number of GNM cells turned into necrosis, accompanying with the destruction of mitochondria and endoplasmic reticula.. TNP-470 has a strong tumor cytotoxic effect on GNM cells, which may be due to its destructibility on mitochondria and endoplasmic reticula of GNM cells. TNP-470 can alter the surface structure of GNM cell membrane, which suggests that TNP-470 may interrupt the metastasis of GNM cells.

Topics: Angiogenesis Inhibitors; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cyclohexanes; Gingival Neoplasms; Humans; Lymphatic Metastasis; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

The anti-tumour effect of the angiogenic inhibitor TNP470, sigma-(chloro-acetyl-carbamoyl) fumagillol, a synthetic analogue of fumagillin, was studied in vitro and in vivo using KB cells, one of the human head and neck carcinoma cell lines that produce interleukin(IL)-8. In the in vitro study, the combination treatment of TNP470 and anti-IL-8 antibody significantly reduced the proliferation of KB cells. In the in vivo studies, TNP470 administration by any route (intratumoral: i.t., intraperitoneal: i.p., intravenous: i.v.) reduced the tumour volume significantly, compared to the control group. Among the groups administered TNP470, the anti-tumour effect was strongest in the it group. Furthermore, the concurrent treatment of anti-IL-8 antibody and TNP470 also maximally reduced the tumour volume. The combination therapy of TNP470 and anti-IL-8 antibody was very effective. These results suggest that combination therapy of TNP470 and anti-IL-8 antibody could be beneficial for solid tumours, such as head and neck cancer.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Combined Modality Therapy; Cyclohexanes; Depression, Chemical; Flow Cytometry; Humans; Injections, Intralesional; Interleukin-8; Male; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

To evaluate the efficacy of the use of tirapazamine (TPZ), especially combined with mild hyperthermia (40 degrees C, 60 min), in the treatment of solid tumors following an anti-angiogenic treatment with TNP-470. In addition, we assessed the effect of TPZ and/or mild hyperthermia (MHT) combined with conventional radiotherapy or chemotherapy on TNP-470 treated tumors.. C3H/He mice bearing SCC VII tumors subcutaneously received TNP-470 at two doses of 100 mg/kg after tumor cell inoculation. At the same time, the tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received TPZ administration combined with or without MHT, gamma-ray irradiation combined with or without TPZ and/or MHT, or cisplatin injection with or without TPZ and/or MHT. Another group of mice received a series of test doses of gamma-rays while alive or after being killed to obtain hypoxic fractions (HFs) in the tumors at various time points after the above-mentioned cytotoxic treatment point. After each treatment, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (or quiescent [Q] cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells.. TPZ administration combined with TNP-470 treatment and MHT increased the MN frequency more markedly than treatment with TPZ alone, and this tendency was more remarkable in Q cells than total cells. In both total and Q cells, combined treatment with TPZ and MHT produced significant increases in MN frequencies whether gamma-rays were delivered to TNP-470 treated tumors or cisplatin was injected into the TNP-470 administered mice. Although not significantly, the HFs of total and Q cell populations within solid tumors increased after TNP-470 treatment.. Combined treatment with TPZ and MHT, whether other cytotoxic treatments such as gamma-ray irradiation or chemotherapy using cisplatin were combined or not, was useful for sensitizing tumor cells in vivo including Q cells even after TNP-470 treatment.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Cell Survival; Cisplatin; Combined Modality Therapy; Cyclohexanes; DNA, Neoplasm; Dose-Response Relationship, Drug; Hyperthermia, Induced; Mice; Mice, Inbred C3H; Micronucleus Tests; O-(Chloroacetylcarbamoyl)fumagillol; Radiation-Sensitizing Agents; Regression Analysis; Sesquiterpenes; Tirapazamine; Triazines

We investigated the effects of the angiogenesis inhibitor TNP-470 on human lung squamous cell carcinoma cell lines H226B and H226Br both in vivo and in vitro. H226B was established from human lung squamous cell carcinoma and H226Br was established from a brain metastatic lesion of H226B in nude mice. Nude mice inoculated with these cells were treated with 30 mg / kg of TNP-470 subcutaneously every other day. At this dose, TNP-470 only significantly suppressed the growth of H226Br tumor, but not H226B tumor. Attempts to use a high dose of TNP-470 (100 mg / kg) resulted in a severe loss of body weight. Immunohistochemical studies showed marked tumor vascularization in H226Br tumor, but the formation of new blood vessels was suppressed by 30 mg / kg of TNP-470. Investigation of the mechanism of anti-angiogenic effects of TNP-470 in vivo showed that the expression and the activity of platelet-derived endothelial cell growth factor / thymidine phosphorylase (PD-ECGF / dThdPase) in H226Br tumor was significantly suppressed by 30 mg / kg of TNP-470. Furthermore, TNP-470 inhibited cell growth of cultured H226Br dose-dependently at concentrations of 1 microg / ml. Immunoblot analysis revealed H226Br cells gave a stronger PD-ECGF signal than H226B cells, and the expression of PD-ECGF / dThdPase in H226Br was also suppressed by treatment with TNP-470 at 0.1 microg / ml. No change in basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) was noted in these cell lines. Our results suggested that TNP-470 acts, at least in part, by downregulation of PD-ECGF / dThdPase in this cell line.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Body Weight; Carcinoma, Squamous Cell; Cell Division; Cyclohexanes; Down-Regulation; Female; Growth Inhibitors; Humans; Lung Neoplasms; Mice; Mice, Inbred ICR; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Thymidine Phosphorylase; Tumor Cells, Cultured

The antitumor effect of the angiogenesis inhibitor TNP470, O-(chloro-acetyl-carbamoyl) fumagillol, a synthetic analogue of fumagillin, was studied in vitro and in vivo on, cell line KB which produced interleukin (IL)-8. In vitro, TNP470 reduced the production of IL-8 from KB cells, the same as anti-IL-8 antibody (Ab.) The combination of anti-IL-8 Ab (10 micrograms/ml) and TNP470 (10 ng/ml) significantly inhibited the proliferation of KB cells, compared to no treatment (p < 0.05). Proliferation of KB cells was also significantly more suppressed by simultaneous treatment of cisplatin and TNP470 (1 mg/ml), than cisplatin alone. The in vivo antitumor effect of TNP470 was studied using anti-IL-8 Ab, anti-vascular endothel growth factor (VEGF) Ab, and TNP470, in administered by different routes, i.e., intratumoral (i.t.), intraperitoneal (i.p.), and intravenous. TNP470 (10 mg/ml) showed an antitumor effect, and intratumoral administration of TNP470 was the most effective route. Combined administration of anti-IL-8 Ab (i.p.) and TNP470 (i.t.) reduced tumor volume more than anti-IL-8 Ab alone did. These results suggest that the combination of TNP470, cisplatin, and anti-IL-8 Ab could be a beneficial treatment for solid tumors of the head and neck.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Cyclohexanes; Head and Neck Neoplasms; Humans; Interleukin-8; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

Clinical and histopathological results in human tumors indicate that the connection of solid tumors to the vascular system precedes the exponential tumor growth and further progression. Acknowledging the concept of tumor angiogenesis, the search for angiogenesis inhibiting agents as potential drugs in cancer treatment began rather early. In the present preclinical nude mice model, the antitumoral effect of TNP-470 on xenotransplanted squamous cell carcinoma was tested. The chosen dosage of 30 mg/kg and 60 mg/kg resulted in a significant growth inhibition (P = 0.006 and P = 0.01) compared to the control group. The available in vivo and in vitro data lead to the conclusion that the concept of angiogenesis inhibition will have some impact on treatment of solid tumors in the near future.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Cyclohexanes; Dose-Response Relationship, Drug; Humans; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tongue Neoplasms; Tumor Cells, Cultured

Anti-tumor effects of an anti-angiogenic agent, TNP-470, on the growth of oral squamous cell carcinomas (SCCs) were evaluated in vivo and in vitro. The growth of an oral SCC cell line, HSC-2, inoculated subcutaneously in severe combined immuno-deficiency (SCID) mice was inhibited in a dose-dependent manner by the treatment with this agent. A reduction of microvessels surrounding tumor tissues treated with TNP-470 was observed by immunohistochemistry. Significant side-effects were not observed except for weight loss during the period of treatment with high dose (50 mg/kg) of TNP-470. The direct effects of TNP-470 on oral SCC cell lines were also evaluated in culture. The growth of all eight SCC cell lines tested was inhibited by TNP-470, but the sensitivity of the oral SCC cell lines to TNP-470 was about 1700 times less than that of endothelial cells. These results suggest that TNP-470 inhibits the growth of oral SCC by anti-angiogenic activities and that it may be effective as a new therapy of oral cancer.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Division; Cyclohexanes; Drug Screening Assays, Antitumor; Immunohistochemistry; Mice; Mice, SCID; Mouth Neoplasms; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

Topics: Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Clinical Trials, Phase I as Topic; Cyclohexanes; Female; Humans; Lung Neoplasms; Middle Aged; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Uterine Cervical Neoplasms