Effect Of TNP-470 Research Articles

Implication of Vascular Endothelial Growth Factor (VEGF) in Human Head and Neck Cancer

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.

Anti-tumor effect of radiation response by combined treatment with angiogenesis inhibitor, TNP-470, in oral squamous cell carcinoma

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.

TNP-470 fails to block the onset of angiogenesis and early tumor establishment in an intravital minimal disease model

The angiogenesis inhibitor TNP-470 (AGM-1470) has shown encouraging results in animal models of established tumors. However, results of recent clinical trials using TNP-470 have been disappointing. Since little is known about the effects of TNP-470 at the minimal disease stage, we analyzed the effects of TNP-470 on the early stages of tumor establishment. Twenty thousand green fluorescent protein (GFP)-transfected murine CT-26 (colonic carcinoma) or Panc-02-H0 (pancreatic adenocarcinoma) cells were inoculated in dorsal skin-fold chambers in BALB/c or C57BL6 mice. Tumor area and microvessel density (MVD) were quantified by intravital microscopy (IVM). Body weight was also monitored. Effects were compared with those in a conventional model involving subcutaneous (s.c.) inoculation of 10(6) tumor cells, followed by measurement of tumor volume, endogenous plasma VEGF/endostatin (ELISA) and proliferation/apoptosis/microvessel density (Ki-67/TUNEL/CD-34). TNP-470 was injected s.c. over the 10-day experimental period (30 mg/kg every 2 days [n=6] to 100 mg/kg/day [n=5 dorsal skin-fold chamber model, n=4 s.c. tumor model]). At 30 mg/kg/every second day neither CT-26 nor PANC-02-H0 tumors were inhibited in neither of the two models. TNP-470 dosage was escalated in CT-26-bearing animals until an antiangiogenic effect could be observed. In the IVM model, only TNP-470 100 mg/kg/day reduced MVD (P=0.006), but failed to block the onset of angiogenesis and tumor area increase. Body weight decreased by 25% (P<0.05). In the subcutaneous tumor model, tumor growth was reduced (P=0.045) but not blocked, while vascular endothelial growth factor (VEGF)/endostatin synthesis and Ki67/TUNEL/CD-34 were not significantly affected. While capable of reducing tumor growth in a conventional model, treatment with TNP-470 does not block the onset of angiogenesis and tumor establishment in a model of minimal disease. When used as a single agent TNP-470 does not control minimal tumor disease in experimental colonic carcinoma.

TNP-470 Inhibits Oxidative Stress, Nitric Oxide Production and Nuclear Factor Kappa B Activation in a Rat Model of Hepatocellular Carcinoma

The objective of this study is to determine if treatment with the angiogenesis inhibitor TNP-470 results in impairment of oxidative stress, inhibition of nuclear factor kappa B (NF-κB) activation and decrease of nitric oxide production in an experimental model of rat hepatocarcinogenesis. Tumour was induced by diethylnitrosamine and promoted by two-thirds hepatectomy plus acetaminofluorene administration. Experiments were carried out at 28 weeks after initiating the treatment. TNP-470 was administered at 30 mg/kg, three times per week from 20 to 28 weeks. Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Liver concentrations of thiobarbituric acid reactive substances, reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly higher than those of controls and there was a significant increase in the GSSG/GSH ratio. Tumour growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of (NF-κB) and proteolysis of IkappaB. All these effects were absent in animals receiving TNP-470. Our results indicate that TNP-470 inhibits oxidative stress, nitric oxide production and NF-κB activation induced by experimental hepatocarcinogenesis. These changes would contribute to the beneficial effects of TNP-470 in cancer treatment.

Effect of TNP-470 (AGM-1470) on the Growth of Rat Rhabdomyosarcoma Tumors of Different Sizes

Potential anticancer therapy with the fumagillin analog TNP-470 was investigated in the present project using subcutaneously growing rhabdomyosarcomas in rats. Specifically, influences of different tumor sizes at the start of treatment as well as dose/schedules were evaluated with this angiogenesis inhibitor. The results show a significant (p = ≤0.01) reduction of the growth rate, even for relatively large-sized (>7 cm3) tumors, when 50 mg/kg TNP-470 was used every other day for up to 3 or 5 injections. With 30 mg/kg TNP-470 injections, effects were seen only with tumors measuring <7 cm3. The histologic examinations demonstrate an increase in necrosis, both in the center and in the peripheral part of TNP-470-treated tumors. Overall, both tumor volume and drug dose determine treatment outcome with the rat rhabdomyosarcoma. The results suggest that angiogenesis inhibitors could represent a valid component in the treatment of progressive tumor growth, also of large tumors as often encountered in clinics. The antivasculature therapy might also improve hypoxia/necrosis–related therapeutic approaches.

Therapeutic effect of TNP-470 on spontaneous liver metastasis of colon tumors in the rabbit.

Even though angiogenesis inhibitor is thought to be one of the promising agents in tumor dormancy therapy, its optimal administration is still unknown. Therefore, the efficient protocol using TNP-470 was examined regarding its treatment affect against spontaneous liver metastases of colon tumors in the rabbit. A spontaneous liver metastases model was established in the rabbit by the inoculation of VX-2 tumors into the subserosal space of the colon. The therapeutic effect of TNP-470 was then investigated by monitoring both the number of metastatic nodules in the liver and the microvessel density (MVD) in the tumor by immunohistochemical staining using anti-CD31 monoclonal antibody. TNP-470 did not show any effect on the primary tumor. It was able to reduce the metastatic spread to liver when it was administered at the microscopic metastatic stage. Treatment at this stage, however, was not able to sufficiently control the disease. These results indicated that TNP-470 could efficiently cause the tumor to enter into a dormant state by inhibiting angiogenesis when it was used at the initial stage of the metastatic process in the liver. Regarding its clinical application, TNP-470 might be useful for preventing the metachronous liver metastases of colorectal cancer when it is administered as adjuvant therapy after curative surgery.

Contribution of angiogenesis to the progression of colon cancer: possible inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and hepatic metastasis.

The contribution of angiogenesis to tumor growth and hepatic metastasis of colorectal cancer was investigated by means of immunohistochemical study and in vitro and in vivo experiments. Colorectal cancer specimens from 30 patients with hepatic metastasis and 39 patients without hepatic metastasis were studied by staining with antibodies against factor VIII-related antigen. Microvessel count in patients with liver metastasis was significantly higher than in those without liver metastasis (p<0.005). The effect of TNP-470 was evaluated with in vitro and in vivo experiments using human colon cancer cell line, LM and the highly hepatic metastasis cell line, LM-H5. The effect of TNP-470 on the proliferation of the cancer cells and human umbilical vein endothelial cells (HUVECs) was examined. TNP-470 inhibited more sensitively the proliferation of HUVECs than cancer cells in vitro. IC50 was approximately 3 pg/ml in HUVECs and approximately 2 microg/ml in cancer cells. The effect of TNP-470 on the growth of xenografts and liver metastases by LM-H5 in nude mice was examined. TNP-470 (30 mg/kg) was administered by subcutaneous injection every third day for 4 weeks. TNP-470 inhibited both the growth of xenograft and the hepatic metastasis. The number of metastatic foci in the liver was 78.2+/-30.1 in the control group and 20.6+/-16.5 in the treated group. These results suggest that TNP-470 is a potent agent to inhibit tumor growth and hepatic metastasis of colon cancer.

Inhibition by an angiogenesis inhibitor, TNP-470, of the growth of a human hepatoblastoma heterotransplanted into nude mice

Background/Purpose: The effect of TNP-470, an angiogenesis inhibitor, on the growth of a hepatoblastoma transplanted into nude mice was examined. Methods: A hepatoblastoma obtained from a 3-year-old girl was serially transplanted into nude mice subcutaneously, and the transplant tumors of the seventh and eighth generations were used for experiments. Expression of various markers in the tumors was examined immunohistochemically. TNP-470 was injected subcutaneously every other day into tumor-bearing mice from 3 weeks after tumor transplantation. The proliferation of tumor cells and endothelial cells was estimated by means of the bromodeoxyuridine labeling index. Results: The original hepatoblastoma showed the histology of the epithelial type, consisting of both the fetal and embryonal subtypes and was positively stained with anti-α-fetoprotein (AFP), anti-cytokeratin-19 and polyclonal anticarcinoembryonic antigen antibodies, and an antihuman hepatocyte antibody (hepatocyte paraffin 1). The transplant tumors consisted of solid nests of tumor cells with numerous vascular lakes of various sizes, and showed positive staining with all antibodies that reacted positively with the original hepatoblastoma. Injections of TNP-470 at the doses of 15 mg and 30 mg/kg body weight suppressed the tumor growth and the increase in the serum level of AFP dose dependently. Injections of TNP-470 also suppressed the proliferation of tumor cells and endothelial cells in the tumors. Conclusions: Hepatoblastomas maintained in nude mice retained the immunohistochemical characteristics of the original hepatoblastoma, and TNP-470 suppressed the growth of hepatoblastomas transplanted into nude mice. TNP-470 may be worth investigating further as to its usefulness as a therapy for hepatoblastomas. J Pediatr Surg 35:1198-1204. Copyright © 2000 by W.B. Saunders Company.

Combined treatment with TNP-470 and 5-fluorouracil effectively inhibits growth of murine colon cancer cells in vitro and liver metastasis in vivo.

The combined effects of TNP-470 (TNP), a semisynthetic analogue of fumagillin, and 5-fluorouracil (5FU), a representative chemotherapeutic agent for colorectal cancer, were investigated using murine colon 26 adenocarcinoma (CT 26) cells. In a cell-proliferation study in vitro, 50% inhibitory concentrations (IC50) were determined to be 5.2 microg/ml and 240 ng/ml for TNP and 5FU, respectively. When CT 26 cells were treated with TNP and 5FU in combination, a remarkable cytotoxic effect was obtained. Isobologram analysis revealed synergism of these two agents in inhibition of the cell growth. In vivo, using a dorsal air sac assay, we found that TNP significantly inhibited the CT 26-induced angiogenesis. In addition, the combination of TNP and 5FU exerted a synergistic anti-tumor effect in a model of hepatic metastasis by portal injection of CT 26 cells. Since TNP is known to exert inhibitory effects on tumor cell growth through suppression of cell cycle progress from the G1 to S phases as well as neovascularization, it is speculated that the treatment with TNP enhanced the anti-tumor effect of 5FU through suppression of the cell cycle and tumor-derived angiogenesis. Taken together, these results suggest that combined treatment with TNP and 5FU is potentially useful for inhibition of tumor cell growth and liver metastasis of colorectal cancer.

Effects of an anti-angiogenic agent, TNP-470, on the growth of oral squamous cell carcinomas.

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.

Time until initiation of tumor growth is an effective measure of the anti-angiogenic effect of TNP-470 on human glioblastoma in nude mice.

We examined the effect of the anti-angiogenic compound TNP-470 on early tumor growth characteristics following subcutaneous implantation of 1 mm3 tissue blocks of human glioblastoma U87, in nude mice. The mice received daily injections with TNP-470, 7 mg/kg, from one day before until either 3, 7, 11, or 15 days after inoculation. The time from inoculation until initiation of exponential tumor growth was determined along with the post-therapeutic growth delay and the initial tumor doubling time (TD) for each individual tumor (n=103) on the basis of tumor volume growth curves. We found that: i) the onset of growth of U87 xenografts was effectively inhibited by concurrent treatment with TNP-470 beyond the first three days after inoculation, ii) this effect was fully reversible upon termination of therapy, and iii) the post-therapeutic growth delay was independent of the accumulated dose. These findings demonstrate that the in vivo effect of TNP-470 on tumor growth is tumor inhibitory rather than cytotoxic. The lack of effect of the anti-angiogenic compound, TNP-470, in the early 3-day schedule is consistent with the existence of an early avascular phase which precede the angiogenesis-dependent tumor growth.

Antitumor effect of the angiogenesis inhibitor TNP-470 on human digestive organ malignancy.

The antitumor and antimetastatic effects of TNP-470, an angiogenesis inhibitor, on human gastrointestinal tumors xenotransplanted into nude mice were investigated. When two gastric cancer (MT-2 and MT-5) and two colon cancer (TK-4 and TK-13) xenografts are transplanted orthotopically into nude mice, liver metastasis develops 6 weeks after transplantation. TNP-470 30 mg/kg had a significant inhibitory effect on primary tumor growth of gastric cancers when given on alternate days from 7 days after transplantation. However, when given from 10 days or 14 days after transplantation, no inhibitory effect on the growth of any tumor xenograft was observed. In contrast, liver metastasis of each xenograft type was inhibited significantly by TNP-470. The effect of TNP-470 on prognosis was investigated using a hepatic metastatic model of rat hepatoma. Although all untreated rats that received AH-130 cell implants died within one month of massive hepatic metastasis, >50% of rats treated with TNP-470 survived for 4 months. The number of apoptotic cells in hepatic metastatic foci was significantly increased by TNP-470 administration. These results suggest that TNP-470 may provide a new approach to the treatment of digestive organ malignancies.

Angiocytotoxic Therapy in Human Non-Small Cell Lung Cancer Cell Lines: Advantage of Combined Effects of TNP-470 and SN-38

The combined effects of TNP-470, a promising antiangiogenic agent, and SN-38, a camptothecin derivative, were evaluated in four human cultured cell lines derived from non-small cell lung cancer (NSCLC). Cytotoxicity experiments were determined by using a tetrazolium salt (MTT) assay. The inhibitory effects of TNP-470 on cell proliferation were dose related and the 50% inhibitory concentrations on these cell lines were 47.3-139.8 microM. Evaluation of drug interactions with isobologram and the combination index values showed that sequential exposure to SN-38 followed by TNP-470 produced synergistic effects in the four cell lines tested. Our findings suggest that such an angiocytotoxic chemotherapy might be promising for the treatment of NSCLC.

TNP-470 (a fungus-derived inhibitor of angiogenesis) reduces proliferation of cultured fibroblasts isolated from primary pterygia: a possible drug therapy for pterygia

PURPOSE. To study drug therapy for pterygium, especially the effect of a fumagillin analog, TNP-470, a potent anti-angiogenic compound, on the growth of cultured fibroblasts obtained from primary pterygia and normal human conjunctiva. METHODS. Cultured pterygium fibroblasts (PF) were exposed to different concentrations of TNP-470 every other day for 7 days (Treatment A) and to a single dose before 4 days of culture (Treatment B). Human normal conjunctival fibroblasts (HCF) were treated with TNP-470 every other day for 7 days. The cells were observed daily by phase contrast microscopy. Cell proliferation was assessed by counting cells with a hemocytometer. Trypan blue uptake was used to determine cell viability at harvest. RESULTS. TNP-470 induced a significant inhibition of PF and HCF proliferation in a dose-dependent manner (P <. 0001). At the lowest dose of TNP-470 (100 pg/ml), the cumulative inhibitory effect of TNP-470 was more potent than the sustained inhibitory effect observed by treatment B in one high dose. Nevertheless, the cytotoxic effect was dose-dependent and more marked after treatment A than after treatment B. After washing out of the drug, partial reversibility was observed at doses lower than 5 mg/ml with a significant increase of viability. HCF were less sensitive to TNP-470 and doses less than 5 mg/ml were not cytotoxic. CONCLUSION. TNP-470 appears to have a marked inhibitory effect on PF proliferation, and it may be of considerable value in the prevention of pterygium growth and recurrence.

Antitumor effect of the angiogenesis inhibitor TNP-470 and its combination effect with 5-FU and PSK in colon 26 tumors

TNP-470 is known to inhibit angiogenesis. In this study, we investigated the antitumor effect of TNP-470 in colon 26 tumors. A dose dependent growth inhibition of tumor and liver metastasis was observed. An additive administration of PSK enhanced inhibitory effect of the tumor. Combined treatment of TNP-470 and 5-FU significantly prolonged the life span compared to the TNP-470 and 5-FU group. It is suggested that the combination of TNP-470 and 5-FU might be effective for the treatment of colon cancer and PSK might increase the antitumor effect from the viewpoint of immunity and angiogenetic mechanism.

Suppression of Cyclin D1 mRNA Expression by the Angiogenesis Inhibitor TNP-470 (AGM-1470) in Vascular Endothelial Cells

The effects of TNP-470 on DNA synthesis and the expression of c-myc and cyclin D1 mRNAs were investigated in human umbilical vein endothelial (HUVE) cells synchronized by serum depletion and stimulated with bFGF and serum. DNA synthesis occurring 16 h after stimulation was inhibited when TNP-470 was present from 2 to 6 h after stimulation. C-myc mRNA expression occurring 2 h after stimulation was not inhibited by the addition of TNP-470. Cyclin D1 mRNA expression occurring 6 h after stimulation was suppressed in the presence of TNP-470 from 2 to 6 h after stimulation. On the other hand, cyclin D1 expression was not suppressed in the TNP-470-insensitive human tumor cell line WiDr. These results suggest that the inhibition of HUVE cells by TNP-470 is due to the suppression of cyclin D1 expression in mid G1 phase.

Cytostatic inhibition of endothelial cell growth by the angiogenesis inhibitor TNP-470 (AGM-1470)

Recently, we reported the anti-angiogenic action along with anti-tumour activity of TNP-470 (AGM-1470). In this study, the effect of TNP-470 on the growth of human umbilical vein endothelial (HUVE) cells was examined. TNP-470 inhibited the growth of HUVE cells in a biphasic manner. The inhibition was cytostatic in the first phase (complete inhibition at 300 pg ml-1 to 3 micrograms ml-1 with an IC50 of 15 pg ml-1) and cytotoxic in the second phase (> or = 30 micrograms ml-1). The cytostatic inhibition of HUVE cell growth by TNP-470 was durable after washing out TNP-470 in culture. Incorporation of thymidine but not uridine and leucine by HUVE cells was inhibited in the first phase, while that of all three compounds was inhibited in the second phase. Human and rat endothelial cells among various types of cells were the most sensitive to the cytostatic inhibition, while differences in the cytotoxic inhibition were minimal. These results suggest that TNP-470 exerts its specific anti-angiogenic action by inhibiting cytostatically growth of endothelial cells in a relatively specific manner.

Effect of angiogenesis inhibitor TNP-470 on vascular formation in pituitary tumors induced by estrogen in rats.

TNP-470 (TNP), an analog of fumagillin, inhibits cell proliferation and tumorigenesis in estrogen-induced pituitary tumors in rats. This study found that treatment with TNP or TNP and bromocriptine suppressed vascular formation in these pituitary tumors more than bromocriptine alone. TNP had a greater suppressive effect on vascular formation in pituitary tumors than in normal pituitary gland. Laminin and basic fibroblast growth factor (FGF) were detected around new blood vessels in normal pituitary gland and pituitary tumor. TNP did not completely suppress laminin or basic FGF expression. The effect of TNP on pituitary tumor may involve inhibition of vascular formation by mechanisms mediated by other factors in addition to basic FGF. Treatment with bromocriptine and TNP acts indirectly and directly on vascular formation and may provide a useful chemotherapeutic modality for pituitary tumors.