o-(chloroacetylcarbamoyl)fumagillol and Carcinoma--Lewis-Lung

Cancer-related deaths are caused principally by recurrence and metastasis arising from residual disease, whose therapeutic responses has been suggested to be substantially different from primary tumors. However, experimental animal models designed for evaluating the therapeutic responses of residual disease are mostly lacking. To overcome this deficiency, we have developed a preclinical model that recapitulates the progression for advanced nonsmall cell lung cancer (NSCLC). An archived Lewis lung carcinoma mouse tumor, propagated only through serial in vivo transplantation and never adapted to cell culture, was stably labeled using lentivirus-encoded biomarkers, consistently expressed through an RNA polymerase II promoter. Labeled tumors were inoculated into syngeneic immunocompetent mice to ensure superior tumor-host interactions. Primary tumors were resected on reaching a predetermined size, followed by treatment in a setting akin to postsurgical first-line adjuvant chemotherapy and routine imaging to monitor the progression of pulmonary metastasis. We discovered that efficacious treatment, instead of reducing disease growth rates, significantly prolonged disease-free survival and overall survival. As in the clinic, cisplatin-based regimes were more effective in this model. However, the response of metastases to specific agents could not be predicted from, and often opposed, their effects on subcutaneous "primary" tumors, possibly due to their distinct growth kinetics and host interactions. We here introduce a clinically relevant model of residual metastatic disease that may more accurately predict the therapeutic response of recurrent, metastatic disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Lewis Lung; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Cisplatin; Cyclohexanes; Disease Models, Animal; Disease Progression; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm, Residual; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Survival Rate; Treatment Outcome

Many drugs vary in potency and/or toxicity according to the time of day when they are administered. In this study, we investigated whether antitumor efficacy of angiogenesis inhibitor, TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], could be improved by optimizing the dosing schedule. Tumor-bearing mice were housed under standardized light/dark cycle conditions (lights on at 7:00 AM, off at 7:00 PM) with food and water ad libitum. The antitumor effect of TNP-470 (30 mg/kg s.c.) was more potent in mice injected with the drug at the early light phase than it was when administered at the early dark phase. The diurnal change in the antitumor effect of TNP-470 was parallel to that in its antiangiogenic activity. The variation in the effects of TNP-470 was closely related to the diurnal variations in its inhibitory action on methionine aminopeptidase activity in tumor masses. There was a significant dosing time-dependent change in the concentration of TNP-470 in plasma. The higher concentration of TNP-470 in plasma was observed when its antitumor and antiangiogenic activities were increased. These results suggest that therapeutic efficacy of TNP-470 can be enhanced by choosing the most appropriate time of day to administer the drug.

Topics: Aminopeptidases; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Carcinoma, Lewis Lung; Chronotherapy; Cyclohexanes; Male; Melanoma, Experimental; Methionyl Aminopeptidases; Mice; Mice, Inbred ICR; O-(Chloroacetylcarbamoyl)fumagillol; Sarcoma, Experimental; Sesquiterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Inflammatory conditions are associated with tumor development. IL-1beta is a multifunctional and proinflammatory cytokine that affects nearly all types of cells. To investigate the role of IL-1beta in tumor growth in vivo, we transduced the retroviral vector coding human IL-1beta gene into mouse Lewis lung carcinoma (LLC) cells and subsequently inoculated the transformant (LLC/IL-1beta) to syngeneic C57BL/6 mice. Tumors derived from LLC/IL-1beta grew faster (240%, day 18, vs null-vector control LLC/neo; p < 0.01) and showed more abundant vasculature (250%, vs LLC/neo; p < 0.05), whereas LLC/IL-1beta cells, LLC/neo cells, and wild-type LLC cells did not show any significant difference in the growth rate in vitro. As compared with LLC/neo cells, LLC/IL-1beta cells secreted 2-fold the amount of vascular endothelial growth factor and >10-fold the amount of macrophage-inflammatory protein-2 (CXCL2), one of whose main functions is angiogenesis. Although LLC/IL-1beta itself did not secrete hepatocyte growth factor (HGF), the tumor derived from LLC/IL-1beta cells also contained a >4-fold higher concentration of HGF, another angiogenic factor. In situ hybridization of HGF mRNA in LLC/IL-1beta tumor sections demonstrated that stromal fibroblasts and infiltrating cells overexpressed HGF mRNA. Moreover, when cultured in the presence of HGF in vitro, LLC/IL-1beta cells secreted even larger amounts of vascular endothelial growth factor and macrophage-inflammatory protein-2. The antiangiogenic agent TNP-470 and anti-CXCR2 Ab inhibited the tumor growth of LLC/IL-1beta cells in vivo. These results indicated that secreting IL-1beta into the tumor milieu induces several angiogenic factors from tumor and stromal cells and thus promotes tumor growth through hyperneovascularization.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Animals; Antibodies; Carcinoma, Lewis Lung; Cell Communication; Cell Division; Chemokine CXCL2; Chemokines; Cyclohexanes; Endothelial Growth Factors; Gene Transfer Techniques; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Inflammation; Interleukin-1; Lymphokines; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; O-(Chloroacetylcarbamoyl)fumagillol; Receptors, Interleukin-8B; Sesquiterpenes; Species Specificity; Stromal Cells; Tumor Cells, Cultured; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

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

Several previously identified inhibitors of angiogenesis have been epoxide-containing fungus-derived metabolites. We therefore hypothesized that novel epoxide-containing low molecular weight compounds structurally resembling known antiangiogenic agents may also exhibit antiangiogenic activity. Cytochalasin E was found to be a potent and selective inhibitor of bovine capillary endothelial (BCE) cell proliferation. Cytochalasin E differed from other cytochalasins by the presence of an epoxide. The epoxide was required for activity, because acid-catalyzed hydrolysis of the epoxide abrogated the specificity and potency of cytochalasin E. Phalloidin staining indicated that disruption of actin stress fibers by cytochalasin E occurred only at relatively high concentrations. Lower concentrations of cytochalasin E preferentially inhibited BCE cell proliferation without disrupting actin stress fibers. In vivo, cytochalasin E inhibited angiogenesis induced by basic fibroblast growth factor by 40% to 50% in the mouse cornea assay and inhibited the growth of Lewis lung tumors by approximately 72%. Cytochalasin E is a potent antiangiogenic agent that may hold promise for the treatment of cancer and other types of pathologic angiogenesis.

Topics: 3T3 Cells; Aminopeptidases; Angiogenesis Inhibitors; Animals; Aspergillus; Carcinoma, Lewis Lung; Cell Division; Cornea; Cyclohexanes; Cytochalasins; Endothelial Growth Factors; Endothelium, Vascular; Fibroblast Growth Factor 2; Humans; Lymphokines; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Mycotoxins; Neovascularization, Pathologic; Neovascularization, Physiologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

With the introduction of new drugs, the efficacy of chemotherapy in non-small-cell lung cancer has been improving. The combination of paclitaxel and carboplatin has shown activity in this disease but is far from curative.. The antiangiogenic agent regimen of TNP-470/minocycline was added to treatment with paclitaxel and carboplatin alone and in combination in animals bearing the Lewis lung carcinoma.. Administration of the antiangiogenic regimen prior to, during and after the cytotoxic therapy increased the tumor growth delay 1.6-fold and decreased the number of lung metastases to 20% of the number observed in the control animals. [14C]Paclitaxel, platinum from carboplatin and [14C]albumin levels were determined over a 24-h time course in tumors and normal tissues of animals bearing the Lewis lung carcinoma and pretreated with TNP-470/minocycline or not pretreated. There were higher levels of [14C]paclitaxel, platinum from carboplatin and [14C]albumin in the tumors and some normal tissues of the animals that had received TNP-470/minocycline compared with those that had not received TNP-470/minocycline, especially at the earlier time points. Administration of TNP-470/minocycline to animals bearing the EMT-6 mammary carcinoma increased the cytotoxicity of high-dose paclitaxel toward EMT-6 tumor cells and toward bone marrow CFU-GM. Administration of TNP-470/minocycline to animals bearing the EMT-6 mammary carcinoma also increased the cytotoxicity of carboplatin toward the EMT-6 tumor cells but did not affect the toxicity of carboplatin toward the bone marrow CFU-GM.. The addition of TNP-470/minocycline to treatment with paclitaxel and carboplatin resulted in increased antitumor activity and efficacy and further investigation of this combination is warranted.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Cells; Carboplatin; Carcinoma, Lewis Lung; Cell Survival; Colony-Forming Units Assay; Cyclohexanes; Drug Synergism; Drug Therapy, Combination; Female; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Minocycline; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Paclitaxel; Sesquiterpenes

TNP-740, minocycline, suramin and genistein have demonstrated antiangiogenic activity in various experimental systems. The effect of these agents alone and in two agent combinations on the number of intratumoral vessels and response to cytotoxic anticancer therapies was assessed in animals bearing the Lewis lung carcinoma. Treatment with each of the antiangiogenic agents alone and in two agent combinations decreased the number of intratumoral vessels visualized by CD31 or Factor VIII staining to 30% to 50% of the number in the untreated control tumors. In general, the antiangiogenic agents are more effective adjuvants to cytotoxic therapies when used as two agent combinations than as single agents. The most effective antiangiogenic combinations were: TNP-470/minocycline > TNP-470/genistein > TNP-470/suramin. The increases in the response of the primary tumor to cyclophosphamide, adriamycin, CDDP, BCNU, x-rays or 5-fluorouracil and the lung metastases occur to about the same level with the addition of antiangiogenic agents to the therapies. With the treatment combination TNP-470/minocycline/cyclophosphamide 40% of the animals were cured. The results of these studies indicate that antiangiogenic agents can be very useful additions to treatment regimens for solid tumors.

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Lewis Lung; Carmustine; Cell Division; Cisplatin; Cyclohexanes; Cyclophosphamide; Doxorubicin; Drug Synergism; Genistein; Isoflavones; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Minocycline; Neoplasm Invasiveness; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Random Allocation; Sesquiterpenes; Suramin

A remarkable approach to a specific tumor angiogenesis model in vivo is the use of alginate implants encapsulating tumor cells. However, this previously reported approach has often been questioned because of doubts regarding the relevance of hemoglobin at the alginate implant as a parameter of vascularization. In the present investigation, we examined whether or not the use of the blood pool agents FITC-dextran of high molecular weight would significantly improve the determination of vascularization at the alginate implant. In our experiments, we found a rapid distribution of FITC-dextran within the blood circulation of mice after i.v. bolus injection. The amount of FITC-dextran within alginate implants strongly correlated with the number of LL2 carcinoma cells or B16/F10 cells encapsulated. Even a low number of 10(3) cells per alginate implant led to a significantly increased accumulation of FITC-dextran. A more than 10-fold stimulation above that of controls was found with alginate implants containing 10(4) LL2 or B16/F10 tumor cells. Using the investigational compound AGM-1470 in different treatment schedules, we found that quantification of alginate implant anglogenesis with FITC-dextran is a sensitive method for the determination of angiogenesis inhibition. In conclusion, our results demonstrated that the use of FITC-dextran enables highly sensitive, quantitative measurement of blood vessel formation by alginate implants.

Topics: Alginates; Animals; Antibiotics, Antineoplastic; Carcinoma, Lewis Lung; Carcinoma, Renal Cell; Cyclohexanes; Dextrans; Drug Carriers; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Glucuronic Acid; Hemostatics; Hexuronic Acids; Kidney Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Microspheres; Molecular Weight; Neoplasms; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

The inhibition effect of angiogenesis inhibitor TNP-470 on tumor growth and metastasis was studied using Lewis lung carcinoma.. Lewis lung carcinoma cells(2.4 x 10(6)/mouse) were inoculated subcutaneously to 20 mice, they were randomized into 2 groups. From the 2nd day, the treated group was given 40 mg/kg of TNP-470 s.c. q.o.d. (8 times) and the control group was given vehicle only (3% ethanol). On the 22nd day, the weight of the subcutanous tumors and the lung metastasis rates of the 2 groups were detected. The results were analysed by Student-t and chi 2 test.. The tumor weight of the control and treated group was 3.77 +/- 1.05 g and 1.98 +/- 0.96 g, respectively (P = 0.0009). The lung metastasis rate of the control and treated group was 80%(8/10) and 30%(3/10), respectively (P = 0.03).. These results suggest that the angiogenesis inhibitor TNP-470 has a strong inhibitory effect both on growth of the primary tumor and metastasis of Lewis lung carcinoma.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Lewis Lung; Cell Division; Cyclohexanes; Female; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Random Allocation; Sesquiterpenes

In previous animal studies, interleukin 12 (IL 12) was shown to inhibit the growth of a wide spectrum of tumors in vivo but to have no direct effect on tumor cells in vitro. Also, contrary to the expectation of a T-cell-mediated effect, the antitumor activity of IL 12 was not completely abrogated in tests of T-cell-deficient mice. These observations suggest that IL 12 may possess antiangiogenic properties that account for its tumor-inhibitory effects in vivo.. Our goal was to investigate the hypothesis that IL 12 has antiangiogenic properties.. A model of basic fibroblast growth factor-induced corneal neovascularization in mice was used to evaluate the effects of IL 12 and interferon gamma (IFN gamma) on angiogenesis in vivo. Different strains of male mice, e.g., immunocompetent C57BL/6 mice, severe combined immune-deficient (SCID) mice, natural killer cell-deficient beige mice, and T-cell-deficient nude mice, were treated with IL 12 (1 microgram/day) intraperitoneally for 5 consecutive days. The extent of neovascularization in response to a basic fibroblast growth factor pellet and the inhibition of neovascularization by IL 12 or IFN gamma were assessed by measuring the maximal vessel length and the corneal circumference involved in new blood vessel formation. The antitumor activities of IL 12 and of the angiogenesis inhibitor AGM-1470 were evaluated in Lewis lung carcinoma-bearing mice. In vitro proliferation studies were performed on bovine capillary endothelial cells, mouse pancreatic islet endothelial cells, and mouse hemangioendothelioma cells.. IL 12 treatment almost completely inhibited corneal neovascularization in C57BL/6, SCID, and beige mice. This potent suppression of angiogenesis was prevented by the administration of IFN gamma-neutralizing antibodies, suggesting that the suppression was mediated through IFN gamma. In addition, the administration of IFN gamma reproduced the antiangiogenic effects observed during treatment with IL 12. Treatment with IL 12 and AGM-1470 combined did not increase toxicity and showed a trend toward enhanced antitumor efficacy in Lewis lung carcinoma-bearing mice.. IL 12 strongly inhibits neovascularization. This effect is not mediated by a specific cell type of the immune system. Instead, IL 12 has been shown to induce IFN gamma, which, in turn, appears to play a critical role as a mediator of the antiangiogenic effects of IL 12.. Recognition of the mechanisms of the antiangiogenic properties of IL 12 may be crucial in planning its clinical applications, including a possibility of coadministration with other inhibitors of neovascularization.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Lewis Lung; Cells, Cultured; Cornea; Cyclohexanes; In Vitro Techniques; Interferon-gamma; Interleukin-12; Male; Mice; Mice, Inbred C57BL; Mice, SCID; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Recombinant Proteins; Sesquiterpenes

In cancer patients, dormant micrometastases are often asymptomatic and clinically undetectable, for months or years, until relapse. We have studied dormant lung metastases under angiogenesis suppression in mice. The metastases exhibited rapid growth when the inhibition of angiogenesis was removed. Tumour cell proliferation, as measured by bromodeoxyuridine incorporation and immunohistochemical staining proliferating cell nuclear antigen, was not significantly different in dormant and growing metastases. However, tumour cells of dormant metastases exhibited a more than threefold higher incidence of apoptosis. These data show that metastases remain dormant when tumour cell proliferation is balanced by an equivalent rate of cell death and suggest that angiogenesis inhibitors control metastatic growth by indirectly increasing apoptosis in tumour cells.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Lewis Lung; Cell Division; Cyclohexanes; Fibrosarcoma; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Organ Size; Proliferating Cell Nuclear Antigen; S Phase; Sesquiterpenes; Tumor Cells, Cultured

Although the antiangiogenic agent TNP-470 does not, in general, increase the cytotoxicity of anti-cancer therapies in cell culture, the antiangiogenic agents TNP-470 and minocycline individually and especially in combination have been shown to increase the tumor growth delay produced by several standard cytotoxic therapies in the Lewis lung carcinoma. In an effort to understand the mechanism by which the antiangiogenic agent combination TNP-470/minocycline potentiates the antitumor activity of cytotoxic therapeutic agents in vivo, the biodistribution of [14C]-cyclophosphamide and cis-diamminedichloroplatinum(II) was determined 6 h after cytotoxic drug administration in animals bearing Lewis lung carcinoma pretreated with TNP-470/minocycline and in animals without pretreatment. Higher levels of 14C and platinum were found in 9 tissues (including tumor) except blood in animals pretreated with TNP-470/minocycline. The increased drug levels in the tumors may be sufficient to account for the increased tumor growth delays observed previously. DNA alkaline elution of tumors from animals pretreated with TNP-470/minocycline showed increased DNA cross-linking by both cyclophosphamide and cis-diamminedichloroplatinum(II). The possible implications of these results are discussed.

Topics: Animals; Antineoplastic Agents; Carcinoma, Lewis Lung; Carmustine; Cell Hypoxia; Cell Survival; Cisplatin; Cyclohexanes; Cyclophosphamide; DNA, Neoplasm; Drug Combinations; Male; Melphalan; Mice; Mice, Inbred C57BL; Minocycline; O-(Chloroacetylcarbamoyl)fumagillol; Platinum; Sesquiterpenes; Tumor Cells, Cultured