Hisatsugu Goto

Multifunctional interleukin‐1β promotes metastasis of human lung cancer cells in SCID mice via enhanced expression of adhesion‐, invasion‐ and angiogenesis‐related molecules

We examined whether interleukin‐1 (IL‐1), a multifunctional proinflammatory cytokine, progresses or regresses metastasis of lung cancer. Exogenous IL‐lβ enhanced expression of various cytokines (IL‐6, IL‐8, and vascular endothelial growth factor (VEGF)) and intracellular adhesion molecule‐1 (ICAM‐1) by A549, PC14, RERF‐LC‐AI, and SBC‐3 cells expressing IL‐1 receptors. A549 cells transduced with human IL‐1β‐gene with the growth‐hormone signaling‐peptide sequence (A549/IL‐1β) secreted a large amount of IL‐1β protein. Overexpression of IL‐1β resulted in augmentation of expression of the cytokines, ICAM‐1, and matrix metallo‐proteinase‐2 (MMP‐2). A549/IL‐1β cells intravenously inoculated into severe combined immunodeficiency (SCID) mice distributed to the lung more efficiently and developed lung metastasis much more rapidly than did control A549 cells. Treatment of SCID mice with anti‐IL‐1β antibody inhibited formation of lung metastasis by A549/IL‐1β cells. Moreover, A549/IL‐1β cells inoculated in the subcutis grew more rapidly, without necrosis, than did control A549 cells, which produced smaller tumors with central necrosis, suggesting involvement of angiogenesis in addition to enhanced binding in the high metastatic potential of A549/IL‐1β cells. Histological analyses showed that more host‐cell infiltration, fewer apoptotic cells, more vascularization, and higher MMP activity were observed in tumors derived from A549/IL‐1β cells, compared with tumors derived from control A549 cells. These findings suggest that IL‐1β facilitates metastasis of lung cancer via promoting multiple events, including adhesion, invasion and angiogenesis. (Cancer Sci 2003; 94: 244–252)

ZD6474, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, inhibits growth of experimental lung metastasis and production of malignant pleural effusions in a non-small cell lung cancer model.

ZD6474 is a novel, orally active inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase, with some additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. The purpose of this study was to determine the potential of ZD6474 in the control of established experimental lung metastasis and pleural effusions produced by human non-small cell lung cancer (NSCLC) cells. PC14PE6 (adenocarcinoma) and H226 (squamous cell carcinoma) cells express high levels of EGFR and only PC14PE6 cells overexpress VEGF. Neither ZD6474 nor the EGFR tyrosine kinase inhibitor gefitinib inhibit proliferation of PC14PE6 or H226 cells in vitro. Both PC14PE6 and H226 cells inoculated intravenously into nude mice induced multiple lung nodules after 5-7 weeks. In addition, PC14PE6 cells produced bloody pleural effusions. Daily oral treatment with ZD6474 did not reduce the number of lung nodules produced by PC14PE6 or H226 cells, but did reduce the lung weight and the size of lung nodules. ZD6474 also inhibited the production of pleural effusions by PC14PE6 cells. Histological analyses of lung lesions revealed that ZD6474 treatment inhibited activation of VEGFR-2 and reduced tumor vascularization and tumor cell proliferation. Therapeutic effects of ZD6474 were considered likely to be due to inhibition of VEGFR-2 tyrosine kinase because gefitinib was inactive in this model. These results indicate that ZD6474, an inhibitor of VEGFR-2, may be useful in controlling the growth of established lung metastasis and pleural effusions by NSCLC.

The role of surfactant protein A in bleomycin-induced acute lung injury.

RATIONALE Surfactant protein A (SP-A) is a collectin family member that has multiple immunomodulatory roles in lung host defense. SP-A levels are altered in the bronchoalveolar lavage (BAL) fluid and serum of patients with acute lung injury and acute respiratory distress syndrome, suggesting the importance of SP-A in the pathogenesis of acute lung injury. OBJECTIVES Investigate the role of SP-A in the murine model of noninfectious lung injury induced by bleomycin treatment. METHODS Wild-type (WT) or SP-A deficient (SP-A(-/-)) mice were challenged with bleomycin, and various indices of lung injury were analyzed. MEASUREMENTS AND MAIN RESULTS On challenge with bleomycin, SP-A(-/-) mice had a decreased survival rate as compared with WT mice. SP-A(-/-) mice had a higher degree of neutrophil-dominant cell recruitment and the expression of the inflammatory cytokines in BAL fluid than did WT mice. In addition, SP-A(-/-) mice had increased lung edema as assessed by the increased levels of intravenously injected Evans blue dye leaking into the lungs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and active caspase-3 staining suggested the increased apoptosis in the lung sections from SP-A(-/-) mice challenged with bleomycin. SP-A also specifically reduced bleomycin-induced apoptosis in mouse lung epithelial 12 cells in vitro. Moreover, intratracheal administration of exogenous SP-A rescued the phenotype of SP-A(-/-) mice in vivo. CONCLUSIONS These data suggest that SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity in the lung in response to acute noninfectious challenges.

A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a+) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

E7080, a Multi–Tyrosine Kinase Inhibitor, Suppresses the Progression of Malignant Pleural Mesothelioma with Different Proangiogenic Cytokine Production Profiles

Purpose: Malignant pleural mesothelioma (MPM) is a biologically heterogeneous malignant disease with a poor prognosis. We reported previously that the anti–vascular endothelial growth factor (VEGF) antibody, bevacizumab, effectively inhibited the progression of VEGF-high-producing (but not VEGF-low-producing) MPM cells in orthotopic implantation models, indicating the need for novel therapeutic strategies to improve the poor prognosis of this disease. Therefore, we focused on the multi–tyrosine kinase inhibitor E7080 and assessed its therapeutic efficacy against MPM cells with different proangiogenic cytokine production profiles. Experimental Design: The efficacy of E7080 was assayed in orthotopic implantation of severe combined immunodeficient mouse models with three human MPM cell lines (MSTO-211H, NCI-H290, and Y-MESO-14). Results: With regard to proangiogenic cytokine production profiles, MSTO-211H and Y-MESO-14 cells were MPM cells producing high levels of fibroblast growth factor-2 and VEGF, respectively. NCI-H290 cells produced low levels of fibroblast growth factor-2 and VEGF compared with the other two cell lines. E7080 potently suppressed the phosphorylation of VEGF receptor-2 and FGF receptor 1 and, thus, inhibited proliferation of endothelial cells, but not that of the MPM cell lines, in vitro. Orthotopically inoculated MSTO-211H cells produced only thoracic tumors, whereas NCI-H290 and Y-MESO-14 cells also developed pleural effusions. Treatment with E7080 potently inhibited the progression of these three MPM cell lines and markedly prolonged mouse survival, which was associated with decreased numbers of tumor-associated vessels and proliferating MPM cells in the tumor. Conclusions: These results strongly suggest broad-spectrum activity of E7080 against MPM with different proangiogenic cytokine production profiles in humans. (Clin Cancer Res 2009;15(23):7229–37)

Antitumor Vascular Strategy for Controlling Experimental Metastatic Spread of Human Small-Cell Lung Cancer Cells with ZD6474 in Natural Killer Cell–Depleted Severe Combined Immunodeficient Mice

Background: Small-cell lung cancer is often characterized by rapid growth and metastatic spread. Because tumor growth and metastasis are angiogenesis dependent, there is great interest in therapeutic strategies that aim to inhibit tumor angiogenesis. Methods: The effect of ZD6474, an orally available inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor tyrosine kinases, was studied in experimental multiple-organ metastasis models with human small-cell lung cancer cell lines (SBC-3 or SBC-5) in natural killer cell–depleted severe combined immunodeficient mice. Results: Intravenously inoculated SBC-5 cells produced experimental metastases in the liver, lung, and bone whereas SBC-3 cells produced the metastases in the liver, systemic lymph nodes, and kidneys. Daily oral treatment with ZD6474 (50 mg/kg), started on day 14 (after the establishment of micrometastases), significantly reduced the frequency of large (>3 mm) metastatic colonies (in the liver and lymph nodes) and osteolytic bone lesions. ZD6474 treatment did not significantly reduce the frequency of small (<2-3 mm) metastatic lesions found in the lung (SBC-5) or kidney (SBC-3), consistent with an antiangiogenic mechanism of action. Immunohistochemical analysis of SBC-5 metastatic deposits in the liver showed that ZD6474 treatment inhibited VEGFR-2 activation and induced apoptosis of tumor-associated endothelial cells, resulting in decreasing tumor microvessel density. ZD6474 treatment was also associated with a decrease in tumor cell proliferation and an increase in tumor cell apoptosis. The antitumor effects of ZD6474 were considered likely to be due to inhibition of VEGFR-2 tyrosine kinase because gefitinib, a small-molecule inhibitor of epidermal growth factor receptor tyrosine kinase, was inactive in these models. Conclusions: These results suggest that ZD6474 may be of potential therapeutic value in inhibiting the growth of metastatic small-cell lung cancer in humans. Phase II trials with ZD6474 are currently ongoing in a range of solid tumors.

Novel orthotopic implantation model of human malignant pleural mesothelioma (EHMES-10 cells) highly expressing vascular endothelial growth factor and its receptor.

Malignant pleural mesothelioma (MPM) is closely related to exposure to asbestos, and a rapid increase in the number of MPM patients in Japan is estimated in the years 2010–2050. The purpose of the present study was to establish a clinically relevant animal model that shows human patient‐like progression of MPM. Here, we demonstrate that a human MPM cell line (EHMES‐10) inoculated orthotopically (thoracic cavity) into severe combined immunodeficiency (SCID) mice produces highly vascularized thoracic tumors with pleural dissemination and bloody pleural effusions by 5 weeks, suggesting a patient‐like progression of this cell line after orthotopic inoculation. EHMES‐10 cells overexpressed vascular endothelial growth factor (VEGF), a molecule responsible for malignant effusions, and its receptor. Treatment with cisplatin, but not gemcitabine, significantly inhibited the production of pleural effusions, but it was not effective for thoracic tumors, consistent with chemotherapy refractory characteristics of MPM in patients. Our patient‐like orthotopic model using EHMES‐10 cells overexpressing VEGF and its receptor may be useful for examining the molecular pathogenesis of MPM and may contribute to the development of novel treatment strategies for MPM. (Cancer Sci 2006; 97: 183 –191)

A third‐generation matrix metalloproteinase (MMP) inhibitor (ONO‐4817) combined with docetaxel suppresses progression of lung micrometastasis of MMP‐expressing tumor cells in nude mice

The lung is the common target organ of hematogenous metastasis that restricts the prognosis of cancer patients. MMPs play a pivotal role in metastasis by promoting tumor invasion and angiogenesis; therefore, a large number of MMPIs have been developed. Our purpose was to determine the therapeutic efficacy of a selective‐spectrum MMPI, ONO‐4817 (inhibits MMP‐2 and MMP‐9 but not MMP‐1), against established lung micrometastasis in combination with a cytotoxic anticancer drug, DOC, in a nude mouse model. Human non‐small cell lung cancer PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells, expressing MMP‐2, MMP‐9 and/or MMP‐1, were injected i.v. into nude mice on day 0. Mice received a single injection of DOC on day 7 (after establishment of micrometastasis) and/or ONO‐4817 mixed with food from day 7 to the end of experiments. Monotherapy with ONO‐4817 or DOC inhibited formation of lung metastasis by PC14PE6 and H226 cells. In addition, combined use of ONO‐4817 with DOC significantly suppressed the tumor burden of H226 and PC14PE6 cells in the lung and prolonged the survival of PC14PE6‐bearing mice compared to ONO‐4817 or DOC alone. These therapies did not affect the body weight or food intake of tumor‐bearing mice. FIZ revealed that lung lesions, but not nontumor parenchyma of the lung, expressed gelatinolytic activity and that treatment with ONO‐4817 abrogated the gelatinolytic activity in lung lesions. These results suggest that the combined use of MMPIs with cytotoxic anticancer drugs may be helpful in the control of established lung micrometastasis by tumor cells expressing MMPs.

Molecular mechanisms of angiogenesis in non-small cell lung cancer, and therapeutics targeting related molecules

Angiogenesis, neovascularization from pre‐existing vasculature, is necessary to supply oxygen and nutrition for tumor growth in both primary and distant organs. It consists of sprouting and non‐sprouting (the enlargement, splitting, and fusion of pre‐existing vessels) processes, and both can occur concurrently. Growth of solid tumors, including non‐small cell lung cancer (NSCLC), is usually dependent on angiogenesis, which is regulated by complex mechanisms involving various angiogenesis‐related molecules. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), one of the most potent angiogenic molecules, regulates both angiogenesis and vascular permeability, and hence promotes tumor progression and development of malignant pleural effusions in NSCLC. Signals via epidermal growth factor receptor (EGFR) promote not only the tumor cell cycle, but also the process of angiogenesis. Therefore, these molecules are potential targets for anti‐tumor vasculature therapy. Many agents targeting tumor vasculature have been developed, and several compounds have shown anti‐tumor potential in pre‐clinical studies. Their efficacy against NSCLC is currently being evaluated in clinical trials.

Sensitization of Tumor-Associated Endothelial Cell Apoptosis by the Novel Vascular-Targeting Agent ZD6126 in Combination with Cisplatin

Purpose: ZD6126 is a novel vascular-targeting agent that selectively disrupts the tubulin cytoskeleton of endothelial cells. In the immature vessels characteristic of tumor vasculature, this leads to endothelial cell contraction, blood vessel congestion, and, consequently, tumor cell death. ZD6126 has been shown to delay tumor growth in a range of xenograft models. The antitumor effect of ZD6126 can be increased in combination with cisplatin or radiation therapy, although the precise mechanism of this enhancement has not been demonstrated. ZD6126 treatment has also been shown to inhibit lung metastasis, and the present study has explored the potential to increase the antimetastatic effect of ZD6126 by combining with cisplatin, and the underlining mechanism has been investigated. Experimental Design: Human lung adenocarcinoma PC14PE6 cells were injected into the tail vein of nude mice. Five weeks after injection animals were treated with ZD6126 (200 mg/kg i.p.), cisplatin (6 mg/kg i.v.), or a combination of the two agents. The animals were sacrificed 24 hours later, and the extent of lung metastases and the presence of apoptotic cells were assessed. Results: Histologic analysis revealed that the ZD6126/cisplatin combination resulted in a 2 to 4-fold increase in the total number of tumor-associated apoptotic cells compared with either treatment alone. ZD6126 alone induced apoptosis of tumor-associated endothelial cells in tumors, and the extent of apoptosis was increased 2-fold in combination with cisplatin. The lung weight was significantly reduced, and the number of metastatic nodules significantly was lower in the combined treatment group than in the control group. Conclusions: These data suggest that the antimetastatic effect of the vascular-targeting agent ZD6126 can be increased by use in combination with cisplatin, which increases the incidence of endothelial cell apoptosis.