Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Antitumor activity of α-pinene in T-cell tumors.

T-cell acute leukemia and lymphoma have a poor prognosis. Although new therapeutic agents have been developed, their therapeutic effects are suboptimal. α-Pinene, a monoterpene compound, has an antitumor effect on solid tumors; however, few comprehensive investigations have been conducted on its impact on hematologic malignancies. This report provides a comprehensive analysis of the potential benefits of using α-pinene as an antitumor agent for the treatment of T-cell tumors. We found that α-pinene inhibited the proliferation of hematologic malignancies, especially in T-cell tumor cell lines EL-4 and Molt-4, induced mitochondrial dysfunction and reactive oxygen species accumulation, and inhibited NF-κB p65 translocation into the nucleus, leading to robust apoptosis in EL-4 cells. Collectively, these findings suggest that α-pinene has potential as a therapeutic agent for T-cell malignancies, and further investigation is warranted.

Aminopeptidase N/CD13 as a potential therapeutic target in malignant pleural mesothelioma.

Angiogenesis is a crucial factor in the progression of malignant pleural mesothelioma (MPM) and antiangiogenic strategies might be effective against MPM. Aminopeptidase N (APN)/CD13 promotes tumour angiogenesis and is associated with poor prognosis; however, its clinical significance in MPM remains unclear.In 37 consecutive patients with surgically resected MPM, we evaluated the association between immunohistochemical APN/CD13 expression in resected tumours and survival. Additionally, the antitumour and antiangiogenic effects of MT95-4, a fully humanised anti-APN/CD13 monoclonal antibody, were evaluated in mice orthotopically implanted with EHMES-10 (abundantly expressing APN/CD13) and MSTO-211H (scarcely expressing APN/CD13) MPM cells.High tumour APN/CD13 expression was associated with poor prognosis in MPM patients (p=0.04), and MT95-4 treatment reduced tumour growth and angiogenesis in mice harbouring EHMES-10 but not MSTO-211H cells. Furthermore, in mice harbouring EHMES-10 cells, MT95-4 combined with cisplatin more effectively suppressed tumour progression than cisplatin alone.Taken together, these results suggest that APN/CD13 is implicated in the aggressiveness of MPM. Here, MT95-4 treatment reduced tumour progression likely by inhibiting angiogenesis, suggesting APN/CD13 as a potential molecular target for MPM treatment. Additionally, combination treatment with MT95-4 and cisplatin could represent a promising approach to treating MPM exhibiting high APN/CD13 expression.

MT95-4, a fully humanized antibody raised against aminopeptidase N, reduces tumor progression in a mouse model.

Aminopeptidase N (APN/CD13) is involved in tumor cell invasion and tumor angiogenesis and is considered a promising therapeutic target in the treatment of cancer. To develop a novel monoclonal antibody-based cancer therapy targeting APN/CD13, we established a fully humanized anti-APN/CD13 monoclonal antibody, MT95-4. In vitro, MT95-4 inhibited APN/CD13 enzymatic activity on the tumor cell surface and blocked tumor cell invasion. B16 mouse melanoma cells stably expressing human APN/CD13 were also established and were inoculated s.c. or injected i.v. into nude mice. We found that expression of human APN/CD13 in murine melanoma cells increased the size of subcutaneous tumors, extent of lung metastasis and degree of angiogenesis in the subcutaneous tumors; these tumor-promoting and angiogenesis-promoting characteristics were reduced by the i.p. administration of MT95-4. To further verify the specificity of MT95-4 for neutralization of APN/CD13 activity, MT95-4 was administered into NOD/SCID mice inoculated s.c. with H1299 or PC14 cells, which exhibit high expression of APN/CD13, or with A549 cells, which exhibit weak expression of APN/CD13. MT95-4 reduced tumor growth and angiogenesis in mice bearing H1299-derived and PC14-derived tumors, but not in mice bearing A549-derived tumors. These results suggested that the antitumor and anti-angiogenic effects of MT95-4 were dependent on APN/CD13 expression in tumor cells. Given that MT95-4 is the first fully humanized monoclonal antibody against APN/CD13, MT95-4 should be recognized as a promising candidate for monoclonal antibody therapy against tumors expressing APN/CD13.

Clinicopathological significance of aminopeptidase N/CD13 expression in human gastric carcinoma.

BACKGROUND/AIMS: Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme occurring in a wide variety of cells. Recently, APN/CD13 has been reported to be involved in tumor invasion and metastasis. However, precise functions in tumor cells, and its role in gastric carcinoma remain unclear. METHODOLOGY: To evaluate the role of APN/CD13 in gastric carcinoma, we conducted immunohistochemical staining for APN/CD13 in 121 gastric carcinoma specimens, using anti-APN monoclonal antibody. The relationship between APN/CD13 expression and various prognostic factors of gastric carcinoma were investigated. RESULTS: Of the 121 patients with gastric carcinoma studied, 48 were strongly positive, 36 were weakly positive, and 37 were negative. Overall survival rate of the patients with negative APN/CD13 expression was significantly lower than that of the patients with positive APN/CD13 expression. APN/CD13 expression was negatively associated with lymph node metastasis. Multivariate analysis showed APN/CD13 expression to be a significant prognostic factor. CONCLUSIONS: Decreased expression of APN/CD13 was associated with a poor prognosis. Hence, our results demonstrate that the immunohistochemical detection of APN/CD13 could provide useful information as one of the prognostic factors in gastric cancer.

Adenoviral transduction of MRP-1/CD9 and KAI1/CD82 inhibits lymph node metastasis in orthotopic lung cancer model.

Conventional therapies still remain less effective for metastasis of lung cancer, thus leading to a poor prognosis for this disorder. Although the processes involved in metastasis have not yet been clearly elucidated, our previous studies have shown that higher expression levels of MRP-1/CD9 and KAI1/CD82 in cancer cells are significantly correlated with less metastatic potency. To determine whether the gene transfer of these tetraspanins into lung tumor cells may be a useful strategy to regulate metastasis, we adopted an orthotopic lung cancer model produced by the intrapulmonary implantation of Lewis lung carcinoma (LLC) cells and evaluated the metastatic growth in the mediastinal lymph nodes using two different methods of gene delivery as follows: (a) the implantation of LLC cells preinfected with adenovirus encoding either MRP-1/CD9 cDNA, KAI1/CD82 cDNA, or LacZ gene into the mouse lung and (b) the intratracheal administration of these adenoviruses into the mice orthotopically preimplanted with LLC cells. In both cases, we found that the delivery of either MRP-1/CD9 or KAI1/CD82 cDNA dramatically reduced the metastases to the mediastinal lymph nodes in comparison with those of LacZ gene delivery, without affecting the primary tumor growth at the implanted site. These results reemphasize the important role of MRP-1/CD9 and KAI1/CD82 in the suppression of the metastatic process and also show the feasibility of gene therapy when using these tetraspanins for lung cancer to prevent metastasis to the regional lymph nodes. This strategy may therefore be clinically applicable as a prophylactic treatment to suppress the occurrence of lymph node metastasis.

Clinical significance of aminopeptidase N in non-small cell lung cancer.

PURPOSE: The aim of our study is to investigate the mechanism of metastasis, to detect novel metastasis-associated molecules, and to evaluate the molecules from the point of view of clinical application. A monoclonal antibody MH8-11, which we established, recognizes a glycoprotein that is identical to aminopeptidase N (APN/CD13). APN/CD13 degrades the extracellular matrix, while it is also involved in cell motility and improves angiogenesis. EXPERIMENTAL DESIGN: We investigated the expression of APN/CD13 in 194 cases of non-small cell lung cancer (NSCLC) by immunohistochemical analyses and reverse transcription-PCR assay to determine the significance of this prognostic factor; 95 tumors were stage I, 36 were stage II, 39 were stage IIIA, and 24 were stage IIIB. Moreover, we investigated that the relationship between the expression of APN/CD13 and angiogenesis and prognosis for patients with NSCLC. RESULTS: We found a correlation between the expression of APN/CD13 and angiogenesis (r = 0.659; P < 0.0001). In the 194 patients with NSCLC, we found 68 patients to be APN/CD13+ and 126 patients to be APN/CD13-. The 5-year survival rate in patients with APN/CD13+ tumors was significantly lower than in those whose tumors had negative APN/CD13 (48.3% versus 67.1%; P = 0.0001). CONCLUSION: Our data suggest the expression of APN/CD13 for patients with NSCLC to be associated with a poor prognosis and angiogenesis. This is the first study to show the relationship between the expression of APN/CD13 and the prognosis of patients with NSCLC. The inhibition of APN/CD13 may be an effective new molecular target therapy for patients with NSCLC.

Circulating aminopeptidase N/CD13 is an independent prognostic factor in patients with non-small cell lung cancer.

PURPOSE: Aminopeptidase N, also known as CD13, has important roles in tumor metastasis and angiogenesis. Its expression in tumor tissue has been reported to be associated with poor prognosis. However, the clinical significance of circulating aminopeptidase N/CD13 in patients with solid tumors is unknown. We previously developed an aminopeptidase N/CD13-specific monoclonal antibody (mAb) MH8-11, which inhibits cell motility and angiogenesis in vitro. The aim of this study was to evaluate the clinical significance of circulating aminopeptidase N/CD13 protein detected by mAb MH8-11 in patients with non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: We used electrochemiluminescence immunoassay with mAb MH8-11 to determine circulating aminopeptidase N/CD13 levels in 90 healthy volunteers and 90 patients with NSCLC. Circulating aminopeptidase N/CD13 levels were measured in sera taken before treatment and evaluated for a relationship with clinical outcomes. RESULTS: A significant correlation was found between tumor progression and serum aminopeptidase N/CD13 concentrations (r=0.23, P=0.029). High serum aminopeptidase N/CD13 levels (n=17) were associated with advanced stage (P=0.004) or poor performance status (P=0.001). The overall survival rate for patients with high serum aminopeptidase N/CD13 levels (n=17) was significantly less than that of patients with low serum aminopeptidase N/CD13 levels (n=73, P<0.0001). In a multivariate survival analysis in patients with NSCLC, serum aminopeptidase N/CD13 levels had an independent influence on survival (relative risk, 4.1; 95% confidence interval, 1.9-8.8). CONCLUSIONS: Our data suggest that a high level of circulating aminopeptidase N/CD13 at diagnosis is an independent prognostic factor in patients with NSCLC.

MRP-1/CD9 gene transduction downregulates Wnt signal pathways.

Motility-related protein-1 (MRP-1/CD9) is a transmembrane glycoprotein that has been implicated in cell adhesion, motility, proliferation, and differentiation. It has a functional role as a tumor metastatic suppressor. During tumor progression, a reduction of MRP-1/CD9 gene expression results in tumor cells with a high metastatic potential. However, the mechanism of action of MRP-1/CD9 is still unclear. We studied changes of gene expression in relation to MRP-1/CD9 gene transduction into tumor cell lines, HT1080 and A549, using microarray assays and real-time PCR. Consequently, we have demonstrated that MRP-1/CD9 gene transduction can downregulate expression of several Wnt family genes, such as Wnt1, Wnt2b1 and Wnt5a, and their target genes, including WISP-1 (Wnt-1 induced secreted protein 1), WISP-3, c-Myc, vascular endothelial growth factor-A, and matrix metalloproteinase-26. Western blot analyses also showed that MRP-1/CD9 gene transduction downregulated expression of Wnt1 protein and its target proteins. In addition, a neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited the downregulation of Wnt signal pathways in MRP-1/CD9-transfected cells. The present study has revealed that the MRP-1/CD9 signal is located upstream of the Wnt signal pathways. Therefore, MRP-1/CD9 could suppress cell transformation including epithelial to mesenchymal transition through downregulation of Wnt1, and might suppress tumor metastasis through downregulation of Wnt5a.

The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism.

Mice deficient in the plasminogen activator inhibitor-1 gene (PAI-1-/- mice) are relatively protected from developing pulmonary fibrosis from bleomycin administration. We hypothesized that one of the protective mechanisms may be the ability of the plasminogen system to enhance hepatocyte growth factor (HGF) effects, which have been reported to be anti-fibrotic in the lung. HGF is known to be sequestered in tissues by binding to extracellular matrix components. Following bleomycin administration, we found that HGF protein levels were higher in bronchoalveolar lavage fluid from PAI-1-/- mice compared to wild-type (PAI-1+/+) mice. This increase could be suppressed by administering tranexamic acid, which inhibits plasmin activity. Conversely, intratracheal instillation of urokinase into bleomycin-injured PAI-1+/+ mice to activate plasminogen caused a significant increase in HGF within bronchoalveolar lavage and caused less collagen accumulation in the lungs. Administration of an anti-HGF neutralizing antibody markedly increased collagen accumulation in the lungs of bleomycin-injured PAI-1-/- mice. These results support the hypothesis that increasing the availability of HGF, possibly by enhancing its release from extracellular matrix by a plasmin-dependent mechanism, is an important means by which activation of the plasminogen system can limit pulmonary fibrosis.

Association of CC chemokine receptor 7 with lymph node metastasis of esophageal squamous cell carcinoma.

PURPOSE: CC chemokine receptor 7 (CCR7) plays a critical role in the migration of activated dendritic cells to regional lymph nodes. Recent studies have shown that CCR7 is involved in metastasis in some malignant diseases. The role of CCR7 in esophageal squamous cell carcinoma (SCC) has not yet been clarified. EXPERIMENTAL DESIGN: We performed reverse transcription-PCR analysis for CCR7 in 20 esophageal SCC cell lines and immunohistochemical analysis of 96 esophageal SCC samples. We then performed a cell migration assay, F-actin polymerization, and a phagokinetic assay on esophageal SCC cell lines in the presence of CCL21, a ligand of CCR7. RESULTS: CCR7 mRNA was detected in 9 of 20 esophageal SCC cell lines. Immunoreactive CCR7 was found mainly in esophageal cancer cells. High CCR7 expression was significantly correlated with esophageal SCC lymphatic permeation, lymph node metastasis, tumor depth, and tumor-node-metastasis stage and was associated with poor survival. In vitro studies demonstrated that CCL21 significantly increased the cell migration ability of esophageal SCC cell lines, and pseudopodia formation was induced by CCL21 stimulation. Furthermore, CCL21 markedly enhanced the motility of esophageal carcinoma cell lines by the phagokinetic assay. CONCLUSIONS: The results suggested that the CCR7/CCL21 receptor ligand system may play a role in the lymph node metastasis of esophageal SCC.

Benign acquired bronchoesophageal fistula in an adult.

A 45-year-old woman was admitted to our hospital with a diagnosis of bronchoesophageal fistula, after a barium esophagography performed in an annual medical check-up had demonstrated an esophageal diverticulum in the middle of the thoracic esophagus, having a narrow bridge between the right intermediate bronchi. The patient had a history of tuberculosis in her childhood, and chest radiography showed multiple calcified hilar lymph nodes. The fistula was observed on gastroenteroscopy and on bronchofiberscopy. The patient was treated surgically, with ligation and resection of the fistula. The fistulous tract was attached to a calcified hilar lymph node, and both the diverticulum and fistula were concluded to have been caused by the tuberculous lymphadenitis in her childhood. It is very uncommon to have bronchoesophageal fistula, caused by tuberculosis in childhood, diagnosed and treated surgically at more than forty years later.

Clinical significance of aminopeptidase N/CD13 expression in human pancreatic carcinoma.

PURPOSE: We previously established a novel murine monoclonal antibody(MH8-11) that recognized aminopeptidase N (APN)/cluster of differentiation antigen 13 (CD13). This monoclonal antibody inhibited human umbilical vein endothelial cells migration and capillary-like tube formation of human umbilical vein endothelial cells on Matrigel. In this study, we investigated the expression of APN/CD13 and the intratumor microvessel density (IMD) as the number of microvessel counts in 50 patients with pancreatic carcinoma. EXPERIMENTAL DESIGN: We investigated APN/CD13 gene expression using the reverse transcriptase-PCR. We also used immunohistochemistry with MH8-11 to investigate APN/CD13 protein expression. Moreover, we investigated the relationship between APN/CD13 expression and tumor angiogenesis by measuring the IMD. RESULTS: APN/CD13 gene expression detected by reverse transcriptase-PCR was positive in 50.0% (25 of 50) of the tumors, and APN/CD13 protein positive was detected by immunohistochemistry in 48.0% (24 of 50). APN/CD13 gene expression agreed well with the immunohistochemical findings (90.0% concordance). APN/CD13 was also significantly associated with an increase of the IMD (r = 0.71, P = 0.0003). However, APN/CD13 expression was not associated with various prognostic factors. The median survival time of patients with APN/CD13 expression was significantly shorter than that of patients without APN/CD13 expression (P = 0.009), and multivariate analysis showed that the APN/CD13 status was a significant independent factor (P = 0.016). CONCLUSIONS: Our data suggest that APN/CD13 may be a new prognostic marker for patients with pancreatic carcinoma and may have a relationship with the angiogenesis for this cancer.

Aminopeptidase N is involved in cell motility and angiogenesis: its clinical significance in human colon cancer.

BACKGROUND & AIMS: The molecular basis of cell motility is highly complex and is controlled by a number of molecular systems, whereas angiogenesis is an important biological component of tumor progression. The aims of this study were to investigate the possible involvement of proteins at the cell surface in controlling cell motility and angiogenesis, and to identify the cell surface molecules involved in gastrointestinal tumors. METHODS: We addressed these issues using functional monoclonal antibodies, which inhibit cell motility, endothelial cell migration, and tube formation. Furthermore, we investigated the relationship between this antigen and colon cancer, and showed the prognostic significance in human colon cancer. RESULTS: We established a murine monoclonal antibody MH8-11, which inhibits cell motility and in vitro angiogenesis. This epitope was a 165-kilodalton protein, and the sequencing analysis revealed that it was almost identical to aminopeptidase N (APN)/cluster of differentiation (CD) 13. APN/CD13 expression was associated with tumor status (P = 0.025). The disease-free and overall survival rate for patients with positive APN/CD13 expression tumors was significantly lower than that for patients with negative APN/CD13 expression tumors (P = 0.014, 0.033, respectively). Among 47 node-positive patients, the survival rate of patients with negative APN/CD13 expression was better than that of those with positive APN/CD13 expression. CONCLUSIONS: Our data suggest that APN/CD13 is involved in cell motility and angiogenesis, and APN/CD13 expression may be a useful indicator of a poor prognosis for node-positive patients with colon cancer.

CD151 enhances cell motility and metastasis of cancer cells in the presence of focal adhesion kinase.

We have examined the role of the protein CD151 in cell motility, invasion and metastasis of cancer cells by using CD151-overexpressing cells prepared by transfection of CD151 cDNA into three cancer cell lines established from different origins; a human colon cancer RPMI4788, a human glioblastoma A172 and a human fibrosarcoma HT1080. Invasion into Matrigel and cell motility of all 3 CD151-overexpressing cancer cells were enhanced significantly when compared to control parental cells. Pulmonary metastasis of 2 metastatic CD151-overexpressing cancer cell lines, RPMI4788/CD151 and HT1080/CD151, was higher than that of control parental cells and was markedly inhibited by anti-CD151 monoclonal antibody (MAb), SFA1.2B4. To examine whether focal adhesion kinase (FAK) is associated with promotion of cell motility and invasion of cancer cells through CD151, we transfected human CD151 cDNA into FAK (+/+) or FAK (-/-) fibroblasts that were isolated from embryos in FAK-deficient mice and compared invasion into Matrigel and cell motility between each CD151-transfected cells and controls. The invasion into Matrigel and cell motility of CD151-transfected FAK (+/+) fibroblasts increased significantly above those of parental cells and were inhibited by anti-CD151 MAb, whereas those of CD151-transfected FAK (-/-) fibroblasts were not enhanced at all and were not blocked by anti-CD151 MAb. These findings indicate that the CD151 molecule enhances cell motility, invasion and metastasis of cancer cells and that FAK is needed for these events through CD151.

Integrin alpha3 expression as a prognostic factor in colon cancer: association with MRP-1/CD9 and KAI1/CD82.

Recently, we established that a murine monoclonal antibody (MAb) MH8-4 inhibits the motility of the colon cancer cell line RPMI4788 and that it recognizes integrin alpha3. In addition, we have also cloned the motility-related protein-1 (MRP-1)/cluster of differentiation 9 (CD9) as a metastasis suppressor molecule. We investigated integrin alpha3 expression in 114 resected colon cancers using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate whether these experimental results are of relevance in the prognosis of actual colon cancers. Furthermore, we investigated the correlation of integrin alpha3 with MRP-1/CD9 and KAI1/CD82. Sixty patients (52.6%) were evaluated as integrin alpha3-positive and 54 patients (47.4%) as integrin alpha3-negative. Integrin alpha3 expression was associated with tumor status, lymph node status and pathologic stage. The overall and disease-free survival rates for patients whose tumors were positive for integrin alpha3 were significantly higher than for those with integrin alpha3-negative tumors (p < 0.001 and p < 0.001, respectively). This same tendency was observed in node-negative patients (p = 0.007 and p = 0.001, respectively). Integrin alpha3 was found to be the significant prognostic factor in a multivariate analysis using the Cox proportional hazards model (p = 0.036). A correlation was found between integrin alpha3 with MRP-1/CD9 and KAI1/CD82 for stage I tumors. However, no correlation was found in stage III tumors. Our data seem to suggest that low expression of integrin alpha3 is a useful indicator of a poor prognosis for colon cancer patients and that colon cancer progresses following collapse of the complex formed by integrin alpha3 with MRP-1/CD9 and KAI1/CD82.