Jeon Hwang-Bo

p38 MAPK-Mediated Bmi-1 Down-Regulation and Defective Proliferation in ATM-Deficient Neural Stem Cells Can Be Restored by Akt Activation

A-T (ataxia telangiectasia) is a genetic disease caused by a mutation in the Atm (A-T mutated) gene that leads to neurodegeneration. Despite an increase in the numbers of studies in this area in recent years, the mechanisms underlying neurodegeneration in human A-T are still poorly understood. Previous studies demonstrated that neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm(-/-) mouse brains show defective self-renewal and proliferation, which is accompanied by activation of chronic p38 mitogen-activated protein kinase (MAPK) and a lower level of the polycomb protein Bmi-1. However, the mechanism underlying Bmi-1 down-regulation and its relevance to defective proliferation in Atm(-/-) NSCs remained unclear. Here, we show that over-expression of Bmi-1 increases self-renewal and proliferation of Atm(-/-) NSCs to normal, indicating that defective proliferation in Atm(-/-) NSCs is a consequence of down-regulation of Bmi-1. We also demonstrate that epidermal growth factor (EGF)-induced Akt phosphorylation renders Bmi-1 resistant to the proteasomal degradation, leading to its stabilization and accumulation in the nucleus. However, inhibition of the Akt-dependent Bmi-1 stabilizing process by p38 MAPK signaling reduces the levels of Bmi-1. Treatment of the Atm(-/-) NSCs with a specific p38 MAPK inhibitor SB203580 extended Bmi-1 posttranscriptional turnover and H2A ubiquitination in Atm(-/-) NSCs. Our observations demonstrate the molecular basis underlying the impairment of self-renewal and proliferation in Atm(-/-) NSCs through the p38 MAPK-Akt-Bmi-1-p21 signaling pathway.

Recombinant canstatin inhibits angiopoietin-1-induced angiogenesis and lymphangiogenesis

We describe the effect of recombinant canstatin, the NC1 domain of the α2 chain of Type IV collagen, on suppression of angiogenesis and lymphangiogenesis both in vitro and in vivo. Recombinant canstatin produced from stably transformed Drosophila S2 cells reduced the expression of angiopoietin‐1 in hypoxia mimetic agent, CoCl2‐treated CT‐26 cells. Recombinant canstatin inhibited proliferation, tube formation and migration of human angiopoietin‐1 (rhAngpt‐1)‐treated human umbilical vein endothelial cells (HUVEC) and lymphatic endothelial cells (LEC). Recombinant canstatin suppressed the expression of Tie‐2 and vascular endothelial growth factor‐3 (VEGFR‐3) transcripts in rhAngpt‐1‐treated HUVEC and LEC, respectively. The inhibitory effect of recombinant canstatin on tumor growth was also investigated using a heterotopic CT‐26 colon carcinoma animal (BALB/c mice) model. Recombinant canstatin reduced the final volume and weight of tumors, and blood and lymphatic vessel densities of tumors, which were evaluated by CD‐31 and LYVE‐1 immunostaining. Immunohistochemical analysis showed that recombinant canstatin dramatically reduced the expression of angiopoietin‐1 in CT‐26 colon carcinoma‐induced tumor, but not the expression of VEGF‐C. Tie‐2 and VEGFR‐3 expressions were also reduced in recombinant canstatin‐treated tumors. These results indicate that recombinant canstatin has anti‐tumoral activities against CT‐26 colon carcinoma cells. Recombinant canstatin reduces the expression of angiopoietin‐1 in hypoxia‐induced CT‐26 cells and inhibits the angiogenic and lymphangiogenic signaling induced by angiopoietin‐1. Recombinant canstatin probably inhibits angiogenesis and lymphangiogenesis via suppression of the integrin‐dependent FAK signaling induced by angiopoietin‐1/Tie‐2 and/or VEGFR‐3.

Expression and immunogenicity of recombinant polypeptide VP1 of human hepatitis A virus in stably transformed fruitfly (Drosophila melanogaster) Schneider 2 cells

We describe the secretory expression and immunogenicity of the recombinant HAV (hepatitis A virus) structural polypeptide VP1 from stably transformed Drosophila melanogaster S2 (Schneider 2) cells. Southern‐blot analysis indicated that transformed S2 cells contained multiple copies of the HAV VP1 gene in the genome. Recombinant VP1 was secreted into a culture medium with a molecular mass of 42–49 kDa. A maximum production level of 6.24 mg of recombinant VP1/litre was obtained in a T‐flask culture of Drosophila S2 cells 5 days after induction with 0.5 mM CuSO4. The recombinant HAV VP1 protein elicited the production of specific IgA in the small intestine by oral immunization and production of specific IgG in the serum by intraperitoneal immunization. Our findings show that secretory recombinant VP1 from transformed Drosophila S2 cells can be used as an effective experimental immunogen for research in vaccine development.

3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells.

Abstract3-O-Acetyloleanolic acid, a pentacyclic triterpenoid isolated from cowpea seeds, inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. HUVECs. The induced apoptosis was characterized by detection of cell surface annexin V and sub-G1 populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate increased after treatment with 3-O-acetyloleanolic acid. The sub-G1 cell populations were also increased in treated HUVECs. 3-O-Acetyloleanolic acid induced activation of caspase 3, a critical mediator of apoptosis signaling. It also significantly inhibited angiogenesis in an in vivo Matrigel plug assay. 3-O-Acetyloleanolic acid thus exhibits anti-angiogenic effects and induces apoptosis in HUVECs and the results suggest that it has a potential use for suppression of the tumor growth stimulated by angiogenesis.

Recombinant canstatin inhibits VEGF‐A‐induced lymphangiogenesis and metastasis in an oral squamous cell carcinoma SCC‐VII animal model

We describe the inhibitory effects of recombinant canstatin on tumor growth and lymphangiogenesis induced by an oral squamous cell carcinoma (SCC) using an orthotropic oral SCC animal model. Recombinant canstatin treatment decreased final tumor volumes and weights, as well as densities of blood and lymphatic vessels. Lung metastasis of oral SCC was significantly reduced in recombinant canstatin‐treated animals. Recombinant canstatin reduced vascular endothelial growth factor (VEGF)‐A expression in SCC‐VII cells treated with the hypoxia mimetic agent, CoCl2. VEGF‐A induced in vivo lymphatic vessel formation in a Matrigel plug, but this was remarkably reduced in a recombinant canstatin‐treated Matrigel. Recombinant canstatin suppressed the expression of vascular endothelial growth factor receptors (VEGFR)‐1 and ‐2 stimulated by VEGF‐A. Based on immunohistochemical analysis, recombinant canstatin significantly reduced the expression of VEGF‐A, VEGFR‐1, and ‐2 in SCC‐VII‐induced tumors. Recombinant canstatin did not affect the expression of VEGF‐C or VEGFR‐3. In addition, recombinant canstatin suppressed the VEGF‐A‐induced phosphorylation of VEGFR‐1 and ‐2. Our results indicate that recombinant canstatin exhibits antitumoral and antilymphangiogenic activities against oral SCC cells. Antilymphangiogenic signaling by recombinant canstatin is probably mediated by the suppression of the integrin αvβ3/VEGFR‐1 and/or ‐2 signaling induced by VEGF‐A. Our results also suggest that recombinant canstatin has a high potential to inhibit oral SCC‐induced tumors and lymphatic metastasis.

Corosolic Acid Exhibits Anti‐angiogenic and Anti‐lymphangiogenic Effects on In Vitro Endothelial Cells and on an In Vivo CT‐26 Colon Carcinoma Animal Model

We describe the anti‐angiogenic and anti‐lymphangiogenic effects of corosolic acid, a pentacyclic triterpenoid isolated from Cornus kousa Burg. A mouse colon carcinoma CT‐26 animal model was employed to determine the in vivo anti‐angiogenic and anti‐lymphangiogenic effects of corosolic acid. Corosolic acid induced apoptosis in CT‐26 cells, mediated by the activation of caspase‐3. In addition, it reduced the final tumor volume and the blood and lymphatic vessel densities of tumors, indicating that it suppresses in vivo angiogenesis and lymphangiogenesis. Corosolic acid inhibited the proliferation and tube formation of human umbilical vein endothelial cells and human dermal lymphatic microvascular endothelial cells. In addition, corosolic acid decreased the proliferation and migration of human umbilical vein endothelial cells stimulated by angiopoietin‐1. Pretreatment with corosolic acid decreased the phosphorylation of focal adhesion kinase (FAK) and ERK1/2, suggesting that corosolic acid contains anti‐angiogenic activity that can suppress FAK signaling induced by angiopoietin‐1. Copyright

Two New Cyototoxic Cardenolides from the Whole Plants of Adonis multiflora Nishikawa & Koki Ito

A phytochemical investigation of the whole plants of Adonis multiflora Nishikawa & Koki Ito. resulted in the isolation and identification of two new cardenolides—adonioside A (1) and adonioside B (6)—as well as four known cardenolides: tupichinolide (2) oleandrine (3), cryptostigmin II (4), and cymarin (5). Their structures were elucidated on the basis of NMR, MS, and IR spectroscopic analyses. Compounds 1, 2, 5, and 6 showed significant cytotoxicity against six human cancer cell lines (HCT-116, HepG2, HeLa, SK-OV-3, and SK-MEL-5, and SK-BR-3).

Drosophila melanogaster S2 cells are more suitable for the production of recombinant COX-1 than Trichoplusia ni BTI TN-5B1-4 cells

Recombinant human cyclooxygenase-1 (COX-1) was expressed from stably transfected Trichoplusia ni BTI TN-5B1-4 (TN-5B1-4) and Drosophila melanogaster S2 cells. Two kinds of recombinant COX-1 with molecular weights (MWs) of 68 and 74 kDa were expressed in the intracellular fractions of stably transfected TN-5B1-4/ COX-1 and S2/COX-1 cells, due to glycosylation. The recombinant COX-1 secreted to medium fractions has a MW of 72 kDa. Recombinant COX-1 in the intracellular fractions was purified to homogeneity using a one-step Ni-NTA affinity fractionation method. Recombinant COX-1 purified from TN-5B1-4/COX-1 and S2/COX-1 cells contained 11,389 and 33,850 Unit/mg of specific peroxidase activity, respectively. The maximum productions of intracellular recombinant COX-1 were 1.7 and 5.6 μg/107 cells in the T-flask cultures of TN-5B1-4/COX-1 and S2/COX-1 cells, respectively. Taken together, our findings indicate that S2 cells can be more suitable system to produce recombinant COX-1, compared to TN-5B1-4 cells.

3- O -Acetyloleanolic acid inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in an oral cancer sentinel lymph node animal model

BackgroundSentinel lymph node metastasis is a common and early event in the metastatic process of head and neck squamous cell carcinoma (HNSCC) and is the most powerful prognostic factor for survival of HNSCC patients. 3-O-acetyloleanolic acid (3AOA), a pentacyclic triterpenoid compound isolated from seeds of Vigna sinensis K., has been reported to have potent anti-angiogenesis and anti-tumor activities. However, its effects on tumor-related lymphangiogenesis and lymph node metastasis are not yet understood.MethodsThe in vitro inhibitory effects of 3AOA on VEGF-A-induced lymphangiogenesis were investigated via in vitro experiments using mouse oral squamous cell carcinoma (SCCVII) cells and human lymphatic microvascular endothelial cells (HLMECs). The in vivo inhibitory effects of 3AOA on VEGF-A-induced lymphangiogenesis and sentinel lymph node metastasis were investigated in an oral cancer sentinel lymph node (OCSLN) animal model.Results3AOA inhibited tumor-induced lymphangiogenesis and sentinel lymph node metastasis in an OCSLN animal model, and reduced expression of VEGF-A, a lymphangiogenic factor in hypoxia mimetic agent CoCl2-treated SCCVII cells. 3AOA inhibited proliferation, tube formation, and migration of VEGF-A-treated HLMECs. The lymphatic vessel formation that was stimulated in vivo in a by VEGF-A Matrigel plug was reduced by 3AOA. 3AOA suppressed phosphorylation of vascular endothelial growth factor (VEGFR) -1 and − 2 receptors that was stimulated by VEGF-A. In addition, 3AOA suppressed phosphorylation of the lymphangiogenesis-related downstream signaling factors PI3K, FAK, AKT, and ERK1/2. 3AOA inhibited tumor growth, tumor-induced lymphangiogenesis, and sentinel lymph node metastasis in a VEGF-A-induced OCSLN animal model that was established using VEGF-A overexpressing SCCVII cells.Conclusion3AOA inhibits VEGF-A-induced lymphangiogenesis and sentinel lymph node metastasis both in vitro and in vivo. The anti-lymphangiogenic effects of 3AOA are probably mediated via suppression of VEGF-A/VEGFR-1 and VEGFR-2 signaling in HLMECs, and can be a useful anti-tumor agent to restrict the metastatic spread of oral cancer.

Red pigment produced by Zooshikella ganghwensis inhibited the growth of human cancer cell lines and MMP-1 gene expression

A red pigment produced by Zooshikella ganghwensis, was purified by solvent extraction and fractionation. The pigment was identified to be prodigiosin on the basis of spectroscopic data such as 1H-NMR, 13C-NMR, DEPT, 2D-NMR, IR, and FAB-MS. Prodigiosin inhibited matrix metalloproteinase-1 gene expression and the growth of human cancer cell lines, SK-BR-3, HCT-116, SK-OV-3, HeLa, HepG2, and SK-MEL-5.