Effects of 6,8-Diprenylgenistein on VEGF-A-Induced Lymphangiogenesis and Lymph Node Metastasis in an Oral Cancer Sentinel Lymph Node Animal Model.

BACKGROUND: The major determining factor of prognosis of oral squamous cell carcinoma is cervical lymph node metastasis. 6,8-Diprenylgenistein (6,8-DG), an isoflavonoid isolated from Cudrania tricuspidata has been reported to have anti-microbial and anti-obesity activities. However, its effects on lymphangiogenesis and lymph node metastasis in oral cancer have not yet been reported. METHODS: To investigate the in vitro inhibitory effects of 6,8-DG on VEGF-A-induced lymphangiogenesis, we performed the proliferation, tube formation, and migration assay using human lymphatic microvascular endothelial cells (HLMECs). RT-PCR, Western blot, immunoprecipitation, ELISA and co-immunoprecipitation assays were used to investigate the expression levels of proteins, and mechanism of 6,8-DG. The in vivo inhibitory effects of 6,8-DG were investigated using an oral cancer sentinel lymph node (OCSLN) animal model. RESULTS: 6,8-DG inhibited the proliferation, migration and tube formation of rhVEGF-A treated HLMECs. In addition, the in vivo lymphatic vessel formation stimulated by rhVEGF-A was significantly reduced by 6,8-DG. 6,8-DG inhibited the expression of VEGF-A rather than other lymphangiogenic factors in CoCl2-treated SCCVII cells. 6,8-DG inhibited the expression and activation of VEGFR-2 stimulated by rhVEGF-A in HLMECs. Also, 6,8-DG inhibited the activation of the lymphangiogenesis-related downstream signaling factors such as FAK, PI3K, AKT, p38, and ERK in rhVEGF-A-treated HLMECs. Additionally, 6,8-DG inhibited the expression of the hypoxia-inducible factor (HIF-1α), which is involved in the expression of VEGF-A in CoCl2-treated SCCVII cells, and 6,8-DG inhibited VEGF-A signaling via interruption of the binding of VEGF-A and VEGFR-2 in HLMECs. In the VEGF-A-induced OCSLN animal model, we confirmed that 6,8-DG suppressed tumor-induced lymphangiogenesis and SLN metastasis. CONCLUSION: These data suggest that 6,8-DG inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in vitro and in vivo. Furthermore, the inhibitory effects of 6,8-DG are probably mediated by inhibition of VEGF-A expression in cancer cells and suppression of the VEGF-A/VEGFR-2 signaling pathway in HLMEC. Thus, 6,8-DG could be novel and valuable therapeutic agents for metastasis prevention and treatment of oral cancer.

Robustaflavone induces G0/G1 cell cycle arrest and apoptosis in human umbilical vein endothelial cells and exhibits anti-angiogenic effects in vivo.

We investigated the anti-angiogenic and pro-apoptotic effects of robustaflavone (RF), a naturally occurring biflavonoid, on human umbilical vein endothelial cells (HUVECs). RF inhibited HUVEC proliferation and showed cytotoxicity that inhibited HUVEC viability. RF-induced apoptosis was characterized by flow cytometry and caspase 3 analysis. We found that RF increased the number of sub-G1 cells and terminal deoxynucleotidyl transferase dUTP nick end-labeled cells. Additionally, RF induced caspase 3 and poly (ADP-ribose) polymerase activation. Potential molecular targets were identified using a human apoptosis antibody array. RF upregulated Bax, Bad, cleaved caspase 3, p21, and phosphorylated p53 levels. RF induced mitochondrial membrane potential loss and the release of cytochrome c and apoptosis-inducing factor. Cell cycle arrest at G0/G1 phase and the downregulation of Cdk4, Cdk6, and cyclin D1 expression were induced by RF. In vivo anti-angiogenic effects were investigated using a tumor allograft animal model and a Matrigel plug assay. RF reduced the volumes and weights of CT-26 cell-derived tumors. The blood vessel density was significantly decreased in RF-treated tumors. RF also inhibited VEGF-A-stimulated blood vessel formation in vivo in Matrigel plugs. These results suggest that RF can potentially inhibit angiogenesis-dependent tumor growth and metastasis.

3-O-Acetyloleanolic acid inhibits angiopoietin-1-induced angiogenesis and lymphangiogenesis via suppression of angiopoietin-1/Tie-2 signaling.

Tumor angiogenesis and lymphangiogenesis are important processes in tumor progression and metastasis. The inhibitory effects of 3-O-acetyloleanolic acid (3AOA), a pentacyclic triterpenoid compound isolated from Vigna sinensis K., on tumor-induced angiogenesis and lymphangiogenesis in vitro and in vivo were studied. Angiopoietin-1 is an important angiogenic and lymphangiogenic factor secreted from colon carcinoma CT-26 cells under hypoxia conditions. 3AOA inhibited proliferation, migration, and tube formation of angiopoietin-1-treated human umbilical vein endothelial cells (HUVEC) and human lymphatic microvascular endothelial cells (HLMEC). 3AOA reduced angiogenesis and lymphangiogenesis in angiopoietin-1-stimulated Matrigel plugs. Also, 3AOA inhibited tumor growth and tumor-induced angiogenesis and lymphangiogenesis in an angiopoietin-1-induced CT-26 allograft colon carcinoma animal model. 3AOA inhibited activation of the angiopoietin-1 receptor Tie-2 and activation of the downstream signaling factors FAK, AKT, and ERK1/2 that are involved in the angiopoietin-1/Tie-2-signaling pathway. Thus, 3AOA has an inhibitory effect on angiogenesis and lymphangiogenesis induced by angiopoietin-1 both in vitro and in vivo, and the inhibitory effect of 3AOA is probably due to suppression of angiopoietin-1/Tie-2 signaling in HUVEC and HLMEC.

Human colorectal cancer antigen GA733-2-Fc fused to endoplasmic reticulum retention motif KDEL enhances its immunotherapeutic effects.

OBJECTIVE: The aim of this is to compare the immunotherapeutic effects of human colorectal cancer antigen GA733-2 fused to the Fc fragment of antibody (GA733-2-Fc) and to Fc and endoplasmic reticulum (ER) retention motif KDEL (GA733-2-Fc-KDEL). MATERIALS AND METHODS: Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were produced from infiltrated Nicotiana benthamiana leaves and purified by affinity chromatography. Glycan structures were determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The allergic and immunogenic responses of recombinant GA733-2-Fc and GA733-2-Fc-KDEL were estimated in an intraperitoneally immunized mouse. The tumor regression effect of recombinant GA733-2-Fc and GA733-2-Fc-KDEL was examined using a colorectal carcinoma CT-26 animal model. RESULTS: Recombinant GA733-2-Fc contained plant-specific glycan structures including ß(1,2)-xylose and α(1,3)-fucose whereas recombinant GA733-2-Fc-KDEL contained oligomannose type glycan structures. Mice immunized intraperitoneally with recombinant GA733-2-Fc and GA733-2-Fc-KDEL elicited strong GA733-2-Fc-specific immunoglobulin G (IgG) and IgA serum antibody responses. Recombinant GA733-2-Fc-KDEL reduced the production of GA733-2-Fc-specific IgE. Recombinant GA733-2-Fc-KDEL increased the production of interferon-γ. Intraperitoneal preimmunization with recombinant GA733-2-Fc and GA733-2-Fc-KDEL regressed tumor growth in a colorectal carcinoma CT-26 animal model. The tumor regression effect induced by recombinant GA733-2-Fc-KDEL was greater than that induced by recombinant GA733-2-Fc. The human and mouse colorectal carcinoma cell binding activities of recombinant GA733-2-Fc-KDEL-immunized sera were higher than those of recombinant GA733-2-Fc. CONCLUSIONS: Our results suggest that GA733-2-Fc conjugated to ER-retention motif KDEL is a more efficient antigen to prevent tumor growth induced by colorectal carcinoma and minimize an allergic response.

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

BACKGROUND: Sentinel 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. METHODS: The 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. RESULTS: 3AOA 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. CONCLUSION: 3AOA 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.

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 αvß3/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.

Tumstatin induces apoptosis mediated by Fas signaling pathway in oral squamous cell carcinoma SCC-VII cells.

Oral squamous cell carcinoma is a cancer originating in the tissues lining the mouth and lips. The present study investigated the effects of recombinant tumstatin, an anti-angiogenic agent with distinct antitumor activity, on oral squamous cell carcinoma SCC-VII cells. Apoptosis was characterized by YO-PRO-1 staining, sub-G1 population, and DNA fragmentation analysis. Apoptotic mechanism of tumstatin was also investigated. The antitumor activity of tumstatin was further evaluated using an SCC-VII animal model. Recombinant tumstatin was found to decrease the viability of SCC-VII cells in a dose-dependent manner. The number of cells stained with the apoptotic marker YO-PRO-1, the sub-G1 cell population and the level of apoptotic DNA fragmentation increased in the SCC-VII cells following treatment with recombinant tumstatin. In addition, recombinant tumstatin treatment increased the expression of the Fas gene at the transcript and protein levels, and the inhibition of cell viability by recombinant tumstatin was suppressed by a neutralizing anti-Fas antibody. Furthermore, treatment with recombinant tumstatin decreased the volume and weight of tumors in C3H/HeJ mice implanted with SCC-VII cells. In conclusion, the results indicated that tumstatin induced apoptosis that is mediated by the Fas signaling pathway in SCC-VII cells and inhibited tumor growth in an SCC-VII animal model.

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).

The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses.

In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is the most highly contagious infectious disease that has been affecting domestic poultry in recent years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic strains. However, few studies have reported viruses circulating in the south-central region of Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013-2014, and their genetic diversity was analyzed. A common sequence motif for HPAI virus was identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a) or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of HPAI H5N1: 1.1.2 (n=1), 2.3.2.1a (n=1), and 2.3.2.1c (n=45). The phylogenetic analysis indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus clades isolated in 2012-2013 but are less closely related to the clades detected from the Tyva Republic, Bulgaria, Mongolia, Japan, and Korea in 2009-2011. Detection of the coexistence of virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local importance and highlights concerns regarding their spread, both northwards and southwards, as well as the potential for reassortment. The obtained data highlight the importance of regular identification of viral evolution and the development and use of region-specific vaccines.

Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa).

We expressed the heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae under the control of the rice (Oryza sativa) globulin (Glb) promoter. Binding of recombinant LTB and CTB proteins was confirmed based on GM1-ganglioside binding enzyme-linked immunosorbent assays (GM1-ELISA). Real-time PCR of three generations (T3, T4, and T5) in homozygous lines (LCI-11) showed single copies of LTB, CTB, bar and Tnos. LTB and CTB proteins in rice transgenic lines were detected by Western blot analysis. Immunogenicity trials of rice-derived CTB and LTB antigens were evaluated through oral and intraperitoneal administration in mice, respectively. The results revealed that LTB- and CTB-specific IgG levels were enhanced in the sera of intraperitoneally immunized mice. Similarly, the toxin-neutralizing activity of CTB and LTB in serum of orally immunized mice was associated with elevated levels of both IgG and IgA. The results of the present study suggest that the combined expression of CTB and LTB proteins can be utilized to produce vaccines against enterotoxigenic strains of Escherichia coli and Vibrio cholera, for the prevention of diarrhea.

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.

Cynandione A attenuates lipopolysaccharide-induced production of inflammatory mediators via MAPK inhibition and NF-κB inactivation in RAW264.7 macrophages and protects mice against endotoxin shock.

Cynanchum wilfordii has been traditionally used in eastern Asia for the treatment of various diseases such as gastrointestinal diseases and arteriosclerosis. Cynandione A (CA), an acetophenone, is one of major constituents from roots of C. wilfordii. In the present study, the anti-inflammatory activities of CA were investigated in lipopolysaccharide (LPS)-treated RAW264.7 macrophages and LPS-administered C57BL/6 N mice. CA significantly decreased LPS-induced production of nitric oxide and prostaglandin E2 in a dose-dependent manner, while CA up to 200 µM did not exhibit cytotoxic activity. Our data also showed that CA significantly attenuated expression of iNOS and COX-2 in LPS-stimulated macrophages. CA inhibited phosphorylation of IκB-α and MAP kinases such as ERK and p38. Furthermore, we demonstrated that CA inhibited translocation of NF-κB to the nucleus, transcription of the NF-κB minimal promoter and NF-κB DNA binding activity. Administration of CA significantly decreased the plasma levels of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß in LPS-injected mice and improved survival of septic mice with lethal endotoxemia. These results demonstrate that CA has effective inhibitory effects on production of inflammatory mediators via suppressing activation of NF-κB and MAPK signaling pathways, suggesting that CA may be used as a potential anti-inflammatory agent for the prevention and treatment of inflammatory diseases.

Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Cell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.

Expression and immunogenic analysis of recombinant polypeptides derived from capsid protein VP1 for developing subunit vaccine material against hepatitis A virus.

Three recombinant polypeptides, VP1-His, VP1-3N-His, and 3D2-His, were produced by Escherichia coli expression system. Recombinant VP1-His, VP1-3N-His, and 3D2-His were expressed as bands with molecular weights of 32, 38, and 30 kDa, respectively. These were purified by affinity chromatography using Ni-NTA Fast-flow resin and/or ion-exchange chromatography using DEAE-Sepharose Fast-flow resin. Intraperitoneal immunizations of recombinant polypeptides successfully elicited the productions of VP1-His, VP1-3N-His, and 3D2-His specific IgG antibodies (IgG subclass distribution of IgG1>IgG2a>IgG2b>IgG3) in sera and induced the secretions of cytokines IFN-γ and IL-6 in spleen cells. Sera from recombinant VP1-His-, VP1-3N-His-, and 3D2-His-immunized mice neutralized the propagation of HAV. The highest neutralizing activity was shown in sera from recombinant VP1-3N-His-immunized mice. These results suggest that recombinant VP1-3N-His can be a useful source for developing hepatitis A virus (HAV) subunit vaccine candidates.

Neolignans from the fruits of Magnolia obovata and their inhibition effect on NO production in LPS-induced RAW 264.7 cells.

Three new neolignans, named 9-methoxyobovatol (6), magnobovatol (7), and 2-hydroxyobovaaldehyde (9), along with six known ones, magnolol (1), honokiol (2), isomagnolol (3), obovatol (4), obovatal (5), and obovaaldehyde (8), were isolated from the fruits of Magnolia obovata using silica gel and ODS column chromatography. From the results of spectroscopic data including EIMS, IR, 1H- and 13C-NMR, DEPT, and 2D-NMR (gCOSY, gHSQC, gHMBC), the chemical structures were determined. All isolated compounds were evaluated for inhibition activity on nitric oxide production in LPS-induced RAW 264.7 cells, and compounds 1-4, 6, 7, and 9 showed significant activity with IC50 values of 15.8 ± 0.3, 3.3 ± 1.2, 14.1 ± 0.9, 6.2 ± 1.2, 14.8 ± 2.3, 14.2 ± 1.2, and 14.8 ± 3.2 µM, respectively, without any visible toxic effect.

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

3-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.

Whole-genome sequence analysis of a Korean G11P[25] rotavirus strain identifies several porcine-human reassortant events.

A rare rotavirus, RVA/Human-wt/KOR/CAU12-2/2012/G11P[25], was isolated from a 16-year-old female with fever and diarrhea during the 2012 rotavirus surveillance in South Korea using a cell culture system, and its full genome sequence was determined and analyzed. Strain CAU12-2 exhibited a G11-P[25]-I12-R1-C1-M1-A1-N1-T1-E1-H1 genotype constellation. Phylogenetic analysis of this strain revealed that it is a human-porcine reassortant of two distant relatives of the G11 strains circulating in the world. The VP7 and VP4 genes are most closely related to those of human G11P[25] viruses (Dhaka6, KTM368, and N-38 strains) identified in South Asia, whereas the VP1 gene originated from a porcine G11P[7] virus (YM strain) that was identified in South America. The VP6 gene was found to belong to the new genotype I12. This study indicates that the G11-P[25]-I12 genotype was introduced into the South Korean population by interspecies transmissions of human and animal rotaviruses, followed by multiple reassortment events.

A new phenanthrene derivative and two diarylheptanoids from the roots of Brassica rapa ssp. campestris inhibit the growth of cancer cell lines and LDL-oxidation.

Brassica rapa ssp. campestris (Brassicaceae) is a conical, deep purple, edible root vegetable commonly known as a turnip. We initiated phytochemical and pharmacological studies to search for biological active compounds from the roots of B. rapa ssp. campestris. We isolated a novel phenanthrene derivative, 6-methoxy-1-[10-methoxy-7-(3-methylbut-2-enyl)phenanthren-3-yl]undecane-2,4-dione, named brassicaphenanthrene A (3) along with two known diarylheptanoid compounds, 6-paradol (1) and trans-6-shogaol (2), through the repeated silica gel (SiO2), octadecyl silica gel, and Sephadex LH-20 column chromatography. The chemical structures of the compounds were determined by spectroscopic data analyses including nuclear magnetic resonance, mass spectrometry, ultraviolet spectroscopy, and infra-red spectroscopy. All compounds exhibited high inhibitory activity against the growth of human cancer lines, HCT-116, MCF-7, and HeLa, with IC50 values ranging from 15.0 to 35.0 µM and against LDL-oxidation with IC50 values ranging from 2.9 to 7.1 µM.

Pomolic acid induces apoptosis in SK-OV-3 human ovarian adenocarcinoma cells through the mitochondrial-mediated intrinsic and death receptor-induced extrinsic pathways.

The cytotoxic effect of pomolic acid (PA), a pentacyclic triterpene isolated from flowers of Osmanthus fragrans var. aurantiacus Makino, was investigated in SK-OV-3 human ovarian adenocarcinoma cells. PA dose-dependently inhibited the viability of SK-OV-3 cells. PA-induced apoptosis was further characterized by detection of cell surface annexin V and sub-G1 apoptotic cell populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate (FITC) increased following treatment with PA. The sub-G1 cell populations also increased in PA-treated SK-OV-3 cells. PA induced the activation of caspase-8, -9 and -3, critical mediators of apoptosis signaling. PA decreased the mitochondrial transmembrane potential (ΔΨ(m)), resulting in the activation of caspase-9. In addition, PA increased the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis signaling-related death receptor 5 (DR5), mediating caspase-8-involved extrinsic pathway. Taken together, our results indicate that PA induces apoptosis in SK-OV-3 cells, which is mediated by the mitochondrial-mediated intrinsic and death receptor-induced extrinsic pathways.

Helicobacter pylori decreases p27 expression through the delta opioid receptor-mediated inhibition of histone acetylation within the p27 promoter.

Chronic Helicobacter pylori infection is associated with the decreased expression of the gastric tumour suppressor protein p27. Because transcription of the gene p27 may be regulated epigenetically through histone acetylation, which is mediated by G-protein coupled delta opioid receptor (DOR) stimulation, we examined whether H. pylori regulates the DOR/histone acetylation/p27 promoter pathway. The levels of acetylated histone and p300, a gene-specific histone acetyltransferase within the p27 promoter, were measured using ChIP assays. The expression of phospho-DOR was evaluated by Western blot and immunohistochemical analyses. Growth curves were constructed, and cell proliferation was assessed after BrdU incorporation. Low p27 expression in acutely H. pylori-infected AGS gastric epithelial cells and in chronically H. pylori-infected AGS-derived HS3C cells was associated with approximate 20% and 40% decreases in p27 mRNA expression, respectively, when compared to p27 mRNA levels in uninfected AGS parental cells. The low p27 mRNA levels following H. pylori infection were associated with a 15-60% reduction in p27 promoter histone H4 acetylation. The recruitment of p300 to the p27 promoter was also markedly decreased by H. pylori infection. The expression of phospho-DOR was decreased by H. pylori infection in cell lines in vitro and in H. pylori-infected human gastric mucosa in vivo. The level of cellular p27 inversely correlated with cell proliferation in HS3C cells. These results demonstrate that H. pylori decreases p27 expression by modulating the DOR and thereby inhibiting histone acetylation of the p27 promoter. These findings link low gastric p27 expression levels with increased instances of gastric carcinogenesis associated with H. pylori infection.