Delta/Notch-like Epidermal Growth Factor-Related Receptor (DNER), a Potential Prognostic Marker of Gastric Cancer Regulates Cell Survival and Cell Cycle Progression.

Upregulation of the expression of Delta/notch-like epidermal growth factor-related receptor (DNER) and its oncogenic role have been reported in several cancers, including gastric, breast, and prostate cancers. This study aimed to investigate the oncogenic role of DNER and the mechanisms behind its oncogenic role in gastric cancer. Analysis of the RNASeq data of gastric cancer tissues obtained from the TCGA database revealed that the expression of DNER was associated with the pathology of advanced gastric cancer and the prognosis of patients. DNER expression was increased upon stem cell-enriching cancer spheroid culture. Knockdown of DNER expression inhibited cell proliferation and invasion, induced apoptosis, enhanced chemosensitivity, and decreased spheroid formation of SNU-638 gastric cancer cells. DNER silencing elevated the expression of p53, p21cip/waf, and p27, and increased G1 phase cells at the expense of S phase cells. Knockdown of p21cip/waf expression in the DNER-silenced cells partially restored cell viability and S phase progression. DNER silencing also induced the apoptosis of SNU-638 cells. While both cleaved caspases-8 and 9 were detected in adherent cells, only cleaved caspase-8 was found to have increased in spheroid-cultured cells, suggesting a distinct activation pattern of caspase activation depending on the growth condition. Knockdown of p53 expression rescued the DNER-silenced cells from apoptosis and partially restored cell viability. In contrast, overexpression of the Notch intracellular domain (NICD) decreased the expression of p53, p21cip/waf, and cleaved caspase-3 in DNER-silenced cells. Moreover, NICD expression fully reverted the cell viability reduction, arrest in the G1 phase, and elevated apoptosis caused by DNER silencing, thereby suggesting activation of Notch signaling by DNER. Expression of a membrane-unbound mutant of mDNER also decreased cell viability and induced apoptosis. On the other hand, TGF-ß signals were found to be involved in DNER expression in both adherent and spheroid-cultured cells. DNER could therefore be a link connecting TGF-ß signaling to Notch signaling. Taken together, DNER regulates cell proliferation, survival, and invasive capacity of the gastric cancer cells through the activation of Notch signaling, which may facilitate tumor progression into an advanced stage. This study provides evidences suggesting that DNER could be a potential prognostic marker, a therapeutic target, and a drug candidate in the form of a cell-free mutant.

Competitive Hybridization of a Microarray Identifies CMKLR1 as an Up-Regulated Gene in Human Bone Marrow-Derived Mesenchymal Stem Cells Compared to Human Embryonic Fibroblasts.

Mesenchymal stem cells (MSCs) have been widely applied to the regeneration of damaged tissue and the modulation of immune response. The purity of MSC preparation and the delivery of MSCs to a target region are critical factors for success in therapeutic application. In order to define the molecular identity of an MSC, the gene expression pattern of a human bone marrow-derived mesenchymal stem cell (hBMSC) was compared with that of a human embryonic fibroblast (hEF) by competitive hybridization of a microarray. A total of 270 and 173 genes were two-fold up- and down-regulated with FDR < 0.05 in the hBMSC compared to the hEF, respectively. The overexpressed genes in the hBMSC over the hEF, including transcription factors, were enriched for biological processes such as axial pattern formation, face morphogenesis and skeletal system development, which could be expected from the differentiation potential of MSCs. CD70 and CD339 were identified as additional CD markers that were up-regulated in the hBMSC over the hEF. The differential expression of CD70 and CD339 might be exploited to distinguish hEF and hBMSC. CMKLR1, a chemokine receptor, was up-regulated in the hBMSC compared to the hEF. RARRES2, a CMKLR1 ligand, stimulated specific migration of the hBMSC, but not of the hEF. RARRES2 manifested as ~two-fold less effective than SDF-1α in the directional migration of the hBMSC. The expression of CMKLR1 was decreased upon the osteoblastic differentiation of the hBMSC. However, the RARRES2-loaded 10% HA-silk scaffold did not recruit endogenous cells to the scaffold in vivo. The RARRES2−CMKLR1 axis could be employed in recruiting systemically delivered or endogenous MSCs to a specific target lesion.

Modulation of Spheroid Forming Capacity and TRAIL Sensitivity by KLF4 and Nanog in Gastric Cancer Cells.

The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

The Role of Melanotransferrin (CD228) in the regulation of the differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells (hBM-MSC).

Melanotransferrin (CD228), firstly reported as a melanoma-associated antigen, is a membrane-bound glycoprotein of an iron-binding transferrin homolog. CD228 was found to be expressed significantly higher in human bone marrow-derived mesenchymal stem cells (hBM-MSC) than in human embryonic fibroblasts (FB) by RT-PCR, western blotting and flow cytometry. The expression of CD228 declined in aged hBM-MSC as osteogenesis-related genes did. We examined a possible role for CD228 in the regulation of osteogenesis and adipogenesis of hBM-MSC. Surprisingly, siRNA-mediated CD228 knockdown increased the expression of the transcription factor DLX5 and enhanced osteogenesis of hBM-MSC evidenced by an increased expression of the runt-related transcription factor 2 (RUNX2), osterix (Osx), and osteocalcin (OC), as well as higher alkaline phosphatase (ALP) activity and extracellular calcium deposition. Interestingly, hBM-MSC transfected with CD228 siRNA also showed an increase in intracellular lipid level during adipogenesis, indicated by oil red O staining of differentiated adipocytes. Overall, our study unveils CD228 as a cell surface molecule expressed by young hBM-MSC, but not by FB. It also provides evidence to suggest a role for CD228 as a negative regulator of osteogenesis and of lipid accumulation during adipogenesis in hBM-MSC in vitro.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to ß-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

In the Wnt canonical pathway, Wnt3A has been known to stabilize ß-catenin. In the non-canonical Wnt signaling pathway, Wnt is known to activate Rho GTPases. The correlation between canonical and non-canonical pathways by Wnt signaling, however, has not been well elucidated. Here, we identified that Wnt3A promoted superoxide generation, leading to Tyr42 phosphorylation of RhoA through activations of c-Src and Rho-dependent coiled coil kinase 2 (ROCK2) and phosphorylation of p47phox, a component of NADPH oxidase. Wnt3A also induced accumulation of ß-catenin along with activations of RhoA and ROCK1. Concurrently, ROCK1 was able to phosphorylate GSK-3ß at Ser9, which phosphorylated Src at Ser51 and Ser492 residues, leading to Src inactivation through dephosphorylation of Tyr416 during the late period of Wnt3A treatment. Meanwhile, p-Tyr42 RhoA bound to ß-catenin via the N-terminal domain of ß-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/ß-catenin complex. Notably, p-Tyr42 RhoA as well as ß-catenin was associated with the promoter of Vim, leading to increased expression of vimentin. In addition, stomach cancer patients harboring higher expressed p-Tyr42 Rho levels revealed the much poorer survival probability. Therefore, we propose that p-Tyr42 RhoA is crucial for transcriptional regulation of specific target genes in the nucleus by binding to their promoters and involved in tumorigenesis.

Synergistic apoptosis of human gastric cancer cells by bortezomib and TRAIL.

Resistance against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death of cancer cells is a major obstacle in clinical application of TRAIL. Variable response to TRAIL of gastric cancer cells, synergy of TRAIL with bortezomib and potential mechanisms behind the phenomena were investigated in this study. The response to TRAIL varied among six gastric cancer cell lines, which correlated with the expression of apoptotic TRAIL receptors. Analysis of TCGA gene expression data showed that DR4 expression correlated with DR5 in gastric cancer. Although higher expression of DR4 was significantly associated with lower T, N and TNM stages, neither DR4 nor DR5 expression meaningfully influenced overall survival rate. Combined treatment of TRAIL with bortezomib resulted in strong synergistic response with enhanced activation of caspases-8, -9 and -3, and increased Annexin V-binding cell fractions in TRAIL-resistant SNU-216 cells. Bortezomib increased the expression of p21cip1/waf1, but p21cip1/waf1 silencing did not restore cell viability significantly. Bortezomib also increased DR5 expression and knockdown of DR5 expression significantly recovered cell viability reduced by the combination treatment. Bortezomib decreased phosphorylation of ERK1/2, but increased that of JNK. Treatment with either an ERK1/2 inhibitor U0126 or a JNK inhibitor SP600125 rescued SNU-216 from dying of bortezomib or combined treatment. However, upregulation of DR5 by bortezomib was knocked down only by inhibition of ERK1/2 activation significantly, but not by JNK activity inhibition. In summary, upregulation of DR5 by bortezomib is of critical significance in the synergy of bortezomib with TRAIL in apoptosis of TRAIL-resistant SNU-216 and that activity of ERK1/2 is required in the bortezomib-induced DR5 overexpression.

Melatonin alleviates oxidative stress-inhibited osteogenesis of human bone marrow-derived mesenchymal stem cells through AMPK activation.

Oxidative stress plays an important role in the pathogenesis of aging-related osteoporosis through the increased bone resorption or reduced bone formation. Melatonin, which can exert beneficial actions through antioxidant, anti-inflammatory, and bone-preserving effects, shows promise in preventing oxidative stress-inhibited osteogenesis. However, specific mechanisms by which melatonin rescues oxidative stress-inhibited osteogenesis of human mesenchymal stem cells (MSCs) have not been fully elucidated yet. We therefore investigated whether activation of AMPK by melatonin regulates the antagonistic crosstalk between oxidative stress and osteogenic differentiation in human MSCs. Melatonin treatment significantly enhanced osteogenic differentiation of human MSCs through activation of AMPK and upregulation of FOXO3a and RUNX2 which were known as master transcription factors responsible for the mechanistic link between oxidative stress and osteogenic phenotype. Osteogenic differentiation determined by calcium deposition was significantly increased by melatonin treatment against oxidative stress. In addition, melatonin treatment reconstituted activation of AMPK and expression of FOXO3a and RUNX2 inhibited by oxidative stress. Overall, these results demonstrate that melatonin enhances osteogenic differentiation of human MSCs and restores oxidative stress-inhibited osteogenesis through AMPK activation in human MSCs, suggesting that activation of AMPK by melatonin may represent a promising new therapeutic strategy for treating metabolic bone diseases such as osteoporosis.

Enantiomeric CopA3 dimer peptide suppresses cell viability and tumor xenograft growth of human gastric cancer cells.

The CopA3 dimer peptide is a coprisin analog that has an anticancer effect against human cancer cells in vitro. In this study, we investigated the anticancer activity of the enantiomeric CopA3 dimer peptide in human gastric cancer cell lines as well as in an in vivo tumor xenograft model. Enantiomeric CopA3 reduced gastric cancer cell viability and exhibited cytotoxicity against cancer cells. Enantiomeric CopA3-induced cell death was mediated by specific interactions with phosphatidylserine and phosphatidylcholine, membrane components that are enriched in cancer cells, in a calcein leakage assay. Moreover, acridine orange/ethidium bromide staining, flow cytometric analysis, and Western blot analysis showed that enantiomeric CopA3 induced apoptotic and necrotic gastric cancer cell death. The antitumor effect was also observed in a mouse tumor xenograft model in which intratumoral inoculation of the peptide resulted in a significant decrease in the SNU-668 gastric cancer tumor volume. In addition, periodic acid-Schiff and hematoxylin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed apoptotic and necrotic cell death in tumor masses treated with greater than 150 µg CopA3. Collectively, these results indicate that the enantiomeric CopA3 dimer peptide induces apoptosis and necrosis of gastric cancer cells in vitro and in vivo, indicating that the peptide is a potential candidate for the treatment of gastric cancer, which is a common cause of cancer and cancer deaths worldwide.

Anticancer Activity of the Antimicrobial Peptide Scolopendrasin VII Derived from the Centipede, Scolopendra subspinipes mutilans.

Previously, we performed de novo RNA sequencing of Scolopendra subspinipes mutilans using high-throughput sequencing technology and identified several antimicrobial peptide candidates. Among them, a cationic antimicrobial peptide, scolopendrasin VII, was selected based on its physicochemical properties, such as length, charge, and isoelectric point. Here, we assessed the anticancer activities of scolopendrasin VII against U937 and Jurkat leukemia cell lines. The results showed that scolopendrasin VII decreased the viability of the leukemia cells in MTS assays. Furthermore, flow cytometric analysis and acridine orange/ethidium bromide staining revealed that scolopendrasin VII induced necrosis in the leukemia cells. Scolopendrasin VII-induced necrosis was mediated by specific interaction with phosphatidylserine, which is enriched in the membrane of cancer cells. Taken together, these data indicated that scolopendrasin VII induced necrotic cell death in leukemia cells, probably through interaction with phosphatidylserine. The results provide a useful anticancer peptide candidate and an efficient strategy for new anticancer peptide development.

Anticancer activity of CopA3 dimer peptide in human gastric cancer cells.

CopA3 is a homodimeric α-helical peptide derived from coprisin which is a defensin-like antimicrobial peptide that was identified from the dung beetle, Copris tripartitus. CopA3 has been reported to have anticancer activity against leukemia cancer cells. In the present study, we investigated the anticancer activity of CopA3 in human gastric cancer cells. CopA3 reduced cell viability and it was cytotoxic to gastric cancer cells in the MTS and LDH release assay, respectively. CopA3 was shown to induce necrotic cell death of the gastric cancer cells by flow cytometric analysis and acridine orange/ethidium bromide staining. CopA3-induced cell death was mediated by specific interactions with phosphatidylserine, a membrane component of cancer cells. Taken together, these data indicated that CopA3 mainly caused necrosis of gastric cancer cells, probably through interactions with phosphatidylserine, which suggests the potential utility of CopA3 as a cancer therapeutic.

Knockdown of anterior gradient 2 expression extenuates tumor-associated phenotypes of SNU-478 ampulla of Vater cancer cells.

BACKGROUND: Anterior gradient 2 (AGR2) has been implicated in tumor-associated phenotypes such as cell viability, invasion and metastasis in various human cancers. However, the tumor promoting activity of AGR2 has not yet been determined in biliary tract cancers. Thus, we examined the expression of AGR2 and its tumor-promoting activity in biliary tract cancer cells in this study. METHODS: Expression of AGR2 mRNA and protein was analyzed by real time RT-PCR and western blotting, respectively. MTT assay was employed to measure cell viability and pulsed BrdU incorporation by proliferating cells was monitored by flow cytometry. Soft agar colony formation assay and transwell invasion assay were employed to determine anchorage-independent growth and in vitro invasion of the tumor cells, respectively. In vivo tumor formation was examined by injection of tumor cells into immunocompromised mice subcutaneously. Statistical analysis was performed with 2-tailed unpaired Student's t-test for continuous data and with one-way ANOVA for multiple group comparisons. Bonferroni tests were used for post hoc 2-sample comparisons. RESULTS: AGR2 mRNA was detected in SNU-245, SNU-478, and SNU-1196 cell lines, and its protein expression was confirmed in SNU-478 and SNU-245 cell lines by western blot analysis. Knockdown of AGR2 expression with an AGR2-specific short hairpin RNA (shRNA) in SNU-478, an ampulla of Vater cancer cell line resulted in decreased cell viability and in decreased anchorage-independent growth by 98%. The AGR2 knockdown also increased the sensitivity of the cells to chemotherapeutic drugs, including gemcitabine, 5-fluorouracil and cisplatin. In addition, SNU-478 cells expressing AGR2-shRNA failed to form detectable tumor xenografts in nude mice, whereas control cells formed tumors with an average size of 179 ± 84 mm3 in 3 weeks. Overexpression of AGR2 in SNU-869 cells significantly increased cell viability through enhanced cell proliferation and the number of Matrigel™-invading cells compared with AGR2-negative SNU-869 cells. CONCLUSIONS: Our findings implicate that AGR2 expression augments tumor-associated phenotypes by increasing proliferative and invasive capacities of the ampulla of Vater cancer cells.

The osteogenic or adipogenic lineage commitment of human mesenchymal stem cells is determined by protein kinase C delta.

BACKGROUND: Mesenchymal stem cells (MSCs) have the potential to differentiate into specialized cell lineages such as osteoblasts and adipocytes in vitro. There exists a reciprocal relationship between osteogenic and adipogenic differentiation of MSCs that an osteogenic phenotype occurs at the expense of an adipogenic phenotype and vice versa, which in turn influence one another's phenotype through negative feedback loops. Thus, it is important to understand what signaling molecules modulate the lineage commitment of MSCs. Protein kinase C (PKC) plays a central role in cellular signal transduction for mediating diverse biological functions, and dysregulation of PKC activity is involved in various metabolic diseases including cancer, diabetes, and heart disease. Although the role of individual PKC isoforms has been investigated in various fields, the potential role of PKC in bone metabolism is not completely understood. In this study, we investigated the potential role of PKCδ in osteogenic lineage commitment of human bone marrow-derived mesenchymal stem cells (hBMSCs). RESULTS: We observed that expression and phosphorylation of PKCδ were increased during osteogenic differentiation of hBMSCs. Pharmacological inhibition and genetic ablation of PKCδ in hBMSCs resulted in a significant attenuation of osteogenic differentiation as evidenced by reduced ALP activity and ECM mineralization, as well as down-regulation of the expression of osteoblast-specific genes. These effects were also accompanied by induction of adipogenic differentiation and up-regulation of the expression of adipocyte-specific genes involved in lipid synthesis in osteogenic induction of hBMSCs. Additionally, the activation of AMPK, which is a key cellular energy sensor, induced osteogenesis of hBMSCs. However, the inhibition of AMPK activity by compound C did not affect the activation of PKCδ at all, indicating that there is no direct correlation between AMPK and PKCδ in osteogenesis of hBMSCs. CONCLUSIONS: These results suggest that PKCδ is a critical regulator for the balance between osteogenesis and adipogenesis of hBMSCs and thus has a potential novel therapeutic target for the treatment of metabolic bone diseases.

Expression of anterior gradient 2 is decreased with the progression of human biliary tract cancer.

Biliary tract cancers include cancers of the gallbladder and extrahepatic bile ducts, and its prognosis is poor. The anterior gradient 2 (AGR2) is a protein disulfide isomerase and is highly expressed in various human cancers, such as breast, prostate and pancreas cancers. AGR2 is expressed in normal cholangiocytes and its expression is maintained during biliary carcinogenesis. However, the clinical significance of AGR2 expression in biliary tract cancer has not yet been assessed. Thus, we examined the expression of AGR2 protein in biliary tract tumors using immunohistochemistry and its association with various clinicopathologic parameters. This study included 100 patients who underwent surgery for biliary tract cancers: 46 men and 54 women with a mean and median age of 64.2 and 65.0 years, respectively. AGR2 expression was detected in ductal epithelial cells of the normal biliary tract and in 95% of biliary tract cancer tissues. While the AGR2 expression was not associated with cancer location, patient age, patient sex, degree of regional lymph node metastasis (N-status), or residual status, the AGR2 expression level was decreased with increased tumor size (T-status, p = 0.006) and progression of tumor stage (p = 0.009). Moreover, well-differentiated cancers tended to show higher AGR2 expression than poorly differentiated cancers (p = 0.068); in fact, AGR2 expression was not associated with patient survival (Kaplan-Meier analysis, p = 0.415). Thus, AGR2 is of limited value as a prognostic marker for biliary tract cancer. In conclusion, the expression of AGR2 is decreased with the progression of biliary tract cancer.

AEG-1/MTDH/LYRIC, the beginning: initial cloning, structure, expression profile, and regulation of expression.

Since its initial identification as a HIV-1-inducible gene in 2002, astrocyte elevated gene-1 (AEG-1), subsequently cloned as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), has emerged over the past 10 years as an important oncogene providing a valuable prognostic marker in patients with various cancers. Recent studies demonstrate that AEG-1/MTDH/LYRIC is a pleiotropic protein that can localize in the cell membrane, cytoplasm, endoplasmic reticulum (ER), nucleus, and nucleolus, and contributes to diverse signaling pathways such as PI3K-AKT, NF-κB, MAPK, and Wnt. In addition to tumorigenesis, this multifunctional protein is implicated in various physiological and pathological processes including development, neurodegeneration, and inflammation. The present review focuses on the discovery of AEG-1/MTDH/LYRIC and conceptualizes areas of future direction for this intriguing gene. We begin by describing how AEG-1, MTDH, and LYRIC were initially identified by different research groups and then discuss AEG-1 structure, functions, localization, and evolution. We conclude with a discussion of the expression profile of AEG-1/MTDH/LYRIC in the context of cancer, neurological disorders, inflammation, and embryogenesis, and discuss how AEG-1/MTDH/LYRIC is regulated. This introductory discussion of AEG-1/MTDH/LYRIC will serve as the basis for the detailed discussions in other chapters of the unique properties of this intriguing molecule.

Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis.

Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent.

Antimicrobial activity of the synthetic peptide scolopendrasin ii from the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

Differential susceptibility of gastric cancer cells to TRAIL-induced apoptosis.

Understanding the molecular basis of the differential sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis is required to predict therapeutic outcomes and to improve the effectiveness of TRAIL-based therapy. This study aimed to compare the responsiveness of gastric cancer cells to TRAIL treatment and to investigate the molecular basis of the differential TRAIL sensitivity of four gastric cancer cell lines. The TRAIL sensitivity of the four cell lines was ranked in the following order: SNU-16 ≈ SNU-620 > SNU-5 >> SNU-1. The level of Annexin V binding and the activation profile of caspase-3, -8 and -9 corroborated the differential TRAIL susceptibility of the cell lines. To determine the molecular basis of the differential sensitivity to TRAIL, we examined the expression of signaling components involved in TRAIL-mediated apoptosis. The mRNA level and surface expression of death receptor 4 (DR4) were significantly decreased in the SNU-1 cells compared to the other cell lines. Bid cleavage and X-linked inhibitor of apoptosis (XIAP) degradation were significantly increased in the SNU-16 and SNU-620 cells compared to the SNU-5 and SNU-1 cells, although Bid and XIAP were expressed at similar levels across the four cell lines. The expression and degradation of FLICE-inhibitory protein (FLIP) upon TRAIL treatment was independent of TRAIL sensitivity. In conclusion, the differential susceptibility of the four gastric cancer cells to TRAIL may be ascribed to the differential expression of DR4 and the proper augmentation of the death signal by the truncation of Bid and degradation of XIAP.

Effects of the synthetic coprisin analog peptide, CopA3 in pathogenic microorganisms and mammalian cancer cells.

A synthetic coprisin analog peptide, 9-mer dimer CopA3 (CopA3) was designed based on a defensin-like peptide, Coprisin, isolated from the bacteria-immunized dung beetle Copris tripartitus. Here, CopA3 was investigated for its antimicrobial activity and cancer cell growth inhibition. CopA3 showed antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in 2~32 µM ranges, and inhibited the cell viabilities of pancreatic and hepatocellular cancer cells, except MIAPaca2, Hep3B, and HepG2 cells, in a dose-dependent manner. The average IC(50) values of CopA3 against pancreatic and hepatocellular cancer cells were 61.7 µM and 67.8 µM, respectively. The results indicate that CopA3 has potential in the treatments of pancreatic and hepatocellular cancers as well as microorganism infection disease.

Effect of glucagon-like peptide-1 gene expression on graft function in mouse islet transplantation.

This study investigated the effect of local glucagon-like peptide-1 (GLP-1) production within mouse islets on cytoprotection in vitro and in vivo by gene transfer of GLP-1. Transduction of recombinant adenovirus vector expressing GLP-1 (rAd-GLP-1) induced a significant increase in bioactive GLP-1 in the mouse islet culture, whereas transduction with adenovirus vector expressing ß-galactosidase (rAd-LacZ), as a control, had no effect on GLP-1 secretion. Islets transduced with rAd-GLP-1 were protected from H(2) O(2) -induced cell damage in vitro. In addition, glucose-stimulated insulin secretion was significantly increased in rAd-GLP-1-transduced islets. When transplanted under the kidney capsule of diabetic syngeneic mice, islet grafts retrieved 4 or 7 days after transplantation revealed that the rAd-GLP-1-transduced group had significantly more Ki67-positive cells as compared with the rAd-LacZ-transduced group. Regarding blood glucose control, diabetic mice transplanted with a marginal mass of rAd-GLP-1-transduced islets became normoglycemic more rapidly and 78% of the recipients were normoglycemic at 35 days post-transplant, whereas only 48% of the mice transplanted with rAd-LacZ-transduced islets were normoglycemic (P < 0.05). In conclusion, delivery of the GLP-1 gene to islets enhanced islet cell survival during the early post-transplant period, and preserved islet mass and functions over time in the transplants.

Oncogene AEG-1 promotes glioma-induced neurodegeneration by increasing glutamate excitotoxicity.

Aggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene that is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition, AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.