A novel delivery platform for therapeutic peptides

Although many peptides have therapeutic effects against diverse disease, their short half-lives in vivo hurdle their application as drug candidates. To extend the short elimination half-lives of therapeutic peptides, we developed a novel delivery platform for therapeutic peptides using an anti-hapten antibody and its corresponding hapten. We selected cotinine because it is non-toxic, has a well-studied metabolism, and is physiologically absent. We conjugated WKYMVm-NH₂, an anti-sepsis therapeutic peptide, to cotinine and showed that the conjugated peptide in complex with an anti-cotinine antibody has a significantly improved in vivo half-life while retaining its therapeutic efficacy. We suggest that this novel delivery platform for therapeutic peptides will be very useful to develop effective peptide therapeutics.     

Inhibitory role of polyunsaturated fatty acids on lysophosphatidic acid-induced cancer cell migration and adhesion

Polyunsaturated fatty acids (PUFAs) have important pharmacological effects on mammalian cells. Here, we show that carboxyl group-containing PUFAs inhibit lysophosphatidic acid (LPA)-induced focal adhesion formation, thereby inhibiting migration and adhesion. Carboxyl group-containing PUFAs inhibit LPA-induced calcium mobilization, whereas ethyl ester-group containing PUFAs have no effect. In addition, carboxyl group-containing PUFAs functionally inhibit LPA-dependent RhoA activation. Given these results, we suggest that PUFAs may inhibit LPA-induced calcium/RhoA signaling pathways leading to focal adhesion formation. Carboxyl group-containing PUFAs may have a functional role in this regulatory mechanism.     

Spirosoma radiotolerans sp. nov., a Gamma-Radiation-Resistant Bacterium Isolated from Gamma Ray-Irradiated Soil

A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A^T, was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A^T belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74^T, 93.4 % with Spirosoma rigui WPCB118^T, 92.8 % with Spirosoma luteum SPM-10^T, 92.7 % with Spirosoma spitsbergense SPM-9^T, and 91.9 % with Spirosoma panaciterrae Gsoil 1519^T. Strain DG5A^T revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C_16:1 ω7c/C_16:1 ω6c (36.90 %), C_16:1 ω5c (29.55 %), and iso-C_15:0 (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A^T was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A^T presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A^T (=KCTC 32455^T = JCM19447^T).     

Specificity Protein 1 Expression Contributes to Bcl-w-Induced Aggressiveness in Glioblastoma Multiforme

We already had reported that Bcl-w promotes invasion or migration in gastric cancer cells and glioblastoma multiforme (GBM) by activating matrix metalloproteinase-2 (MMP-2) via specificity protein 1 (Sp1) or β-cateinin, respectively. High expression of Bcl-w also has been reported in GBM which is the most common malignant brain tumor and exhibits aggressive and invasive behavior. These reports propose that Bcl-w-induced signaling is strongly associated with aggressive characteristic of GBM. We demonstrated that Sp1 protein or mRNA expression is induced by Bcl-w using Western blotting or RT-PCR, respectively, and markedly elevated in high-grade glioma specimens compared with low-grade glioma tissues using tissue array. However, relationship between Bcl-w-related signaling and aggressive characteristic of GBM is poorly characterized. This study suggested that Bcl-w-induced Sp1 activation promoted expression of glioma stem-like cell markers, such as Musashi, Nanog, Oct4 and sox-2, as well as neurosphere formation and invasiveness, using western blotting, neurosphere formation assay, or invasion assay, culminating in their aggressive behavior. Therefore, Bcl-w-induced Sp1 activation is proposed as a putative marker for aggressiveness of GBM.     

Expression of Vesicular Glutamate Transporters VGLUT1 and VGLUT2 in the Rat Dental Pulp and Trigeminal Ganglion following Inflammation

Background

There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund’s adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis.

Results

The density of VGLUT2− immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group.

Conclusions

These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.     

Establishment and characterization of bortezomib-resistant U266 cell line: Constitutive activation of NF-κB-mediated cell signals and/or alterations of ubiquitylation-related genes reduce bortezomib-induced apoptosis

Bortezomib has been known as the most promising anti-cancer drug for multiple myeloma (MM). However, recent studies reported that not all MM patients respond to bortezomib. To overcome such a stumbling-block, studies are needed to clarify the mechanisms of bortezomib resistance. In this study, we established a bortezomib-resistant cell line (U266/velR), and explored its biological characteristics. The U266/velR showed reduced sensitivity to bortezomib, and also showed crossresistance to the chemically unrelated drug thalidomide. U266/velR cells had a higher proportion of CD138 negative subpopulation, known as stem-like feature, compared to parental U266 cells. U266/velR showed relatively less inhibitory effect of prosurvival NF-κB signaling by bortezomib. Further analysis of RNA microarray identified genes related to ubiquitination that were differentially regulated in U266/velR. Moreover, the expression level of CD52 in U266 cells was associated with bortezomib response. Our findings provide the basis for developing therapeutic strategies in bortezomib-resistant relapsed and refractory MM patients. [BMB Reports 2014; 47(5): 274-279]     

A novel antagonist to the inhibitors of apoptosis (IAPs) potentiates cell death in EGFR-overexpressing non-small-cell lung cancer cells

In the effort to develop an efficient chemotherapy drug for the treatment of non-small-cell lung cancer (NSCLC), we analyzed the anti-tumorigenic effects of a novel small molecule targeting the inhibitor of apoptosis (IAPs), HM90822B, on NSCLC cells. HM90822B efficiently decreased IAP expression, especially that of XIAP and survivin, in several NSCLC cells. Interestingly, cells overexpressing epidermal growth factor receptor (EGFR) due to the mutations were more sensitive to HM90822B, undergoing cell cycle arrest and apoptosis when treated. In xenograft experiments, inoculated EGFR-overexpressing NSCLC cells showed tumor regression when treated with the inhibitor, demonstrating the chemotherapeutic potential of this agent. Mechanistically, decreased levels of EGFR, Akt and phospho-MAPKs were observed in inhibitor-treated PC-9 cells on phosphorylation array and western blotting analysis, indicating that the reagent inhibited cell growth by preventing critical cell survival signaling pathways. In addition, gene-specific knockdown studies against XIAP and/or EGFR further uncovered the involvement of Akt and MAPK pathways in HM90822B-mediated downregulation of NSCLC cell growth. Together, these results support that HM90822B is a promising candidate to be developed as lung tumor chemotherapeutics by targeting oncogenic activities of IAP together with inhibiting cell survival signaling pathways.Resistance to apoptosis is a hallmark of many solid tumors, including lung cancer, and is, therefore, an important target mechanism for controlling cancer proliferation. The inhibitor of apoptosis (IAP) is a family of proteins containing one or more conserved cysteine and histidine-rich baculoviral IAP repeat (BIR) in their N-terminal domains and a C-terminal RING (really interesting new gene) domain. The BIR domains of IAPs form zinc figure-like structures that bind to active caspases to block caspase activity, while the RING domain acts as an ubiquitin ligase to facilitate proteasome degradation of caspases. Several IAPs have been identified in mammals, including X-linked IAP (XIAP), cellular IAP-1 and -2 (cIAP-1 and cIAP-2) and survivin. Among these IAP proteins, XIAP is a central regulator of both the death receptor- and mitochondria-mediated apoptosis pathways. Consistent with their role in the inhibition of apoptosis, XIAP and survivin are highly expressed in a diverse array of tumors and are often associated with resistance to apoptosis and low sensitivity to chemotherapy drugs in some tumor types.^{1, 2, 3}Recent studies have shown that inhibition of the expression level or function of survivin and/or XIAP with anti-sense RNA, short interfering RNA (siRNA), dominant-negative mutants, or small molecules induces apoptotic cell death in tumor cells but not in normal cells.⁴ Several chemical IAP antagonists, such as AT-406, LCL-161, GDC-0152, TL-32711, LBW242 and HGS-1029, which mimic the interactions of IAP proteins with secondary mitochondria-derived activator of caspase (SMAC) N-terminal peptide (an endogenous antagonist of IAP proteins), have been developed and are currently being evaluated in clinical settings.^{5, 6, 7, 8} The elucidation of the mechanism of antagonism and identification of biomarkers that indicate apoptotic cell death in tumors are key issues in the development of IAP antagonists. As such, the role of IAPs in regulating the apoptotic response and as molecular targets for achieving selective therapeutic effects in tumor cells has attracted great attention in an effort to identify peptide antagonists or small-molecule inhibitors.Lung cancer is the leading cause of cancer-related death worldwide, with more than one million mortalities each year. Almost 85% of all lung cancer cases are diagnosed as non-small-cell lung cancers (NSCLC), which are further classified histologically as adenocarcinoma, squamous cell carcinoma or large cell carcinoma. Platinum-based chemotherapy represents the recommended standard first-line systemic treatment for advanced NSCLC, although the results of this approach are limited to a modest increase in survival rates. Epidermal growth factor receptor (EGFR) is often hyper-activated in many lung cancers due to the presence of a mutation in the kinase domain, causing the activation of multiple cell survival signals, especially Akt and mitogen-activated protein kinase (MAPK) pathways. This finding has led to the development of targeted therapeutics against the kinase, such as erlotinib and gefitinib, which becomes one of the most promising strategies for cancer treatment. The targeted therapeutics has often failed, however, due to the development of resistance through multiple mechanisms, indicating that additional adjuvants are necessary to achieve effective results.In this study, we investigated the therapeutic potential of HM90822B, originally synthesized to inhibit IAP activity, on NSCLC cells and in a xenograft mouse model and analyzed the cellular effects of the drug to elucidate its mechanism of action. Our results showed that HM90822B inhibits cell growth resulting in cell cycle arrest and apoptosis by targeting XIAP and survivin in conjunction with the inhibition of EGFR-MAPK pathway, primarily AKT, p38 and c-jun phosphorylation. These results indicate that the IAP inhibitor HM90822B is a promising therapeutics for the treatment of NSCLC.     

Production and Deformation of Clonorchis sinensis Eggs during In Vitro Maintenance

Clonorchis sinensis is a carcinogenic human liver fluke. The present study monitored eggs produced by long-term maintained adult worms of C. sinensis to confirm their egg productivity in vitro. The worms from infected rabbits were incubated in vitro in 1× Locke’s solution and broth media (RPMI-1640, DMEM and IMDM). Numbers of expelled eggs were counted sequentially and their morphological changes were monitored by microscopy after 1, 30, 60, and 90 days of cultivation. On the 1–3 days of cultivation, the eggs counted maximum 4,756±202 eggs/worm/day in IMDM medium. The number of eggs gradually decreased less than 1,000 at 7–14 days and below 100 at 21days but continued to pass eggs after 56 days in all media. Length of the eggs were reduced about 1 µm at 30 days, and the length/width ratio was maintained around 1.8 at 30 days but decreased to 1.7 at 60 days and 1.5 at 90 days. Faust-Meleney index (FMI) decreased as the cultivation duration increased and lowest FMI (5662.9±974.7) observed in IMDM media at day 90 (P = 0.001). Microscopic findings of the eggs recognized the miracidium in most of eggs at 60 days but not in those at 90 days. Instead, the eggs contained dark granules or vacuoles in the deformed shell at 90 days. Scanning electron microscopy revealed partial loss of wrinkles on the deformed egg surface and prominent abopercular knob. Eggs viability decreased as the cultivation progressed and showed significant positive correlation with FMI and length/width ratio. In conclusion, the cultivated worms pass only the eggs which are preformed in their uterus before cultivation. One gravid C. sinensis contains about 37,000 eggs in its uterus and produces about 4,000 eggs every day. The deformed eggs with FMI less than 7,000 and length/width ratio lower than 1.7 are non-viable.     

Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages

Protopine is an isoquinoline alkaloid contained in plants in northeast Asia. In this study, we investigated whether protopine derived from Hypecoum erectum L could suppress lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (Raw 264.7 cells). Protopine was found to reduce nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E(2) (PGE(2)) production by LPS-stimulated Raw 264.7 cells, without a cytotoxic effect. Pre-treatment of Raw 264.7 cells with protopine reduced the production of pro-inflammatory cytokines. These inhibitory effects were caused by blocking phosphorylation of mitogen-activated protein kinases (MAP kinases) and also blocking activation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).     

Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats

Reference ranges of standard experimental parameters are useful for comparisons in toxicology. The aim of this study was to collect data from 13-week repeated toxicity studies in Crl:CD (SD) rats, a strain widely used for toxicity and efficacy research, for establishing domestic reference values. Data on body weight, food consumption; urinalysis, hematological, and blood biochemical parameters; and organ weights were collected from 11 toxicity studies in 220 Crl:CD (SD) rats (110 males and 110 females). The studies had been performed at a single testing facility over the last 5 years and involved animals sourced from a single breeder. The findings were collated as means, standard deviations, percentages, and ranges. Urine volume, uterus weight, eosinophil, and basophil counts, and triglyceride, total bilirubin, and gamma-glutamyl transpeptidase levels showed standard deviations of 30% or more. These historical control data would help to interpret the effects of test substances in routine toxicity and efficacy studies.