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Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in colon cancers, but its role in cancer progression remains to be elucidated. In this work, we found that the extracellular domain of EpCAM (EpEX) activated EGFR and downstream ERK1/2 signaling to promote colon cancer cell migration and proliferation, as well as tumor growth. Mechanistically, we discovered that EpEX-EGFR-ERK1/2 signaling positively regulated intramembrane proteolysis (RIP) of EpCAM and shedding of the intracellular domain (EpICD). Treatment with an EGFR inhibitor ablated the EpEX-induced phosphorylation of ERK1/2 and AKT. Additionally, treatment with inhibitors of either EGFR or MEK decreased EpEX-induced EpICD shedding and further revealed that EpICD is necessary for nuclear accumulation of β-catenin and the induction of HIF1α target gene expression in vitro and in vivo. Moreover, an anti-EpCAM neutralizing monoclonal antibody, EpAb2-6, inhibited the nuclear translocation of EpICD and β-catenin and induced apoptosis in colon cancer cells. Importantly, analysis of colorectal cancer tissues showed that nuclear accumulation of EpICD was highly correlated with metastasis and poor prognosis, suggesting that it may play an important functional role in cancer progression. Thus, we provide novel insights into the mechanisms and functions of EpEX-mediated signaling, which may be considered as a promising target for the treatment of colon cancer.

While there has been extensive development of anti-cancer drugs for treatment of prostate cancer, the therapeutic efficacy of such drugs remains inadequate in many cases. Here, we performed in vitro biopanning of the PC3 human prostate carcinoma cell line to select prostate cancer-specific peptides by phage display. We successfully identified specific peptides targeting prostate cancer cells, and their specificity was confirmed by cellular ELISA and flow cytometry. Moreover, we found that the phage clones also recognize other prostate cancer cell lines and surgical specimens from prostate cancer patients. The tumor targeting ability of these phages was validated in a xenograft model, in which high accumulation of targeting phage was observed. To investigate whether selected peptides are able to target tumors and enhance drug delivery into cancer cells, we synthesized peptide-PEGylated lipids and post-inserted them into preformed liposomal doxorubicin and vinorelbine. The results of our cellular uptake and MTT assays indicate that peptide-conjugated liposomes exhibit enhanced drug intracellular delivery and cytotoxicity. The conjugation of targeting peptide to imaging agents, such as quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs), results in more precise delivery of these agents to tumor sites. Furthermore, administration of liposomal doxorubicin and vinorelbine conjugated with targeting peptides was found to markedly increase the inhibition of human prostate tumor growth in mouse xenograft and orthotopic models. These results indicate that targeting peptide, SP204, has significant potential for targeted therapy and molecular imaging in prostate cancer.

It is more challenging to design peptide drugs than small molecules through molecular docking and in silico analysis. Here, we developed a structure-based approach with various computational and analytical techniques to optimize cancer-targeting peptides for molecular imaging and therapy. We first utilized a peptide-binding protein database to identify GRP78, a specific cancer cell-surface marker, as a target protein for the lead, L-peptide. Subsequently, we used homologous modeling and molecular docking to identify a peptide-binding domain within GRP78 and optimized a series of peptides with a new protein-ligand scoring program, HotLig. Binding of these peptides to GRP78 was confirmed using an oriented immobilization technique for the Biacore system. We further examined the ability of the peptides to target cancer cells through in vitro binding studies with cell lines and clinical cancer specimens, and in vivo tumor imaging and targeted chemotherapeutic studies. MicroSPECT/CT imaging revealed significantly greater uptake of (188)Re-liposomes linked to these peptides as compared with non-targeting (188)Re-liposomes. Conjugation with these peptides also significantly increased the therapeutic efficacy of Lipo-Dox. Notably, peptide-conjugated Lipo-Dox significantly reduced stem-cell subpopulation in xenografts of breast cancer. The structure-based optimization strategy for peptides described here may be useful for developing peptide drugs for cancer imaging and therapy.

The objective of this study was to evaluate radiolabeled DOTA-SP90 as a radiotracer for breast cancer. The in vitro competition assay showed that radiolabeled DOTA-SP90 had significant binding affinity to BT-483 cancer cells. Biodistribution, nanoSPECT/CT and nanoPET/CT imaging results indicated that radiolabeled DOTA-SP90 can accumulate in tumors. In addition, radiolabeled DOTA-SP90 peptides can also detect metastatic tumors. Therefore, radiolabeled SP90 peptide may provide the potential capability as diagnostic agent for breast cancer patients.

Background Even though new molecular targeted agents are now being developed, they are still premature in application of clinical use for triple negative breast cancers (TNBCs). Therefore, it is highly demanded to explore the effective antitumor agents against TNBCs. Novel non-camptothecin topoisomerase I inhibitors are discovered and evaluated in this study. Methods Human MDA-MB-231 (BCRC-60425), BT-549 (ATCC® HTB-122™) and MCF-7 (BCRC-60436) were used in this study. Top1 assay was determined by gel mobility assay to validate the Top1-mediated DNA cleavages at different concentrations of non-camptothecin compounds. Cell viability analysis (MTT assay), comet assay and flow cytometry analysis were used to evaluate their growth inhibitory activity, DNA damage, and induction of cell arrest. Mechanistic pathways were studied and validated through western blot analysis, flow cytometry and confocal microscopy analysis. Binding interactions between top1 and non-camptothecin compound were analyzed by computational analysis (Discovery Studio). Results The IC50 values (growth inhibitory activity) of these non-camptothecin compounds are in the micromolar to nanomolar range (1.8 μm to 190 nM) against MDA-MB-231, BT-549 and MCF-7 cell lines. These compounds significantly inhibited the process in which supercoiled DNA strand transforms into its relaxed state and showed antitumor spectra similar to camptothecin rather than doxorubicin. Comet tails were observed to increase significantly with various doses of compounds in a dose-dependent manner. In addition, their mode of actions were shown to involve G2/M arrest of the cell cycle along with a dose-dependent increase in protein levels of cleaved caspase-3 and cleavage of cPARP. Except apoptosis pathway, non-camptothecin compounds also induce necrosis, and autophagy. Favorable ADMET characteristics of non-camptothecin compounds were observed using ADMET Descriptors. Conclusions Our results have provided evidence for therapeutic intervention in the treatment of TNBC using new top1 inhibitors, non-camptothecin compounds. Legal entity responsible for the study Wen-Shan Li. Funding Ministry of Science and Technology, Taiwan and Academia Sinica, Taiwan. Disclosure All authors have declared no conflicts of interest.

Background EpCAM is a type I transmembrane glycoprotein with an extracellular domain (EpEX) and an intracellular domain (EpICD), which have 265 and 26 amino acid residues, respectively. EpCAM is highly expressed in advanced epithelial cancers and tumor-initiating cells, but its role in cancer progression remains to be elucidated. Methods To identify cell signaling pathways that are stimulated by EpEX, we used a Human Phospho-RTK Array Kit to screen for phosphorylation of RTKs. Inhibition of EpEX-induced EGFR-PI3K-AKT signaling was analyzed by small molecule inhibitors or shRNA knockdown. Results Here, we found that EpEX activated AKT signaling, thereby inducing the phosphorylation and nuclear exclusion of FOXO3a. The EGFR inhibitor, AG1478, and MEK inhibitor, U0126, both decreased the production of EpICD, which was found to be necessary for nuclear accumulation of β-catenin protein and expression of HIF1α target genes in vitro and in mouse xenograft models. We also demonstrated that treatment with an anti-EpCAM neutralizing antibody, EpAb2-6, decreased the ADAM17 and γ-secretase activity, the EpCAM-downstream gene expression, and tumor colony and sphere formation. We also found that EpAb2-6 inhibited EpEX-activated EGFR-PI3K-AKT signaling and induced apoptotic signaling through FOXO3a activation of HTRA2 gene expression. Importantly, we also showed that EpAb2-6 inhibited the nuclear translocation of EpICD and oncogenic signaling through β-catenin. Finally, in both metastatic and orthotopic animal models of colorectal cancer, EpAb2-6 therapy exhibited an antitumor effect and markedly extended the survival time of mice. Conclusions Our results demonstrate that EpEX contributes to malignancy by functioning as a growth factor, which activates EpICD-mediated signaling, thereby enhancing colon cancer cell survival. To the best of our knowledge, hEpAb2-6 is the first humanized anti-EpCAM antibody to directly trigger apoptosis in cancer cells. Thus, we provide novel insight into EpEX-EGFR signaling, which can be considered as a promising target for treatment of colon cancer. Legal entity responsible for the study The authors. Funding Academia Sinica. Disclosure All authors have declared no conflicts of interest.

A new Cu6 cluster, [Cu6O2(L)2(OAc)6] (1) (HL = 2-(5-(pyrazin-2-yl)-4H-1,2,4-triazol-3-yl)pyrazine), was been synthesized via the reaction of pyrazine-2-carbonitrile and [Cu2(OAc)4]·2H2O in THF under solvothermal conditions, in which the HL ligand was in situ generated through coupled, rearranged and delaminated processes of pyrazine-2-carbonitrile and hydrazine. The structure of the complex 1 consisted a [Cu6(μ4-O)2]8+ bitetrahedral core. Variable temperature magnetic susceptibility measurements of complex 1 that it has a ground state spin value of S = 0, as the results of significant intramolecular antiferromagnetic interactions.

The synthesis of four lanthanide coordination polymers, [Ln(Dcam)(NO 3 )(MeOH) 2 ] n (Ln = Pr· 1 , Nd· 2 , Sm· 3 , and Eu· 4 ; d -H 2 cam = (+) d -camphoric acid) under solvothermal conditions is reported. Single-crystal X-ray diffraction analyses revealed that the compounds Pr· 1 –Eu· 4 are isostructural and crystallize in the monoclinic space group P 2 1 / n . The structures of the compounds are consisted a dimeric sub-building unit (SUB) of [Ln 2 (O 2 CR) 4 (NO 3 ) 2 (MeOH) 4 ], which is covalently-linked by cam 2− to a 2D grid layer and the layer is extended into a 3D supramolecular network by hydrogen bonding. The room temperature-photophysical properties of compounds Eu· 4 were investigated and the direct current (dc) magnetic susceptibility of compounds Pr· 1 –Eu· 4 were collected. The findings indicate that compounds Pr· 1 –Eu· 4 are capable of both antiferromagnetic and spin–orbital interactions.

This work describes the development of a capillary electrophoresis (CE) method for the simultaneous separation of acetophenone (AP), 2-hydroxyacetophenone (2-HAP), 3-hydroxyacetophenone (3-HAP) and 4-hydroxyacetophenone (4-HAP) in synthetic mixtures using 10 mmol/L of sodium tetraborate buffer (pH 9.5). The aim of this work is to demonstrate the effectiveness of CE to separate AP and its monohydroxy isomers and to define how the separations are affected by buffers, buffer pH, sample matrices and separation voltage. This method was successfully used for the trace level separation and determination of 2-HAP, 3-HAP and 4-HAP in synthetic mixture and 4-HAP in spiked plasma samples.

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.

Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.

It is known that solid tumors recruit new blood vessels to support tumor growth, but the molecular diversity of receptors in tumor angiogenic vessels might also be used clinically to develop better targeted therapy. In vivo phage display was used to identify peptides that specifically target tumor blood vessels. Several novel peptides were identified as being able to recognize tumor vasculature but not normal blood vessels in severe combined immunodeficiency (SCID) mice bearing human tumors. These tumor-homing peptides also bound to blood vessels in surgical specimens of various human cancers. The peptide-linked liposomes containing fluorescent substance were capable of translocating across the plasma membrane through endocytosis. With the conjugation of peptides and liposomal doxorubicin, the targeted drug delivery systems enhanced the therapeutic efficacy of the chemotherapeutic agent against human cancer xenografts by decreasing tumor angiogenesis and increasing cancer cell apoptosis. Furthermore, the peptide-mediated targeting liposomes improved the pharmacokinetics and pharmacodynamics of the drug they delivered compared with nontargeting liposomes or free drugs. Our results indicate that the tumor-homing peptides can be used specifically target tumor vasculature and have the potential to improve the systemic treatment of patients with solid tumors.

To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC-TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV-infected (EBV+) cells. In the animals bearing EBV+ and EBV- NPC xenografts, the MDM2+ cells only appeared in clusters in both EBV+ and EBV- tumors with stronger expression in EBV+ cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV-LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2-Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2-Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis.

Studies of the pathogenesis of dengue hemorrhagic fever (DHF), a potentially life-threatening disease, have revealed the importance of initial high levels of virus replication. However, the possible involvement of virus during the transition from fever to defervescence, a critical stage in determining the severity of disease, has not been appreciated. Using quantitative reverse transcription-polymerase chain reaction, we examined the levels of plasma dengue viral load during both fever and defervescence periods in patients from a DEN-3 outbreak in southern Taiwan in 1998. Higher levels of plasma dengue viral RNA were found in DHF patients than in DF patients. During defervescence, while the level of plasma dengue viral RNA was undetectable in most DF patients, it remains high in all DHF patients. Using a modified immunoprecipitation assay, we demonstrated for the first time that the plasma dengue viruses persisting during defervescence were in the immune complexes for most DHF patients. These findings suggest that continued active viral replication or delay in the clearance of viremia contributes to the pathogenesis of DHF. Moreover, high levels of plasma dengue viral RNA during defervescence may serve as a disease marker for DHF.

Many nasopharyngeal carcinoma (NPC) biopsy specimens contain Epstein-Barr virus (EBV). However, the response of NPC cells to EBV infection in vitro and in vivo is not well characterized. In this experiment we infected NPC cells with EBV particles through endocytosis of a complex of EBV immunoglobulin A (IgA) secretory component (SC) protein to observe the response of host cells to the foreign viral infection in vitro. We found that EBV particles were endocytosed and stabilized in NPC nuclei 24 hours after infection; the EBV genomes were then gradually decreased after serial passages within 3 to 4 weeks by the following pathway: the EBV genomes first moved toward the nuclear envelope from the center of the nucleus; after crossing the nuclear envelope, they moved into the cytoplasm and toward the plasma membrane and were discharged by exocytosis. At the 10th day of EBV infection, EBV-latent membrane protein-1 and Epstein-Barr nuclear antigen (EBNA)-1 protein expressions could be detected, but not EBV-viral capsid antigen. Observation of EBNA-1 protein and host growth factor and cytokine gene expressions in the weeks after incubation revealed that the EBNA-1 protein expression was decreased proportionally with decrease of EBV genome. The mRNA expression of epithelial growth factor receptor, transforming growth factor (TGF)-alpha, interleukin (IL)-1beta, IL-6, and granulocyte-macrophage colony-stimulating factor increased within 1 to 2 weeks after infection, and gradually recovered to the original level at 3 to 4 weeks, whereas the mRNAs of TGFbeta1, TGFbeta receptor type I (TGFbetaRI), TGFbetaR type II, IL-8, and tumor necrosis factor-alpha remained unchanged. It is concluded that in vitro EBV infection in NPC cells results in increase of certain growth factor and cytokine gene expressions in host cells. The change in gene expression returns to the original level approximately 3 to 4 weeks after infection because of exocytosis of EBV DNA by the infected cells through an unidentified mechanism.

We have examined the relationship between brain-derived neurotrophic factor gene expression in the hippocampus and memory retention as well as long-term potentiation of rats. One-way inhibitory avoidance learning was adopted as the behavioural paradigm. Results revealed that brain-derived neurotrophic factor messenger RNA levels in the dentate gyrus of the hippocampus were markedly increased at 1 h, 3 h and 6 h post-training in rats showing good retention performance when compared with the poor retention controls. Direct injection of brain-derived neurotrophic factor antisense oligonucleotide into the dentate gyrus of the hippocampus before memory consolidation takes place markedly impaired retention performance in rats. It also significantly decreased brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. The same antisense treatment also markedly reduced the amplitude and slope of excitatory postsynaptic potential as well as the brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. These results suggest that hippocampal brain-derived neurotrophic factor gene expression plays an important role in the memory consolidation process and in the expression of long-term potentiation in rats. These results provide the first evidence to relate brain-derived neurotrophic factor gene expression and memory function in vertebrates. It further suggests that brain-derived neurotrophic factor gene expression is involved in behavioural plasticity.

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.