Endy Widya Putranto

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells

Angiogenesis is essential for tumor development and metastasis. Among several angiogenic factors, vascular endothelial growth factor receptor (VEGF) is important for tumor-derived angiogenesis and commonly overexpressed in solid tumors. Thus, many antitumor strategies targeting VEGF have been developed to inhibit cancer angiogenesis, offering insights into the successful treatment of solid cancers. However, there are a number of issues such as harmful effects on normal vascularity in clinical trials. Taking this into consideration, we employed Cordyceps militaris as an antitumor approach due to its biological safety in vivo. The herbal medicinal mushroom Cordyceps militaris has been reported to show potential anticancer properties including anti-angiogenic capacity; however, its concrete properties have yet to be fully demonstrated. In this study, we aimed to elucidate the biological role of Cordyceps militaris extract in tumor cells, especially in regulating angiogenesis and tumor growth of a human malignant melanoma cell line. We demonstrated that Cordyceps militaris extract remarkably suppressed tumor growth via induction of apoptotic cell death in culture that links to the abrogation of VEGF production in melanoma cells. This was followed by mitigation of Akt1 and GSK-3β activation, while p38α phosphorylation levels were increased. Extract treatment in mouse model xenografted with human melanoma cells resulted in a dramatic antitumor effect with down-regulation of VEGF expression. The results suggest that suppression of tumor growth by Cordyceps militaris extract is, at least, mediated by its anti-angiogenicity and apoptosis induction capacities. Cordyceps militaris extract may be a potent antitumor herbal drug for solid tumors.

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

Background: RAGE receptor plays a critical role in many inflammatory disorders. Results: Functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated cell survival. Conclusion: DAP10 membrane adaptor is critically involved in RAGE-mediated survival signaling upon S100A8/A9 binding. Significance: This is the first report demonstrating that RAGE-mediated survival signaling is critically regulated by DAP10 interaction. The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

MCAM, as a novel receptor for S100A8/A9, mediates progression of malignant melanoma through prominent activation of NF-κB and ROS formation upon ligand binding

The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-κB activation and ROS formation. Notably, MCAM not only activated NF-κB more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.

Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid β. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

Identification of an S100A8 Receptor Neuroplastin-β and its Heterodimer Formation with EMMPRIN

We previously reported a positive feedback loop between S100A8/A9 and proinflammatory cytokines mediated by extracellular matrix metalloproteinase inducer, an S100A9 receptor. Here, we identify neuroplastin-β as an unreported S100A8 receptor. Neuroplastin-β and extracellular matrix metalloproteinase inducer form homodimers and a heterodimer, and they are co-localized on the surface of cultured normal human keratinocytes. Knockdown of both receptors suppressed cell proliferation and proinflammatory cytokine induction. Upon stimulation with S100A8, neuroplastin-β recruited GRB2 and activated extracellular signal-regulated kinase, resulting in keratinocyte proliferation. Keratinocyte proliferation in response to inflammatory stimuli was accelerated in involucrin promoter-driven S100A8 transgenic mice. Further, S100A8 and S100A9 were strongly up-regulated and co-localized in lesional skin of atopic dermatitis patients. Our results indicate that neuroplastin-β and extracellular matrix metalloproteinase inducer form a functional heterodimeric receptor for S100A8/A9 heterodimer, followed by recruitment of specific adaptor molecules GRB2 and TRAF2, and this signaling pathway is involved in activation of both keratinocyte proliferation and skin inflammation in atopic skin. Suppression of this pathway might have potential for treatment of skin diseases associated with chronic inflammation such as atopic dermatitis.

Active Secretion of Dimerized S100A11 Induced by the Peroxisome in Mesothelioma Cells.

S100A11, a small Ca2+ binding protein, acts extracellularly as a mediator of cancer progression. That raises the question of how a protein that lacks the classical secretory signal is able to be secreted outside cells without being damaged. Some insights into this question have been obtained, and there has been accumulating evidence indicating a pivotal role of a non-classical vesicle-mediated pathway using lysosomes or peroxisomes for the protein secretion. To obtain a more precise insight into the secretory mechanism of S100A11, we first screened representative cancer cells exhibiting significantly active secretion of S100A11. From the results of profiling, we turned our attention to aggressive cancer mesothelioma cells. In mesothelioma cells, we found that abundant dimeric S100A11 was produced selectively in the peroxisome after transportation of monomeric S100A11 through an interaction with PEX14, a peroxisome membrane protein, resulting in peroxisomal secretion of dimerized S100A11. In an extracellular environment in vitro, dimerized S100A11 promoted mesothelial cell invasion indirectly with the help of fibroblast cells. Overall, the results indicate that the peroxisome functions as an essential vesicle for the production of dimerized S100A11 and the subsequent secretion of the protein from mesothelioma cells and that peroxisome-mediated secretion of dimerized S100A11 might play a critical role in mesothelioma progression in a tumor microenvironment.

ß-1,3-galactosyl-O-glycosyl-glycoprotein ß-1,6-N-acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility.

We previously identified novel S100A8/A9 receptors, extracellular matrix metalloproteinase inducer (EMMPRIN), melanoma cell adhesion molecule (MCAM), activated leukocyte cell adhesion molecule (ALCAM), and neuroplastin (NPTN) β, that are critically involved in S100A8/A9-mediated cancer metastasis and inflammation when expressed at high levels. However, little is known about the presence of any cancer-specific mechanism(s) that modifies these receptors, further inducing upregulation at protein levels without any transcriptional regulation. Expression levels of glycosyltransferase-encoding genes were examined by a PCR-based profiling array followed by confirmation with quantitative real-time PCR. Cell migration and invasion were assessed using a Boyden chamber. Western blotting was used to examine the protein level, and the RNA level was examined by Northern blotting. Immunohistochemistry was used to examine the expression pattern of β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-N-acetylglucosaminyltransferase 3 (GCNT3) and MCAM in melanoma tissue. We found that GCNT3 is overexpressed in highly metastatic melanomas. Silencing and functional inhibition of GCNT3 greatly suppressed migration and invasion of melanoma cells, resulting in the loss of S100A8/A9 responsiveness. Among the novel S100A8/A9 receptors, GCNT3 favorably glycosylates the MCAM receptor, extending its half-life and leading to further elevation of S100A8/A9-mediated cellular motility in melanoma cells. GCNT3 expression is positively correlated to MCAM expression in patients with high-grade melanomas. Collectively, our results showed that GCNT3 is an upstream regulator of MCAM protein and indicate the possibility of a potential molecular target in melanoma therapeutics through abrogation of the S100A8/A9-MCAM axis.

Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction

Pancreatic ductal adenocarcinoma (PDAC) is currently one of the most intractable malignancies with a typical scirrhous pattern in histology. Due to its abundant tumor stroma and scant vascularization, chemotherapeutic agents are considered inefficiently permeable to cancer nests, making it highly difficult to cure the patients with PDAC. However, PDAC is also considered to owe its intractability to other critical factors such as cellular interaction between tumor cells and tumor microenvironment as well as architectural barriers, which increases in therapeutic resistance. Here, we report a specific cellular interaction between PDAC cells and mesenchymal stem cells (MSCs) intermingled in PDAC stroma, which facilitates cancer invasion. Secretory phenotype profiling revealed that production of Amphiregulin (AREG) and MMP-3 were specifically upregulated under the coexistence of BxPC3 cells with human MSCs (approximately four to ten folds in AREG, and twenty to sixty-folds in MMP-3 compared to that of BxPC3 cells alone), whereas MMP-9 expression was decreased (less than one-tenth comparing with that of BxPC3 cells alone). Blockage of AREG production by its specific siRNA removed MSC-mediated driving force of BxPC3 invasiveness. Immunohistochemical analysis of tissue samples obtained both from PDAC patients and PDAC imitating mouse xenografted models revealed that significant coexpression of AREG and its receptor EGFR were detected on the cancer cells at invasive front. These results strongly suggested that cellular interaction between cancer cells and MSCs in the PDAC stroma might be critical to cancer progression, especially in the process of local invasion and the early stage development of metastasis.