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It is known that VEGF receptors (VEGFR) and integrins interact with each other to regulate angiogenesis. We reported previously that the fasciclin 1 (FAS1) domain–containing protein, TGFBIp/βig-h3 (TGF-β–induced protein) is an angiogenesis regulator that inhibits both endothelial cell migration and growth via αvβ3 integrin. In an attempt to target the interaction between VEGFR-2 and αvβ3 integrin, we determined whether the FAS1 domain region of TGFBIp/βig-h3 (FAS1 domain protein) can block the interaction between the two receptors, leading to the suppression of angiogenesis. In this study, we showed that FAS1 domain protein inhibits VEGF165-induced endothelial cell proliferation and migration via αvβ3 integrin, resulting in the inhibition of VEGF165-induced angiogenesis. We also defined a molecular mechanism by which FAS1 domain protein blocks the association between αvβ3 integrin and VEGFR-2, showing that it binds to αvβ3 integrin but not to VEGFR-2. Blocking the association of these major angiogenic receptors with FAS1 domain protein inhibits signaling pathways downstream of VEGFR-2. Collectively, our results indicate that FAS1 domain protein, in addition to its inhibitory effect on αvβ3 integrin–mediated angiogenesis, also inhibits VEGF165-induced angiogenesis. Thus, FAS1 domain protein can be further developed into a potent anticancer drug that targets two principal angiogenic pathways. Mol Cancer Res; 10(8); 1010–20. ©2012 AACR.

PDF file - 259K, S1 Cell cytotoxicity assay. S2 FAS1-domain protein inhibits VEGF165-induced 9 association of alphavbeta3 integrin with VEGFR-2 and VEGFR-2 phosphorylation via alphavbeta3 10 integrin. S3 FAS1-domain protein inhibits both vitronectin- and 32 collagen-i 1 nduced VEGFR-2 phosphorylation and association of alphavbeta3 integrin with 2 VEGFR-2. S4 Serum-starved VEGFR-2/beta5/HEK293 cells were plated on collagen I-coated in serum-free 16 medium supplemented with FAS1-domain protein (10 muM) for 30 min, followed by 17 stimulation for 5 min with VEGF165 (20 ng/ml) or vehicle alone

Supplementary Table S1 from Targeting Bladder Tumor Cells In vivo and in the Urine with a Peptide Identified by Phage Display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXRX/RC showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer. (Mol Cancer Res 2007;5(1):11–19)

Vijaya Sarangthem,1,2,* Aena Yi,1,* Yunjae Kim,1 Alnawaz Rehemtulla,3 Byung-Heon Lee,1 Young Hyun Jeon,4 Thoudam Debraj Singh,5 Rang-Woon Park1 1Department of Biochemistry and Cell Biology, School of Medicine, and Cell & Matrix Research Institute, Kyungpook National University, Daegu, 41944, Republic of Korea; 2Department of Pathology, All India Institute of Medical Sciences (AIIMS), New Delhi, 110029, India; 3Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA; 4Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu, Republic of Korea; 5Department of Medical Oncology Laboratory, All India Institute of Medical Sciences (AIIMS), New Delhi, 110029, India*These authors contributed equally to this workCorrespondence: Rang-Woon ParkDepartment of Biochemistry and Cell Biology, School of Medicine, and Cell & Matrix Research Institute, Kyungpook National University, Daegu, 41944, Republic of KoreaTel +82 53 420 4822Fax +82 53 422 1466Email nwpark@knu.ac.krThoudam Debraj SinghDepartment of Medical Oncology Laboratory, All India Institute of Medical Sciences (AIIMS), New Delhi, 110029, IndiaEmail debraj.thoudam@gmail.comBackground: Thermal-responsive self-assembled elastin-like polypeptide (ELP)-based nanoparticles are an emerging platform for controlled delivery of therapeutic peptides, proteins and small molecular drugs. The antitumor effect of bioengineered chimeric polypeptide AP1-ELP-KLAK containing an interleukin-4 receptor (IL-4R) targeting peptide and pro-apoptotic peptide (KLAKLAK) was evaluated in glioblastoma (GBM) in vitro and in vivo.Methods and Results: Herein, the therapeutic effect of AP1-ELP-KLAK was tested in advanced, and less curable glioblastoma cells with higher expression of IL-4R. Glioblastoma cell lines stably expressing different reporter systems i.e., caspase-3 sensor (surrogate marker for cellular apoptosis) or effluc/enhanced firefly luciferase (cellular viability) were established to measure cell death non-invasively. Bioluminescence imaging (BLI) of D54/effluc and U97MG/effluc treated with AP1-ELP-KLAK exhibited higher cell death up to 2∼ 3-fold than the control. Treatment with AP1-ELP-KLAK resulted in time-dependent increase of caspase-3 sensor BLI activity in D54/C cells and D54/C tumor-bearing mice. Intravenous injection of AP1-ELP-KLAK dramatically reduced tumor growth by inducing cellular apoptosis in D54/effluc tumor-bearing mice. Further, the immuno-histological examination of the excised tumor tissue confirmed the presence of apoptotic cells as well as caspase-3 activation.Conclusion: Collectively, AP1-ELP-KLAK effectively induced cellular apoptosis of glioblastoma cells and non-invasive imaging provides a window for real-time monitoring of anti-tumor effect with the provision of improving therapeutic efficacy in a glioblastoma mice model.Keywords: tumor targeting, ELP, caspase-sensor, apoptosis, IL-4 receptor, glioblastoma

The development of reliable methods to diagnose acute kidney injury is essential to allow the adoption of early therapeutic interventions and evaluate their effectiveness. Based on the fact that kidney injury molecule-1 (KIM-1) expression levels in kidneys are markedly upregulated early after a damage event, here we developed a noninvasive KIM-1-based molecular imaging technique to detect kidney injury. First, we took advantage of a phage-display platform to select small peptides demonstrating a specific high binding affinity to KIM-1. The promising candidate was conjugated with fluorescent probes, and its imaging potential was validated

Cholangiocarcinoma (CCA) has a poor prognosis and high chemoresistance. Interleukin-4 receptor (IL-4R) is overexpressed in several cancer cells and plays a crucial role in tumor progression and drug resistance. IL4RPep-1, an IL-4R-binding peptide, has been identified by phage display and used for tumor targeting. In this study, we exploited IL4RPep-1 to guide the tumor-specific delivery of a proapoptotic peptide to chemoresistant CCA, thereby inhibiting tumor growth. Immunohistochemistry of human primary CCA tissues showed that IL-4R levels were upregulated in moderately to poorly differentiated types, and higher levels of IL-4R are correlated with lower survival rates in patients with CCA. IL4RPep-1 was observed to preferentially bind with high IL-4R-expressing KKU-213 human CCA cells, whereas it barely bound with low IL-4R-expressing KKU-055 cells. A hybrid of IL4RPep-1 and a proapoptotic peptide (KLAKLAK)2 (named as IL4RPep-1-KLA) induced cytotoxicity and apoptosis in KKU-213 cells and increased those levels induced by 5-fluorouracil (5-FU). IL4RPep-1-KLA was internalized in the cells and colocalized with mitochondria. Whole-body fluorescence imaging and immunohistochemical analysis of tumor tissues showed the homing of IL4RPep-1-KLA as well as IL4RPep-1 to KKU-213 tumor in mice. Systemic administration of IL4RPep-1-KLA efficiently inhibited KKU-213 tumor growth, whereas treatment with 5-FU alone did not significantly inhibit tumor growth in mice. No significant systemic side effects including liver toxicity and immunotoxicity were observed in mice during peptide treatments. These findings suggest that IL4RPep-1-KLA holds potential as a targeted therapeutic agent against chemoresistant CCA.

Acquired drug resistance is a common occurrence and the main cause of melanoma treatment failure. Melanoma cells frequently developed resistance against cisplatin during chemotherapy, and thus, targeting delivery systems have been devised to decrease drug resistance, increase therapeutic efficacy, and reduce side effects. We genetically engineered a macromolecular carrier using the recursive directional ligation method that specifically targets cisplatin-resistant (Cis-R) melanoma. This carrier is composed of an elastin-like polypeptide (ELP) and multiple copies of Cis-R melanoma-targeting ligands (M-peptide). The designed M16E108 contains 16 targeting ligands incorporated within an ELP and has an ideal thermal phase transition at 39 °C. When treated to melanoma cells, M16E108 specifically accumulated in Cis-R B16F10 melanoma cells and accumulated to a lesser extent in parental B16F10 cells. Consistently, M16E108 exhibited efficient homing and longer retention in tumor tissues in Cis-R melanoma-bearing mice than in parental B16F10 melanoma-bearing mice. Thus, M16E108 was found to display considerable potential as a novel agent that specifically targets cisplatin-resistant melanoma.

In order to improve clinical outcomes for novel drug delivery systems, distinct optimization of size, shape, multifunctionality, and site-specificity are of utmost importance. In this study, we designed various multivalent elastin-like polypeptide (ELP)-based tumor-targeting polymers in which multiple copies of IL-4 receptor (IL-4R)-targeting ligand (AP1 peptide) were periodically incorporated into the ELP polymer backbone to enhance the affinity and avidity towards tumor cells expressing high levels of IL-4R. Several ELPs with different molecular sizes and structures ranging from unimer to micelle-forming polymers were evaluated for their tumor accumulation as well as in vivo bio-distribution patterns. Different percentages of cell binding and uptake were detected corresponding to polymer size, number of targeting peptides, or unimer versus micelle structure. As compared to low molecular weight polypeptides, high molecular weight AP1-ELP showed superior binding activity with faster entry and efficient processing in the IL-4R-dependent endocytic pathway. In addition, in vivo studies revealed that the high molecular weight micelle-forming AP1-ELPs (A86 and A100) displayed better tumor penetration and extensive retention in tumor tissue along with reduced non-specific accumulation in vital organs, when compared to low molecular weight non-micelle forming AP1-ELPs. It is suggested that the superior binding activities shown by A86 and A100 may depend on the multiple presentation of ligands upon transition to a micelle-like structure rather than a larger molecular weight. Thus, this study has significance in elucidating the different patterns underlying unimer and micelle-forming ELP-mediated tumor targeting as well as the in vivo biodistribution.

Expression and function of odorant receptors (ORs), which account for more than 50% of G protein–coupled receptors, are being increasingly reported in nonolfactory sites. However, ORs that can be targeted by drugs to treat diseases remain poorly identified. Tumor-derived lactate plays a crucial role in multiple signaling pathways leading to generation of tumor-associated macrophages (TAMs). In this study, we hypothesized that the macrophage OR Olfr78 functions as a lactate sensor and shapes the macrophage–tumor axis. Using Olfr78(+/+) and Olfr78(−/−) bone marrow–derived macrophages with or without exogenous Olfr78 expression, we demonstrated that Olfr78 sensed tumor-derived lactate, which was the main factor in tumor-conditioned media responsible for generation of protumoral M2-TAMs. Olfr78 functioned together with Gpr132 to mediate lactate-induced generation of protumoral M2-TAMs. In addition, syngeneic Olfr78-deficient mice exhibited reduced tumor progression and metastasis together with an increased anti- versus protumoral immune cell population. We propose that the Olfr78–lactate interaction is a therapeutic target to reduce and prevent tumor progression and metastasis.

Fibrin, one of the components of the extracellular matrix (ECM), acts as a transport barrier within the core of tumors by constricting the blood vessels and forming clots, leading to poor intratumoral distribution of anticancer drugs. Our group previously developed a microplasmin-based thrombolytic ferritin nanocage that efficiently targets and dissolves clots without causing systemic fibrinolysis or disrupting hemostatic clots. We hypothesized that the thrombolytic nanocage-mediated degradation of fibrin clots in the tumor ECM can lead to enhanced intratumoral drug delivery, especially for nanosized anticancer drugs. Fibrin clot deposition worsens after surgery and chemotherapy, further hindering drug delivery. Moreover, the risk of venous thromboembolism (VTE) also increases. Here, we used thrombolytic nanocages with multivalent clot-targeting peptides and fibrin degradation enzymes, such as microplasmin, to dissolve fibrin in the tumor microenvironment and named them fibrinolytic nanocages (FNCs). These FNCs target tumor clots specifically and effectively. FNCs efficiently dissolve fibrin clots inside of the tumor vessels, suggesting that they can mitigate the risk of VTE in cancer patients. Coadministration of FNC and doxorubicin led to improved chemotherapeutic activity in a syngeneic mouse melanoma model. Furthermore, the FNCs increased the distribution of Doxil/doxorubicin nanoparticles within mouse tumors. These results suggest that fibrinolytic cotherapy might help improve the therapeutic efficacy of anticancer nanomedicines. Thus, microplasmin-based fibrinolytic nanocages are promising candidates for this strategy due to their hemostatic safety and ability to home in on the tumor., Cancer: Improving treatment by dissolving clots Engineered “nanocages” that deliver clot-busting enzymes to tumors can improve the potency of chemotherapy and potentially reduce the risk of life-threatening complications. Many tumors form capillary-constricting clots composed of the protein fibrin, which impede drug penetration and create the potential for fatal venous thromboembolism. Researchers led by Soyoun Kim of Kyungpook National University, Daegu, South Korea, have devised a promising intervention that uses fibrin-degrading enzymes to clear these clogged vessels. The enzyme is delivered in nanocages composed of the protein ferritin coupled to short peptides that selectively bind fibrin, essentially targeting the treatment to clots. When they administered these nanocages to a mouse model of melanoma, the researchers observed a significantly improved response to chemotherapy and greater tumor penetration. This selective fibrin degradation should also eliminate the conditions that predispose patients to thrombembolism.

The lack of molecular targets and targeting probes remains a major drawback for targeted imaging and drug delivery in lung cancer. In this study, we exploited in vivo phage display to identify a novel targeting probe that homes to the tumor in a K-rasLA2 mutant mouse lung cancer model. Compared with other candidate peptides selected from 5 rounds of phage display, the CRQTKN peptide homed to tumor nodules in the lung of mutant mice at higher levels. Photoacoustic tomography of mutant mice detected lung tumors via tumor homing of the near-infrared fluorescence dye-labeled CRQTKN peptide. Ex vivo photoacoustic images of isolated organs further demonstrated tumor homing of the CRQTKN peptide, whereas minimal accumulation was observed in control organs, such as the liver. Compared with untargeted liposomes and doxorubicin, doxorubicin-loaded liposomes whose surface was modified with the CRQTKN peptide more efficiently delivered doxorubicin and reduced the number or size of tumor lesions in K-rasLA2 mutant mice. Analysis of hematologic parameters and liver and kidney function showed no significant systemic side effects by the treatments. Affinity-based identification was used to detect TNF receptor superfamily member 19L (TNFRSF19L), which was upregulated in lung tumors of mutant mice, as the receptor for the CRQTKN peptide. In conclusion, these results suggest that the CRQTKN peptide is a promising targeting probe for photoacoustic-guided detection and drug delivery to lung cancer, and acts by binding to TNFRSF19L. Significance: These findings present a new tumor-targeting probe for photoacoustic-guided detection and drug delivery.

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology., Extracellular vesicles are released from cells and permit communication between different cell types. Here, the authors identify that the FDA approved antibiotic sulfisoxazole, can block the release of these vesicles in breast cancer cells resulting in reduced cell growth in vitro and in vivo.

Purpose This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo. Materials and methods Biopanning of a phage-displayed peptide library was performed on KIM-1-coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1-overexpressing and KIM-1-low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1-overexpressing and KIM1-low expressing tumors in mice was examined by whole-body fluorescence imaging. Results A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1-overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells. Co-localization and competition assays using an anti-KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1-overexpressing A498 renal tumor compared to KIM1-low expressing HepG2 liver tumor in mice. Conclusion The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1‒overexpressing tumors.

The interaction of programmed cell death 1 ligand 1 (PD-L1) with its receptor, programmed cell death 1 (PD-1), inhibits T cell responses. Monoclonal antibodies that block this interaction have been shown effective as immunotherapy. However, only a subset of cancers exhibits a durable response to PD-1/PD-L1 blockade. Moreover, antibody-based immune checkpoint blockade is costly and is occasionally accompanied by systemic side effects. To overcome these limitations of antibody-based immune checkpoint blockade, an immune checkpoint-blocking ferritin nanocage displaying 24 PD-L1 binding peptides (PD-L1pep1) on its surface was designed and constructed. These ferritin nanocages displaying PD-L1pep1 (PpNF) specifically bind to PD-L1 expressed on cancer cells or to purified PD-L1 with a ~30 nM binding affinity. The addition of PpNF to co-cultures of T cells and cancer cells inhibited PD-1/PD-L1 interactions and restored T cell activities. In a mouse model of syngeneic colon cancer, PpNF specifically targeted tumors and showed antitumor activity. Moreover, PpNF nanocages encapsulating the chemotherapeutic drug doxorubicin had more potent antitumor activity than a monoclonal antibody against PD-L1. These results demonstrate that ferritin nanocages displaying surface PD-L1pep1 can be efficiently applied for immunotherapy, especially when encapsulating small chemotherapeutic drugs. These nanocages may have promise as an immunotherapeutic nanomedicine against various solid tumors.

CD44v6, a splice variant of the cell surface glycoprotein CD44, acts as a co-receptor for c-Met and is upregulated in tumors with high metastatic potential. Methods: We screened a phage-displayed peptide library for peptides that selectively bind to CD44v6-overexpressing cells and exploited them to block CD44v6 and deliver a pro-apoptotic peptide to tumors for cancer therapy. Results: CNLNTIDTC (NLN) and CNEWQLKSC (NEW) peptides bound preferentially to CD44v6-high cells than to CD44v6-low cells. The binding affinities of NLN and NEW to CD44v6 protein were 253 ± 79 and 85 ± 18 nM, respectively. Peptide binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments. Conclusions: These results suggest that NLN and NEW are promising metastasis-inhibiting peptide therapeutics and targeting moieties for CD44v6-expressing metastases.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes accompanied by increased infiltration of immune cells. Previous studies have demonstrated that hispidulin (4',5,7-trihydroxy-6-methoxyflavone, HPD) has various pharmacological benefits such as anti-fungal, anti-inflammation, and anti-allergic effects. This study investigated the effectiveness of HPD to treat psoriasis using an imiquimod (IMQ)-induced mouse model and activated keratinocytes. IMQ was topically applied to the back skin of mice for six consecutive days, and the mice were orally administered HPD. Based on the histological observation and immunological analysis, oral administration of HPD suppressed psoriatic characteristics including skin thickness, psoriasis area severity index, transepidermal water loss, and neutrophil infiltration. HPD alleviated pathologically increased levels of immunoglobulin G2a, myeloperoxidase, and tumor necrosis factor-α. Splenic Th1 and Th17 cell populations were also reduced by HPD in the murine model. In addition, in activated keratinocytes, HPD inhibited gene expression of Th1- and Th17-associated cytokines and chemokines, and phosphorylation of mitogen-activated protein kinases and nuclear factor-κB. In summary, HPD alleviates psoriasis skin inflammation in vivo and in vitro. Therefore, we suggest that HPD would be a potent therapeutic candidate for the treatment of psoriasis.

TRAIL is considered a promising target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors, DR4 or DR5. Although recombinant human TRAIL has shown high potency and specificity for killing cancer cells in preclinical studies, it has failed in multiple clinical trials for several reasons, including a very short half-life mainly caused by instability of the monomeric form of TRAIL and rapid renal clearance of the off-targeted TRAIL. To overcome such obstacles, we developed a TRAIL-active trimer nanocage (TRAIL-ATNC) that presents the TRAIL ligand in its trimer-like conformation by connecting it to a triple helix sequence that links to the threefold axis of the ferritin nanocage. We also ligated the tumor-targeting peptide, IL4rP, to TRAIL-ATNC to enhance tumor targeting. The developed TRAIL-ATNCIL4rP showed enhanced agonistic activity compared with monomeric TRAIL. The in vivo serum half-life of TRAIL-ATNCIL4rP was ~ 16-times longer than that of native TRAIL. As a consequence of these properties, TRAIL-ATNCIL4rP exhibited efficacy as an anti-tumor agent in vivo against xenograft breast cancer as well as orthotopic pancreatic cancer models, highlighting the promise of this system for development as novel therapeutics against cancer.

Accurate and timely visualization of apoptotic status in response to radiation is necessary for deciding whether to continue radiation or change to another mode of treatment. This is especially critical in patients with colorectal cancer, which requires a delicate combination of surgery, radiation, and chemotherapy in order to achieve optimal outcome. In this study, we investigated the potential of phosphatidylserine-recognizing peptide 1 (PSP1) as an apoptosis-targeting probe, which identifies phosphatidylserine on cell surfaces. We first screened colon cancer cell lines for their sensitivity to radiation and selected two cell lines: HCT116 and HT29. Cell binding assay using fluorescence-activated cell sorting and optical imaging showed that HCT116 cells had better binding to PSP1 than HT29 cells. Thus, mouse xenograft model using HCT116 cells was generated and was topically irradiated with either single or fractionated dose of radiation followed by systemic administration of PSP1 for subsequent molecular optical imaging. We confirmed that the PSP1 probe was selectively bound to apoptosis-induced tumor in a radiation dose-dependent manner. We also observed that fractionated radiation regimen, which is recently being used in clinical situation, was more effective in inducing tumor apoptosis than corresponding single-dose radiation treatment. We then evaluated the correlation between tumor targeting of PSP1 and suppression effect of tumor development and found that tumor volume and fluorescence intensity were correlated before (correlation coefficient r2 = 0.534) and after (r2 = 0.848) radiation therapy. Our study shows that PSP1 peptide is an efficient index probe for deciding “go or no-go” for radiation therapy in colorectal cancer.

Blockade of programmed cell death ligand-1 (PD-L1) restores T-cell activity and enhances anti-tumor immunity. Screening a phage-displayed peptide library for peptides that selectively bind to PD-L1-overexpressing cells identified two peptides, CLQKTPKQC and CVRARTR (PD-L1Pep-1 and PD-L1Pep-2, respectively) that appeared to block PD-L1. PD-L1Pep-1 and PD-L1Pep-2 preferentially bound to high PD-L1-expressing cells over low PD-L1-expressing cells; binding was further enhanced by interferon-γ, an inducer of PD-L1 expression. Binding affinities of PD-L1Pep-1 and PD-L1Pep-2 were approximately 373 and 281 nM, respectively. Cellular binding of the PD-L1-binding peptides was reduced by silencing PD-L1 gene expression or competition with anti-PD-L1 antibody. PD-L1Pep-1 and PD-L1Pep-2 induced the internalization and downregulated cell surface levels of PD-L1. The PD-L1-binding peptides restored cytokine secretion and T-cell proliferation to cells inhibited by co-culture with tumor cells or culture on PD-L1-coated plates. Intravenously injected PD-L1Pep-1 and PD-L1Pep-2 efficiently homed to tumor tissues, inhibited tumor growth, and increased CD8+/FoxP3+ ratio in mice. The PD-L1-binding peptides in combination with doxorubicin or PD-L1-targeted liposomal doxorubicin inhibited tumor growth and increased CD8+/FoxP3+ ratio more efficiently than doxorubicin alone and untargeted liposomal doxorubicin, respectively. These results suggest that PD-L1Pep-1 and PD-L1Pep-2 block PD-L1 and reinvigorate T-cell activity, inhibiting tumor growth by enhancing anti-tumor immunity.

Adoptive transfer of cytotoxic T lymphocytes (CTLs) has been used as an immunotherapy in melanoma. However, the tumor homing and therapeutic efficacy of transferred CTLs against melanoma remain unsatisfactory. Interleukin-4 receptor (IL-4R) is commonly up-regulated in tumors including melanoma. Here, we studied whether IL-4R-targeted CTLs exhibit enhanced tumor homing and therapeutic efficacy against melanoma. CTLs isolated from mice bearing melanomas were non-genetically engineered with IL4RPep-1, an IL-4R-binding peptide, using a membrane anchor composed of dioleylphosphatidylethanolamine. Compared to control CTLs, IL-4R-targeted CTLs showed higher binding to melanoma cells and in vivo tumor homing. They also exerted a more rapid and robust effector response, including increased cytokine secretion and cytotoxicity against melanoma cells and enhanced reprogramming of M2-type macrophages to M1-type macrophages. Moreover, IL-4R-targeted CTLs efficiently inhibited melanoma growth and reversed the immunosuppressive tumor microenvironment . These results suggest that non-genetically engineered CTLs targeting IL-4R have potential as an adoptive T cell therapy against melanoma.

M2-polarized, pro-tumoral tumor-associated macrophages (TAMs) express the interleukin-4 receptor (IL4R) at higher levels compared with M1-polarized, anti-tumoral macrophages. In this study, we harnessed M1 macrophage-derived exosomes engineered to foster M1 polarization and target IL4R for the inhibition of tumor growth by reprogramming TAMs into M1-like macrophages. M1 exosomes were transfected with NF-κB p50 siRNA and miR-511-3p to enhance M1 polarization and were surface-modified with IL4RPep-1, an IL4R-binding peptide, to target the IL4 receptor of TAMs (named IL4R-Exo(si/mi). IL4R-Exo(si/mi) were internalized and downregulated target gens in M2 macrophages and decreased M2 markers, while increasing M1 markers, more efficiently compared with untargeted and control peptide-labeled exosomes and exosomes from non-immune, normal cells. Whole-body fluorescence imaging showed that IL4R-Exo(si/mi) homed to tumors at higher levels compared with the liver, unlike untargeted and control peptide-labeled exosomes. Systemic administration of IL4R-Exo(si/mi) inhibited tumor growth, downregulated target genes, and decreased the levels of M2 cytokines and immune-suppressive cells, while increasing the levels of M1 cytokines and immune-stimulatory cells, more efficiently than untargeted and control peptide-labeled exosomes. These results suggest that IL4R-Exo(si/mi) inhibits tumor growth by reprogramming TAMs into M1-like macrophages and increasing anti-tumor immunity, thus representing a novel cancer immunotherapy.

Cellular cross-talk between tumors and M2-polarized tumor-associated macrophages (TAM) favors tumor progression. Upregulation of IL4 receptor (IL4R) is observed in diverse tumors and TAMs. We tested whether an IL4R-targeted proapoptotic peptide could inhibit tumor progression. The IL4R-binding peptide (IL4RPep-1) preferentially bound to IL4R-expressing tumor cells and M2-polarized macrophages both in vitro and in 4T1 breast tumors in vivo. To selectively kill IL4R-expressing cells, we designed an IL4R-targeted proapoptotic peptide, IL4RPep-1-K, by adding the proapoptotic peptide (KLAKLAK)2 to the end of IL4RPep-1. IL4RPep-1-K exerted selective cytotoxicity against diverse IL4R-expressing tumor cells and M2-polarized macrophages. Systemic administration of IL4RPep-1-K inhibited tumor growth and metastasis in 4T1 breast tumor-bearing mice. Interestingly, IL4RPep-1-K treatment increased the number of activated cytotoxic CD8+ T cells while reducing the numbers of immunosuppressive regulatory T cells and M2-polarized TAMs. No significant systemic side effects were observed. These results suggest that IL4R-targeted proapoptotic peptide has potential for treating diverse IL4R-expressing cancers. Mol Cancer Ther; 16(12); 2803–16. ©2017 AACR.

Tumor-associated macrophages (TAMs) are M2-polarized protumoral macrophages, whereas inflammatory macrophages are M1-polarized antitumoral macrophages. Interleukin-4 receptor (IL-4R) is expressed in M2 macrophages at higher levels than M1 macrophages. In this study, we aimed to inhibit tumor growth using M1 macrophage-derived exosomes engineered to target IL-4R and reprogram TAMs into M1 macrophages. M1 exosomes were transfected with NF-κB p50 siRNA and miR-511-3p as cargoes to enhance M1 polarization (Exo-si/mi) and surface-modified with IL4RPep-1, an IL-4R-binding peptide, to target IL-4R of M2 macrophages (IL4R-Exo-si/mi). The engineered exosomes showed sizes similar with parental exosomes, and IL4RPep-1 was retained on the exosomes and not degraded in the presence of serum up to 24 h. IL-4R-targeted exosomes were internalized into M2 macrophages and delivered cargoes through IL-4R more efficiently than untargeted exosomes. Whole-body fluorescence imaging showed that IL-4R-targeted exosomes homed to tumors at higher levels than to liver unlike to untargeted exosomes. Intravenous administration of IL4R-Exo-si/mi decreased the levels of M2 markers and immune-suppressive cells including TAMs, while increasing the levels of M1 markers and immune-stimulatory cells including M1 macrophages in tumor and spleen and subsequently inhibited tumor growth more efficiently than Exo-si/mi as well as M1 exosomes. These results suggest that IL-R-targeted exosomes carrying NF-κB p50 siRNA and miR-511-3p inhibit tumor growth by reprogramming TAMs into M1 macrophages and increasing anti-tumor immunity and hold a potential for caner immunotherapy. Citation Format: Byungheon Lee, Gowri Rangaswamy Gunassekaran. Reprogramming of tumor-associated macrophages to M1-like macrophages using M1 macrophage-derived exosomes engineered to target IL-4 receptor and enhance M1 polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2722.

// Se Young Jang 1, * , Gyeonghwa Kim 2, * , Soo Young Park 1 , Yu Rim Lee 1 , Sang Hoon Kwon 1 , Hyeong Seok Kim 1 , Jun Sik Yoon 1 , Jun Seob Lee 1 , Young-Oh Kweon 1 , Heon Tak Ha 3 , Jae Min Chun 3 , Young Seok Han 3 , Won Kee Lee 4 , Jun Young Chang 5 , Jung Gil Park 6 , Byungheon Lee 2 , Won Young Tak 1 and Keun Hur 2 1 Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, Republic of Korea 2 Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, Republic of Korea 3 Department of Surgery, Kyungpook National University School of Medicine, Daegu, Republic of Korea 4 Biostatistics, Medical Research Collaboration Center in Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Republic of Korea 5 Department of Neurology, Gyeongsang National University Changwon Hospital, Changwon, Republic of Korea 6 Department of Internal Medicine, College of Medicine, Yeungnam University, Daegu, Republic of Korea * These authors contributed equally to this work Correspondence to: Keun Hur, email: KeunHur@knu.ac.kr Won Young Tak, email: wytak@knu.ac.kr Keywords: hepatocellular carcinoma, long noncoding RNA ATB, survival, prognosis, epithelial mesenchymal transition Received: June 28, 2017 Accepted: September 04, 2017 Published: September 20, 2017 ABSTRACT Hepatocellular carcinoma (HCC) is a worldwide health problem and it is important to understand the mechanistic roles of the biomolecules involved in its pathogenesis. Long non-coding RNAs (lncRNAs) are frequently and aberrantly expressed in various human cancers and are known to play a role in cancer pathogenesis. The aim of this study was to analyze the expression of lncRNA-ATB in HCC and investigate the implications for prognoses. In total, 100 samples of HCC tissues and their corresponding, adjacent, non-cancerous liver tissues were collected. Total RNAs were extracted and the expression levels of lncRNA-ATB were measured by qRT-PCR. The association of lncRNA expression with clinicopathological features and patient survival were then analyzed. LncRNA-ATB was significantly upregulated in HCC tissues compared with the levels in corresponding non-cancerous tissues. Expression of lncRNA-ATB was significantly associated with portal vein thrombosis, intrahepatic or extrahepatic metastases, mUICC stage, and the BCLC stage. Large tumors (> 5 cm, HR = 3.851, 95% CI = 1.431–10.364, p = 0.008) and higher lncRNA-ATB expression (HR = 4.158, 95% CI = 1.226–14.107, p = 0.022) were the significant prognostic factors for overall survival. With this novel evidence of the involvement of lncRNA-ATB in HCC pathogenesis and clinical features, lncRNA-ATB can be concluded to have potential as a biomarker for the prognosis of HCC and as a targeted therapy for afflicted patients.

Ursolic acid (UA), a pentacyclic triterpenoid, is a common natural substance known to be effective in the treatment of inflammation, oxidative stress, and ulcers in arthritis. This study examined the effects of ursolic acid-3-acetate (UAA), a derivative of UA, on rheumatoid arthritis (RA) and verified the underlying mechanism of action by using a type-II collagen-induced arthritis (CIA) mice model and tumor necrosis factor (TNF)-α-stimulated RA synovial fibroblasts. The oral administration of UAA showed a decrease in clinical arthritis symptoms, paw thickness, histologic and radiologic changes, and serum IgG1 and IgG2a levels. UAA administration reduced Th1/Th17 phenotype CD4+ T lymphocyte expansion and inflammatory cytokine production in draining lymph nodes. In addition, UAA effectively reduced the expression and production of inflammatory mediators, including cytokines and matrix metalloproteinase-1/3 in the knee joint tissue and RA synovial fibroblasts, through the downregulation of IKKα/β, ΙκBα, and nuclear factor-κB. Our findings showed that UAA modulated helper T cell immune responses and matrix-degrading enzymes. The effects of UAA were comparable with those of the positive control drug, dexamethasone. In summary, all the evidence presented in this paper suggest that UAA could be a therapeutic candidate for the treatment of RA.

This study was performed to evaluate whether microRNAs (miRNAs) in circulating exosomes may serve as biomarkers of drug-induced liver, kidney, or muscle-injury. Quantitative PCR analyses were performed to measure the amounts of liver-specific miRNAs (miR-122, miR-192, and miR-155), kidney-specific miR-146a, or muscle-specific miR-206 in plasma and exosomes from mice treated with liver, kidney or muscle toxicants. The levels of liver-specific miRNAs in circulating plasma and exosomes were elevated in acetaminophen-induced liver injury and returned to basal levels by treatment with antioxidant N-acetyl-cysteine. Circulating miR-146a and miR-206 were increased in cisplatin-induced nephrotoxicity and bupivacaine-induced myotoxicity, respectively. Taken together, these results indicate that circulating plasma and exosomal miRNAs can be used as potential biomarkers specific for drug-induced liver, kidney or muscle injury.

Bioluminescence imaging is a non-invasive tool for in vivo real-time monitoring of infectious disease progression in animal models. However, no bioluminescence imaging assay has been developed to monitor Acinetobacter baumannii infections. In the current study, bioluminescent strains of A. baumannii ATCC 17978 and its isogenic ΔompA mutant were constructed by integrating the promoter of the ompA gene and the luxCDABE luciferase gene into the bacterial chromosome. In an acute murine pneumonia model, bioluminescence of the two reporter strains was clearly visible in the lungs and the bioluminescent signal increased over time. Bioluminescence was correlated with bacterial burden and histopathology in reporter strain-infected mice, suggesting that bioluminescent bacteria are useful for monitoring A. baumannii infections in animal models.

Mast cells play a crucial role in allergic diseases via the release of inflammatory mediators, particularly histamine and pro-inflammatory cytokines. Avenanthramide (Avn) C, a polyphenol found mainly in oats, is known to exhibit various biological properties. In this study, we aimed to evaluate the effectiveness of Avn C from germinated oats against mast cell-mediated allergic inflammation. For the in vitro study, RBL-2H3, mouse bone marrow-derived mast cells and rat peritoneal mast cells were used. Avn C (1–100 nM) inhibited the immunoglobulin (Ig)E-stimulated mast cells degranulation by suppressing phosphorylation of phosphoinositide 3-kinase and phospholipase Cγ1 and decreasing intracellular calcium levels. It inhibited IgE-stimulated secretion of inflammatory cytokines via suppression of FcεRI-mediated signaling proteins Lyn, Syk, Akt, and nuclear factor-κB. To verify the effects of Avn C in vivo, ovalbumin-induced active systemic anaphylaxis (ASA) and IgE-mediated passive cutaneous anaphylaxis (PCA) models were used. Oral administration of Avn C dose-dependently attenuated the ASA reactions, as evidenced by the inhibition of hypothermia and reduction of elevated serum histamine, IgE, and interleukin-4 levels. Avn C also inhibited the PCA reactions, such as ear swelling and plasma extravasation. Our results suggested that Avn C from germinated oats might be a possible therapeutic candidate for mast cell-mediated allergic inflammation.

Peptides have advantages over antibodies in terms of deep tissue penetration, low immunogenicity, and cost-effective production, but they have short circulation time and poor stability in vivo. Peptides have been extensively used as targeting moieties for the delivery of drug-loaded nanoparticles and function as targeted therapeutics in cancer treatment. Here, we review peptides that are exploited as targeted therapeutics in cancer therapy and apoptosis imaging probes for the monitoring of treatment responses.

Exosomes are small extracellular membrane vesicles released from endosomes of various cells and could be found in most body fluids. The main functions of exosomes have been recognized as important mediators of intercellular communication and as potential biomarkers of various disease states. This study investigated whether exogenous exosomes from mice with acetaminophen (APAP)-induced liver injury can damage the recipient hepatic cells or promote hepatotoxicity in mice. We observed that exogenous exosomes derived from APAP-exposed mice were internalized into the primary mouse hepatocytes or HepG2 hepatoma cells and significantly decreased the viability of these recipient cells. They also elevated mRNA transcripts and proteins associated with the cell death signaling pathways in primary hepatocytes or HepG2 cells via exosomes-to-cell communications. In addition, confocal microscopy of ex vivo liver section showed that exogenously added exosomes were accumulated in recipient hepatocytes. Furthermore, plasma reactive oxygen species and hepatic TNF-α/IL-1β production were elevated in APAP-exosomes recipient mice compared to control-exosomes recipient mice. The levels of apoptosis-related proteins such as phospho-JNK/JNK, Bax, and cleaved caspase-3 were increased in mouse liver received APAP-exosomes. These results demonstrate that exogenous exosomes from APAP-exposed mice with acute liver injury are functional and stimulate cell death or toxicity of the recipient hepatocytes and mice.

Perfluorooctanoic acid (PFOA) has wide applications, including as a raw material for converted paper and packaging products. With the widespread use of PFOA, concerns regarding its potential environmental and health impacts have increased. In spite of the known hepatotoxicity and genotoxicity of PFOA, correlation with PFOA and allergic inflammation is not well known. In this study, the effect of PFOA on the degranulation of mast cells and mast cell-mediated allergic inflammation in the presence of FceRI cross-linking was evaluated. In immunoglobulin (Ig) E-stimulated mast cells, PFOA increased the release of histamine and β-hexosaminidase by the up-regulation of intracellular calcium levels. PFOA enhanced gene expression of several pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 by the activation of nuclear factor (NF)-κB in IgE-stimulated mast cells. Also, PFOA exacerbated allergic symptoms via hypothermia, and an increase of serum histamine, TNF-α, IgE and IgG1 in the ovalbumin-induced systemic anaphylaxis. The present data indicate that PFOA aggravated FcɛRI-mediated mast cell degranulation and allergic symptoms. Copyright © 2016 John Wiley & Sons, Ltd.

Elastin-like polypeptide (ELP)-based drug delivery has been utilized for various applications including cancer therapies for many years. Genetic incorporation of internalization ligands and cell-targeting peptides along with ELP polymer enhanced tumor accumulation and retention time as well as stability and activities of the drug conjugates. Herein, we described a unique delivery system comprised of genetically engineered ELP incorporated with multiple copies of IL-4 receptor targeting peptide (AP1) periodically and proapoptotic peptide (KLAKLAK)(2) referred to as AP1-ELP-KLAK. It triggered thermal-responsive self-assembly into a nanoparticle-like structure at physiological body temperature and stabilized its helical conformation, which is critical for its membrane-disrupting activities. Increased IL-4 receptor specific cellular internalization was associated with the enhanced cytotoxic effect of (KLAKLAK) 2 peptide. Additionally, multivalent presentation of targeting ligands by AP1-ELP-KLAK significantly enhanced intratumoral localization and prolonged the retention time compared to ELP-KLAK, non-targeted control. Systemic administration of AP1-ELP-KLAK significanltly inhibited tumor growth by provoking cell apoptosis in various tumor xenograft models without any specific organ toxicity. Thus, our newly designed AP1-ELP-KLAK polymer nanoparticle is a promising candidate for effective cancer therapy and due to the simple preparative procedures of ELPs, this platform can be used as a good carrier for tumor-specific delivery of other therapeutics.

Mast cells are major effector cells for allergic responses that act by releasing inflammatory mediators, such as histamine and pro-inflammatory cytokines. Accordingly, different strategies have been pursued to develop anti-allergic and anti-inflammatory candidates by regulating the function of mast cells. The purpose of this study was to determine the effectiveness of elaeocarpusin (EL) on mast cell-mediated allergic inflammation. We isolated EL from Elaeocarpus sylvestris L. (Elaeocarpaceae), which is known to possess anti-inflammatory properties. For this study, various sources of mast cells and mouse anaphylaxis models were used. EL suppressed the induction of markers for mast cell degranulation, such as histamine and β-hexosaminidase, by reducing intracellular calcium levels. Expression of pro-inflammatory cytokines, such as tumor necrosis factor-α and IL-4, was significantly decreased in activated mast cells by EL. This inhibitory effect was related to inhibition of the phosphorylation of Fyn, Lyn, Syk, and Akt, and the nuclear translocation of nuclear factor-κB. To confirm the effect of EL in vivo, immunoglobulin E-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin-induced active systemic anaphylaxis (ASA) models were induced. EL reduced the PCA reaction in a dose dependent manner. In addition, EL attenuated ASA reactions such as hypothemia, histamine release, and IgE production. Our results suggest that EL is a potential therapeutic candidate for allergic inflammatory diseases that acts via the inhibition of mast cell degranulation and expression of proinflammatory cytokines.

Expression of various molecules on the surface of cancer cells compared to normal cells creates a platform for the generation of various drug vehicles for targeted therapy. Multiple interactions between ligands and their receptors mediated by targeting peptide-modified polymer could enable simultaneous delivery of a drug selectively to target tumor cells, thus limiting side effects resulting from non-specific drug delivery. In this study, we synthesized a novel tumor targeting system by using two key elements: (1) Bld-1 peptide (SNRDARRC), a recently reported bladder tumor targeting peptide identified by using a phage-displayed peptide library, and (2) ELP, a thermally responsive polypeptide. B5V60 containing five Bld-1 peptides and non-targeted ELP77 with a thermal phase-transition over 37 °C were analyzed to determine their bioactivities. Further studies confirmed the superior binding ability of B5V60 to bladder tumor cells and the cellular accumulation of B5V60 in cancer cells was dependent on the expression level of sialyl-Tn antigen (STn), a tumor-associated carbohydrate antigen. Additionally, B5V60 displayed excellent localization in bladder tumor xenograft mice after intravenous injection and was strictly confined to sialyl-Tn antigen-overexpressing tumor tissue. Thus, our newly designed B5V60 showed high potential as a novel carrier for STn-specific targeted cancer therapy or other therapeutic applications.

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are anti-inflammatory and immunomodulatory cytokines that play crucial roles in cancer progression. However, the clinical significance of the expression of these cytokines and their receptors (IL-4R) in oral cavity squamous cell carcinoma (OSCC) is unknown. Therefore, we evaluated the expression of IL-4R in OSCC specimens by immunohistochemistry (IHC) and analysed its relationship to recurrence and survival.A total of 186 patients with OSCC were enrolled, and the expression of IL-4Rα and IL-13Rα1 on their primary tumour specimens was evaluated by IHC and correlated to clinicopathologic parameters, recurrence and survival.High expression of IL-4Rα and IL-13Rα1 was observed in 60 (32.3%) and 165 (88.7%) patients, respectively. IL-4Rα expression was inversely correlated with parameters reflecting primary tumour burden, including tumour size, tumour stage and depth of invasion at the initial diagnosis (P0.05). High expression of IL-4Rα also correlated with a greater risk of recurrence (P = 0.002), but was unrelated to cancer-specific survival (CSS, P = 0.118). Conversely, high IL-13Rα1 expression correlated with reduced recurrence (P0.001) and increased CSS (P0.001) in OSCC patients.High expression of IL-4Rα correlated with increased recurrence, while high IL-13Rα1 expression had an inverse relationship to recurrence and CSS in OSCC patients.

Affinity maturation of protein-targeting peptides is generally accomplished by homo- or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low-affinity peptides into high-affinity peptides would clearly be advantageous for advancing peptide-based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β-hairpin scaffold and combinatorial phage-display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold-assisted approach is highly efficient and effective in generating high-affinity peptides from their low-affinity counterparts.

Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity ( K d = 57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide ( K d = 125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide–peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.

The use of thrombolytic therapies is limited by an increased risk of systemic hemorrhage due to lysis of hemostatic clots. We sought to develop a plasmin-based thrombolytic nanocage that efficiently dissolves the clot without causing systemic fibrinolysis or disrupting hemostatic clots. Here, we generated a double chambered short-length ferritin (sFt) construct that has an N-terminal region fused to multivalent clot targeting peptides (CLT: CNAGESSKNC) and a C-terminal end fused to a microplasmin (μPn); CLT recognizes fibrin-fibronectin complexes in clots, μPn efficiently dissolves clots, and the assembly of double chambered sFt (CLT-sFt-μPn) into nanocage structure protects the activated-μPn from its circulating inhibitors. Importantly, activated CLT-sFt-μPn thrombolytic nanocage showed a prolonged circulatory life over activated-μPn and efficiently lysed the preexisting clots in both arterial and venous thromboses models. Thus, CLT-sFt-μPn thrombolytic nanocage platform represents the prototype of a targeted clot-busting agent with high efficacy and safety over existing thrombolytic therapies.

Interleukin (IL)-13 is an immunoregulatory, anti-inflammatory cytokine that is produced by numerous immune cells, and plasma membrane receptor for IL-13 (IL-13R) is known to be expressed in various human malignancies and in immune cells.The authors evaluated the expression of IL-13R alpha 1 (IL-13Rα1, an IL-13R subtype) by immunohistochemistry in tissue microarrays of 1213 invasive breast cancer (IBC) samples to determine the prognostic value of IL-13Rα1 expression.High IL-13Rα1 expression was observed in 619 (51%) cases and was found to be associated with an older (≥50 years) age (p = 0.022), lymph node metastasis (p = 0.015), ductal and micropapillary histologic subtypes (p 0.001), lymphovascular invasion (p = 0.012), HER2 positivity (p 0.001), and a high (20%) Ki-67 index (p = 0.039). No significant correlation was found between IL-13Rα1 expression and clinicopathological variables, including tumor size, histological grade, hormone receptor expressions, and tumor-infiltrating lymphocyte levels. Patients with high IL-13Rα1 expression showed poorer overall survival (p = 0.044) and disease-free survival (DFS, p = 0.001) than those with low/negative expression. Subgroup analysis revealed an association between IL-13Rα1 expression and survival for HER2-negative, but not for HER2-positive tumors. Multivariate analysis showed high IL-13Rα1 expression was an independent negative prognostic factor of DFS (p = 0.019).The results of this study suggest the IL-13 and IL-13Rα1 interaction promotes cancer cell growth and metastasis, and IL-13Rα1 expression is a potential prognostic marker in IBC.

IL-4 receptor (IL-4R) is commonly up-regulated on tumor cells, and interactions between the receptor and Interleukin-4 (IL-4) can induce the expression of anti-apoptotic proteins, including Bcl-xL. This contributes to tumor cell survival and their resistance to chemotherapy. In this study, we exploited IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumor cells in order to sensitize them to chemotherapy. To target IL-4R, an IL-4R-binding peptide, IL4RPep-1, was attached to branched polyethyleneimine-superparamagnetic iron oxide nanoparticles (BPEI-SPION). These nanoparticles were then complexed with Bcl-xL-targeting siRNA. IL-4R-targeted BPEI-SPION/Bcl-xL siRNA more efficiently reduced Bcl-xL gene expression and enhanced cytotoxicity of doxorubicin in MDA-MB231 breast tumor cells compared to untargeted BPEI-SPION/Bcl-xL siRNA. The siRNA was released from the complexes after 15 h of incubation at pH 5.5 and was stable in the complexes up to 72 h in the serum. The IL-4R-targeted BPEI-SPION/siRNA was internalized by cells through IL-4R, successfully escaped the endosomes, and was dispersed into the cytoplasm. Near-infrared fluorescence and magnetic resonance imaging demonstrated that in vivo tumor homing and accumulation of IL-4R-targeted BPEI-SPION/siRNA were both higher than untargeted BPEI-SPION/siRNA. The IL-4R-targeted BPEI-SPION/Bcl-xL siRNA, in combination with doxorubicin, significantly inhibited tumor growth in mice compared to untargeted BPEI-SPION/Bcl-xL siRNA. These results suggest that the IL-4R-targeted delivery of Bcl-xL siRNA to IL-4R-expressing tumors can sensitize tumors to chemotherapy and enhance the efficacy of anti-tumor therapeutics.

// Jong-Lyel Roh 1 , Eun Hye Kim 1 , Jin Young Park 1 , Ji Won Kim 1 , Minsu Kwon 1 and Byung-Heon Lee 2 1 Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea 2 Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea Correspondence: Jong-Lyel Roh, email: // Keywords : piperlongumine, reactive oxygen species, head and neck cancer, cisplatin, cell death Received : June 06, 2014 Accepted : August 26, 2014 Published : August 27, 2014 Abstract Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in head-and-neck cancer (HNC) cells in vitro and in vivo . The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC cells and normal cells by measuring growth, death, cell cycle progression, reactive oxygen species (ROS) production, and protein expression, and in tumor xenograft mouse models. Piperlongumine killed HNC cells regardless of p53 mutational status but spared normal cells. It increased ROS accumulation in HNC cells, an effect that can be blocked by the antioxidant N -acetyl-L-cysteine. Piperlongumine induced selective cell death in HNC cells by targeting the stress response to ROS, leading to the induction of death pathways involving JNK and PARP. Piperlongumine increased cisplatin-induced cytotoxicity in HNC cells in a synergistic manner in vitro and in vivo . Piperlongumine might be a promising small molecule with which to selectively kill HNC cells and increase cisplatin antitumor activity by targeting the oxidative stress response.