Semaphorin 3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer.

We previously reported that novel targeted "hybrid peptide" in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cells. In this study, we have generated a novel type hybrid peptide, semaphorin 3A lytic (Sema3A-lytic), which is composed of two functional amino acid domains: a sequence derived from Sema3A that binds to neuropilin-1 (NRP1) and a cytotoxic lytic peptide. We found that this hybrid peptide had cytotoxic activity against NRP1-positive pancreatic cancer cell lines such as BxPC-3 and Panc-1, whereas the peptide did not affect the viability of normal cells in vitro. It was also found by affinity analysis that Sema3A peptide binds to NRP1, and two arginines (372R and 377R) in Sema3A peptide are involved in the interaction with NRP1 protein. In addition, confocal microscopy analysis revealed that Sema3A-lytic peptide could not penetrate normal cells regardless of the presence of NRP1 mRNA, suggesting that the ability of Sema3A-lytic peptide to concentrate adjacent to the cell membrane by binding to NRP1 with the target-binding moiety contributes to its selective cytotoxic activity. These results indicate that Sema3A-lytic hybrid peptide would be a possible anti-cancer agent for treatment of human pancreatic cancer.

A novel hybrid peptide targeting EGFR-expressing cancers.

Several potential molecular-targeted anticancer drugs focus on the inhibition of receptor tyrosine kinase and tumour growth, but these tyrosine kinase inhibitors (TKI) have been reported that the mutations of kinase-related signal molecule genes in cancer cells lead to the drug resistance. To overcome this issue, we have designed a novel targeting anticancer 'hybrid-peptide' EGFR-lytic peptide, in which epidermal growth factor receptor (EGFR) binding peptide is conjugated with a newly designed lytic-type peptide containing cationic-rich amino acids that disintegrates the cell membrane to kill cancer cells. In this report, cytotoxic activity of EGFR-lytic peptide was investigated in various human cancer and normal cell lines. It was found that the resulting conformational change in the novel lytic peptide enabled it to bind selectively to the membrane of cancer cells, and due to its acquired synergistic action, hybrid peptide demonstrated selective destruction of cancer cells as swiftly as 10 min after exposure. Treatment with EGFR-lytic peptide exerted a sufficient in vitro cytotoxic activity against TKI-resistant cancer cells with K-ras mutations. Moreover, in vivo analyses revealed that this peptide displayed significant antitumour activity in mouse xenograft models of both human K-ras mutation negative and positive cancers. Thus, hybrid peptide can be a unique and powerful tool for a new cancer-targeted therapy.

Antedrug budesonide by intrapulmonary treatment attenuates bleomycin-induced lung injury in rats with minimal systemic adverse effects.

Corticosteroids are routinely used in patients with pulmonary fibrosis, yet they have several adverse effects. To improve this situation, we used an animal model of pulmonary injury and early fibrosis and investigated whether the combination of an intrapulmonary inhalation device with antedrug budesonide (BUD) administered to the lung had greater efficacy and fewer systemic adverse effects compared to long-acting dexamethasone (DEX). BUD or DEX was administrated either intrapulmonary or intravenously to bleomycin-treated rats. Anti-inflammatory and antifibrotic effects were evaluated according to inflammatory cell count, total protein concentration and soluble collagen concentration in bronchoalveolar lavage fluid. The systemic immunosuppressive effects were also assessed by measuring body, spleen and thymus weight. BUD and DEX were compared with respect to their pharmacokinetic profiles in plasma and lung. Intrapulmonary treatment of BUD attenuates various inflammatory and early fibrotic indices with minimal systemic adverse effects compared with DEX. The area under the curve (AUC) of BUD by intrapulmonary spray was 6.6-fold higher than the AUC of DEX in the lung. This study suggests that antedrug BUD by intrapulmonary treatment has local anti-inflammatory and antifibrotic effects with minimal systemic adverse effects.

Pancreatic cancer therapy with a novel pump for controlled drug release.

Enhancing antitumor activity and minimizing treatment side effects are important issues in cancer therapy. One method to deal with these issues is the utilization of a drug delivery system (DDS). In this study, we developed a novel drug administration pump, a mechanically controlled DDS (M-DDS). The antitumor activity of 5-fluorouracil (5-FU) (15 or 30 mg/kg/day) was evaluated in comparison with systemic intraperitoneal (i.p.) administration for 7 days in a rat model of human pancreatic cancer. The M-DDS was superior to i.p. administration in enhancing antitumor activity and also prolonging median survival from 69 to 85 days at the lower drug dose (15 mg/kg/day). In addition, toxicities in liver, kidney and spleen were found in animals receiving i.p. administration, whereas rats receiving M-DDS treatment did not show these toxicities. The concentration of 5-FU in tumors 1 day after the completion of treatment was considerably higher in rats receiving M-DDS treatment. These results suggest that this novel M-DDS may be a powerful tool for the treatment of pancreatic cancer in combination with conventional chemotherapeutic drugs, offering strong antitumor activity with fewer toxicities. This novel M-DDS, consisting of a control circuit and drug reservoir/pump unit, may be a useful tool for the treatment not only of pancreatic cancer but also of various other accessible cancers for which there is no effective treatment, such as bile-duct and brain tumors.

Use of telomelysin (OBP-301) in mouse xenografts of human head and neck cancer.

We previously reported that telomerase-specific replication-component adenovirous, Telomelysin (OBP-301) has cytotoxic activity to the YCUT892, KCCT873, KCCT891, KCCL871, YCUM862, HN12, and KCCOR891 cell lines in vitro, and investigated the association between cytotoxic activity and adenoviral receptor expression. In this study, we evaluated the most appropriate way to administer telomelysin (OBP-301) in the treatment of squamous cell carcinoma of the head and neck (SCCHN), and assessed the effect of OBP-301 in large subcutaneous KCCT873 human SCCHN tumors in immunodeficient mice. We also compared antitumor responses following three intratumoral (i.t.) injections of OBP-301 given daily, every 2 days or weekly. To investigate the mechanism of the antitumor effect, we evaluated cellular infiltration in treated tumors. OBP-301 showed remarkable antitumor activity against large KCCT873 tumors, and three treatment schedules produced similar antitumor effects. The weekly regimen also significantly reduced the growth of large tumors. Immunochemistry revealed that macrophages, but not natural killer cells, were responsible for tumor regression. A regimen of three weekly injections of OBP-301 has remarkable antitumor effects against large KCCT873 tumors. These results may provide a new platform for treating patients with localized SCCHN.

Telomerase-specific virotherapy in an animal model of human head and neck cancer.

Telomerase-specific replication-competent adenovirus, Telomelysin (OBP-301), has a human telomerase reverse transcriptase promoter that regulates viral replication and efficiently kills human cancer cells. The objectives of this study are to examine the effects of OBP-301 in squamous cell carcinoma of the head and neck cells in vitro and in xenografted animals in vivo. OBP-301 was found to be cytotoxic to the YCUT892, KCCT873, KCCT891, KCCL871, YCUM862, HN12, and KCCOR891 cell lines in vitro. However, the level of cytotoxicity is not correlated with the expression levels of adenoviral receptors, which may be required for adenoviral infection in squamous cell carcinoma of the head and neck cells. OBP-301 shows remarkable antitumor activity against established s.c. KCCT873 tumors in immunodeficient animals in a dose-dependent manner. In addition, no significant toxicity was observed in animals receiving treatment. These results suggest that OBP-301 is a novel therapeutic agent with promise for the treatment of human head and neck cancers.

Targeted anticancer immunotoxins and cytotoxic agents with direct killing moieties.

Despite the progress of the bioinformatics approach to characterize cell-surface antigens and receptors on tumor cells, it remains difficult to generate novel cancer vaccines or neutralizing monoclonal antibody therapeutics. Among targeted cancer therapeutics, biologicals with targetable antibodies or ligands conjugated or fused to toxins or chemicals for direct cell-killing ability have been developed over the last 2 decades. These conjugated or fused chimeric proteins are termed immunotoxins or cytotoxic agents. Two agents, DAB389IL-2 (ONTAKTM) targeting the interleukin-2 receptor and CD33-calicheamicin (Mylotarg), have been approved by the FDA for cutaneous T-cell lymphoma (CTCL) and relapsed acute myeloid leukemia (AML), respectively. Such targetable agents, including RFB4(dsFv)-PE38 (BL22), IL13-PE38QQR, and Tf-CRM107, are being tested in clinical trials. Several agents using unique technology such as a cleavable adapter or immunoliposomes with antibodies are also in the preclinical stage. This review summarizes the generation, mechanism, and development of these agents. In addition, possible future directions of this therapeutic approach are discussed.

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology.

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.

Regulation of dendritic branching and spine maturation by semaphorin3A-Fyn signaling.

A member of semaphorin family, semaphorin3A (Sema3A), acts as a chemorepellent or chemoattractant on a wide variety of axons and dendrites in the development of the nervous systems. We here show that Sema3A induces clustering of both postsynaptic density-95 (PSD-95) and presynaptic synapsin I in cultured cortical neurons without changing the density of spines or filopodia. Neuropilin-1 (NRP-1), a receptor for Sema3A, is present on both axons and dendrites. When the cultured neurons are exposed to Sema3A, the cluster size of PSD-95 is markedly enhanced, and an extensive colocalization of PSD-95 and NRP-1 or actin-rich protrusion is seen. The effects of Sema3A on spine morphology are blocked by PP2, an Src type tyrosine kinase inhibitor, but not by the PP3, the inactive-related compound. In the cultured cortical neurons from fyn(-/-) mice, dendrites bear few spines, and Sema3A does not induce PSD-95 cluster formation on the dendrites. Sema3A and its receptor genes are highly expressed during the synaptogenic period of postnatal days 10 and 15. The cortical neurons in layer V, but not layer III, show a lowered density of synaptic bouton-like structure on dendrites in sema3A- and fyn-deficient mice. The neurons of the double-heterozygous mice show the lowered spine density, whereas those of single heterozygous mice show similar levels of the spine density as the wild type. These findings suggest that the Sema3A signaling pathway plays an important role in the regulation of dendritic spine maturation in the cerebral cortex neurons.

Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.

Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.