Koichi Ogino

Nobiletin, a Citrus Flavonoid, Improves Memory Impairment and Aβ Pathology in a Transgenic Mouse Model of Alzheimer's Disease

Increasing evidence suggests that the elevation of β-amyloid (Aβ) peptides in the brain is central to the pathogenesis of Alzheimers disease (AD). Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, enhances cAMP/protein kinase A/extracellular signal-regulated kinase/cAMP response element-binding protein signaling in cultured hippocampal neurons and ameliorates Aβ-induced memory impairment in AD model rats. For the first time, we report that this natural compound improves memory deficits in amyloid precursor protein (APP) transgenic mice that overexpress human APP695 harboring the double Swedish and London mutations [APP-SL 7-5 transgenic (Tg) mice]. Our enzyme-linked immunosorbent assay (ELISA) also showed that administration of nobiletin to the transgenic mice for 4 months markedly reduced quantity of guanidine-soluble Aβ1–40 and Aβ1–42 in the brain. Furthermore, consistent with the results of ELISA, by immunohistochemistry with anti-Aβ antibody, it was evidently shown that the administration of nobiletin decreased the Aβ burden and plaques in the hippocampus of APP-SL 7-5 Tg mice. These findings suggest that this natural compound has potential to become a novel drug for fundamental treatment of AD.

Anti-neovascular therapy using novel peptides homing to angiogenic vessels.

Cancer chemotherapy targeted to angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels formed by a dorsal air sac method from a phage-displayed peptide library. Three distinct phage clones that markedly accumulated in murine tumor xenografts presented PRPGAPLAGSWPGTS-, DRWRPALPVVLFPLH- or ASSSYPLIHWRPWAR-peptide respectively. After the determination of the epitope sequences of these peptides, we modified liposomes with epitope penta-peptides. Liposome modified with APRPG-peptide showed high accumulation in murine tumor xenografts, and APRPG-modified liposome encapsulating adriamycin effectively suppressed experimental tumor growth. Finally, specific binding of APRPG-modified liposome to human umbilical endothelial cells, and that of PRP-containing peptide to angiogenic vessels in human tumors, i.e., islet cell tumor and glioblastoma, were demonstrated. The present study indicates the usefulness of APRPG-peptide as a tool for anti-neovascular therapy, a novel modality of cancer treatment.

Anti-neovascular therapy by liposomal drug targeted to membrane type-1 matrix metalloproteinase

Because membrane type‐1 matrix metalloproteinase (MT1‐MMP) is expressed specifically on the angiogenic endothelium as well as tumor cells, an agent possessing the ability to bind to this molecule might be useful as a tool for active targeting of tumor angiogenic vessels. Based on the sequences of peptide substrates of MT1‐MMP, which had been determined by using a phage‐displayed peptide library, we examined the binding ability of peptide‐modified liposomes for endothelial cells and targeting ability for tumor tissues by positron emission tomography (PET). Liposomes modified with stearoyl‐Gly‐Pro‐Leu‐Pro‐Leu‐Arg (GPLPLR‐Lip) showed high binding ability to human umbilical vein endothelial cells and accumulated in the tumor about 4‐fold more than did the unmodified liposomes. Because we reported previously that liposomalized 5′‐O‐dipalmitoylphosphatidyl 2′‐C‐cyano‐2′‐deoxy‐1‐β‐D‐arabino‐pentofuranosylcytosine (DPP‐CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, strongly suppressed tumor growth when delivered in liposomes modified with another angiogenic homing peptide, we examined the antitumor activity of DPP‐CNDAC entrapped in GPLPLR‐Lip. DPP‐CNDAC/GPLPLR‐Lip showed significant tumor growth suppression compared to DPP‐CNDAC/unmodified liposomes. These results suggest that DPP‐CNDAC‐liposomes modified with MT1‐MMP‐targeted peptide are useful for cancer anti‐neovascular therapy (ANET), namely, tumor growth suppression by damage to angiogenic endothelial cells.

Anti-neovascular therapy by liposomal DPP-CNDAC targeted to angiogenic vessels

We previously reported that liposomalized 5′‐O‐dipalmitoylphosphatidyl 2′‐C‐cyano‐2′‐deoxy‐1‐β‐D‐arabino‐pentofuranosylcytosine (DPP‐CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy. On the other hand, for anti‐neovascular therapy, we recently isolated peptides homing to angiogenic vessels from a phage‐displayed random peptide library, and observed that peptide‐modified liposomal adriamycin strongly suppressed tumor growth, perhaps through damaging angiogenic endothelial cells. In the present study, we modified DPP‐CNDAC‐liposomes with one of the angiogenic homing peptides, APRPG, and examined their antitumor activity. Three doses of APRPG‐modified DPP‐CNDAC‐liposomes (15 mg/kg as CNDAC) strongly inhibited tumor growth compared with the same number of doses of unmodified DPP‐CNDAC‐liposomes. The life span was increased 31.8%, with one completely cured mouse out of the six mice treated. Since the accumulation of liposomes in the tumor tissue was not so much different between APRPG‐liposomes and non‐modified liposomes, the enhanced therapeutic efficacy may be explained as the alteration of targets, i.e. APRPG‐modified DPP‐CNDAC‐liposomes caused tumor growth suppression through damage of angiogenic endothelial cells. Anti‐neovascular therapy promises no drug resistance, and should be effective against essentially any kind of solid tumor; and thus the present results demonstrate another benefit of the therapy, namely, high efficacy of cancer treatment.

Amyloid precursor protein cytoplasmic domain with phospho-Thr668 accumulates in Alzheimer’s disease and its transgenic models: a role to mediate interaction of Aβ and tau

Abnormal accumulation of Aβ and tau in senile plaques (SP) and neurofibrillary tangles (NFTs) is a key event in Alzheimer’s disease (AD). Here, we show that T668-phosphorylated cytoplasmic domain of APP (pT668-ACD) accumulates Aβ and tau in AD and its transgenic models. Anti-pT668 immunostaining of AD brain sections with hydrated autoclave enhancement identified SP neurites and NFTs in which pT668-ACD colocalizes with tau. We produced and examined transgenic (Tg) mice that overexpress human APP695, harboring the double Swedish/London mutation, and develop age-dependently Aβ plaques in the brain. All Aβ plaques contain co-accumulations of pT668-ACD, but co-accumulation of tau appears in only a fraction of Aβ plaques in older animals. We also examined the established tau Tg mice that overexpress the smallest human brain tau isoform and develop neuronal accumulations of tau in older animals. Examination of the old tau Tg mice showed that neuronal cells affected by tau accumulation induce co-accumulation of pT668-ACD. We speculate that in AD brains, extracellular Aβ deposition is accompanied by intracellular accumulation of pT668-ACD, followed by tau accumulation in the SP with dystrophic neurites and that neuronal cells affected by tau accumulation induce co-accumulation of pT668-ACD in NFTs. Thus, pT668-ACD is likely to mediate pathological interaction between Aβ and tau.

GD1α-replica peptides functionally mimic GD1α, an adhesion molecule of metastatic tumor cells, and suppress the tumor metastasis

A novel peptide technology to produce mimicking peptides of carbohydrate moiety (which we propose to name glyco‐replica peptides) is a useful tool to elucidate the functions of glycoconjugate. Carbohydrate moiety of ganglioside GD1α functions as a molecule involved in the adhesion between murine highly metastatic lymphoma RAW117‐H10 cells and hepatic sinusoidal endothelial (HSE) cells. To prepare peptides which mimic the carbohydrate structure of GD1α, phage clones expressing peptides which bound to a monoclonal antibody against GD1α (KA17) were isolated from a phage‐displayed random peptide library. Four phage clones having affinity to the monoclonal antibody KA17 were isolated, and these clones showed inhibitory effect on the binding of KA17 to GD1α. The amino acid sequences of the displayed pentadecamers were determined, and one of the phages displaying sequence WHWRHRIPLQLAAGR bound to HSE cells directly and showed the highest inhibitory effect on the adhesion between RAW117‐H10 cells and HSE cells. The synthesized peptides having the same sequences to the displayed 15mers in the four isolated phage clones also showed the inhibitory effect on the adhesion of RAW117‐H10 cells to HSE cells, and, again, the WHWRHRIPLQLAAGR peptide showed the highest inhibitory effect. Furthermore, intravenous injection of the peptide brought almost complete inhibition of the metastasis of RAW117‐H10 cells to lung and spleen, and about 50% inhibition of the liver metastasis. These results indicate that GD1α plays an important role for metastasis of RAW117‐H10 cells, and the peptides obtained by the present procedure are able to mimic the functional role of the glycoconjugate.

Gangliosides determine the amyloid pathology of Alzheimer's disease.

Gangliosides, GM3 and GM1, are suggested to accelerate the deposition of the amyloid &bgr;-protein as amyloid angiopathy and senile plaques, respectively, in the Alzheimer brain. We investigated the profile of amyloid deposition in the brains of transgenic mice expressing a mutant amyloid precursor protein with a disrupted GM2 synthase gene, in which GM3 accumulates whereas GM1 is lacking. These mice showed a significantly increased level of deposited amyloid &bgr;-protein in the vascular tissues. Furthermore, formation of severe dyshoric-form amyloid angiopathy, in which amyloid extended from the blood vessel walls deeply into the surrounding parenchyma was observed. Our results indicate that the expression of gangliosides is a critical determinant for the amyloid pathology in the Alzheimer brain.

Suppression of tumor growth by novel peptides homing to tumor-derived new blood vessels.

Novel peptides homing to angiogenic vessels were recently isolated from a phage‐displayed random pentadecapeptide library. One of the isolated peptides, ASSSYPLIHWRPWAR, significantly suppressed the migration of VEGF‐stimulated human umbilical vein endothelial cells. Dendoric ASSSYPLIHWRPWAR‐peptide suppressed the formation of new blood vessels in dorsal air sac model mice. Furthermore, ASSSYPLIHWRPWAR‐peptide and the fragment peptides containing WRP, which is revealed to be an epitope sequence, significantly suppressed the tumor growth, although 15‐mer shuffled peptide derived from ASSSYPLIHWRPWAR and pentapeptides with alanine substitution of each residue of WRP did not. Taken together, ASSSYPLIHWRPWAR‐peptide may cause tumor dormancy through inhibition of angiogenesis, and the WRP sequence may be the minimal and essential sequence for this activity.

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells.

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.

Suppression of GD1α ganglioside-mediated tumor metastasis by liposomalized WHW-peptide

GD1α ganglioside‐replica peptides were recently isolated from a phage‐displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117‐H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp‐His‐Trp (WHW) peptide was identified as a minimal sequence of the GD1α‐replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide‐attached liposomes displayed efficient inhibition of liver metastasis of RAW117‐H10 cells as well as of GD1α‐mediated adhesion of RAW117‐H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.