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Laminins are the major constituents of blood vessel basement membranes (BMs). Each laminin is a trimer consisting of three assembled polypeptide chains, α, β and γ. More than 15 laminin isoforms are known to date and the expression of specific isoforms may change in certain pathological conditions. Here we show that during progression of glial tumors laminin-9 (α4β2γ1) is switched to laminin-8 (α4β1γ1), which is dramatically increased in glial brain tumors. Laminin-8 overproduction by glial tumor cells facilitates spread of glioma. Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time. Laminin-8 may be thus used as a predictor of tumor recurrence, patient survival and as a potential molecular target for glioma therapy.
PMCID: PMC3506377  PMID: 16146715
Laminin-8; Laminin-9; Basement Membrane; Extracellular Matrix; Angiogenesis; Human; Cancer; Tumor; Neoplasm; Glioma; Glioblastoma Multiforme; Recurrence; Survival; Invasion; Morpholino antisense; Review
2.  Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis 
Angiogenesis  2006;9(4):183-191.
We have previously shown that laminin-8, a vascular basement membrane component, was over-expressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 α4 and β1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.
PMCID: PMC3487708  PMID: 17109197
Tumor angiogenesis; Glioma; Laminin-8; Multiple drug targeting; Poly(malic acid)
3.  Polycefin, a New Prototype of a Multifunctional Nanoconjugate Based on Poly(β-l-malic acid) for Drug Delivery 
Bioconjugate chemistry  2006;17(2):317-326.
A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic β-poly(l-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin α4 and β1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. l-valine containing, pH-sensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl ester-activated β-poly-(l-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 α4 and β1 chains at the same time. Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.
PMCID: PMC3487710  PMID: 16536461
4.  Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity 
Cancer research  2011;71(4):1454-1464.
Biodegradable nanopolymers are believed to offer great potential in cancer therapy. Here, we report the characterization of a novel, targeted, nanobiopolymeric conjugate based on biodegradable, nontoxic, and nonimmunogenic PMLA [poly(β-l-malic acid)]. The PMLA nanoplatform was synthesized for repetitive systemic treatments of HER2/neu-positive human breast tumors in a xenogeneic mouse model. Various moieties were covalently attached to PMLA, including a combination of morpholino antisense oligonucleotides (AON) directed against HER2/neu mRNA, to block new HER2/neu receptor synthesis; anti-HER2/neu antibody trastuzumab (Herceptin), to target breast cancer cells and inhibit receptor activity simultaneously; and transferrin receptor antibody, to target the tumor vasculature and mediate delivery of the nanobiopolymer through the host endothelial system. The results of the study showed that the lead drug tested significantly inhibited the growth of HER2/neu-positive breast cancer cells in vitro and in vivo by enhanced apoptosis and inhibition of HER2/neu receptor signaling with suppression of Akt phosphorylation. In vivo imaging analysis and confocal microscopy demonstrated selective accumulation of the nanodrug in tumor cells via an active delivery mechanism. Systemic treatment of human breast tumor-bearing nude mice resulted in more than 90% inhibition of tumor growth and tumor regression, as compared with partial (50%) tumor growth inhibition in mice treated with trastuzumab or AON, either free or attached to PMLA. Our findings offer a preclinical proof of concept for use of the PMLA nanoplatform for combination cancer therapy.
PMCID: PMC3428373  PMID: 21303974
5.  134 Expression and Localization of CYSLT2 Receptor in Human Nasal Mucosa 
The World Allergy Organization Journal  2012;5(Suppl 2):S61-S62.
We have previously reported the localization of CysLT1 receptor by using immunohistochemistry and in situ hybridization (Shirasaki H et al. Clin Exp Allergy. 2002;32:1007–1012).
To clarify the expression of CysLT2 receptor in human nasal mucosa, we investigated CysLT2 receptor mRNA expression and its protein localization in human nasal mucosa, by polymerase chain reaction (PCR) and immunohistochemistry.Human turbinates were obtained after turbinectomy from 6 patients with nasal obstruction refractory to medical therapy. Total RNA was isolated from human nasal mucosa, and CysLT2 receptor mRNA was detected in these tissues by using reverse transcriptase-PCR analysis. To identify the cells expressing CysLT2 receptor protein, double immunostaining was performed using anti-CysLT2 receptor antibody and anti-CD31 (endothelial cell) antibody.
Reverse transcriptase-PCR analysis of total nasal RNA demonstrated the expression of CysLT2 receptor mRNA. The immunohistochemical studies revealed that anti-CysLT2 receptor antibody mainly labeled blood vessels.
The results suggest a primary role for CysLT2 receptor as the vascular responses in upper respiratory tract.
PMCID: PMC3512797
6.  Use of a Population-Based Cancer Registry to Calculate Twenty-Year Trends in Cancer Incidence and Mortality in Fukui Prefecture 
Journal of Epidemiology  2010;20(3):244-252.
There have been only a limited number of trend analyses of incidence and mortality using population-based cancer registry data in Japan, and the national statistics regarding incidence are estimated data. In the present study, data from the Fukui Prefecture cancer registry, which is the most accurate in Japan, were used to observe trends in incidence and mortality rates.
Cancer incidence and mortality rates from 1984 through 2004 were obtained from the Fukui Prefecture cancer registry. Joinpoint analysis developed for the US National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) Program was used to compute and graphically present annual percentage changes in age-adjusted incidence and mortality in Fukui Prefecture.
On joinpoint analysis, there were slight increases in incidence at all cancer sites combined for both sexes from 1986, but the trend was not significant in Fukui. Mortality in women appeared to significantly decrease, while mortality in men, which had been increasing until 1999, began to significantly decrease thereafter. In an analysis by anatomical site, both the incidence and mortality of stomach cancer significantly decreased in both sexes. However, the incidence and mortality of breast and prostate cancers significantly increased. The mortality of liver and lung cancers also increased in both sexes.
Cancer mortality has been declining in recent years, and the reduction in mortality from stomach cancer has significantly affected the trends in Fukui. Urgent cancer control planning by the Fukui local government is necessary, especially for cancers of the liver, lung, prostate, and breast.
PMCID: PMC3900848  PMID: 20431235
incidence; mortality; population-based cancer registry
7.  Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery 
Nanomedicine (London, England)  2008;3(2):247-265.
Nanoconjugates are emerging as promising drug-delivery vehicles because of their multimodular structure enabling them to actively target discrete cells, pass through biological barriers and simultaneously carry multiple drugs of various chemical nature. Nanoconjugates have matured from simple devices to multifunctional, biodegradable, nontoxic and nonimmunogenic constructs, capable of delivering synergistically functioning drugs in vivo. This review mainly concerns the Polycefin family of natural-derived polymeric drug-delivery devices as an example. This type of vehicle is built by hierarchic conjugation of functional groups onto the backbone of poly(malic acid), an aliphatic polyester obtained from the microorganism Physarum polycephalum. Particular Polycefin variants target human brain and breast tumors implanted into animals specifically and actively and could be detected easily by noninvasive imaging analysis. Delivery of antisense oligonucleotides to a tumor-specific angiogenic marker using Polycefin resulted in significant inhibition of tumor angiogenesis and increase of animal survival.
PMCID: PMC2734385  PMID: 18373429
biodegradable; brain cancer; breast cancer; imaging analysis; multiple antibodies; multiple drug delivery; multitargeting; Polycefin; poly(malic acid); tumor angiogenesis
8.  Nanoconjugate based on polymalic acid for tumor targeting 
Chemico-biological interactions  2007;171(2):195-203.
A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(β-L-malic acid) (Mw 50,000, Mw/Mn 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection.
Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5 mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0–180 min up to 24 h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p < 0.003). In brain tumors drug accumulated selectively in 24 h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal. © 2007 Elsevier Ireland Ltd. All rights reserved.
PMCID: PMC2329596  PMID: 17376417
Brain glioma; Breast cancer; EPR effect; Fluorescence imaging; Drug delivery system; Poly(malic acid)
9.  Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly(β-L-malic acid) 
Tumor-specific targeting using achievements of nanotechnology is a mainstay of increasing efficacy of anti-tumor drugs. To improve drug targeting we covalently conjugated for the first time two different monoclonal antibodies, an anti-mouse transferrin receptor antibody and a mouse autoimmune anti-nucleosome antibody 2C5, onto the drug delivery nanoplatform, poly(β-L-malic acid). The active anti-tumor drug components attached to the same carrier molecule were antisense oligonucleotides to vascular protein laminin-8. The resulting drug, a new Polycefin variant, was administered intravenously into glioma-bearing xenogeneic animals. The drug delivery system was targeted across mouse endothelial system by the anti-mouse transferring receptor antibody and to the tumor cell surface by the anti-nucleosome antibody 2C5. The targeting efficacies of the Polycefin variants bearing either two antibodies or each single antibody were compared in vitro and in vivo. ELISA confirmed the co-existence of two antibodies on the same nanoplatform molecule and their functional activities. Fluorescence imaging analysis after 24 h of intravenous injection demonstrated significantly higher tumor accumulation of Polycefin variants with the tandem configuration of antibodies than with single antibodies. The results suggest improved efficacy for tandem configuration of antibodies than for single configurations carried by a drug delivery vehicle.
PMCID: PMC2394675  PMID: 17630012
Enhanced tumor targeting; Antibody tandem configuration; Nanobiopolymer; Brain tumor; Poly(β-L-malic acid)
10.  Overexpression of β1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases 
Breast Cancer Research  2005;7(4):R411-R421.
Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the α4 chain from predominantly laminin-9 (α4β2γ1) to predominantly laminin-8 (α4β1γ1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.
In the present study the expressions of laminin isoforms α1–α5, β1–β3, γ1, and γ2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9.
Laminin α4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin β2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin β1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (α5β2γ1) to laminin-10 (α5β1γ1) during breast tumor progression. Additionally, laminin-2 (α2β1γ1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (α3β3γ2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression.
These results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch from laminin-9 and laminin-11 to laminin-8 and laminin-10 first occurs in carcinomas in situ and becomes more pronounced with progression of carcinomas to the invasive stage. Similar to high-grade brain gliomas, the expression of laminin-8 (and laminin-10) in breast cancer tissue may be a predictive factor for tumor neovascularization and invasion.
PMCID: PMC1175051  PMID: 15987446
11.  Characterization of Staphylococcus aureus Coagulase Type VII Isolates from Staphylococcal Food Poisoning Outbreaks (1980–1995) in Tokyo, Japan, by Pulsed-Field Gel Electrophoresis 
Journal of Clinical Microbiology  2000;38(10):3746-3749.
Staphylococcus aureus coagulase type VII strains have been the strains most frequently isolated from staphylococcal food poisoning outbreaks in Tokyo, Japan. We applied pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with SmaI to characterize 129 coagulase type VII strains. These were isolated from 129 cases occurring in outbreaks in 35 districts during a 16-year period (1980–1995). The 129 outbreak strains were classified into three types, designated A (n = 115), B (n = 10), and C (n = 4). Types A and C were further divided into 33 (A1 to A33) and 4 (C1 to C4) subtypes, respectively. Strains of the same subtypes were isolated from food poisoning cases in the same districts at time intervals of 1 or 2 to 5 years. PFGE typing appears to be a useful method for subdividing strains of S. aureus coagulase type VII. A combination of coagulase typing and PFGE typing would provide more detailed information than the former method alone in epidemiologic investigations of staphylococcal food poisoning.
PMCID: PMC87468  PMID: 11015395

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