HER-2-Targeted Boron Neutron Capture Therapy with Carborane-integrated Immunoliposomes Prepared via an Exchanging Reaction.

Boron neutron capture therapy (BNCT) is a promising modality for cancer treatment because of its minimal invasiveness. To maximize the therapeutic benefits of BNCT, the development of efficient platforms for the delivery of boron agents is indispensable. Here, carborane-integrated immunoliposomes were prepared via an exchanging reaction to achieve HER-2-targeted BNCT. The conjugation of an anti-HER-2 antibody to carborane-integrated liposomes successfully endowed these liposomes with targeting properties toward HER-2-overexpressing human ovarian cancer cells (SK-OV3); the resulting BNCT activity toward SK-OV3 cells obtained using the current immunoliposomal system was 14-fold that of the l-BPA/fructose complex, which is a clinically available boron agent. Moreover, the growth of spheroids treated with this system followed by thermal neutron irradiation was significantly suppressed compared with treatment with the l-BPA/fructose complex.

HER-2-targeted boron neutron capture therapy using an antibody-conjugated boron nitride nanotube/ß-1,3-glucan complex.

The development of boron agents with integrated functionality, including biocompatibility, high boron content, and cancer cell targeting, is desired to exploit the therapeutic efficacy of boron neutron capture therapy (BNCT). Here, we report the therapeutic efficacy of BNCT using a HER-2-targeted antibody-conjugated boron nitride nanotube/ß-1,3-glucan complex. The anticancer effect of BNCT using our system was 30-fold that of the clinically available boron agent l-BPA/fructose complex.

Phospholipid-Coated Boronic Oxide Nanoparticles as Boron Agents for Boron Neutron Capture Therapy.

Minimally invasive boron neutron capture therapy (BNCT) is an elegant approach for cancer treatment. The highly selective and efficient deliverability of boron agents to cancer cells is the key to maximizing the therapeutic benefits of BNCT. In addition, enhancement of the frequencies to achieve boron neutron capture reaction is also significant in improving therapeutic efficacy by providing a highly concentrated boron agent in each boron nanoparticle. As the density of the thermal neutron beam remains low, it is unable to induce high-efficiency cell destruction. Herein, we report phospholipid-coated boronic oxide nanoparticles as agents for BNCT that can provide a highly concentrated boron atom in each nanoparticle. The current system exhibited in vitro BNCT activity seven times higher than that of commercial boron agents. Furthermore, the system could penetrate cancer spheroids deeply, efficiently suppressing thermal neutron irradiation-induced growth.

Carborane bearing pullulan nanogel-boron oxide nanoparticle hybrid for boron neutron capture therapy.

Boron neutron capture therapy shows is a promising approach to cancer therapy, but the delivery of effective boron agents is challenging. To address the requirements for efficient boron delivery, we used a hybrid nanoparticle comprising a carborane = bearing pullulan nanogel and hydrophobized boron oxide nanoparticle (HBNGs) enabling the preparation of highly concentrated boron agents for efficient delivery. The HBNGs showed better anti-cancer effects on Colon26 cells than a clinically boron agent, L-BPA/fructose complex, by enhancing the accumulation and retention amount of the boron agent within cells in vitro. The accumulation of HBNGs in tumors, due to the enhanced permeation and retention effect, enabled the delivery of boron agents with high tumor selectivity, meeting clinical demands. Intravenous injection of boron neutron capture therapy (BNCT) using HBNGs decreased tumor volume without significant body weight loss, and no regrowth of tumor was observed three months after complete regression. The therapeutic efficacy of HBNGs was better than that of L-BPA/fructose complex. BNCT with HBNGs is a promising approach to cancer therapeutics.

Tumor-targeting hyaluronic acid/fluorescent carborane complex for boron neutron capture therapy.

In cancer therapeutics, boron neutron capture therapy (BNCT) requires a platform for selective and efficient 10B delivery into tumor tissues for a successful treatment. However, the use of carborane, a promising candidate with high boron content and biostability, has significant limitations in the biomedical field due to its poor water-solubility and tumor-selectivity. To overcome these hurdles, we present in this study a fluorescent nano complex, combining fluorescent carborane and sodium hyaluronate for high boron concentration and tumor-selectivity. Tumor cells actively internalized the complex through binding hyaluronan to CD44, overexpressed on the tumor cell surface. Furthermore, the subcellular distribution of this complex could also be detected due to its fluorescent properties. Moreover, after thermal neutron irradiations, the complex produced excellent cytotoxicity, equal to or greater than that of the clinically-used BPA-fructose. Therefore, this novel complex could be potentially more suitable for BNCT than the boron agent.

S-allyl-glutathione improves experimental liver fibrosis by regulating Kupffer cell activation in rats.

S-allyl-glutathione (SAG) is one of the metabolites of diallyl sulfide (DAS), a component of garlic. DAS has shown preventative effects on carcinogenesis in animal models. However, whether synthetic SAG can improve liver fibrosis has not been investigated. We examined the potential preventive effects of SAG on acute and chronic models of liver fibrosis by chronic carbon tetrachloride (CCl4) administration. SAG inhibited liver fibrogenesis induced by CCl4 in a dose-dependent manner and reduced heat shock protein-47 (HSP47), a collagen-specific chaperone, and other fibrosis markers. In fibrosis regression models, after administration of either CCl4 for 9 wk or dimethyl nitrosamine (DMN) for 6 wk, SAG markedly accelerated fibrolysis in both models. In the regression stage of DMN-treated liver, SAG normalized the ratio of M2 phenotype (expression of mannose receptor) in Kupffer cells (KCs). Consistent with these results, the culture supernatants of SAG-treated M2-phenotype KCs inhibited collagen-α1(I) chain (COL1A1) mRNA expression in primary culture-activated rat hepatic stellate cells (HSCs). However, SAG did not directly inhibit HSC activation. In an acute model of CCl4 single injection, SAG inhibited hepatic injury dose dependently consistent with the inhibited the elevation of the bilirubin and ALT levels. These findings suggest that SAG could improve the fibrogenic and fibrolysis cascade via the regulation of excess activated and polarized KCs. SAG may also serve as a preventive and therapeutic agent in fibrosis of other organs for which current clinical therapy is unavailable. NEW & NOTEWORTHY S-allyl-glutathione (SAG) is a metabolite of diallyl sulfide, a component of garlic. SAG increased hepatic glutathione levels and GSH-to-GSSG ratio in normal rats. SAG treatment before or after liver fibrosis from chronic CCl4 administration improved liver fibrosis and regression. SAG decreased heat shock protein-47 (HSP47), a collagen-specific chaperone, and other fibrosis markers in CCl4-treated livers. SAG-treated Kupffer cell conditioned medium also inhibited collagen-α1(I) chain (COL1A1) mRNA expression and other markers in primary culture hepatic stellate cells.

Therapeutic administration of an ingredient of aged-garlic extracts, S-allyl cysteine resolves liver fibrosis established by carbon tetrachloride in rats.

S-allyl cysteine (SAC) is the most abundant compound in aged garlic extracts (AGEs). AGE has been reported to ameliorate the oxidative damage implicated in a variety of diseases. However, the effects of SAC have not been established in liver cirrhosis. The aim of this study was to examine the effect of therapeutic administration of SAC in liver cirrhosis by chronic carbon tetrachloride (CCl4) administration in rats. SAC or other cysteine compounds were administered from 4 weeks when liver fibrosis was confirmed to be in process. CCl4 administration elevated plasma alanine aminotransferase, plasma lipid peroxidation, liver hydroxyproline, and liver transforming growth factor (TGF)-ß at 12 weeks. SAC prevented these changes induced by CCl4. Furthermore, SAC improved survival in a dose-dependent manner following consecutive CCl4 administration. The inhibitory mechanisms may be associated with a decrease in the profibrogenic cytokine, TGF-ß as well as the antioxidative properties of SAC.

Anti-inflammatory effect of water-soluble complex of 1'-acetoxychavicol acetate with highly branched ß-1,3-glucan on contact dermatitis.

The anti-inflammatory effect on contact dermatitis of the water solubilized 1'-Acetoxychavicol Acetate (ACA) by complexation with ß-1,3-glucan isolated form Aureobasidium pullulans black yeast is reported. It is well-known that ACA possesses a function to inhibit the activation of NF-κB by which genes encoding proinflammatory cytokines, chemokines, and growth factors are regulated. However, because ACA is quite insoluble in water, its usefulness has been extremely limited. On the other hand, a triple-helical polysaccharide ß-1,3-glucan can include hydrophobic compounds into intrastrand hydrophobic cavity and solubilize poorly water-soluble compounds. In this study, solubilization of ACA by complexation with highly branched ß-1,3-glucan was achieved. The effect of anti-inflammatory response of water-soluble ACA complex with ß-1,3-glucan was confirmed in vitro and in vivo.

The synergistic effect of 1'-acetoxychavicol acetate and sodium butyrate on the death of human hepatocellular carcinoma cells.

It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. In this study, we evaluated the combination of 1'-acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate, a major short chain fatty acid, on the growth of HepG2 human hepatocellular carcinoma cells and found that treatment had a synergistic inhibitory effect. The number of HepG2 cells was synergistically decreased via apoptosis induction when cells were treated with both ACA and sodium butyrate. In ACA- and sodium butyrate-treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and sodium butyrate was diminished when cells were pretreated with catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP-activated protein kinase (AMPK), a cellular energy sensor, plays an essential role in controlling processes related to tumor development. In ACA- and sodium butyrate-treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation. In normal hepatocyte cells, treatment with ACA and sodium butyrate did not decrease cell numbers or increase ROS levels. In conclusion, combined treatment with ACA and sodium butyrate synergistically induced apoptotic cell death via an increase in intracellular ROS and phosphorylation of AMPK. Our findings may provide new insight into the development of novel combination therapies against hepatocellular carcinoma.

S-Allyl cysteine improves nonalcoholic fatty liver disease in type 2 diabetes Otsuka Long-Evans Tokushima Fatty rats via regulation of hepatic lipogenesis and glucose metabolism.

It is important to prevent and improve diabetes mellitus and its complications in a safe and low-cost manner. S-Allyl cysteine, an aged garlic extract with antioxidant activity, was investigated to determine whether S-allyl cysteine can improve type 2 diabetes in Otsuka Long-Evans Tokushima Fatty rats with nonalcoholic fatty liver disease. Male Otsuka Long-Evans Tokushima Fatty rats and age-matched Long-Evans Tokushima Otsuka rats were used and were divided into two groups at 29 weeks of age. S-Allyl cysteine (0.45% diet) was administered to rats for 13 weeks. Rats were killed at 43 weeks of age, and detailed analyses were performed. S-Allyl cysteine improved hemoglobinA1c, blood glucose, triglyceride, and low-density lipoprotein cholesterol levels. Furthermore, S-allyl cysteine normalized plasma insulin levels. S-Allyl cysteine activated the mRNA and protein expression of both peroxisome proliferator-activated receptor α and γ, as well as inhibiting pyruvate dehydrogenase kinase 4 in Otsuka Long-Evans Tokushima Fatty rat liver. Sterol regulatory element-binding protein 1c and forkhead box O1 proteins were normalized by S-allyl cysteine in Otsuka Long-Evans Tokushima Fatty rat liver. In conclusions, these findings support the hypothesis that S-allyl cysteine has diabetic and nonalcoholic fatty liver disease therapeutic potential as a potent regulating agent against lipogenesis and glucose metabolism.

HVJ-E/importin-ß hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery.

In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-ß conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importin-ß. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-ß conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/importin-ß-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection.

Protein recognition of hetero-/homoleptic ruthenium(II) tris(bipyridine)s for α-chymotrypsin and cytochrome c.

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.

1'-Acetoxychavicol acetate enhances the phase II enzyme activities via the increase in intranuclear Nrf2 level and cytosolic p21 level.

(1'S)-acetoxychavicol acetate ((S)-ACA) exhibits chemopreventive effects on chemically induced tumor formation. It has been shown that ACA inhibited the development of azoxymethane-induced colon carcinogenesis through its suppression of cell proliferation in the colonic mucosa and its induction of glutathione S-transferase and quinone oxidoreductase 1 in vivo. In this study, we investigated how ACA induced these enzymes by using rat intestine epithelial cells (IEC6) in vitro. ACA induced glutathione S-transferase (GST) and NAD (P)H: quinone oxidoreductase 1 (NQO1) activities, increased intracellular glutathione (GSH) level, and upregulated intranuclear Nrf2 and cytosolic p21. It suggested that activation of phase II enzymes via Nrf2 associated with p21 is one of possible mechanisms of ACA to prevent advance of carcinogenesis.

Biological activity of water-soluble inclusion complexes of 1'-acetoxychavicol acetate with cyclodextrins.

1'-Acetoxychavicol acetate (ACA), isolated from the rhizomes and the seeds of the Zingiberaceae plant, has a variety of biological activities such as antitumor, antiallergic and repellent effects. However, ACA seems to have some disadvantages which may limit for future possible clinical applications, for example, its poor water solubility. Furthermore, ACA is not stable in aqueous solutions and undergoes hydrolysis and/or isomerization. To improve the solubility and stability of ACA in water, we prepared the inclusion complexes with various ß-cyclodextrins (ß-CDs).In aqueous solution, the association constants of ACA with various CDs were estimated at 662±95 (ß-CD), 336±70 (methyl-ß-CD, Meß-CD), and 322±44M(-1) (hydroxypropyl-ß-CD, HPß-CD), respectively, by a spectrofluorometric displacement method based on competition between a guest and a fluorescent probe for CDs. It was revealed that almost all ACAs existed as a free molecule in the CD-containing aqueous solution. However, in the case of preparing the inclusion complexes of CDs with ACA by a solid phase 'high-speed vibration milling' technique, the average inclusion rates of the obtained water-soluble complexes were calculated as 88±13% (ß-CD), 70±1% (Meß-CD), and 63±2% (HPß-CD), respectively, by (1)H NMR analysis. To characterize the structures of the CD·ACA complexes, 2,3,6-trimethyl-ß-CD (TMeß-CD)·ACA complex was prepared as a model compound (inclusion rate: 40%). As a result of 2D ROESY experiments, it was considered that the aromatic ring of ACA is located in the narrow side of the hydrophobic cavity of the TMeß-CD and both 1'- and 4-acetoxy groups of ACA positioned in the vicinity of the secondary and primary methoxy groups of TMeß-CD, respectively. Furthermore, we examined the apoptogenic activity of CD·ACA complexes to evaluate whether or not the bioactivities of ACA were affected by their inclusion. Although the cytotoxicity of all CD·ACA complexes in human epithelial carcinoma HeLa cells and murine adenocarcinoma colon26 cells were diminished as compared with the ACA alone, only HPß-CD·ACA maintained high levels of activity. In addition, HPß-CD·ACA, and Meß-CD·ACA showed suppressive effect for the transcription factor NF-κB activation on LPS-activated murine macrophage RAW264.7 cells and the former was more active complex. Furthermore, HPß-CD·ACA inhibited the in vivo tumor growth of tumor-bearing mice, although the activity was slightly weak compared with that of free ACA. These results indicate that HPß-CD is the best host molecule for ACA to form a water-soluble complex with the similar biological activity of free ACA.

Tumor accumulation of ε-poly-lysines-based polyamines conjugated with boron clusters.

Boron Neutron Capture Therapy (BNCT) is one of the potent cancer radiotherapies using nuclear reaction between (10)B atoms and the neutron. Whether BNCT will succeed or not depends on tumor selective delivery of (10)B compounds. ε-Poly-L-lysine is a naturally occurring polyamine characterized by the peptide linkages between the carboxyl and ε-amino groups of L-lysine. Because of high safety ε-PLL is applied practically as a food additive due to its strong antimicrobial activity. In this study, we focus on a development of a novel polymeric delivery system for BNCT using biodegradable ε-PLL conjugated with (10)B-containing clusters (BSH). This polymeric boron carrier will be expected to deliver safely and efficiently into tumor tissues based on Enhanced Permeability and Retention (EPR) effect.

Enhanced internalization and endosomal escape of dual-functionalized poly(ethyleneimine)s polyplex with diphtheria toxin T and R domains.

A new multifunctional gene delivery system was constructed with diphtheria toxin's functional domains. Used functional domains are T domain for endosomal escape and R domain for efficient internalization into cell. In order to conjugate these domains into PEI polyplex, diphtheria toxin T and R domains-streptavidin fusion protein (DTRS) was prepared. The conjugation of the DTRS with biotinylated PEI polyplex (DTRS-polyplex) lead to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex in CHO-K1 cell. It was demonstrated that DTRS-polyplex had high endosomal escape efficiency and internalization efficiency by several measurements, such as in vitro intracellular trafficking observation and the internalization inhibition with several inhibitors. These results suggest that this multifunctional non-viral vector may contribute to the future cancer gene therapy.

Comparison of glutathione reductase activity and the intracellular glutathione reducing effects of 13 derivatives of 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells.

In a previous study, we showed that (1'S)-acetoxychavicol acetate ((S)-ACA) caused a rapid decrease in glutathione (GSH) levels less than 15 min after exposure. (S)-ACA-induced cell death was reversed by the addition of N-acetylcysteine. In the current study, we investigated the inhibitory activities of 13 derivatives of (S)-ACA on tumor cell viability, intracellular GSH level and GR activity. Correlations were found among a decrease in cell viability, intracellular GSH levels and the activity of GR in Ehrlich ascites tumor cells treated with the various ACA analogues. A test of the 13 derivatives revealed that the structural factors regulating activity were as follows: (1) the para or 1'-position of acetoxyl group (or other acyl group) was essential, (2) the presence of a C2'-C3' double or triple bond was essential, and (3) the S configuration of the 1'-acetoxyl group was preferable.

Cytochrome c-binding "proteo-dendrimers" as new types of apoptosis inhibitors working in HeLa cell systems.

The suppressive effects of synthetic dendrimers on mitochondrial apoptosis were first demonstrated in human epithelial carcinoma HeLa cells. The employed proteo-dendrimers included polyanionic hepta(glutamic acids), a fluorescent zinc porphyrinate core, hydrophilic polyether surface and nonpeptide hydrophobic dendrons, and electrostatically interacted with cytochrome c in aqueous solutions. The ceramide analogue, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (C2-ketoCer) induced mitochondrial apoptosis into HeLa cells and the cell viability was significantly recovered by pretreatment with some dendrimers. Among a series of proteo-dendrimers, the second-generation dendrimer 2a had the lowest cytotoxicity and the highest solubility. When the cells were treated with this dendrimer, a decrease in the protein levels of active caspase-3 and proteolytically cleaved PARP was remarkably observed. Since cytochrome c release from the mitochondria to the cytoplasm was unaffected in the presence of dendrimer 2a, the observed suppressive effects probably indicate that the proteo-dendrimer trapped cytochrome c, not only in the aqueous solutions but also in the living cells..

The conjugation of diphtheria toxin T domain to poly(ethylenimine) based vectors for enhanced endosomal escape during gene transfection.

The endosomal escape is a well-known serious obstacle for non-viral gene delivery. This is because of an acidic and enzymatic degradation of the contents of the endosome/lysosome. Therefore, the internalized gene needs to be efficient released into the cytosol to obtain the efficiently transfection efficiency. On the other hand, the diphtheria toxin T domain fuses with endosome membrane by pH decrease, then enhances the endosomal escape of the diphtheria toxin C fragment. In this study, we constructed diphtheria toxin T domain-conjugated poly(ethylenimine)s (PEI) polyplex for enhancing the endosomal escape of exogenous gene. The conjugation of diphtheria toxin T domain with PEI/pDNA polyplex leads to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex. The pH-responsive increase in hydrophobicity of the diphtheria toxin T domain might not only trigger the perturbation of the endocytic vesicle membrane but might also increase the membrane permeability.

Inhibitory effect of enamel matrix derivative on osteoblastic differentiation of rat calvaria cells in culture.

BACKGROUND AND OBJECTIVE: The effect of enamel matrix derivative (EMD) on bone differentiation remains unclear. Transforming growth factor beta1 (TGF-beta1) is reported to be contained in EMD. The aim of this study was to clarify the effect of EMD on osteoblastic cell differentiation and the possible role of TGF-beta1. MATERIAL AND METHODS: Fetal rat carvarial cells were treated with 10, 50 or 100 microg/ml EMD for 5-17 days. Alkaline phosphatase (ALP) activity and bone nodule formation were measured, and mRNA expressions of bone matrix proteins and core binding factor were analysed. RESULTS: Enamel matrix derivative inhibited ALP activity from the early stage of culture (29-44% inhibition) on days 5 and 10 and decreased bone nodule formation by 37-67% on day 17. These effects of EMD were concentration dependent. Enamel matrix derivative inhibited mRNA expression of osteocalcin and core binding factor. A high level of the active form of TGF-beta1 protein was detected in the conditioned medium treated with 100 microg/ml EMD. Treatment with TGF-beta1 antibody partly restored the inhibitory effect of EMD on ALP activity. CONCLUSION: Enamel matrix derivative inhibited the osteoblastic differentiation of rat carvarial cells and this was partly mediated by an increase in the activated form of TGF-beta1, suggesting that EMD may function initially to inhibit osteoblastic differentiation to allow a predominant formation of other periodontal tissues.