Identification of a Novel Deactivating Small-Molecule Compound for Fibrogenic Hepatic Stellate Cells.

BACKGROUND: Liver fibrosis progresses to decompensated liver cirrhosis, for which medical needs remain unmet. We recently developed IC-2, a small-molecule compound that suppresses Wnt/ß-catenin signaling, and found that IC-2 also suppresses liver fibrosis. In this study, we performed three-step screening of newly synthesized IC-2 derivatives to identify other small-molecule compounds that suppress liver fibrosis. METHODS: The screening system consisted of three steps: a cell viability assay, a transcription factor 4 (TCF4) reporter assay, and induction of α-smooth muscle actin (α-SMA) and collagen 1α1 (Col1A1) expression in response to each compound. Screening using human LX-2 hepatic stellate cells (HSCs) was performed to target HSCs, which are the driver cells of liver fibrosis. RESULTS: In the first step, since 9b and 9b-CONH2 at 100 µM did not have any effects on cell viability, they were omitted in the next screening. Additionally, the conditions that led to > 40% inhibition of the controls were also excluded in subsequent screening. The second step was performed under 31 conditions for 19 small-molecule compounds. Sixteen small-molecule compounds caused significant reduction of TCF4 activity relative to that of 0.1% DMSO. Of the 16 compounds, the 10 showing the greatest suppression of TCF4 activity were selected for the third step. Expressions of mRNA for α-SMA and Col1A1 were significantly reduced by seven and three small-molecule compounds, respectively. The greatest reductions in the α-SMA and Col1A1 mRNA expressions were observed in the cells treated with IC-2-F. Protein expressions of α-SMA and Col1A1 caused by IC-2-F were also comparable to those caused by IC-2. CONCLUSION: IC-2-F was identified as a novel deactivating small-molecule compound for HSCs in vitro. These data suggest that IC-2-F is a promising medicine for liver fibrosis.

WNT/ß-catenin signal inhibitor IC-2-derived small-molecule compounds suppress TGF-ß1-induced fibrogenic response of renal epithelial cells by inhibiting SMAD2/3 signalling.

Renal fibrosis compromises kidney function, and it is a risk factor for chronic kidney disease (CKD). CKD ultimately progresses to end-stage kidney disease that can be cured only by kidney transplantation. Owing to the increasing number of CKD patients, effective treatment strategies are urgently required for renal fibrosis. TGF-ß is a well-established fibrogenic factor that signals through SMAD2/3 signaling pathway. It was shown that there is a cross-talk between TGF-ß/SMAD and WNT/ß-catenin signaling pathways in renal tubular epithelial cells, and that a WNT/ß-catenin inhibitor, ICG-001, ameliorates TGF-ß1induced renal fibrosis. IC-2, a derivative of ICG-001, has been shown to potently induce hepatocyte differentiation of human mesenchymal stem cells by inhibiting WNT/ß-catenin signaling. In the present study, we examined the effect of ICG-001, IC-2, and IC-2 derivatives (IC-2-506-1, IC-2-506-2, IC-2-506-3, IC-2-Ar-Cl, IC-2-OH, IC-2-OTBS, and IC-2-F) on TGF-ß1-induced SMAD activation and fibrogenic response in immortalized human renal tubular epithelial HK-2 cells. All these compounds inhibited LiCl-induced WNT/ß-catenin reporter activation to a similar extent, whereas ICG-001, IC-2-OTBS, and IC-2-F almost completely suppressed TGF-ß1-induced SMAD reporter activation without apparent cytotoxicity. Phosphorylation of SMAD2/3 by TGF-ß1 was more potently inhibited by IC-2-OTBS and IC-2-F than by ICG-001 and IC-2. IC-2-F suppressed TGF-ß1-induced COL1A1 protein expression, whereas IC-2-506-1 and IC-2-OTBS suppressed TGF-ß1-induced epithelial-mesenchymal transition. These results demonstrated that IC-2 derivatives suppress the TGF-ß1-induced fibrogenic response of tubular epithelial cells and thus could be promising therapeutic agents for the treatment of renal fibrosis.

Guanidinylation of Chitooligosaccharides Involving Internal Cyclization of the Guanidino Group on the Reducing End and Effect of Guanidinylation on Protein Binding Ability.

In order to synthesize a promising material for developing a novel peptide/protein delivery system, guanidinylation of chitooligosaccharides with 1-amidinopyrazole hydrochloride was investigated herein. The production of guanidinylated chitooligosaccharides was demonstrated by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and elemental analyses. Interestingly, we found that the reducing end in the guanidinylated chitooligosaccharides was converted to a cyclic guanidine structure (2-[(aminoiminomethyl)amino]-2-deoxy-d-glucose structure). This reaction was carefully proven by the guanidinylation of d-glucosamine. Although this is not the first report on the synthesis of the 2-[(aminoiminomethyl)amino]-2-deoxy-d-glucose, it has provided a rational synthetic route using the high reactivity of the reducing end. Furthermore, we found that the interaction between chitooligosaccharides and bovine serum albumin is weak when in a neutral pH environment; however, it is significantly improved by guanidinylation. The guanidinylated chitooligosaccharides are useful not only for the development of a novel drug delivery system but also as a chitinase/chitosanase inhibitor and an antibacterial agent.

Reversal of established liver fibrosis by IC-2-engineered mesenchymal stem cell sheets.

Chronic hepatitis viral infection, alcoholic intoxication, and obesity cause liver fibrosis, which progresses to decompensated liver cirrhosis, a disease for which medical demands cannot be met. Since there are currently no approved anti-fibrotic therapies for established liver fibrosis, the development of novel modalities is required to improve patient prognosis. In this study, we clarified the anti-fibrotic effects of cell sheets produced from human bone marrow-derived mesenchymal stem cells (MSCs) incubated on a temperature-sensitive culture dish with the chemical compound IC-2. Orthotopic transplantation of IC-2-engineered MSC sheets (IC-2 sheets) remarkably reduced liver fibrosis induced by chronic CCl4 administration. Further, the marked production of fibrolytic enzymes such as matrix metalloproteinase (MMP)-1 and MMP-14, as well as thioredoxin, which suppresses hepatic stellate cell activation, was observed in IC-2 sheets. Moreover, the anti-fibrotic effect of IC-2 sheets was much better than that of MSC sheets. Finally, knockdown experiments revealed that MMP-14 was primarily responsible for the reduction of liver fibrosis. Here, we show that IC-2 sheets could be a promising therapeutic option for established liver fibrosis.

Hair growth-promoting activities of chitosan and surface-deacetylated chitin nanofibers.

In this study, the effects of chitosan and surface-deacetylated chitin nanofibrils (SDACNFs) on hair growth were evaluated. In human follicle dermal papilla cells in vitro, chitosan and SDACNFs were shown to increase cell growth on day 3 after the initiation of treatment, together with an increase in the production of fibroblast growth factor-7 (FGF-7) by these cells on day 3. Furthermore, in an in vivo study in mice, chitosan and SDACNF application promoted hair growth. The number of anagen follicles significantly increased compared with that in the control group, whereas the number of telogen follicles significantly decreased in the chitosan and SDACNF groups. In the chitosan and SDACNFs groups, moreover, the expression levels of FGF-7 and Sonic hedgehog were significantly upregulated in hair follicles. Overall, our results demonstrated that chitosan and SDACNFs promoted hair growth and therefore may have applications as novel therapeutic agents for the treatment of hair loss in patients.

Guanidinylated chitosan inspired by arginine-rich cell-penetrating peptides.

We introduce a simple method for producing guanidinylation of chitosan (CS) with 1­amidinopyrazole hydrochloride (AP). Production of GCS is proved via NMR, IR, and elemental analyses. In a reaction using 4.5 eq of AP for 7 days at room temperature, we obtained GCS with 42.3% of degree of guanidinylation (DG). The DG value can be controlled by the reaction time and AP amount. Furthermore, remarkable enhancement of the interaction between GCS and bovine serum albumin by guanidinylation was observed. This simple guanidinylation method for CS could provide novel additives for protein/peptide delivery systems.

Hepatic cell sheets engineered from human mesenchymal stem cells with a single small molecule compound IC-2 ameliorate acute liver injury in mice.

INTRODUCTION: We previously reported that transplantation of hepatic cell sheets from human bone marrow-derived mesenchymal stem cells (BM-MSCs) with hexachlorophene, a Wnt/ß-catenin signaling inhibitor, ameliorated acute liver injury. In a further previous report, we identified IC-2, a newly synthesized derivative of the Wnt/ß-catenin signaling inhibitor ICG-001, as a potent inducer of hepatic differentiation of BM-MSCs. METHODS: We manufactured hepatic cell sheets by engineering from human BM-MSCs using the single small molecule IC-2. The therapeutic potential of IC-2-induced hepatic cell sheets was assessed by transplantation of IC-2- and hexachlorophene-treated hepatic cell sheets using a mouse model of acute liver injury. RESULTS: Significant improvement of liver injury was elicited by the IC-2-treated hepatic cell sheets. The expression of complement C3 was enhanced by IC-2, followed by prominent hepatocyte proliferation stimulated through the activation of NF-κB and its downstream molecule STAT-3. Indeed, IC-2 also enhanced the expression of amphiregulin, resulting in the activation of the EGFR pathway and further stimulation of hepatocyte proliferation. As another important therapeutic mechanism, we revealed prominent reduction of oxidative stress mediated through upregulation of the thioredoxin (TRX) system by IC-2-treated hepatic cell sheets. The effects mediated by IC-2-treated sheets were superior compared with those mediated by hexachlorophene-treated sheets. CONCLUSION: The single compound IC-2 induced hepatic cell sheets that possess potent regeneration capacity and ameliorate acute liver injury.

Retraction: Azuma, K. et al. Chitin, Chitosan, and Its Derivatives for Wound Healing: Old and New Materials. J. Funct. Biomater. 2015, 6, 104⁻142.

The Journal of Functional Biomaterials Editorial Office have been made aware that some parts of the article [1] are duplicated from other publications[...].

Wood-mimetic skins prepared using horseradish peroxidase catalysis to induce surface wrinkling of chitosan film upon drying.

We previously developed bio-based wrinkled surfaces induced by wood-mimetic skins upon drying in which microscopic wrinkles were fabricated on a chitosan (CS) film by immersing it in a phenolic acid solution, followed by horseradish peroxidase (HRP)-catalyzed surface reaction and drying. However, the detailed structure of the resulting wood-mimetic skins, including crosslinking mode and thickness, has not been clarified due to the difficulty of the analysis. Here, we prepare wrinkled films using ferulic acid (FE), vanillic acid (VA), and homovanillic acid (HO) and characterize their structures to clarify the unknown characteristics of wood-mimetic skin. Chemical and structural analyses of wood-mimetic skins prepared using VA and HO indicate that the crosslinking structure in the skin is composed of ionic bonds between CS and an oligophenolic residue generated by the HRP-catalyzed reaction on the CS surface. Moreover, the quantity of these ionic bonds is related to the skin hardness and wrinkle size. Finally, SEM and TOF-SIMS analyses indicate that the skin thickness is on the submicron order (<200nm).

A Novel Small-molecule WNT Inhibitor, IC-2, Has the Potential to Suppress Liver Cancer Stem Cells.

BACKGROUND/AIM: The presence of cancer stem cells (CSCs) contributes to metastasis, recurrence, and resistance to chemo/radiotherapy in hepatocellular carcinoma (HCC). The WNT signaling pathway is reportedly linked to the maintenance of stemness of CSCs. In the present study, in order to eliminate liver CSCs and improve the prognosis of patients with HCC, we explored whether small-molecule compounds targeting WNT signaling pathway suppress liver CSCs. MATERIALS AND METHODS: The screening was performed using cell proliferation assay and reporter assay. We next investigated whether these compounds suppress liver CSC properties by using flow cytometric analysis and sphere-formation assays. A mouse xenograft model transplanted with CD44-positive HuH7 cells was used to examine the in vivo antitumor effect of IC-2. RESULTS: In HuH7 human HCC cells, 10 small-molecule compounds including novel derivatives, IC-2 and PN-3-13, suppressed cell viability and WNT signaling activity. Among them, IC-2 significantly reduced the CD44-positive population, also known as liver CSCs, and dramatically reduced the sphere-forming ability of both CD44-positive and CD44-negative HuH7 cells. Moreover, CSC marker-positive populations, namely CD90-positive HLF cells, CD133-positive HepG2 cells, and epithelial cell adhesion molecule-positive cells, were also reduced by IC-2 treatment. Finally, suppressive effects of IC-2 on liver CSCs were also observed in a xenograft model using CD44-positive HuH7 cells. CONCLUSION: The novel derivative of small-molecule WNT inhibitor, IC-2, has the potential to suppress liver CSCs and can serve as a promising therapeutic agent to improve the prognosis of patients with HCC.

Preparation and biocompatibility of a chitin nanofiber/gelatin composite film.

The development of chitin-based materials with favorable mechanical properties and biocompatibility is an important research goal owing to the wide-ranging practical applications. In this study, a composite film was prepared using chitin nanofibers and gelatin. The CNF/gelatin composite film was highly viscous and had a fine nanofiber structure. The transmittances indicated high transparency, regardless of nanofiber content. The water content of the CNF/gelatin composite film increased linearly as the gelatin content increased. Although the CNF/gelatin composite film did not induce severe inflammation, it strongly induced fibroblast proliferation, indicating high biocompatibility. Based on these results, the films are suitable for biological applications, e.g., tissue engineering, medicines, and cosmetics.

Oral Administration of Surface-Deacetylated Chitin Nanofibers and Chitosan Inhibit 5-Fluorouracil-Induced Intestinal Mucositis in Mice.

This study investigated the prophylactic effects of orally administered surface-deacetylated chitin nanofibers (SDACNFs) and chitosan against 5-fluorouracil (5-FU)-induced intestinal mucositis, which is a common side effect of 5-FU chemotherapy. SDACNFs and chitosan abolished histological abnormalities associated with intestinal mucositis and suppressed hypoproliferation and apoptosis of intestinal crypt cells. These results indicate that SDACNF and chitosan are useful agents for preventing mucositis induced by anti-cancer drugs.

Biobased Wrinkled Surfaces Induced by Wood Mimetic Skins upon Drying: Effect of Mechanical Properties on Wrinkle Morphology.

We previously developed biobased wrinkled surfaces based on wood mimetic skins in which microscopic wrinkles were fabricated on a chitosan film by immersion in a phenolic acid solution, horseradish peroxidase-catalyzed surface reaction, and drying. Here, we prepared a diverse range of wrinkled films by immersion treatment at 30, 40, 50, and 60 °C in p-coumaric acid and then investigated the correlation between wrinkle morphology and mechanical properties. Wrinkle wavelengths gradually decreased as the immersion temperature increased as well as the previous report. In order to clarify the mechanisms responsible for the different wrinkle morphologies, the films were subjected to elastic moduli measurement and GPC analysis after immersion treatment. These experiments provided evidence that the chitosan around the film surface decomposed along with the immersion process. The decomposition was accelerated by higher immersion temperature, suggesting that higher temperatures led to the formation of softer skins, inducing smaller wrinkles. In fact, wrinkle morphologies with this system were predominately determined by the hardness of the wood mimetic skins. This phenomenon is consistent with the fundamentals of surface wrinkling in nature. This study is the first to demonstrate that artificial wrinkling triggered by water evaporation can be controlled by precise control of the surface hardness of soft material.

Protein/CaCO3/Chitin Nanofiber Complex Prepared from Crab Shells by Simple Mechanical Treatment and Its Effect on Plant Growth.

A protein/CaCO3/chitin nanofiber complex was prepared from crab shells by a simple mechanical treatment with a high-pressure water-jet (HPWJ) system. The preparation process did not involve chemical treatments, such as removal of protein and calcium carbonate with sodium hydroxide and hydrochloric acid, respectively. Thus, it was economically and environmentally friendly. The nanofibers obtained had uniform width and dispersed homogeneously in water. Nanofibers were characterized in morphology, transparency, and viscosity. Results indicated that the shell was mostly disintegrated into nanofibers at above five cycles of the HPWJ system. The chemical structure of the nanofiber was maintained even after extensive mechanical treatments. Subsequently, the nanofiber complex was found to improve the growth of tomatoes in a hydroponics system, suggesting the mechanical treatments efficiently released minerals into the system. The homogeneous dispersion of the nanofiber complex enabled easier application as a fertilizer compared to the crab shell flakes.

Preparation of chitin nanofibers by surface esterification of chitin with maleic anhydride and mechanical treatment.

Esterification with maleic anhydride significantly improved the mechanical disintegration of chitin into uniform 10-nm nanofibers. Nanofibers with 0.25° of esterification were homogeneously dispersed in basic water due to the carboxylate salt on the surface. Esterification proceeded on the surface and did not affect the relative crystallinity. A cast film of the esterified chitin nanofibers was highly transparent, since the film was free from light scattering.

Wnt/Beta-Catenin Signal Inhibitor HC-1 Sensitizes Oral Squamous Cell Carcinoma Cells to 5-Fluorouracil through Reduction of CD44-Positive Population.

BACKGROUND: Oral squamous cell carcinoma is a prevalent and frequently lethal malignancy worldwide. Existence of treatment-resistant cancer stem cells is considered to be associated with tumor formation, recurrence and metastasis. Wnt/beta-catenin signal is one of the crucial signaling pathways for cancer stem cells. Wnt/beta-catenin signal inhibitor may reduce the population of cancer stem cells and improve therapeutic effects on the cancers. METHODS: The effects of three derivatives of Wnt/beta-catenin signal inhibitors, HC-1, IC-2 and PN3-13, which we recently developed, on oral squamous cell carcinoma cell line HSC2, were examined by luciferase reporter assay, WST assay, cell sorting assay and apoptosis assay. RESULTS: The reporter assay showed that these small molecule compounds reduced Wnt/beta-catenin transcriptional activity in HSC2 cells. Of these compounds, IC-2 and PN3-13 inhibited cell viability in a dose-dependent manner, whereas HC-1 did not at even higher concentrations. Notably, however, the cell-sorting assay revealed that HC-1 significantly reduces the CD44-positive population of oral squamous cell carcinoma cells, compared to other compounds without affecting cell viability. In addition, HC-1 increases the cytotoxicity of HSC2 cells to 5-fluorouracil. The combination treatment of HC-1 with 5-fluorouracil significantly increased the apoptotic cells whereas treatment by either compound did not. CONCLUSION: These data suggest that HC-1 is an effective compound to target cancer stem cells, and the combination treatment of HC-1 and 5-fluorouracil can stimulate the tumor suppressive effect on oral squamous cell carcinoma cells.

Chitin nanofibrils suppress skin inflammation in atopic dermatitis-like skin lesions in NC/Nga mice.

We evaluated the effect of chitin nanofibril (CNF) application via skin swabs on an experimental atopic dermatitis (AD) model. AD scores were lower, and hypertrophy and hyperkeratosis of the epidermis were suppressed after CNF treatment. Furthermore, inflammatory cell infiltration in both the epidermis and dermis was inhibited. CNFs also attenuated histological scores. The suppressive effects of CNFs were equal to those of corticosteroid application; however, chitin did not show these effects. CNF application might have anti-infllammatory effects via suppression of the activation of nuclear factor-kappa B, cyclooxygenase-2, and inducible nitric oxide synthase. In an early-stage model of experimental AD, CNFs suppressed AD progression to the same extent as corticosteroids. They also suppressed skin inflammation and IgE serum levels. Our findings indicate that CNF application could aid in the prevention or treatment of AD skin lesions.

Biomineralization of calcium phosphate crystals on chitin nanofiber hydrogel for bone regeneration material.

We previously reported a chitin nanofiber hydrogel from squid pen ß-chitin by a simple NaOH treatment. In the present study, a calcium phosphate/chitin nanofiber hydrogel was prepared for bone tissue engineering. Calcium phosphate was mineralized on the hydrogel by incubation in a solution of diammonium hydrogen phosphate solution followed by calcium nitrate tetrahydrate. X-ray diffractometry and Fourier transform infrared spectroscopy showed the formation of calcium phosphate crystals. The morphology of the calcium phosphate crystals changed depending on the calcification time. After mineralization, the mechanical properties of the hydrogel improved due to the reinforcement effect of calcium phosphate crystal. In an animal experiment, calcium phosphate/chitin nanofiber hydrogel accelerated mineralization in subcutaneous tissues. Morphological osteoblasts were observed.

Characterization of Chitosan Nanofiber Sheets for Antifungal Application.

Chitosan produced by the deacetylation of chitin is a cationic polymer with antimicrobial properties. In this study, we demonstrate the improvement of chitosan properties by nanofibrillation. Nanofiber sheets were prepared from nanofibrillated chitosan under neutral conditions. The Young's modulus and tensile strength of the chitosan NF sheets were higher than those of the chitosan sheets prepared from dissolving chitosan in acetic acid. The chitosan NF sheets showed strong mycelial growth inhibition against dermatophytes Microsporum and Trichophyton. Moreover, the chitosan NF sheets exhibited resistance to degradation by the fungi, suggesting potentials long-lasting usage. In addition, surface-deacetylated chitin nanofiber (SDCNF) sheets were prepared. The SDCNF sheet had a high Young's modulus and tensile strength and showed antifungal activity to dermatophytes. These data indicate that nanofibrillation improved the properties of chitosan. Thus, chitosan NF and SDCNF sheets are useful candidates for antimicrobial materials.

Human mesenchymal stem cell-engineered hepatic cell sheets accelerate liver regeneration in mice.

Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapy. Based on our hypothesis that suppression of Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we developed human mesenchymal stem cell-engineered hepatic cell sheets by a small molecule compound. Screening of 10 small molecule compounds was performed by WST assay, TCF reporter assay, and albumin mRNA expression. Consequently, hexachlorophene suppressed TCF reporter activity in time- and concentration-dependent manner. Hexachlorophene rapidly induced hepatic differentiation of human MSCs judging from expression of liver-specific genes and proteins, PAS staining, and urea production. The effect of orthotopic transplantation of human mesenchymal stem cell-engineered hepatic cell sheets against acute liver injury was examined in one-layered to three-layered cell sheets system. Transplantation of human mesenchymal stem cell-engineered hepatic cell sheets enhanced liver regeneration and suppressed liver injury. The survival rates of the mice were significantly improved. High expression of complement C3 and its downstream signals including C5a, NF-κB, and IL-6/STAT-3 pathway was observed in hepatic cell sheets-grafted tissues. Expression of phosphorylated EGFR and thioredoxin is enhanced, resulting in reduction of oxidative stress. These findings suggest that orthotopic transplantation of hepatic cell sheets manufactured from MSCs accelerates liver regeneration through complement C3, EGFR and thioredoxin.