Yoshinosuke Hamada

Crystallinity and Solubility Characteristics of Hydroxyapatite Adsorbed Amino Acid

Hydroxyapatite (HAp) was synthesized in the presence of a variety of amino acids in order to investigate the effect of amino acid on the crystallinity and the solubility characteristics of HAp in the HAp-synthesizing condition. In the results of X-ray diffraction analysis, HAp synthesized in the presence of glycine (HAp-Gly), serine (HAp-Ser), aspartic acid (HAp-Asp) and glutamic acid (HAp-Glu) showed poor crystallinity compared with HAp synthesized in the absence of amino acid (HAp-con). The results of Fourier transform infrared spectroscopy suggested the adsorption of these amino acids on HAp. Scanning electron microphotographs showed that the size and morphology of HAp adsorbed amino acids changed significantly. Furthermore, the solubility of these HAps increased significantly compared to HAp-con, each differing in four amino acids. However, other amino acids did not affect the crystallinity and morphology of HAp and had no significant change in their solubility. Collectively, this study suggests that the crystallinity and the solubility of synthesized HAp are different owing to the variation of amino acids in the HAp synthesizing condition. It is expected that digestion-regulated HAp materials could be synthesized by using amino acid in their synthesizing condition.

Aged Garlic Extract Inhibits Angiogenesis and Proliferation of Colorectal Carcinoma Cells

Because colorectal cancer is likely to develop in many people at some point during their lives, prevention has become a high priority. Diet and nutrition play an important role during the multistep colon carcinogenic process. Garlic has been traditionally used as a spice and is well known for its medicinal properties; several studies have indicated its pharmacologic functions, including its anticarcinogenic properties. However, the mechanisms by which garlic can prevent colorectal cancer remain to be elucidated. This study investigated the effect of aged garlic extract (AGE) on the growth of colorectal cancer cells and their angiogenesis, which are important microenvironmental factors in carcinogenesis. AGE suppressed the proliferation of 3 different colorectal cancer cell lines-HT29, SW480, and SW620-in the same way, but its effects on the invasive activities of these 3 cell lines were different. the invasive activities of SW480 and SW620 cells were inhibited by AGE, whereas AGE had no effect on the invasive activity of Ht29 cells. The action of AGE appears to be dependent on the type of cancer cell. On the other hand, AGE enhanced the adhesion of endothelial cells to collagen and fibronectin and suppressed cell motility and invasion. AGE also inhibited the proliferation and tube formation of endothelial cells potently. These results suggest that AGE could prevent tumor formation by inhibiting angiogenesis through the suppression of endothelial cell motility, proliferation, and tube formation. AGE would be a good chemopreventive agent for colorectal cancer because of its antiproliferative action on colorectal carcinoma cells and inhibitory activity on angiogenesis.

Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts

Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and alphavbeta3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.

A Dominant-Negative FGF1 Mutant (the R50E Mutant) Suppresses Tumorigenesis and Angiogenesis

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent (“FGF1 decoy”).

Effects of far infrared ray on Hela cells and WI-38 cells

Abstract The ability of cells to proliferate and adhere is an important step of cancer metastasis. Far infrared ray (FIR) with wavelengths between 4 and 14 μm is called the growth ray that is efficiently absorbed by a living organism. Therefore, the use of these substances in cancer therapy at body temperature (37 °C) will be significant. Bamboo charcoal powder was used as a FIR emission material. Here, we performed WST assay and adhesion assay in order to examine the effects of the FIR using Hela cells and fetal lung fibroblasts (WI-38). The multiplication rate of Hela cells showed a significant difference for a 6-day culture, though the multiplication rate of WI-38 cells showed no significant difference at a 3- and 6-day culture. These results show the FIR plays an important role in tumor cells in vitro.

Transplantation of myoblast sheets that secrete the novel peptide SVVYGLR improves cardiac function in failing hearts

AIMS Transplantation of myoblast sheets is a promising therapy for enhancing cardiac function after heart failure. We have previously demonstrated that a 7-amino-acid sequence (Ser-Val-Val-Tyr-Gly-Leu-Arg) derived from osteopontin (SV peptide) induces angiogenesis. In this study, we evaluated the long-term therapeutic effects of myoblast sheets secreting SV in a rat infarction model. METHODS AND RESULTS Two weeks after ligation of the left anterior descending coronary artery, the animals were divided into the following three groups: a group transplanted with wild-type rat skeletal myoblast sheets (WT-rSkMs); a group transplanted with SV-secreting myoblast sheets (SV-rSkMs); and a control group (ligation only). We evaluated cardiac function, histological changes, and smooth muscle actin (SMA) expression through transforming growth factor-β (TGF-β) signalling. The ejection fraction and fractional shortening were significantly better, and the enlargement of end-systolic volume was also significantly attenuated in the SV-rSkM group. Left ventricular remodelling, including fibrosis and hypertrophy, was significantly attenuated in the SV-rSkM group, and SV secreted by the myoblast sheets promoted angiogenesis in the infarcted border area. Furthermore, many clusters of SMA-positive cells were observed in the infarcted areas in the SV-rSkM group. In vitro SMA expression was increased when SV was added to the isolated myocardial fibroblasts. Moreover, SV bound to the TGF-β receptor, and SV treatment activated TGF-β receptor-Smad signalling. CONCLUSION The SV-secreting myoblast sheets facilitate a long-term improvement in cardiac function. The SV can induce differentiation of fibroblasts to myofibroblasts via TGF-β-Smad signalling. This peptide could possibly be used as a bridge to heart transplantation or as an ideal peptide drug for cardiac regeneration therapy.

Enhanced Expression of Integrin αvβ3 Induced by TGF-β Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1) in TGF-β-Induced Epithelial-Mesenchymal Transition (EMT) in Mammary Epithelial Cells

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.

The role of MT2-MMP in cancer progression

The role of MT2-MMP in cancer progression remains to be elucidated in spite of many reports on MT1-MMP. Using a human fibrosarcoma cell, HT1080 and a human gastric cancer cell, TMK-1, endogenous expression of MT1-MMP or MT2-MMP was suppressed by siRNA induction to examine the influence of cancer progression in vitro and in vivo. In HT1080 cells, positive both in MT1-MMP and MT2-MMP, the migration as well as the invasion was impaired by MT1-MMP or MT2-MMP suppression. Also cell proliferation in three dimensional (3D) condition was inhibited by MT1-MMP or MT2-MMP suppression and tumor growth in the nude mice transplanted with tumor cells were reduced either MT1-MMP or MT2-MMP suppression with a prolongation of survival time in vivo. MT2-MMP suppression induces more inhibitory effects on 3D proliferation and in vivo tumor growth than MT1-MMP. On the other hand, TMK-1 cells, negative in MT1-MMP and MMP-2 but positive in MT2-MMP, all the migratory, invasive, and 3D proliferative activities in TMK-1 are decreased only by MT2-MMP suppression. These results indicate MT2-MMP might be involved in the cancer progression more than or equal to MT1-MMP independently of MMP-2 and MT1-MMP.

Transplantation of elastin-secreting myoblast sheets improves cardiac function in infarcted rat heart

Myoblast sheet transplantation for cardiac failure is a promising therapy to enhance cardiac function via paracrine mechanism. However, their efficacies of treatment showed a gradual decline. The gene modification of the implanted myoblast is important in improving the long-term results of the treatment. Elastin fiber enhances the extensibility of the infarcted wall and can prevent left ventricular dilation. We therefore hypothesized that the elastin gene modification of the implanted myoblast could strengthen and maintain the long-term improvement effects of cardiac function. In this study, we evaluated long-term follow-up benefits of functional myoblast sheets that secrete elastin in an infarcted model. The animal models were divided into three groups: a group transplanted with nontransfected, wild-type, skeletal myoblast-type sheets (WT-rSkM); group transplanted with myoblast sheets that secreted elastin fragments (ELN-rSkM); and a control group (ligation only). Cardiac function was examined by echocardiography, and cardiac remodeling after infarction was evaluated by histological examination. The cardiac function was significantly improved and the left ventricle end-diastolic dimensions were significantly reduced in the ELN-rSkM group. Histological analysis showed that left ventricular remodeling was attenuated in the ELN-rSkM group and that elastic fiber was formed in the epicardial area of ELN-rSkM group. The functionalization of myoblast sheet by elastin gene transfer showed the long-term improvement of cardiac function. Expressed recombinant elastin fiber prevented the dilation of the left ventricular chamber after myocardial infarction. The functional myoblast sheet transplantation maintained the treatment effect by the paracrine effect of myoblast and the formed recombinant elastin.

SVVYGLR motif of the thrombin-cleaved N-terminal osteopontin fragment enhances the synthesis of collagen type III in myocardial fibrosis

Osteopontin (OPN) is involved in various physiological processes such as inflammatory and wound healing. However, little is known about the effects of OPN on these tissues. OPN is cleaved by thrombin, and cleavage of the N-terminal fragment exposes a SVVYGLR sequence on its C-terminus. In this study, we examined the effects of the thrombin-cleaved OPN fragments on fibroblasts and myocardial fibrosis, particularly the role of the SVVYGLR sequence. The recombinant thrombin-cleaved OPN fragments (N-terminal fragment [N-OPN], C-terminal fragment [C-OPN], and the N-terminal fragment lacking the SVVYGLR sequence [ΔSV N-OPN]) were added to fibroblasts, and the cellular motility, signal activity, and production of collagen were evaluated. A sustained-release gel containing an OPN fragment or SVVYGLR peptide was transplanted into a rat model of ischemic cardiomyopathy and the quantities and ratio of collagen type I (COL I) and type III (COL III) were estimated. N-OPN significantly promoted fibroblast migration. Smad signal activity, expression of smooth muscle actin (SMA), and the production of COL III were enhanced by N-OPN and SVVYGLR peptide. Conversely, ΔSV N-OPN and C-OPN had no effect. In vivo, the expression level of N-OPN was associated with COL III distribution, and the COL III/COL I ratio was significantly increased by the sustained-release gel containing N-OPN or SVVYGLR peptide. The cardiac function was also significantly improved by the N-OPN- or SVVYGLR peptide-released gel treatment. The N-terminal fragment of thrombin-cleaved OPN-induced Smad signal activation, SMA expression, and COL III production, and its SVVYGLR sequence influences this function.