Published papers

We have investigated the effect of calcium spirulan (Ca-SP) isolated from a blue-green alga, Spirulina platensis, which is a sulfated polysaccharide chelating calcium and mainly composed of rhamnose, on invasion of B16-BL6 melanoma, Colon 26 M3.1 carcinoma and HT-1080 fibrosarcoma cells through reconstituted basement membrane (Matrigel). Ca-SP significantly inhibited the invasion of these tumor cells through Matrigel/fibronectin-coated filters. Ca-SP also inhibited the haptotactic migration of tumor cells to laminin, but it had no effect on that to fibronectin. Ca-SP prevented the adhesion of B16-BL6 cells to Matrigel and laminin substrates but did not affect the adhesion to fibronectin. The pretreatment of tumor cells with Ca-SP inhibited the adhesion to laminin, while the pretreatment of laminin substrates did not. Ca-SP had no effect on the production and activation of type IV collagenase in gelatin zymography. In contrast, Ca-SP significantly inhibited degradation of heparan sulfate by purified heparanase. The experimental lung metastasis was significantly reduced by co-injection of B16-BL6 cells with Ca-SP. Seven intermittent i.v. injections of 100 microg of Ca-SP caused a marked decrease of lung tumor colonization of B16-BL6 cells in a spontaneous lung metastasis model. These results suggest that Ca-SP, a novel sulfated polysaccharide, could reduce the lung metastasis of B16-BL6 melanoma cells, by inhibiting the tumor invasion of basement membrane probably through the prevention of the adhesion and migration of tumor cells to laminin substrate and of the heparanase activity.

The partially modified retro- and retro-inverso peptides of the Arg-Gly Asp (RGD) sequence of fibronectin, in which the direction of the Arg residue is reversed and/or the chirality of the amino acid residue is inverted, i.e., mainly R(rev)-COCH2CO-D and DR(rev)-COCH2CO-D, have been synthesized to examine their antimetastatic effects in murine lung or liver metastasis models, as well as their inhibitory effect on tumor cell invasion in vitro. R(rev)-COCH2CO-D inhibited lung metastasis produced by i.v. coinjection with B16-BL6 melanoma more potently than did other pseudo-peptides or the original RGDS peptide. Rrev-COCH2CO-D also showed antimetastatic effects against several different types of tumor cells such as B16-BL6 melanoma, Colon26 M3.1 carcinoma, and L5178Y-ML25 lymphoma cells, in a dose-dependent manner, and multiple administrations had a therapeutic effect on spontaneous lung metastasis. The invasion of melanoma cells into reconstituted basement membrane Matrigel in vitro was suppressed by R(rev)-COCH2CO-D more effectively than by RGDS. These results indicate that the antimetastatic effect by R(rev)-COCH2CO-D was in part due to the inhibition of tumor invasion. The RGDS peptide decomposed when incubated with fresh plasma in vitro, whereas R(rev)-COCH2CO-D was not affected by this treatment. Thus, the reversion of the Arg-Gly linkage in the RGD sequence resulted in protease resistance leading to the retardation of the clearance of the peptide in vivo, and consequently augmented its antimetastatic and antiinvasive properties. Designed peptide analogues may provide various advantages and be useful for preventing cancer metastasis.

We have investigated the inhibitory effect of oral administration of Juzen-taiho-to, a Kampo (Chinese herbal) medicine, on progressive growth of a mouse fibrosarcoma. Spontaneously regressive QR-32 tumor cells were able to grow progressively in vivo when coimplanted s.c. with a foreign body, gelatin sponge, whereas QR-32 cells alone gradually grew for over 15 days after inoculation and thereafter regressed for up to 25 days. Oral administration of Juzen-taiho-to (40 mg/day/mouse) for 7 days after inoculation of QR-32 cells with gelatin sponge resulted in significant inhibition of tumor growth and prolongation of the survival of the tumor-bearing mice. This growth-inhibitory effect of Juzen-taiho-to observed on day 25 was dose-dependent over the dose range from 4 to 40 mg/day. Treatment with Juzen-taiho-to for 7 days before tumor inoculation with gelatin sponge also significantly suppressed tumor growth examined on day 25, as did the administration of bismuth subnitrate, which is well known to induce metallothionein, an antioxidant. On the other hand, inoculation of progressed tumor cells (QRsP) resulted in growth without gelatin sponge, leading to death in syngeneic mice. Administration of Juzen-taiho-to for 7 days after inoculation of QRsP cells resulted in a decrease of the tumor growth and prolongation of the survival of mice, but the effect was less than that on the growth of QR-32 regressor tumor after coimplantation with gelatin sponge. These results suggest that the inhibitory effect of Juzen-taiho-to is partly associated with prevention of gelatin sponge-elicited progressive growth, probably mediated by endogenous factors including antioxidant substances, in addition to the augmentation of host-mediated antitumor activity.

We have investigated the inhibitory mechanism of the initial arrest of L5178Y-ML25 lymphoma cells in a target organ (liver) by using recombinant fibronectin fragments with cell- and/or heparin-binding domains (C-274, H-271 or the fusion fragment CH-271). Pretreatment of hepatic sinusoidal endothelial (HSE) cell monolayers with lymphoma cells or their conditioned medium for 4 to 6 h resulted in the enhancement of lymphoma cell adhesion to HSE cell monolayer. The increased tumor adhesiveness was completely abolished by preincubation of the conditioned medium with anti interleukin-1 beta monoclonal antibody (mAb). Synthetic sialyl Le(x) (SLe(x)) as a ligand for endothelial cell leukocyte adhesion molecule-1 (ELAM-1) adhesion receptor and anti ELAM-1 mAb blocked the conditioned medium-induced enhancement of tumor-endothelial cell interaction, while pretreatment of the activated HSE cell monolayer with anti vascular cell adhesion molecule-1 (VCAM-1) mAb did not affect the enhanced tumor cell adhesion. These results indicate that tumor cell interaction with the stimulated HSE cells is mediated by ELAM-1 molecules on HSE cells. However, the expression of SLe(x) and SLe(a) on the tumor surface was not observed by flow cytometric analysis. ELAM-1-mediated enhancement of tumor cell adhesion to HSE monolayer was also inhibited in a concentration-dependent manner by CH-271 fusion polypeptide or the sulfated chitin derivative sulfated carboxymethyl-chitin, which can bind to the heparin-binding domain of CH-271. In addition, CH-271 inhibited not only tumor-endothelium interaction but also tumor cell invasion into reconstituted basement membrane Matrigel in vitro.

The kinetics of lymphocyte function associated antigen 1 (LFA-1) expression on spleen cells (SPC) and liver non-parenchymal cells (NPC), and intercellular adhesion molecule 1 (ICAM-1) expression on hepatocytes (HC) was examined in acute liver injury mice induced by a DTH reaction to picryl chloride (PCl). The peak expression of LFA-1 on SPC was seen at 6 hr after eliciting liver injury, and then that of LFA-1 on NPC and ICAM-1 on HC appeared at 12 hr. Thereafter, the serum ALT elevation reached to a peak at 18 hr. A splenectomy before the PCl elicitation significantly reduced the ALT elevation. Both SPC and NPC from liver injury mice induced a remarkable release of ALT from HC in vitro, in parallel with their LFA-1 expression. The pre-treatment of NPC or SPC with anti-LFA-1 mAb, irrespective of the presence of complement, completely blocked the ALT release. Also, when HC was prebound with anti-ICAM-1 mAb, neither NPC nor SPC showed a cytotoxicity against the HC. Furthermore, the treatment of NPC with either anti-Thy1.2 or anti-CD4 mAb in the presence but not absence of complement, showed a complete abolishment of ALT release. Anti-CD8 mAb plus complement also tended to inhibit ALT release. The twofold increase in CD4+ LFA-1+ and mild increase in CD8+ LFA-1+ populations were also confirmed in NPC at 12 hr. These results suggest that PCl elicitation in liver may trigger an increased expression of LFA-1 on SPC and NPC and ICAM-1 on HC. LFA-1/ICAM-1 interaction between liver-infiltrating NPC, mainly including CD4+ and CD8+ T cells, and HC may be an essential step for the hepatocyte damage in PCl-DTH liver injury.

Increasing evidence suggests that mesenchymal stem cells (MSCs) play anti-inflammatory roles during innate immune responses. However, little is known about the effect of MSCs or their secretions on the ligand response of Toll-like receptor (TLR) 7 and TLR8, receptors that recognize viral single-stranded RNA (ssRNA). Macrophages play a critical role in the innate immune response to ssRNA virus infection; therefore, we investigated the effect of MSC-conditioned medium on cytokine expression in macrophages following stimulation with TLR7/8 ligands. After stimulation with TLR7/8 ligand, bone marrow-derived macrophages cultured with MSCs or in MSC-conditioned medium expressed lower levels of tumor necrosis factor (TNF) α and interleukin (IL) 6 and higher levels of IL-10 compared to macrophages cultured without MSCs or in control medium, respectively. The modulations of cytokine expression were associated with prostaglandin E2 (PGE2) secreted by the MSCs. PGE2 enhanced extracellular signal-related kinase (ERK) signaling and suppressed nuclear factor- κ B (NF- κ B) signaling. Enhanced ERK signaling contributed to enhanced IL-10 production, and suppression of NF- κ B signaling contributed to the low production of TNF- α . Collectively, these results indicate that MSCs and MSC-conditioned medium modulate the cytokine expression profile in macrophages following TLR7/8-mediated stimulation, which suggests that MSCs play an immunomodulatory role during ssRNA virus infection.

Increasing evidence indicates that obesity is a risk factor for increased severity of influenza virus infection. However, its precise immunological mechanism is not fully understood. To investigate this, diet-induced obese (DIO) mice were established by feeding C57BL/6 male mice a high-fat diet for 16 weeks. DIO and lean control mice were infected intranasally with 3000 pfu of influenza A virus (IAV) (PR8/H1N1). Interestingly, we found adipose tissue located along the bronchus in naïve DIO mice. In addition, the Nos2 level was significantly higher and Arg1 level was significantly lower in lung macrophages of naïve DIO mice, consistent with an M1-skewed phenotype. The survival rate and body weight of DIO mice infected with IAV were significantly lower than those of lean control mice and associated with higher viral load in the lungs of DIO mice. Histopathological analysis demonstrated higher numbers of inflammatory cells in the lungs of DIO mice after IAV infection. Levels of cytokines, including TNF-α, IL-6, IL-10, and type I IFN (IFN-α and IFN-β), in bronchoalveolar lavage fluid (BALF) were altered after IAV infection; in particular, IFN-α and IFN-β levels were significantly suppressed in the BALF of DIO mice. In vitro, bone marrow-derived macrophages were stimulated with ligands of toll-like receptor (TLR) 7/8, a pattern recognition receptor for single-stranded RNA, and levels of TNF-α, IL-6, and IL-10 were similarly altered. In addition, levels of IFN-α and IFN-β were significantly lower in culture supernatants of alveolar macrophages sorted from naïve DIO mice and infected with IAV, compared to those in macrophages sorted from lean control mice. Collectively, these results suggest that macrophages may be the main contributors to poor outcomes of influenza virus infection in obesity.

Macrolide antibiotics are used in treating Pseudomonas aeruginosa chronic biofilm infections despite their unsatisfactory antibacterial activity, because they display several special activities, such as modulation of the bacterial quorum sensing and immunomodulatory effects on the host. In this study, we investigated the effects of the newly synthesized P. aeruginosa quorum-sensing autoinducer analogs (AIA-1, -2) on the activity of azithromycin and clarithromycin against P. aeruginosa. In the killing assay of planktonic cells, AIA-1 and -2 enhanced the bactericidal ability of macrolides against P. aeruginosa PAO1; however, they did not affect the minimum inhibitory concentrations of macrolides. In addition, AIA-1 and -2 considerably improved the killing activity of azithromycin and clarithromycin in biofilm cells. The results indicated that AIA-1 and -2 could affect antibiotic tolerance. Moreover, the results of hydrocarbon adherence and cell membrane permeability assays suggested that AIA-1 and -2 changed bacterial cell surface hydrophobicity and accelerated the outer membrane permeability of the hydrophobic antibiotics such as azithromycin and clarithromycin. Our study demonstrated that the new combination therapy of macrolides and AIA-1 and -2 may improve the therapeutic efficacy of macrolides in the treatment of chronic P. aeruginosa biofilm infections.

We have examined the effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on invasive ability of murine melanoma cell lines with different metastatic potential in a Matrigel invasion assay. alpha-MSH potently blocked the invasion of B16-BL6 cells with highly metastatic potential in a concentration-dependent manner, whereas it was less effective in inhibiting the invasion of weakly metastatic B16-F1 cells. Pretreatment of B16-BL6 cells with alpha-MSH resulted in a decrease of the adhesiveness to fibronectin and laminin substrates in a time-dependent fashion. As assessed by zymographic analysis, alpha-MSH partially inhibited the production of matrix metalloproteinase (MMP)-2 and -9 from both cell lines to a similar degree without affecting the degradative activity of these MMPs. alpha-MSH was more potent in inhibiting the migration of B16-BL6 cells towards both fibronectin- and laminin-coated substrates than that of B16-F1 cells. The growth and morphology of B16-BL6 cells were not changed after a 7-day incubation with alpha-MSH. The number of lung tumor colonies markedly decreased when B16-BL6 cells were coinjected intravenously with 10(-6) M alpha-MSH. However, alpha-MSH had no effect on the experimental lung metastases by B16-F1 cells. These results suggest that alpha-MSH suppressed the invasive and metastatic properties of B16 melanoma cells, and the degree of inhibition was associated with metastatic potential of B16 melanoma cells.

Tumor invasion into the extracellular matrix (ECM) and basement membrane (BM) is a crucial step of tumor metastasis. In order to investigate the possible therapeutic procedure for the tumor invasion, we investigated the anti-invasive activities of several synthetic serine protease inhibitors. FOY-305, a serine protease inhibitor, showed no cytotoxic activity against human HT-1080 fibrosarcoma cells at concentrations ranging from 0.1 to 100 micrograms/ml, while its analogs ONO-3403 and FO-349 showed slight cytotoxic activities at the concentration of 100 micrograms/ml. These compounds inhibited the activity of urokinase-type plasminogen activator (u-PA) which is one of serine proteases and considered to be associated with tumor invasion and metastasis in fibrin zymography. FOY-305 more potently inhibited the invasion of HT-1080 cells into the reconstituted BM Matrigel, as well inhibited u-PA activity, compared with ONO-3403 and FO-349. These results suggest that the anti-invasive activity of these compounds is consistent with their anti-fibrinolytic activities. In addition, the combined treatment of FOY-305 with FC-336 processing anti-invasive and anti-MMP properties resulted in marked enhancement of anti-invasive activity. In conclusion, FOY-305 inhibited the invasion of tumor cells through interference with the u-PA activity of tumor cells, and this inhibitory activity was augmented by the combination with a MMP inhibitor.

We have investigated the anti-metastatic effect of Celosia argentea seed extracts (CAE), which have traditionally been used as a therapeutic drug for eye and hepatic diseases in China and Japan. Intraperitoneal (i.p.) administration of CAE for 7 d before tumor inoculation significantly inhibited liver metastasis caused by intraportal injection of colon 26-L5 carcinoma cells in a dose-dependent manner. CAE also showed concentration dependent mitogenic activity on BALB/c whole splenocytes, whereas incubation of the non-adherent fraction of splenocytes with CAE did not induce this activity. CAE has the ability to induce interleukin (IL)-12 production from macrophages in vitro. Following i.p. administration of CAE the maximal levels of IL-12 and interferon (IFN)-gamma production in serum were achieved at 2-3 and 6 h, respectively. Experiments using macrophage- or NK cell-deficient mice revealed that CAE-induced IL-12 in serum was not mediated by macrophages and that IFN-gamma production was mainly dependent on natural killer (NK) cells. Since CAE was inactive when the contributions of macrophages were removed in our system, its inhibitory mechanism is likely to be mainly associated with the activation of macrophages to an anti-metastatic state rather than NK cells. CAE administration resulted in increased production of IL-2, IFN-gamma and decreased production of a Th2 cytokine (IL-4) from splenocytes stimulated by PMA and A23187. Thus, the anti-metastatic effect by CAE is based on its immunomodulating properties including induction of cytokines such as IL-12, IL-2 and IFN-gamma leading to a Th1 dominant immune state and activating macrophages to the tumoricidal state. This may provide a basis for the inhibition of cancer metastasis.

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of several infectious oral diseases, including dental caries, periodontal disease, and oral candidiasis. Houttuynia cordata (HC, Saururaceae) is a widely used traditional medicine, for both internal and external application. A decoction of dried HC leaves (dHC) has long been consumed as a health-promoting herbal tea in Japan. We have recently reported that a water solution of HC poultice ethanol extract (wHCP) exerts antimicrobial and antibiofilm effects against several important oral pathogens. It also exhibits anti-inflammatory effects on human keratinocytes. In our current study, we examined the effects of dHC on infectious oral pathogens and inflammation. Our results demonstrated that dHC exerts moderate antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, Fusobacterium nucleatum (involved in dental plaque formation), and Candida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated by Porphyromonas gingivalis lipopolysaccharide (a cause of periodontal disease), without cytotoxic effects. This suggests that dHC exhibits multiple activities in microorganisms and host cells. dHC can be easily prepared and may be effective in preventing infectious oral diseases.

We have investigated the inhibitory effect of the N-terminal modified Arg-Gly-Asp-Ser (RGDS) analogues, AcDRGDS and AcDRLDS, on tumor cell adhesion to the components of extracellular matrix and basement membrane, and also tested the antimetastatic effect of their conjugates with trimesic acid, Ar(DRGDS)3 and Ar(DRLDS)3. AcDRGDS significantly inhibited tumor cell adhesion to fibronectin, vitronectin and RGDS substrates, but not to CS1 substrate which is a ligand for the alpha 4 beta 1 tumor surface integrin receptor. In contrast, AcDRLDS variant peptide significantly inhibited tumor cell adhesion to laminin, in addition to RGDS-mediated adhesion to fibronectin and vitronectin. AcDRLDS also inhibited tumor cell adhesion to CS1 as well as the RGDS sequence within the fibronectin molecule in a concentration-dependent manner, although the inhibitory effect was less than that of the CS1 (EILDV) peptide. Ar(DRLDS)3 inhibited the laminin- and fibronectin-mediated invasion and migration of tumor cells, whereas Ar(DRGDS)3 selectively inhibited fibronectin-mediated invasion and migration. Ar(DRGDS)3 and Ar(DRLDS)3 were much more effective in inhibiting experimental lung or liver metastases of various types of murine and human tumors than the original RGDS-containing peptides or Ar(COONa)3. Multiple administrations of Ar(DRGDS)3 or Ar(DRLDS)3 potently inhibited spontaneous lung metastasis produced by intra-footpad injection of B16-BL6 cells without affecting the primary tumor size at the time of surgical excision, as compared with RGDS peptide or untreated control. Thus, Ar(DRGDS)3 and Ar(DRLDS)3 substantially increased the exhibiting any antimetastatic effect of the peptides without direct cytotoxicity.

Tumor metastasis into distinct organs and tissues, of which many patients with malignancies die, is regulated in multiple steps. Using a murine metastasis model, in which highly metastatic B16-BL6 melanoma cells were inoculated i.v. into syngeneic C57BL/6 mice, the administration of a recombinant human tissue inhibitor of metalloproteinases-2 (r-hTIMP-2) once a day on days -1 to 3 after the implantation significantly inhibited the formation of metastatic foci in the lungs. The antimetastatic effect of r-hTIMP-2 was detected irrespective of administration route [i.v., i.p., s.c., and i.m. routes] and in a dose-dependent manner. The i.m.-injection of r-hTIMP-2 during the early phase after tumor inoculation is suggested to be essential for antimetastasis. In another model using spontaneously metastasing B16-BL6 cells, multiple i.m.-injections of r-hTIMP-2 also resulted in a reduced but not statistically significant number of pulmonary metastases. In addition to these antimetastatic effects, a slight inhibitory effect on tumor cell growth was observed in vitro and in vivo. In conclusion, the antimetastasis by r-hTIMP-2 may be due to inhibition of the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) and, in part, to the suppression of the tumor cell growth.

Intraportal vein injection of highly metastatic L5 cells consistently resulted in liver metastases (increases in the number of tumor colonies in the liver), whereas inoculation of P cells rarely did. L5 cells invaded the basement membrane Matrigel in greater numbers than did P cells, suggesting that the metastatic potential of L5 cells is partly related to enhanced invasive properties. The enhanced adhesion of L5 cells to fibronectin-, laminin- and Matrigel-coated substrates, as well as their haptotactic migration to fribronectin, may be associated with the preferential expression of VLA-2 and VLA-4 integrins on the surface of these cells detected by flow cytometry. Gelatin zymograms showed that the degradative activity of 72-kD gelatinases was greater in L5 cells than P cells. These results indicate that, in addition to adhesiveness and motility, the invasive ability of L5 cells may also be attributed to enhanced gelatinolytic activity. L5 cells grew more rapidly than P cells in vitro. Thus, an experimental model using highly metastatic colon 26 L5 cells would be useful for analyzing the molecular mechanism of liver metastasis and for evaluating the efficacy of treatment of occult micrometastases which may already have been disseminated at the time of surgery.

Macrolides are used to treat various inflammatory diseases owing to their immunomodulatory properties; however, little is known about their precise mechanism of action. In this study, we investigated the functional significance of the expansion of myeloid-derived suppressor cell (MDSC)-like CD11b+Gr-1+ cells in response to the macrolide antibiotic clarithromycin (CAM) in mouse models of shock and post-influenza pneumococcal pneumonia as well as in humans. Intraperitoneal administration of CAM markedly expanded splenic and lung CD11b+Gr-1+ cell populations in naïve mice. Notably, CAM pretreatment enhanced survival in a mouse model of lipopolysaccharide (LPS)-induced shock. In addition, adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice against LPS-induced lethality via increased IL-10 expression. CAM also improved survival in post-influenza, CAM-resistant pneumococcal pneumonia, with improved lung pathology as well as decreased interferon (IFN)-γ and increased IL-10 levels. Adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice from post-influenza pneumococcal pneumonia. Further analysis revealed that the CAM-induced CD11b+Gr-1+ cell expansion was dependent on STAT3-mediated Bv8 production and may be facilitated by the presence of gut commensal microbiota. Lastly, an analysis of peripheral blood obtained from healthy volunteers following oral CAM administration showed a trend toward the expansion of human MDSC-like cells (Lineage-HLA-DR-CD11b+CD33+) with increased arginase 1 mRNA expression. Thus, CAM promoted the expansion of a unique population of immunosuppressive CD11b+Gr-1+ cells essential for the immunomodulatory properties of macrolides.

We often come across difficult to treat infections-even after administering appropriate antibiotics according to the minimal inhibitory concentration of the causative bacteria. Antibiotic tolerance has recently started to garner attention as a crucial mechanism of refractory infections. However, few studies have reported the correlation between clinical outcomes and antibiotic tolerance. This study aims to clarify the effect of antibiotic tolerance on clinical outcomes of respiratory tract infection caused by Pseudomonas aeuginosa (P. aeruginosa). We examined a total of 63 strains isolated from sputum samples of different patients and conducted a retrospective survey with the medical records of 37 patients with imipenem-sensitive P. aeruginosa infections. Among them, we selected 15 patients with respiratory infections, and they were divided into high-tolerance minimal bactericidal concentration for adherent bacteria (MBCAD)/minimal inhibitory concentration for adherent bacteria (MICAD) ≥ 32 (n = 9) group and low-tolerance MBCAD/MICAD ≤ 16 (n = 6) group for further investigations. The findings indicated that the high-tolerance group consisted of many cases requiring hospitalization. Chest computed tomography findings showed that the disease was more extensive in the high-tolerance group compared to the low-tolerance group. Regarding the bacterial phenotypic characterization, the high-tolerance group significantly upregulated the production of the virulence factors compared to the low-tolerance group. Our study provided evidence that carbapenem tolerance level is a potent prognostic marker of P. aeruginosa infections, and carbapenem tolerance could be a potential target for new antimicrobial agents to inhibit the progression of persistent P. aeruginosa infections.