Suppressive Effect of Juzen-Taiho-To on Lung Metastasis of B16 Melanoma Cells In Vivo

Juzen-Taiho-To (JTT) is well known to be one of Kampo (Japanese herbal) medicine consisted of 10 component herbs and used for the supplemental therapy of cancer patients with remarkably success. However, the precise mechanisms by which JTT could favorably modify the clinical conditions of cancer patients are not well defined. The present study, therefore, was undertaken to examine the possible mechanisms of JTT on prevention of cancer metastasis using experimental mouse model. JTT was well mixed with rodent chow at concentrations of either 0.2 or 1.0%, and administered orally ad libitum, which was started 1 week before tumor cell injection and continue throughout the experiment. Oral administration of JTT at concentration 0.2 and 1.0% into C57BL/6 male mice significantly inhibited tumor metastasis in lungs, which was induced by the intravenous injection of 2 × 105 B16 melanoma cell. JTT at a concentration of 1.0% also significantly suppressed lung metastasis of B16 melanoma cell from hind footpad in C57BL/6 mice. In the second part of experiments, the influence of the depression of natural killer (NK) cell, natural killer T (NKT) cell and several types of cytokines on JTT-mediated inhibition of tumor cell metastasis. Intraperitoneal injection of anti asialo-GM1 antibody against NK cells and anti NK-1.1 monoclonal antibody (mAb) to NKT cells abrogated the inhibitory action of JTT on lung metastasis of B16 melanoma cells. Although intraperitoneal administration of anti-IFN-γ mAb scarcely affected the inhibitory action of JTT on tumor cell metastasis, injection of amrinone, which used for IL-12 suppression, significantly decreased the ability of JTT to prevent tumor cell metastasis. These results strongly suggest that oral administration of JTT caused increase in the production of IL-12, which is responsible for the activation of both NK cell and NKT cell, in the lungs and results in inhibition of B16 melanoma cell metastasis in the lungs

Suppression of matrix metalloproteinase-9 production from neutrophils by a macrolide antibiotic, roxithromycin, in vitro.

BACKGROUND: Macrolide antibiotics such as erythromycin and roxithromycin (RXM) have an anti-inflammatory effect that may account for their clinical benefit in the treatment of chronic airway inflammatory diseases. However, the precise mechanism of this anti-inflammatory effect is not well understood. PURPOSE: The influence of RXM on matrix metalloproteinase (MMP)-9 production from neutrophils in response to lipopolysaccharide (LPS) stimulation was examined in vitro. METHODS: Neutrophils prepared from normal human peripheral blood (1 x 10(5) cells/ml) were treated with various concentrations of RXM for 1 h, and then stimulated with 1.0 microg/ml of LPS in the presence of the agent for 24 h. MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 levels in culture supernatants were examined by enzyme-linked immunosorbent assay. RESULTS: Addition of RXM at more than 5.0 microg/ml into cell cultures caused significant suppression of MMP-9 production, which was increased by LPS stimulation. However, the ability of cells to produce TIMP-1 was not affected by RXM treatment, even when the cells were cultured in the presence of agent at 10.0 microg/mL We then examined the influence of RXM on transcriptional factor, nuclear factor-kappaB and activator protein (AP)-1 activation by LPS stimulation. RXM exerted suppressive action on NF-kappaB (P50 and P65) activation when the cells were cultured for 4 h at more than 5.0 microg/ml of the agent. RXM at more than 2.5 microg/ml also suppressed AP-1 (Fra 1 and Jun B) activation in 4-h cultured cells. We finally examined the influence of RXM on MMP-9 mRNA expression in neutrophils. Addition of RXM into cell cultures at more than 5.0 microg/ml caused significant inhibition of mRNA expression, which was enhanced by LPS stimulation for 12 h. CONCLUSION: These results strongly suggest that RXM inhibits neutrophil transmigration into inflammatory sites and results in favorable modification of the clinical status of inflammatory diseases

Inhibitory action of a macrolide antibiotic, roxithromycin, on co-stimulatory molecule expressions in vitro and in vivo.

OBJECTIVE: The influence of a macrolide antibiotic, roxithromycin (RXM), on co-stimulatory molecule expression was examined in vitro and in vivo. MATERIALS AND METHODS: Spleen cells obtained from BALB/c mice 10 days after immunization with 8.0 microg of hemocyanin absorbed to 4.0 mg of aluminum hydroxide were cultured in the presence of 100.0 microg/ml of hemocyanin and various concentrations of RXM. We first examined the influence of RXM on cell activation by examining the proliferative response of cells and cytokine production. We also examined the influence of RXM on co-stimulatory molecule (CD40, CD80 and CD86) expressions on cultured splenic B-lymphocytes induced by in vitro antigenic stimulation using flow cytometry. In the second part of experiments, non-immunized and immunized mice were treated orally with 2.5 mg/kg of RXM once a day for 4 or 8 weeks. Splenic B lymphocytes were obtained from these mice 24 h after antigenic challenge, and co-stimulatory molecule expressions were examined by flow cytometer. RESULTS: Cell activation induced by in vitro antigenic stimulation was suppressed by RXM when cells were cultured in the presence of more than 5.0 microg/ml of the agent. Addition of RXM at a concentration of 5.0 microg/ml into cell cultures also suppressed co-stimulatory molecule (CD40, CD80 and CD86) expressions on splenic B lymphocytes, which was enhanced by antigenic stimulation in vitro. Oral RXM administration for 4 weeks clearly suppressed the enhancement of CD40 and CD86 (but not CD80) expressions on splenic B lymphocytes induced by antigenic stimulation in vivo. This suppressive activity of RXM on co-stimulatory molecule (CD40 and CD86) expressions was further strengthened by the treatment of mice for 8 weeks. Long-term treatment with oral RXM also suppressed CD80 expressions, which was not suppressed by 4-week treatment. CONCLUSION: The present results suggest that RXM exerts its immunomodulating effects through suppression of both cell activation and co-stimulatory molecule expressions induced by antigenic stimulation. These suppressive activities of RXM might contribute, in part, to the therapeutic mode of action of RXM on inflammatory diseases

Suppression of Matrix Metalloproteinase Production in Nasal Fibroblasts by Tranilast, an Antiallergic Agent, In Vitro

Allergic rhinitis is an inflammatory disease characterized by nasal wall remodeling with intense infiltration of eosinophils and mast cells/basophils. Matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are the major proteolytic enzymes that induce airway remodeling. These enzymes are also important in the migration of inflammatory cells through basement membrane components. We evaluated whether tranilast (TR) could inhibit MMP production from nasal fibroblasts in response to tumor necrosis factor-α (TNF-α) stimulation in vitro. Nasal fibroblasts (NF) were established from nasal polyp tissues taken from patients with allergic rhinitis. NF (2 × 10(5) cells/mL) were stimulated with TNF-α in the presence of various concentrations of TR. After 24 hours, the culture supernatants were obtained and assayed for MMP-2, MMP-9, TIMP-1, and TIMP-2 levels by ELISA. The influence of TR on mRNA expression of MMPs and TIMPs in cells cultured for 12 hours was also evaluated by RT-PCR. TR at more than 5 × 10(−5) M inhibited the production of MMP-2 and MMP-9 from NF in response to TNF-α stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected. TR also inhibited MMP mRNA expression in NF after TNF-α stimulation. The present data suggest that the attenuating effect of TR on MMP-2 and MMP-9 production from NF induced by inflammatory stimulation may underlie the therapeutic mode of action of the agent in patients with allergic diseases, including allergic rhinitis

Suppression of nitric oxide production from nasal fibroblasts by metabolized clarithromycin in vitro

<p>Abstract</p> <p>Background</p> <p>Low-dose and long-term administration of 14-membered macrolide antibiotics, so called macrolide therapy, has been reported to favorably modify the clinical conditions of chronic airway diseases. Since there is growing evidence that macrolide antibiotic-resistant bacteria's spreaders in the populations received macrolide therapy, it is strongly desired to develop macrolide antibiotics, which showed only anti-inflammatory action. The present study was designed to examine the influence of clarithromycin (CAM) and its metabolized materials, M-1, M-4 and M-5, on free radical generation from nasal polyp fibroblasts (NPFs) through the choice of nitric oxide (NO), which is one of important effector molecule in the development of airway inflammatory disease <it>in vitro</it>.</p> <p>Methods</p> <p>NPFs (5 × 10⁵cells/ml) were stimulated with 1.0 μg/ml lipopolysaccharide (LPS) in the presence of agents for 24 hours. NO levels in culture supernatants were examined by the Griess method. We also examined the influence of agents on the phosphorylation of MAPKs, NF-κB activation, iNOS mRNA expression and iNOS production in NPFs cultured for 2, 4, 8, and 12 hours, respectively.</p> <p>Results</p> <p>The addition of CAM (> 0.4 μg/ml) and M-4 (> 0.04 μg/ml) could suppress NO production from NPFs after LPS stimulation through the suppression of iNOS mRNA expression and NF-κB activation. CAM and M-4 also suppressed phosphorylation of MAPKs, ERK and p38 MAPK, but not JNK, which are increased LPS stimulation. On the other hand, M-1 and M-5 could not inhibit the NO generation, even when 0.1 μg/ml of the agent was added to cell cultures.</p> <p>Conclusion</p> <p>The present results may suggest that M-4 will be a good candidate for the agent in the treatment of chronic airway inflammatory diseases, since M-4 did not have antimicribiological effects on gram positive and negative bacteria.</p

Influence of Epinastine Hydrochloride, an H1-Receptor Antagonist, on the Function of Mite Allergen-Pulsed Murine Bone Marrow-Derived Dendritic Cells In Vitro and In Vivo

There is established concept that dendritic cells (DCs) play essential roles in the development of allergic immune responses. However, the influence of H1 receptor antagonists on DC functions is not well defined. The aim of the present study was to examine the effect of epinastine hydrochloride (EP), the most notable histamine H1 receptor antagonists in Japan, on Dermatophagoides farinae (Der f)-pulsed mouse bone marrow-derived DCs in vitro and in vivo. EP at more than 25 ng/mL could significantly inhibit the production of IL-6, TNF-α and IL-10 from Der f-pulsed DCs, which was increased by Der f challenge in vitro. On the other hand, EP increased the ability of Der f-pulsed DCs to produce IL-12. Intranasal instillation of Der f-pulsed DCs resulted in nasal eosinophilia associated with a significant increase in IL-5 levels in nasal lavage fluids. Der f-pulsed and EP-treated DCs significantly inhibited nasal eosinophila and reduced IL-5. These results indicate that EP inhibits the development of Th2 immune responses through the modulation of DC functions and results in favorable modification of clinical status of allergic diseases

Suppressive Effect of Juzentaihoto on Vascularization Induced by B16 Melanoma Cells In Vitro and In Vivo

Juzentaihoto (JTT) is well known to be one of Japanese herbal medicines, and used for the supplemental therapy of cancer patients with remarkable success. The present study, therefore, was undertaken to examine the possible therapeutic mechanisms of JTT on cancer using B16 melanoma cell (B16 cell)/experimental mouse system. JTT was well mixed with rodent chow at 3.0% concentrations, and was administered orally ad libitum. Administration of JTT was started one week before tumor cell injection and continued throughout the experiment. Administration of JTT into mice significantly inhibited tumor metastasis in lungs after intravenous injection of 2 × 105 B16 cells in a volume of 50 μL. JTT also significantly suppressed enlargement of tumor size in hind footpad after the subcutaneous injection of 2 × 105 (50 μL) B16 cells. In the second part of experiments, the chamber that containing B16 cells was buried in the murine back. In JTT administrated group, vascular endothelial growth factor (VEGF) of chamber internal fluid significantly decreased, and vascularization of chamber circumference was also inhibited. These results strongly suggest that oral administration of JTT caused decrease in the generation of VEGF, which is responsible for vascularization, and results in inhibition of B16 cell metastasis