Suppressive effect of Shichimotsu-koka-to (Kampo medicine) on pulmonary metastasis of B16 melanoma cells

Shichimotsu-koka-to (SKT) is a Kampo (traditional Japanese herbal) medicine, which is used in Japan to treat hypertension and atherosclerosis. We investigated the inhibitory effect of SKT on experimental pulmonary metastasis of B16 melanoma cells. The intake of SKT at a dose of 430 mg/kg for 6 weeks from 2 weeks before tumor inoculation significantly reduced the number of metastatic surface nodules in the lung and extended the life span. When the duration of SKT intake was examined, survival time was not affected by preintake before B16 melanoma cell inoculation and was slightly extended by postintake after B16 melanoma cell inoculation, although the life span was prolonged by intake throughout the experiment. To address the mechanism underlying the antimetastatic effect of SKT, we studied whether SKT modulated macrophage function, which is involved in killing tumor cells. The intake of SKT for 6 weeks dose dependently increased nitric oxide (NO) production by macrophages following stimulation with lipopolysaccharide. The elevated NO was found to serve as a cytotoxic mediator against B16 melanoma cells in co-culture with macrophages. On the contrary, B16 melanoma-conditioned medium reduced NO production by macrophages. However, SKT treatment reversed the reduction in NO production by the conditioned medium significantly. These findings may suggest that macrophage function-modulating activity by SKT appears to underlie its antimetastatic activity, which leads to a decrease in the number of lung metastatic surface nodules and the extension of life span.     

三肽化合物酪丝亮肽对巨噬细胞吞噬及肿瘤细胞杀伤功能的影响

目的：研究三肽化合物酪丝亮肽(YSL)对巨噬细胞(M(?))吞噬功能、肿瘤细胞杀伤功能和细胞因子分泌功能的影响。方法：通过碳粒廓清实验观察YSL对小鼠M(?)吞噬功能的影响,采用MTT法观察YSL对腹腔M(?)杀伤人肝癌细胞BEL-7402和小鼠黑色素瘤细胞B16-F10作用的影响,用ELISA法观察YSL对M(?)株RAW264．7分泌细胞因子IL-1β和NO功能的影响。结果：YSL 80,160,320μg·kg~(-1)·d~(-1)均能够明显增强小鼠M(?)的吞噬功能(P＜0．01),YSL各浓度(0．1,1,10,100 mg·L~(-1))在体外明显增强小鼠腹腔M(?)对肿瘤细胞BEL-7402和B16-F10的杀伤作用(P＜0．01)。YSL可以促进细胞毒效应分子IL- 1β和NO的分泌合成,NO和IL-1β水平分别在YSL作用12 h,24 h时达到高峰。结论：YSL可以增强M(?)吞噬功能,增强对肿瘤细胞的杀伤作用,促进细胞因子IL-1β和NO分泌。     

Effect of itraconazoles on the production of pro-inflammatory substances in mouse macrophage-like cells

BACKGROUND, MATERIALS AND METHODS: Synthetic triazoles are widely used for the treatment of fungal infection. In order to understand their possible anti-inflammatory action, we investigated the effect of itraconazole and its hydroxylated derivative (hydroxyitraconazole) on the production of various pro-inflammatory substances by mouse macrophage-like RAW264.7 cells. RESULTS: These compounds did not apparently show any growth inhibitory or stimulatory effects over a wide range of concentrations (0.2-50 μg/ml). Itraconazoles dose-dependently increased the production of prostaglandin E? (PGE?) and tumor necrosis factor-α (TNF-α) without affecting the production of interleukin-1β (IL-1β) and nitric oxide (NO). LPS treatment significantly enhanced the production of NO, PGE?, TNF-α and IL-1β. The addition of itraconazoles to LPS-stimulated RAW264.7 cells significantly reduced the production of NO, but rather enhanced the production of PGE?, TNF-α and IL-1β. ESR spectroscopy demonstrated that itraconazoles did not significantly scavenge NO and superoxide anion radicals, indicating that the inhibition of NO production by itraconazoles is not due to their radical-scavenging activity. Hydroxyitraconazole was slightly more cytostatic, and more efficiently inhibited NO production, but enhanced the production of other pro-inflammatory substances. CONCLUSION: These data suggest that itraconazoles regulate NO and other pro-inflammatory substances differently in activated macrophages.     

Anti-inflammatory and immunomodulatory properties of 2-amino-3H-phenoxazin-3-one

Accumulating evidence suggests that nitric oxide (NO) and prostaglandin E(2) (PGE(2)) are involved in the pathogenesis of various chronic inflammatory diseases and cancer. During the course of a screening program to identify natural anti-inflammatory substances, we isolated the compound 2-amino-3H-phenoxazin-3-one (APO) from an extract of the edible brown mushroom Agaricus bisporus IMBACH. APO inhibited NO production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharide (LPS) and interferon (IFN)-gamma (LPS/IFN-gamma) at low concentrations (IC(50)=1.5 microM) through reduced inducible NO synthase protein expression. PGE(2) production by LPS/IFN-gamma-stimulated macrophages was inhibited by APO at much lower concentrations (IC(50)=0.27 microM) than those required for the inhibition of NO production. Mechanistic analysis showed that APO inhibited both cyclooxygenase (COX)-1 and COX-2 enzyme activities with almost equal selectivity. Secretion of NO and the pro-inflammatory cytokine IL-6 by IFN-gamma-activated RAW264.7 cells, a murine macrophage-like cell line, was also dose-dependently reduced by APO. Furthermore, APO increased the secretion of the anti-inflammatory cytokine IL-4 by antigen-stimulated T cells and promoted the polarization of CD4(+) Th cells toward the anti-inflammatory Th2 phenotype at equimolar concentrations that inhibited NO production. Our results suggested that APO induced polarization toward the Th2 subset, at least in part through the down-regulation of IL-12 production. Thus, APO appears to have potent anti-inflammatory and immunoregulatory properties that may provide a promising therapeutic strategy for the treatment of T cell-mediated inflammatory autoimmune diseases as well as for bacteria-induced chronic-inflammatory diseases.     

双环醇对脂多糖诱导巨噬细胞iNOS蛋白的表达和NF-κB活化的抑制作用

目的炎症是肝炎病毒或其它因素所致的肝损伤的一个共同特征,该文目的系研究抗肝炎新药双环醇对与炎症反应相关分子的调节作用。方法以无毒性浓度双环醇预先与巨噬细胞株RAW264.7和小鼠腹腔巨噬细胞温孵1h后,加入一定量脂多糖(LPS)共同培养适当时间以诱导上述细胞的活化,培养上清中NO2-的含量和TNF-α的水平分别用Griess试剂及L929细胞株生物法测定,用Westernblot方法测定iNOS蛋白的表达和NF-κB的活化。结果双环醇在0.1～0.5mmol.L-1剂量依赖性降低1mg.L-1LPS引起的RAW264.7和小鼠腹腔巨噬细胞NO的释放以及TNF-α的分泌,双环醇0.5mmol.L-1能够明显抑制1mg.L-1LPS引起的iNOS蛋白的表达和NF-κB的活化。结论双环醇对与炎症相关的调控因子iNOS蛋白的表达和NF-     

Cytotoxicity studies of Dynasan 114 solid lipid nanoparticles (SLN) on RAW 264.7 macrophages-impact of phagocytosis on viability and cytokine production

Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of cytokines such as interleukin 6 (IL-6), IL-12 and tumour necrosis factor-alpha (TNF-alpha). Results were compared with previously obtained data when peritoneal mouse macrophages were used. SLN produced with stabilizers/surfactants such as poloxamer 188, sodium cholate, Lipoid S75, Tween 80, Poloxamine 908 and sodium dodecylsulfate were shown to be nontoxic towards RAW 264.7 cells. Cytokine production was reduced and stimulation, expressed in elevated cytokine levels, could not be found. Using cetylpyridinium chloride (CPC) as stabilizing surfactant, SLN became cytotoxic in a concentration-dependent manner. Not only were the viabilities reduced but also cytokine production. Cytotoxic effects of CPC stabilized SLN could be antagonized using cytochalasin B to block phagocytosis. D114-SLN produced with pharmaceutically accepted surfactants for intravenous injection (poloxamer 188, Lipoid S75, sodium cholate, Tween 80) were very well tolerated by the cells. Even sodium dodecylsulfate-stabilized D114-SLN did not exert toxic effects. Comparison of the RAW 264.7 data with previously obtained data from toxicity studies of D114-SLN towards peritoneal mouse macrophages showed similar results. This offers the possibility of using the RAW 264.7 cell line for cytotoxicity studies of colloidal drug carrier systems, rather than using laboratory animals as source of macrophages for these kinds of studies.     

Antitumor activity of the Korean Mistletoe Lectin is attributed to activation of macrophages and NK cells

Inhibitory effect of the lectins (KML-C) isolated from Korean mistletoe (KM; Viscum album coloratum) on tumor metastases produced by murine tumor cells (B16-BL6 melanoma, colon 26-M3.1 carcinoma and L5178Y-ML25 lymphoma cells) was investigated in syngeneic mice. An intravenous (i.v.) administration of KML-C (20-50 ng/mouse) 2 days before tumor inoculation significantly inhibited lung metastases of both B16-BL6 and colon 26-M3.1 cells. The prophylactic effect of 50 ng/mouse of KML-C on lung metastasis was almost the same with that of 100 microg/mouse of KM. Treatment with KML-C 1 day after tumor inoculation induced a significant inhibition of not only the experimental lung metastasis induced by B16-BL6 and colon 26-M3.1 cells but also the liver and spleen metastasis of L5178Y-ML25 cells. Furthermore, multiple administration of KML-C given at 3 day-intervals after tumor inoculation led to a significant reduction of lung metastasis and suppression of the growth of B16-BL6 melanoma cells in a spontaneous metastasis model. In an assay for natural killer (NK) cell activity, i.v. administration of KML-C (50 ng/mouse) significantly augmented NK cytotoxicity against Yac-1 tumor cells 2 days after KML-C treatment. In addition, treatment with KML-C (50 ng/mouse) induced tumoricidal activity of peritoneal macrophages against B16-BL6 and 3LL cells. These results suggest that KML-C has an immunomodulating activity to enhance the host defense system against tumors, and that its prophylactic and therapeutic effect on tumor metastasis is associated with the activation of NK cells and macrophages.     

Inhibitory effects of hydrolyzable tannins from Melastoma dodecandrum Lour. on nitric oxide production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide and interferon-gamma.

An extract of Melastoma dodecandrum LOUR. with 80% aqueous acetone (MDL) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide (LPS) and recombinant mouse interferon-gamma (IFN-gamma). On further fractionation of the extract, the majority of the inhibitory activity was recovered in the 50% methanol extracts, which contained hydrolyzable tannins. Among the latter, casuarinin, casuarictin, pedunclagin and nobotannin B exhibited strong inhibitory activities toward NO production, with ID50 values between 2.0 and 5.1 microM. Both MDL and the purified tannins significantly reduced the induction of the inducible nitric oxide synthase (iNOS) protein in the course of macrophage activation with LPS and IFN-gamma. In addition, the NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by these tannins, with IC50 values around 30-130 microM, but not by MDL. These results suggest that MDL has the pharmacological ability to suppress NO production by activated macrophages and that the hydrolyzable tannins have major inhibitory activities.     

Analysis of the mechanism for the anti-inflammatory effect of the anti-rheumatic drug auranofin

Effects of auranofin, an orally active chrysotherapeutic agent, were examined on the production of prostaglandin E2 (PGE2) and nitric oxide (NO) in rat peritoneal macrophages and in RAW 264.7 cells, a murine macrophage-like cell line. Auranofin (1-10 microM) inhibited PGE2 production in rat peritoneal macrophages stimulated with 12-O-tetra-decanoylphorbol 13-acetate (TPA, 16.2 nM) at 8-20 h, but did not affect PGE2 production at 4 h. However, in non-stimulated rat peritoneal macrophages, auranofin increased PGE2 production at 4 h and had no effect on PGE2 production at 8-20 h. It was proved that auranofin (1-10 microM) increased COX (cyclooxygenase)-1-dependent PGE2 production and inhibited COX-2-dependent PGE2 production in rat peritoneal macrophages. Auranofin showed no effect on the enzyme activities of the purified COX-1 and COX-2 proteins. Furthermore, auranofin did not affect the COX-1 protein level, but inhibited the TPA-induced expression of COX-2 protein. Therefore, it was suggested that auranofin inhibited PGE2 production by inhibiting the COX-2 protein induction in TPA-stimulated macrophages. In RAW 264.7 cells, auranofin (0.3-3 microM) inhibited lipopolysaccharide-induced NO synthesis by inhibiting the induction of NO synthase (NOS) protein expression. Auranofin did not affect the enzyme activity of iNOS (inducible NOS). Finally, using rat peritoneal macrophages, the effects of auranofin on PGE2 production and NO production were determined. Auranofin (10 microM) strongly inhibited the production of PGE2 and NO, and the induction of COX-2 protein and NOS protein by TPA. Indomethacin, a COX inhibitor, partially inhibited NO production at the concentration at which PGE2 production was completely inhibited. On the other hand, L-NG-monomethyl-L-arginine acetate (L-NMMA), a NOS inhibitor, partially inhibited PGE2 production. NO production was completely inhibited at the same concentration as shown above. These findings suggest that PGE2 production and NO production partially affect each other. Therefore, the inhibition of PGE2 production by auranofin might be partly due to the inhibition of NO production, and the inhibition of NO production by auranofin be partly due to the inhibition of PGE2 production. In conclusion, auranofin inhibits both PGE2 production and NO production by inhibiting the upregulation of mRNA levels of COX-2 and NOS.     

Immunomodulatory effect of arctigenin, a lignan compound, on tumour necrosis factor-alpha and nitric oxide production, and lymphocyte proliferation

We have investigated the immunomodulatory effects of arctigenin, a dibenzyl butyrolactone lignan compound, on tumour necrosis factor (TNF)-alpha and nitric oxide (NO) production, and lymphocyte proliferation. Arctigenin inhibited strongly TNF-alpha production by lipopolysaccharide-stimulated murine macrophage RAW264.7 and differentiated human macrophage U937 with IC50 values of 5.0 and 3.9 microM, respectively, without displaying cytotoxicity. The TNF-alpha inhibitory effect of arctigenin in lipopolysaccharide-triggered RAW264.7 cells was increased by co-treatment with several known TNF-alpha inhibitors. It also potently attenuated T and B cell proliferation stimulated by concanavalin A and lipopolysaccharide in a dose-dependent manner with IC50 values of 2.9 and 14.6 microM, respectively. In contrast, the compound showed a different pattern in lipopolysaccharide- and interferon (IFN)-gamma-induced NO production from RAW264.7 cells. Arctigenin inhibited NO release by IFN-gamma signal, whereas it significantly enhanced lipopolysaccharide-triggered NO production in RAW264.7 cells. The results suggested that arctigenin may regulate immune responses in activated macrophages and lymphocytes including TNF-alpha and NO production and lymphocyte proliferation.     

Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo.

The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2-200 microg/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a concentration-dependent manner. In vivo administration of T. indica extract (100-500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-gamma induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity.     

Antitumor and antimetastatic activity of an antibiotic, ascofuranone, and activation of phagocytes

Ascofuranone demonstrated antitumor activity against FM3A murine mammary carcinoma, implanted in the peritoneal cavity of syngeneic mice, C3H/He. It was more effective by treatment prior to implantation than by that after implantation. Treatment with ascofuranone also increased splenic cytotoxicity and phagocytic activity of host animal cells. Moreover, ascofuranone induced inflammatory cells in the peritoneal cavity which are mainly composed of polymorphonuclear leukocytes and macrophages. These cells are more potent in cytotoxicity against FM3A cells than with resident peritoneal cells. The antitumor activity of ascofuranone was suppressed by ip administration of silica, just prior to tumor implantation. These results suggest that the prophylactic antitumor activity of ascofuranone is expressed through the activation of phagocytes. Ascofuranone also suppressed pulmonary metastasis of B16 melanoma and Lewis lung carcinoma. Treatment after tumor implantation failed to suppress the metastasis. Single treatment of ascofuranone 4 days prior to implantation decreased the metastasis of Lewis lung carcinoma but not that of B16, whereas single treatment of ascofuranone 24 hours prior to the tumor implantation decreased the metastasis of B16 but not that of Lewis lung carcinoma.     

Anti-inflammatory and cytotoxic activities of five Veronica species

Biological activities of five Veronica species (Scrophulariaceae), V. cymbalaria, V. hederifolia, V. pectinata var. glandulosa, V. persica and V. polita were studied for their anti-inflammatory and cytotoxic activities. Their methanol extracts showed both the inhibitory activity of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages and cytotoxic activity against KB epidermoid carcinoma and B16 melanoma. When the methanol extracts were fractionated between water and chloroform, water fractions significantly inhibited NO production without any cytotoxicity, while chloroform fractions showed cytotoxicity dose-dependently. When the radical scavenging activity was determined using 2,2-diphenyl-1-picryl-hydrazyl (DPPH), water fractions of the five Veronica species scavenged free radicals effectively, suggesting that the inhibitory effect of this species on NO production was due to their radical scavenging activity. On the other hand, chloroform fractions of Veronica species except for V. cymbalaria showed similar cytotoxic activity against KB and B16 melanoma cells.     

Immunomodulation by ethanolic extract of Boerhaavia diffusa roots

We have earlier reported that ethanolic extract of Boerhaavia diffusa, a plant used in Indian traditional system of medicine, significantly inhibits the cell proliferation. This led us to evaluate the immunomodulatory properties of this plant extract on various in vitro tests such as human natural killer (NK) cell cytotoxicity, production of nitric oxide (NO) in mouse macrophage cells, RAW 264.7, interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), intracytoplasmic interferon-gamma (IFN-gamma) and expression of various cell surface markers on human peripheral blood mononuclear cells (PBMCs). Ethanolic extracts of B. diffusa roots inhibited human NK cell cytotoxicity in vitro, production of NO in mouse macrophage cells, IL-2 and TNF-alpha in human PBMCs. Intracytoplasmic IFN-gamma and cell surface markers such as CD16, CD25, and HLA-DR did not get affected on treatment with B. diffusa extract. Our study demonstrates immunosuppressive potential of ethanolic extract of B. diffusa.     

Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris

In this study, the signaling mechanism of the polysaccharides isolated from fruiting body of Cordyceps militaris (CM) was investigated in macrophages to evaluate its immuno-stimulating properties. We found that CM was capable of upregulation of NO, ROS, TNF-α and phagocytic uptake in mouse peritoneal macrophages and RAW264.7 macrophages. Macrophages activation by CM seemed to occur via activation of NF-κB and all three MAPKs pathways through dectin-1 and TLR2 macrophage receptors. Additionally, we showed that CM suppressed in vivo growth of melanoma in an experimental mouse model. Based on these data, we suggested that CM may potentially regulate the immune response.     

重组人纤连蛋白CH50对黑色素瘤生长及转移的影响

目的：研究重组人纤连蛋白ＣＨ５０对小鼠黑色素瘤生长的影响及其抗肿瘤机制。方法 进行小鼠动物实验及黑色素瘤Ｂ１６细胞实验。结果：ＣＨ５０在体内明显抑制黑色素瘤生长及实验性肺转移,ＣＨ５０在体外能附粘黑色素瘤Ｂ１６细胞,抑制Ｂ１６细胞粘附层粘蛋白并明显提高腹腔巨噬细胞对Ｂ１６细胞杀伤活性,结论：ＣＨ５０抑制小鼠黑色素瘤的生长和转移,ＣＨ５０的抗肿瘤机制与其粘附肿瘤细胞,提高巨噬细胞杀瘤性活性有关。