Young Jin Jeon

The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis.

It was previously reported that polysaccharides (PL) isolated from Phellinus linteus strongly stimulated cell-mediated and humoral immunity. This study was undertaken to investigate the immunochemotherapeutic activity of PL against tumor growth and metastasis. PL alone significantly prolonged the survival rate of B16F10-implanted mice, inhibited tumor growth in NCI-H23-implanted nude mice, and reduced the frequency of pulmonary metastasis of B16F10 melanoma. Adriamycin significantly inhibited tumor growth, but only slightly inhibited metastasis. The combination therapy with PL and adriamycin was more effective in inhibiting tumor growth, but not metastasis. PL did not induce direct toxicity in cancer cells, which is characteristic of immunotherapeutics. In conclusion, PL might be of use in immunochemotherapy of cancer because of its effective activities on tumor growth and metastasis through the immunopotentiation of the patients without toxicity.

Dexamethasone inhibits IL-1β gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-κB/Rel and AP-1 activation

In the present study, the mechanism by which dexamethasone (DEX) inhibited IL-1beta gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in LPS-induced IL-1beta mRNA expression was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Since the promoter in IL-1beta gene contains binding motifs for NF-kappaB/Rel, AP-1, NF-IL6, and CREB/ATF, which appear to be important in LPS-mediated IL-1beta induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-kappaB/Rel and AP-1 in chloramphenicol acetyltransferase activity, while neither NF-IL6 nor CREB/ATF activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-kappaB/Rel and AP-1 proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay (EMSA). DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. DEX treatment of RAW 264.7 cells did not inhibit the nuclear translocation of c-jun and c-fos. We found that the inhibition of IL-1beta production by DEX is not related to p38, which is important in the IL-1beta induction. These results suggest that DEX may inhibit IL-1beta gene expression by a mechanism involving the blocking of LPS-induced NF-kappaB/Rel and AP-1 activation.

Activation of NF-κB/Rel in angelan-stimulated macrophages

In our previous studies we showed that the primary target cell of angelan, a polysaccharide purified from Angelica gigas Nakai, is a macrophage (Han et al., 1998). In the present study we examined the effect of angelan on iNOS, IL-1β, and TNF-α transcription in mouse macrophage line RAW 264.7. We show that angelan produces a marked induction of iNOS, IL-1β, and TNF-α transcription by RAW 264.7 cells. Since these gene transcriptions have been recently shown to be under the control of NF-κB/Rel family of transcription factors, we assessed the effect of angelan on NF-κB/Rel using a electrophoretic mobility shift assay. Treatment of RAW 264.7 cells with angelan produced strong induction of NF-κB/Rel binding. Treatment of RAW 264.7 cells with angelan slightly induced AP-1 binding activity, whereas Oct binding was not affected by angelan. Angelan stimulated macrophages to activate NF-κB/Rel, whereas neither B-cells nor T-cells were affected by the angelan. In conclusion, we demonstrate that the stimulation effect of angelan on macrophage is mediated by specific activation of NF-κB/Rel.

Cytotoxicity of urushiols isolated from sap of Korean lacquer tree (Rhus vernicifera stokes)

Cytotoxicities of four urushiols, congeners isolated from the sap of Korean lacquer tree (Rhus vernicifera Stokes), to 29 human cancer cell lines originated from 9 organs were evaluated. Their values of 50% growth inhibition were below 4 μg/ml, and showed cell line specific cytotoxicity. The present result is the first report on the cytotoxicity of urushiols suggesting that they would have an anticancer activity to human cancer cells.

Suppression of IL-8 gene expression by radicicol is mediated through the inhibition of ERK1/2 and p38 signaling and negative regulation of NF-κB and AP-1

We show that radicicol, an anti-fungal agent, inhibits interleukin-8 (IL-8) production by the human monocyte line THP-1 in response to phorbol-12-myristate-13-acetate/lipopolysaccharide (PMA/LPS). IL-8 is a potent chemokine and needs for an optimal immune response--such as inflammation by activation of neutrophils. The decrease in PMA/LPS-induced IL-8 mRNA expression was demonstrated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Since the promoter in IL-8 gene contains binding motifs for NF-KB, AP-1. and NF-IL6, which appear to be important in IL-8 induction, the effects of radicicol on the activation of these transcription factors were examined. Treatment of radicicol to THP-1 cells produced a strong inhibition of NF-KB and AP-1, while NF-IL6 was not significantly affected by radicicol. Western blot analysis showed that radicicol inhibited the phosphorylation and phosphotransferase activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38. PD98059 and SB203580, known as a specific inhibitor of MEKI and p38 kinase, respectively, inhibited IL-8 gene expression showing that both of the kinase pathways are involved in IL-8 regulation in human monocytes. Collectively, this series of experiments indicates that radicicol inhibits IL-8 gene expression by blocking ERK1/2 and p38 signaling.

Differential activation of murine macrophages by angelan and LPS

In our previous studies, we showed that angelan, a polysaccharide purified from Angelica gigas Nakai, is a potent LPS-mimetic in murine macrophages [Jeon, Y.J., Han, S.B., Ahn, K.S., Kim, H.M., 1999. Activation of NF-kB/Rel in angelan-stimulated macrophages. Immunopharmacology 43, 1-9]. Angelan stimulates murine macrophage to produce cytokines including iNOS and activate NF-kappaB/Rel. In the present study, we investigated the role of CD14 and complement receptor type 3 (CR3) in mediating NO production and NF-kappaB/Rel activation induced by angelan and LPS. Three major differences between angelan and LPS were observed. First, angelan does not require serum proteins for NO response and NF-kappaB/Rel activation, while the activation by LPS requires serum proteins. Second, blocking of either CD14 or CR3 decreased angelan-induced NO response, while LPS-mediated NO production was inhibited by anti-CD14 mAb only. Third, angelan induced strong NF-kappaB/Rel and slight AP-1 DNA binding, whereas LPS potently activated both NF-kappaB/Rel and AP-1. Both angelan and LPS degraded IkappaB proteins and subsequently induced the mobilization of NF-kappaB/Rel proteins (p65, c-rel and p50) into nucleus. This suggests that macrophages display a common signaling machinery leading to the NF-kappaB/Rel activation in response to different stimulants. In conclusion, angelan and LPS use the membrane receptor CD14 and CR3 differentially for signaling NF-kappaB/Rel activation and NO production.

Experimental evidences and signal transduction pathways involved in the activation of NF-κB/Rel by angelan in murine macrophages

In our previous studies, we showed that angelan, a polysaccharide purified from Angelica gigas Nakai, activated macrophages to induce the translocation of NF-kappa B/Rel into nucleus and DNA binding to its cognate site in the promoter of iNOS gene [Immunopharmacology 43 (1999) 1; Immunopharmacology 49 (2000) 275]. In the present study, we showed that angelan induces the transcriptional activation of NF-kappa B/Rel and investigated the intracellular signal transduction pathways involved in the angelan-induced NF-kappa B/Rel activation by murine macrophages. Treatment of RAW 264.7 cells with angelan resulted in significant activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38, while stress-activated protein kinase/c-Jun NH2 terminal kinase (SAPK/JNK) was not activated by angelan. The specific p38 inhibitor SB203580 abrogated the angelan-induced NF-kappa B/Rel activation, whereas the selective MAPK/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD98059 did not affect the NF-kappa B/Rel induction. Treatment of RAW 264.7 cells with both anti-CD14 Ab and anti-CR3 Ab significantly blocked angelan-induced NF-kappa B/Rel activation. In conclusion, we demonstrate that angelan induces NF-kappa B/Rel activation through the CD14 and CR3 membrane receptor and p38 kinase that is critically involved in the signal transduction leading to NF-kappa B/Rel activation in murine macrophages.

Paclitaxel causes mouse splenic lymphocytes to a state hyporesponsive to lipopolysaccharide stimulation.

Multiple immune system actions have been ascribed to paclitaxel (taxol), a novel anticancer drug, including the capacity to induce macrophage antitumor cytotoxic molecule production. In the present studies, we demonstrated that paclitaxel produced a selective inhibition of lipopolysaccharide (LPS)-induced B cell proliferation. Similarly, in vitro polyclonal antibody-forming cell responses also were found to be inhibited by paclitaxel. Conversely, paclitaxel exhibited no inhibitory effects on concanavalin A (Con A)-induced T cell proliferation. To study the pathway leading to paclitaxel-induced immunosuppression, we analyzed Raf-1/ERK and JNK/p38 MAPK pathways, both of which have been reported to be involved in LPS signaling. Our results indicate that taxol treatment inhibits Raf-1 kinase activation while having no effect on ERK activation suggesting that ERK activation is distinct from upstream Raf-1 kinase in taxol-induced immunomodulation. Furthermore, paclitaxel pretreatment caused down-regulation of stress-activated MAPKs, JNK and p38 MAPK in lipopolysaccharide (LPS)-stimulated mouse splenic lymphocytes, demonstrating that spleen cells are induced to a state hyporesponsive to LPS stimulation by pre-exposing them to paclitaxel. Taken together, these results suggest that down-regulation of JNK/p38 MAP kinase may contribute to paclitaxel-induced immunosuppression in mouse splenic lymphocytes.

Activation of mitogen-activated protein kinase pathways by angelan in murine macrophages

In our previous studies, we showed that angelan, a polysaccharide purified from Angelica gigas Nakai, specifically activated macrophages to induce cytokines including inducible nitric oxide synthase (iNOS) which has strong anti-tumor activities [Immunopharmacology, 1999; 43: 1.]. In the present study, we investigated the intracellular signal transduction pathways involved in the angelan-induced iNOS synthesis by murine macrophages. Protein tyrosine phosphorylation was induced within 5 min by angelan, and the blocking of protein tyrosine kinases (PTKs) inhibited down-stream pathways leading to iNOS production in response to angelan. Treament of RAW 264.7 cells with angelan resulted in significant activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38, while stress-activated protein kinase/c-Jun NH2 terminal kinase (SAPK/JNK) was not activated by angelan. The specific p38 inhibitor SB203580 abrogated the angelan-induced iNOS synthesis, whereas the selective mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD98059 did not affect the iNOS induction. In conclusion, we demonstrate that PTK and p38 MAPK activation are required to transduce signals leading to iNOS expression in angelan-stimulated murine macrophages.

Suppression of IL-2 and IL-4 gene expression by nodularin through the reduced NF-AT binding activity

Nodularin is a cyclic peptide produced by cyanobacteria. In the present study, the inhibitory effect of nodularin on T lymphocyte functions was demonstrated. Direct addition of nodularin to B6C3F1 mouse splenocyte cultures produced a concentration-dependent inhibition of the lymphoproliferative response to concanavalin A stimulation. Nodularin inhibited PMA plus ionomycin (Io)-induced IL-2 mRNA expression in murine splenocytes and thymocytes as determined by quantitative/competitive RT-PCR. To further characterize the mechanism for the transcriptional regulation of IL-2, the binding activity of transcription factors, NF-AT, AP-1, NF-kappaB, and Oct, was evaluated by electrophoretic mobility shift assays in mouse splenocytes. Nodularin reduced the NF-AT binding activity in PMA/Io-induced splenocytes, but no significant effect was observed on AP-1, NF-kappaB, or Oct binding activity. Nodularin also inhibited IL-4 mRNA expression in PMA/Io-stimulated murine splenocytes. These results suggest that T lymphocyte is a possible cellular target of nodularin, and the inhibitory effect of nodularin on T-cell specific transcription factor NF-AT induces T-cell dysfunction, which leads to a diminution in IL-2 and IL-4 gene transcription.