Mi-Hyoung Kim

Protective action of the immunomodulator ginsan against carbon tetrachloride-induced liver injury via control of oxidative stress and the inflammatory response

The aim of the present study was to evaluate immunomodulator ginsan, a polysaccharide extracted from Panax ginseng, on carbon tetrachloride (CCl(4))-induced liver injury. BALB/c mice were injected i.p. with ginsan 24 h prior to CCl(4) administration. Serum liver enzyme levels, histology, expression of antioxidant enzymes, and several cytokines/chemokines were subsequently evaluated. Ginsan treatment markedly suppressed the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic histological necrosis increased by CCl(4) treatment. Ginsan inhibited CCl(4) induced lipid peroxidation through the cytochrome P450 2E1 (CYP2E1) downregulation. The hepatoprotective effect of ginsan was attributed to induction of anti-oxidant protein contents, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) as well as restoration of the hepatic glutathione (GSH) concentration. The marked increase of proinflammatory cytokines (IL-1beta, IFN-gamma) and chemokines (MCP-1, MIP-2beta, KC) in CCl(4) treated mice was additionally attenuated by ginsan, thereby preventing leukocyte infiltration and local inflammation. Our results suggest that ginsan effectively prevent liver injury, mainly through downregulation of oxidative stress and inflammatory response.

An effective strategy for increasing the radiosensitivity of Human lung Cancer cells by blocking Nrf2-dependent antioxidant responses

Radiotherapy and chemotherapeutic agents can effectively induce apoptosis through generation of reactive oxygen species (ROS). Cancer cells frequently express high levels of ROS-scavenging enzymes, which confer resistance to ROS-mediated cell death. Keap1 (Kelch-like ECH-associated protein 1) sequesters and promotes the degradation of the antioxidant response element-binding transcription factor Nrf2 (nuclear factor erythroid-2-related factor 2). In non-small-cell lung cancer (NSCLC) cell lines and NSCLC patients, Keap1 is often present as a biallelic mutant that results in constitutive activation of Nrf2 function, which contributes to cytoprotection against oxidative stress and xenobiotics. To identify small molecules that inhibit antioxidant responses and increase apoptotic death after radiotherapy, we screened a chemical library containing 8000 synthetic compounds using a cell-based luciferase assay system. 4-(2-Cyclohexylethoxy)aniline (IM3829) inhibited the increase in Nrf2-binding activity and expression of the Nrf2 target genes induced by treatment with tertiary butylhydroquinone or radiation. Combined treatment with IM3829 and radiation significantly inhibited clonogenic survival of H1299, A549, and H460 lung cancer cells. IM3829 significantly increased ROS accumulation in irradiated cells compared with cells exposed to radiation alone and led to apoptotic cell death, as confirmed by caspase-3 and PARP cleavage. In mice bearing H1299 or A549 lung cancer xenografts, IM3829 together with radiation inhibited tumor growth more effectively than radiation alone. Our findings suggest that IM3829 could be a promising radiosensitizer in lung cancer patients, particularly those with high expression of Nrf2.

Radioprotective effects of an acidic polysaccharide of Panax ginseng on bone marrow cells

An acidic polysaccharide of Panax ginseng (APG), so called ginsan is known to have important immunomodulatory activities. It was recently reported that APG has radioprotective effects in mice but the detailed mechanism was not fully elucidated. This study examined the effects of APG on bone marrow cells (BMs). The phenotypical and functional changes in APG-treated BMs after gamma radiation were studied. The benefit of APG on BMs damaged by gamma radiation was determined by measuring the cell viability. Using 2 different assays, a pretreatment with APG significantly increased the viability of BMs against gamma radiation. APG-treated BMs had a significantly higher amount of IL-12, which is a major cytokine for immune responses, compared with the medium-treated BMs. The expression of MHC class II molecules of APG-treated BMs was also increased, and APG-treated BMs showed significantly higher levels of allogeneic CD4+ T lymphocyte proliferation. Furthermore, APG-treated mice had a larger number of BMs after gamma radiation than the control mice, and the BMs of APG-treated mice were successfully cultured into dendritic cells, which are the representative antigen-presenting cells. Overall, this study shows that APG alters the phenotype of BMs, increases the viability and alloreactivity of BMs after gamma radiation both in vitro and in vivo. Therefore, APG may be a good candidate radioprotective agent for BMs.

Immunomodulatory Activity of Ginsan, a Polysaccharide of Panax Ginseng, on Dendritic Cells

Ginsan, a Panax ginseng polysaccharide that contains glucopyranoside and fructofuranoside, has immunomodulatory effects. Although several biologic studies of ginsan have been performed, its effects on dendritic cells (DCs), which are antigen-presenting cells of the immune system, have not been studied. We investigated the immunomodulatory effects of ginsan on DCs. Ginsan had little effect on DC viability, even when used at high concentrations. Ginsan markedly increased the levels of production by DCs of IL-12 and TNF-alpha, as measured by ELISA. To examine the maturation-inducing activity of ginsan, we measured the surface expression levels of the maturation markers MHC class II and CD86 (B7.2) on DCs. It is interesting that ginsan profoundly enhanced the expression of CD86 on DC surfaces, whereas it increased that of MHC class II only marginally. In (3)H-thymidine incorporation assays, ginsan-treated DCs stimulated significantly higher proliferation of allogeneic CD4(+) T lymphocytes than did medium-treated DCs. Taken together, our data demonstrate that ginsan stimulates DCs by inducing maturation. Because DCs are critical antigen-presenting cells in immune responses, this study provides valuable information on the activities of ginsan.

The inhibitory effect of ginsan on TGF‐β mediated fibrotic process

Transforming growth factor‐beta (TGF‐β) plays a central role in the development of fibrosis by stimulating extracellular matrix accumulation, and signals either directly or indirectly through types I, II, and III (TβRI, II, and III) TGF‐β receptor complexes. Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. Here, we examine whether ginsan regulates the fibrogenic process by interfering with TGF‐β signaling pathways. TGF‐β treatment of murine or human normal lung fibroblasts enhanced the levels of several fibrotic markers, including smooth muscle alpha actin (α‐SMA), collagen‐1, and fibronectin. Interestingly, ginsan treatment either before or after TGF‐β administration led to significant reductions in all of α‐SMA, collagen‐1, and fibronectin expression levels. Ginsan not only inhibited phosphorylation of Smad2 and Smad3, but also attenuated pERK and pAKT signaling induced by TGF‐β. Moreover, ginsan restored TβRIII protein expression, which was significantly downregulated by TGF‐β, but reduced TβRI and TβRII protein levels. In a murine model of bleomycin (BLM)‐induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α‐SMA, and TGF‐β. These data collectively suggest that ginsan acts as an effective anti‐fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF‐β signaling pathways. J. Cell. Physiol. 226: 1241–1247, 2011.

Radioprotective effects of fucoidan on bone marrow cells: improvement of the cell survival and immunoreactivity.

Fucoidan is a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and has a variety of biological effects including mobilization of hematopoietic progenitor cells. Recently, we demonstrated that fucoidan stimulates the antigen-presenting functions of dendritic cells. In this study, we investigated the radioprotective effects of fucoidan on bone marrow cells (BMCs), which are the main cellular reservoir for the hematopoietic and immune system. To evaluate the effects of fucoidan, we assayed cell viability and immune responses. In a viability assay, fucoidan significantly increased the viability of BMCs. Based on the results of flow cytometric analysis, the increased viability of fucoidan-treated BMCs was attributed to the inhibition of radiation-induced apoptosis. Furthermore, fucoidan altered the production of immune-related cytokines from BMCs and increased the capability of BMCs to induce proliferation of allogeneic splenocytes. Taken together, our study demonstrated that fucoidan has radioprotective effects on BMCs with respect to cell viability and immunoreactivity. These results may provide valuable information, useful in the field of radiotherapy.

Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2.

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.

Acriflavine enhances radiosensitivity of colon cancer cells through endoplasmic reticulum stress-mediated apoptosis.

Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by γ-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), eIF-2α (eukaryotic initiation factor 2α), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1α by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer.

Analysis of immune cell populations and cytokine profiles in murine splenocytes exposed to whole-body low-dose irradiation.

Purpose: In contrast to high-dose therapeutic irradiation, definitive research detailing the physiological effects of low-dose irradiation is limited. Notably, the immunological response elicited after low-dose irradiation remains controversial. Materials and methods: Female C57BL/6 mice were whole- body-irradiated with a single or three daily fractions up to a total dose of 0.1, 1, or 10 cGy. Blood and spleen were harvested 2, 7 and 14 days after irradiation. Results: The splenic CD4+ T cell subpopulations were temporarily increased at 2 days after single or fractionated irradiation, whereas the percentage of dendritic cells (DC) and macrophages was decreased. Whereas CD8+ T cell populations were decreased in single-dose irradiated mice at day 7, early and sustained reduction of CD8+ T cell numbers was observed in fractionated- dose-irradiated mice from day 2 until day 14. In addition, single-dose irradiation resulted in a Th1 cytokine expression profile, whereas fractionated-dose irradiation drove a Th2 shift. Additionally, increased expression of immune-related factors was observed at early time-points with single-dose irradiation, in contrast to the dose-independent induction following fractionated-dose irradiation. Conclusions: Our results demonstrate that low-dose irradiation modulates the immune response in mice, where the sensitivity and kinetics of the induced response vary according to the dosing method.

Quantitative proteomic analysis of single or fractionated radiation-induced proteins in human breast cancer MDA-MB-231 cells

BackgroundRadiotherapy is widely used to treat cancer alone or in combination with surgery, chemotherapy, and immunotherapy. However, damage to normal tissues and radioresistance of tumor cells are major obstacles to successful radiotherapy. Furthermore, the immune network around tumors appears to be connected to tumor progression and recurrence.MethodsWe investigated the cytosolic proteins produced by irradiated tumor cells by using a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture. MDA-MB-231 breast cancer cells were treated with a single or fractionated 10 Gray dose of 137Cs γ-radiation, which was selected based on cell viability.ResultsRadiation-induced proteins were differentially expressed based on the fractionated times of radiation and were involved in multiple biological functions, including energy metabolism and cytoskeleton organization. We identified 46 proteins increased by at least 1.3-fold, and high ranks were determined for cathepsin D, gelsolin, arginino-succinate synthase 1, peroxiredoxin 5, and C-type mannose receptor 2.ConclusionThese results suggest that a number of tumor-derived factors upregulated by γ-radiation are promising targets for modulation of the immune response during radiation treatment.