Jiyeon Ahn

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.

The immunomodulator ginsan induces resistance to experimental sepsis by inhibiting Toll‐like receptor‐mediated inflammatory signals

Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. In this study, we show that pretreatment of ginsan (25 μg/kg) protected mice from lethality induced by Staphylococcus aureus challenge. This survival benefit was associated with enhanced bacterial clearance from circulation, spleen and kidney. The phagocytic activity of macrophages treated with ginsan was significantly enhanced against S. aureus. However, the production of proinflammatory cytokines, such as TNF‐α, IL‐1β, IL‐6, IFN‐γ, IL‐12, and IL‐18, was markedly down‐regulated in ginsan‐treated mice compared with those of control‐infected mice. The expression of Toll‐like receptor (TLR) 2 and the adaptor molecule MyD88, which was greatly increased in septic macrophages, was significantly reduced by ginsan treatment in vitro. Similarly, the expression of phospho‐JNK1/2, phospho‐p38 MAPK, and NF‐κB was decreased in the same culture system. These results illustrate that the antiseptic activity of ginsan can be attributed to enhanced bacterial clearance, and reduced proinflammatory cytokines via the TLR signaling pathway.

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.

Chemoprotective and Adjuvant Effects of Immunomodulator Ginsan in Cyclophosphamide-Treated Normal and Tumor Bearing Mice

Ginsan is a polysaccharide extracted from Panax ginseng that is known to have multiple immunomodulatory effects. This study evaluates the chemoprotective effect of ginsan on normal mice and the adjuvant effect on tumor bearing mice in combination with cyclophosphamide (CP). Ginsan (100 mg/kg) was injected 24 h before or after a sublethal dose of a CP treatment. The mice pre-treated with ginsan all died within 10 days whereas up to 53% of the mice post-treated with ginsan increased survival to day 30 compared with only 10% in the CP alone treated group on day 30. The post-treatment of ginsan accelerated the recovery of the bone marrow cells and blood neutrophils by approximately 1.3- and 1.75-fold compared to CP treated control mice at 5 days after CP administration, respectively. These marked differences in activity between the pre- and post-treatment of ginsan with CP was clarified by examining the mRNA expression levels of several cytokines in spleen cells and the self-renewal potential of hematopoietic progenitor cells, CFU-s. The post-treatment with ginsan increased the mRNA expression levels of TNF-α, IL-1β, IL-6, SCF, and GM-CSF with respect to that of the CP alone or ginsan pre-treated group. Similarly, the number of CFU-s was significantly higher in the mice post-treated with ginsan. The inhibition of tumor growth and survival elongation was also observed when ginsan was administered 24 h after the CP treatment. These results show that the post-treatment with ginsan had an immunomodulating and adjuvant effect in combination with CP, which indicates its wide applications in reducing the adverse effects of chemotherapy and improving the general conditions of patients.

Niclosamide enhances ROS-mediated cell death through c-Jun activation

Radiotherapy is an effective treatment modality in the clinical treatment of cancers, and has been combined with chemotherapy in order to improve therapeutic efficacy. Therefore, we aimed to develop small molecules that enhance the cytotoxic effects of radiotherapy. In this study, we provide evidence that niclosamide is an effective radiosensitizer in non-small cell lung cancer cells. Using a cell-based high-throughput viability screen of 1040 compounds in combination with γ-ionizing radiation (IR), we found niclosamide, an FDA-approved antihelminthic agent, had a radiosensitizing effect on H1299 human lung cancer cells. Pretreatment with niclosamide enhanced IR- induced cell death of H1299 in a dose-dependent manner via apoptosis compared with IR or niclosamide alone. The combined treatment induced significantly more phosphorylation of p38 MAPK and c-Jun in H1299 cells than IR or niclosamide alone. Since IR induces apoptosis through generation of reactive oxygen species (ROS), hydrogen peroxide (H2O2) was employed as another ROS generator and we found that niclosamide also sensitized cells to H2O2. Niclosamide pretreatment also induced c-Jun and its phosphorylation in the presence of H2O2, thereby enhancing apoptosis. N-acetyl-L-cysteine (NAC) treatment abolished both cell death and c-Jun activation induced by the combination treatments. Knockdown of c-Jun also decreased PARP cleavage and clonogenic cell survival in niclosamide- and IR-treated H1299 cells. Our findings suggest that niclosamide could be a promising radiosensitizer in lung cancer patients through activation of the p38 MAPK-c-Jun axis.

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.

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.

Effectiveness of slow freezing and vitrification for long-term preservation of mouse ovarian tissue.

This study was conducted to evaluate the interaction between cryo-damage and ART outcome after cryopreservation of mouse ovarian tissues with different methods. Either a vitrification or a slow freezing was employed for the cryopreservation of B6CBAF1 mouse ovaries and follicle growth and the preimplantation development of intrafollicular oocytes following parthenogenesis or IVF were monitored. Both cryopreservation protocols caused significant damage to follicle components, including vacuole formation and mitochondrial deformities. Regardless of the cryopreservation protocols employed, a sharp (P < 0.0001) decrease in follicle viability and post-thaw growth was detected. When IVF program was employed, significant (P < 0.05) decrease in cleavage and blastocyst formation was notable in both modes of cryopreservation. However, such retardation was not found when oocytes were parthenogenetically activated. In the IVF oocytes, slow freezing led to better development than vitrification. In conclusion, a close relationship between cryopreservation and ART methods should be considered for the selection of cryopreservation program.

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.