A new FGFR inhibitor disrupts the TGF-ß1-induced fibrotic process.

Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti-fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3-(2-chloro-6-fluorobenzyl)-1,6,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-1918), markedly inhibited transforming growth factor (TGF)-ß-stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α-smooth muscle actin, on human lung fibroblasts. However, IM-1918 neither decreased Smad-2 and Smad-3 nor affected p38MAPK and JNK. Instead, IM-1918 reduced Akt and extracellular signal-regulated kinase 1/2 phosphorylation increased by TGF-ß. Additionally, IM-1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin-induced murine lung fibrosis model, IM-1918 profoundly reduced fibrotic areas and decreased collagen and α-smooth muscle actin accumulation. These results suggest that IM-1918 can be applied to treat lung fibrosis.

Induction of galectin-1 by TGF-ß1 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-ß1 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-ß1-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-ß1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.

The inhibitory effect of ginsan on TGF-ß mediated fibrotic process.

Transforming growth factor-beta (TGF-ß) 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 (TßRI, II, and III) TGF-ß 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-ß signaling pathways. TGF-ß 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-ß 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-ß. Moreover, ginsan restored TßRIII protein expression, which was significantly downregulated by TGF-ß, but reduced TßRI and TßRII protein levels. In a murine model of bleomycin (BLM)-induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α-SMA, and TGF-ß. These data collectively suggest that ginsan acts as an effective anti-fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF-ß signaling pathways.

IM-412 inhibits transforming growth factor beta-induced fibroblast differentiation in human lung fibroblast cells.

Pulmonary fibrosis is a type of interstitial lung disease that causes progressive scarring in lung tissues. Although there have been many studies on fibrosis, there is no standard treatment for fibrotic disease. Thus, there is an urgent need for the development of effective anti-fibrotic drugs. Transforming growth factor beta (TGF-beta) is a major fibrotic mediator known to stimulate fibrosis. To identify small molecules that inhibit TGF-beta responses, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells. Among 8000 chemical compounds containing biologically active natural products and synthetic or clinically used compounds, we found that 3-(2-chlorobenzyl)-1,7-dimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-412) significantly decreased TGF-beta stimulated reporter activity in a dose-dependent manner. In addition, IM-412 inhibited TGF-beta-induced expression of the fibrotic markers alpha-smooth muscle actin (alpha-SMA) and fibronectin, and collagen accumulation in CCD-18Lu human normal lung fibroblasts without cell cytotoxicity. IM-412 decreased Smad2 and -3 phosphorylation as well as JNK and ERK activity. Moreover, expression levels of TGF-beta receptor I (TbetaRI) and receptor II (TbetaRII) were down-regulated by IM-412 in a dose-dependent manner. Thus, our findings indicate that the small molecule IM-412 attenuated TGF-beta-mediated fibroblast differentiation through inhibition of the overall TGF-beta response and may be a promising novel agent for the treatment of pathological fibrotic conditions.

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.

Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells.

Fucoidan is a polysaccharide purified from the brown seaweed Fucus vesiculosus. Although some effects of fucoidan on immune functions have been elucidated, there have been no studies concerning the immunomodulatory effects of fucoidan on dendritic cells (DCs), which are powerful antigen-presenting cells. In this study, fucoidan increased the viability of DCs, the production of interleukin-12 and tumor necrosis factor-alpha, and the expression of major histocompatibility complex class I, class II, CD54, and CD86 molecules. Furthermore, fucoidan-treated DCs showed decreased antigen uptake and increased proliferation of allogeneic splenocytes. In a study of the transcriptional regulation effects of fucoidan, translocation of p65 molecules of nuclear factor-kappaB (NF-kappaB) from the cytosol to the nucleus was clearly observed in fucoidan-treated DCs. Taken together, the results suggest that fucoidan has immunostimulating and maturing effects on DCs, via a pathway involving at least NF-kappaB.

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.

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 antigenpresenting 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.

Induction of immunogenic cell death by radiation-upregulated karyopherin alpha 2 in vitro.

Accumulating evidence suggests the potential for radiation therapy to generate antitumor immune responses against tumor cells by inducing immunogenic cell death and phenotypic changes. We recently found that ionizing radiation upregulated karyopherin α2 (KPNA2) in HT-29 colorectal tumor cells using quantitative proteomic analysis. To determine whether this increased KPNA2 could function as a damage-associated molecular pattern to induce antitumor immune responses, mouse bone-marrow-derived dendritic cells (BMDCs) were treated with KPNA2. KPNA2 enhanced the surface expression of CD40, CD54, CD80, CD86, and MHC class I/II on BMDCs. DCs treated with KPNA2 exhibited increased secretion of pro-inflammatory cytokines such as IL-1ß, IL-6, IL-12, IL-23, and TNF-α. Co-culture of CD4(+) T cells and KPNA2-treated DCs resulted in induction of Th1/17 cytokines (IFN-γ and IL-17) and reduction of TGF-ß production. Moreover, KPNA2-treated DCs were capable of increasing granzyme B and perforin expression in cytotoxic T lymphocytes. These results demonstrated that radiation-induced dying colorectal cancer cells released considerable amounts of KPNA2 that induce the maturation and activation of DCs for synergistic antitumor effect of radiation.

IM-412 inhibits the invasion of human breast carcinoma cells by blocking FGFR-mediated signaling.

Triple-negative breast cancer (TNBC) is an aggressive cancer with a poor prognosis due to its epithelial­to-mesenchymal transition (EMT) phenotype. Cancer patients often experience several detrimental effects of cancer treatment, such as chemoresistance, radioresistance and the maintenance of cancer stem cells due to EMT. Thus, EMT signaling is considered to be a valuable therapeutic target for cancer treatment, and its inhibition is being attempted as a new treatment option for TNBC patients. Previously, we showed that 3-(2-chlorobenzyl)-1,7-dimethyl-1H-imidazo[2,1-f]purine­2,4(3H,8H)-dione (IM-412) inhibits transforming growth factor-ß (TGF-ß)-induced differentiation of human lung fibroblasts through both Smad-dependent and -independent pathways. In the present study, we examined the inhibitory effect of IM-412 on EMT pathways and invasiveness in TNBC cells since the TGF-ß signaling pathway is a typical signaling pathway that functions in EMT. IM-412 not only potently suppressed the migration and invasion of MDA-MB-231 cells, but also lowered the expression of mesenchymal markers and EMT-activating transcription factors in these cells. IM-412 inhibited the activation of several signaling proteins, including Smad2/Smad3, p38MAPK, Akt and JNK, and it also attenuated the phosphorylation of FGFR1 and FGFR3. Collectively, our findings suggest that the synthetic compound IM-412 suppressed the EMT process in MDA-MB-231 cells and thereby effectively inhibited the migration and invasion of these cancer cells. Thus, IM-412 could serve as a novel therapeutic agent for malignant cancers.

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

BACKGROUND: Radiotherapy 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. METHODS: We 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 (137)Cs γ-radiation, which was selected based on cell viability. RESULTS: Radiation-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. CONCLUSION: These 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.

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.

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.

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.

Sustained expression of NADPH oxidase 4 by p38 MAPK-Akt signaling potentiates radiation-induced differentiation of lung fibroblasts.

Radiation-induced fibrosis (RIF) is a long-term adverse effect of curative radiotherapy; however, the distinct molecular mechanisms of RIF in neighboring normal tissue are not fully understood. We investigated the mechanisms underlying radiation-induced fibroblast differentiation into myofibroblasts. Lung fibroblasts produced reactive oxygen species (ROS) immediately after irradiation, the level of which remained increased for 24 h. The NADPH oxidase inhibitor, diphenyleneiodonium (DPI), suppressed ROS production and significantly decreased the radiation-induced expression of alpha-smooth muscle actin (alpha-SMA) and fibronectin (FN). The mRNA and protein expression of Nox4 was increased by radiation, and siRNA knockdown of Nox4 reduced alpha-SMA and FN levels. Increased phosphorylation of p38MAPK, Erk, and PI3k/Akt was observed after irradiation. Inhibitors of p38 MAPK and Akt, but not of Erk, reduced radiation-induced fibroblast differentiation and Nox4 expression. Notably, DPI partially decreased phosphorylation of p38MAPK and Akt, suggesting that p38MAPK, Akt, and Nox4 may cooperate in a positive feedback loop. Nox4 expression was also increased during bleomycin-induced fibroblast differentiation, and downregulation of Nox4 reduced alpha-SMA levels and extracellular matrix (ECM) accumulation. These results demonstrate that interfering Nox4 activation can be a potential strategy to disrupt fibrotic process.

The role of Bcl-xL and nuclear factor-kappaB in the effect of taxol on the viability of dendritic cells.

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-kappaB (NF-kappaB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-(L)-phenylalanine chloromethyl ketone confirmed that NF-kappaB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.

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.