• Clinical and Experimental Pediatrics
• /
• v.46 no.12
• /
• pp.1186-1193
• /
• 2003

Purpose : Fragile sites are points on chromosomes which tend to break non-randomly when exposed to specific chemical agents or conditions of tissue culture. The chromosomal break induced by the antineoplastic drug, 1-${\beta}$-D-arabinofuranosyl-cytosine(Ara-c), was investigated to study the laboratory conditions in which the incidence of chromosomal break could be enhanced. Besides, the fragile sites induced by Ara-C were investigated and compared to the already known locations of the specific chromosomal alterations observed in specific neoplasms. Methods : T-lymphocytes from theree normal males and three females were cultured for 48 hours. Cells from each individual were exposed to the Ara-C for an additional 24 hours. After the caffeine was added during the last six hours culture, the metaphase chromosomes were prepared following the conventional method. A site was considered fragile if it was found to break two or more per 100 chromosomal breaks in more than four of six individuals tested. Results : Ara-C induced 252.1 chromosomal breaks per 100 mitotic cells and this result was significantly higher than that of the control, which induced 25.2 breaks(P<0.05). The incidence of the chromosomal break by Ara-C was higher, if cultured in the MEM-FA, which has no folic acid, than in the RPMI 1640 which contains enough folic acid(P<0.05). The most common break site by Ara-C was 3p14.2(FRA3B). There were 20 fragile sites induced by Ara-C. Among these 20 fragile sites, seven coincided with the locations of the mapped oncogenes, JUN, SKI, REL, N-MYC, FHIT, MET, ETS-1, and FOS. Conclusion : S phase specific chemotherapeutic agent, Ara-C, induced the expression of the chromosomal fragile sites effectively using the T-lymphocyte in vitro. Some of the fragile sites by Ara-C highly coincided with the oncogenes and neoplasm specific chromosome breakpoints. In this regard, the fragile sites reported here could provide the unknown neoplasm related chromosomal alternation points.

• The Journal of Pediatrics of Korean Medicine
• /
• v.26 no.4
• /
• pp.10-23
• /
• 2012

Objectives: Gagamyanggyeoksan (G-YGS) has been used to suppress allergic reaction, however, the cellular target of G-YGS and its mode of action remain unclear. The present study was designed to investigate the effect of extracted G-YGS on the PMA and lonomycin (PI)-induced activation of RBL-2H3. Methods: For this investigation, We examined IL-4, IL-13 mRNA expression by Real-Time PCR, IL-4, IL-13 production by ELISA analysis and manifestations of GATA-1, GATA-2, NF-AT1, NF-AT2, AP-1 and NF-${\kappa}B$ p65 transcription factors by western blotting, OVA-specific IgE, IL-4, IL-13 by mouse be sensitive to OVA. Results: Here we showed that treatment of RBL-2H3 mast cells with G-YGS, suppressed PI-induced production of Th2 cytokines including IL-4 and IL-13 in a dose dependent manner. The mRNA expression of IL-4 were completely abolished by G-YGS at the concentration of $100{\mu}g/ml$. Data from a stable cell lines consistently expressing IL-4. And the mRNA expression of IL-13 were abolished by G-YGS at the $200{\mu}g/ml$. But there is no difference between the $50{\mu}g/ml$, the $100{\mu}g/ml$ and the comparison. Results from the western blot analysis of transcription factors involving IL-4 and IL-13 expression indicated that it prominently decreased the expression of mast cell specific transcricption factors including GATA-1, GATA-2, NF-AT2, c-Jun, NF-${\kappa}B$ p65 but not c-Fos. And G-YGS suppressed IgE, IL-4, IL-13 in mouse be sensitive to OVA. Conclusions We suggested the anti-allergic activities of G-YGS might be mediated by down-regulation of Th2 cytokines such as IL-4 and IL-13 through the regulation of transcription factors as GATA-1, GATA-2, NF-AT2, c-Jun, NF-${\kappa}B$ p65.

• The Journal of Pediatrics of Korean Medicine
• /
• v.29 no.4
• /
• pp.39-66
• /
• 2015

Objectives The purpose of this study is to investigate the effects of Sesamum indicum extracted (SEI) on atopic dermatitis in an in-vitro and in-vivo experiment using a MC/9 murine mast cells and a NC/Nga mouse. Methods In-vitro experiment, IL-4, IL-5, IL-6, IL-13, TNF-${\alpha}$ and GM-CSF mRNA expression were evaluated by Real-time PCR, IL-13, MIP-$1{\alpha}$ production by ELISA and manifestations of NFAT-1, NFAT-2, c-jun, c-fos, NF-${\kappa}B$ p65 transcription factors by western blotting. In-vivo experiment, we measured WBC, Eosinophil, Neutrophil, and serum IL-5, IL-13 in NC/Nga atopic dermatitis mouse, IL-5, IL-13, IFN-${\gamma}$, IL-4 in the spleenocyte culture supernatant by ELISA, the absolute cell numbers of CD4+, CD8+, +Gr-1+CD11b, B220+CD23+ in the axillary lymph node (ALN), peripheral blood mononuclear cells (PBMCs) and dorsal skin tissue, IL-5, IL-13 by Real-time PCR, the distribution of tissue inflammation and cellular infiltration by H&E and toluidine blue. Results SEI decreased IL-4, IL-5, IL-6, IL-13, GM-CSF, TNF-${\alpha}$ mRNA expression, IL-13, MIP-$1{\alpha}$ production and the expression of transcription factors including NFAT-1, c-jun, NF-${\kappa}B$ p65 in MC/9 murine mast cells. SEI orally administration decreased cell number of WBC, Eosinophil, the level of serum IgE, total cell number of ALN and dorsal skin tissue, absolute cell number of CD4+, CD8+, B220+CD23+ in the ALN. SEI orally administration also increased absolute cell number of CD8+/CD3+ and decreased Gr-1+/CD11b+ in PBMCs, decreased CD4+ in dorsal skin tissue, inhibited IL-5, IL-13 mRNA expression. Infiltration levels of inflammatory immune cells, mast cells and thickness of epidermis decreased in dorsal skin tissue. Conclusions SEI can regulate allergic inflammatory response suppressed the gene expression and production of cytokines that mediate allergic reactions, and will be able to be effectively utilized in the treatment of atopic dermatitis future.

• The Journal of Pediatrics of Korean Medicine
• /
• v.28 no.3
• /
• pp.31-46
• /
• 2014

Objectives Forsythiae Fructus treatment has been used for inflammatory and allergic diseases in Korean Medicine. Nevertheless, the mechanism of action and the cellular targets are not understood well. The pathogenesis of allergic diseases are associated with Th2 cytokines such as IL-13, MIP-$1{\alpha}$, IL-13, IL-5, GM-CSF, IL-4, TNF-${\alpha}$ and IL-6, which are secreted by the mast cells. This study was conducted to investigate the effects of Forsythiae Fructus extracts (FF) on Th2 cytokines expression and signal transduction in MC/9 mast cells. Methods In the study, MC/9 mast cells were stimulated with DNP-IgE for 24 hours and then treated separately with CsA $10{\mu}g/m{\ell}$ and varying doses of FF for one hour. MC/9 mast cells stimulated with DNP-IgE was the control group, a treatment with CsA was the positive control group and a treatment with varying doses FF was the experimental groups. The mRNA levels of IL-13, IL-5, GM-CSF, IL-4, TNF-${\alpha}$, IL-6 were analyzed with Real-time PCR. The levels of IL-13, MIP-$1{\alpha}$ were measured using enzyme-linked immunosorbent assays(ELISA). NFAT, AP-1 and NF-${\kappa}B$ p65 were examined by Western blot analysis. Results 1. FF were observed to suppress the mRNA expression of IL-13, IL-5, GM-CSF, IL-4, TNF-${\alpha}$, IL-6 in comparison to DNP-IgE control group. 2. FF also has inhibited the IL-13, MIP-$1{\alpha}$ production significantly in comparison to DNP-IgE control group. 3. Western blot analysis of transduction factors involving Th2 cytokines expression has revealed a prominent decrease of the mast cell specific transduction factors including NFAT-1, NFAT-2, c-Jun, and NF-${\kappa}B$ p65 but c-Fos. Conclusions In conclusion, the anti-allergenic activities of FF may be strongly related to the regulation of transcription factors NFAT-1, NFAT-2, c-Jun, and NF-${\kappa}B$ p65 causing inhibition of Th2 cytokines in mast cells.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.28 no.1
• /
• pp.29-34
• /
• 2014

Bone homeostasis is maintained by balance between bone resorbing-osteoclasts and bone forming-osteoblasts. Excessive osteoclastic bone resorption plays a critical role in bone destruction in pathological bone diseases such as osteoporosis, rheumatoid arthritis, and periodontal disease. Many compounds derived from natural products have pharmacological applications and have therapeutic value for treating or preventing several bone diseases characterized by excessive bone resorption. To discover new compounds that can act as anti-resorptive agents, we screened for natural compounds that regulate osteclast differentiation, and found that water extract of Ziziphus Jujuba Mill (WEZJ) has inhibitory effects on osteoclast differentiation. In this study, WEZJ clearly inhibits the osteoclast differentiation in the presence of receptor activator of nuclear factor kB (RANKL), macrophage colony-stimulating factor (M-CSF) without cytoxicity by blocking activation of nuclear factor of activated T cells (NFAT)c1, and c-Fos. In signaling pathway, the phosphorylation of Akt, p38, c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK) and the expression of osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphates (TRAP), Integrin av, Integrin b3, Cathepsin K are suppressed, too. These result suggest that WEZJ may have therapeutic value for treating or preventing several bone diseases characterized by excessive bone destruction.

• Journal of Acupuncture Research
• /
• v.17 no.2
• /
• pp.169-186
• /
• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• BMB Reports
• /
• v.54 no.5
• /
• pp.266-271
• /
• 2021

Estrogen-related receptor γ (ERRγ), a member of the orphan nuclear receptor family, is a key mediator in cellular metabolic processes and energy homeostasis. Therefore, ERRγ has become an attractive target for treating diverse metabolic disorders. We recently reported that ERRγ acts as a negative regulator of osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In the present study, we explored the effects of an ERRγ-specific modulator, GSK5182, on ERRγ-regulated osteoclast differentiation and survival. Interestingly, GSK5182 increased ERRγ protein levels much as does GSK4716, which is an ERRγ agonist. GSK5182 inhibited osteoclast generation from bone-marrow-derived macrophages without affecting cytotoxicity. GSK5182 also attenuated RANKL-mediated expression of cFos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), pivotal transcription factors for osteoclastogenesis. Arrested osteoclast differentiation was associated with reduced RANK expression, but not with the M-CSF receptor, c-Fms. GSK5182 strongly blocked the phosphorylation of IκBα, c-Jun N-terminal kinase, and extracellular signal-regulated kinase in response to RANKL. GSK5182 also suppressed NF-κB promoter activity in a dose-dependent manner. In addition to osteoclastogenesis, GSK5182 accelerated osteoclast apoptosis by caspase-3 activation. Together, these results suggest that GSK5182, a synthetic ERRγ modulator, may have potential in treating disorders related to bone resorption.

• Environmental Mutagens and Carcinogens
• /
• v.26 no.1
• /
• pp.12-19
• /
• 2006

Cadmium, a human carcinogen, can induce toxicity in various cell lines and organs. Despite extensive research, the mechanisms of cadmium-induced cell toxicity and estrogenic potential in human are not clear. This study was performed to investigate cadmium-induced toxicity on human breast cancer cells: MCF-7 cells, an estrogen receptor (ER) positive breast cancer cells, and MDA-MB-231 cells, an ER negative breast cancer cells. MCF-7 cells was proved to be more sensitive than the other cell lines (IC50 = $50\;{\mu}M$ at MCF-7 cells and $120{\mu}M$ at MDA-MB-231). The expression of JNK and AP-1 transcription factors such as c-Jun and c-Fos dependent transcription were increased by cadmium treatment. Inhibition of ER activation by ER antagonist (tamoxifen or ICI 182,780) significantly recovered the viablity and inhibited apoptotic cell death. This suggested that cadmium-induced cell death in ER (+) cells was mediated by JNK/AP-1 pathway and this pathway was more stimulated by ER activated by cadmium. Co-treatment of antioxidants such as selenium (Se), butylated hydroxyanisole (BHA), glutathione (GSH), or N-acetyl-L-cysteine (NAC) recovered the cadmium-induced cell death in MCF-7 cells. Cadmium-induced lipid peroxidation was decreased by GSH, NAC, or BHA in MCF-7 cells. The expression of SOD protein was decreased by cadmium ($100{\mu}M$) but recovered by GSH, NAC, BHA, or Se. Our data showed that the cadmium-induced cell toxicity in human breast cancer cells could be protected by the antioxidants (Se, BHA, NAC, GSH, or NAC) and ER antagonist (tamoxifen or ICI 182,780). Therefore, toxicity of cadmium in breast cancer were mediated by oxidative stress and $ER{\alpha}$.

• Journal of Life Science
• /
• v.15 no.5 s.72
• /
• pp.802-808
• /
• 2005

Human Disabled-2 (Dab2) is a candidate tumor suppressor gone that regulates cell growth by c-Fos suppression in normal cells. In many cancer cells, Dab2 expression is lost or greatly diminished in $\∼85\%$ of the breast and ovarian cancers. In this study, we have examined the methylation status of CpG island on Dab2 gene promoter using bisulfite-assisted genomic sequencing and methylation specific PCR (MSP) method in human breast cancer cell line, MDA MB-231 cells. In normal human uterus endometrial cells, Dab2 was completely unmethylated. In contrast, Dab2 was methylated on CpG dinucleotides near the TATA_ box in MDA MB-231 cells. following MDA MB-231 cells by treatment with 5-azacytidine, Dab2 gene were demethylated and reexpressed. Result of this study suggested that silencing of Dab2 gene is correlated to CpG island methylation in human breast cancer cell line, MBA MD-231 cells.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.27 no.4
• /
• pp.431-436
• /
• 2013

Bone homeostasis is maintained by co-ordination of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between osteoclasts and osteoblasts leads to many bone diseases such as osteoporosis, rheumatoid arthritis. Taxillus chinensis is a herb that has been widely used to improve bone health. However, the effect and mechanism of Taxillus chinensis extract on osteoclast differentiation and bone resportion has been unknown. Thus, We investigated the effect of Taxillus chinensis on expression of receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL)-induced osteoclast differentiation and bone resorption. Also, the action of Taxillus chinensis on mechanisms relating to osteoclast differentiation was studied. In this results, we identified that Taxillus chinensis significantly inhibited RANKL-induced osteoclast differentiation and bone resportion. Moreover, Taxillus chinensis was suppressed the activation of NF-${\kappa}B$ in bone marrow macrophage treated RANKL and M-CSF. Taxillus chinensis was down-regulated the mRNA expression of c-Fos, nuclear factor of activated T-cells (NFAT)c1, osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP). The cell adhesion-related molecules such as integrin ${\alpha}v$ and integrin ${\beta}3$, and the filamentous actin (F-actin) rings of mature osteoclasts-related molecules such as dendritic cell-specific transmembrane preotein (DC-STAMP) and cathepsin K are also suppressed. Taken together, these results indicated that Taxillus chinensis will be a good candidate to treat osteoclast-mediated bone diseases.