• Molecules and Cells
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• v.41 no.2
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• pp.119-126
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• 2018

microRNA (miR)-612 shows anticancer activity in several types of cancers, yet its function in melanoma is still unclear. This study was undertaken to investigate the expression of miR-612 and its biological relevance in melanoma cell growth, invasion, and tumorigenesis. The expression and prognostic significance of miR-612 in melanoma were examined. The effects of miR-612 overexpression on cell proliferation, colony formation, tumorigenesis, and invasion were determined. Rescue experiments were conducted to identify the functional target gene(s) of miR-612. miR-612 was significantly downregulated in melanoma tissues compared to adjacent normal tissues. Low miR-612 expression was significantly associated with melanoma thickness, lymph node metastasis, and shorter overall, and disease-free survival of patients. Overexpression of miR-612 significantly decreased cell proliferation, colony formation, and invasion of SK-MEL-28 and A375 melanoma cells. In vivo tumorigenic studies confirmed that miR-612 overexpression retarded the growth of A375 xenograft tumors, which was coupled with a decline in the percentage of Ki-67-positive proliferating cells. Mechanistically, miR-612 targeted Espin in melanoma cells. Overexpression of Espin counteracted the suppressive effects of miR-612 on melanoma cell proliferation, invasion, and tumorigenesis. A significant inverse correlation (r = -0.376, P = 0.018) was observed between miR-612 and Espin protein expression in melanoma tissues. In addition, overexpression of miR-612 and knockdown of Espin significantly increased the sensitivity of melanoma cells to doxorubicin. Collectively, miR-612 suppresses the aggressive phenotype of melanoma cells through downregulation of Espin. Delivery of miR-612 may represent a novel therapeutic strategy against melanoma.

• Molecules and Cells
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• v.41 no.2
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• pp.140-149
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• 2018

The $TIS21^{/BTG2/PC3}$ gene belongs to the antiproliferative gene (APRO) family and exhibits tumor suppressive activity. However, here we report that TIS21 controls lipid metabolism, rather than cell proliferation, under fasting condition. Using microarray analysis, whole gene expression changes were investigated in liver of TIS21 knockout (TIS21-KO) mice after 20 h fasting and compared with wild type (WT). Peroxisome proliferator-activated receptor alpha ($PPAR{\alpha}$) target gene expression was almost absent in contrast to increased lipid synthesis in the TIS21-KO mice compared to WT mice. Immunohistochemistry with hematoxylin and eosin staining revealed that lipid deposition was focal in the TIS21-KO liver as opposed to the diffuse and homogeneous pattern in the WT liver after 24 h starvation. In addition, cathepsin E expression was over 10 times higher in the TIS21-KO liver than that in the WT, as opposed to the significant reduction of thioltransferase in both adult and fetal livers. At present, we cannot account for the role of cathepsin E. However, downregulation of glutaredoxin 2 thioltransferase expression might affect hypoxic damage in the TIS21-KO liver. We suggest that the $TIS21^{/BTG2}$ gene might be essential to maintain energy metabolism and reducing power in the liver under fasting condition.

• Molecules and Cells
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• v.41 no.6
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• pp.575-581
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• 2018

Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal stem cells (hMSCs) from bone marrow in PMOP remains unknown. In this study, we investigated the biological function of SOX5 and explore its molecular mechanism in hMSCs from patients with PMOP. Our findings showed that the mRNA and protein expression levels of SOX5 were upregulated in hMSCs isolated from bone marrow samples of PMOP patients. We also found that SOX5 overexpression decreased the alkaline phosphatase (ALP) activity and the gene expression of osteoblast markers including Collagen I, Runx2 and Osterix, which were increased by SOX5 knockdown using RNA interference. Furthermore, $TNF-{\alpha}$ notably upregulated the SOX5 mRNA expression level, and SOX5 knockdown reversed the effect of $TNF-{\alpha}$ on osteogenic differentiation of hMSCs. In addition, SOX5 overexpression increased Kruppel-like factor 4 (KLF4) gene expression, which was decreased by SOX5 silencing. KLF4 knockdown abrogated the suppressive effect of SOX5 overexpression on osteogenic differentiation of hMSCs. Taken together, our results indicated that $TNF-{\alpha}$-induced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through KLF4 signal pathway, suggesting that SOX5 might be a novel therapeutic target for PMOP treatment.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.348-358
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• 2007

Metallothionein, a cysteine-rich stress response protein that is naturally induced by a variety of immunologic stressors, has been shown to suppress autoimmune disorders through mechanisms not yet fully defined. In the present study, we examined the underlying mechanisms by which metallothionein might mediate such regulation of autoimmunity. $Na\ddot{i}ve\;CD4^+$ T cells from metallothionein-deficient mice differentiated to produce significantly less IL-10, $TGF-{\gamma}$, and repressor of GATA, but more $IFN-{\gamma}$ and T-bet, when compared with those from wild-type mice. The levels of IL-4 and GATA-3 production were not different between the two groups of mice. Conversely, treatment with exogenous metallothionein during the priming phase drove $na\ddot{i}ve$ wild-type $CD4^+\;T$ cells to differentiate into cells producing more IL-10 and $TGF-{\beta}$, but less $IFN-{\gamma}$ than untreated cells. Metallothionein-primed cells were hyporesponsive to restimulation, and suppressive to T cell proliferation in an IL-10-dependent manner. Lymphocytes from metallothionein-deficient mice displayed significantly elevated levels of AP-1 and JNK activities in response to stimulation compared with those from wild-type controls. Importantly, transgenic mice overexpressing metallothionein exhibited significantly reduced susceptibility to collagen-induced arthritis and enhanced IL-10 level in the serum, relative to their nontransgenic littermates. Taken together, these data suggest that metallothionein is able to promote the generation of IL-10-and $TGF-{\beta}$-producing type 1 regulatory T-like cells by downregulating JNK-dependent AP-1 activity. Thus, metallothionein may play an important role in the regulation of Th1-dependent autoimmune arthritis, and may represent both a potential target for therapeutic manipulation and a critical element in the diagnostic assessment of disease potential.

• Korean Journal of Pharmacognosy
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• v.42 no.3
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• pp.253-259
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• 2011

Macrophages play a vital role in the innate immune system involving defensive cytokines such as TNF (tumor necrosis factor)-${\alpha}$ and nitric oxide (NO). Therefore, we try to elucidate the anti-inflammatory activity of Chaga mushroom (Inonotus Obliquus, IO) in murine macrophages. Raw 264.7 cells and peritoneal macrophages of mice were cultured with or without LPS/LPS + IFN-${\gamma}$ in the presence of IO aqueous extracts (IOE 0.2, 2, 20, 100 ${\mu}g$/mL) for 24 hr and 48 hr, respectively. Exposure of IOE caused the decrease of NO production and increase of TNF-${\alpha}$ production in dose-dependent manner in activated peritoneal macrophage in vitro. To further investigate anti-inflammatory effects of IO ex vivo, we orally administrated capsaicin (PC, 3 mg/kg/day) and IOE (100, 200, 400 mg/kg/day) for 4 consecutive days to C57BL/6 mice (7~9 weeks old, female), then observed the NO secretion and cytokine (TNF-${\alpha}$) production of LPS/LPS + INF-${\gamma}$-stimulated peritoneal macrophages. IOE inhibits NO secretion in dose-dependent manner both ex vivo and in vitro and increases the production of TNF-${\alpha}$ in vitro. In addition, we found that IOE possessed suppressive effects of LPS-stimulated TNF-${\alpha}$, IL-$1{\beta}$, COX-2, as well as iNOS expressions in Raw 264.7 cells. These findings indicate that IOE suppress not only the LPS-induced NO overproduction of murine peritoneal macrophages, but also iNOS, COX-2, TNF-${\alpha}$, and IL-$1{\beta}$ overexpression of LPS-induced Raw 264.7 cells. Consequently, our results suggest that IO may have the anti-inflammatory effects via suppression of the inflammatory cytokines and mediators, and be useful for the treatment of inflammatory diseases.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.2
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• pp.202-211
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• 2011

The stems and branchs of Euryale ferox Salisbury (EF), are used in Chinese herbal medicine for latent-heat-clearing, antipyretic, detoxicant and anti-inflammatory ailments. This plant is used worldwide for the treatment of many types of inflammatory disease including respiratory infections, diabetes mellitus, rheumatoid arthritis and play an important role in the immune reaction. Topical natural antioxidants are a useful strategy for the prevention of oxidative stress mediated inflammatory disease. Plants produce significant amounts of antioxidants to prevent the oxidative stress caused by photons and oxygen, therefore they represent a potential source of new compounds with antioxidant activity. This study was designed to evaluate whether EFEA (ethylacetate fraction of EF) may ameliorate oxidative stress and inflammatory status through the antioxidative mechanism in LPS-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with EFEA significantly reduced LPS-stimulated inflammatory response in a dose-dependent manner. In conclusion, the EF extracts have anti-inflammatory effects in vitro system, which can be used for developing pharmaceutical drug against oxidative stress and chronic inflammatory disease.

• Biomolecules & Therapeutics
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• v.25 no.2
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• pp.130-139
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• 2017

$CXCR5^+$ T follicular helper (Tfh) cells are associated with aberrant autoantibody production in patients with antibody-mediated autoimmune diseases including lupus. Follicular regulatory T (Tfr) cells expressing CXCR5 and Bcl6 have been recently identified as a specialized subset of $Foxp3^+$ regulatory T (Treg) cells that control germinal center reactions. In this study, we show that retroviral transduction of CXCR5 gene in $Foxp3^+$ Treg cells induced a stable expression of functional CXCR5 on their surface. The Cxcr5-transduced Treg cells maintained the expression of Treg cell signature genes and the suppressive activity. The expression of CXCR5 as well as Foxp3 in the transduced Treg cells appeared to be stable in vivo in an adoptive transfer experiment. Moreover, Cxcr5-transduced Treg cells preferentially migrated toward the CXCL13 gradient, leading to an effective suppression of antibody production from B cells stimulated with Tfh cells. Therefore, our results demonstrate that enforced expression of CXCR5 onto Treg cells efficiently induces Tfr cell-like properties, which might be a promising cellular therapeutic approach for the treatment of antibody-mediated autoimmune diseases.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.63-70
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• 2019

Myeloid-derived suppressor cells (MDSCs) that are able to suppress T cell function are a heterogeneous cell population frequently observed in cancer, infection, and autoimmune disease. Immune checkpoint molecules, such as programmed death 1 (PD-1) expressed on T cells and its ligand (PD-L1) expressed on tumor cells or antigen-presenting cells, have received extensive attention in the past decade due to the dramatic effects of their inhibitors in patients with various types of cancer. In the present study, we investigated the expression of PD-1 on MDSCs in bone marrow, spleen, and tumor tissue derived from breast tumor-bearing mice. Our studies demonstrate that PD-1 expression is markedly increased in tumor-infiltrating MDSCs compared to expression in bone marrow and spleens and that it can be induced by LPS that is able to mediate $NF-{\kappa}B$ signaling. Moreover, expression of PD-L1 and CD80 on $PD-1^+$ MDSCs was higher than on $PD-1^-$ MDSCs and proliferation of MDSCs in a tumor microenvironment was more strongly induced in $PD-1^+$ MDSCs than in $PD-1^-$ MDSCs. Although we could not characterize the inducer of PD-1 expression derived from cancer cells, our findings indicate that the study on the mechanism of PD-1 induction in MDSCs is important and necessary for the control of MDSC activity; our results suggest that $PD-1^+$ MDSCs in a tumor microenvironment may induce tumor development and relapse through the modulation of their proliferation and suppressive molecules.

• Toxicological Research
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• v.35 no.4
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• pp.371-387
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• 2019

Although the dried root of Saposhnikovia divaricata (Turcz.) Schischk. (Umbelliferae) is a popular medicinal plant in East Asia, there has been no systemic toxicological evaluation of a water extract of Saposhnikoviae Radix (SRE). In this experiment, an oral acute and 13-week subchronic toxicological evaluations of SRE (500-5,000 mg/kg body weight) were performed in both sexes of Crl:CD(SD) rats. Based on the results from mortality, clinical signs, effects on body weight and organ weight, clinical biochemistry, hematology, urinalysis, and histopathology, significant acute, 4-week repeated dose range finding (DRF) and 13-week subchronic toxicity of SRE was not observed in either sex of rats; thus, the no observed adverse effect level (NOAEL) was 5,000 mg (kg/day). To identify anti-hyperuricemia potential of SRE, the suppressive effect of SRE was determined in mice challenged with potassium oxonate (PO; 250 mg/kg) via intraperitoneal injection for 8 days (each group; n = 7). SRE supplementation suppressed the uric acid level in urine through significant xanthine oxidase (XO) inhibitory activity. Kidney dysfunctions were observed in PO-challenged mice as evidenced by an increase in serum creatinine level. Whereas, SRE supplementation suppressed it in a dose-dependent manner. Collectively, SRE was safe up to 5,000 mg (kg/day) based on NOAEL found from acute and 13-week subchronic toxicological evaluations. SRE had anti-hyperuricemia effect and lowered the excessive level of uric acid, a potential factor for gout and kidney failure.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.481-488
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• 2022

Background: Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods: Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results: Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion: Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.