Ki-Hyung Kim

Curcumin Inhibits Immunostimulatory Function of Dendritic Cells: MAPKs and Translocation of NF-κB as Potential Targets

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the effect of curcumin on the maturation and immunostimulatory function of dendritic cells (DC) largely remains unknown. In this study, we examined whether curcumin can influence surface molecule expression, cytokine production, and their underlying signaling pathways in murine bone marrow-derived DC. DC were derived from murine bone marrow cells and used as immature or LPS-stimulated mature cells. The DC were tested for surface molecule expression, cytokine production, dextran uptake, the capacity to induce T cell differentiation, and their underlying signaling pathways. Curcumin significantly suppressed CD80, CD86, and MHC class II expression, but not MHC class I expression, in the DC. The DC also exhibited impaired IL-12 expression and proinflammatory cytokine production (IL-1β, IL-6, and TNF-α). The curcumin-treated DC were highly efficient at Ag capture, via mannose receptor-mediated endocytosis. Curcumin inhibited LPS-induced MAPK activation and the translocation of NF-κB p65. In addition, the curcumin-treated DC showed an impaired induction of Th1 responses and a normal cell-mediated immune response. These novel findings provide new insight into the immunopharmacological role of curcumin in impacting on the DC. These novel findings open perspectives for the understanding of the immunopharmacological role of curcumin and therapeutic adjuvants for DC-related acute and chronic diseases.

Curcumin Attenuates TNF‐α‐induced Expression of Intercellular Adhesion Molecule‐1, Vascular Cell Adhesion Molecule‐1 and Proinflammatory Cytokines in Human Endometriotic Stromal Cells

Curcumin, a naturally occurring polyphenolic compound from Curcuma longa, has long been used in folk medicine as an antiinflammatory remedy in Asian countries. Endometriosis is a chronic gynecological inflammatory disorder in which immune system deregulation may play a role in its initiation and progression. A number of mediators, including cell adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1); proinflammatory cytokines such as tumour necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), IL‐6 and IL‐8; and chemokines such as monocyte chemotactic protein‐1 (MCP‐1), play key roles in the pathogenesis of endometriosis. The aim of our study was to explore the effect of curcumin on the expression of these critical molecules in human ectopic endometriotic stromal cells isolated from women with endometriosis. Endometriotic stromal cells treated with curcumin showed marked suppression of TNF‐α‐induced mRNA expression of ICAM‐1 and VCAM‐1. Curcumin treatment also significantly decreased the TNF‐α‐induced cell surface and total protein expression of ICAM‐1 and VCAM‐1 in a dose‐dependent manner. In addition, treatment of endometriotic stromal cells with curcumin markedly inhibited TNF‐α‐induced secretion of IL‐6, IL‐8 and MCP‐1. Furthermore, curcumin inhibited the activation of transcription factor NF‐κB, a key regulator of inflammation, in human endometriotic stromal cells. These findings suggest that curcumin may have potential therapeutic uses in the prevention and treatment of endometriosis. Copyright

Chloroform extract of aged black garlic attenuates TNF-α-induced ROS generation, VCAM-1 expression, NF-κB activation and adhesiveness for monocytes in human umbilical vein endothelial cells

Aged black garlic is a type of fermented garlic (Allium sativum) which has been used in Oriental countries for a long time because of various biological properties of garlic derivatives. The current study explored the potential of the chloroform extract of aged black garlic (CEABG) in attenuating the activities of adhesion molecules in tumor necrosis factor‐α (TNF‐α)‐stimulated human umbilical vein endothelial cells (HUVECs). The study was performed on HUVECs that were pretreated with 30 μg/mL of CEABG before TNF‐α treatment. Treatment of HUVECs with CEABG significantly inhibited TNF‐α‐induced reactive oxygen species (ROS) formation. HUVECs treated with CEABG showed markedly suppressed TNF‐α‐induced mRNA expression of VCAM‐1, but little alteration in ICAM‐1 and E‐selectin mRNA expression. CEABG treatment also significantly decreased the TNF‐α‐induced cell surface and total protein expression of VCAM‐1 without affecting ICAM‐1 and E‐selectin expression. In addition, treatment of HUVECs with CEABG markedly reduced THP‐1 monocyte adhesion to TNF‐α‐stimulated HUVECs. Furthermore, CEABG significantly inhibited NF‐κB transcription factor activation in TNF‐α‐stimulated HUVECs. The data provide new evidence of the antiinflammatory properties of CEABG that may have a potential therapeutic use for the prevention and treatment of vascular diseases such as atherosclerosis through mechanisms involving the inhibition of VCAM‐1 expression and NF‐κB activation in vascular endothelial cells. Copyright

TAZ Mediates Lysophosphatidic Acid-Induced Migration and Proliferation of Epithelial Ovarian Cancer Cells

Background: Transcriptional co-activator with PDZ-binding motif (TAZ), a downstream effector of the Hippo pathway, has been reported to regulate organ size, tissue homeostasis, and tumorigenesis by acting as a transcriptional co-activator. Lysophosphatidic acid (LPA) is a bioactive lipid implicated in tumorigenesis and metastasis of ovarian cancer through activation of G protein-coupled receptors. However, the involvement of TAZ in LPA-induced tumorigenesis of ovarian cancer has not been elucidated. Methods: In order to demonstrate the role of TAZ in LPA-stimulated tumorigenesis, the effects of LPA on TAZ expression and cell migration were determined by Western blotting and chemotaxis analyses in R182 human epithelial ovarian cancer cells. Results and Conclusion: Treatment of R182 cells with the LPA receptor inhibitor Ki16425 blocked LPA-induced cell migration. In addition, transfection of R182 cells with small interfering RNA specific for LPA receptor 1 resulted in abrogation of LPA-stimulated cell migration. LPA induced phosphorylation of ERK and p38 MAP kinase in R182 cells and pretreatment of cells with the MEK-ERK pathway inhibitor U0126, but not the p38 MAPK inhibitor SB202190, resulted in abrogation of LPA-induced cell migration. Pretreatment of R182 cells with U0126 attenuated LPA-induced mRNA levels of TAZ and its transcriptional target genes, such as CTGF and CYR61, without affecting phosphorylation level of YAP. These results suggest that MEK-ERK pathway plays a key role in LPA-induced cell migration and mRNA expression of TAZ in R182 cells, without affecting stability of TAZ protein. In addition, small interfering RNA-mediated silencing of TAZ expression attenuated LPA-stimulated migration of R182 cells. These results suggest that TAZ plays a key role in LPA-stimulated migration of epithelial ovarian cancer cells.

FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells

Ovarian cancer has the highest mortality rate of all gynecological cancers with a high recurrence rate. It is important to understand the nature of recurring cancer cells to terminally eliminate ovarian cancer. The winged helix transcription factor Forkhead box P1 (FOXP1) has been reported to function as either oncogene or tumor-suppressor in various cancers. In the current study, we show that FOXP1 promotes cancer stem cell-like characteristics in ovarian cancer cells. Knockdown of FOXP1 expression in A2780 or SKOV3 ovarian cancer cells decreased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment, whereas overexpression of FOXP1 in A2780 or SKOV3 ovarian cancer cells increased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment. In addition, overexpression of FOXP1 increased promoter activity of ABCG2, OCT4, NANOG, and SOX2, among which the increases in ABCG2, OCT4, and SOX2 promoter activity were dependent on the presence of FOXP1-binding site. In xenotransplantation of A2780 ovarian cancer cells into nude mice, knockdown of FOXP1 expression significantly decreased tumor size. These results strongly suggest FOXP1 functions as an oncogene by promoting cancer stem cell-like characteristics in ovarian cancer cells. Targeting FOXP1 may provide a novel therapeutic opportunity for developing a relapse-free treatment for ovarian cancer patients.

Proteomic Identification of ADAM12 as a Regulator for TGF-β1-Induced Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells

Background Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs. Methods and Results Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells. Conclusions These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.

Autotaxin Regulates Maintenance of Ovarian Cancer Stem Cells through Lysophosphatidic Acid‐Mediated Autocrine Mechanism

Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat‐stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non‐CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere‐forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC‐associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1‐specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA‐stimulated CSC properties. Autotaxin, an LPA‐producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA‐producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA‐dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin‐LPA‐LPA receptor 1‐AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs. Stem Cells 2016;34:551–564

Hypoxia-NOTCH1-SOX2 signaling is important for maintaining cancer stem cells in ovarian cancer

Hypoxia and NOTCH signaling have been reported to be associated with the self-renewal and drug resistance of cancer stem cells (CSCs). However, the molecular mechanisms by which hypoxia and NOTCH signaling stimulate the self-renewal and drug resistance of ovarian CSCs are poorly understood. In the present study, we identified SOX2 as a key transcription factor for CSC-like characteristics in the downstream of hypoxia-induced NOTCH signaling in epithelial ovarian cancer cells. Hypoxic treatment or overexpression of intracellular domain of NOTCH1 (NICD1) in ovarian cancer cells increased sphere formation, drug resistance, and expression of CSC-associated genes such as SOX2, ALDH, and ABC transporters. Hypoxic treatment increased the expression of NICD1, and hypoxic treatment or NICD1 overexpression increased SOX2 promoter activity, which was inhibited by deletion of HIF-1 or CSL binding sites. Furthermore, DAPT treatment decreased the effect of hypoxic treatment, and SOX2 knockdown decreased the effect of hypoxic treatment and NICD overexpression on sphere formation and drug resistance in established ovarian cancer cell lines and primary ovarian cancer cells. These results suggest that hypoxia-NOTCH1-SOX2 signaling axis is important for activation of ovarian CSCs, which may provide a novel opportunity for developing therapeutics to eradicate CSCs in ovarian cancer patients.

Hexane extract of aged black garlic reduces cell proliferation and attenuates the expression of ICAM-1 and VCAM‑1 in TNF-α-activated human endometrial stromal cells.

Increasing evidence indicates the potentially crucial roles of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the pathological process underlying endometriosis. The present study aimed to investigate the effects of a hexane extract of aged black garlic (HEABG) on the proliferation and expression of ICAM-1 and VCAM-1 in tumor necrosis factor-α (TNF-α)-activated human endometrial stromal cells (HESCs) isolated from patients with endometriosis. HESCs were isolated from endometriotic tissues obtained from women with advanced endometriosis who underwent laparoscopic surgery for ovarian endometrioma (n=18). Cell proliferation and cell cycle analysis were assessed by WST-1 assay and flow cytometry, respectively. The expression of ICAM-1 and VCAM-1 was measured by flow cytometry, immunofluorescence staining, immunoblotting and quantitative reverse transcriptase-PCR. The secretion of interleukin-6 (IL-6) was determined by enzyme-linked immunosorbent assay (ELISA). The activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) was detected by electrophoretic mobility shift assay (EMSA) and the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK was analyzed by immunoblotting. Cell proliferation and cell cycle progression were significantly suppressed by HEABG in the TNF-α-induced HESCs through the inhibition of the ERK and JNK signaling pathways. Remarkably, the treatment of the HESCs with HEABG potently suppressed the TNF-α-induced ICAM-1 and VCAM-1 transcript and protein expression by inhibiting the activation of NF-κB and AP-1 transcription factors. Our results suggest that HEABG may be effective in the prevention and treatment of endometriosis in humans.

Prevalence of hepatitis C antibody in patients with chronic liver disease and hepatocellular carcinoma in Korea

Summary. To investigate the contribution of hepatitis C virus (HCV) to chronic liver disease and hepatocellular carcinoma (HCC) in Korea, antibodies to HCV (anti‐HCV) were tested by enzyme immunoassay in 1759 patients with chronic liver disease and HCC, and in 808 healthy adults. The prevalence of anti‐HCV was 1.6% in 808 controls. Anti‐HCV was present in 32 (7.7%) of 418 hepatitis B surface antigen (HBsAg)‐positive and 128 (53.1%) of 241 HBsAg‐negative patients with chronic hepatitis, 16 (6.0%) of 265 HBsAg‐positive and 90 (30.5%) of 295 HBsAg‐negative patients with liver cirrhosis, and 16 (4.8%) of 330 HBsAg‐positive and 61 (29.0%) of 210 HBsAg‐negative patients with HCC. Antibodies to hepatitis B core antigen (anti‐HBc) were present in 80–88% of patients who were seropositive for anti‐HCV and seronegative for HBsAg. Among the sera from 114 patients with HBsAg‐negative and anti‐HCV‐positive chronic liver diseases, HBV DNA and HCV RNA were detected by polymerase chain reaction (PCR) in 54 (47.4%) and 61 (53.3%), respectively. Both HBV DNA and HCV RNA were detected in 4 (4.4%) samples. The mean age of the patients with both HBsAg and anti‐HCV was not different from that of patients who were seropositive for HBsAg alone. These findings indicate that current and/or past HBV infection is still the main cause of chronic liver disease in Korea. Although multivariate analysis showed that anti‐HCV is a risk factor for chronic hepatitis, cirrhosis of the liver and HCC, PCR data for HBV DNA and HCV RNA indicate that HCV infection plays only a minor role in HBsAg‐positive as well as in HBsAg‐negative liver disease and does not accelerate the development of HCC in HBV carriers.