Interleukin 1β Up-Regulates mRNA Expression of Inducible Nitric Oxide Synthase in 3T3-L1 Preadipocytes: Role of JAKs/STATs, PKCs, and Src / 계명의대학술지

Recent evidence suggests obesity as a low or systemic chronic inflammation. (Pre)adipocytes in the adipose tissue (AT) express and secrete a variety of cytokines and adipokines. Inducible nitric oxide synthase (iNOS) is an inflammatory enzyme involved in the production of NO. Until now, the inducer(s) of iNOS expression in (pre)adipocytes remains unclear. In this study, we investigated the effects of proinflammatory cytokines (interleukin-1β (IL-1β), IL-10, IL-12, interferon-γ (IFN-γ), adipokines (retinol-binding protein 4 (RBP4), adiponectin, leptin, and resistin), and lipopolysaccharide (LPS), a bacterial cell wall component, on the expression of iNOS in 3T3-L1 preadipocytes. Notably, treatment with IL-1β at 20 ng/mL for 4 h markedly increased iNOS mRNA expression in 3T3-L1 preadipocytes, but that with IL-10 (10 ng/mL), IL-12 (5 ng/mL), IFN-γ (10 ng/mL), RBP4 (5 g/mL), adiponectin (100 ng/mL), leptin (100 ng/mL), and resistin (100 ng/mL), and LPS (1 g/mL) for 4 h had little or no effect on it. Results of dose-response and time-course experiments confirmed the ability of IL-1β at 20 ng/mL for 4 h to maximally induce iNOS mRNA expression in 3T3-L1 preadipocytes. Importantly, pharmacological inhibition studies demonstrated that treatment with AG490 (an inhibitor of Janus-activated kinases (JAKs) and signal transducer and activator of transcription proteins (STATs)), GO6976 (an inhibitor of PKCs), or PP1 (an Src kinase inhibitor) suppressed IL-1β-induced iNOS mRNA expression in 3T3-L1 preadipocytes, pointing out the involvement of JAKs/STATs, PKCs, and Src in the process. This work advocates that IL-1β is a major pro-inflammatory cytokine contributing to inflammation in the AT by up-regulating iNOS.

Interleukin 1β Up-Regulates mRNA Expression of Inducible Nitric Oxide Synthase in 3T3-L1 Preadipocytes: Role of JAKs/STATs, PKCs, and Src / 계명의대학술지

Recent evidence suggests obesity as a low or systemic chronic inflammation. (Pre)adipocytes in the adipose tissue (AT) express and secrete a variety of cytokines and adipokines. Inducible nitric oxide synthase (iNOS) is an inflammatory enzyme involved in the production of NO. Until now, the inducer(s) of iNOS expression in (pre)adipocytes remains unclear. In this study, we investigated the effects of proinflammatory cytokines (interleukin-1β (IL-1β), IL-10, IL-12, interferon-γ (IFN-γ), adipokines (retinol-binding protein 4 (RBP4), adiponectin, leptin, and resistin), and lipopolysaccharide (LPS), a bacterial cell wall component, on the expression of iNOS in 3T3-L1 preadipocytes. Notably, treatment with IL-1β at 20 ng/mL for 4 h markedly increased iNOS mRNA expression in 3T3-L1 preadipocytes, but that with IL-10 (10 ng/mL), IL-12 (5 ng/mL), IFN-γ (10 ng/mL), RBP4 (5 g/mL), adiponectin (100 ng/mL), leptin (100 ng/mL), and resistin (100 ng/mL), and LPS (1 g/mL) for 4 h had little or no effect on it. Results of dose-response and time-course experiments confirmed the ability of IL-1β at 20 ng/mL for 4 h to maximally induce iNOS mRNA expression in 3T3-L1 preadipocytes. Importantly, pharmacological inhibition studies demonstrated that treatment with AG490 (an inhibitor of Janus-activated kinases (JAKs) and signal transducer and activator of transcription proteins (STATs)), GO6976 (an inhibitor of PKCs), or PP1 (an Src kinase inhibitor) suppressed IL-1β-induced iNOS mRNA expression in 3T3-L1 preadipocytes, pointing out the involvement of JAKs/STATs, PKCs, and Src in the process. This work advocates that IL-1β is a major pro-inflammatory cytokine contributing to inflammation in the AT by up-regulating iNOS.

Cucurbitacin E’s Anti-Cancer Effects on HCT116 Human Colon Cancer Cells by Controlling Expression and Phosphorylation Levels of Caspase-9, eIF-2α, and ATF-4 / 계명의대학술지

Cucurbitacin E is a pivotal member of the cucurbitacin family and has been shown to have anti-cancer effects. However, until now, the anti-cancer effect and mode of action of cucurbitacin E in human colon cancer cells remain unclear. In this study, we investigated whether cucurbitacin E inhibits the growth of HCT116 human colorectal cancer cells. Treatment of cucurbitacin E at 1 mM markedly reduced the survival of HCT116 cells. Moreover, treatment of cucurbitacin E at 1 mM caused nuclear DNA fragmentation in HCT116 cells, pointing out its apoptosis-inducing effect. Treatment of cucurbitacin E at 1 mM also led to the activation of caspase-9 and poly(ADP‑ribose) polymerase (PARP) cleavage without affecting expression of death receptor (DR)-4/5 in HCT116 cells. Furthermore, while treatment of cucurbitacin E at 1 mM had no effect on expression of Mcl-1, it largely increased expression and phosphorylation of eukaryotic translation initiation factor-2α (eIF-2α) and activating transcription factor-4 (ATF-4) in HCT116 cells. Treatment of cucurbitacin E at 1 mM further up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), but not c-Jun N-terminal kinase1/2 (JNK-1/2), in HCT116 cells. However, treatment with PD98059, an inhibitor of ERK-1/2, that strongly blocked activation of ERK-1/2 had no effect on reduction of survival of HCT116 cells treated with cucurbitacin E at 1 mM. Taken together, these findings demonstrate that cucurbitacin E at 1 mM has strong anti-survival and pro-apoptotic effects on HCT116 cells, which are mediated through control of the expression and phosphorylation levels of caspase-9, PARP, eIF-2α, and ATF-4.

Cryptotanshinone Inhibits Lipid Accumulation in Differentiating 3T3-L1 Preadipocytes by Down-regulating C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3 / 계명의대학술지

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.

Cryptotanshinone Inhibits Lipid Accumulation in Differentiating 3T3-L1 Preadipocytes by Down-regulating C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3 / 계명의대학술지

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.

Cryptotanshinone Inhibits Lipid Accumulation in Differentiating 3T3-L1 Preadipocytes by Down-regulating C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3 / 계명의대학술지

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.

Prognostic Significance of Claudin 4 in Completely Resected Adenocarcinoma of the Lung

BACKGROUND: The development of diagnostic techniques and an awareness of health examinations can bring about an early diagnosis of lung cancer. However, appropriate postoperative management and adjuvant chemotherapy remain under debate in postoperative therapeutic strategy. The present study was conducted to assess the clinicopathologic factors that influence recurrence and prognosis after complete resection of lung cancer. METHODS: The present study analyzed 62 patients with lung cancer who underwent complete resection of diagnosed adenocarcinoma between 1994 and 2007. In addition to conventional factors, which include staging factor and histological evaluation, the present study also performed univariate and multivariate analyses to consider claudin, a cell adhesion molecule, as a prognostic factor by immunohistochemical staining. RESULTS: There was no correlation between conventional factors, including lymphatic and vascular invasion, and recurrence. However, there was a significant correlation between high expression of claudin 4 and cancer recurrence. In particular, there was a correlation between high expressions of claudin 1, 4, and 5 and a reduction of disease-free survival. CONCLUSION: Increased expressions of claudin 4 were negative prognostic factors in adenocarcinoma of the lung and thus could be used to identify high-risk patients for adjuvant chemotherapy, even if they had early-stage lung cancer. The present findings collectively suggest that consideration of claudin as a prognostic factor in the active postoperative treatment in patients at high risk will lead to better therapeutic outcomes with fewer side effects.

Down-regulation of IL-1beta-induced COX-2 Expression in A549 Lung Cancer Cells at Transcriptional Level by Leptomycin B Involves Inhibition of the IkappaB-alpha/NF-kappaB Pathway but Independent of CRM1

PURPOSE: Overexpression of COX-2, an enzyme responsible fro the synthesis of prostaglandins, is well linked to human chronic lung diseases. The mechanism by which COX-2 expression is increased or enhanced in cancer cells remains largely unknown. Any compound which can reduce COX-2 expression may be considered as an anti-cancer agent. MATERIALS AND METHODS: Leptomycin B (LMB) is a metabolite of Streptomyces and a specific inhibitor of CRM1 nuclear export receptor. A549 is a human lung cancer cell line. To evaluate the effect of LMB on COX-2 expression induced by IL-1beta, a pro-inflammatory cytokine, in A549 cells, Western blot and RT-PCR assays were applied to measure COX-2 protein and mRNA expressions in response to IL-1beta, respectively. Luciferase experiments were done to measure promoter activity of COX-2, NF-kappaB or AP-1. CRM1 siRNA trasfection experiment was performed to knock-down endogenous CRM1. Biochemical protein fractionation method was also carried out to see intracellular localization of proteins. RESULTS: LMB at 9 nM strongly suppressed IL-1beta-induced expression of COX-2 protein that was attributable to decreased COX-2 transcript and promoter activity, but not mRNA stability. Distinctly, knock-down of CRM1 had no effect on COX-2 expression by IL-1beta. Moreover, LMB did not affect IL-1beta-induced phosphorylation of ERK-1/2, JNK- 1/2, and p38 MAPK or AP-1 promoter activity. In contrast, LMB blocked IL-1beta- mediated cytosolic IkappaB-alpha degradation, p65 NF-kappaB nuclear translocation, and NF-kappaB promoter activity. CONCLUSION: LMB potently down-regulates IL-1beta- induced COX-2 at transcriptional level in A549 cells, in part, through modulation of the IkappaB-alpha/NF-kappaB pathway but independent of CRM1, MAPKs and AP-1.

Curcumin inhibits the expression of COX-2 in UVB-irradiated human keratinocytes (HaCaT) by inhibiting activation of AP-1: p38 MAP kinase and JNK as potential upstream targets

Ultraviolet B (UVB) irradiation of skin induces an acute inflammation. Cyclooxygenase-2 (COX-2) protein plays key roles in acute inflammation in UVB-irradiated keratinocyte cell line HaCaT. Recently, curcumin has been regarded as a promising anti-inflammatory agent due to its ability to inhibit COX-2 expression. However, it remains largely unknown whether curcumin inhibits the UVB-induced COX-2 expression in HaCaT cells. This study was undertaken to clarify the effect of curcumin on the expression of COX-2 in UVB- irradiated HaCaT cells and further determined the molecular mechanisms associated with this process. In this study, we have found that the expression of COX-2 mRNA and protein were up-regulated in UVB-irradiated HaCaT cells in a dose- and time-dependent manner. Interestingly, treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 MAPK and JNK in HaCaT cells. The DNA binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX- 2 expression by suppressing p38 MAPK and JNK activities in UVB-irradiated HaCaT cells. We propose that curcumin may be applied as an effective and novel sunscreen drug for the protection of photoinflammation.

Expression of Human beta-defensin 2 mRNA by Lipopolysaccharide in Human Corneal Epithelial Cells

Recently the transcriptional up-regulation of human beta-defensin 2 (HBD-2) by lipopolysaccharide (LPS) was found to be associated with NF-kappaB binding site. Although the general mechanisms of NF-kappaB activation by LPS stimulation are well understood, less is known about the signal transduction pathway leading to LPS-induced NF-kappaB activation in human corneal epithelial (HCE) cells. The aim of this study was to investigate the intracellular signals involved in LPS-induced HBD-2 mRNA expression in HCE cells. Pretreatments of inhibitors for NF-kappaB, protein tyrosine kinase, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) attenuated the LPS-induced NF-kappaB DNA binding activity and HBD-2 mRNA expression. Furthermore, pretreatments with inhibitors for protein kinase C (PKC), phosphatidylcholine-phospholipase C, phosphatidylinositol-phospholipase C, or phosphatidate phosphohydrolase prevented LPS-induced HBD-2 mRNA expression and HBD-2 prmoter-driven luciferase activity. However, the increased expression of HBD-2 mRNA and the increased DNA binding activity of NF-kappaB induced by LPS were not changed by the blockage of extracellular signal-regulated kinase (ERK) and of addition of antioxidants. Forskolin, a protein kinase A (PKA) agonist did not induce HBD-2 mRNA expression. These data demonstrate that LPS-induced HBD-2 mRNA expression via NF-kappaB is, at least in part, dependent on PKC, p38 MAPK, JNK, and protein tyrosine kinase status, but appears to be independent on PKA, ERK and ROS in HCE cells. Taken together, there may be more than one signaling pathways that lead to LPS-induced up-regulation of HBD-2 mRNA expression in HCE cells.