• The Korea Journal of Herbology
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• v.31 no.1
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• pp.1-5
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• 2016

Objectives : The purpose of this study was to investigate inhibitory effects of steamed Polygonatum odoratum extract (POE) on insulin resistance in rat skeletal muscle cells, L6 cells.Methods : Polygonatum odoratum (P. odoratum) extract was extracted with ethyl acetate. Activity of α-glucosidase in POE was measured for blood glucose regulation. MTT assay was examined for cell toxicity. Western blot analysis for measurement of adiponectine, peroxisome proliferator-activated receptorγ (PPARγ), insulin receptor substrate (IRS), glucose transporter 4 (Glut-4) and phosphorylation of serine/threonine-specific protein kinase (Akt) expressions were performed. Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor.Results : The results revealed that POE inhibited α-glucosidase activity. Treatment of POE in L6 cells inhibited the differentiation of L6 cells compared to those of vehicl control. Additionally, protein expressions of adiponectine, PPARγ, IRS and Glut-4 were significantly regulated compared to those of vehicle control (p < 0.05), respectively. Futhermore, phosphorylation of Akt was increased in L6 cells treated with POE compared to that of vehicle control (p < 0.05). pAkt expression was significantly accentuated with Akt inhibitor (LY294002).Conclusions : These results suggest that POE may have potential as a natural agent for prevention/improvement of diabetes, especially, regulation of blood glucose. Therefore, further additional study should be conducted to elucidate in depth the pharmaceutical efficacy of these.

• Tuberculosis and Respiratory Diseases
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• v.65 no.4
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• pp.343-350
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• 2008

PD-L1 is expressed in a variety of antigen-presenting cells and provides T cell tolerance via ligation with its receptor PD-1 and B7-1 on T cells. Stimulation with lipopolysaccharide (LPS) can increase the level of PD-L1 expression in B cells and macrophages, which suggests that this molecule plays a role in the immunosuppression observed in severe sepsis. The aim of this study was to identify which of the downstream pathways of TLR4 are involved in the up-regulation of PD-L1 by LPS in macrophages. Flow cytometry was used to examine the expression of PD-L1 in RAW 264.7 macrophages stimulated with LPS. The following chemical inhibitors were used to evaluate the role of each pathway: LY294002 for PI3K/Akt, SB202190 for p38 MAPK, and U0126 for MEK. LPS induced the expression of PD-L1 in a time- and dose-dependent manner. Transfection of siRNA for TLR4 suppressed the induction of PD-L1. Pretreatment with LY294002 and SB202190 decreased the level of PD-L1 expression but U0126 did not. Overall, the PI3K/Akt and p38 MAPK pathways are involved in the up-regulation of PD-L1 expression in RAW 264.7 macrophages stimulated with LPS.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.12
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• pp.1708-1716
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• 2016

The Akt/mammalian target of the rapamycin (mTOR) pathway is activated in the majority of human cancers. Activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy. In this study, we evaluated the apoptotic effect of ethanol extract of Artemisia annua L. through down-regulation of Akt signal pathways and the mitochondrial pathway in hepato-carcinoma cells (HepG2). A. annua extract is known as a medicinal herb that is effective against cancer. We evaluated anti-proliferative activity by MTT-based viability assay and apoptotic effect by Annexin-V/PI staining, mitochondrial membrane potential (MMP), and caspase-3/7 activity as determined by flow cytometry. A. annua treatment led to loss of MMP, resulting in cytochrome c-inducible activation of caspase-3/7. Treatment with A. annua extract reduced activities of Akt/mTOR/anti-apoptotic proteins (such as Bcl-2 and $Bcl-X_L$), leading to increased activation of tumor suppressor p53 and pro-apoptotic proteins (such as Bax and Bak). We applied LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR) to determine the relationship between signal transduction of proteins associated with apoptosis. LY294002 and rapamycin significantly reduced cell viability and increased apoptosis. These results indicate that Bcl-2 and caspase-3 are key regulators in A. annua extract-induced apoptosis in HepG2 cells and are controlled through the Akt/mTOR signaling pathway.

• Journal of Life Science
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• v.21 no.1
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• pp.89-95
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• 2011

p53 is tumor suppressor gene that regulates apoptosis such as caspase-dependent and p21-mediated signaling pathways. PI3K/Akt is known to be over-activated in cancer cells. Akt activates many survival-related signals such as mTOR and COX-2. Inactivation of Akt would result in non-inhibition of p53 as well as induced apoptosis. In this study, we showed that curcumin and EGCG activate p53 via inhibition of the Akt signaling pathway. Treatments using curcumin and EGCG in different concentrations for 24 hr and 48 hr inhibited proliferation of HCT116 colon cancer cells and increased apoptotic cell death. Also, our data showed that curcumin and EGCG increased the p53 expression and decreased the p-Akt. Treatment of LY294002 (Akt inhibitor) resulted in decreased cell proliferation of cancer cells, while LY294002 treated with curcumin or EGCG showed a greater decrease of cell proliferation. In addition, inhibition of Akt induced p53 activation in HCT116 colon cancer cells. These results suggest that curcumin and EGCG induce apoptosis by inhibiting Akt and increase p53 in HCT116 colon cancer cells.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.5
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• pp.731-738
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• 2016

Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that $100{\mu}g/mL$ LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ's expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway.

• Biomedical Science Letters
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• v.22 no.2
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• pp.60-64
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• 2016

In the pathogenesis of inflammatory diseases such as allergies, S100A8 acts as an important molecule and T lymphocytes are essential cytokine-releasing cells. In this study, we investigated the effect of S100A8 on release of cytokines, specifically MCP-1, IL-6, and IL-8 in T cells, and its associated signaling mechanism. S100A8 increased secretion of MCP-1, IL-6, and IL-8 in a time- and dose-dependent manner. Elevated secretion of MCP-1, IL-6, and IL-8 due to S100A8 was inhibited by the TLR4 inhibitor TLR4i, the PI3K inhibitor LY294002, the $PKC{\delta}$ inhibitor rottlerin, the ERK inhibitor PD98059, the p38 MAPK inhibitor SB202190, the JNK inhibitor SP600125, and the NF-${\kappa}B$ inhibitor BAY-11-7085. S100A8 induced phosphorylation of ERK, p38 MAPK, and JNK in a time-dependent manner, and activation was suppressed by TLR4i, LY294002, and rottlerin. S100A8 induced NF-${\kappa}B$ activation by $I{\kappa}-B{\alpha}$ degradation, and NF-${\kappa}B$ activity was suppressed by PD98059, SB202190, and SP600125. These results indicate that S100A8 induces cytokine release via TLR4. Study of PI3K, $PKC{\delta}$, MAPKs, and NF-${\kappa}B$ will contribute to elucidation of the S100A8-invovled mechanism.

• Journal of Physiology & Pathology in Korean Medicine
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• v.32 no.3
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• pp.157-164
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• 2018

This study was designed to investigate the mechanism of the synergistic anticancer effect of Astragalus membranaceus (AM) and Adenophora triphylla var. japonica (AT) in H1299 human lung carcinoma cells. A combined treatment of ethanol extract of AM (EAM) and AT (EAT) explosively increased the reactive oxygen species (ROS) generation in H1299 cells compared to the single treatment of each of them. Co-treatment of N-acetyl-L-cysteine (NAC) with EAM and EAT markedly enhanced the cell viability and suppressed apoptosis in H1299 cells, suggesting that ROS generation contributed to the anticancer effect of EAM and EAT. Interestingly, the combined treatment of EAM and EAT down-regulated p-AKT in H1299 cells, which was abrogated by NAC treatment. These results clearly indicated that ROS generation mediated the inactivation of AKT. Co-treatment of LY294002 with EAM and EAT significantly reduced the cell viability at a concentration which EAM and EAT didn't show any cytotoxicity. In addition, the recovery of cell viability by co-treatment of NAC with EAM and EAT was quite reversed by LY294002 treatment, which confirmed that the inactivation of AKT played a pivotal role in ROS-mediated apoptosis. Taken together, our results demonstrated that the synergistic anticancer effect of EAM and EAT was mediated by ROS generation and inactivation of AKT. We provide a valuable preclinical data for the development of more effective combination of AM and AT to treat lung cancer.

• Korean Journal of Acupuncture
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• v.28 no.3
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• pp.43-51
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• 2011

Objectives : In this study, we investigated the effect of Fructus Ligustri Lucidi $H_2O$ fraction (FLLW) on cell proliferation, and the phosphorylation of ERKs and Akt in human dermal fibroblast neonatal (HDFn). Methods : After treatment of HDFn with FLLW, MTT assay was performed to quantitatively determine cellular viability. The ERK and Akt pathways were analyzed in vitro by Western blot in a HDFn. HDFn proliferation after FLLW and minoxidil treatment in the absence or presence of PD98059, a MEK inhibitor, LY294002, and a PI3K inhibitor, was examined by Western blot or MTT assay. Results : FLLW increased cell proliferation in a dose-dependent manner and minoxidil used as positive control also induced cell proliferation in HDFn. FLLW increased the phosphorylation of ERK and Akt. In addition, minoxidil, too, induced the phosphorylation of ERK and Akt in HDFn. PD98059 and LY294002 significantly attenuated FLLW-inducible p-ERK and p-Akt expression and proliferation in cultured HDFn. Conclusions : Our results suggest that FLLW stimulates the growth of fibroblast cells through ERK and Akt pathways. Therefore, FLLW is a potential agent for the inducer of fibroblast growth.

• The Journal of Korean Society for Radiation Therapy
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• v.14 no.1
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• pp.165-174
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• 2002

Vascular endothelial growth factor (VEGF) has been identified as a peptide growth factor specific for vascular endothelial cells. In this study, we examined the effect of VEGF on radiation induced apoptosis and receptor/second messenger signal transduction pathway for VEGF effect in human umbilical vein endothelial cells (HUVECs). VEGF was found to protect HUVECs against the lethal effects of ionizing radiation by inhibiting the apoptosis induced in these cells by radiation exposure. VEGF (1-30 ng/ml) dose dependently inhibited apoptosis by irradiation. Pre-treatment with Flt-1 and Flk-l/KDR receptor blocked the VEGF-in duced antiapoptotic effect. Phosphatidylinositol 3'-kinase (PI3-kinase) specific inhibitor, Wortman in and LY294002, blocked the VEGF-induced antiapoptotic effect. These data suggest that VEGF may play an important role in survival of HUVECs due to the prevention of apoptotic cell death caused by some stresses such as ionizing radiation.

• Biomedical Science Letters
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• v.13 no.4
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• pp.279-285
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• 2007

Although retinoic acid has been known as either anti-inflammatory or pro-inflammatory molecule, depending on the cell type, its exact role in mature human neutrophils has not been fully explored. In this study, we investigate the effects of retinoic acid on neutrophil apoptosis and the associated mechanism and found that 9-cis retinoic acid (9CRA) significantly inhibits the spontaneous apoptosis of neutrophils. Its effect is increased by co-treatment with $TNF-\alpha$ (P<0.05). The 9CRA-induced inhibition is blocked by the following enzyme inhibitors: Ly 294002, phosphoinoside (PI)-3 kinase inhibitor, U73122, a phospholipase C (PLC) inhibitor, PP2, Src family protein inhibitor, SB202190, p38 MAPK inhibitor, and BAY-11-7085, NF-kB inhibitor. This study also demonstrates that all-trans retinoic acid suppresses spontaneous apoptosis, similar to the mechanism of inhibition exhibited by 9CRA. Phosphorylation of p38 MAPK decreases by 9CRA treatment. $Ik-B{\alpha}$ is degraded until 30 minutes after a time-dependent 9CRA treatment, but degradation can be inhibited by Ly 294002. These results indicate that 9CRA decreases p38 MAPK activation, induces NF-kB activation via PI-3 kinase, and also blocks cleavage of caspase 3. As these findings suggest, 9CRA has a molecular mechanism which may help pro-inflammatory response by blocking neutrophil apoptosis.