• BMB Reports
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• 제49권8호
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• pp.437-442
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• 2016

We aimed to study the role of protein L-isoaspartyl methyltransferase (PIMT) in neuronal differentiation using basic fibroblast growth factor (bFGF)-induced neuronal differentiation, characterized by cell-body shrinkage, long neurite outgrowth, and expression of neuronal differentiation markers light and medium neurofilaments (NF). The bFGF-mediated neuronal differentiation of PC12 cells was induced through activation of mitogen-activated protein kinase (MAPK) signaling molecules [MAPK kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p90RSK], and phosphatidylinositide 3-kinase (PI3K)/Akt signaling molecules PI3Kp110β, PI3Kp110γ, Akt, and mTOR. Inhibitors (adenosine dialdehyde and S-adenosylhomocysteine) of protein methylation suppressed bFGF-mediated neuronal differentiation of PC12 cells. PIMT-eficiency caused by PIMT-specific siRNA inhibited neuronal differentiation of PC12 cells by suppressing phosphorylation of MEK1/2 and ERK1/2 in the MAPK signaling pathway and Akt and mTOR in the PI3K/Akt signaling pathway. Therefore, these results suggested that PIMT was critical for bFGF-mediated neuronal differentiation of PC12 cells and regulated the MAPK and Akt signaling pathways.

• Molecules and Cells
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• 제28권1호
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• pp.37-42
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• 2009

Iron binding lactoferrin (Lf) is involved in the control of cell cycle progression. However, the molecular basis underlying the effects of Lf on cell cycle control, as well as its target genes, remains incompletely understood. In this study, we have demonstrated that a relatively low level of ironsaturated Lf, Lf($Fe^{3+}$), can stimulate S phase cell cycle entry, and requires Akt activation in MCF-7 cells. Lf($Fe^{3+}$) immediately induced Akt phosphorylation at Ser473, which subsequently induced the phosphorylation of two G1-checkpoint Cdk inhibitors, $p21^{Cip/WAF1}$ and $p27^{kip1}$. The Lf($Fe^{3+}$)-induced phosphorylation of Cdk inhibitors impaired their nuclear import behavior, thereby inducing cell cycle progression. However, the treatment of cells with a PI3K inhibitor, LY294002, almost completely blocked Lf($Fe^{3+}$)-stimulated cell cycle progression. LY294002 treatment abrogated Lf($Fe^{3+}$)-induced Akt activation, and prevented the cytoplasmic localization of $p27^{kip1}$. Higher levels of $p21^{Cip/WAF1}$ were also detected in the cytoplasmic sub-cellular compartment as a measure of cellular response to Lf($Fe^{3+}$). Consequently, the degree of phosphorylation of retinoblastoma protein was enhanced in response to Lf($Fe^{3+}$). Therefore, we conclude that Lf($Fe^{3+}$), as a potential antagonist of Cdk inhibitors, can facilitate the functions of E2F during progression to S phase via the Akt signaling pathway.

• Archives of Pharmacal Research
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• 제27권1호
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• pp.68-76
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• 2004

Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.

• The Korean Journal of Physiology and Pharmacology
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• 제23권2호
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• pp.103-111
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• 2019

The study is to investigate effects of andrographolide on experimental autoimmune myocarditis (EAM). Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. The EAM rats were treated with either andrographolide (25, 50, 100 mg/kg/day) or vehicle for 21 days. An antigen-specific splenocytes proliferation assay was performed by using the cells from control rats immunized with cardiac myosin. Survival rates, myocardial pathology and myocardial functional parameters (left ventricle end-diastolic pressure, ${\pm}dP/dt$ and left ventricular internal dimension) of EAM rats received andrographolide were significantly improved. Andrographolide treatment caused an decrease in the infiltration of $CD3^+$ and $CD14^+$ positive cells in myocardial tissue. Moreover, andrographolide treatment caused a reduction in the plasma levels of tumor necrosis factor-alpha, interleukin-17 (IL-17) and myosin-antibody, and an increase in the level of IL-10 in EAM rats. Oral administration of andrographolide resulted in the decreased expression of p-PI3K, p-Akt without any change of PI3K and Akt. Further results indicate andrographolide significantly inhibited myosin-induced proliferation in splenocytes, and this effect was inhibited by co-treatment of SC79 (Akt activator). Our data indicate andrographolide inhibits development of EAM, and this beneficial effect may be due to powerful anti-inflammatory activity and inhibitory effect on PI3K/Akt pathway.

• Journal of Ginseng Research
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• 제43권1호
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• pp.154-160
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• 2019

Background: Compound K (CK) is an active metabolite of ginseng saponin, ginsenoside Rb1, that has been shown to have ameliorative properties in various diseases. However, its role in inflammation and the underlying mechanisms are poorly understood. In this report, the antiinflammatory role of CK was investigated in macrophage-like cells. Methods: The CK-mediated antiinflammatory mechanism was explored in RAW264.7 and HEK293 cells that were activated by lipopolysaccharide (LPS) or exhibited overexpression of known activation proteins. The mRNA levels of inflammatory genes and the activation levels of target proteins were identified by quantitative and semiquantitative reverse transcription polymerase chain reaction and Western blot analysis. Results: CK significantly inhibited the mRNA expression of inducible nitric oxide synthase and tumor necrosis factor-${\alpha}$ and morphological changes in LPS-activated RAW264.7 cells under noncytotoxic concentrations. CK downregulated the phosphorylation of AKT1, but not AKT2, in LPS-activated RAW264.7 cells. Similarly, CK reduced the AKT1 overexpression-induced expression of aldehyde oxidase 1, interleukin-$1{\beta}$, interferon-${\beta}$, and tumor necrosis factor-${\alpha}$ in a dose-dependent manner. Conclusion: Our results suggest that CK plays an antiinflammatory role during macrophage-mediated inflammatory actions by specifically targeting the AKT1-mediated signaling pathway.

• 대한한방내과학회지
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• 제27권1호
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• pp.149-165
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• 2006

Objectives: This study was done to evaluate the effects of Saengkankunbi-tang on cell-viability, proliferation, cell-cycle, apoptosis and DNA replication on HepG2 cell and to find out by which molecular-biological mechanism by which Saengkankunbi-tang operates. Methods : The MTT assay, cell counting assay, [3H]-thymidine incorporation assay, flow cytometric analysis, tryphan blue exclusion assay, western blot analysis, quantative RT-PCR were taken. Results : Saengkankunbi-tang had no effect on proliferation, cell-cycle and DNA replications of HepG2 cells, while it improved cell viability and reduced apoptosis, and it activated Akt and NFKB. But, it did not produce an effect on cell viability and apoptosis when P13K/Akt pathway was blocked by LY294002 nor when $NF{\kapa}B$ activation was blocked by DN-$I{\kapa}B$. Conclusion : These results suggests that Saengkankunbi-tang improves cell viability and reduces apoptosis of HepG2 cells, by activating $NF{\kapa}B$ through PI3K/Akt pathway.

• Biomolecules & Therapeutics
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• 제22권3호
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• pp.200-206
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• 2014

N-(p-Coumaryol) tryptamine (CT), a phenolic amide, has been reported to exhibit anti-oxidant and anti-inflammatory activities. However, the underlying mechanism by which CT exerts its pharmacological properties has not been clearly demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of CT in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. CT significantly inhibited LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and $PGE_2$, and protein expressions of iNOS and COX-2. In addition, CT significantly suppressed LPS-induced secretion of pro-inflammatory cytokines such as TNF-${\alpha}$ and IL-$1{\beta}$. To elucidate the underlying anti-inflammatory mechanism of CT, involvement of MAPK and Akt signaling pathways was examined. CT significantly attenuated LPS-induced activation of JNK/c-Jun, but not ERK and p38, in a concentration-dependent manner. Interestingly, CT appeared to suppress LPS-induced Akt phosphorylation. However, JNK inhibition, but not Akt inhibition, resulted in the suppression of LPS-induced responses, suggesting that JNK/c-Jun signaling pathway significantly contributes to LPS-induced inflammatory responses and that LPS-induced Akt phosphorylation might be a compensatory response to a stress condition. Taken together, the present study clearly demonstrates CT exerts anti-inflammatory activity through the suppression of JNK/c-Jun signaling pathway in LPS-challenged RAW264.7 macrophage cells.

• Journal of Ginseng Research
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• 제47권2호
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• pp.274-282
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• 2023

Background: Ginsenoside compound K (CK) stimulated activation of the PI3K-Akt signaling is one of the major mechanisms in promoting cell survival after stroke. However, the underlying mediators remain poorly understood. This study aimed to explore the docking protein of ginsenoside CK mediating the neuroprotective effects. Materials and methods: Molecular docking, surface plasmon resonance, and cellular thermal shift assay were performed to explore ginsenoside CK interacting proteins. Neuroscreen-1 cells and middle cerebral artery occlusion (MCAO) model in rats were utilized as in-vitro and in-vivo models. Results: Ginsenoside CK interacted with recombinant human PTP1B protein and impaired its tyrosine phosphatase activity. Pathway and process enrichment analysis confirmed the involvement of PTP1B and its interacting proteins in PI3K-Akt signaling pathway. PTP1B overexpression reduced the tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) after oxygen-glucose deprivation/reoxygenation (OGD/R) in neuroscreen-1 cells. These regulations were confirmed in the ipsilateral ischemic hemisphere of the rat brains after MCAO/R. Ginsenoside CK treatment reversed these alterations and attenuated neuronal apoptosis. Conclusion: Ginsenoside CK binds to PTP1B with a high affinity and inhibits PTP1B-mediated IRS1 tyrosine dephosphorylation. This novel mechanism helps explain the role of ginsenoside CK in activating the neuronal protective PI3K-Akt signaling pathway after ischemia-reperfusion injury.

• 생명과학회지
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• 제25권9호
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• pp.984-992
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• 2015

혈근초(Sanguinaria canadensis)에서 처음 분리된 sanguinarine은 항산화, 항암 및 면역 증강 등의 효능이 있는 것으로 알려진 alkaloid 계열 물질 중의 하나이다. 본 연구에서는 인체간암 HepG2 세포를 대상으로 sanguinarine의 apoptosis 유도 효능 및 관련 기전 해석을 시도하였다. 본 연구의 결과에 의하면 sanguinarine은 HepG2 간암세포의 증식을 처리 농도 의존적으로 억제하였으며, 이는 apoptosis 유도와 연관성이 있었다. Sanguinarine에 의한 apoptosis 유도에는 Fas 및 Bax의 발현 증가, 미토콘드리아에서 세포질로의 cytochrome c 유리 및 MMPl (Δψm)의 소실을 동반하였다. Sanguinarine은 intrinsic 및 extrinsic apoptosis pathway의 활성에 관여하는 initiator caspase인 caspase-9와 -8의 활성과 effector caspase인 caspase-3의 활성 및 PARP 단백질의 단편화를 유발하였다. Sanguinarine은 또한 ROS의 생성을 촉진시켰으며, N-acetylcysteine 처리에 의한 ROS의 생성을 차단하였을 경우, sanguinarine에 의한 apoptosis 효능이 완벽하게 차단되었다. 아울러 sanguinarine은 Akt의 인산화를 억제한 반면, MAPKs의 인산화를 촉진시켰으며, 특히 PI3K와 ERK의 선택적 억제제는 sanguinarine에 의한 HepG2 간암세포의 증식을 더욱 억제시켰다. 따라서 sanguinarine에 의한 HepG2 간암세포의 apoptosis 유발에는 ROS 생성 의존적인 intrinsic 및 extrinsic signaling pathway가 동시에 활성화되며, PI3K/Akt 및 ERK 신호계가 관여함을 알 수 있었다.

• 대한본초학회지
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• 제31권1호
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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.