• Asian Pacific Journal of Cancer Prevention
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• 제15권11호
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• pp.4555-4561
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• 2014

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and Methods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and $IC_{50}$ values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. Results: The $IC_{50}$ for imatinib on K562 was 362nM compared to 3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down-regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.

• 대한예방한의학회지
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• 제15권3호
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• pp.127-140
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• 2011

Objectives : The research was conducted to confirm the effect of Acanthopanax senticosus harms(ASH) on the anti-tumor activities in AGS human gastric cancer cells. Methods : To examine the potential anti-tumor effect of ASH, we performed many experiments. After processing AGS cancer cells with varying concentrations 80% ethanol ASH extract, analyses by MTT, flow cytometer(FACS) and western blot were used. Results : AGS cancer cells showed decreased cell proliferation and increased contents of S phase when treated with ASH. Moreover, the Western blot experiment showed that ASH affected S phase cell cycle-related molecules(Cyclin A, p21 and p16) in AGS cells. ASH also inhibited EGFR-STAT3 pathway in AGS human gastric cancer cells. Conclusion : Based on these results, we observed that ASH arrested the cell cycle at S phase and inhibited the phosphorylation of EGFR and STAT3 proteins which reduce the cell cycle and the manifestation of the genes that are related to inhibiting cell growth in AGS cells. It can be concluded that ASH can be used in developing medicine for gastric cancer.

• 대한본초학회지
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• 제35권4호
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• pp.9-16
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• 2020

Objective : To identify the effects of the water extract of Liriope platyphylla tuber (Liriopis tuber, LT) on the activation of astocytes, we investigated the regulatory effects of LT extract on H₂O₂-induced oxidative damage in C6 rat astrocytes. Methods : LT extract was extracted with boiling water. C6 cell line were treated with LT extract at 1, 2, and 3 mg/㎖ or without for 30 min and then stimulated with H₂O₂ at 5 ㎛ for 24 hr. The cell viability was measured by MTT assay. The expression of glial fibrillary acidic protein (GFAP), signal transducer and activator of transcription 3 (STAT3), phospho-STAT3 (pSTAT3), cyclooxygenase (COX-2), Nuclear factor-κB (NF-κB), superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1), catalase, Akt, phospho-Akt (p-Akt) phosphoinositide 3-kinases (PI3K), and protein kinase C alpha (PKCα) proteins were determined by Western blot, respectively. GFAP expression was also observed with immunocytochemistry under a fluorescence microscope. Results : LT extract induced cell proliferation in H₂O₂-stimulated C6 cells. LT extract significantly inhibited the expression of GFAP, NF-κB and COX-2 and increased the expression of HO-1 and the phosphorylation of STAT3 in H₂O₂-stimulated C6 cells. LT extract also significantly increased the phosphorylation of Akt and decreased the expression of PKCα in a dose-dependent manner in H₂O₂-stimulated C6 cells. Conclusions : LT extract can regulate H₂O₂-induced activation of astrocytes through inhibiting the expression of NF-κB, COX-2 and regulating Akt / HO-1, STAT3 or PKCα signaling pathway.

• 한국식품영양과학회지
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• 제41권9호
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• pp.1226-1234
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• 2012

본 연구에서는 염증반응을 조절하는 다양한 신호전달체계를 중심으로 분자생물학적 방법을 통해 piceatannol의 항염증 기전을 규명하였다. LPS로 염증반응을 유도한 Raw 264.7 대식세포에서 piceatannol은 iNOS의 발현 억제를 통해 NO의 생성을 감소시키고 염증성 사이토카인(TNF-${\alpha}$, IL-6, IL-$1{\beta}$)의 생성을 감소시켰다. 염증반응을 조절하는 신호전달체계 중 piceatannol은 LPS에 의해 유도된 $I{\kappa}B$의 분해와 p65의 핵으로의 이동을 억제하고, LPS에 의해 유도된 SAPK/JNK의 인산화를 억제하였다. 또한 piceatannol은 LPS와 IL-6(LPS에 의해 증가됨)에 의한 STAT3의 활성화를 억제하였다. 뿐만 아니라 piceatannol은 Nrf2의 핵 내 축적을 야기하고 ARE의 transcriptional activity를 증가시켜 HO-1의 발현을 증가시켰다. 본 연구의 결과, piceatannol은 NF-${\kappa}B$와 AP-1, STAT3 신호전달의 억제를 통해, 그리고 HO-1의 발현 증가를 통해 항염증 효과를 나타내었다(Fig. 8).

• Biomolecules & Therapeutics
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• 제17권4호
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• pp.370-378
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• 2009

N-myc downstream-regulated gene 2 (NDRG2) has recently been found to be a tumor suppressor gene. Although it has been reported that NDRG2 expression in breast cancer cells decreases cell proliferation by inhibiting STAT3 activation via SOCS1 induction, the molecular mechanism of chemotherapeutic agent-induced apoptosis is not well known. To elucidate the effect of NDRG2 on the apoptotic pathway induced by doxorubicin, we established stable cell lines expressing NDRG2 and investigated the effect of NDRG2 expression on the doxorubicin-induced apoptosis. While STAT3 activation was remarkably inhibited by NDRG2 overexpression, the expression level of p21 was increased by NDRG2 expression. We confirmed that NDRG2-expressing cells treated with doxorubicin suppressed STAT3 activation and upregulated p21 expression. NDRG2 expression considerably enhanced TUNEL positive apoptotic cells, poly-ADP ribose polymerase (PARP) cleavage, release of cytochrome c to cytosol, and caspase-3 activity in doxorubicin-induced apoptosis. Bid expression in a resting state and after treatment with doxorubicin increased in MDA-MB-231-NDRG2 cells compared to MDA-MB-231-mock cells. Meanwhile, Bcl-$x_L$ expression decreased in MDA-MB-231-NDRG2 cells compared to MDA-MB-231-mock cells in a resting state and in doxorubicin-treated cells. Collectively, these data suggest that suppression of STAT3 activation by NDRG2 influences the sensitivity to doxorubicin-induced apoptosis of breast cancer cells and this may provide a potential therapeutic benefit to overcome the resistance against doxorubicin in breast cancer.

• 대한약침학회지
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• 제18권1호
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• pp.19-26
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• 2015

Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC) have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/Bcl2 Antagonist X (Bcl2/Bax) ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$) activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-${\kappa}B$ expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

• Molecules and Cells
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• 제45권4호
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• pp.257-272
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• 2022

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

• Journal of Ginseng Research
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• 제40권1호
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• pp.18-27
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• 2016

Background: It is not clear whether ginseng affects cyclosporine A (CsA)-induced desirable immunosuppressive action. In this study, we evaluated the immunological influence of combined treatment of ginseng with CsA. Methods: Using CD4+ T cells from mouse spleens stimulated with the T cell receptor (TCR) or allogeneic antigen-presenting cells (APCs), we examined the differentiation of naïve T cells into T helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs), and their cytokine production during treatment by Korean Red Ginseng extract (KRGE) and/or CsA. The influence of KRGE on the allogeneic T cell response was evaluated by mixed lymphocyte reaction (MLR). We also evaluated whether signal transducer and activator of transcription 3 (STAT3) and STAT5 are implicated in this regulation. Results: Under TCR stimulation, KRGE treatment did not affect the population of CD4+interferon gamma ($IFN{\gamma}$)+ and CD4+interleukin (IL)-4+ cells and their cytokine production compared with CsA alone. Under the Th17-polarizing condition, KRGE significantly reduced the number of CD4+IL-17+ cells and CD4+/phosphorylated STAT3 (p-STAT3)+ cells, but increased the number of CD4+CD25+forkhead box P3 (Foxp3)+ cells and CD4+/p-STAT5+ cells compared with CsA alone. In allogeneic APCs-stimulated CD4+ T cells, KRGE significantly decreased total allogeneic T cell proliferation. Consistent with the effects of TCR stimulation, KRGE reduced the number of CD4+IL-17+ cells and increased the number of CD4+CD25+Foxp3+ cells under the Th17-polarizing condition. Conclusion: KRGE has immunological benefits through the reciprocal regulation of Th17 and Treg cells during CsA-induced immunosuppression.

• Clinical and Experimental Reproductive Medicine
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• 제36권3호
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• pp.187-197
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• 2009

목 적: 단백질 인산화는 세포신호전달에 매우 중요한 현상으로서, 수많은 조절인자들이 난자성숙에 관여하게 된다. 그러나 이들 중에서 어떤 단백질이 인산화되어 난자성숙을 조절하는지는 잘 알려져 있지 않다. 따라서 체세포의 신호전달과정에서 인산화를 통해 중요한 기능을 한다고 알려져 있는 일곱 가지 단백질들이 생쥐의 난자성숙과정에서 어떻게 인산화 되고 있는지 알아보고자 한 개 샘플에서 일곱 개의 변화를 한꺼번에 측정할 수 있는 bead-based multiplex phosphorylation assay를 이용하여 본 연구를 수행하였다. 연구방법: ICR 생쥐에 PMSG를 주사하고 46시간 후에 cumulus-oocyte complex (COCs) 형태로 미성숙 난자를 채취한 후 체외배양 하면서, 배양 2시간 후에 GVBD를, 배양 8시간 후에 MI을, 배양 16시간 후에 MII 단계의 난자를 얻었고 체내에서 배란한 MII 단계의 난자는 수란관에서 얻었다. 각 단계의 난자를 100개씩 모아서 mitogen-activated protein kinase (MAPK)에 속하는 세가지 단백질인 ERK1/2, JNK, p38 MAPK와 Akt, GSK-$3{\alpha}/{\beta}$, $I{\kapa}B{\alpha}$, STAT3 등 총 일곱 단백질의 인산화를 Bio-Plex System을 이용하여 같은 시료에서 동시에 측정하였으며 세 번의 반복실험을 통하여 얻어진 결과를 통계적으로 분석하였다. 결 과: 생쥐의 난자성숙과정에서 측정된 일곱 가지 단백질 중에서 인산화가 현저히 증가하는 단백질로는 ERK1/2, JNK, p38 MAPK와 STAT3로서 미성숙 난자에 비해서 3배에서 20배까지 인산화되는 결과를 보였다. 반면에 GSK-$3{\alpha}/{\beta}$, $I{\kappa}B{\alpha}$의 인산화의 변화는 미약하였으며, Akt의 경우에는 변화가 전혀 없었다. 난자성숙 과정에서 분석 대상 단백질들의 인산화는 GVBD 단계에서 활성화되기 시작하여 MI에서 현저히 높게 증가하며 MII까지 높게 유지되었다. 결 론: 본 연구는 난자성숙과정에서 일곱 가지 단백질의 인산화를 동시에 측정한 최초의 보고로서 이 방법은 난자와 같이 적은 양의 시료에서의 여러 개의 단백질 인산화를 동시에 분석하는데 유용할 것으로 생각된다. 본 연구결과, 세 가지 MAPK 단백질인 ERK1/2, JNK, p38 MAPK 외에도 STAT3가 난자성숙에 있어서 매우 중요한 조절자로 생각되었다. 또한 Akt의 473번 serine기의 인산화는 난자성숙에 관여하지 않음을 알 수 있었다.

• KSBB Journal
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• 제15권2호
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• pp.134-138
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• 2000

알콜증류폐액에 빵효모를 배양하는 중에 glucose와 ammo-nium을 자동첨가함으로써 폐액배양액의 균체농도를 높여서 알콜증류폐액의 이용성을 증가시키고자 하였다. 배양중에 glucose를 공급하여 줌으로서 공급하지 않은 경우보다 1.3배 더 높은 균체농도를 얻었으며, glucose와 (NH4)2SO4를 공급하여 줌으로써 5.8배 더 높은 균체농도를 얻었다. Glucose의 경우에는 배양액의 DO를 제어파라미터로 하는 제어방법을 사용하여 자동첨가하고, ammonium의 경우에는 pH조정제로서 NH4OH을 사용함으로서 pH제어기에 의하여 자동으로 공급되도록 배양하였다. 배양결과 증식의 정체없이 배양 22.5시간후에 최대 12.0 g/L의 건조균체량에 도달하였으며, 균의 최대비증식속도는 0.18 hr-1였고, 대당균체수율이 약 0.54g/gdldjTdmamfh glucose의 첨가가 경제적이라고 생각되었다. 증균배양으로 폐액배양액의 COD를 약 22% 더 감소시킬 수 있었다.