• BMB Reports
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• 제40권2호
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• pp.196-204
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• 2007

Our previous study has shown that sodium selenite can cause apoptosis in acute promyelocytic leukemia-derived NB4 cells in a caspase-dependent manner, but the detailed mechanism is unknown. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK) in mediating sodium selenite -induced apoptosis in NB4 cell. Though no apparent elevation of ERK activity was observed during the apoptosis in NB4 cells caused by 20 μM sodium selenite treatment, PD98059 and U0126, specific chemical inhibitors of the MEK/ERK signaling pathway, were shown to strongly prevent the apoptosis process, while ERK activator TPA enhanced the process. It is also known that p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 had slight effects on apoptosis. Further study indicated that ERK exerted its proapoptotic effect only at the early stage of apoptosis and played an antiapoptotic role at the later stages. Taken together, our findings suggest that ERK plays an active role in mediating sodium seleniteinduced apoptosis in NB4 cells .

• Tuberculosis and Respiratory Diseases
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• 제69권4호
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• pp.271-278
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• 2010

Background: Recent studies have demonstrated that the epidermal growth factor receptor (EGFR) genotype is the most important predictive marker to EGFR-tyrosine kinase inhibitors (TKIs) and first-line gefitinib treatment will be approved in the near future for use in non-small cell lung cancer (NSCLC) patients with the EGFR mutation. Direct sequencing is known to be the standard for detecting EGFR mutations; however, it has limited sensitivity. Peptide nucleic acids (PNA)-mediated PCR clamping method is a newly introduced method for analyzing EGFR mutations with increased sensitivity and stability. Methods: A total of 71 NSCLC patients were analyzed for EGFR mutations using the PNA-mediated PCR clamping technique. Sixty-nine patients were analyzed for clinicopathologic correlation with EGFR genotype; 2 patients with indeterminate results were excluded. In order to determine EGFR-TKI drug response, 57 patients (42 gefitinib, 15 erlotinib) were included in the analysis. Results: The EGFR mutation rate was 47.8%. Being female, a non-smoker, and having adenocarcinoma were favorable clinicopathologic factors, as expected. However, more than a few smokers (33.3%), male (28.1%), and patients with non-adenocarcinoma (28.6%) had the EGFR mutation. Having a combination of favorable clinicopathologic factors did not increase the EGFR mutation rate significantly. Drug response to EGFR-TKIs showed significant differences depending on the EGFR genotype; ORR was 14.3% for wild type vs 69.0% for mutant type; DCR is 28.6% for wild type vs 96.6% for mutant type. The median EGFR-TKI treatment duration is 7.6 months for mutant type group and 1.4 months for wild type group. Conclusion: EGFR genotype determined using the PNA-mediated PCR clamping method is significantly correlated with the clinical EGFR-TKI responses and PNA-mediated PCR.

• Proceedings of the Korean Society of Applied Pharmacology
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.186-186
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• 1994

Many agonists have been known to activate the hydrolysis of membrane phospholipids through the bindings with corresponding receptors on the various cells. Diacylglycerol and inositol 1,4,5-trisphosphate(IP3) generated by the action of phosphoinositide-specific phospholipase C (PI-PLC) are well known second messengers for the activation of protein kinase C and the mobilization of Ca2+ in many cells. Three types of PI-PLC isozyme (${\alpha}$,${\gamma}$, and $\delta$) and several subtrpes for each type have been identified from mammalian sources by purification of enzymes and cloning of their cDNAs. Each type PI-PLC isozyme is coupled to different receptors and mediators, for example, ${\beta}$-types are coupled to the seven-transmembrane-receptors via Gq family of G-proteins and ${\beta}$-types directly to the receptor tyrosine kinases. Specific modulators for the signaling pathway through each type of PI-PLC should be very useful as potential potential candidates for lend substances in developing novel drugs. To establish the sensitive and convenient screening systems for searching modulators on PI-PLC mediated signaling, two kinds of approaches have been tried. (1) Establishment of in vitro assay condition for each type of PI-PLC isozyme: Overexpression by using vaccinia virus and purification of each isozyme was carried out for the preparation of large amounts of enaymes. Optimum and sensitive assay condition for the measurements of PI-ELC activities were established. (2) Development of the cell lines in which each type of PI-PLC is permanently overexpressed: A fibroblast cell line (3T3${\gamma}$1-7) in which PI-PLC-${\gamma}$1 was overexpressed by using pZip-neo expression vector was developed and used for the measurement of PDGF-induced IP3 formation. The responses for IP3 formed in 3T3${\gamma}$1-7 cells by the treatment of PDGF is 8 times more sensitive than those in control cells. 3T3${\gamma}$l-7 cell is useful for the screening of the inhibitors on the PDGF-induced cellular responses from large number of samples in a small volume(50 ${\mu}$l) and short time(5-15 min). Using these systems, we screened hundreds of herb-extracts for the inhibition of PDGF-induced IP3 formation and selected several extracts that showed the inhibition as the candidates for isolation and characterization of active substances. The determination of the acting point of selected extracts or fractions in the PDGF signaling pathway has been analyzing.

• Parasites, Hosts and Diseases
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• 제56권2호
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• pp.135-145
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• 2018

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, $CK2{\beta}$, VEGF, GCL, GST and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1170-1176
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• 2015

Ginsenosides, the major active component of ginseng, are traditionally used to treat various diseases, including cancer, inflammation, and obesity. Among these, compound K (CK), an intestinal bacterial metabolite of the ginsenosides Rb₁, Rb₂, and Rc from Bacteroides JY-6, is reported to inhibit cancer cell growth by inducing cell-cycle arrest or cell death, including apoptosis and necrosis. However, the precise effect of CK on breast cancer cells remains unclear. MCF-7 cells were treated with CK ($0-70{\mu}M$) for 24 or 48 h. Cell proliferation and death were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Changes in downstream signaling molecules involved in cell death, including glycogen synthase kinase $3\beta$ ($GSK3\beta$), $GSK3\beta$, $\beta$-catenin, and cyclin D1, were analyzed by western blot assay. To block $GSK3\beta$ signaling, MCF-7 cells were pretreated with $GSK3\beta$ inhibitors 1 h prior to CK treatment. Cell death and the expression of $\beta$-catenin and cyclin D1 were then examined. CK dose- and time-dependently inhibited MCF-7 cell proliferation. Interestingly, CK induced programmed necrosis, but not apoptosis, via the $GSK3\beta$ signaling pathway in MCF-7 cells. CK inhibited $GSK3\beta$ phosphorylation, thereby suppressing the expression of $\beta$-catenin and cyclin D1. Our results suggest that CK induces programmed necrosis in MCF-7 breast cancer cells via the $GSK3\beta$ signaling pathway.

• Tuberculosis and Respiratory Diseases
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• 제75권1호
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• pp.9-17
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• 2013

Background: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells. Methods: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control. Results: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-$G_1$ phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process. Conclusion: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

• The Journal of Internal Korean Medicine
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• 제37권1호
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• pp.65-76
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• 2016

Objectives: The purpose of this study was to identify the inhibitory effects of Hwangheuk-san (HHS), a Korean multi-herb formula comprising four medicinal herbs, on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, using the human bladder cancer 5637 cell line.Methods: Cell viability, motility, and invasion were assessed by 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphnyl-2H-tetrazolium bromide (MTT), wound healing migration, and Transwell assays, respectively. Gene expression was detected by Western blot analysis. In addition, the activities of matrix metalloproteinases (MMPs) and the values for transepithelial electrical resistance (TER) were analyzed using a Gelatinase Activity Assay Kit and an Epithelial Tissue Voltohmmeter, respectively.Results: Our data indicated that within the concentration range that was not cytotoxic, HHS effectively inhibited the cell motility and invasiveness of 5637 cells. HHS markedly decreased the expression and activity of MMP-2 and MMP-9, which was associated with unregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2. Further investigation revealed that phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT was decreased in HHS-treated 5637 cells, and a PI3K/AKT inhibitor synergistically reduced the inhibition of migration and invasion and also inactivated MMP-2 and MMP-9. Moreover, HHS increased the tightening of tight junctions (TJs), which was demonstrated by an increase in the TER, and reduced the expression the levels of claudin family members (claudin-3 and -4), which are major components involved in the tightening of TJs.Conclusions: The present findings demonstrated that HHS attenuated the migration and invasion of bladder cancer 5637 cells by modulating the activity of the PI3K/Akt signaling pathway and also through TJ tightening.

• Journal of Life Science
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• 제20권10호
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• pp.1443-1450
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• 2010

Honokiol is a small molecular weight ligand originally isolated from the Chinese medicinal herb Magnolia officinalis, a plant used in traditional Chinese and Japanese medicine [9]. In a previous study, the effects of honokiol were shown to have anti-angiogenic, anti-invasive and anti-proliferative activities in a variety of cancers [1,3,4,11,13,17,24,29,30]. We showed previously that direct production of nitric oxide (NO) by treatment of NO donor, sodium nitroprusside (SNP), led to apoptosis in rabbit articular chondrocytes [15,16]. This study confirmed that NO-induced apoptosis was suppressed by honokiol treatment in a dose-dependent manner as determined by cell phenotype, MTT assay, Western blot analysis and FACS analysis in articular chondrocytes. Treatment of honokiol inhibited SNP-induced expression of p53 as well as DNA fragmentation in articular chondrocytes, but increased expressionof pro-caspase-3. Inhibition of SNP-induced apoptosis by honokiol treatment was rescued by LY294002, the specific inhibitors of phosphoinositide 3-kinase (PI-3K) in articular chondrocytes. Our results indicate that honokiol inhibits NO-induced apoptosis via PI-3K/AKT pathway in rabbit articular chondrocytes.

• Journal of Life Science
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• 제19권10호
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• pp.1438-1443
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• 2009

Renal fibrosis is a final common manifestation of every type of chronic kidney disease. Plasminogen activator inhibitor (PAI)-1 is induced by lipopolysaccharide (LPS) and is known to play an essential role in the progress of renal fibrosis. In this paper, we found that an isoprenoid antibiotic, ascofuranone (AF), suppresses expression of profibrotic factors, PAI-1 and promoter activity of PAI-1 induced by LPS in rat kidney fibroblast cells. We therefore investigated signaling pathway mediated inhibitory effects of LPS-induced PAI-1 by AF in rNRK-49F cells. PAI-1 expression is suppressed by treatment with kinase inhibitors for MEK-1/2, as it isin inhibition of PAI-1 expression by AF, and AF inhibits phosphorylation of ERK-1/2. This study suggest that AF suppresses expression of PAI-1 through the inhibition of an ERK-1/2-dependent signal transduction pathway. The data indicates the possibility that AF can be used to prevent the development and progression of renal fibrosis.

• Parasites, Hosts and Diseases
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• 제55권5호
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• pp.491-503
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• 2017

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine ($5{\mu}M$) at $20{\mu}M$ and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at $1-5{\mu}M$, but host cells were destroyed at $10-20{\mu}M$. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.