• Nutrition Research and Practice
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• 제14권2호
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• pp.127-133
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• 2020

BACKGROUND/OBJECTIVES: Non-small cell lung cancer is mostly recognized among other types of lung cancer with a poor prognosis by cause of chemotherapeutic resistance and increased metastasis. Luteolin has been found to decrease cell metastasis. However, its underlying mechanisms remain unresolved. The objective of this study was to examine the effect (and its mechanism) of luteolin on the migration and invasion of human non-small cell lung cancer A549 cells. MATERIALS/METHODS: Cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Wound healing and transwell assays were evaluated to assess migration and invasion, respectively. Western blot analysis and immunofluorescence were further performed to investigate the role of luteolin and its mechanisms of action. RESULTS: Administration with up to 40 μM luteolin showed no cytotoxic activity on lung cancer A549 cells or non-cancer MRC-5 cells. Additionally, luteolin at 20-40 μM significantly suppressed A549 cells' migration, invasion, and the formation of filopodia in a concentration-dependent manner at 24 h. This is similar with western blot analysis, which revealed diminished the phosphorylated focal adhesion kinase (pFAK), phosphorylated non-receptor tyrosine kinase (pSrc), Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 (Cdc42), and Ras homolog gene family member A (RhoA) expression levels. CONCLUSIONS: Overall, our data indicate that luteolin plays a role in controlling lung cancer cells' migration and invasion via Src/FAK and its downstream Rac1, Cdc42, and RhoA pathways. Luteolin might be considered a promising candidate for suppressing invasion and metastasis of lung cancer cells.

• Genomics & Informatics
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• 제9권3호
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• pp.102-113
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• 2011

C-terminal SRC kinase (CSK) is a ubiquitously expressed, cytosolic enzyme that phosphorylates and inactivates several SRC family protein tyrosine kinases. Recent genomewide association studies have implicated CSK in the regulation of blood pressure. The current study aim is to determine the blood pressure association of the genes regulated by CSK down-regulation. The CSK mRNA expression was downregulated in vascular smooth muscle cells using small interfering RNA (siRNA). CSK mRNA levels fell by 90% in cells that were treated with CSK siRNA; the RNA from these cells was examined by microarray using the Illumina HumanRef-8 v3 platform, which comprises 24,526 reference mRNA probes. On treatment with CSK siRNA, 19 genes were downregulated by more than 2-fold and 13 genes were upregulated by more than 2-fold. Three (CANX, SLC30A7, and HMOX1) of them revealed more than 3 fold differential expression. Interestingly, the HMOX1 SNPs were associated with diastolic blood pressure in the 7551 Koreans using Korea Association REsource data, and the result was supported by the other reports that HMOX1 linked to blood vessel maintenance. Among the remaining 29 differentially expressed genes, seven (SSBP1, CDH2, YWHAE, ME2, PFTK1, G3BP2, and TUFT1) revealed association with both systolic and diastolic blood pressures. The CDH2 gene was linked to blood pressures. Conclusively, we identified 32 differentially expressed genes which were regulated by CSK reduction, and two (HOMX1 and CDH2) of them might influence the blood pressure regulation through CSK pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.259-265
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• 2017

Excessive influx and the subsequent rapid cytosolic elevation of $Ca^{2+}$ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic $Ca^{2+}$ level in normal as well as pathological conditions. Delayed rectifier $K^+$ channels ($I_{DR}$ channels) play a role to suppress membrane excitability by inducing $K^+$ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under $Ca^{2+}$-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of $I_{DR}$ channels to hyperexcitable conditions induced by high $Ca^{2+}$ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high $Ca^{2+}$-treatment significantly increased the amplitude of $I_{DR}$ without changes of gating kinetics. Nimodipine but not APV blocked $Ca^{2+}$-induced $I_{DR}$ enhancement, confirming that the change of $I_{DR}$ might be targeted by $Ca^{2+}$ influx through voltage-dependent $Ca^{2+}$ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated $I_{DR}$ enhancement was not affected by either $Ca^{2+}$-induced $Ca^{2+}$ release (CICR) or small conductance $Ca^{2+}$-activated $K^+$ channels (SK channels). Furthermore, PP2 but not H89 completely abolished $I_{DR}$ enhancement under high $Ca^{2+}$ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for $Ca^{2+}$-mediated $I_{DR}$ enhancement. Thus, SFKs may be sensitive to excessive $Ca^{2+}$ influx through VDCCs and enhance $I_{DR}$ to activate a neuroprotective mechanism against $Ca^{2+}$-mediated hyperexcitability in neurons.

• BMB Reports
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• 제34권1호
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• pp.33-38
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• 2001

The human PTK6 (also known as Brk) polypeptide, which is deduced from its full-length cDNA, represents a non-receptor protein tyrosine kinase (PTK). It contains SH3, SH2, and tyrosine kinase catalytic domains that are closely related to Src family members. We generated an antihuman PTK6 antibody by immunizing rabbits with a PTK6-specific oligopeptide conjugated to BSA, which corresponds to 11 amino acid residues near the C-terminus. An immunoblot analysis with the antibody detected an expected 52-kDa band in various mammalian transformed cell lines. Immunoprecipitation and immunoblot analyses demonstrated that PTK6 is phosphorylated on the tyrosine residues) and interacts with approximately a 23-kDa tyrosine-phosphorylated polypeptide (most likely a substrate of PTK6) in breast carcinoma T-47D cells. An immunofluorescence analysis demonstrated that PTK6 is localized throughout the cytoplasm of T-47D cells. These results support a possible role for PTK6 in the intracellular signal transduction through tyrosine phosphorylation.

• Animal cells and systems
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• 제14권3호
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• pp.155-160
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• 2010

Extracellular signal-regulated kinases 1/2 (ERK1/2) play important roles in a variety of biological processes including cell growth and differentiation. We have previously reported that GAR-3 activates ERK1/2 via phospholipase C and protein kinase C, presumably through pertussis toxin (PTX)-insensitive Gq proteins, in Chinese hamster ovary (CHO) cells. Here we provide evidence that GAR-3 also activates ERK1/2 through PTX-sensitive G proteins, phosphatidylinositol 3-kinase (PI 3-kinase), and Src family kinases in CHO cells. We further show that in human embryonic kidney (HEK293) cells, epidermal growth factor receptor and Ras are required for efficient ERK1/2 activation by GAR-3. Taken together, our data indicate that GAR-3 evokes ERK1/2 activation through multiple signaling pathways in cultured mammalian cells.

• Biomolecules & Therapeutics
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• 제20권4호
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• pp.393-398
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• 2012

Recently, we reported that defective autophagy may contribute to the inhibition of the growth in response to PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a selective SFK inhibitor, in multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr). In this study, we demonstrated that PP2 induces LC3 conversion via a mechanism that is uncoupled from autophagy and increases apoptosis in Ras-NIH 3T3/Mdr cells. PP2 preferentially induced autophagy in Ras-NIH 3T3 cells rather than in Ras-NIH 3T3/Mdr cells as determined by LC3-I to LC3-II conversion and GFP-LC3 fluorescence microscopy. Beclin 1 knockdown experiments showed that, regardless of drug resistance, PP2 induces autophagy via a Beclin 1-dependent mechanism. PP2 induced a conformational change in Beclin 1, resulting in the enhancement of the pro-autophagic activity of Beclin 1, in Ras-NIH 3T3 cells. Further, PI3K inhibition induced by wortmannin caused a significant increase in apoptosis in Ras-NIH 3T3 cells, as demonstrated by flow cytometric analysis of Annexin V staining, implying that autophagy inhibition through PI3K increases apoptosis in response to PP2 in Ras-NIH 3T3 cells. However, despite the fact that wortmannin abrogates PP2-induced GFP-LC3 punctae formation, some LC3 conversion remains in Ras-NIH 3T3/Mdr cells, suggesting that LC3 conversion may occur in an autophagy-independent manner. Taken together, these results suggest that PP2 induces LC3 conversion independent of PI3K, concomitant with the uncoupling of LC3 conversion from autophagy, in multidrug-resistant cells.

• 약학회지
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• 제56권1호
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• pp.26-34
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• 2012

Mast cells play an important role in early and late phase allergic reactions through allergen and IgE-dependent release of histamine, proteases, prostaglandins, and several multifunctional cytokines. In this study, we investigated whether Cudrania tricuspidata extract (CTE) suppresses IgE-mediated allergic responses in mast cells, an allergic animal model, and its mechanism of action in mast cells. We found that CTE inhibited IgE-mediated degranulation and cytokine production in rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMC), as well as passive cutaneous anaphylaxis (PCA) in mice. With regard to its mechanism of action, CTE suppressed the activating phosphorylation of spleen tyrosine kinase (Syk), a key enzyme in mast cell signaling processes and that of LAT, a downstream adaptor molecule of Syk in $Fc{\varepsilon}RI$-mediated signal pathways. CTE also suppressed the activating phosphorylation of mitogen-activated protein (MAP) kinases and Akt. The present results strongly suggest that the anti-allergic activity of CTE is mediated through inhibiting degranulation and allergic cytokine secretion by inhibition of Syk kinase in mast cells. Therefore, CTE may be useful for the treatment of allergic diseases.

• BMB Reports
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• 제47권9호
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• pp.483-487
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• 2014

Transmembrane 4 L six family member 5 (TM4SF5), as a membrane glycoprotein with 4 transmembrane domains, is similar to the tetraspanins in terms of membrane topology and plays important roles in tumorigenesis and tumor metastasis. Especially, TM4SF5 appears to form a massive protein-protein complex consisting of diverse membrane proteins and/or receptors in addition to cytosolic signaling molecules to regulate their signaling activities during the pathological processes. TM4SF5 is shown to interact with integrins ${\alpha}2$, ${\alpha}5$, and ${\beta}1$, EGFR, IL6R, CD151, focal adhesion kinase (FAK), and c-Src. This review focuses on the significance of the interactions with regards to TM4SF5-positive tumorigenesis and metastasis.

• Interdisciplinary Bio Central
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• 제4권2호
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• pp.3.1-3.7
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• 2012

Epidermal growth factor receptor (EGFR) is a member of the ErbB family (ErbB1-4) of receptor tyrosine kinases (RTKs). EGFR controls numerous physiological functions, including cell proliferation, migration, differentiation and survival. Importantly, aberrant signaling by EGFR has been linked to human cancers in which EGFR and its various ligands are frequently overexpressed or mutated. EGFR coordinates activation of multiple downstream factors and is subject of various regulatory processes as it mediates biology of the cell it resides in. Therefore, many studies have been devoted to understanding EGFR biology and targeting the protein for the goal of controlling tumor in clinical settings. Endocytic regulation of EGFR offers a promising area for targeting EGFR activity. Upon ligand binding, the activated receptor undergoes endocytosis and becomes degraded in lysosome, thereby terminating the signal. En route to lysosome, the receptor becomes engaged in activating various signaling pathways including PI-3K, MAPK and Src, and endocytosis may offer both spatial and temporal regulation of downstream target activation. Therefore, endocytosis is an important regulator of EGFR signaling, and increasing emphasis is being placed on endocytosis in terms of cancer treatment and understanding of the disease. In this review, EGFR signaling pathway and its intricate regulation by endocytosis will be discussed.

• Animal cells and systems
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• 제5권2호
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• pp.145-151
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• 2001

p62 is a phosphotyrosine-independent ligand of the SH2 domain of $p56^{Ick}$, a T-cell specific Src family tyrosine kinase. Recently p62 has been shown to interact with a number of proteins, such as $PKC\varsigma$ and ubiquitin, and implicated in important cellular functions such as cell proliferation. Since the two p62 interacting proteins, $p56^{Ick}$ and $PKC\varsigma$, have been reported to play roles in cell death, 1 have addressed the potential role of p62 during apoptosis in Jurkat cells in this study. Herein 1 show that p62 was specifically cleaved into two peptides by a caspase-3-like activity during Fas-receptor mediated apoptosis in Jurkat cells. This cleavage generated two fragments with molecular weights of about 35 kDa that differed in subcellular localizations. The N-terminal cleaved fragment was present in the detergent-insoluble fraction whereas the C-terminal fragment was found in the detergent-soluble fraction. In addition, the C-terminal fragment appeared to be subjected to further degradation as apoptosis prolonged. Moreover, overexpression of p62 in Jurkat cells attenuated the Fas receptor mediated apoptosis, suggesting that p62 is involved in apoptotic signal transduction pathway in lymphocytes.