• Animal cells and systems
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• v.14 no.3
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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.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.1044-1050
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• 2020

Abelmoschus manihot (Linn.) is a medicinal herbal plant that is commonly used to treat chronic kidney disease and hepatitis. However, its effect on cell proliferation has not been clearly revealed. In this report, we sought to determine the effect of the flower extract of A. manihot (FA) on cell proliferation. Based on our findings, FA increased the proliferation of human diploid fibroblast (HDF) and HEK293 cells. Through cell cycle analysis, FA was found to increase the number of HDF cells in the S phase and G2/M phase. FA also increased the expression of cyclin D1 and enhanced the migration of HDF cells. By administering FA to HDF cells with ≥30 passages, a decrease in the number of senescence-associated β galactosidase-positive cells was observed, thereby indicating that FA can ameliorate cellular senescence. Collectively, our findings indicate that FA increases cyclin D1 expression and regulates cell proliferation.

• Animal cells and systems
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• v.15 no.2
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• pp.123-130
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• 2011

Transient receptor potential melastatin 7 (TRPM7) channels are novel $Ca^{2+}$-permeable non-selective cation channels that are ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in the survival of gastric cancer cells. Here we show evidence suggesting that TRPM7 channels play an important role in $Zn^{2+}$- mediated cellular injury. Using a combination of electrophysiology, pharmacological analysis, small interfering RNA (siRNA) methods and cell death assays, we showed that activation of TRPM7 channels augmented $Zn^{2+}$-induced apoptosis of AGS cells, the most common human gastric adenocarcinoma cell line. The $Zn^{2+}$-mediated cytotoxicity was inhibited by the non-specific TRPM7 blockers $Gd^{3+}$ or 2 aminoethoxydiphenyl borate (2-APB) and TRPM7 specific siRNA. In addition, we showed that overexpression of TRPM7 channels in HEK293 cells increased $Zn^{2+}$- induced cell injury. Thus, TRPM7 channels may represent a novel target for physiological disorders where $Zn^{2+}$ toxicity plays an important role.

• BMB Reports
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• v.40 no.4
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• pp.467-474
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• 2007

Autosomal recessive polycystic kidney disease (ARPKD) is one of the important genetic disorders in pediatric practice. Mutation of the polycystic kidney and hepatic disease gene 1 (PKHD1) was identified as the cause of ARPKD. The gene encodes a 67-exon transcript for a large protein of 4074 amino acids termed fibrocystin, but its function remains unknown. The neoplastic-like in cystic epithelial proliferation and the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis overactivity are known as the most important characteristics of ARPKD. Since the misregulation of $Ca^{2+}$ signaling may lead to aberrant structure and function of the collecting ducts in kidney of rat with ARPKD, present study aimed to investigate the further mechanisms of abnormal proliferation of cystic cells by inhibition of PKHD1 expression. For this, a stable PKHD1-silenced HEK-293T cell line was established. Then cell proliferation rates, intracellular $Ca^{2+}$ concentration and extracellular signal-regulated kinase 1/2 (ERK1/2) activity were assessed after treatment with EGF, a calcium channel blocker and agonist, verapamil and Bay K8644. It was found that PKHD1-silenced HEK-293T cell lines were hyperproliferative to EGF stimulation. Also PKHD1-silencing lowered the intracellular $Ca^{2+}$ and caused EGF-induced ERK1/2 overactivation in the cells. An increase of intracellular $Ca^{2+}$ in PKHD1-silenced cells repressed the EGF-dependent ERK1/2 activation and the hyperproliferative response to EGF stimulation. Thus, inhibition of PKHD1 can cause EGF-induced excessive proliferation through decreasing intracellular $Ca^{2+}$ resulting in EGF-induced ERK1/2 activation. Our results suggest that the loss of fibrocystin may lead to abnormal proliferation in kidney epithelial cells and cyst formation in ARPKD by modulation of intracellular $Ca^{2+}$.

• Food Science and Preservation
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• v.20 no.5
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• pp.691-698
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• 2013

The biological activities of Smilax china L. rhizome (SCR), hot water (SCRW) and 70% ethanol extract (SCRE) were analyzed. The total phenolic contents of SCRW and SCRE were 51.7 and 100.5 mg/g, respectively. The measured flavonoid content of SCRW ($67.7{\mu}g/g$) was almost double that of SCRE ($31.7{\mu}g/g$). SCRE ($IC_{50}=42.4{\mu}g/mL$) exhibited stronger antioxidant activity in the DPPH system than the positive control ${\alpha}$-tocopherol ($71.3{\mu}g/mL$) or butylated hydroxy anisole ($53.8{\mu}g/mL$) did. SCRE ($IC_{50}=50.3{\mu}g/mL$) also showed stronger ABTS radical scavenging activity, as did ${\alpha}$-tocopherol ($67.1{\mu}g/mL$). The SOD-like activity and Tyrosinase inhibition activity of SCRW and SCRE showed almost the same pattern. The best SOD-like activity and tyrosinase inhibition activity were measured as 24.9% and 20.3% in SCRW at $1,000{\mu}g/mL$, respectively. The cytotoxic effects of the SCR extracts were analyzed via MTT assay on human cancer and normal cells. SCRW and SCRE did not show cytotoxicity up to the concentration of $1,000{\mu}g/mL$ against the normal human cell line HEK293. Against human breast cancer cells (MCF-7), SCRW inhibited MCF-7 growth (by 27.6%) better than the anticancer drug cyclophosphamide (15.5%) at $1,000{\mu}g/mL$. SCRE ($1,000{\mu}g/mL$) inhibited the growth of human lung cancer cells A549 (37.6%) and human stomach cancer cells AGS (53.6%) more effective than did SCRW (21.0% and 35.4%) or CPA (22.2% and 31.7%). These results suggest the potential use of SCRE and SCRW as an excellent antioxidant and antiproliferative substance, respectively.

• BMB Reports
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• v.50 no.3
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• pp.144-149
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• 2017

Ceramides are the major sphingolipid metabolites involved in cell survival and apoptosis. When HepG2 hepatoma cells were treated with celecoxib, the expression of the genes in de novo sphingolipid biosynthesis and sphingomyelinase pathway was upregulated and cellular ceramide was elevated. In addition, celecoxib induced endoplasmic reticulum (ER) stress in a time-dependent manner. SPTLC2, a subunit of serine palmitoyltransferase, was overexpressed by adenovirus. Adenoviral overexpression of SPTLC2 (AdSPTLC2) decreased cell viability of HEK293 and HepG2 cells. In addition, AdSPTLC2 induced apoptosis via the caspase-dependent apoptotic pathway and elevated cellular ceramide, sphingoid bases, and dihydroceramide. However, overexpression of SPTLC2 did not induce ER stress. Collectively, celecoxib activates de novo sphingolipid biosynthesis and the combined effects of elevated ceramide and transcriptional activation of ER stress induce apoptosis. However, activation of de novo sphingolipid biosynthesis does not activate ER stress in hepatoma cells and is distinct from the celecoxib-mediated activation of ER stress.

• Molecules and Cells
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• v.19 no.1
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• pp.23-30
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• 2005

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying $ataxin-1-{\Delta}114$, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of $ataxin-1-{\Delta}114$ aggregates and the amount of d2EGFP were drastically reduced in cells containing $ataxin-1-{\Delta}114$. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.2044-2051
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• 2017

The main pathological hallmark of Alzheimer's disease is the deposition of amyloid-beta ($A{\beta}$) peptides in the brain. $A{\beta}$ has been widely used to mimic several aspects of Alzheimer's disease. However, several characteristics of amyloid-induced Alzheimer's disease pathology are not well established, especially in mice. The present study aimed to develop a new Alzheimer's disease model by investigating how $A{\beta}$ can be effectively aggregated using prokaryotes and eukaryotes. To express the $A{\beta}42$ complex in HEK293 cells, we cloned the $A{\beta}42$ region in a tandem repeat and incorporated the resulting construct into a eukaryotic expression vector. Following transfection into HEK293 cells via lipofection, cell viability assay and western blotting analysis revealed that exogenous $A{\beta}42$ can induce cell death and apoptosis. In addition, recombinant His-tagged $A{\beta}42$ was successfully expressed in Escherichia coli BL21 (DE3) and not only readily formed $A{\beta}$ complexes, but also inhibited the proliferation of SH-SY5Y cells and E. coli. For in vivo testing, recombinant His-tagged $A{\beta}42$ solution ($3{\mu}g/{\mu}l$ in $1{\times}PBS$ containing $1mM\;Ni^{2+}$) was injected stereotaxically into the left and right lateral ventricles of the brains of C57BL/6J mice (n = 8). Control mice were injected with $1{\times}PBS$ containing $1mM\;Ni^{2+}$ following the same procedure. Ten days after the sample injection, the Morris water maze test confirmed that exogenous $A{\beta}$ caused an increase in memory loss. These findings demonstrated that $Ni^{2+}$ is capable of complexing the 50-kDa amyloid and that intracerebroventricular injection of $A{\beta}42$ can lead to cognitive impairment, thereby providing improved Alzheimer's disease models.

• Analytical Science and Technology
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• v.27 no.5
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• pp.223-227
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• 2014

${\delta}$-Aminolevulinic acid (ALA) is a compound which is widely present in the biosphere and plays an important role in the living body as an intermediate of the tetrapyrrole compound biosynthesis pathway that leads to heme in mammals and chlorophyll in plants. ALA is of interest as a biodegradable mediator, a growth regulator, a precursor of heme proteins, and an effective agent used in therapy of cancer. It has been recently reported that ALA is commonly used in dermatology, due to good effects of skin therapy. Although for the last few decades a substantial amount of research has been focused on the elucidation of the mechanism of ALA and the improvement of its therapeutic activity, it's effect on the cell functions and growth was not cleared. Here, we identified that ALA treatment could attenuate cell proliferation of HEK293T and HaCaT cells. In addition, ALA treatement could induce apoptosis of HeLa cells. These results suggest that apoptosis induced by ALA treatment might be responsible for inhibition of cell proliferation. These results propose the possibility of the improved therapeutic strategy making ALA one of the effective drugs used in human cancers.

• Journal of Life Science
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• v.26 no.8
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• pp.963-969
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• 2016

Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motor proteins essential for the intracellular transport of organelles and protein complexes in cells. Kinesin 1 is a member of those KIFs that transport various cargoes, including organelles, synaptic vesicles, neurotransmitter receptors, cell signaling molecules, and mRNAs through interaction between its light chain subunit and the cargoes. Kinesin light chains (KLCs) are non-motor subunits that associate with the kinesin heavy chain (KHC) dimer. KLCs interact with many different binding proteins, but their particular binding proteins have not yet been fully identified. We used the yeast two-hybrid assay to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. We found an interaction between the TPR domain of KLC1 and Wiskott-Aldrich syndrome protein family member 1 (WAVE1), a member of the WASP/WAVE family involved in regulation of actin cytoskeleton. WAVE1 bound to the six TPR domain-containing regions of KLC1 and did not interact with KHCs (KIF5A, KIF5B, and KIF5C) in the yeast two-hybrid assay. The carboxyl (C)-terminal verprolin-cofilin-acidic (VCA) domain of WAVE1 is essential for interaction with KLC1. Also, other WAVE isoforms (WAVE2 and WAVE3) interacted with KLC1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, WAVE1 co-localized with KLC1 and co-immunoprecipitated with KLC1 and KIF5B. These results suggest that kinesin 1 motor protein may transport WAVE complexes or WAVE-coated cargoes in cells.