• Journal of Animal Science and Technology
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• v.66 no.1
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• pp.204-218
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• 2024

Elsholtzia fruticosa (EF) is present in tropical regions throughout South Asian countries as well as the Himalayas. Although it has been used as a traditional medicine to treat digestive, respiratory, and inflammatory issues, its effect on preadipocyte differentiation is unknown. In this study, we examined the effects of a methanol extract prepared from EF on the differentiation of 3T3-L1 preadipocytes. Cell differentiation was assessed by microscopic observation and oil-red O staining. The expression of adipogenic and lipogenic genes, including PPARγ and C/EBPα, was measured by western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), to provide insight into adipogenesis and lipogenesis mechanisms. The results indicated that EF promotes the differentiation of 3T3-L1 preadipocytes, with elevated lipid accumulation occurring in a concentration-dependent manner without apparent cytotoxicity. EF enhances the expression of adipogenic and lipogenic genes, including PPARγ, FABP4, adiponectin, and FAS, at the mRNA and protein levels. The effect of EF was more pronounced during the early and middle stages of 3T3-L1 cell differentiation. Treatment with EF decreased C/EBP homologous protein (CHOP) mRNA and protein levels, while increasing C/EBPα and PPARγ expression. Treatment with EF resulted in the upregulation of cyclin E and CDK2 gene expression within 24 h, followed by a decrease at 48 h, demonstrating the early-stage impact of EF. A concomitant increase in cyclin-D1 levels was observed compared with untreated cells, indicating that EF modulates lipogenic and adipogenic genes through intricate mechanisms involving CHOP and cell cycle pathways. In summary, EF induces the differentiation of 3T3-L1 preadipocytes by increasing the expression of adipogenic and lipogenic genes, possibly through CHOP and cell cycle-dependent mechanisms.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7825-7829
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• 2014

Objective: To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. Methods: 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. Results: The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. Conclusion: Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.

• Journal of Marine Life Science
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• v.7 no.2
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• pp.145-153
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• 2022

Mercury (Hg) is a major concern in marine environment because of their bioaccumulation and biomagnification properties, and adverse effects to aquatic organisms at even a trace amount. However, little information on the effects of Hg, compared to other heavy metals, is available in marine small crustaceans. Here, we investigated the transcriptional modulation of metabolism-related genes in the brackish water flea, Diaphanosoma celebensis after exposure to sublethal concentration (0.2, 0.4, 0.8 ㎍/l) of HgCl₂ for 48 h. Relative mRNA expression levels of five detoxification enzyme-coding genes (cytochrome P450; cyp360A1, cyp361A1, cyp4AP3, cyp4C122, and cyp370C5) and six digestive enzyme-coding genes [alpha amylase (AMY), alpha amylase related protein (AMY-like), trypsin (TRYP), chymotrypsin-like protein (CHY), lipase (LIP), pancreatic lipase-related protein (PLRP)] were analyzed using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). As results, Hg increased the mRNA level of cyp370C5 (clan2) and cyp4AP3 (clan4) in a concentration dependent manner. A significant increase in TRYP mRNA was also concentration-dependently observed after exposure to Hg. These findings suggest that cyp370C5 and cyp4AP3 play a key role in Hg detoxification in D. celebensis, and Hg can affect energy metabolism by modulating the transcription of digestive enzyme. This study will provide better understanding the molecular effects of Hg in marine small crustacean.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.821-831
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• 2022

American plum line pattern virus (APLPV), a member of the genus Ilarvirus in the family Bromoviridae, is one of the plant quarantine pathogens in Korea. In this study, 15 candidate primer sets were designed and examined to develop a reverse transcription polymerase chain reaction (RT-PCR) assay for plant quarantine inspection of APLPV. Using APLPV-infected and healthy samples, the primer sets were assessed for APLPV detection. To confirm the occurrence of nonspecific reactions, six ilarviruses (Apple mosaic virus, Asparagus virus 2, Blueberry shock virus, Prune dwarf virus, Prunus necrotic ringspot virus, and Tobacco streak virus) and 10 target plants (Prunus mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera) were examined. Finally, two primer sets were selected. These primer sets could generate the expected amplicons even with at least 1 ng of the total RNA template in concentration-dependent amplifications. In addition, a positive clone was developed for use as a positive control in the abovementioned RT-PCR assay.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.257-267
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• 2017

Background: Heat-processed ginseng, sun ginseng (SG), has been reported to have improved therapeutic properties compared with raw forms, such as increased antidiabetic, anti-inflammatory, and antihyperglycemic effects. The aim of this study was to investigate the antiobesity effects of SG through the suppression of cell differentiation and proliferation of mouse 3T3-L1 preadipocyte cells and the lipid accumulation in Caenorhabditis elegans. Methods: To investigate the effect of SG on adipocyte differentiation, levels of stained intracellular lipid droplets were quantified by measuring the oil red O signal in the lipid extracts of cells on differentiation Day 7. To study the effect of SG on fat accumulation in C. elegans, L4 stage worms were cultured on an Escherichia coli OP50 diet supplemented with $10{\mu}g/mL$ of SG, followed by Nile red staining. To determine the effect of SG on gene expression of lipid and glucose metabolism-regulation molecules, messenger RNA (mRNA) levels of genes were analyzed by real-time reverse transcription-polymerase chain reaction analysis. In addition, the phosphorylation of Akt was examined by Western blotting. Results: SG suppressed the differentiation of 3T3-L1 cells stimulated by a mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI), and inhibited the proliferation of adipocytes during differentiation. Treatment of C. elegans with SG showed reductions in lipid accumulation by Nile red staining, thus directly demonstrating an antiobesity effect for SG. Furthermore, SG treatment down-regulated mRNA and protein expression levels of peroxisome proliferator-activated receptor subtype ${\gamma}$ ($PPAR{\gamma}$) and CCAAT/enhancer-binding protein-alpha ($C/EBP{\alpha}$) and decreased the mRNA level of sterol regulatory element-binding protein 1c in MDI-treated adipocytes in a dose-dependent manner. In differentiated 3T3-L1 cells, mRNA expression levels of lipid metabolism-regulating factors, such as amplifying mouse fatty acid-binding protein 2, leptin, lipoprotein lipase, fatty acid transporter protein 1, fatty acid synthase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were increased, whereas that of the lipolytic enzyme carnitine palmitoyltransferase-1 was decreased. Our data demonstrate that SG inversely regulated the expression of these genes in differentiated adipocytes. SG induced increases in the mRNA expression of glycolytic enzymes such as glucokinase and pyruvate kinase, and a decrease in the mRNA level of the glycogenic enzyme phosphoenol pyruvate carboxylase. In addition, mRNA levels of the glucose transporters GLUT1, GLUT4, and insulin receptor substrate-1 were elevated by MDI stimulation, whereas SG dose-dependently inhibited the expression of these genes in differentiated adipocytes. SG also inhibited the phosphorylation of Akt (Ser473) at an early phase of MDI stimulation. Intracellular nitric oxide (NO) production and endothelial nitric oxide synthase mRNA levels were markedly decreased by MDI stimulation and recovered by SG treatment of adipocytes. Conclusion: Our results suggest that SG effectively inhibits adipocyte proliferation and differentiation through the downregulation of $PPAR{\gamma}$ and $C/EBP{\alpha}$, by suppressing Akt (Ser473) phosphorylation and enhancing NO production. These results provide strong evidence to support the development of SG for antiobesity treatment.

• IMMUNE NETWORK
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• v.12 no.2
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• pp.58-65
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• 2012

Background: A cell line with transfected Wilms' tumor protein 1 (WT1) is has been used for the preclinical evaluation of novel treatment strategies of WT1 immunotherapy for leukemia due to the lack of appropriate murine leukemia cell line with endogenous WT1. However, silencing of the transgene occurs. Regarding the effects of hypomethylating agents (HMAs) on reactivation of silenced genes, HMAs are considered to be immune enhancers. Methods: We treated murine WT1- transfected C1498 (mWT1-C1498) with increasing doses of decitabine (DAC) and azacitidine (AZA) to analyze their effects on transgene reactivation. Results: DAC and AZA decreased the number of viable cells in a dose- or time-dependent manner. Quantification of WT1 mRNA level was analyzed by real-time polymerase chain reaction after mWT1-C1498 treated with increasing dose of HMA. DAC treatment for 48 h induced 1.4-, 14.6-, and 15.5-fold increment of WT1 mRNA level, compared to untreated sample, at 0.1, 1, and $10{\mu}M$, respectively. Further increment of WT1 expression in the presence of 1 and $10{\mu}M$ DAC was evident at 72 h. AZA treatment also induced up-regulation of mRNA, but not to the same degree as with DAC treatment. The correlation between the incremental increases in WT1 mRNA by DAC was confirmed by Western blot and concomitant down-regulation of WT1 promoter methylation was revealed. Conclusion: The in vitro data show that HMA can induce reactivation of WT1 transgene and that DAC is more effective, at least in mWT1-C1498 cells, which suggests that the combination of DAC and mWT1-C1498 can be used for the development of the experimental model of HMA-combined WT1 immunotherapy targeting leukemia.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.3
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• pp.483-489
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• 2002

APA-01, which is an aqueous extract of five Chinese herbs, is a modified formula of Huoxiang-Zhengqi-San. The effect of new herb extract on immune response was investigated. The parameter examined to assess apparent immune response of APA-01 in mice included changes of body weight, relative weight of immune organs, cell proliferation and cytokine gene expression. The body weight and relative weight of immune organs were not significantly changed among the tested groups. In the spleen cell prolijeration assay, APA-01 increased the cell proliferation in a dose-dependent manner. Methotrexate (MTX), an agent of immune suppression, inhibited the spleen cell proliferation (IC/sub 50/: 800㎍/㎖). However, APA-01 significantly inhibited the suppression of mouse spleen cell proliferation. Therefore, it seems that APA-01 has a reducing effect of immune suppression. Immunomodulatory effect of APA-01 was further investigated using reverse transcription polymerase chain reaction (RT-PCR) in mouse spleen cells. In RT-PCR test, APA-01 enhanced the expression of cyclooxygenase-2 (COX-2) mRNA in a dose-dependent manner. In spite of immune suppression by MTX, COX-2 mRNA was induced by co-treatment with APA-01. These results suggest that APA-01 stimulates the proliferation of spleen cells, regulates the expression of COX-2 mRNA, and accelerates the recovery of inhibition of spleen cell proliferation induced by MTX, thus providing the immunological basis for clinical benefit of APA-01.

• Journal of Marine Life Science
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• v.6 no.2
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• pp.73-79
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• 2021

Bisphenol A is a representative endocrine disruptor and continuously detected in aquatic environment due to wide use, resulting in adverse effects on growth, development, and reproduction in diverse organisms as well as human. Structural analogs have been developed to substitute BPA are also suspected to have endocrine disrupting effects. In the present study, the time-dependent expression patterns of ecdysteroid synthesis (nvd, cyp314a1), receptors (EcRA, EcRB, USP, ERR), and downstream signaling pathway - related genes (HR3, E75, Vtg, VtgR) were investigated using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) in the brackish water flea Diaphanosoma celebensis exposed to Bisphenol analogs (BPs; BPA, BPF, and BPS) for 6, 12, and 24 h. As results, the expression of nvd, cyp314a1, EcRs, USP, ERR and E75 mRNA was upregulated at 6 h exposure to BPF, which is earlier than BPA and BPS (12 h). On the other hand, HR3, E75 and VtgR mRNA levels were elevated at 6 h earlier at BPS and BPF than at BPA (12 h), but Vtg mRNA level was slightly changed within 24 h. These findings suggest that like BPA, BPF and BPS can also modulate the transcription of ecdysteroid pathway - related genes with different mechanisms, and have a potential as endocrine disruptors. This study will provide a better understanding the molecular mode of action of bisphenols on ecdysteroid pathway in the brackish water flea.

• Journal of Life Science
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• v.33 no.1
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• pp.73-81
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• 2023

In the present study, we prepared hot water extracts and the subsequent organic solvent fractions of methanol extracts of Houttuynia cordata (HC) and Lespedeza cuneata (LC), and investigated their anti-inflammatory effects on lipopolysaccharide-stimulated RAW264.7 cells. Among the treated samples, hexane, chloroform, and ethyl-acetate fractions of HC and LC inhibited nitric oxide (NO) production in a dose-dependent manner, and decreased inducible nitric oxide synthase (iNOS) protein expression. And, we analyzed the flavonoid contents of the ethyl-acetate fraction of HC and LC, and chose apigenin for the further experiments because apigenin was one of flavonoids commonly found in HC and LC. Apigenin dramatically inhibited NO production in a dose-dependent manner without affecting cell viability and decreased iNOS and cyclooxygenase-2 (COX-2) expression. In addition, apigenin suppressed the phosphorylation of p38 and Jun N-terminal kinase (JNK) indicating that apigenin exerts anti-inflammatory activity via the mitogen-activated protein kinase (MAPK) signaling pathway. Subsequently, we conducted RNA-sequencing analysis to detect differentially expressed genes upon apigenin treatment. Among the down-regulated genes, four cytokine genes (interleukin (IL)-1α, IL-1β, IL-6, and colony stimulating factor 2 (CSF2)) were selected for the further analysis, and the reduction of their expression by apigenin was confirmed with quantitative real-time polymerase chain reaction. Overall, our results suggest that Houttuynia cordata and Lespedeza cuneata have the anti-inflammatory effects and apigenin can be the one of key molecules responsible for their anti-inflammatory activities.

• Biomolecules & Therapeutics
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• v.18 no.2
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• pp.178-183
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• 2010

Costunolide is an active compound isolated from the stem bark of Magnolia sieboldii, and is considered a potential therapeutic for the treatment of various cancers. In this study, we investigated the underlying mechanism whereby costunolide induces the apoptosis of human leukemia cells. Using apoptosis analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) results obtained during this study show that costunolide is a potent inducer of apoptosis and that it is triggered due to the premature activation of Cdc2. $G_1$-synchronized cells, which cannot undergo mitosis, were found to be more sensitive to costunolide, and Cdc2 mRNA levels were increased by costunolide treatment. Furthermore, the Cdk inhibitors, olomucine and butyrolactone I, were found to suppress costunolide-induced apoptosis. In addition, the PKC activator TPA rescued cells from cell death by costunolide, and this was prevented by the PKC inhibitor staurosporin. The present study suggests that costunolide induces the apoptosis of HL-60 leukemic cells by modulating cyclin-dependent kinase Cdc2.