• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2425-2430
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• 2015

Clinical resistance to chemotherapeutic agents is one of the major hindrances in the treatment of human cancers. EHZ2 is involved in drug resistance and is overexpressed in drug-resistant cancer cell lines. In this study, we investigated the effects of EHZ2 on cisplatin -resistance in A549/DDP and AGS/DDP cells. EHZ2 mRNA and protein were found to be significantly overexpressed in A549/DDP and AGS/DDP cells, compared to parental cells. EHZ2 siRNA successfully silenced EHZ2 mRNA and protein expression. Proliferation was inhibited and drug resistance to cisplatin was improved. Flow cytometry showed that silencing of EHZ2 arrested A549/DDP and AGS/DDP cells in the G0/G1 phase, increasing apoptosis, rh-123 fluorescence intensity and caspase-3/8 activities. Silencing of EHZ2 also significantly reduced the mRNA and protein expression levels of cyclin D1 and MDR1,while up-regulating p15, p21, p27 and miR-218 in A549/DPP cells. Furthermore, silencing of EHZ2 also significantly increased the expression level of tumor suppressor factor miR-218. We also found down-regulating EHZ2 expression increased methylation in A549/DDP and AGS/DDP cells. This study demonstrates that drug resistance can be effectively reversed in human cisplatin-resistant lung and gastric cancer cells through delivery of siRNAs targeting EHZ2.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.287-294
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• 2014

The transcription factor E2F1 is active during G1 to S transition and is involved in the cell cycle and progression. A recent study reported that increased E2F1 is associated with DNA damage and tumor development in several tissues using transgenic models. Here, we show that E2F1 expression is regulated by tristetraprolin (TTP) in prostate cancer. Overexpression of TTP decreased the stability of E2F1 mRNA and the expression level of E2F1. In contrast, inhibition of TTP using siRNA increased the E2F1 expression. E2F1 mRNA contains three AREs within the 3'UTR, and TTP destabilized a luciferase mRNA that contained the E2F1 mRNA 3'UTR. Analyses of point mutants of the E2F1 mRNA 3'UTR demonstrated that ARE2 was mostly responsible for the TTP-mediated destabilization of E2F1 mRNA. RNA EMSA revealed that TTP binds directly to the E2F1 mRNA 3'UTR of ARE2. Moreover, treatment with siRNA against TTP increased the proliferation of PC3 human prostate cancer cells. Taken together, these results demonstrate that E2F1 mRNA is a physiological target of TTP and suggests that TTP controls proliferation as well as migration and invasion through the regulation of E2F1 mRNA stability.

• Biomolecules & Therapeutics
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• 제21권1호
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• pp.21-28
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• 2013

Microglia play a role in maintaining and resolving brain tissue homeostasis. In pathological conditions, microglia release pro-inflammatory cytokines and cytotoxic factors, which aggravate the progression of neurodegenerative diseases. Autophagy pathway might be involved in the production of pro-inflammatory cytokines and cytotoxic factors in microglia, though details of the mechanism remain largely unknown. In the present study, we examined the role of the autophagy pathway in activated BV2 microglia cells. In BV2 cells, rapamycin treatment activated the formation of anti-LC3-labeled autophagosomes, whereas the ATG5 depletion using siRNA-ATG5 prevented the formation of LC3-labeled autophagosomes, indicating that BV2 cells exhibit an active classical autophagy system. When treated with LPS, BV2 cells expressed an increase of anti-LC3-labeled dots. The levels of LC3-labeled dots were not suppressed, instead tended to be enhanced, by the inhibition of the autophagy pathway with siRNA-ATG5 or wortmannin, suggesting that LPS-induced LC3-labeled dots in nature were distinct from the typical autophagosomes. The levels of LPS-induced expression of iNOS and IL6 were suppressed by treatment with rapamycin, and conversely, their expressions were enhanced by siRNA-ATG5 treatment. Moreover, the activation of the autophagy pathway using rapamycin inhibited cell death of LPS-stimulated microglia. These results suggest that although microglia possess a typical autophagy pathway, the glial cells express a non-typical autophagy pathway in response to LPS, and the activation of the autophagy pathway suppresses the expression of iNOS and IL6, and the cell death of LPS-stimulated microglia.

• Journal of Ginseng Research
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• 제46권3호
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• pp.444-453
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• 2022

Background: Compound K (CK) is among the protopanaxadiol (PPD)-type ginsenoside group, which produces multiple pharmacological effects. Herein, we examined the effects of CK on muscle atrophy under hyperlipidemic conditions along with its pro-myogenic effects. Further, the molecular pathways underlying the effects of CK on skeletal muscle have been justified. Methods: C2C12 myotubes were treated with palmitate and CK. C2C12 myoblasts were differentiated using CK for 4-5 days. For the in vivo experiments, CK was administered to mice fed on a high-fat diet for 8 weeks. The protein expression levels were analyzed using western blotting analysis. Target protein suppression was performed using small interfering (si) RNA transfection. Histological examination was performed using Jenner-Giemsa and H&E staining techniques. Results: CK treatment attenuated ER stress markers, such as eIF2a phosphorylation and CHOP expression and impaired myotube formation in palmitate-treated C2C12 myotubes and skeletal muscle of mice fed on HFD. CK treatment augmented AMPK along with autophagy markers in skeletal muscle cells in vitro and in vivo experiments. AMPK siRNA or 3-MA, an autophagy inhibitor, abrogated the impacts of CK in C2C12 myotubes. CK treatment augmented p38 and Akt phosphorylation, leading to an enhancement of C2C12 myogenesis. However, AMPK siRNA abolished the effects of CK in C2C12 myoblasts. Conclusion: These findings denote that CK prevents lipid-induced skeletal muscle apoptosis via AMPK/autophagy-mediated attenuation of ER stress and induction of myoblast differentiation. Therefore, we may suggest the use of CK as a potential therapeutic approach for treating muscle-wasting conditions associated with obesity.

• Biomolecules & Therapeutics
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• 제18권3호
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• pp.262-270
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• 2010

CD95 receptor is a member of tumor necrosis factor receptor family that mediates apoptosis in many cell types when bound by CD95 ligand or cross-linked by agonistic anti-CD95 antibodies. To determine the role of neutral sphingomyelinase (nSMase) on CD95-mediatd apoptosis, human Jurkat T lymphocytes were exposed to recombinant human CD95 ligand. Treatment with CD95 ligand induced cell death in a concentration and time-dependent manner. CD95-induced cell death was suppressed by inhibitors of SMase such as AY9944 or desipramine. Transfection with human nSMase1 siRNA plasmid into CD95 ligand-treated cells significantly prevented CD95-mediated cell death. CD95-mediated elevation of intracellular ceramide level detected by FACS analysis with anti-ceramide antibody was also decreased by nSMase1 siRNA. Knock-down of nSMase1 expression also blocked cytochrome c release into cytosol and caspase-3 cleavage in CD95-treated cells. Taken together, these results suggest that nSMase1 may play an important role in CD95-mediated apoptotic cell death in Jurkat T cells.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.1823-1827
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• 2012

The gene encoding the Nin one binding (NOB1) protein which plays an essential role in protein degradation has been investigated for possible tumor promoting functions. The present study was focused on NOB1 as a possible therapeutic target for breast cancer treatment. Lentivirus mediated NOB1 siRNA transfection was used to silence the NOB1 gene in two established breast cancer cell lines, MCF-7 and MDA-MB-231, successful transfection being confirmed by fluorescence imaging. NOB1 deletion caused significant decline in cell proliferation was observed in both cell lines as investigated by MTT assay. Furthermore the number and size of the colonies formed were also significantly reduced in the absence of NOB1. Moreover NOB1 gene knockdown arrested the cell cycle and inhibited cell cycle related protein expression. Collectively these results indicate that NOB1 plays an essential role in breast cancer cell proliferation and its gene expression could be a therapeutic target.

• Korea Journal of Cosmetic Science
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• 제1권1호
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• pp.31-43
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• 2019

Melanosomes are specific melanin-containing intracellular organelles of epidermal melanocytes. In epidermal melanocytes, there are three kinds of key player proteins. Rab27a, melanophilin or Slac2-a and Myosin 5a form a tripartite complex connects the melanosome. Mature melanosomes make movements through the tripartite protein complex along actin filaments.In this study, we found that the haplamine (6-Methoxyflindersine) induced melanosome aggregation around the nucleus in epidermal melanocyte. In an attempt to elucidate the inhibitory effect of haplamine on melanosome transport, effect of haplamineon the expression level of Rab27a, melanophilin and Myosin 5a was measured. The results indicated that haplamine up to 5��M effectively suppressed mRNA and protein expression level of melanophilin.To determine the upstream regulator of melanophilin regulated by haplamine, we checked the level of MITF, c-JUN and USF1. Those are possible transcription factor of melanophilin. Among them,treatment of USF1 siRNA decreased mRNA and protein expression level of USF1 as well as melanophilin. Also, treatment of haplamine decreased mRNA and protein expression level of melanophilin as well as USF1 in a dose-dependent manner. Consequently, we found the inhibitory effect of haplamine on melanosome transport in melan-a melanocyte. Treatment of haplamine reduced melanophilin expression level which is a key protein of melanosome transport. We identified that USF1 could be a major transcription factor of melanophilin regulated by haplamine.

• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.181-189
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• 2019

Curcumin, an active ingredient of Curcuma longa L., can reduce the concentration of low-density lipoproteins in plasma, in different ways. We had first reported that curcumin exhibits hypocholesterolemic properties by improving the apolipoprotein B (apoB) mRNA editing in primary rat hepatocytes. However, the role of curcumin in the regulation of apoB mRNA editing is not clear. Thus, we investigated the effect of curcumin on the expression of multiple editing components of apoB mRNA cytidine deamination to uridine (C-to-U) editosome. Our results demonstrated that treatment with $50{\mu}M$ curcumin markedly increased the amount of edited apoB mRNA in primary mouse hepatocytes from 5.13%-8.05% to 27.63%-35.61%, and significantly elevated the levels of the core components apoB editing catalytic polypeptide-1 (APOBEC-1), apobec-1 complementation factor (ACF), and RNA-binding-motif-protein-47 (RBM47), as well as suppressed the level of the inhibitory component glycine-arginine-tyrosine-rich RNA binding protein. Moreover, the increased apoB RNA editing by $50{\mu}M$ curcumin was significantly reduced by siRNA-mediated APOBEC-1, ACF, and RBM47 knockdown. These findings suggest that curcumin modulates apoB mRNA editing by coordinating the multiple editing components of the edito-some in primary hepatocytes. Our data provided evidence for curcumin to be used therapeutically to prevent atherosclerosis.

• Tuberculosis and Respiratory Diseases
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• 제67권4호
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• pp.303-310
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• 2009

Background: Elevated expression of cyclooxygenase-2 (COX-2) and Polo-like kinase-1 (PLK-1) is observed in a wide variety of cancers. Augmented expression of COX-2 and enhanced production of prostaglandin $E_2(PGE_2)$ are associated with increased tumor cell survival and malignancy; COX-2 has been implicated in the control of human non-small cell lung carcinoma (NSCLC) cell growth. PLK-1 siRNA induced the cell death of lung cancer cells and the systemic administration of PLK-1 siRNA/atelocollagen complex inhibited the growth of lung cancer in a liver metastatic murine model. COX-2 and PLK-1 are involved in proliferation and in cell cycle regulation, and there is a significant correlation between their interaction in prostate carcinoma. Methods: In this study, we investigated the pattern of COX-2 and PLK-1 expression in NSCLC, after treatment with IL-1$\beta$, COX-2 inhibitor and PLK-1 siRNA. Results: Expression of PLK-1 was decreased in A549 COX-2 sense cells, and was increased in A549 COX-2 anti-sense cells. Knock out of PLK-1 expression by PLK-1 siRNA augmented COX-2 expression in A549 and NCl-H157 cells. When A549 and NCI-H157 cells were treated with COX-2 inhibitor on a dose-dependent basis, PLK-1 and COX-2 were reduced. However, when the expression of COX-2 was induced by IL-1$\beta$, the production of PLK-1 decreased. Conclusion: These results demonstrate that COX-2 and PLK-1 are regulated and inhibited by each other in NSCLC, and suggest that these proteins have a reverse relationship in NSCLC.

• Journal of Animal Science and Technology
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• 제53권3호
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• pp.269-274
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• 2011

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.