• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.198-205
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• 2004

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Recent industrialized industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA. Some flavonoids such as genistein, daidzein, chrysin, naringenin and morin were also investigeted. These flavonoids decreased B(k)F infuced luciferase activity at low concentration. But, these flavonoids exhibited stimulatory effect at high concentration.

• KSBB Journal
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• v.28 no.1
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• pp.1-6
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• 2013

Curcumin has diverse anticancer activities that lead to tumor growth inhibition of cancer cells and induction of apoptosis. Curcumin is involved in the regulation of multiple genes via transcription factors including NF-${\kappa}B$, STATs, AP1, and SP. Notch signaling plays critical roles in maintaining the balance between cell proliferation, differentiation and apoptosis, and thereby may contribute to the development of various cancers involving breast cancer. This study was to investigate the effects of curcumin on Notch1 gene expression and to explore the underlying mechanism. Here, we found that curcumin decreased the levels of Notch1 mRNA and protein in MDA-MB-231 human breast cancer cells, along with the downregulation of Sp family genes (Sp1, Sp2, Sp3, and Sp4). The repressive effect of curcumin on Notch1 gene transcription was confirmed by performing Notch1 promoter-driven reporter assay and three Sp-binding sites were identified on Notch1 promoter that may act as curcumin-respose elements. Moreover, treatment with mitramycin A, a specific Sp inhibitor, decreased the levels of Notch1 mRNA and protein in human breast cancer cells. Taken together, our results indicate that Notch1 gene expression is downregulated by curcumin, at least in part, through the suppression of Sp family, which may lead to apoptosis in human breast cancer cells.

• Genomics & Informatics
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• v.12 no.4
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• pp.240-246
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• 2014

Mutation in HNF1B, the hepatocyte nuclear factor-$1{\beta}$ (HNF-$1{\beta}$) gene, results in maturity-onset diabetes of the young (MODY) 5, which is characterized by gradual impairment of insulin secretion. However, the functional role of HNF-$1{\beta}$ in insulin secretion and glucose metabolism is not fully understood. We identified a family with early-onset diabetes that fulfilled the criteria of MODY. Sanger sequencing revealed that a heterozygous P159L (CCT to CTT in codon 159 in the DNA-binding domain) mutation in HNF1B was segregated according to the affected status. To investigate the functional consequences of this HNF1B mutation, we generated a P159L HNF1B construct. The wild-type and mutant HNF1B constructs were transfected into COS-7 cells in the presence of the promoter sequence of human glucose transporter type 2 (GLUT2). The luciferase reporter assay revealed that P159L HNF1B had decreased transcriptional activity compared to wild-type (p < 0.05). Electrophoretic mobility shift assay showed reduced DNA binding activity of P159L HNF1B. In the MIN6 pancreatic ${\beta}$-cell line, overexpression of the P159L mutant was significantly associated with decreased mRNA levels of GLUT2 compared to wild-type (p < 0.05). However, INS expression was not different between the wild-type and mutant HNF1B constructs. These findings suggests that the impaired insulin secretion in this family with the P159L HNF1B mutation may be related to altered GLUT2 expression in ${\beta}$-cells rather than decreased insulin gene expression. In conclusion, we have identified a Korean family with an HNF1B mutation and characterized its effect on the pathogenesis of diabetes.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.10.1-10.12
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• 2012

Introduction: We investigated frameshift suppressor tRNAs previously reported to use five-base anticodon-codon interactions in order to provide a collection of frameshift suppressor tRNAs to the synthetic biology community and to develop modular frameshift suppressor logic devices for use in synthetic biology applications. Results and Discussion: We adapted eleven previously described frameshift suppressor tRNAs to the BioBrick cloning format, and built three genetic logic circuits to detect frameshift suppression. The three circuits employed three different mechanisms: direct frameshift suppression of reporter gene mutations, frameshift suppression leading to positive feedback via quorum sensing, and enzymatic amplification of frameshift suppression signals. In the course of testing frameshift suppressor logic, we uncovered unexpected behavior in the frameshift suppressor tRNAs. The results led us to posit a four-base binding hypothesis for the frameshift suppressor tRNA interactions with mRNA as an alternative to the published five-base binding model. Conclusion and Prospects: The published five-base anticodon/codon rule explained only 17 of the 58 frameshift suppression experiments we conducted. Our deduced four-base binding rule successfully explained 56 out of our 58 frameshift suppression results. In the process of applying biological knowledge about frameshift suppressor tRNAs to the engineering application of frameshift suppressor logic, we discovered new biological knowledge. This knowledge leads to a redesign of the original engineering application and encourages new ones. Our study reinforces the concept that synthetic biology is often a winding path from science to engineering and back again; scientific investigations spark engineering applications, the implementation of which suggests new scientific investigations.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.3 s.47
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• pp.389-392
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• 2004

We screened several materials to stimulate IGF-1 promoter activity using luciferase reporter assay and found that Amomi Semen extract (ASE) among them is the most powerful stimulator We also studied about the anti-wrinkle effect of ethanolic extract of Amoni Semen in vitro and in vivo. Semi-quantitative RT-PCR showed that the extract elevated the presence level of IGF-1 mRNA. And $[^3H]$ proline incorporation and semi-quantitative RT-PCR showed that the extract increased the expression of type-I collagen compared with vehicle in vitro and in vivo, respectively. Significant inhibition of MMP-1 expression was determined by ELISA and Western blot. Finally, topical treatment of the extract on hairless mouse's dorsal skin expanded the volume of collagen and dermal thickness. These results suggest that Amomi Semen may be a good candidate for improving extracellular matrix through the increase of collagen expression and inhibition of MMP-1 expression. Moreover, this study enables us to guess that IGF-1 stimulated by the extract may be involved in the mechanism of anti-wrinkle effect of it.

• The Korean Journal of Zoology
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• v.38 no.4
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• pp.550-556
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• 1995

The c-myc proto-oncogene is Involved In the control of normal cell proliferation and differentiation of many cell lineages. Although it has heen suggested that c-myc may play an important role in the mammalian early development, it Is unclear whether the embryonic c-myc mRNA is originated from zygotic gene expression or stored maternal message. Thus, we have construded expression vectors, In which the 5, flanking sequences including c-myc promoter region and a large non-coding exon I are fused 'sith E. coli lacZ gene that encedes $\beta$-galactosldase as a reporter. As c-myc exon I contains a modulatory sequence, we designed t, vo types of vectors (pcmyc.Gall and pcmyc-Ga12) to examine the role of exon I in c-myc expression. The former contains the complete exon I and the later has a deletion in 40 bp of modulator sequence located In the exon I of c-myc These vectors were microInjected into fertilized one-cell embryos and $\beta$-galactosidase activity was examined by X-gal staining during early embryogenesis. $\beta$-galactosidase activity derived from c-myc promoter was decreased at two-cell stage. The expression level directed by pcmyc- Ga12 was similar to that of pcmyc-Gal1, indicating that the medulatory sequence in exon I may not be Involved at least In the regulation of embryonic c-myc expression. In summary, the present study indicates that the c-myc promoter is functional at the early stage embryo, and the regulation of c-myc expression is under the control of "zygotic" clock of preimplantation mouse embryos.e embryos.

• Archives of Pharmacal Research
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• v.29 no.8
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• pp.633-644
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• 2006

In last couple of decades the use of natural compounds like flavonoids as chemopreventive agents has gained much attention. Our current study focuses on identifying chemopreventive flavonoids and their mechanism of action on human prostate cancer cells. Human prostate cancer cells (PC3), stably transfected with activator protein 1 (AP-1) luciferase reporter gene were treated with four main classes of flavonoids namely flavonols, flavones, flavonones, and isoflavones. The maximum AP-1 luciferase induction of about 3 fold over control was observed with $20\;{\mu}M$ concentrations of quercetin, chrysin and genistein and $50\;{\mu}M$ concentration of kaempferol. At higher concentrations, most of the flavonoids demonstrated inhibition of AP-1 activity. The MTS assay for cell viability at 24 h showed that even at a very high concentration $(500\;{\mu}M)$, cell death was minimal for most of the flavonoids. To determine the role of MAPK pathway in the induction of AP-1 by flavonoids, Western blot of phospho MAPK proteins was performed. Four out of the eight flavonoids namely kaempferol, apigenin, genistein and naringenin were used for the Western Blot analysis. Induction of phospho-JNK and phospho-ERK activity was observed after two hour incubation of PC3-AP1 cells with flavonoids. However no induction of phospho-p38 activity was observed. Furthermore, pretreating the cells with specific inhibitors of JNK reduced the AP-1 luciferase activity that was induced by genistein while pretreatment with MEK inhibitor reduced the AP-1 luciferase activity induced by kaempferol. The pharmacological inhibitors did not affect the AP-1 luciferase activity induced by apigenin and naringenin. These results suggest the possible involvement of JNK pathway in genistein induced AP-1 activity while the ERK pathway seems to play an important role in kaempferol induced AP-1 activity.

• The Korea Journal of Herbology
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• v.30 no.6
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• pp.7-15
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• 2015

Objectives : Rosa laevigata Michx. has been used for the treatment of renal disease in traditional Korean medicine. In this study, we investigated cytoprotective effect of R. laevigata water extract (RLE) against oxidative stress induced by arachidonic acid (AA) + iron.Methods : To evaluate the protective effects of RLE against AA + iron-induced oxidative stress in HepG2 cell, cell viability and changes on apoptosis-related proteins were assessed by MTT and immunoblot analyses. The effects of RLE on reduced glutathione level, production of reactive oxygen species and mitochondrial membrane potential were also monitored. Furthermore, to verify underlying molecular mechanism, NF-E2-related factor 2 (Nrf2) was examined by immunoblot analysis. Additionally, Nrf2 transactivation and its downstream target genes expression were also determined by reporter gene and realtime RT-PCR analyses.Results : RLE pretreatment (30-300 μg/ml) prevented cells from AA + iron-mediated cell death in a concentration dependent manner. In addition, 100 μg/ml RLE inhibited AA + iron-induced glutathione depletion, reactive oxygen species production and mitochondrial dysfunction. RLE accumulated nuclear Nrf2 and also transactivated Nrf2, which was evidenced by antioxidant response element- and glutathione S-transferase A2-driven luciferase activities and mRNA level of glutamate-cysteine ligase catalytic subunit, NAD(P)H:quinone oxidoreductase 1 and sestrin 2. Moreover, protective effect of RLE against AA + iron was abolished in Nrf2 knockout cells.Conclusions : These results indicate that RLE has the ability to protect hepatocyte against oxidative stress through Nrf2 activation.

• Molecules and Cells
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• v.38 no.2
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• pp.130-137
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• 2015

MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3'-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3'-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.

• Molecules and Cells
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• v.41 no.5
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• pp.390-400
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• 2018

Studies have revealed that miR-103a-3p contributes to tumor growth in several human cancers, and high miR-103a-3p expression is associated with poor prognosis in advanced gastric cancer (GC) patients. Moreover, bioinformatics analysis has shown that miR-103a-3p is upregulated in The Cancer Genome Atlas (TCGA) stomach cancer cohort. These results suggest that miR-103a-3p may function as an oncogene in GC. The present study aimed to investigate the role of miR-103a-3p in human GC. miR-103a-3p expression levels were increased in 33 clinical GC specimens compared with adjacent nontumor stomach tissues. Gain- and loss-of-function studies were performed to identify the correlation between miR-103a-3p and tumorigenesis in human GC. Inhibiting miR-103a-3p suppressed GC cell proliferation and blocked the S-G2/M transition in MKN-45/SGC-7901 cells, whereas miR-103a-3p overexpression improved GC cell proliferation and promoted the S-G2/M transition in vitro. Bioinformatics and dual-luciferase reporter assays confirmed that ATF7 is a direct target of miR-103a-3p. Analysis of the TCGA stomach cancer cohort further revealed that miR-103a-3p expression was inversely correlated with ATF7 expression. Notably, silencing ATF7 showed similar cellular and molecular effects as miR-103a-3p overexpression, namely, increased GC cell proliferation, improved CDK2 expression and decreased P27 expression. ATF7 overexpression eliminated the effects of miR-103a-3p expression. These findings indicate that miR-103a-3p promotes the proliferation of GC cell by targeting and suppressing ATF7 in vitro.