• IMMUNE NETWORK
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• 제14권6호
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• pp.321-327
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• 2014

TGF-${\beta}$ induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-${\beta}$ signal-transducing transcription factors, mediate germline (GL) ${\alpha}$ transcription induced by TGF-${\beta}1$, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-${\beta}$-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-${\beta}1$-induced, Smad3/4-mediated $GL{\alpha}$ transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-${\beta}1$-induced, Smad3/4-mediated $GL{\alpha}$ promoter activity, expression of endogenous $GL{\alpha}$ transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-${\beta}1$-induced, Smad3/4-mediated $GL{\alpha}$ promoter activity. We found that PIASy overexpression suppresses the $GL{\alpha}$ promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of $GL{\alpha}$ transcription and IgA switching induced by TGF-${\beta}1$/Smad3/4, while PIASy acts as a repressor.

• Journal of Ginseng Research
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• 제48권1호
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• pp.40-51
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• 2024

Background: Ginsenoside 20(S)-Rg3 shows promising tumor-suppressive effects in ovarian cancer via inhibiting NF-kB signaling. This study aimed to explore the downstream tumor suppressive mechanisms of ginsenoside Rg3 via this signaling pathway. Materials and methods: A systematical screening was applied to examine the expression profile of 41 kinesin family member genes in ovarian cancer. The regulatory effect of ginsenoside Rg3 on KIF20A expression was studied. In addition, we explored interacting proteins of KIF20A and their molecular regulations in ovarian cancer. RNA-seq data from The Cancer Genome Atlas (TCGA) was used for bioinformatic analysis. Epithelial ovarian cancer cell lines SKOV3 and A2780 were used as in vitro and in vivo cell models. Commercial human ovarian cancer tissue arrays were used for immunohistochemistry staining. Results: KIF20A is a biomarker of poor prognosis among the kinesin genes. It promotes ovarian cancer cell growth in vitro and in vivo. Ginsenoside Rg3 can suppress the transcription of KIF20A. GST pull-down and co-immunoprecipitation (IP) assays confirmed that KIF20A physically interacts with BTRC (β-TrCP1), a substrate recognition subunit for SCF^β-TrCP E3 ubiquitin ligase. In vitro ubiquitination and cycloheximide (CHX) chase assays showed that via interacting with BTRC, KIF20A reduces BTRC-mediated CDC25A poly-ubiquitination and enhances its stability. Ginsenoside Rg3 treatment partly abrogates KIF20A overexpression-induced CDC25A upregulation. Conclusion: This study revealed a novel anti-tumor mechanism of ginsenoside Rg3. It can inhibit KIF20A transcription and promote CDC25A proteasomal degradation in epithelial ovarian cancer.

• Molecules and Cells
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• 제39권11호
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• pp.821-826
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• 2016

The ${\beta}$-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, ${\beta}$-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of ${\beta}$-catenin. The level of ${\beta}$-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to ${\beta}$-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and ${\beta}$-catenin in HEK293T cells. To our knowledge, this is the first study to report that ${\beta}$-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated ${\beta}$-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active ${\beta}$-catenin via degradation, which stabilized SIAH-1 and increased its interaction with ${\beta}$-catenin. These results suggest that activated STAT3 regulates active ${\beta}$-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of ${\beta}$-catenin in HEK293T cells.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8271-8279
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• 2016

Background: Hepato-carcinogenesis is multifaceted in its molecular aspects. Among the interplaying agents are altered gap junctions, the proteasome/autophagy system, and mitochondria. The present experimental study was designed to outline the roles of these players and to investigate the tumor suppressive effects of curcumin with or without mesenchymal stem cells (MSCs) in hepatocellular carcinoma (HCC). Materials and Methods: Adult female albino rats were divided into normal controls and animals with HCC induced by diethyl-nitrosamine (DENA) and $CCl_4$. Additional groups treated after HCC induction were: Cur/HCC which received curcumin; MSCs/HCC which received MSCs; and Cur+MSCs/HCC which received both curcumin and MSCs. For all groups there were histopathological examination and assessment of gene expression of connexin43 (Cx43), ubiquitin ligase-E3 (UCP-3), the autophagy marker LC3 and coenzyme-Q10 (Mito.Q10) mRNA by real time, reverse transcription-polymerase chain reaction, along with measurement of LC3II/LC3I ratio for estimation of autophagosome formation in the rat liver tissue. In addition, the serum levels of ALT, AST and alpha fetoprotein (AFP), together with the proinflammatory cytokines $TNF{\alpha}$ and IL-6, were determined in all groups. Results: Histopathological examination of liver tissue from animals which received DENA-$CCl_4$ only revealed the presence of anaplastic carcinoma cells and macro-regenerative nodules. Administration of curcumin, MSCs; each alone or combined into rats after induction of HCC improved the histopathological picture. This was accompanied by significant reduction in ${\alpha}$-fetoprotein together with proinflammatory cytokines and significant decrease of various liver enzymes, in addition to upregulation of Cx43, UCP-3, LC3 and Mito.Q10 mRNA. Conclusions: Improvement of Cx43 expression, nonapoptotic cell death and mitochondrial function can repress tumor growth in HCC. Administration of curcumin and/or MSCs have tumor suppressive effects as they can target these mechanisms. However, further research is still needed to verify their effectiveness.

• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5471-5476
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• 2015

2-deoxy-D-Glucose (2DG) causes cytotoxicity in cancer cells by disrupting thiol metabolism. It is an effective component in therapeutic strategies. It targets the metabolism of cancer cells with glycolysis inhibitory activity. On the other hand, MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 activating enzyme), inactivates SCF E3 ligase and causes accumulation of its substrates which triggers apoptosis. Combination of these components might provide a more efficient approach to treatment. In this research, 2DG and MLN4924 were co-applied to breast cancer cells (MCF-7 and SKBR-3) and cytotoxic and apoptotic activity were evaluated the by Micro culture tetrazolium test (MTT), TUNEL and ELISA methods. Caspase3 and Bcl2 genes expression were evaluated by real time Q-PCR methods. The results showed that MLN4924 and MLN4924/2DG dose-dependently suppressed the proliferation of MCF7 and SKBR-3 cells. Cell survival of breast cancer cells exposed to the combination of 2DG/MLN4924 was decreased significantly compared to controls (p<0.05), while 2DG and MLN4924 alone had less pronounced effects on the cells. The obtained results suggest that 2DG/MLN4924 is much more efficient in breast cancer cell lines with enhanced cytotoxicity via inducing a apoptosis cell signaling gene, caspase-3.

• 생약학회지
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• 제35권4호통권139호
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• pp.368-374
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• 2004

Cervical cancer is one of the leading causes of female death. Viral oncoproteins E6 and E7 are selectively retained and expressed in carcinoma cells infected with HPV (Human papillomavirus) type 16. The HPV is cooperated in immotalization and transformation of primary keratinocyte. E6 and E7 oncoproteins interfere the functions of tumor suppressor proteins p53 and retinoblasoma protein (pRb), respectively. Among a lots of natural products, Artemisia scoparia Waldstein et Kitamura has inhibitory effects on the binding between E6 oncoprotein and tumor suppressor p53, or the binding between E6 and E6 associated protein (E6AP), an E3 ubiquitin-protein ligase. HPV oncoprotein inhibitors from Artemisia scoparia W. were isolated by solvent partition and column chromatography (Silica gel, RP-18) and the inhibitory compounds were finally purified by HPLC using an ELISA screening system based on the binding between E6 and E6AP. The aim of this study is to identify the structure of inhibitory compounds and to investigate whether these compounds have inhibitory effects on the functions of E6 oncoprotein. We investigated whether 3,5-di-O-caffeoylquinic acid (DCQA) extracted from Artemisia scoparia W. Could inhibit the function of E6 oncoprutein. DCQA inhibited the in vitro binding of E6 and E6AP which are essential for the binding and degradation of the tumor suppressor p53 and also inhibited the proliferation of human cervical cancer cell lines (SiHa and CaSKi) in a dose response manner. These results suggest that DCQA inhibited the function of E6 oncoprotein, suggesting that it can be used as a potential drug for the treatment of cervical cancers infected with HPV.

• IMMUNE NETWORK
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• 제9권6호
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• pp.248-254
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• 2009

[ $TGF-{\beta}1$ ]is well known to induce Ig germ-line ${\alpha}$ ($GL{\alpha}$) transcription and subsequent IgA isotype class switching recombination (CSR). Homeodomain protein TG-interacting factor (TGIF) and E3-ubiquitin ligases TGIF interacting ubiquitin ligase 1 (Tiul1) are implicated in the negative regulation of $TGF-{\beta}$ signaling. In the present study, we investigated the roles of Tiul1 and TGIF in $TGF{\beta}1$-induced IgA CSR. We found that over-expression of Tiul1 decreased $TGF{\beta}1$-induced $GL{\alpha}$ promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Likewise, overexpression of TGIF also diminished $GL{\alpha}$ promoter activity and further strengthened the inhibitory effect of Tiul1, suggesting that Tiul1 and TGIF can down-regulate $TGF{\beta}1$-induced $GL{\alpha}$ expression. In parallel, overexpression of Tiul1 decreased the expression of endogenous IgA CSR-predicitive transcripts ($GLT_{\alpha},\;PST_{\alpha},\;and\;CT_{\alpha}$) and $TGF{\beta}1$-induced IgA secretion, but not $GLT_{\gamma3}$ and IgG3 secretion. Here, over-expressed TGIF further strengthened the inhibitory effect of Tiul1. These results suggest that Tiul1 and TGIF act as negatively regulators in $TGF{\beta}1$-induced IgA isotype expression.

• Molecules and Cells
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• 제40권12호
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• pp.945-953
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• 2017

We report the biological functions of a zebrafish homologue of RING-finger protein 152 (rnf152) during embryogenesis. rnf152 was initially identified as a brain-enriched E3 ligase involved in early embryogenesis of zebrafish. Expression of rnf152 was ubiquitous in the brain at 24 hpf but restricted to the eyes, midbrain-hindbrain boundary (MHB), and rhombomeres at 48 hpf. Knockdown of rnf152 in zebrafish embryos caused defects in the eyes, MHB, and rhombomeres (r1-7) at 24 hpf. These defects in rnf152-deficient embryos were analyzed by whole-mount in situ hybridization (WISH) using neuroD, deltaD, notch1a, and notch3 probes. NeuroD expression was abolished in the marginal zone, outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the eyes at 27 hpf. Furthermore, deltaD and notch1a expression was remarkably reduced in the ONL, INL, subpallium, tectum, cerebellum, and rhombomeres (r1-7) at 24 hpf, whereas notch3 expression was reduced in the tectum, cerebellum, and rhombomeres at 24 hpf. Finally, we confirmed that expression of Notch target genes, her4 and ascl1a, also decreased significantly in these areas at 24 hpf. Thus, we propose that Rnf152 is essential for development of the eyes, midbrain and hindbrain, and that Delta-Notch signaling is involved.

• Toxicological Research
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• 제33권3호
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• pp.211-218
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• 2017

Cytochrome P450 1B1 (CYP1B1) acts as a hydroxylase for estrogen and activates potential carcinogens. Moreover, its expression in tumor tissues is much higher than that in normal tissues. Despite this association between CYP1B1 and cancer, the detailed molecular mechanism of CYP1B1 on cancer progression in HeLa cells remains unknown. Previous reports indicated that the mRNA expression level of Herc5, an E3 ligase for ISGylation, is promoted by CYP1B1 suppression using specific small interfering RNA, and that ISGylation may be involved in ubiquitination related to ${\beta}-catenin$ degradation. With this background, we investigated the relationships among CYP1B1, Herc5, and ${\beta}-catenin$. RT-PCR and western blot analyses showed that CYP1B1 overexpression induced and CYP1B1 inhibition reduced, respectively, the expression of $Wnt/{\beta}-catenin$ signaling target genes including ${\beta}-catenin$ and cyclin D1. Moreover, HeLa cells were treated with the CYP1B1 inducer $7,12-dimethylbenz[{\alpha}]anthracene$ (DMBA) or the CYP1B1 specific inhibitor, tetramethoxystilbene (TMS) and consequently DMBA increased and TMS decreased ${\beta}-catenin$ and cyclin D1 expression, respectively. To determine the correlation between CYP1B1 expression and ISGylation, the expression of ISG15, a ubiquitin-like protein, was detected following CYP1B1 regulation, which revealed that CYP1B1 may inhibit ISGylation through suppression of ISG15 expression. In addition, the mRNA and protein expression levels of Herc5 were strongly suppressed by CYP1B1. Finally, an immunoprecipitation assay revealed a direct physical interaction between Herc5 and ${\beta}-catenin$ in HeLa cells. In conclusion, these data suggest that CYP1B1 may activate $Wnt/{\beta}-catenin$ signaling through stabilization of ${\beta}-catenin$ protein from Herc5-mediated ISGylation for proteosomal degradation.

• 한국수정란이식학회지
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• 제25권4호
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• pp.281-286
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• 2010

This study was to evaluate the protein profile of seminal plasma using 2-DE in Hanwoo. Seminal plasma was harvested from five mature Hanwoo, and seminal plasma protein was extracted by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $300\;{\mu}l$. Immobilized pH gradient (IPG) strip was used 18 cm and 3~11 NL. SDS-PAGE was used 12% acrylamide gel. Each gels were visualized by comassie brilliant blue and silver staining. These spots were analyzed by MALDI-TOF MS and searched on NCBInr. The result, 20 proteins of 36 protein spots were searched through peptide sequencing on the NCBInr. 8 proteins profiled by 2-DE were proved through previous bovine studies and the name of each protein was albumin, nucleobindin, clusterin, TIMP-2, spermadhesin Z13, spermadhesin-1 and BSP proteins (BSP 30 kDa and BSP A1/A2). 12 new proteins were ATP synthase, protein MAK16 homolog, Transmembrane protein 214, E3 ubiquitin-protein ligase BRE1A, dual serine/threonine and tyrosine protein kinase, tissue factor pathway inhibitor 2, alpha-actinin-4, RUN domain-containing protein 3B, catenin alpha-1, protein-glutamine gamma-glutamyltransferase 2, plakophilin-1 and inter-alpha-trypsin inhibitor heavy chain H1 has not been previously described in the bovine seminal plasma study. These proteins may be contribute to define the type of proteins affecting fertility of male and improve the fertilizing ability of semen in Hanwoo.