• Journal of Life Science
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• v.28 no.6
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• pp.639-647
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• 2018

A new heat shock protein 70 was identified in red-spotted grouper (Epinephelus akaara) based on an expression analysis. The cDNA of red-spotted grouper Hsp70 (designated RgHsp70) was cloned by the rapid amplification of cDNA ends (RACE) techniques. The full-length of RgHsp70 cDNA was 2,152 bp, consisting of a 5'-terminal untranslated region (UTR) of 105 bp, a 3'-terminal UTR of 274 bp, and an open reading frame (ORF) of 1,773 bp that encode a polypeptide of 590 amino acids with a theoretical molecular weight of 64.9 kDa and an estimated isoelectric point of 5.2. Multiple alignment and phylogenetic analyses revealed that the RgHsp70 gene shares a high similarity with other Hsp70 fish genes. RgHsp70 contained all three classical Hsp70 family signatures. The results indicated the RgHsp70 is a member of the heat shock protein 70 family. RgHsp70 mRNA was predominately expressed in the liver, with reduced expression noted in the head-kidney tissues. The expression analysis of different water temperatures (21, 18, 15 and $12^{\circ}C$) for sampled livers revealed that expression gradually increased at $12^{\circ}C$ compared to $21^{\circ}C$. In this study, the effects of water temperature lowering on the physiological conditions were investigated, and the results revealed that novel RgHsp70 may be an important molecule involved in stress responses.

• Molecules and Cells
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• v.24 no.2
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• pp.210-214
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• 2007

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.

• Biomedical Science Letters
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• v.29 no.1
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• pp.11-25
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• 2023

In hepatocellular carcinoma (HCC), chromosome 4 open-reading frame 47 (C4orf47) has not been so far investigated for its prognostic value or association with infiltrating immune cells. We performed bioinformatics analysis on HCC data and analyzed the data using online databases such as TIMER, UALCAN, Kaplan-Meier plotter, LinkedOmics, and GEPIA2. We found that C4orf47 expression in HCC was higher compared to normal tissues. High C4orf47 expression was associated with a worse prognosis in HCC. The correlation between C4orf47 and infiltrating immune cells is positively associated with CD4+T cells, B cells, neutrophils, macrophages, and dendritic cells in HCC. Moreover, high C4orf47 expression was correlated with a poor prognosis of infiltrating immune cells. Analysis of C4orf47 gene co-expression networks revealed that 12501 genes were positively correlated with C4orf47, whereas 7200 genes were negatively correlated. The positively related genes of C4orf47 are associated with a high hazard ratio in different types of cancer, including HCC. Regarding the biological functions of C4orf47 gene, it mainly regulates RNA metabolic process, DNA replication, and cell cycle. The C4orf47 gene may play a prognostic role by regulating the global transcriptome process in HCC. Our findings demonstrate that high C4orf47 expression correlates with poor prognosis and tumor-infiltrating immune cells in HCC. We suggest that C4orf47 is a novel prognostic biomarker and potential immune therapeutic target for HCC.

• Journal of Life Science
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• v.25 no.9
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• pp.976-983
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• 2015

Achyranthoside C dimethyl ester (ACDE) is an oleanolic acid glycoside from Achyranthes japonica which has been used in traditional medicine for the treatment of edema and arthritis. In this study, we investigated the anti-inflammatory effects of ACDE in RAW264.7 macrophages. ACDE significantly induced heme oxygenase-1 (HO-1) gene expression in RAW264.7 cells, while ACDE improved LPS-induced toxicity of cells. And ACDE induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. Further study demonstrated that ACDE-induced expression of HO-1 was inhibited by inhibitors of phosphatidylinositol 3-kinase (PI-3K) (LY294002), c-Jun kinase (JNK) (SP600125), extracellular signal regulated kinase (ERK) (PD98059) and p38 kinase (SB203580). Moreover, ACDE phosphorylated Akt, JNK, ERK, and p38 MAPK. In addition, ACDE inhibited LPS-induced NO secretion as well as inducible NO synthase (iNOS) expression in a dose-dependent manner. The inhibitory effects of ACDE on iNOS expression were abrogated by small interfering RNA (siRNA)-mediated knock-down of HO-1. Therefore, these results suggest that ACDE suppresses the production of pro-inflammatory mediator such as NO by inducing HO-1 expression via PI-3K/Akt/MAPK-Nrf2 signaling pathway. These findings could help us to understand the active principle included in the roots of A. japonica and the molecular mechanisms underlying anti-inflammatory action of ACDE.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.195-204
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• 2021

Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4) regulates the ubiquitination and degradation of c-Jun, mediating the lipopolysaccharide-induced cellular response. However, the upstream signaling pathway that regulates this process is unknown. In this study, we describe how endoplasmic reticulum (ER) stress reversely regulates sequestosome-1 (p62)and c-Jun protein levels. Furthermore, our study reveals that expression of p62 attenuates c-Jun protein levels through the ubiquitinproteasome system. Conversely, siRNA knockdown of p62 elevates c-Jun protein levels. Immunoprecipitation and immunoblotting experiments demonstrate that p62 interacts with c-Jun and CRBN to form a ternary protein complex. Moreover, we find that CRBN knockdown completely abolishes the inhibitory effect of p62 on c-Jun. Using brefeldin A as an inducer of ER stress, we demonstrate that the p62/c-Jun axis participates in the regulation of ER stress-induced apoptosis, and that CRBN is required for this regulation. In summary, we have identified an upstream signaling pathway, which regulates p62-mediated c-Jun degradation. Our findings elucidate the underlying molecular mechanism by which p62/c-Jun axis regulates the ER stress-induced apoptosis, and provide a new molecular connection between ER stress and apoptosis.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.2
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• pp.113-120
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• 2019

Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma ($PPAR-{\gamma}$), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of $PPAR-{\gamma}$ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of $PPAR-{\gamma}$. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.3
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• pp.819-825
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• 2003

pharbitis nil and Taraxacum mongolicum are representative herbs that have been used for cancer treatment in Korean traditional medicine. To understand the molecular basis of the antitumor function, we analyzed the effect of these herbs on proliferation and apoptosis of tumor cells using a gastric cancer cell line AGS. Cell counting assay showed that pharbitis nil strongly inhibit cell proliferation Of AGS whereas Taraxacum mongolicum exhibit no detectable effect on cellular growth. [³H]thymidine uptake analysis also demonstrated that DNA replication of AGS is suppressed in a dose-dependent manner by treatment with pharbitis nil. Additionally, tryphan blue exclusion assay showed that Pharbitis nil induce apoptotic cell death of AGS in a dose-dependent. To explore whether anti antiproliferative and/or proapototic property of Pharbitis nil is associated with their effect on gene expression, we performed RT-PCR analysis of cell cycle- and apoptosis-related genes. Interestingly, mRNA expression levels of c-Jun, c-Fos, c-Myc, and Cyclin D1 were markedly reduced by Pharbitis nil. Taraxacum mongolicum also showed inhibitory action on expression of these growth-promoting protooncogene but there effects are less significant, as compared to Pharbitis nil. Furthermore, it was also found that Pharbitis nil activates expression of the p53 tumor suppressor and its downstream effector p21Waf1, which induce G1 cell cycle arrest and apoptosis. Collectively, our data demonstrate that Pharbitis nil induce growth inhibition and apoptosis of human gastric cancer cells and these effects are accompanied with down-and up-regulation of growth-regulating protooncogenes and tumor suppressor genes, respectively. This observation thus suggests that the anticancer effect of Pharbitis nil might be associated with its regulatory capability of tumor-related gene expression.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.229-233
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• 2010

Pluripotency and self-renewal capacity of human embryonic stem cells (hESCs) are retained by hESCs related genes as OCT4, SOX2 and NANOG. These genes are shown high expression level in diverse cancer cells and have potential role in the carcinogenesis. On the contrary to this, several genes which are up-regulated in the differentiated hESCs are involved to suppress the carcinogenesis or proliferation of cells. We discovered several genes in immortalized lung fibroblast (WI-38 VA13) by suppression subtractive hybridization. Among them, we focused chromosome 6 open reading frame 62 (C6orf62) which is uncharacterized, mapped to 6p22.3 and generated to Hepatitis B virus X-transactivated proteins (HBVx-transactivated proteins, XTP). Aim of this study was to characterize C6orf62 through analyzing of expression pattern in various cell lines. Expression of C6orf62 was significantly upregulated in diverse normal cell lines than cancer cell lines. And C6orf62 was up-regulated in differentiated hESCs (endothelial cells, neural cells) compared to those of undifferentiated hESCs. Also, C6orf62 in WI-38 cells was highly up-regulated during G1/S transition of the cell cycle. Taken together, C6orf62 is shown expression pattern similar to differentiated hESCs-associated genes which down-regulated in cancer cells. Therefore, we assume that C6orf62 may participate to suppress the proliferation and to induce differentiation through regulating the cell cycle.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4871-4876
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• 2014

Background: We sought to evaluate the role of tumor associated macrophages (TAMs) on the promotion of coal tar pitch extract (CTPE)-induced tumorigenesis of human bronchial epithelial cells (BEAS-2B) and tumor metastasis in nude mice, and related mechanisms. Materials and Methods: BEAS-2B cells were first treated with 2.4 mg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured and passaged using trypsin-EDTA. THP-1 cells were used as macrophage-like cells. BEAS-2B cells under different conditions (n=6/group) were injected into the back necks of nude mice, and alterations of tumor xenograft growth, indicative of tumorigenicity, and tumor metastasis were determined. Pathological changes (tumor nests and microvascular lesions) of HE-stained tumor tissues were also evaluated. The expression of AP-1(c-Jun) in xenografts and metastatic tumors was determined using immunohistochemistry. Results: Tumor size and weight in nude mice transplanted with the mixture of CTPE-induced passage 30 BEAS-2B and THP-1 cells (2:1) were increased compared to those from the CTPE-treated BEAS-2B cells at passage 30 alone at different observation time points. Tumor metastasis to lymph nodes and liver was only detected after transplantation of a mixture the two kinds of cells. The numbers of tumor nests and microvascular lesions, and the expression levels of AP-1 (c-Jun) in tumors from the mixture of two kinds of cells were increased apparently in contrast to those in tumor from the CTPE-treated BEAS-2B cells of passage 30 alone. In addition, there was positive correlation between AP-1 (c-Jun) expression level and the number of microvascular lesions, or between AP-1 (c-Jun) expression level and tumor metastasis in these two groups. Conclusions: TAMs not only facilitate tumorigenesis transformation of CTPE-induced BEAS-2B cells, but also promote tumor growth, angiogenesis and metastasis in nude mice in vivo, which may be mediated by AP-1.