• The Korea Journal of Herbology
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• v.31 no.1
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• pp.77-82
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• 2016

Objectives : The study was designed to observe the effect of Polygalae Radix Preparata Cum Glycyrrhizae Radix on 4-Hydroxynonenal (4-HNE)-induced apoptosis in PC-12 cell.Methods : A cytotoxic test on Polygalae Radix Preparata Cum Glycyrrhizae Radix (PG) was conducted and another MTT assay was conducted to observe the cytoprotective effect against 4-HNE that cause oxidative stress. In addition, in order to observe the expression of Bax, Bcl-2, Caspase-3 and TNF-α protein involved with apoptosis, western blot was conducted.Results : The groups treated with 25 ㎍, 50 ㎍ and 100 ㎍ of PG water extract had no toxicity for PC-12 cell. The groups treated with 25 ㎍, 50 ㎍ and 100 ㎍ of PG water extract showed a significant increase of cell survival rate in comparison with the control group injected by only 4-HNE. The groups treated with 25 ㎍ and 50 ㎍ of PG water extract showed a significant supression on increase of Bax protein expression in the control group. The group treated with 100 ㎍ of PG water extract showed a significant promotion on decrease of Bcl-2 protein expression in the control group. The group treated with 50 ㎍ of PG water extract showed a significant supression on increase of Caspase-3 protein expression in the control group. The group treated with 25 ㎍ of PG water extract showed a significant supression on increase of TNF-α protein expression in the control group.Conclusions : These results suggest that Polygalae Radix Preparata Cum Glycyrrhizae Radix is effective in reducing apoptosis by 4-HNE-dameged cell.

• BMB Reports
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• v.38 no.4
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• pp.447-456
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• 2005

TNF-$\alpha$ plays a pivotal role in inflammation processes which are mainly regulated by endothelial cells. While TNF-$\alpha$ induces apoptosis of several cell types like tumor cells, endothelial cells are resistant to TNFa mediated cell death. The cytotoxic effects of TNF-$\alpha$ on most cells are only evident if RNA or protein synthesis is inhibited, suggesting that de novo RNA or protein synthesis protect cells from TNF-$\alpha$ cytotoxicity, presumably by NF-${\kappa}B$ mediated induction of protective genes. However, the cytoprotective genes involved in NF-${\kappa}B$ dependent endothelial cell survival have not been sufficiently identified. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-$\alpha$ inducible genes in human arterial endothelial cells related to cell survival and cell cycle. The TNF-$\alpha$-induced expression of the RNA binding protein $p54^{nrb}$ and the 14-3-3 protein HS1 as shown here for the first time may contribute to the TNF-$\alpha$ mediated cell protection of endothelial cells. These genes have been shown to play pivotal roles in cell survival and cell cycle control in different experimental settings. The concerted expression of these genes together with other genes related to cell protection and cell cycle like DnaJ, $p21^{cip1}$ and the ubiquitin activating enzyme E1 demonstrates the identification of new genes in the context of TNF-$\alpha$ induced gene expression patterns mediating the prosurvival effect of TNF-$\alpha$ in endothelial cells.

• Nutrition Research and Practice
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• v.15 no.5
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• pp.555-567
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• 2021

BACKGROUND/OBJECTIVES: Abeliophyllum distichum is a plant endemic to Korea, containing several beneficial natural compounds. This study investigated the effect of A. distichum leaf extract (ALE) on adipocyte differentiation. MATERIALS/METHODS: The cytotoxic effect of ALE was analyzed using cell viability assay. 3T3-L1 preadipocytes were differentiated using induction media in the presence or absence of ALE. Lipid accumulation was confirmed using Oil Red O staining. The mRNA expression of adipogenic markers was measured using RT-PCR, and the protein expressions of mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor gamma (PPAR𝛾) were measured using western blot. Cell proliferation was measured by calculating the incorporation of Bromodeoxyuridine (BrdU) into DNA. RESULTS: ALE reduced lipid accumulation in differentiated adipocytes, as indicated by Oil Red O staining and triglyceride assays. Treatment with ALE decreased the gene expression of adipogenic markers such as Ppar𝛾, CCAAT/enhancer binding protein alpha (C/ebp𝛼), lipoprotein lipase, adipocyte protein-2, acetyl-CoA carboxylase, and fatty acid synthase. Also, the protein expression of PPAR𝛄 was reduced by ALE. Treating the cells with ALE at different time points revealed that the inhibitory effect of ALE on adipogenesis is higher in the early period treatment than in the terminal period. Furthermore, ALE inhibited adipocyte differentiation by reducing the early phase of adipogenesis and mitotic clonal expansion. This was indicated by the lower number of cells in the Synthesis phase of the cell cycle (labeled using BrdU assay) and a decrease in the expression of early adipogenic transcription factors such as C/ebp𝛽 and C/ebp𝛿. ALE suppressed the phosphorylation of MAPK, confirming that the effect of ALE was through the suppression of early phase of adipogenesis. CONCLUSIONS: Altogether, the results of the present study revealed that ALE inhibits lipid accumulation and may be a potential agent for managing obesity.

• Biomolecules & Therapeutics
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• v.30 no.3
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• pp.265-273
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• 2022

Resistance to chemotherapeutic drugs is a significant problem in the treatment of colorectal cancer, resulting in low response rates and decreased survival. Recent studies have shown that shikonin, a naphthoquinone derivative, promotes apoptosis in colon cancer cells and cisplatin-resistant ovarian cells, raising the possibility that this compound may be effective in drug-resistant colorectal cancer. The aim of this study was to characterize the molecular mechanisms underpinning shikonin-induced apoptosis, with a focus on endoplasmic reticulum (ER) stress, in a 5-fluorouracil-resistant colorectal cancer cell line, SNU-C5/5-FUR. Our results showed that shikonin significantly increased the proportion of sub-G₁ cells and DNA fragmentation and that shikonin-induced apoptosis is mediated by mitochondrial Ca²⁺ accumulation. Shikonin treatment also increased the expression of ER-related proteins, such as glucose regulatory protein 78 (GRP78), phospho-protein kinase RNA-like ER kinase (PERK), phospho-eukaryotic initiation factor 2 (eIF2α), phospho-phosphoinositol-requiring protein-1 (IRE1), spliced X-box-binding protein-1 (XBP-1), cleaved caspase-12, and C/EBP-homologous protein (CHOP). In addition, siRNA-mediated knockdown of CHOP attenuated shikonin-induced apoptosis, as did the ER stress inhibitor TUDCA. These data suggest that ER stress is a key factor mediating the cytotoxic effect of shikonin in SNU-C5/5-FUR cells. Our findings provide an evidence for a mechanism in which ER stress leads to apoptosis in shikonin-treated SNU-C5/5-FUR cells. Our study provides evidence to support further investigations on shikonin as a therapeutic option for 5-fluorouracil-resistant colorectal cancer.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.115-122
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• 2024

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90β and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, β-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

• Journal of Society of Preventive Korean Medicine
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• v.2 no.1
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• pp.13-30
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• 1998

Geraniol (1), olivetol (2), cannabinoids (3 and 4) and 5-fluorou.a.il (5). were tested for their growth inhibitory effects against SK-MEL-3 cell lines using two different 3-(4,5-dimethylthiazol- 2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and sulforhodamine B protein (SRB) assay. These compounds showed inhibitory activity in vitro in the micromolar range against SK-MEL-3 cell lines. In general, the antitumor activity of these compounds (1, 2, 3, 4 and 5) was in a dose-dependent over the micromolar concentration range $1\;to\;100{\mu}M$. The comparison of $IC_{50}$ values of these compounds in tumor cell lines shows that their susceptibility to these compounds decrease in the following order : OLVTL > CBG > CBD > 5-FU > CRNL in MTT assay, CBG > OLVTL > CBD > GRNL > 5-FU in SRB assay. Cannabinoids (3 and 4) and 5-fluorouracil (5) were tested for their cytotoxic effects on NIH 373 fibroblasts using two different MTT assay and SRB assay. These compounds exhibited potent cytotoxic activities in vitro in the micromolar range against NIH 373 fibroblasts. In general, the cytotoxic activities of these compounds (3, 4 and 5) were in a dose-dependent over the micromolar concentration range $1\;to\;100{\mu}M$. The comparison of $CD_{50}$ values of these compounds on NIH 373 fibroblasts shows that their susceptibility to these compounds decrease on the following order ; CBD > 5-FU > CBG in MTT assay and SRB assay. Cannabigerol (3) was shown the least cytotoxic activity on NIH 373 fibroblasts. Cannabigerol (3) exhibited the most growth-inhibitory activity against SK-MEL-3 cell lines.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8947-8950
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• 2014

Immunological functions of heat shock proteins (HSPs) have long been recognized. In this study we aimed to efficiently purify HSP70 from renal cell carcinoma and test it as a tumor antigen for pulsing dendritic cells in vitro. HSP70 was purified from renal cell carcinoma specimens by serial column chromatography on Con A-sepharose, PD-10, ADP-agarose and DEAE-cellulose, and finally subjected to fast protein liquid chromatography (FPLC). Dendritic cells derived from the adherent fraction of peripheral blood mononuclear cells were cultured in the presence of IL-4 and GM-CSF and exposed to tumor HSP70. After 24 hours, dendritic cells were phenotypically characterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclear cells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primary cultured renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitro with tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and HLA-DR maturation markers than those pulsed with tumor cell lysate and comparable to that of dendritic cells pulsed with tumor cell lysate plus TNF-${\alpha}$. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented the highest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70 as the tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cell maturation in the absence of TNF-${\alpha}$. Conspecific HSP70 may effectively be used as a tumor antigen to pulse dendritic cells in vitro.

• Tuberculosis and Respiratory Diseases
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• v.73 no.4
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• pp.204-209
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• 2012

Background: We investigated whether chrysin affected MUC5AC mucin production and gene expression induced by phorbol ester (phorbol 12-myristate 13-acetate, PMA) or epidermal growth factor (EGF) from human airway epithelial cells. Methods: Confluent NCI-H292 cells were pretreated with varying concentrations of chrysin for 30 minutes, and were then stimulated with PMA and EGF for 24 hours, respectively. MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results: Concentrations of $10{\mu}M$ and $100{\mu}M$ chrysin were found to inhibit the production of MUC5AC mucin protein induced by PMA; A concentration of $100{\mu}M$ chrysin also inhibited the production of MUC5AC mucin protein induced by EGF; $100{\mu}M$ chrysin inhibited the expression of MUC5AC mucin gene induced by PMA or EGF. The cytotoxicity of chrysin was checked by lactate dehydrogenase assay, and there was no cytotoxic effect observed for chrysin. Conclusion: These results suggest that chrysin can inhibit mucin gene expression and the production of mucin protein by directly acting on airway epithelial cells.

• Restorative Dentistry and Endodontics
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• v.16 no.2
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• pp.99-117
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• 1991

The purpose of this study is to evaluate the effects of dentin bonding agents on the fibroblasts cultivated from human pulp tissue. The fibroblasts were cultured in DMEM/10%FBS medium. Whatman filter paper discs (6mm diameter) soaked with $2{\mu}l$ of dentin bonding agents were placed on a millipore filter (pore size $0.22{\mu}m$) contained in a 50mm Petri dish, and then, exposed for 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 4 days and 7 days in $37.^{\circ}C$, 5% $CO_2$ incubator. The results of the experiments were analyzed by counting the cells and measuring the protein contents at 1 day, 4 days and 7 days. The results of this study were as follows: l. CLEARFIL NEW BOND, LITE-FIL BOND, GLUMA 3 Primer and GLUMA 4 Sealer showed cytotoxicity compared to the control group in the cell counts and the protein contents. 2. GLUMA 4 Sealer showed the least cytotoxicity among the three dentin bonding agents. 3. The results of the cell count were simialr to the results of protein content measurement. 4. LITE-FIL BOND exhibited marked cytotoxicity during 1 day, but, the cytotoxicity was slightly reduced after 4 and 7 days. 5. In GLUMA 3 Primer group, it was not possible to count the cell numbers and measure the protein contents, but the degeneration of cells was observed under the inverted phase-contrast microscope.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.911-920
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• 2006

[ $CD8^+$ ] T Iymphocytes with the cytotoxic activity and capability to release various cytokines are the major players in immune responses against viral infection and cancer. To identify the proteins specific to resting or activated human CD8$^+$ T cells, human CD8$^+$ T cells were activated with anti-CD3+anti-CD28 mAb in the presence of IL-2. The solubilized proteins from resting and activated human CD8$^+$ T cells were separated by high-resolution two-dimensional polyacrylamide gel electrophoresis, and their proteomes were analyzed. Proteomic analysis of resting and activated T cells resulted in identification of 35 proteins with the altered expression. Mass spectrometry coupled with Profound and SWISS-PROT database analysis revealed that these identified proteins are to be functionally associated with cell proliferation, metabolic pathways, antigen presentation, and intracellular signal transduction pathways. We also identified six unknown proteins predicted from genomic DNA sequences specific to resting or activated CD8$^+$ T cells. Protein network studies and functional characterization of these novel proteins may provide new insight into the signaling transduction pathway of CD8$^+$ T cell activation.