• Journal of Acupuncture Research
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• v.17 no.2
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• pp.169-186
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• 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.

• Environmental Mutagens and Carcinogens
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• v.26 no.4
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• pp.125-132
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• 2006

Previous studies showed that epigallocatechin gallate(EGCG) have substantial effects of suppressing the N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-initiated cell transformation process on the bases of foci formation frequency and loss of anchorage dependency. In this study we tried to clarify the molecular mechanism of suppressing the cell transformation process. Mouse cell line balb/c 3T3 A31-1-1 was exposed 2 days to MNNG followed by 15 days 12-O-tetradecanoylphorbol-13-acetate(TPA) treatment for our transformation process. EGCG was added after the time point of 24 hours exposure to TPA and incubated for 19 days. 2029 genes were selected in our transformation process that showed fold change value of 1.5 or more in the microarray gene expression analysis covering the mouse full genome. These genes were found to be involved mainly in the cell cycle pathway, focal adhesion, adherens junction, TGE-$\beta$ signaling, apoptosis, lysine degradation, insulin signaling, ECM-receptor interaction. Among the genes, we focused on the 631 genes(FC>0.5) reciprocally affected by EGCG treatment. Our study suggest that EGCG down-regulate the gene expressions of up stream signaling factors such as nemo like kinase with MAPK activity and PI3-Kinase, Ras GTPase and down stream factors such as cyclin D1, D2, H, T2, cdk6.

• Molecules and Cells
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• v.40 no.2
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• pp.109-116
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• 2017

Soybean agglutinin (SBA) is an anti-nutritional factor of soybean, affecting cell proliferation and inducing cytotoxicity. Integrins are transmembrane receptors, mediating a variety of cell biological processes. This research aims to study the effects of SBA on cell proliferation and cell cycle progression of the intestinal epithelial cell line from piglets (IPEC-J2), to identify the integrin subunits especially expressed in IPEC-J2s, and to analyze the functions of these integrins on IPEC-J2 cell cycle progression and SBA-induced IPEC-J2 cell cycle alteration. The results showed that SBA lowered cell proliferation rate as the cell cycle progression from G0/G1 to S phase (P < 0.05) was inhibited. Moreover, SBA lowered mRNA expression of cell cycle-related gene CDK4, Cyclin E and Cyclin D1 (P < 0.05). We successfully identified integrins ${\alpha}2$, ${\alpha}3$, ${\alpha}6$, ${\beta}1$, and ${\beta}4$ in IPEC-J2s. These five subunits were crucial to maintain normal cell proliferation and cell cycle progression in IPEC-J2s. Restrain of either these five subunits by their inhibitors, lowered cell proliferation rate, and arrested the cells at G0/G1 phase of cell cycle (P < 0.05). Further analysis indicated that integrin ${\alpha}2$, ${\alpha}6$, and ${\beta}1$ were involved in the blocking of G0/G1 phase induced by SBA. In conclusion, these results suggested that SBA lowered the IPEC-J2 cell proliferation rate through the perturbation of cell cycle progression. Furthermore, integrins were important for IPEC-J2 cell cycle progression, and they were involved in the process of SBA-induced cell cycle progression alteration, which provide a basis for further revealing SBA anti-proliferation and anti-nutritional mechanism.

• Animal Bioscience
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• v.35 no.7
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• pp.1010-1020
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• 2022

Objective: The experiment was conducted to evaluate the effects of maternal undernutrition during late pregnancy on the expressions of genes involved in growth and development in ovine fetal perirenal brown adipose tissue (BAT). Methods: Eighteen ewes with singleton fetuses were allocated to three groups at day 90 of pregnancy: restricted group 1 (RG1, 0.33 MJ metabolisable energy [ME]/kg body weight [BW]^0.75/d, n = 6), restricted group 2 (RG2, 0.18 MJ ME/kg BW^0.75/d, n = 6), and a control group (CG, ad libitum, 0.67 MJ ME/kg BW^0.75/d, n = 6). The fetuses were removed at day 140 of pregnancy. All data were analyzed by using the analysis of variance procedure. Results: The perirenal fat weight (p = 0.0077) and perirenal fat growth rate (p = 0.0074) were reduced in RG2 compared to CG. In fetal perirenal BAT, the protein level of uncoupling protein 1 (UCP1) (p = 0.0001) was lower in RG1 and RG2 compared with CG and UCP1 mRNA expression (p = 0.0265) was decreased in RG2. The protein level of myogenic factor 5 (Myf5) was also decreased in RG2 (p = 0.0001). In addition, mRNA expressions of CyclinA (p = 0.0109), CyclinB (p = 0.0019), CyclinD (p = 0.0015), cyclin-dependent kinase 1 (CDK1) (p = 0.0001), E2F transcription factor 1 (E2F1) (p = 0.0323), E2F4 (p = 0.0101), and E2F5 (p = 0.0018) were lower in RG1 and RG2. There were decreased protein expression of peroxisome proliferator-activated receptor-γ (PPARγ) (p = 0.0043) and mRNA expression of CCAAT/enhancer-binding protein-α (C/EBPα) (p = 0.0307) in RG2 and decreased PPARγ mRNA expression (p = 0.0008) and C/EBPα protein expression (p = 0.0015) in both RG2 and RG1. Furthermore, mRNA expression of bone morphogenetic protein 4 (BMP4) (p = 0.0083) and BMP7 (p = 0.0330) decreased in RG2 and peroxisome proliferator-activated receptor co-activator-1α (PGC-1α) reduced in RG2 and RG1. Conclusion: Our observations support that repression of regulatory factors promoting differentiation and development results in the inhibition of BAT maturation in fetal perirenal fat during late pregnancy with maternal undernutrition.

• Journal of Life Science
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• v.33 no.10
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• pp.767-775
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• 2023

Sulfasalazine is a disease-modifying antirheumatic abiotic agent. It is a derivative of aminosalicylic acid and has been used for the treatment of various inflammatory diseases, such as rheumatoid arthritis, ulcerative colitis, and Crohn's disease, since it was first synthesized in 1941 and approved as a medicine in the United States in 1950. However, its mechanism of action has not yet been clearly identified. In this study, the effects of sulfasalazine on cell survival, apoptosis, and cell cycle progression in macrophages, which are major immune cells that regulate inflammatory responses, were investigated using mouse macrophage RAW 264.7 cells. Sulfasalazine inhibited the viability of RAW 264.7 cells in a dose-dependent manner, starting at a concentration of 0.25 mM. Annexin-V staining was used to confirm that the decrease in cell viability was due to apoptosis, and the number of Annexin-V-positive cells increased significantly at a concentration of 0.25 mM or higher. The effect of sulfasalazine on the expression of key proteins that regulate the G0/G1 phase of the cell cycle was also investigated. Sulfasalazine treatment significantly increased the expression of the cyclin-dependent kinase inhibitors p21 and p27 in RAW 264.7 cells. Although sulfasalazine is frequently used as a control drug in studies on inflammatory diseases, such as inflammatory colitis and rheumatoid arthritis, studies on its effect on macrophages are very limited. Therefore, the results of this study are expected to provide vital information on the use of sulfasalazine as a disease treatment.

• Animal Bioscience
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• v.37 no.3
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• pp.471-480
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• 2024

Objective: The objective of this study was to investigate the regulation relationship of Ten-eleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. Methods: siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/β-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. Results: Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/β-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxyl-methylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/β-catenin signaling pathway. Conclusion: These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/β-catenin signaling pathway.

• Journal of Life Science
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• v.24 no.2
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• pp.137-147
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• 2014

Cortex ulmi pumilae, the cortex of Ulmus davidiana var. japonica, has been used in traditional folk medicine for its anti-inflammatory effect. Although its various bioactivities such as anti-inflammatory, anti-microbial, and anti-cancer, have been reported, the anti-adipogenic activity of cortex ulmi pumilae remains unclarified. In the present study, we investigated the effect of cortex ulmi pumilae extract on adipocyte differentiation in 3T3-L1 preadipocytes. Treatment with cortex ulmi pumilae extract significantly reduced the formation of lipid droplets and triglyceride content in a dose-dependent manner; this is associated with an inhibition of the adipogenic transcription factors, CCAAT/enhancer binding protein ${\alpha}$ ($C/EBP{\alpha}$), $C/EBP{\beta}$, and peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$). In addition, cortex ulmi pumilae extract treatment during the early stage of adipogenesis showed more efficient anti-adipogenic activity than treatment during other stages of adipogenesis. Cortex ulmi pumilae extract also inhibited cell proliferation and induced G1 arrest of 3T3-L1 cells in the early stage of adipogenesis. This was associated with upregulated expression of Cdk inhibitor p21 and downregulated expression of cyclin E and phospho-Rb, indicating that cortex ulmi pumilae extract blocks mitotic clonal expansion by cell cycle regulation. Taken together, these results suggest that cortex ulmi pumilae extract possesses anti-adipogenic activity through the inhibition of adipocyte differentiation by blocking mitotic clonal expansion.

• Journal of Nutrition and Health
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• v.47 no.1
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• pp.1-11
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• 2014

Purpose: Several studies have proven that EGCG, the primary green tea catechin, and glucosamine-6-phosphate (PGlc) reduce triglyceride contents in 3T3-L1 adipocytes. The objective of this study is to evaluate the combination effect of EGCG and PGlc on decline of accumulated fat in differentiated 3T3-L1 adipocytes. Methods: EGCG and PGlc were administered for 6 day for differentiation of 3T3-L1 adipocytes. Cell viability was measured using the CCK assay kit. In addition, TG accumulation in culture 3T3-L1 adipocytes was investigated by Oil Red O staining. We examined the expres-sion level of several genes and proteins associated with adipogenesis and lipolysis using real-time RT-PCR and Western blot analysis. A flow cytometer Calibar was used to assess the effect of EGCG and PGluco on cell-cycle progression of differentiating 3T3-L1 cells. Results: Intracelluar lipid accumulation was significantly decreased by combination treatment with EGCG $60{\mu}M$ and PGlc $200{\mu}g/m$ compared with control and EGCG treatment alone. In addition, use of combination treatment resulted in directly decreased expression of $PPAR{\gamma}$, $C/EBP{\alpha}$, and SREBP1. In addition, it inhibited adipocyte differentiation and adipogenesis through downstream regulation of adipogenic target genes such as FAS, ACSL1, and LPL, and the inhibitory action of EGCG and PGlc was found to inhibit the mitotic clonal expansion (MCE) process as evidenced by impaired cell cycle entry into S phase and the S to G2/M phase transition of confluent cells and levels of cell cycle regulating proteins such as cyclin A and CDK2. Conclusion: Combination treatment of EGCG and PGlc inhibited adipocyte differentiation through decreased expression of genes related to adipogenesis and adipogenic and cell cycle arrest in early stage of adipocyte differentiation.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.163-170
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• 2006

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .