• Journal of Biomedical Engineering Research
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• v.39 no.4
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• pp.168-174
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• 2018

Adipocytes affect obesity through the regulation of lipid metabolism. Physical loading is an important regulator of fat tissue. There are ongoing in vitro studies inducing mechanotransduction on 3T3-L1 preadipocytes with mechanical stimulus in order to treat obesity by inhibiting adipogenesis and provoking cell death. In this study, our goal was to suggest a new therapy for obesity by investigating whether fluid shear stress (FSS) changes transcription factors on 3T3-L1 related with adipogenesis and cell death. FSS loading was applied to 3T3-L1 preadipocytes at 1Pa and 1Hz. After loading, bright field images were taken and an immunofluorescence assay was conducted to observe actin stress fiber formation. Western blot analysis was conducted to identify the activation of the ERK pathway as well as the adipogenic factors, which including C/EBPs and $PPAR{\gamma}$. The expression of osteopontin, a protein related to inflammation in adipose tissue, and cell death related factors, Bax, Bcl-2, and Beclin, were also measured. Results showed that FSS stimulated the formation of actin stress fibers in 3T3-L1 and also that the activation of C/EBPs decreased significantly when compared with the control group. $PPAR{\gamma}$ activation in the 2 hour FSS group was lower than the 1 hour FSS group, which implied that the results were time dependent. Additionally, there were no differences in the expression of cell death factors after FSS loading. In summary, similar to other fibroblasts, the formation of actin stress fibers induced by mechanotransduction may affect the differentiation of 3T3-L1, leading to inhibition of adipogenesis and inflammation.

• The Journal of Korean Academy of Prosthodontics
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• v.46 no.3
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• pp.227-237
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• 2008

STATEMENT OF PROBLEM: Zirconium oxide can be a substitute to titanium as implant materials to solve the esthetic problems of dark color in the gingival portion of implant restorations. PURPOSE: This study was performed to define attachment and growth behavior of osteoblast- like cells cultured on grooved surfaces of zirconium oxide and evaluate the genetic effect of zirconium oxide surfaces using the reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIAL AND METHODS: MC3T3-E1 cells were cultured on (1) commercially pure titanium discs with smooth surface (T group), (2) yttrium-stabilized tetragonal zirconia polycrystal (Y-TZP) with machined surface (ZS group), and (3) Y-TZP with $100{\mu}m$ grooves (ZG group). Cell proliferation activity was evaluated through MTT assay and cell morphology was examined by SEM. The mRNA expression of Runx2, alkaline phosphatase, osteocalcin, TGF-${\beta}1$, IGF-1, G3PDH in E1 cells were evaluated by RT-PCR. RESULTS: From the MTT assay, after 48 hours of adhesion of MC3T3-E1 cells, the mean optical density value of T group and ZG group significantly increased compared to the ZS group. SEM images of osteoblast-like cells showed that significantly more cells were observed to attach to the grooves and appeared to follow the direction of the grooves. After 24 hours of cell adhesion, more spreading and flattening of cells with active filopodia formation occurred. Results of RT-PCR suggest that T group, ZS group, and ZG group showed comparable osteoblast-specific gene expression after 24 hours of cell incubation. CONCLUSION: Surface topography and material of implants can play an important role in expression of osteoblast phenotype markers. Zirconia ceramic showed comparable biological responses of osteoblast-like cells with titanium during a short-time cell culture period. Also, grooves influence cell spreading and guide the cells to be aligned within surface grooves.

• Parasites, Hosts and Diseases
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• v.51 no.3
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• pp.279-287
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• 2013

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.

• BMB Reports
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• v.48 no.5
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• pp.283-288
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• 2015

We found that resveratrol enhances interferon (IFN)-γ-induced tryptophanyl-tRNA-synthetase (TTS) expression in bone marrow-derived dendritic cells (BMDCs). Resveratrol-induced TTS expression is associated with glycogen synthase kinase-3β (GSK-3β) activity. In addition, we found that resveratrol regulates naive CD8⁺ T-cell polarization by modulating GSK-3β activity in IFN-γ-stimulated BMDCs, and that resveratol induces upregulation of TTS in CD8⁺ T-cells in the in vivo tumor environment. Taken together, resveratrol upregulates IFN-γ-induced TTS expression in a GSK-3β-dependent manner, and this TTS modulation is crucial for DC-mediated T-cell modulation. [BMB Reports 2015; 48(5): 283-288]

• Journal of the Korean Society of Tobacco Science
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• v.25 no.2
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• pp.128-136
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• 2003

Although tobacco smoke has been known to have genotoxicity as well as cytotoxicity, the sensitivity of the cell lines used against cigarette smoke is poorly understood. The objective of this study was to evaluate and compare the genotoxicity of several cell lines, which are routinely used in the in vitro assays, with cigarette smoke condensate(CSC) of Kentucky Reference Cigarette 1R4F. In the micronucleus(MN) induction assays, murine(CHO-K1, V79, BALB/c 3T3) cell lines and human(MCF-7, A549) ones were used. As a result, the CSC exhibited cytotoxicity with a concentration-dependent response in all cell lines. EC$_{50}$ of CSC in CHO-K1, V79, BALB/c 3T3, MCF-7 and A549 were 140, 125, 100, 116 and 109 $\mu\textrm{g}$/mL, respectively. On the other hand, the spontaneous micronucleated cell(MNC) frequency was stable and reproducible in every cell lines tested in this study. The dose-response of various cell lines to the induction of MN by CSC was estimated using linear regression analysis. CSC(0～100 $\mu\textrm{g}$/mL) caused a dose-dependent MN induction in CHO-K1, V79, BALB/c 3T3 and MCF-7 cell lines. Putting together all the data obtained and linear regression analysis of the data, we concluded that V79 cells are more susceptible to the accurate assessment of CSC-induced MN than the others.s.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.3
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• pp.169-175
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• 2022

Bovine mammary epithelial (MAC-T) cells are commonly used to study mammary gland development and mastitis. Lipopolysaccharide is a major bacterial cell membrane component that can induce inflammation. Autophagy is an important regulatory mechanism participating in the elimination of invading pathogens. In this study, we evaluated the mechanism underlying bacterial mastitis and mammary cell death following lipopolysaccharide treatment. After 24 h of 50 ㎍/mL lipopolysaccharide treatment, a significant decrease in the proliferation rate of MAC-T cells was observed. However, no changes were observed upon treatment of MAC-T cells with 10 ㎍/mL of lipopolysaccharide for up to 48 h. Thus, upon lipopolysaccharide treatment, MAC-T cells exhibit dose-dependent effects of growth inhibition at 10 ㎍/mL and death at 50 ㎍/mL. Treatment of MAC-T cells with 50 ㎍/mL lipopolysaccharide also induced the expression of autophagy-related genes ATG3, ATG5, ATG10, ATG12, MAP1LC3B, GABARAP-L2, and BECN1. The autophagy-related LC3A/B protein was also expressed in a dose-dependent manner upon lipopolysaccharide treatment. Based on these results, we suggest that a high dose of bacterial infection induces mammary epithelial cell death related to autophagy signals.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.231-237
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• 2010

Objective: To evaluate whether T helper 1 (Th1) immune response is predominant in women with reproductive failures (recurrent spontaneous abortion and recurrent implantation failure) and the activation of T cell is related to Th1 propensity. Methods: Women with a history of recurrent implantation failure or recurrent spontaneous abortion comprise the study group (n=37). Controls are normal fertile women without a history of infertility or pregnancy losses (n=11). Th1/Th2 ratios of interferon (INF)-$\gamma$/interleukin (IL)-10 and tumor necrosis factor (TNF)-$\alpha$/IL-10 expression on $CD3^+/4^+$ cells, CD154, and CD69 expression on T cells are measured by flow cytometric analysis. Results: The ratios of TNF-$\alpha$ to IL-10 expressing on $CD3^+/4^+$ cells (Th1/Th2 cell ratios) are significantly higher in study group ($42.1{\pm}2.3$) as compared with that of controls ($28.7{\pm}2.7$) (p=0.002). The overall trend of CD154 and CD69 expression on T cells are elevated in study group than those of controls. The proportion (%) of $CD3^+/4^+/154^+$ cells ($1.7{\pm}0.5$ vs. $0.3{\pm}0.2$, p=0.038) and the % of $CD3^+/8^+/154^+$ cells ($0.6{\pm}0.2$ vs. $0.1{\pm}0.0$, p=0.024) are significantly higher in study group. The % of $CD3^+/69^+$ cells ($5.6{\pm}1.9$ vs. $1.3{\pm}5.4$, p=0.046) and % of $CD3^+/8^+/69^+$ cells ($4.8{\pm}1.3$ vs. $1.8{\pm}0.2$, p=0.035) among $CD3^+/8^+$ cells are significantly increased in study group. Conclusion: Women with reproductive failures have Th1 propensity with increased T cell activation. These finding means that activated T cell has a harmful effect on early pregnancy and implantation by induction of Th1 immunity.

• Biomolecules & Therapeutics
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• v.19 no.2
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• pp.174-180
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• 2011

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance to paclitaxel in v-H-ras transformed NIH 3T3 fibroblasts (Ras-NIH 3T3). We established a multidrug-resistant cell line (Ras-NIH 3T3/Mdr) from Ras-NIH 3T3 cells by stepwise increases in paclitaxel. Drug sensitivity assays indicated that the $IC_{50}$ value for drug-resistant Ras-NIH 3T3/Mdr cells was more than 1 ${\mu}M$ paclitaxel, 10- or more-fold higher than for the parental Ras-NIH 3T3 cells. Western blot and RT-PCR analysis showed that the drug efflux pump a P-glycoprotein were highly expressed in Ras-NIH 3T3/Mdr cells, while not being detectable in Ras-NIH 3T3 cells. Additionally, verapamil, which appears to inhibit drug efflux by acting as a substrate for P-glycoprotein, completely reversed resistance to paclitaxel in Ras-NIH 3T3/Mdr cell line, indicating that resistance to paclitaxel is associated with overexpression of the multidrug resistance gene. Interestingly, Ras-NIH 3T3/Mdr cells have higher basal Raf-1 activity compared to Ras-NIH 3T3 cells. Unexpectedly, however, the colocalization of Raf-1 and its negative regulator Spry2 was less observed in cytoplasm of Ras-NIH 3T3/Mdr cells due to translocation of Spry2 around the nucleus in the perinuclear zone, implying that Raf-1 may be released from negative feedback inhibition by interacting with Spry2. We also showed that shRNA-mediated knockdown of Raf-1 caused a moderate increase in cell susceptibility to paclitaxel. Thus, the results presented here suggest that a Raf-1-dependent pathway plays an important role in the development of acquired drug-resistance.

• Molecules and Cells
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• v.28 no.2
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• pp.125-130
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• 2009

Tacrolimus is a widely used T cell targeted immunosuppressive drug, known as a calcineurin inhibitor. However, the exact pharmacological effects of tacrolimus on $CD4^+$ T cells have yet to be elucidated. This study investigated the effects of tacrolimus on $CD4^+$ T cell subsets. Mouse or human $CD4^+$ T cells were cultured with immobilized anti-CD3/CD28 antibodies in the presence of tacrolimus. The cell division of $CD4^+$ T cells was analyzed using a flow cytometer according to the expression of Foxp3. The gene expression patterns of tacrolimus-exposed T cells were examined by quantitative PCR. In the case of conventional $CD4^+$ T cells (Tconv cells), tacrolimus inhibited T cell receptor stimulation-induced cell division. In contrast, the cell division of regulatory $CD4^+$ T cells (Treg cells) was even promoted in the presence of tacrolimus, especially in humans. Tacrolimus did not promote conversion of Tconv to Treg cells in mice. Furthermore, tacrolimus modified the expression levels of Foxp3-regulated T cell receptor signal related-genes, PTPN22 and Itk, in human Treg cells. Immunosuppressive effect of tacrolimus may be attributed to the relatively enhanced proliferation of Treg cells in association with altered gene expression levels of TCR signaling molecules.

• Archives of Pharmacal Research
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• v.25 no.4
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• pp.463-468
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• 2002

We have previously reported on the identification of the endogenous transmethylation inhibitor oligosaccharide-linked acyl carrier protein (O-ACP), In this study, the role of the transmethylation reaction on cell cycle progression was evaluated using various transmethylase inhibitors, including O-ACP. O-ACP significantly inhibited the growth of various cancer cell lines, including NIH3T3, ras-transformed NIH3T3, MDA-MB-231, HT-1376, and AGS. In addition, exposure of ras-transformed NIH3T3 to O-ACP caused cell cycle arrest at the $G_0/G_1$ phase, which led to a decrease in cells at the S phase, as determined by flow cytometry. In contrast, transmethylase inhibitors did not affect the expression of $p21^{WAF1/Cip1}$, a well known inhibitor of cyclin dependent kinase, indicating that the cell cycle arrest by transmethylase inhibitors might be mediated by a $p21^{WAF1/Cip1}$-independent mechanism. Therefore, O-ACP, a novel transmethylase inhibitor, could be a useful tool for elucidating the novel role of methylation in cell proliferation and cell cycle progression.