• BMB Reports
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• v.48 no.3
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• pp.127-128
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• 2015

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Hepatic TM4SF5 promotes EMT for malignant growth and migration. Hepatocellular carcinoma (HCC) biomarkers remain unexplored for metastatic potential throughout metastasis. Here, novel TM4SF5/CD44 interaction-mediated self-renewal and circulating tumor cell (CTC) capacities were mechanistically explored. TM4SF5-dependent sphere growth was correlated with $CD133^+$, $CD24^-$, ALDH activity, and a physical association between CD44 and TM4SF5. The TM4SF5/CD44 interaction activated c-Src/STAT3/ Twist1/ B mi1 signaling for spheroid formation, while disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts of less than 5,000 cells/injection, TM4SF5-positive tumors exhibited locally-increased CD44 expression, suggesting tumor cell differentiation. TM4SF5-positive cells were identified circulating in blood 4 to 6 weeks after orthotopic liver-injection. Anti-TM4SF reagents blocked their metastasis to distal intestinal organs. Altogether, our results provide evidence that TM4SF5 promotes self-renewal and CTC properties supported by $CD133^+/TM4SF5^+/CD44^+^{(TM4SF5-bound)}/ALDH^+/CD24^-$ markers during HCC metastasis.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.5
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• pp.629-636
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• 2019

Objective: The study was designed to determine the cytotoxic effect of gallic acid (GA), obtained by the hydrolysis of tannins, on mice TM4 Sertoli cells apoptosis. Methods: In the present study, non-tumorigenic mice TM4 Sertoli cells were treated with different concentrations of GA for 24 h. After treatment, cell viability was evaluated using WST-1, mitochondrial dysfunction, cells apoptosis and necrosis was detected using JC-1, Hoechst 33342 and propidium iodide staining. The expression levels of Cyclin B1, proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (BAX), and Caspase-3 were also detected by quantitative real-time polymerase chain reaction and Western-blotting. Results: The results showed that 20 to $400{\mu}M$ GA inhibited viability of TM4 Sertoli cells in a dose-dependent manner. Treatment with $400{\mu}M$ GA significantly inhibited PCNA and Cyclin B1 expression, however up-regulated BAX and Caspase-3 expression, caused mitochondrial membrane depolarization, activated Caspase-3, and induced DNA damage, thus, markedly increased the numbers of dead cells. Conclusion: Our findings showed that GA could disrupt mitochondrial function and caused TM4 cells to undergo apoptosis and necrosis.

• The Korea Journal of Herbology
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• v.36 no.6
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• pp.17-25
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• 2021

Objectives : Baicalein (3,3', 4', 5, 6-pentahydroxyflavone), a type of flavonoid, is a well-known antioxidant and anti-inflammatory ingredient found in Scutellaria baicalensis root. The aim of this study is to investigate the effect of baicalein on nitric oxide (NO) production in TM3 mouse Leydig cells stimulated by indomethacin (IN). Methods : TM3 cells were treated with IN (0.5 μM) and baicalein at concentrations of 12.5, 25, 50, and 100 μM for 24 hr, 40 hr, 42 hr, 44 hr, and 64 hr. After treatments, cell viabilities were measured with the modified MTT assay. The production of nitric oxide in cells was measured by Griess reagent assay. Results : Baicalein showed no cytotoxicity on IN-stimulated TM3. NO production in IN-stimulated TM3 treated for 24 hr with baicalein at concentrations of 12.5, 25, 50, and 100 μM was 95.8%, 94.86%, 89.97%, and 81.52% of the control group treated with IN only, respectively; NO production for 40 hr was 97.34%, 97.34%, 95.15%, and 87.42%, respectively; NO production for 42 hr was 89.12%, 90.14%, 89.74%, and 90.26%, respectively; NO production for 44 hr was 83.83%, 84.94%, 85.65%, and 86.85%, respectively; NO production for 64 hr was 94.12%, 95.38%, 94.21%, and 94.12%, respectively. Specifically, baicalein at concentrations of 12.5, 25, and 50 have been shown to most efficiently inhibit NO productions in 48 hr of treatment. Conclusions : Baicalein might have anti-toxicant effect on Leydig cells related with its inhibition of NO production in Leydig cells stimulated with IN.

• Development and Reproduction
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• v.25 no.1
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• pp.15-24
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• 2021

Benzo[a]pyrene (B[a]P) is a potent carcinogen and is classified as an endocrine-disrupting chemical. In mammalian testes, Sertoli cells support spermatogenesis. Therefore, if these cells are negatively affected by exposure to xenotoxic chemicals, spermatogenesis can be seriously disrupted. In this context, we evaluated whether mouse testicular TM4 Sertoli cells are susceptible to the induction of cytotoxicity-mediated cell death after exposure to B[a] P in vitro. In the present study, while B[a]P and B[a]P-7,8-diol were not able to induce cell death, exposure to BPDE resulted in cell death. BPDE-induced cell death is accompanied by the activation of caspase-3 and caspase-7. Depolarization of the mitochondrial membrane and cytochrome c release from mitochondria were observed in benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated cells. These results indicate that TM4 cells are susceptible to apoptosis in a caspase-dependent manner. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that aryl hydrocarbon receptor (AhR) expression was almost undetectable in TM4 cells and that its expression was not altered after B[a]P treatment. This indicates that TM4 cells are nearly AhR-deficient. In TM4 cells, the CYP1A1 protein and its activity were not present. From these results, it is clear that AhR may be a prerequisite for CYP1A1 expression in TM4 cells. Therefore, TM4 cells can be referred to as CYP1A1-deficient cells. Thus, TM4 Sertoli cells are believed to have a rigid and protective cellular machinery against genotoxic agents. In conclusion, it is suggested that tolerance to B[a]P cytotoxicity is associated with insufficient AhR and CYP1A1 expression in testicular Sertoli cells.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.4
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• pp.291-299
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• 2023

Background: Leydig cells, crucial for testosterone production, express ion channels like ANO1 that influence hormone secretion. This study investigates the expression and role of the Tandem of P domains in a weak inward rectifying K⁺ channel-related Acid-Sensitive K⁺-1 (TASK-1) channel in these cells, exploring its impact on testicular function and steroidogenesis. Methods: TASK-1 expression in Leydig cells was confirmed using immunostaining, while RT-PCR and Western Blot (WB) validated its expression in the TM3 Leydig cell line. The effect of a TASK-1 channel blocker on cell viability was assessed through live/dead staining and MTT assays. Additionally, the blocker's effect on testosterone secretion was evaluated by measuring testosterone levels. Results: Immunohistochemical analysis revealed a predominant presence of TASK-1, along with c-Kit and ANO-1, in Leydig cells adjacent to seminiferous tubules and also in Sertoli and spermatogenic cells. Expression levels of TASK-1 mRNA and protein were significantly higher in TM3 Leydig cells compared to TM4 Sertoli cells. In addition, blocking TASK-1 in TM3 cells with ML365 induced cell death but did not affect LH-induced testosterone secretion. Conclusions: These findings suggest that TASK-1 in Leydig cells is crucial for their viability and proliferation, highlighting its potential importance in testicular physiology.

• Journal of Physiology & Pathology in Korean Medicine
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• v.29 no.5
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• pp.403-408
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• 2015

Traditionally, 5 kinds of fruits with "ja(子)" in their name, including Rubus coreanus, Schisandra chinensis, Lycinum chineuse, Torilidis fructus, and Cuscuta seed, collectively called Oja(五子), are long known to enhance stamina. In the present study, we replaced tosaja with gyeolmyeongja(Cassiae semen ) and examined the effects of extracts from these fruits on andropause. This study investigated the antioxidant effect and testosterone synthesis of Oja water extract on Leydig TM3 cells. To investigate whether hydrogen peroxide induces oxidative stress in Leydig cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, nitric oxide assay, and testosterone assay were performed on mouse Leydig TM3 cells. The results were obtained as follows: Leydig TM3 cells viability was assessed by a modified MTT assay, and the protection effect of Oja water extract against hydrogen peroxide-induced cell oxidative stress were examined by mitochondrial activity. Oja water extract could efficiently protect cytotoxicity induced by H₂O₂. Oja water extract promoted testosterone synthesis. These results suggest that Oja water extract has protective roles and promotes steroidogenesis in Leydig cells through its anti-oxidant action.

• Journal of Applied Biological Chemistry
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• v.59 no.4
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• pp.305-311
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• 2016

This study investigated the effects of a mixture of fenugreek seeds and Lespedeza cuneata extracts on testosterone synthesis in TM3 cells that were oxidatively stressed with $H_2O_2$. In order to oxidatively stress TM3 cells, the cells were treated with $50{\mu}M$ hydrogen peroxide for 4 hr in serum-free media. Yagwanmun-horopa mixture (YHM) showed neither cytotoxicity nor increment of cell proliferation in the oxidatively stressed TM3 cells in any concentration. When the cells were treated with hydrogen peroxide, testosterone levels decreased, but the testosterone level was returned to that of the control level in the presence of YHM. In order to find out the reasons for the increase of testosterone, the expression of the genes involved in the synthesis or disintegration of testosterone. On the other hand, the levels of $3{\beta}$-HSD4 and 17, 20-desmorase, which are involved in testosterone synthesis, were decreased through the use of hydrogen peroxide and were recovered through YHM treatment. Aromatase and $5{\alpha}$-reductase2, which convert testosterone to estradiol and dihydrotestosterone, respectively, were increased through the use of hydrogen peroxide, and were returned to control level through YHM treatment. These results suggest that YHM does not affect TM3 cell proliferation. However, YHM increases the expression of testosterone-synthesizing enzyme, which was decreased through oxidative stress, and decreases the expression of testosterone- converting enzyme, which was increased through oxidative stress. Therefore, it is reasonable that YHM has strong recovery activity on testosterone to normal level, even in the oxidatively stressed TM3 cells which mimics the andropause state.

• Journal of Life Science
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• v.28 no.6
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• pp.648-655
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• 2018

Enhancement mechanistic actions of testosterone (TS) productions in mouse Leydig TM3 cells by the eritadenine (EA) and/or the Agaricus blazei mycelial liquid culture extract (ABMLCE). Productions of TS in TM3 cells were investigated in normal and oxidative-stressed culture conditions. In the normal culture condition, TM3 cells grown in a Dulbecco's Modified Eagle's Medium (DMEM) were treated with EA (0~100 ppm) and ABMLCE (10 ppm) + EA (0~50 ppm) for 24 hr, and in the oxidative-stressed culture condition, the cells grown in DMEM containing $50{\mu}M$ $H_2O_2$ to induce oxidative stress for 4 h were treated with the same as those in the normal culture condition. TS content, $3{\beta}$-hydroxysteroid dehydrogenase 2 (HSD3B2) enzyme activity, $5{\alpha}$-reductase 2 ($5{\alpha}-R2$) enzyme activity, and free-radical nitric oxide (NO) content in the culture media were measured using their corresponding assay kits. EA, ABMLCE, and ABMLCE + EA significantly, p<0.05, enhanced TS productions in both cultural conditions, relative to control treatment. The activity of the HSD3B2 enzyme, which is involved in the production of precursors for TS production, was elevated by EA, ABMLCE, and ABMLCE + EA treatments in both culture conditions. The activity of the $5{\alpha}-R2$ enzyme, which converts TS to dihydroxytestosterone (DHT), was not significantly affected in either culture condition by EA, ABMLCE, or ABMLCE + EA treatments. The treatments included reduced NO content. These results indicate that EA, ABMLCE, and EA + ABMLCE treatments elevated TS in TM3 cells via the enhancements of HSD3B2 activity and the reduction of NO production, and also imply that EA and ABMLCE or EA + ABMLCE could be useful materials for the production of TS in humans.

• Proceedings of the Zoological Society Korea Conference
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• 2001.04a
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• pp.98.2-98
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• 2001

No Abstract, See Full Text

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.11b
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• pp.168-168
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• 2002

A clear association between occupational tributyltin (TBT) exposure and testicular cell line, TM3 has been observed in mouse. In the present study, we have investigated cytotoxic effects of TBT on Significant cytolethality was observed in TM3 cells in a time- and concentration-dependent manner when measured by MTT assays. (omitted)