• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.53-61
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• 1999

The present study was designed to investigate the effects of gonadotrophins on in vitro growth and maturation in mouse preantral follicles. Ovaries were removed from 12-day-old ICR mice. Follicles were dissociated enzymetically in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I. The follicles were cultured on Transwell-COL membrane inserts in six well cluster dishes for 10 days. The culture medium was $\alpha$MEM medium supplemented with 5% fetal bovine serum and FSH or HMG. After 10 days of growth in vitro, follicles were allowed to mature for 18~20 hr in medium supplemented with 1.5 IU/$m\ell$ hCG. The oocytes were then denuded of their cumulus cells and assessed maturation status. Concentrations of oestradiol and progesterone were measured with a radioimmunoassay. Oocyte diameter was determined with an ocular micrometer. The survival and Metaphase II rates of oocytes were significantly higher in FSH treatment groups than in control group (P＜0.001), but there were no differences among the groups of treated FSH concentration. The survival and Metaphase II rates of oocytes in HMG treatment group (60.9 and 40.6%) were higher than in FSH treatment group (76.6 and 48.2%) and control group (49.2 and 7.1%). The survival and Metaphase II rates of oocytes on both FSH and LH treatment groups were no differences among the ratios of FSH and LH. Diameter of oocyte was no differences among the treatment groups, but smaller than compared to in vivo grown oocyte. Through the entire culture period, secretions of oestradiol and progesterone were significantly less in control group than in HMG and FSH treatment groups. These results suggest that gonadotrophins playa key role in in vitro culture of mouse preantral follicles. Especially, addition of FSH and LH should be more effective than FSH alone.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.4
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• pp.329-338
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• 2020

Rhinorrhea in allergic rhinitis (AR) is characterized by the secretion of electrolytes in the nasal discharge. The secretion of Cl^- and HCO₃^- is mainly regulated by cystic fibrosis transmembrane conductance regulator (CFTR) or via the calcium-activated Cl^- channel anoctamin-1 (ANO1) in nasal gland serous cells. Interleukin-4 (IL-4), which is crucial in the development of allergic inflammation, increases the expression and activity of ANO1 by stimulating histamine receptors. In this study, we investigated ANO1 as a potential therapeutic target for rhinorrhea in AR using an ANO1 inhibitor derived from a natural herb. Ethanolic extracts (30%) of Spirodela polyrhiza (SP_EtOH) and its five major flavonoids constituents were prepared. To elucidate whether the activity of human ANO1 (hANO1) was modulated by SP_EtOH and its chemical constituents, a patch clamp experiment was performed in hANO1-HEK293T cells. Luteolin, one of the major chemical constituents in SP_EtOH, significantly inhibited hANO1 activity in hANO1-HEK293T cells. Further, SP_EtOH and luteolin specifically inhibited the calcium-activated chloride current, but not CFTR current in human airway epithelial Calu-3 cells. Calu-3 cells were cultured to confluency on transwell inserts in the presence of IL-4 to measure the electrolyte transport by Ussing chamber. Luteolin also significantly inhibited the ATP-induced increase in electrolyte transport, which was increased in IL-4 sensitized Calu-3 cells. Our findings indicate that SP_EtOH and luteolin may be suitable candidates for the prevention and treatment of allergic rhinitis. SP_EtOH^- and luteolin-mediated ANO1 regulation provides a basis for the development of novel approaches for the treatment of allergic rhinitis-induced rhinorrhea.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Journal of Gastric Cancer
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• v.17 no.4
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• pp.295-305
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• 2017

Purpose: We previously found that the histone methyltransferase suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3 (SMYD3) is a potential independent predictive factor or prognostic factor for overall survival in gastric cancer patients, but its roles seem to differ from those in other cancers. Therefore, in this study, the detailed functions of SMYD3 in cell proliferation and migration in gastric cancer were examined. Materials and Methods: SMYD3 was overexpressed or suppressed by transfection with an expression plasmid or siRNA, and a wound healing migration assay and Transwell assay were performed to detect the migration and invasion ability of gastric cancer cells. Additionally, an MTT assay and clonogenic assay were performed to evaluate cell proliferation, and a cell cycle analysis was performed by propidium iodide staining. Furthermore, the expression of genes implicated in the ataxia telangiectasia mutated (ATM) pathway and proteins involved in cell cycle regulation were detected by polymerase chain reaction and western blot analyses. Results: Compared with control cells, gastric cancer cells transfected with si-SMYD3 showed lower migration and invasion abilities (P<0.05), and the absence of SMYD3 halted cells in G2/M phase and activated the ATM pathway. Furthermore, the opposite patterns were observed when SMYD3 was elevated in normal gastric cells. Conclusions: To the best of our knowledge, this study provides the first evidence that the absence of SMYD3 could inhibit the migration, invasion, and proliferation of gastric cancer cells and halt cells in G2/M phase via the ATM-CHK2/p53-Cdc25C pathway. These findings indicated that SMYD3 plays crucial roles in the proliferation, migration, and invasion of gastric cancer cells and may be a useful therapeutic target in human gastric carcinomas.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.135-141
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• 2011

Objective: Ovarian angiogenesis plays an important role in folliculogenesis. However, little is known about the expression of angiogenic factors during follicular development according to female age. Stromal cell derived factor-$1{\alpha}$ (SDF-$1{\alpha}$) plays a role in granulosa cell survival and embryo quality as an angiogenic chemokine. Leptin is also involved in folliculogenesis and angiogenesis. This study examined expression of SDF-$1{\alpha}$ and leptin, and their effects on the expression of angiogenic factors in the ovary during follicular development according to female age. Methods: Ovaries were collected from C57BL mice of two age groups (6-9 weeks and 24-26 weeks) at 6, 12, 24, and 48 hours after 5 IU pregnant mare's serum gonadotropin (PMSG) injection. The expression of ovarian SDF-$1{\alpha}$ and leptin mRNA was evaluated by RT-PCR. In the organ culture experiment, the ovaries were cultured in transwell permeable supports with Waymouth's medium treated with various doses of SDF-$1{\alpha}$(50-200 ng/mL) or leptin (0.01-1 ${\mu}g$/mL) for 7 days. Then, mRNA expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and visfatin were examined in the cultured ovaries. Results: Expression of SDF-$1{\alpha}$ and leptin in the ovary was significantly lower in the aged mouse group compared to the young mouse group ($p$ <0.05). Expression of these two factors increased with follicular development after PMSG administration. SDF-$1{\alpha}$ treatment stimulated visfatin expression in a dose-dependent manner, while leptin treatment significantly increased eNOS expression. Conclusion: These results suggest that decrease of ovarian SDF-$1{\alpha}$ and leptin expression may be associated with aging-related reduction of ovarian function. SDF-$1{\alpha}$ and leptin may play a role in follicular development by regulating the expression of angiogenic factors in mouse ovaries.

• Molecules and Cells
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• v.41 no.5
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• pp.423-435
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• 2018

This investigation was aimed at working out the combined role of lncRNA H19, miR-29b and Wnt signaling in the development of colorectal cancer (CRC). In the aggregate, 185 CRC tissues and corresponding para-carcinoma tissues were gathered. The human CRC cell lines (i.e. HT29, HCT116, SW480 and SW620) and normal colorectal mucosa cell line (NCM460) were also purchased. Si-H19, si-NC, miR-29b-3p mimics, miR-29b-3p inhibitor, si-PGRN and negative control (NC) were, respectively, transfected into the CRC cells. Luciferase reporter plasmids were prepared to evaluate the transduction activity of $Wnt/{\beta}-catenin$ signaling pathway, and dual-luciferase reporter gene assay was arranged to confirm the targeted relationship between H19 and miR-29b-3p, as well as between miR-29b-3p and PGRN. Finally, the proliferative and invasive capacities of CRC cells were appraised through transwell, MTT and scratch assays. As a result, overexpressed H19 and down-expressed miR-29b-3p displayed close associations with the CRC patients' poor prognosis (P < 0.05). Besides, transfection with si-H19, miR-29b-3p mimic or si-PGRN were correlated with elevated E-cadherin expression, decreased snail and vimentin expressions, as well as less-motivated cell proliferation and cell metastasis (P < 0.05). Moreover, H19 was verified to directly target miR-29b-3p based on the luciferase reporter gene assay (P < 0.05), and miR-29b-3p also bound to PGRN in a direct manner (P < 0.05). Finally, addition of LiCl ($Wnt/{\beta}-catenin$ pathway activator) or XAV93920 ($Wnt/{\beta}-catenin$ pathway inhibitor) would cause remarkably altered E-cadherin, c-Myc, vimentin and snail expressions, as well as significantly changed transcriptional activity of ${\beta}-catenin/Tcf$ reporter plasmid (P < 0.05). In conclusion, the lncRNA H19/miR-29b-3p/PGRN/Wnt axis counted a great deal for seeking appropriate diagnostic biomarkers and treatment targets for CRC.

• Journal of the Korean Society of Food Science and Nutrition
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• v.44 no.1
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• pp.44-48
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• 2015

To investigate the anti-cancer effect of shiitake mushrooms (Lentinus edodes) cultured in aloe-supplement, we treated extract of shiitake mushroom cultured in aloe-supplement (ESA) to MDA-MB-231 human breast cancer cells. ESA-treated MDA-MB-231 cells showed decreased growth rate in XTT assay. In addition migration/invasion was noticeably inhibited by ESA in TNF-${\alpha}$-treated MDA-MB-231 cells. Western blot analysis showed that the molecular mechanism of cell migration/invasion was mediated by reduced intercellular adhesion molecule-1 expression via p-ERK signal transduction pathways. We found ESA had inhibition activity against cellular growth and migration/invasion. Taken together, ESA has putative anti-cancer activity against human breast cancer.

• Archives of Pharmacal Research
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• v.24 no.6
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• pp.584-589
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• 2001

Isopropryl 2-(1-3-dithiethane-2-ylidene)-2 [N-(4-methyl-thiazole-2-yl) carbamoyl] acetate (YH439) is currently under phase ll clinical trials by the Yuhan Research Center for use as a hepatoprotective agent. Unfortunately, the oral bioavailbility of YH439, which is sparingly soluble in water (i.e., $0.3{\;}\mu\textrm{g}/ml{\;}or{\;}0.91{$\mu}M$ at room temperature), reportedly, is negligibleregardless of the dose administered to rats in the 10-300 mg/kg range. The bioavailability of the compound increased up to 24%, when administered in the form of a micellar solution ($700{\;}\mu\textrm{g}/ml$or 2.1 mM for YH439) at a dose of 10 mg/kg, suggesting that its limited solubility is associated with its negligible bioavailability. In order to obtain additional informmation concerning the bioavailability of YH439, the mechanism(s) involved in gastrointestinal (Gl) absorption were investigated in the present study. For this purpose, the transport of YH430 across a Caco-2 cell monolayer was measured in a $Transwell^{\circledR}$. A permeability of $4.07{\times}10^{-5}{\;}cm/s$ was obtained for the absorptive (i.e., apical to basolateral direction) transport of $0.42{\mu}M$ YH439, implicating that the in vivo Cl absorption is nearly complete. The absorptive transport exhibited a slight concentration-dependency with an intrinsic clearance ($CL_{i}$) of $0.38{\mu}L/{\textrm{cm}^2}/sec$, which accounted for 28.1% of the total intrinsic clearance (i.e., $CL_i$ plus the intrinsic clearance for the linear component) of the transport. Thus, saturation of the absorption process appears to be a minor factor in limiting the bioavailability of the compound. The apparent permeability of YH439 from the basolateral to the apical direction (i.e., efflux, $6.67{\times}10^{-5}{\;}cm/s$) was comparable to that for absorptive transport, but, interestingly, a more distinct concentration-dependency was observed for this transport. However, the efflux does not appear to influence the bioavailability of the compound, as evidenced by the sufficiently high permeability in the absorption direction. Rather, a reportedly extensive first-pass hepatic metabolism appears to be a principal factor in limiting the bioavailability. In this respect, reducing the first-pass metabolism by some means would lead to a higher bioavailability of the compound. Thus, elevation of the absorption rate of YH439 becomes a necessity. From a practical point of view, increasing the concentration of YH439 in the Cl fluid appears to be a feasible way to increase the absorption rate, because the compound is primarily absorbed via a linear mechanism. In summary, the solubilization of YH439, as previously demonstrated for a micellar solution of the compound, appears to be a practical way to increase the oral bioavailability of YH439.

• Molecules and Cells
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• v.44 no.7
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• pp.468-480
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• 2021

Ubiquitin D (UBD) is highly upregulated in many cancers, and plays a pivotal role in the pathophysiological processes of cancers. However, its roles and underlying mechanisms in oral squamous cell carcinoma (OSCC) are still unclear. In the present study, we investigated the role of UBD in patients with OSCC. Quantitative real-time polymerase chain reaction and Western blot were used to measure the expression of UBD in OSCC tissues. Immunohistochemistry assay was used to detect the differential expressions of UBD in 244 OSCC patients and 32 cases of normal oral mucosae. In addition, CCK-8, colony formation, wound healing and Transwell assays were performed to evaluate the effect of UBD on the cell proliferation, migration, and invasion in OSCC. Furthermore, a xenograft tumor model was established to verify the role of UBD on tumor formation in vivo. We found that UBD was upregulated in human OSCC tissues and cell lines and was associated with clinical and pathological features of patients. Moreover, the overexpression of UBD promoted the proliferation, migration and invasion of OSCC cells; however, the knockdown of UBD exerted the opposite effects. In this study, our results also suggested that UBD promoted OSCC progression through NF-κB signaling. Our findings indicated that UBD played a critical role in OSCC and may serve as a prognostic biomarker and potential therapeutic target for OSCC treatment.

• Journal of Ginseng Research
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• v.46 no.6
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• pp.738-749
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• 2022

Background: Ginseng possesses antitumor effects, and ginsenosides are considered to be one of its main active chemical components. Ginsenosides can further be hydrolyzed to generate secondary saponins, and 20(R)-panaxotriol is an important sapogenin of ginsenosides. We aimed to synthesize a new ginsengenin derivative from 20(R)-panaxotriol and investigate its antitumor activity in vivo and in vitro. Methods: Here, 20(R)-panaxotriol was selected as a precursor and was modified into its derivatives. The new products were characterized by ¹H-NMR, ¹³C-NMR and HR-MS and evaluated by molecular docking, MTT, luciferase reporter assay, western blotting, immunofluorescent staining, colony formation assay, EdU labeling and immunofluorescence, apoptosis assay, cells migration assay, transwell assay and in vivo antitumor activity assay. Results: The derivative with the best antitumor activity was identified as 6,12-dihydroxy-4,4,8,10,14-pentamethyl-17-(2,6,6-trimethyltetrahydro-2H-pyran-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl(tert-butoxycarbonyl)glycinate (A11). The focus of this research was on the antitumor activity of the derivatives. The efficacy of the derivative A11 (IC₅₀ < 0.3 µM) was more than 100 times higher than that of 20(R)- panaxotriol (IC₅₀ > 30 µM). In addition, A11 inhibited the protein expression and nuclear accumulation of the hypoxia-inducible factor HIF-1α in HeLa cells under hypoxic conditions in a dose-dependent manner. Moreover, A11 dose-dependently inhibited the proliferation, migration, and invasion of HeLa cells, while promoting their apoptosis. Notably, the inhibition by A11 was more significant than that by 20(R)-panaxotriol (p < 0.01) in vivo. Conclusion: To our knowledge, this is the first study to report the production of derivative A11 from 20(R)-panaxotriol and its superior antitumor activity compared to its precursor. Moreover, derivative A11 can be used to further study and develop novel antitumor drugs.