• Journal of Life Science
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• v.23 no.9
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• pp.1109-1117
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• 2013

Induction of G1 Arrest by Methanol Extract of Lycopus lucidus in Human Lung Adenocarcinoma A549 Cells Lycopus lucidus, a herbaceous perennial, is used as a traditional remedy in East Asia, including China and Korea. It has been reported that L. lucidus has anti-allergic effects, inhibitory effects on cholesterol acyltransferase in high glucose-induced vascular inflammation, and anti-proliferative effects in human breast cancer cells. However, the molecular mechanisms of the anti-cancer effects of L. lucidus have not yet been fully determined. In this study, we evaluated the anti-cancer effect and the mechanism of action of L. lucidus in human lung adenocarcinoma A549 cells using methanol extracts of L. lucidus (MELL). MELL treatment showed cytotoxic activity in a dose-dependent manner and induced G1 arrest in A549 cells. The induction of G1 arrest by MELL was associated with the up-regulation of phospho-CHK2 and the down-regulation of Cdc25A phosphatase. In addition, MELL treatment induced decreased expression of G1/S transition-related proteins, including CDK2, CDK4, CDK6, cyclin D1 and cyclin E. MELL also regulated the mRNA expression of CDK2 and cyclin E. On the other hand, the expression of p53 and the cyclin-dependent kinase inhibitor p21 was not induced by MELL. Collectively, these results suggest that MELL may exert an anti-cancer effect by cell cycle arrest at G1 phase through the ATM/CHK2/Cdc25A/CDK2 pathway in A549 cells.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.42 no.4
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• pp.403-412
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• 2016

The tight junction, one of Intercellular junctions, performs a variety of biological functions by bonding adjacent cells, including the barrier function to control the movement of the electrolyte and water. Recent studies have revealed that unusual expression of tight junction-related genes have been shown to be related in cancer development and progression. Recently, there are many reports that control of tight junction proteins expression is closely related to the skin moisture. In this study, we are focusing on the regulating mechanism of tight junction-associated genes by the steviol and its derivatives. Steviol, used as a sweetner, is known to chemical compound isolated from stevia plant. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was carried out in HaCaT cells (human keratinocyte cell line) in order to determine the cytotoxicity. As a result, while steviol showing cytotoxicity from $250{\mu}M$, steviol derivatives are not cytotoxic more than $250{\mu}M$ concentration. We have observed a change in the tight junction protein via quantitative real-time PCR. Claudin 8 among tight junction proteins is only significantly reduced up to 30% in the presence of steviol. In addition, cell migration was inhibited by steviol, not by stevioside and rebaudioside. Finally, we could observe that steviol, not stevioside and rebaudioside, is able to increase the skin barrier permeability through the transepithelial electric resistance (TEER) measurements. These results suggest that the steviol and its derivatives are specifically acts on the tight junction related gene expression, but steviol derivatives are more suitable as a cosmetic material.

• The Korean Journal of Pharmacology
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• v.25 no.1
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• pp.43-51
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• 1989

Responsiveness of muscarinic and alpha adrenoceptor activation on endothelial cells was studied in isolated canine renal artery rings. Ach (10-100 nM), dose dependently, relaxes endothelial intact rings precontracted with phenylephrine ($IC_{50}$ of Ach was 34.5 nM). Selective mechanical destruction of the endothelium transformed the activity of this substance from vasodilatation to vasoconstriction. Acetylcholine induced relaxations could be selectively inhibited competitively by atropine, but could not be inhibited by cyclooxygenase inhibitor. Methylene blue, however, an inhibitor of soluble guanylate cyclase activity, inhibited Ach as well as sodium nitroprusside (SNP) induced relaxation. Relaxation produced by prostacyclin was not modified by methylene blue. On the other hand, alpha adrenoceptor agonist did not relax but contract canine renal artery rings possessing an intact intima precontracted with U-46619. Clonidine, however, selective alpha-2 adrenergic agonist, is more susceptible than phenylepherine, selective alpha-1 adrenergic agonist, to the inhibitory effect of contraction. These results suggest that in canine renal artery rings, 1) muscarinic receptor is responsible for releasing endothelium dependent relaxation factor (EDRF). 2) alpha-1 and alpha-2 adrenergic receptors are present in canine renal artery. 3) relaxation via EDRF is antagonized by methylene blue, providing further evidence that EDRF acts through a cGMP mechanism.

• The Korean Journal of Pharmacology
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• v.31 no.1 s.57
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• pp.63-74
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• 1995

It has been known that, during hypoxia, the adrenal medulla is activated to release catecholamines (CA) while hypoxia also inhibits high $K^+$ -induced CA secretion in the cultured bovine adrenal chromaffin cells. The present study was attempted to examine the effect of hypoxia on CA secretion evoked by chlinergic stimulation and membrane-depolarization from the isolated perfused rat adrenal glands and also to clarify its mechanism of action. For this purpose, using the isolated rat adrenal glands, the effects of hypoxia on CA release evoked by nicotinic ($N_1$) and muscarinic ($M_1$) receptor agonists, membrane-depolarizing agent, $Ca^{++}$-channel activator, intracellular $Ca^{++}$-releaser and ACh were determined. Experiments were carried out, perfusing Krebs solution pre-equilibrated with a gas mixture of 95% N_2$ and 5% $CO_2$. Hypoxia was maintained for $3{\sim}4$ hours through the experiments. Hypoxia gradually caused a time-dependent seduction in CA secretion evoked by DMPP ($100{\mu}M$), McN-A-343 ($100{\mu}M$), ACh (5.32 mM), Bay-K-8644 ($10{\mu}M$) and high $K^+$ (56 mM) respectively. How-ever, it did not affect CA secretion evoked by cyclopiazonic acid ($10{\mu}M$). Hypoxia itself also did fail to produce any influence on spontaneous secretory response of CA. These experimental results suggest that hypoxia depresses CA release evoked by both cholinergic stimulation and membrane-depolarization from the isolated rat adrenal medulla, and that this inhibitory activity may be due to the result of the direct inhibition of $Ca^{++}$ influx into the chromaffin cells without any effect on the calcium mobilization from the intracellular store.

• Journal of the korean academy of Pediatric Dentistry
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• v.27 no.1
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• pp.15-23
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• 2000

Streptococcus salivarius is a normal inhabitant in the human oral cavity. Streptococcus salivarius 119 in this study was isolated from the oral cavity of child and identified, and its action mechanism on the formation of denal plaque by Streptococcus matans was studied. 1. The optical density of absorbance at 550 nm was 0.327 in the culture of Streptococcus mutans in disposable cuvette, whereas being 0.119 in the combined culture of Streptococcus mutans and Streptococcus salivarius 119. 2. The mean weight of produced artificial plaque on the wires in the beaker was 84.7mg in culture of Streptococcus mutans only, whereas being reduced to 12.3mg in the combined culture of Streptococcus mutans and Streptococcus salivarius 119. 3. When Streptococcus mutans was cultured in the media containing culture supernatant of Streptococcus salivarius 119 in BHI broth, the mean weight of produced artificial plaque was 100.5mg on the wires, whereas being reduced to 20.4mg in the media containing culture supernatant of Streptococcus salivarius 119 in BHIS broth. 4. The viable cells of Streptococcus oralis and Streptococcus salivarius 119 were $4.8\times10^7\;and\;7.5\times10^8$ per ml respectively after each culture, wheras being $4.2\times10^7\;and\;5.8\times10^7$ per ml in the combined culture of Streptococcus oralis and Streptococcus salivarius 119. 5. The polymer produced by Streptococcus salivarius 119 was glucan on the thin layer chromatography. 6. The glucan produced by Streptococcus salivarius 119 was water-soluble glucan containing $1\rightarrow6$ linkages as the main linkage on the thin layer chromatography. These results suggested that isolated Streptococcus salivarius 119 inhibited the formation of artificial plaque by the production of water-soluble glucan.

• Journal of Life Science
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• v.24 no.2
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• pp.173-180
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• 2014

Estrogen can promote or inhibit cellular proliferation depending on tissue cell types and physiological condition and acts through the signal transduction pathways mediated primarily by estrogen receptors. This study examined the effects of fulvestrant (Ful), a well-known antagonist for the estrogen receptor, on the action of $17{\beta}$-estradiol (E2) with respect to the proliferation and apoptosis of Chinese hamster ovarian (CHO) cells. We used different concentrations of E2, Ful, and E2 plus Ful during different treatment durations. Treatment with 15-40 ${\mu}M$ E2 significantly inhibited proliferation in a time-dependent manner, although it had no influence in concentrations up to 1 ${\mu}M$. Interestingly, Ful at 10-40 ${\mu}M$ also inhibited cellular proliferation in both a concentration- and time-dependent manner. In addition, Ful enhanced rather than decreased the inhibitory effect on cellular proliferation by E2 in combined treatment for 10 days. Thus, Ful does not appear to have an antagonistic effect on estrogen's anti-proliferative action in CHO cells. In TUNEL assays to confirm DNA fragmentation by E2 and/or Ful, CHO cells treated with 20 ${\mu}M$ E2 showed a TUNEL-positive reaction in most DAPI-stained nuclei, and cells treated with either 40 ${\mu}M$ Ful or 40 ${\mu}M$ Ful plus 20 ${\mu}M$ E2 also exhibited a TUNEL-positive reaction but at a lower rate compared to the E2-treated cells. These results indicate that Ful does not have an antagonistic effect on estrogen's anti-proliferative action in CHO cells, suggesting that the anti-proliferative and apoptosis-related mechanism(s) through DNA fragmentation by E2 and Ful may be mediated by different signal transduction pathways.

• Journal of Applied Biological Chemistry
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• v.61 no.2
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• pp.205-211
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• 2018

Rebaudioside A is a natural sweetener isolated from Stevia rebaudiana Bertoni, one of the glycosides based on steviol. Recent studies have shown that rebaudioside A inhibits the inflammatory response by inhibiting cytokines secretion such as interleukin-$1{\alpha}/1{\beta}$ in activated RAW264.7 mouse macrophage cells by LPS. However, the inhibitory mechanism of inflammation by rebaudioside A in the presence of LPS has not been fully elucidated. Therefore, in this study, we tried to investigate the anti-inflammatory activity of rebaudioside A at the protein level when RAW264.7 cells were stimulated by LPS. The inducible nitric oxide synthase protein expression level was reduced in the group treated with $250{\mu}M$ rebaudioside A compared to the LPS-treated group. In addition, the mRNA expression level of $NF-{\kappa}B$, which is a representative nuclear transcription factor by inflammatory signal, was also decreased as compared with that of LPS-treated group. In addition, $NF-{\kappa}B$ and inhibitor-${\kappa}B$ ($I-{\kappa}B$) complexes that are known to be dissociated by $I-{\kappa}B$ phosphorylation and ubiquitination were less phosphorylated than LPS treated group in the presence rebaudioside A. Finally, we could find that rebaudioside A was involved in the $NF-{\kappa}B$ pathway through reducing extracellular signal-regulated kinase1/2 phosphorylation in a concentration-dependent manner. These results suggest that rebaudioside A might suppress inflammatory reaction through MAPK and $NF-{\kappa}B$ regulation in LPS-stimulated RAW264.7.

• Applied Microscopy
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• v.30 no.1
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• pp.45-59
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• 2000

Cis-platin is a widely used anticancer drug against certain solid tumors such as malignant ovarian tumor, malignant carcinoma of head and neck, bladder cancer and cervical cancer of uterus, and its major mechanism of action is inhibition of DNA synthesis of the tumor cell. To investigate the inhibitory effects of cis-platin on the ciliogensis of the ciliated cells in the mucosa of oviduct, the author pursued the alterations of $\alpha-tubulin$, which is the main constituent of the microtubles in cilia, after cis-platin treatment. To eliminate the possible variations due to ovarian cycle, female Spargue-Dawley rats ($150\sim200gm$ in B.W.) were pretreated with estradiol benzoate (20 mg/kg, once a day, for 4 consecutive days). Animals were administrated with cis-platin (6 mg/kg, i.p.) and sacrificed at 1day, 3days, 5days and 7days after treatment, respectively. Immunohistochemistry for $\alpha-tubulin$ using mouse anti-rat $\alpha-tubulin$ monoclonal antibody as primary antibody was done. Immunogold electronmicroscopy for intracellular distributions of $\alpha-tubulin$ was also performed with same primary antibody and Goat anti- mouse IgM which is preconjugated with gold particles of 15 nm as secondary antibody. The results obtained were as follows; 1. Strong immunoreactivity of $\alpha-tubulin$ was observed in ciliated cells of oviducts at 1, 3 and 5 days after estradiol pretreatment. 2. Weak immunoreactivity of $\alpha-tubulin$ was observed in ciliated cells of oviducts at 1 and 3 days after cis-platin treatment but it was recovered to strong immunoreactivity in 5 days 3. In immunogold electronmicroscopy, density of gold particles for $\alpha-tubulin$ reactions was decreased in apical cytoplasm, but few changes were observed in basal body or cilia at 1 and 3 days after cis-platin treatment. From these above results, it is indicated that synthesis of $\alpha-tubulin$ in ciliated cells of rat oviduct is inhibited by cis-platin treatment.

• Clinical and Experimental Pediatrics
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• v.48 no.8
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• pp.886-893
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• 2005

Purpose : Kawasaki disease is the most common cause of systemic vasculitis in children less than 5 years of age. Recent immunohistochemistry findings suggest that many vascular growth factors play a role in the formation of the coronary artery lesions. Active remodeling of the coronary artery lesions in Kawasaki disease continues in the form of intimal proliferation and neoangiogenesis for several years after the onset of the disease. Intravenous immunoglobulin(IVIG) and corticosteroid have been used in the treatment of Kawasaki disease but the exact mechanism is not clear. We have investigated that IVIG and corticosteroid inhibited vascular endothelial growth factor(VEGF)-induced tube formation of endothelial cells in vitro on Matrigel assay. Methods : Human umbilical vein endothelial cells(HUVECs) were cultured and seeded on Matrigel coated 24 well plates in medium with or without the following agents : VEGF, VEGF plus IVIG, VEGF plus VEGF antibody, VEGF plus methylprednisolone, VEGF, IVIG plus methylprednisolone for 18 hours. The total length of tube structures in each photograph was quantified. Results : IVIG significantly inhibited the proliferation of HUVECs. The inhibitory effect of IVIG was also reversible. In the meantime, VEGF induced the differentiation of HUVECs into capillary like structures on Matrigel, which was inhibited by VEGF antibody in a dose-dependent manner. Interestingly, IVIG and methylprednisolone inhibited VEGF-induced tube formation of HUVECs. IVIG was more effective in inhibition than methylprednisolone alone. Conclusion : We revealed that VEGF induced the differentiation of HUVECs and this effect was inhibited by IVIG and methylprednisolone.

• The korean journal of orthodontics
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• v.29 no.1 s.72
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• pp.95-106
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• 1999

This study was performed to examine the effect of bisphosphonate, an inhibitor of bone resorption, on the formation of osteoclast and bone resorption during experimental tooth movement. Whether bisphosphonate has a cytotoxicity in high dose was also examined. Eighty-seven male Sprague-Dawley rats, weighing 260-350g, were classified into normal (no appliance + $0.9\%$ NaCl), control (appliance + $0.9\%$ NaCl) and four bisphosphonate-treated (appliance + 0.8, 4, 20 or 100mg/kg) groups. The maxillary left first molar was moved mesially with the tipping movement using 50-70g of force. Bisphosphonate(etidronate disodium) was injected intraperitoneally with a dose of 0.8, 4, 20, or 100 mg/kg simultaneously with the application or the orthodontic force. They were killed at day 1, 3, or 7 after the application or the orthodontic force. The activities of serum acid phosphatase and lactate dehydrogenase (LDH) were assayed, and osteoclasts and the degree of bone resorption were examined histologically. The results obtained were as follows: 1. Acid phosphatase activities were significantly higher in the appliance groups, both control and bisphosphonate-treated (4, 20, and 100 mg/kg) groups, at days 1 and 3 than these in normal. At day 1, bisphosphonate-treated(4, 20mg/kg) groups showed even higher acid phosphatase than control. However, at day 7, no significant difference was noted between the control and bisphosphonate-treated groups. 2. LDH activities in the 4, 20mg/kg bisphosphonate-treated groups were increased during the experimental Periods examined, but there were no significant differences in the 0.8, 100mg/kg bisphosphonate-treated groups. 3. There was no bone resolution at day 1, but severe bone resorption was observed at days 3 and 7 in the control. Bone resorption was reduced by bisphosphonate-treatment at day 3. Bone resolution observed at day 7 was similar between the control and bisphosphonate-treated groups. 4. Few osteoclasts were observed at the alveolar bone in the control and bisphosphonate-treated groups at day 1. At day 3, numerous osteoclasts were shown in the control, the degree of which was reduced in bisphosphonate-treated groups. These results suggest that the inhibition of the osteoclast formation was not the mechanism of bone resorption by the bisphosphonate-treatment during experimenal tooth movement. There was no distinct cytotoxicity with a high dose of bisphosphonate. And the drug should be administrated repeatedly to maintain the inhibitory effect of bone resolution.