• The Journal of Korean Medicine
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• v.29 no.3
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• pp.88-99
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• 2008

Objectives: In order to identify the anti-inflammatory and analgesic properties of Salvia miltiorrhiza (Dan-Sam), widely used in Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-${\alpha}$ and cyclooxygenase (COX)-2 as target genes. Methods: The promoter regions of each gene were generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpn I and Hind III. The final construct was transfected into human myelomonocytic leukemia cells (U-937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, the anti-inflammatory and analgesic effects of several herbal extracts regarded to have the medicinal effects of diminishing body heat and complementing Qi were tested. The chemicals PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-${\alpha}$ and COX-2, respectively. Results: Salvia miltiorrhiza (Dan-Sam) demonstrated significant decrease of TNF-${\alpha}$ and COX-2 mRNA in the in vitro assay system. In MTT assay, Salvia miltiorrhiza (Dan-Sam) did not significantly inhibit the survival and proliferation of human myelomonocytic leukemia cells (U-937). Conclusions: Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.

• Animal cells and systems
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• v.1 no.1
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• pp.81-86
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• 1997

We studied the mechanism of arachidonic acid on the secretion of a neurotransmitter in rat pheochromocytoma PC12 cells. Arachidonic acid inhibited the 70 mM $K^+$-induced secretion of norepinephrine. Arachidonic acid also inhibited the 70 mM $K^+$-induced $Ca^{2+}$ mobilization which is due to the opening of the voltage-sensitive $Ca^{2+}$ channels (VSCC). Both the half maximal inhibitory concentration ($IC_{50}$) of the norepinephrine secretion and VSCC coincided at 30 uM. The major oxidized metabolites of arachidonic acid, prostaglandins did not mimic the inhibitory effect of arachidonic acid. Nordihydroguaiaretic acid (NDGA) and indomethacin which are inhibitors of lipoxygenase and cyclooxygenase, respectively, did not block the inhibitory effect of arachidonic acid. The results suggest that arachidonic acid serves as a signal itself, not in the form of metabolites. The pretreatment of various $K^+$ channel blockers such as 4-aminopyridine, tetraethylarnmonium, glipizide, or glibenclamide also did not show any effect on the inhibitory effect of arachidonic acid. Through these results we suggest that arachidonic acid regulates VSCC directly and affects the secretion of neurotransmitters.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.53-59
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• 2016

This study was to investigate effect of progesterone ($P_4$) on prostaglandin (PG) synthases and plasminogen activators (PAs) system in bovine endometrium during estrous cycle. Endometrium tissues were collected from bovine uterus on follicular and luteal phase and were incubated with culture medium containing 0 (Control), 0.2, 2, 20 and 200 ng/ml $P_4$ for 24 h. The $PGF_{2{\alpha}}$ synthase (PGFS), $PGE_2$ synthase (PGES), cyclooxygenase-2 (COX-2), urokinase PA (uPA), and PA inhibitors 1 (PAI-1) mRNA in bovine endometrium were analyzed using reverse transcription PCR and PA activity was measured using spectrophotometry. In results, COX-2 was higher at 2 ng/ml $P_4$ group than control group in luteal phase (p<0.05), but, it did not change in follicular phase. Contrastively, PGES was significantly increased in 2 ng/ml $P_4$ group compared to control group in follicular phase, but there were no significant differ among the treatments in luteal phase. uPA was no significant difference between $P_4$ treatment groups and control group in both of different phase. PAI-1 was decreased in 20 ng/ml $P_4$ group compared to control group in follicular phase (p<0.05). PA activity was decreased in 2 ng/ml $P_4$ group compared to other groups in follicular and luteal phase (p<0.05). In conclusion, we suggest that $P_4$ may influence to translation and post-translation process of PG production and PA activation in bovine endometrium.

• Biomolecules & Therapeutics
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• v.12 no.4
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• pp.221-227
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• 2004

Nonsteroidal anti-inflammatory drugs (NSAIDs), particularly the highly selective cyclooxygenase (COX)-2 inhibitors have been shown to decrease the growth of tumor, in part, by inhibition of neovascularization. Recently, besides mature endothelial cells, endothelial progenitor cells (EPCs) have been shown to contribute neovascularization in angiogenic tissues. In this study, we addressed a question whether nimesulide, a selective COX-2 inhibitor, could affect differentiation of EPCs into adhesive endothelial cells in vitro. Total mononuclear cells were isolated from cord blood by Ficoll density gradient centrifugation, and then the cells were incubated with nimesulide or vehicle control for 7 days. The number of adherent and spindle-shaped cells decreased by nimesulide treatment in a concentration-dependent fashion at a concentration range of 5 - 200 ${\mu}M$. Moreover, the adherent cells double positive for DiI-ac-LDL uptake and lectin binding significantly decreased upon nimesulide treatment. There was no change of expression of CD31 between treatment and control groups, whereas slight reduction was detected upon treatment in expression of VE-cadherin, ICAM-1, vWF, ${\alpha}v$, and ${\alpha}5$. Nimesulide also reduced cell viability during first 3 days' culture and induced apoptosis in adherent EPCs, resulting in increased annexin-V-positive and propidium iodide-negative cells. Taken together, these results suggest that nimesulide could be applied for the inhibition of new vessel formation, in part, by inhibiting differentiation and survival of EPCs.

• Archives of Pharmacal Research
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• v.24 no.2
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• pp.126-135
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• 2001

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of $DiOC_6$/ demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (${\blacktriangle}{\Psi}_m$). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector caspase-3, Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDCA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDCA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

• Journal of Life Science
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• v.16 no.4
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• pp.691-698
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• 2006

Phospholipase D (PLD) is known to play an important role in a variety of cells. However, little is known about $CoCl_2-mediated$ PLD signaling. In this study we demonstrated for the first time that $CoCl_2$ stimulates PLD activity and increases expression of cyclooxygenase-2 (COX-2), which is known to mediate inflammatory reaction. $CoCl_2-induced$ PLD activity was assessed by measuring the formation of $[^3H]$ phosphatidylbutanol (PtdBut), the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol. To study mechanism of PLD signaling induced by $CoCl_2$, U87 human glioblastoma cells were stimulated by $CoCl_2$ and regulators of PLD activity induced by $CoCl_2$ were investigated using several inhibitors of signaling proteins. Moreover, PLD activation by $CoCl_2$ increased not only expression of COX-2 protein but also COX-2 promoter activity. In summary, these results suggest that $CoCl_2$ increases expression of COX-2 protein via PLD in human U87 glioblastoma cells.

• Journal of Life Science
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• v.32 no.3
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• pp.210-221
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• 2022

This study investigated the mechanisms underlying the anti-cancer effects of non-steroidal anti-inflammatory drugs (NSAIDs) in human cancer cells in combination with either N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor, or MHY2245, a new synthetic sirtuin 1 inhibitor. The results showed both DAPT and MHY2245 as novel chemosensitizers of human colon cancer KM12 and human hepatocellular carcinoma SNU475 cells to NSAIDs involving celecoxib and 2, 5-dimethyl celecoxib. The NSAID-induced cytotoxicity of these cells was significantly increased by DAPT and MHY2245 in a cyclooxygenase-2 independent manner. In addition, DAPT and MHY2245 reduced levels of p62, Notch1 intracellular domain, and multiple cancer stemness (CS)-related markers including Notch1, CD44, CD133, octamer-binding transcription factor 4, mutated p53 and c-Myc. However, the level of activating transcription factor 4 (ATF4) was enhanced, probably indicating the down-regulation of multiple CS-related markers by DAPT or MHY2245-mediated autophagy induction. Moreover, the NSAID-mediated reduction of p62/nuclear factor erythroid-derived 2-like 2 and CS-related marker proteins and the up-regulation of C/EBP homologous protein (CHOP)/ATF4 were accelerated by DAPT and MHY2245. As such, the combination of NSAID and either DAPT or MHY2245 resulted in higher cytotoxicity than NSAID alone by accelerating the down-regulation of multiple CS-related markers and PARP activation, indicating that both inhibitors promote NSAID-mediated autophagic cell death, possibly through the CHOP/ATF4 pathway. In conclusion, either combination strategy may be useful for the effective treatment of human cancer cells expressing CS-related markers.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.447-454
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• 2023

In the environment in which humans live, there are various antigens that invade the human body and interfere with humans leading a healthy life, so the immune system recognizes the antigen then removes them through a complex mechanism. Macrophages are widely distributed immune cells involved in the innate immune system, and produce various immune modulators such as inducible nitric oxide synthase-induced nitric oxide, cyclooxygenase-2 induced prostaglandin E2 and proinflammatory cytokines such as tumor necrosis factor-alpha. On the other hand, Protaetia brevitarsis seulensis larvae are a type of edible insect that have emerged as an alternative to the future food supply problem. The immuno-modulatory effect through the activation of murine macrophage RAW264.7 cell via mitogen-activated protein kinases (MAPKs)/nuclear factor-kappa B (NF-κB) signaling pathways has been reported. Based on this report, in this study, we confirmed how the expression of immune modulators induced by Protaetia brevitarsis seulensis larvae extracts in RAW264.7 cells was changed by treatment with pharmacological inhibitors of toll-like receptor 4 (TLR4), MAPKs and NF-κB signaling pathways. As a result, reduction of immune modulators was confirmed in the c-Jun N-terminal kinase (JNK) inhibitor treatment group and NF-κB inhibitor treatment group among the Protaetia brevitarsis seulensis larvae-treated RAW264.7 cell. Furthermore, in the TLR4 inhibitor-treated group, decreases in phosphorylation of JNK and NF-κB factors were confirmed in Protaetia brevitarsis seulensis larvae-treated RAW264.7 cell, as well as decreases in immune modulators. This results suggest that Protaetia brevitarsis seulensis larvae activates RAW264.7 cells by the engagement of TLR4-JNK/NF-κB signaling pathway.

• Journal of Nutrition and Health
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• v.53 no.5
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• pp.452-463
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• 2020

Purpose: Aster tataricus (AT) is one of the Asteraceae perennial herbs used in traditional Chinese medicine. The herb contains various bioactive substances, such as flavonoids, isoflavonoids, and phenolic compounds in the roots, and exhibits a range of effects including anti-bacterial, anti-oxidant, and anti-inflammatory activities. This study compared the immunomodulatory effects of ethanol and water extracts of whole AT, except the roots, and analyzed the molecular mechanisms for the regulatory effects on cytokine secretion from THP-1 cells. Methods: The effects of AT extract on the cell viability and proliferation of THP-1 cells were analyzed using the Cell Counting Kit-8 method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant of the AT-treated THP-1 cells were measured using an enzyme-linked immunosorbent assay. The protein levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor kappa B (IκBα), and mitogen-activated protein kinase (MAPK) phosphorylation in the cell lysates were determined by western blotting. Results: The water extract and the ethanol extract of AT did not affect the cell viability, and increased the proliferation of THP-1 cells significantly compared to the vehicle. The water extract increased the secretion of IL-1β from THP-1 cells in a dose-dependent manner, but the ethanol extract had no effect. The expression of COX-2 and iNOS protein and the phosphorylation of MAPK and Akt were induced in AT-treated cells. In addition, IκBα was degraded by AT in a concentration-dependent manner. IL-1β secretion by AT was reduced by extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors, while TNF-α secretion was decreased by inhibitors of ERK, p38 MAPK, and JNK. Interestingly, the p38 MAPK inhibitor increased the production of IL-1β by AT further. Conclusion: The water extract of the above-ground parts of AT contains immunomodulatory bioactive substances that stimulate immune cells through the MAPK signaling pathway.

• Radiation Oncology Journal
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• v.23 no.1
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• pp.51-60
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• 2005

Purpose : Selective inhibition of multiple molecular targets may improve the antitumor activity of radiation. Two specific inhibitors of selective cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) were combined with radiation on the HeLa cell line. To investigate cooperative mechanism with selective COX-2 inhibitor and EGFR blocker, in vitro experiments were done. Materials and Methods : Antitumor effect was obtained by growth inhibition and apoptosis analysis by annexin V-Flous method. Radiation modulation effects were determined by the clonogenic cell survival assay. Surviving fractions at 2 Gy ($SF_2$) and dose enhancement ratio at a surviving fraction of 0.25 were evaluated. To investigate the mechanism of the modulation of radiosensitivity, the cell cycle analyses were done by flow cytometry. The bcl-2 and bax expressions were analyzed by western blot. Results : A cooperative effect were observed on the apoptosis of the HeLa ceil line when combination of the two drugs, AG 1478 and NS 398 with radiation at the lowest doses, apoptosis of $22.70\%$ compare with combination of the one drug with radiation, apoptosis of $8.49\%$. In cell cycle analysis, accumulation of cell on $G_0/G_l$ phase and decrement of S phase fraction was observed from 24 hours to 72 hours after treatment with radiation, AG 1478 and NS 398. The combination of NS 398 and AG 1478 enhanced radiosensitivity on a concentration-dependent manner in HeLa cells with dose enhancement ratios of 3.00 and $SF_2$ of 0.12 but the combination of one drug with radiation was not enhanced radlosensitivity with dose enhancement ratios of 1.12 and SF2 of 0.68 (p=0.005). The expression levels of bcl-2 and bax were reduced when combined with AG 1478 and NS 398. Conclusion : Our results indicate that the selective COX-2 inhibitor and EGFR blocker combined with radiation have potential additive or cooperative effects on radiation treatment and may act through various mechanisms including direct inhibition of tumor cell proliferation, suppression of tumor cell cycle progression and inhibition of anti-apoptotic proteins.