• Asian Pacific Journal of Cancer Prevention
• /
• 제16권12호
• /
• pp.4849-4852
• /
• 2015

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been investigated as an effective agent to treat various cancers. Cancer stem cells are resistant to TRAIL treatment, but the mechanism of TRAIL resistance remains unknown. In this study, brain cancer stem cells were isolated by CD133 magnetic sorting, and the number of CD133 positive cells detected by flow cytometry. The self-renewing capacity of brain cancer stem cells was examined by a neurosphere formation assay, and the percentage of cell death after TRAIL treatment was examined by an MTS assay. Expression of DR5, FADD, caspase 8 and BCL2 proteins was detected by western blot. The amount of CD133 positive cells was enriched to 71% after CD133 magnetic sorting. Brain cancer stem cell neurosphere formation was significantly increased after TRAIL treatment. TRAIL treatment also reduced the amount of viable cells and this decrease was inhibited by a caspase 8 inhibitor or by the pan-caspase inhibitor z-VAD (P<0.05). Brain cancer stem cells expressed lower levels caspase 8 protein and higher levels of BCL2 protein when compared with CD133 negative cells (P<0.05). Our data suggest that TRAIL resistance is related to overexpression of BCL2 and low expression of caspase 8 which limit activation of caspase 8 in brain cancer stem cells.

• 대한한의학방제학회지
• /
• 제20권2호
• /
• pp.65-82
• /
• 2012

Objectives : The aim of this study was verification of the anti-oxidative and anti-inflammatory effects of Odukhwan(ODH) and Sasinhwan(SSH) in mouse macrophage, RAW264.7 cells. Methods : To investigate the anti-oxidative effect and scavenging activities of DPPH radical, superoxide anions, nitric oxide and peroxynitrite were measured. Cytotoxic activity of extract of ODH and SSH on RAW264.7 cells was measured using MTS assay. To proof the reductive activity of intracellular oxidation, DCFH-DA assay was performed. The nitric oxide(NO) production was measured and pro-inflammatory cytokines and $PGE_2$ were measured by ELISA kit. The levels of inducible nitric oxide synthase(iNOS), cyclooxygenase-2(COX-2) and nuclear NF-${\kappa}B$ p65 expression were detected by western blot. Results : After those analyses, we bring to a conclusion as follows. Both herbal formulations scavenged DPPH radical and nitric oxide. But ODH had no scavenging activity of superoxide anions and SSH had low scavenging activity in peroxynitrite. And the results indicated that ODH and SSH inhibited the LPS-induced NO, $PGE_2$ production and iNOS, COX-2 expression accompanied by an attenuation of IL-$1{\beta}$ and IL-6 production in RAW264.7 cells. They also have suppression effects of LPS-induced NF-${\kappa}B$ activation. Conclusions : ODH and SSH have anti-oxidative and anti-inflammatory effects and they may be a part of database for development of new anti-oxidative and anti-inflammatory drugs.

• 대한본초학회지
• /
• 제23권4호
• /
• pp.121-134
• /
• 2008

Objectives: Melia toosendan(MT) has been used as a traditional Korean herbal medicine, and today it is used as a medication for colic, side aches, heartache and other disorders of liver. The aim of this study was to determine whether fractionated extracts of MT inhibit free radical generation such as DPPH radical, superoxide radical and nitric oxide, production of nitrite, an index of NO, PGE2, iNOS, COX-2 and proinflammatory cytokines in lipopolysaccharide(LPS)-treated RAW 264.7 macrophages. Methods: MT extract prepared with methanol, and then fractionated with hexane, dichloromethane, ethylacetate, n-butanol and water. Inhibitory effect of MT onto free radical generation was determined by measuring DPPH, superoxide anions and nitric oxide scavenging activities in vitro. Cytotoxic activity of extracts on RAW 264.7 cells was measured using 5-(3-caroboxymeth-oxyphenyl)-2H-tetra-zolium inner salt(MTS) assay. Intracelluar oxidation was analysed by DCF-DA assay. The nitric oxide(NO) production was measured by Griess reagent system. The levels of iNOS and COX-2 expression were confirmed by western blot. And pro inflammatory cytokines were measured by ELISA kit. Results: Our results indicated that fractionated extracts, especially dichloromethane and ethyl acetate extracts, significantly inhibited free radical generation, the LPS-induced $H_2O_2$, NO, $PGE_2$ production and iNOS, COX-2 expression accompanied by an attenuation of TNF-${\alpha}$, IL-1${\beta}$ and IL-6 formation in macrophages. Conclusions: These results indicate that dichloromethane and ethyl acetate extracts of MT have potential as an agent of chronic inflammatory diseases.

• 동의생리병리학회지
• /
• 제25권3호
• /
• pp.459-465
• /
• 2011

The nuclear factor of activated T cells (NFAT) protein induces transcriptions of cytokine genes including IL-2 for T-cell activation. Normally activation of NFAT is important to induce immune responses but excessive NFAT activation provokes immunopathological reactions such as autoimmunity, transplant rejection, and inflammation. Thus, for the treatment of autoimmune diseases drugs repressing the activation of NFAT have been searched. In this study, immnunosuppressive effects of Rosa chinensis Jacq. extracts identified as a potent NFAT inhibitor from a natural product library were examined. NFAT reporter assay, MTS assay, real time PCR, IL-2 ELISA, MLR, and FACS (Fluorescent Activated Cell Sorting) were used to measure inhibitory immunocyte activities of Rosa chinensis Jacq. The variety of natural products have been screened and some were found to show inhibitory activities against the NFAT transcription factor. Among them, extract of Rosa chinensis Jacq. showed an strong inhibitory effect on the activation of NFAT without affecting cell viability. Levels of IL-2 transcripts as well as IL-2 protein were decreased with treatment of Rosa chinensis Jacq. extract. In addition, immunosuppressive activity of Rosa chinensis Jacq. extract was exhibited in the mixed leukocytes reaction. The increasement of CD4+CD25+ (Treg) immunocyte was also detected in the analysis using FACS after applying Rosa chinensis Jacq. extract. Immunosuppressive effects of the Rosa chinensis Jacq. extracts were clearly demonstrated in the present study. In addition, Rosa chinensis Jacq. extract also positively affected regulatory T cell induction. Further investigations in particular on purification of single substance responsible for the immunosuppressive effects from the extract and analysis on possible actions of the extract in interfering cell signaling and cytokine production will be needed.

• Molecules and Cells
• /
• 제39권10호
• /
• pp.742-749
• /
• 2016

The cancer chemo-preventive effects of equol have been demonstrated for a wide variety of experimental tumours. In a previous study, we found that equol inhibited proliferation and induced apoptotic death of human gastric cancer MGC-803 cells. However, the mechanisms underlying equol-mediated apoptosis have not been well understood. In the present study, the dual AO (acridine orange)/EB (ethidium bromide) fluorescent assay, the comet assay, MTS, western blotting and flow cytometric assays were performed to further investigate the pro-apoptotic effect of equol and its associated mechanisms in MGC-803 cells. The results demonstrated that equol induced an apoptotic nuclear morphology revealed by AO/EB staining, the presence of a comet tail, the cleavage of caspase-3 and PARP and the depletion of cIAP1, indicating its pro-apoptotic effect. In addition, equol-induced apoptosis involves the mitochondria-dependent cell-death pathway, evidenced by the depolarization of the mitochondrial membrane potential, the cleavage of caspase-9 and the depletion of Bcl-xL and full-length Bid. Moreover, treating MGC-803 cells with equol induced the sustained activation of extracellular signal-regulated kinase (ERK), and inhibiting ERK by U0126, a MEK/ERK pathway inhibitor, significantly attenuated the equol-induced cell apoptosis. These results suggest that equol induces mitochondria-dependent apoptosis in human gastric cancer MGC-803 cells via the sustained activation of the ERK1/2 pathway. Therefore, equol may be a novel candidate for the chemoprevention and therapy of gastric cancer.

• 동의생리병리학회지
• /
• 제20권3호
• /
• pp.701-709
• /
• 2006

The Rhus verniciflua Stokes (乾漆-RVS) has been used in traditional East Asia medicine for the therapy of gastritis, stomach cancer, although the mechanism for the biological activity is unclear. In the present study aims to investigate RVS extract contributes to growth inhibitory effect and it's the molecular mechanism on the human gastric cancer cells. AGS (gastric cancer cells) and RIEI (normal cells) were treated to different concentrations and periods of RVS extract $(10{\;}{\sim{{\;}100{\;}ug/mil)$. Growth inhibitory effect was analyzed by measuring FACS study and MTS assay. Cell cycle inhibition was confirmed by measuring CDK2 kinase activity by immunoprecipitation and kinase assay. And apoptosis was confirmed by surveying caspase cascades activation using a pan caspase inhibitor Exposure to RVS extract (50 ug/mll) resulted in a synergistic inhibitory effect on cell growth in AGS cells. Growth inhibition was related with the inhibition of proliferation and induction of apoptosis. The extract induces Gl -cell cycle arrest through the regulation of cyclins, the induction of p27kip1, and the decrease CDK2 kinase activity. And upregulated p27kip1 level is caused by protein stability increment by the reduction of S-phase kinase-associated protein 2 (Skp2), a key molecule related with p27kip1 ubiquitination and degradation, and do novo protein synthesis. Besides, 乾漆 extract induces apoptosis through the expression of Bax, poly(ADP-ribose) polymerase (PARP) and activation of caspase-3. RVS extract induces Gl -cell cycle arrest via accumulation of p27kip1 and apoptosis in human gastric cancer cells but not in normal cells, therefore we suggest that the extract can be used as a novel class of anti-cancer drugs.

• 대한한방내과학회지
• /
• 제26권1호
• /
• pp.129-142
• /
• 2005

Objective : Asthma is known as chronic airway inflammatory disease. This inflammation is conducted by various inflammatory cells including eosihophil. Chemotaxis is one way that circulating inflammatory cells invade a specific lesion. This study examines the degree to which Lonicerae Flos inhibits eosinophil chemotaxis at pulmonary epithelium after allergic stimulation. Material and Methods : Water extracts of Lonicerae Flos and pulmonary epithelial cell lines A549(human type II-like epithelial cells) and human eosinophils were used. Cytotoxic effects of Lonicerae Flos via MTS assay were estimated, as well as the effects of Lonicerae Flos on chemokines from prestimulated A549 cells by sandwich ELISA and RT-PCR. Chemotaxis assay was conducted on prestimulated eosinophils treated with Lonicerae Flos. Result : In this study $TNF-{\alpha}$ and IL-4, $IL-l{\beta}$ were seen to induced the accumulation of chemokines mRNA in the pulmonary epithelial cell lines A549 in a dose-dependent manner. Chemokines were inhibited by Liripois Tuber in a dose-dependent manner and especially, IL-8 and ICAM-l were inhibited considerably at $100\;{\mu}g/ml$ concentration of Lonicerae Flos. The eosinophil migration is inhibited in high concentration of Lonicerae Flos in a dose-dependent manner. Conclusion : These findings indicate that the supression of the expression of chemokines can be accomplished by Lonicerae Flos treatment, raising the possibility that Lonicerae Flos might be of therapeutic value in diseases such as asthma.

• 대한한방내과학회지
• /
• 제26권1호
• /
• pp.199-212
• /
• 2005

Objective : Airway inflammation is now regarded as a defining feature of asthma. The importance of eosinophits in the airway inflammation of asthma patients is widely recognized, and eosinophils mobilization in the respiratory epithelium is activated by chemoattractants and cytokines. This study was designed to examine the extent of the ability of Moutan Cortex Radicis to inhibit eosinophil chemotaxis of pulmonary epithelium after allergic stimulation. Material and Methods : Water extracts of Moutan Cortex Radicis and pulmonary epithelial cell lines A549(human type II-like epithelial cells) and human eosinophils were used. Cytotoxic effects of Moutan Cortex Radicis were estimated via MTS assay, and the effects of Moutan Cortex Radicis on chemokines from prestimulated A549 cells were estimated by sandwich ELISA and RT-PCR. Chemotaxis assay on prestimulated eosinophils treated with Moutan Cortex Radicis. was conducted Result : In this study we demonstrated that $TNF-{\alpha}$ and IL-4, $IL-1{\beta}$ induced the accumulation of chemokines' mRNA in the pulmonary epithelial cell lines A549 in a dose-dependent manner. Chemokines of eotaxin, ICAM-1, YCAM-1, IL-8, IL-16 were inhibited by Moutan Cortex Radicis in a dose dependent manner, but RANTES showed no inhibition due to Moutan Cortex Radicis. Eosinophil migration was inhibited at high concentrations of Moutan Cortex Radicis. Conculusion : These findings are indicative of supression of chemokines accomplished by Moutan Cortex Radicis treatment, demonstrating the potential therapeutic value of Moutan Cortex Radicis for treating diseases such as asthma.

• 대한한방부인과학회지
• /
• 제19권2호
• /
• pp.199-213
• /
• 2006

Purpose : This study was aimed to investigate the inhibitory effect of Sparganii Rhizoma on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : This study was evaluated the number of death cells treated with indicated concentration of Sparganii Rhizoma and investigated cell death rate by MTS assay. Furthermore, fluorescence-activated cell sorter analysis and DNA fragmentation assay were used to dissect between necrosis and apoptosis. and then we observed the differential gene expression by western blot analysis. Results :1) The inhibitory effect on the growth of uterine leiomyoma cell treated with Sparganii Rhizoma was increased in a dose dependent manner. 2) As the result of FACS analysis, subG1 phase incrase was observed 23.49% inuterine leiomyoma cell treated with Sparganii Rhizoma at $500\;{\mu}g/ml$ compared to control.. 3) The gene expression of p53, p21 related cell apoptosis was increased according to increasing concentration but p27 was none exchanged. 4) The expression of cyclin A, D and E was decreased in a concentration proportional and then the dephosphorylation of pRb was increased. 5) The character of apoptosis, DNA fragmentation was significantly observed according to increasing concentration. 6) The expression of pro-caspase3 were decreased dependent on treatment concentration and activated PARP took place. Conclusion : The inhibitory effect of Sparganii Rhizoma on the proliferation of human uterine leiomyoma cells was observed with apoptosis and cell cycle arrest. These data suggest that Sparganii Rhizoma might be candidate of medical therapy for uterine leiomyoma.

• 대한본초학회지
• /
• 제35권4호
• /
• pp.37-43
• /
• 2020

Objectives : Allium hookeri is a well-known traditional herbal remedy and its root used for treatment of inflammation and tumor. However, the mechanism of anti-inflammatory effect of Allium hookeri is still unknown. This study aims to examine the mechanism of anti-inflammatory effect of Allium hookeri on mouse macrophage cell line, RAW 264.7 cells. Methods : Anti-oxidant effect of water extract of Allium hookeri (WEAH) was measured by 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay. 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to determine the effect of WEAH on cell viability in RAW 264.7 cells. In addition, anti-inflammatory effect of WEAH was investigated in RAW 264.7 cells. Inflammation of RAW 264.7 cells induced by lipopolysarccharide (LPS) treatment and expression levels of inflammatory cytokine interleukin 1 β (IL-1β) and interleukin 6 (IL-6) gene were analyzed using quantitative reverse transcription PCR (qRT-PCR) analysis. Furthermore, the phosphorylation of inhibitor of nuclear factor kappa B (IκBα) after LPS treatment with WEAH-treated RAW 264.7 cells was confirmed by immunoblot analysis. Results : WEAH showed a strong anti-oxidant effect and no cytotoxicity to RAW 264.7 cells up to 2 mg/㎖ concentration. The LPS-induced mRNA expression levels of IL-1β and IL-6 were decreased by WEAH treatment. Furthermore, the LPS-induced phosphorylation of IκBα is attenuated by WEAH treatment. Conclusions : Through experimental demonstration of anti-oxidant and anti-inflammatory effects of WEAH, we suggest that Allium hookeri is a valuable material for prevention and treatment of various inflammatory diseases.