• The Korean Journal of Physiology and Pharmacology
• /
• v.4 no.5
• /
• pp.369-378
• /
• 2000

This study was undertaken to investigate an involvement of nitroxergic innervation in gastric smooth muscle of rat. Isometric tension study, the measurement of single cell length, NADPH diaphorase stain of smooth muscle layers and neuronal nitric oxide synthase (nNOS) western blotting were performed. Sodium nitroprusside (SNP), a nitric oxide donor, relaxed the muscle strips precontracted by acetylcholine (ACh) in a concentration-dependent manner. Pretreatment of L-arginine decreased the contraction induced by electric field stimulation (EFS). Pretreatment of $N^G-nitro-L-arginine$ methyl ester (L-NAME), a NOS inhibitor, increased the EFS-induced contractions. LY 83583, a guanylate cyclase (GC) inhibitor, reversed the inhibitory actions of L-arginine on the muscle contractions. The effects of L-Arginine, L-NAME and LY 83583 on ACh-induced contractions were not significant. L-arginine reduced the EFS-induced contraction in circular muscle, whereas L-NAME enhanced the EFS-induced contraction in longitudinal strips. By EFS, the phasic contractions appeared approximately $20{\sim}25$ seconds later. L-NAME significantly shortened the delay time to about $2{\sim}3$ seconds. In single cell study, ACh contracted gastric smooth muscle cells, SNP relaxed the cells, and the latter also inhibited the ACh-induced contraction. LY 83583 enhanced the ACh-induced contraction and antagonized SNP-induced relaxation. NADPH diaphorase activity was assessed by a histochemistry, nitroblue tetrazolium (NTB) staining. Positive staining was observed in both circular and longitudinal muscle layers. L-arginine increased the staining, while L-NAME decreased the staining. Western blotting for nNOS proved the presence of nNOS in rat gastric smooth muscle. EFS and additional $Ca^{2+}$ increased nNOS protein expression. These results suggest that in rat stomach, both circular and longitudinal muscle layers are innervated with nitroxergic nerves which relax the gastric smooth muscle via NO-cGMP pathway.

• Journal of Periodontal and Implant Science
• /
• v.25 no.3
• /
• pp.694-702
• /
• 1995

Using the Korean red ginseng saponin, which is known to world-wide and thd effects of it have been investigated by many reserachers for years. Ginseng saponin, one of the major components of Korea ginseng root, has many various biologic effects, such as cytotoxic effect, tumorcidal activity, protein biosynthesis and membrane modifying effect. The purpose of this study was to evaluate effects of ginseng saponin on the alkaline phosphatase activity of ROS cells in culture. After ROS cells were seeded into a 96-well plate, 96-well plate cultured until confluence was obtained. To evaluate cytotoxic effect of total saponin in cultured ROS cells, the plates were added to each total saponin concentration (0-1mg/ml). After 48hr., cells were counted by stain with 0.2% trypan blue at randomly selected field microscopically. Also, to evaluate alkaline phosphatase(ALP) activity of total saponin in cultured ROS cell, the plate was added to each total saponin concentration (0-1mg/ml) and ALP activity was assayed. To evaluate time-course of ALP activity, $31.25{\mu}g/ml$ of saponin added to 96-well plate. After culture of 6, 12, 24 and 48hr., ALP activity test was performed. To evaluate effect of cycloheximide in ALP activity, 96-well plate was added to saponin and cycloheximide. In control group, the plate was added saponin only. The results were as follows. 1. After the various concentration of total saponin was added in the medium, 500 and $1000{\mu}g/ml$ of total saponin showed cytotoxic effect of ROS(P<0.005). 2. In contrast to control group, 7.6, 15.6, 31.25, 62.5 and $250{\mu}g/ml$ of total saponin increased ALP activity significantly. 3. Otherwise, 500 and $1000{\mu}g/ml$ of total saponin decreased ALP activity significantly(P<0.005). 4. As the time span increases, $31.25{\mu}g/ml$ of total saponin increased ALP activity. 5. Cycloheximide decreased saponin-indueced ALP actitity in ROS(P<0.005). These results suggest that Ginseng total saponin stimulates the ALP activity of rat osteoblastic cells.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.43 no.2
• /
• pp.231-237
• /
• 2014

This research showed that delphinidin, an anthocyanin present in blueberry, induced apoptosis in MDA-MB-231 breast cancer cells as well as mediated anti-cancer effects in vivo. As a result, growth of cancer cells as assessed by MTT assay decreased in a concentration-dependent manner. Chromatin condensation by DAPI staining significantly increased in a concentration-dependent manner, indicating apoptosis. The level of p53-protein increased while those of anti-apoptotic molecules (Bcl-2, p-$GSK3{\beta}$) decreased in the western blot. Tumor size decreased in cells treated with 10 mg/kg of delphinidin compared with the control group in vivo. Cell apoptosis assessed by TUNEL assay significantly increased, and tumor inhibitory effect was confirmed. Therefore, delphinidin can be developed for cancer preventive medicine due to its growth inhibitory effects and induction of apoptosis in human breast cancer cells.

• Korean Journal of Head & Neck Oncology
• /
• v.10 no.2
• /
• pp.200-205
• /
• 1994

Proliferating cell nuclear antgen(PCNA) plays an important role in DNA synthesis in nucleoli and is highly conserved non-histone nuclear protein composed of 261 amino acid. and is considered to correlated with the cells proliferative state, because it is synthesized particulary during the proliferative period of late Gland S-phase. Therefore, PCNA index meaningfully increases in the active or proliferative kinetic cells. By the use of recently developed monoclonal antibodies against PCNA, the immunohistochemical staining methods can make possible. These staining methods are the useful and productive one for ascertaining the cell's proliferating abillity. Moreover, immunohistochemical staining method with a antiPCNA antibody has particulrar advantages as follows. By means of these methods, we can stain the tissue that was already fixed in formalin or paraffin wax. We can see with naked eye that which cell is, where is differentiated through a microscope. Lastly, it maintains the whole tissue architecture and makes a search for the correlation. As we have seen above, the immunohistochemical staining methods for PCNA have been studied as an impotant factor that can find the cell proliferative kinetics in malignancy and biologic behavior of tumors. To investigate of the proliferative activity in thyroid nodule, Authors evaluated cell proliferative activity by immunostaing for PNCA in 45 pathologically confirmed solitary thyroid nodule. The results were as follows. 1) The benign nodules were 25 cases(Adenomatous Goiter: 20 cases, Follicular adenoma: 5 cases) and malignant nodules were 20 cases(Papillary Ca : 14 cases, Follicular Ca : 4 cases, Anaplastic Ca : 2 cases). 2) The Most prevalent age groups were 4th decade(11 cases), and the next group was 5th decade. 3) The average PCNA labelling indices were as follows. Adenomatous goiter(I6.9%), Follicular adenoma(37.6%), papillary Ca(26.3%), Follicular Ca(8.8%) and Anaplastic Ca(86.7%). There were no significant differences in benign(20.4) and malignant nodules (28.8%) except anaplastic Ca(p=0.3226). 4) When the average tumor size 2cm in papillary Ca, the PCNA indices were 26.0% (below 2cm) : 26.6% (above 2cm) (p=0.9642). The PCNA incidies were 23.9% (with lymphatic spread) : 28.7% (without lymphatic spread) (p=0.7056). There were no signlficant differences in the above cases. In conclusion, there were no significant differences in cell proliferative activity by staining for PCNA between benign and malignat nodules except anaplastic Ca.

• The Korea Journal of Herbology
• /
• v.23 no.1
• /
• pp.75-83
• /
• 2008

Objectives : The present study was carried out to investigate the effect of Hyperici Japonici Herba on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mice by ovalbumin(OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dessiciated with collagenase(1${\mu}g$/ml). Eosinophils were activated by rIL-3/rmIL-5 co-treatments. The lung cells were treated with extract of Hyperici Japonici Herba(EHH), incubated for 48 hr at $37^{\circ}C$, and analyzed by flow cytometer. ELBA, RT-PCR, immunocytochemistry stain. Results : The cell number ratio of granulocyte, $CD3e^-$/$CCR3^+$, $CD3e^+$/$CD69^+$, $CD4^+$, $CD23^+$/$B220^+$ cells was increased in rmIL-5/rIL-3 treated control group compared to the normal group. Cells numbers in the experimental animal group treated with EHH was all decreased. In ELISA analysis, IL-4, IL-5, IL-13 protein levels and histamine release level were greatly increased in the control group compared to the normal animal group, then significantly decreased in the experimental group with 100 ${\mu}g$/ml of EHH treatment. In RT-PCR analysis, the HT value of IL-4, IL-5, IL-13, CCR3, Eotaxin were increased in the control group compared to the normal animal group, then decreased in the experimental group with 100 ${\mu}g$/ml of EHH treatment. And eosinophil proliferation levels were 18847${\pm}$1527(cpm) in the control group, 4676${\pm}$972(cpm) in the positive control group, and 8675${\pm}$159(cpm), 11352${\pm}$1005(cpm), 14325${\pm}$677(cpm) in the experimental group with 100 ${\mu}g$/ml, 10 ${\mu}g$/ml, 1 ${\mu}g$/ml of EHH treatment. Conclusions : The present data suggested that Hyperici Japonici Herba may have an effects on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of EHH.

• Molecules and Cells
• /
• v.37 no.6
• /
• pp.449-456
• /
• 2014

The use of synovial fluid-derived mesenchymal stem cells (SFMSCs) obtained from patients with degenerative arthropathy may serve as an alternative therapeutic strategy in osteoarthritis (OA) and rheumatoid arthritis (RA). For treatment of OA and RA patients, autologous transplantation of differentiated MSCs has several beneficial effects for cartilage regeneration including immunomodulatory activity. In this study, we induced chondrogenic differentiation of SFMSCs by inhibiting protein kinase A (PKA) with a small molecule and microRNA (miRNA). Chondrogenic differentiation was confirmed by PCR and immunocytochemistry using probes specific for aggrecan, the major cartilaginous proteoglycan gene. Absorbance of alcian blue stain to detect chondrogenic differentiation was increased in H-89 and/or miRNA-23b-transfected cells. Furthermore, expression of matrix metalloproteinase (MMP)-9 and MMP-2 was decreased in treated1 cells. Therefore, differentiation of SFMSCs into chondrocytes through inhibition of PKA signaling may be a therapeutic option for OA or RA patients.

• Journal of Oral Medicine and Pain
• /
• v.30 no.2
• /
• pp.231-238
• /
• 2005

Anti-cancer drugs have been shown to target diverse cellular functions in mediation cell death in chemosensitive tumors. Most antineoplastic drugs used in chemotherapy of leukemias and solid tumors induce apoptosis in drug-sensitive target cells. However, the precise molecular requirements that are central for drug-induced cell death are largely unknown. Etoposide is used for the treatment of lung and testicular cancer. This study was performed to examine whether etoposide promote apoptosis in human oral squamous carcinoma cells (OSC9) as well as in lung and testicular cancer. Etoposide had a significant dose- and time-dependent inhibitory effect on the viability of OSC9 cells. TUNEL assay showed the positive reaction on condensed nuclei. Hoechst stain demonstrated that etoposide induced a change in nuclear morphology. The expression of p53 was increased at 48 hour, suggesting that the nuclear of OSC9 cell was damaged, thereby inducing apoptosis. Etoposide treatment induced caspase-3 cleavage and activation. Intact PARP protein 116-kDa and 85-kDa cleaved product were observed. The activated caspase-3 led cleavage of the PARP. These results demonstrate that etoposide-induced apoptosis in OSC9 cells is associated with caspase-3 activation.

• Tuberculosis and Respiratory Diseases
• /
• v.81 no.1
• /
• pp.80-87
• /
• 2018

Background: Asthma is a disease of chronic airway inflammation with heterogeneous features. Neutrophilic asthma is corticosteroid-insensitive asthma related to absence or suppression of $T_H2$ process and increased $T_H1$ and/or $T_H17$ process. Macrolides are immunomodulatory drug that reduce airway inflammation, but their role in asthma is not fully known. The purpose of this study was to evaluate the role of macrolides in neutrophilic asthma and compare their effects with those of corticosteroids. Methods: C57BL/6 female mice were sensitized with ovalbumin (OVA) and lipopolysaccharides (LPS). Clarithromycin (CAM) and/or dexamethasone (DXM) were administered at days 14, 15, 21, 22, and 23. At day 24, the mice were sacrificed. Results: Airway resistance in the OVA+LPS exposed mice was elevated but was more attenuated after treatment with CAM+DXM compared with the monotherapy group (p<0.05 and p<0.01). In bronchoalveolar lavage fluid study, total cells and neutrophil counts in OVA+LPS mice were elevated but decreased after CAM+DXM treatment. In hematoxylin and eosin stain, the CAM+DXM-treated group showed less inflammation additively than the monotherapy group. There was less total protein, interleukin 17 (IL-17), interferon ${\gamma}$, and tumor necrosis factor ${\alpha}$ in the CAM+DXM group than in the monotherapy group (p<0.001, p<0.05, and p<0.001). More histone deacetylase 2 (HDAC2) activity was recovered in the DXM and CAM+DXM challenged groups than in the control group (p<0.05). Conclusion: Decreased IL-17 and recovered relative HDAC2 activity correlated with airway resistance and inflammation in a neutrophilic asthma mouse model. This result suggests macrolides as a potential corticosteroid-sparing agent in neutrophilic asthma.

• Korean Journal of Head & Neck Oncology
• /
• v.19 no.1
• /
• pp.25-33
• /
• 2003

Background and Objectives: The Herpes Simplex type 2 Defective Infectious Single Cycle virus (DISC virus) is attenuated virus originally produced as viral vaccines but are also efficient gene transfer vehicle. The main goals of this study were to examine the efficiencies of the gene transfer using DISC vectors for various head and neck squamous cell carcinoma cell lines and to evaluate the efficacy of vaccination with DISC virus carrying a immunomodulatory genes (GM-CSF) as cancer therapy in a organotopic oral cavity squamous cell cancer model. Materials and Methods : We determinated the gene transfer efficiency of DISC virus by x-gal stain method and proved gene and protein expression of DISC-GMCSF transfected SCCVII cells by RT-PCR and ELISA method. Also we evaluated the ex vivo vaccination effects of SCCVII/GMCSF (DISC-GMCSF transfected SCCVII vaccine) vaccine on preventing the recurrence of micro-residual tumor. After the vaccination of SCCVII/GMCSF, specific cytotoxic T-cell responses was evaluated by CTL assay. Results: At an MOI of 10 DISC virus showed 64-88% of transfection rates in various head and neck squamous cancer cell lines. SCCVII cells transduced by DISC virus vector (MOI=10) carrying the GM-CSF gene, produced 4.5 nanogram quantities of GM-CSF per $10^6$ cells. In vivo vaccination using tumor cells transduced ex vivo with DISC-GMCSF resulted in better protection rate against subsequent tumor recurrence in organotopic oral cavity cancer model. Although tumor free survival rate was not statistically significantly increased in vaccination group (p=0.078), tumor specific cytotocic T-cell responses were significantly increased in SCCVII/GMCSF vaccination group. Conclusion: These data demonstrate that; 1) The DISC virus vector is capable of efficient gene transfer to various head and neck squamous cancer cell lines, 2) GM-CSF secreting genetically modified tumor vaccine (SCCVII/GMCSF) efficiently protected against tumor recurrence in organotopic micro-residual oral cavity cancer model and produced tumor specific cytotoxic T-cell response. DISC virus-mediated, cytokine gene transfer may prove to be useful as a clinical therapy for head and neck cancers.

• Tuberculosis and Respiratory Diseases
• /
• v.69 no.1
• /
• pp.16-23
• /
• 2010

Background: Most lung cancer patients receive systemic chemotherapy at an advanced stage disease. Cisplatin-based chemotherapy is the main regimen for treating advanced lung cancer. Recently, autophagy has become an important mechanism of cellular adaptation under starvation or cell oxidative stress. The purpose of this study was to determine whether or not autophagy can occurred in cisplatin-treated lung cancer cells. Methods: H460 cells were incubated with RPMI 1640 and treated in $5{\mu}M$ or $20{\mu}M$ cisplatin concentrations at specific time intervals. Cells surviving cisplatin treatment were measured and compared using an MTT cell viability assay to cells that underwent apoptosis with autophagy by nuclear staining, apoptotic or autophagic related proteins, and autophagic vacuoles. The development of acidic vascular organelles was using acridine orange staining and fluorescent expression of GFP-LC3 protein in its transfected cells was observed to evaluate autophagy. Results: Lung cancer cells treated with $5{\mu}M$ cisplatin-treated were less sensitive to cell death than $20{\mu}M$ cisplatin-treated cells in a time-dependent manner. Nuclear fragmentation at $5{\mu}M$ was not detected, even though it was discovered at $20{\mu}M$. Poly (ADP-ribose) polymerase cleavages were not detected in $5{\mu}M$ within 24 hours. Massive vacuolization in the cytoplasm of $5{\mu}M$ treated cells were observed. Acridine orange stain-positive cells was increased according in time-dependence manner. The autophagosome-incorporated LC3 II protein expression was increased in $5{\mu}M$ treated cells, but was not detected in $20{\mu}M$ treated cells. The expression of GFP-LC3 were increased in $5{\mu}M$ treated cells in a time-dependent manner. Conclusion: The induction of autophagy occurred in $5{\mu}M$ dose of cisplatin-treated lung cancer cells.