• The Korean Journal of Physiology and Pharmacology
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• v.8 no.6
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• pp.339-344
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• 2004

Recent data have shown the importance of oxidative stresses in the pathogenesis of inflammatory bowel disease, crohn's disease and ulcerative colitis. $H_2O_2$, reactive oxygen species (ROS) donor, has been reported to act as a signaling molecule involved in a variety of cellular functions such as apo/ptosis and proliferation. In the present study, we investigated viability of cultured ileal smooth muscle cells (ISMC) after stimulation with $H_2O_2$. Trypan blue method revealed that the cell viability of ISMC treated with 1 mM $H_2O_2$ was not different from that of controls at up to 2 h time point, while treatment of ISMC with 1 mM $H_2O_2$ for 48 h finally induced significant decrease in the cell viability. Therefore, we evaluated whether $H_2O_2$ was capable of ERKs activation in ISMC for the short-term exposure and examined whether tyrosine kinase was involved in the process of ERK activation by $H_2O_2$ in ISMC. We also investigated the effects of $H_2O_2$ on activation of SAPK/JNK and p38 MAP kinase in ISMC. Thus, ISMC were cultured and exposed to $H_2O_2$, and western blot analysis was performed with phosphospecific MAP kinase antibodies. Robust activation of ERK occurred within 30 min of 1 mM $H_2O_2$ treatment. $H_2O_2-induced$ ERK activation was attenuated by a tyrosine kinase inhibitor, genistein, indicating that tyrosine kinase was probably involved in the ERK activation by $H_2O_2$. $H_2O_2$ was a moderate activator of SAPK/JNK, while p38 MAP kinase was not activated by $H_2O_2$. We suggest that ERK activation induced by short-term $H_2O_2$ treatment plays a critical role in cellular protection in the early stage of response to oxidative stress. The present study suggests the necessity of identification of MAPK signaling pathways affected by ROS, since it could ultimately elucidate cellular consequences involved in initiation and perpetuation of intestinal tissue damage in the diseases such as crohn's disease and ulcerative colitis, resulted from excessive ROS.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9341-9346
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• 2014

Acanthopanax trifoliatus (L) Merr (AT) is commonly used as an herbal medicine and edible plant in some areas of China and other Asian countries. AT is thought to have anticancer effects, but potential mechanisms remain unknown. To assess the anticancer properties of AT, we exposed prostate cancer cells to AT extracts and assessed cell proliferation and signaling pathways. An ethanol extract of AT was suspended in water followed by sequential extraction with petroleum ether, ethyl acetate and n-butanol. PC-3 cells were treated with different concentrations of each extract and cell viability was determined by the MTT and trypan blue exclusion assays. The ethyl acetate extract of the ethanol extract had a stronger inhibitory effect on growth and a stronger stimulatory effect on apoptosis than any of the other extracts. Mechanistic studies demonstrated that the ethyl acetate extract suppressed the transcriptional activity of NF-${\kappa}B$, increased the level of caspase-3, and decreased the levels of phospho-Erk1/2 and phospho-Akt. This is the first report on the anticancer activity of AT in cultured human prostate cancer cells. The results suggest that AT can provide a plant-based medicine for the treatment or prevention of prostate cancer.

• The Journal of Internal Korean Medicine
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• v.12 no.2
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• pp.96-112
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• 1991

Attempts were made to see the antitumor effects of Sigbunhwan widely used in Oh-jug(五積) employing tumor cells Lines such as K562 derived from erythroleukemia, Raji from lympoma and MO-4 from blastogenic tumor. Different concentrations of Sigbunhwan and combined therapy of Sigbunhwan and Bigihwan were treated to those tumor cells lines and then live cells were counted by Trypan blue assay and $^{3}H-Thymidine$ uptake assay. The results obtained were as follows. 1. $^{3}H-Thymidine$ uptake of various tumor cells lines when treated with high concentrations of Sigbunhwan for 48hours showed that the rate of DNA synthesis decreased 76% to 90% by the treatment of 1% Sigbunhwan but this inhibition was rather decreased when Sigbunhwan concentration was increased to 10, 15 and 20%.(Fig 3) 2. When Sigbunhwan was combined with Bigihwan which was also an antitumor drug, the effectiveness of tumor cells dealth was somewhat inceased showing a generally similar pattern to that of Bigihwan alone used.(Fig 4) This combination therapy also showed that higher concentrations of antitumor agent were no more·effective or rather harmful according to the tumor cells lines having different growth rate.(Fig 5,6) 3. The antitumor effects of combined Sigbunhwan and Bigihwan was decreased if the concentrations of this combination therapy was increased to 10 times showing relatively sluggish decrease in K562 and MO-4 but a sharp inhibitory effect in Raji which grows slowly.(Fig 7). 4. When Sigbunhwan was treated at low concentrations, K562 was more inhibited by 0.75% to 1.0% of Sigbunhwan while Raji was more inhibited by 0.25% to 0.5% of that.(Fig 8) 5. When Sigbunhwan was treated together with Bigihwan at low concentrations, the tumor cells death rate was 75% to 89% in Baji, 31% to 95% in MO-4 and 41 to 89% in K562, showing this combination therapy was more effect to Raji derived from lymphoma.(Fig 9) 6. The number of live tumor cells was correlated with optical density of MTT assay when measured with 2% Sigbunhwan treatment to tumor cells lines for 24 hours.(Fig 10) 7. 7 days treatment of 0.25% Sigbunhwan was compared with one day treatment of 1% suggesting long term treatment more effective.(Fig 11)

• The Korea Journal of Herbology
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• v.21 no.4
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• pp.27-36
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• 2006

Objectives : This study was performed for the investigation of anticancer effects of methanol extract of Rheum Rhizoma (MeOH-RR) on a human liver cancer cell line (HepG2). Methods : To study the cytotoxic effect of MeOH-RR on HepG2 cells, the cell viability was determined by XTT reduction method and trypan blue exclusion assay. The cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of procaspase-3, -8 and -9 were examined by western blot analysis. Furthermore, MeOH-RR-induced apoptosis was confirmed by DNA fragmentation. The release of cytochrome c from mitochondria to cytosol, the level of Bcl-2 and Bax were examined by western blot analysis. Results : MeOH-RR reduced proliferation of HepG2 cells in a dose-dependent manner at 24 h and 48 h treatment. MeOH-RR induced the activation of caspase-3, -8, and -9 and the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3. Furthermore, treatment with MeOH-RR resulted in internucleosomal DNA fragmentation, evidenced by the formation of a DNA ladder on agarose gel, a hallmark of cells undergoing apoptosis. MeOH-RR downregulated Bcl-2, upregulated Bax, and increased the release of cytochrome c from the mitochondria into cytosol in a dose-dependent manner. Moreover, MeOH-RP increased caspase-3 activity. Conclusion : There results suggest that MeOH-RR induce apoptosis via mitochondrial pathway and caspase-3-dependent pathway in HepG2 cells. There results suggest that MeOH-RR is potentially useful as a chemotherapeutic agent in human liver cancer.

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

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6511-6516
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• 2012

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.

• The Journal of Korean Physical Therapy
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• v.21 no.3
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• pp.87-93
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• 2009

Purpose: TThis study examined the effect of repetitive magnetic stimulation (RMS) on the viability and proliferative response of human adipose tissue-derived stromal cells (hATSCs) in vitro. Methods: The hATSCs were cultured primarily from human adipose tissue harvested by liposuction and incubated in a $37^{\circ}C$ plastic chamber. The cells were exposed to a repetitive magnetic field using a customized magnetic stimulator (Biocon-5000, Mcube Technology). The RMS parameters were set as follows: repetition rate=10Hz, 25Hz (stimulus intensity 100%= 0.1 Tesla, at 4cm from the coil), stimulated time= 1, 5, and 20 minutes. Twenty four hours after one application of RMS, the hATSCs were compared with the sham stimulation, which were kept under the same conditions without the application of RMS. The cells were observed by optical microscopy to determine the morphology and assessed by trypan blue staining for cell proliferation. The apoptosis and viability of the hATSCs were also analyzed by fluorescence-activated cell sorting (FACS) analysis of Annexin V and MTT assay. Results: After RMS, the morphology of the hATSCs was not changed and the apoptosis of hATSCs were not increased compared to the sham stimulation. The viability of the cells was similar to the cells given the sham stimulation. Interestingly, the level of hATSC proliferation was significantly higher in all RMS groups. Conclusion: The application of RMS may not cause a change in morphology and viability of hATSCs but can increase the level of cell proliferation in vitro. RMS might be useful as an adjuvant tool in combination with stem cell therapy without adverse effects.

• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.251-261
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• 2000

V. vulnificus is an estuarine bacterium which causes septicemia and shock in susceptible patients. The organism produces a hemolytic cytolysin (VvH), which has a membrane damaging effect on erythrocytes. To clarify the mechanisms by which VvH might contribute to virulence, we examined its effect on macrophages. When mouse peritoneal macrophages were harvested and co-cultured with hemolysin-positive V. vulnificus strains (100 bacteria/cell), about 60% of the macrophages were killed; macrophages were not killed when co-cultured V. vulnificus strain CVD 707, a VvH-negative deletion mutant. Exposure of macrophages to filtered culture supernatants (2.5 HU/ml) and purified VvH (3 HU/ml) resulted in an increase in dead cells (80 and 90%, respectively), as determined by the trypan blue dye exclusion method and LDH release from macrophages was also increased (70 and 65.5%, respectively). The cytotoxic effect of VvH on macrophages was both the dose- and time-dependent. The VvH caused damage to the macrophage membrane and was blocked significantly by preincubation with cholesterol (p<0.01). Fetal bovine serum showed remarkable inhibition of VvH synthesis by V. vulnificus and inhibited VvH activity in culture supernatant. Cell viability was increased by 35% (p<0.01) and LDH release decreased by 28% (p<0.01) when macrophages were incubated with V. vulnificus (100 bacterial cell) in DMEM-10% FBS for 2 hr. Bacterial clearance activity of mice against V. vulnificus CVD 707 was decreased by pretreatment with 10 HU of VvH. This result suggests that the VvH can impair the membrane of macrophages and may playa role in the pathogenesis of V. vulnificus septicemia.

• Journal of Periodontal and Implant Science
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• v.26 no.1
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• pp.216-229
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• 1996

Interferon(IFN) is a sort of glycoproteins that are produced by activated lymphocyte, monocyte and fibroblast. IFN has anti-viral effects, immuno-defensive mechanism and regulating properties to the several kinds of cells that includes affect on the bone formation and resorption. The effect of IFN on the osteoclast & other tissue cells has been studied in a number of researchers with the limited reports on the osteoblast. The purpose of this study was to evaluate the effects of IFN on the osteoblastic function. The MC3T3/El cell(Mouse osteoblast) was incubated in ${\alpha}-minimum$ essential medium containing 10% FBS. To detect the cytotoxic effect of $IFN-{\gamma}$ on osteoblast, the cells were cultured in 96-well plate to which $IFN-{\gamma}$ of various concentrations were added for 2 days. After staining with trypan blue, total cells and living cells were counted under microscope. To determine the activity of alkaline phosphataset(ALP), various concentrations of $IFN-{\gamma}$ were treated to culture medium, and biochemical assay was performed. $IFN-{\gamma}$ and $IFN-{\gamma}$ plus cycloheximide were added to culture medium separately and then ALP activity were determined. To detect the effect of the $IFN-{\gamma}$ on the bone formation of osteoblast, long-term culture was performed, and calcified nodule formation were observed using von Kossa's staining. After the addition of $IFN-{\gamma}$ with various concentrations to the medium, no cytotoxic effect of $IFN-{\gamma}$ was detected at any concentration. The significant increase in ALP activity of osteoblast were found the concentration of $IFN-{\gamma}$ 500-2500U/ml and the culture time of 24-48 hours respectively. The enhancement of ALP activity by $IFN-{\gamma}$ of osteoblast was decreased significantly by the treatment of cycloheximide. After long-term culture of osteoblast, the nodule formation was found to be increased in number and density by the addition of 500 U/ml $IFN-{\gamma}$. These results suggest that $IFN-{\gamma}$ was affected on the bone formation of osteoblast. Forthemore this kind of study or $IFN-{\gamma}$ to osteoblast will be held continuously.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.9 no.1
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• pp.15-37
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• 2003

Objective: We are aimed to identify anti-tumor effects of Curcuma longa L. on the stomach cancer cells through molecular biologic methods. Material & Methods: We used AGS as human stomach cancer cells obtained from American Type Culture Collection. The boiled extract of Curcuma longa L. $5{\mu}l$ (Sample I), $10{\mu}l$ (Sample II) was treated to cultural media(ml) for 0, 6, 12, 24, 48 hours. We measured the killing effect on stomach cancer cells through Trypan blue exclusion test and the suppressive effect on viability of stomach cancer cells via MTT assay. For identification of its anticancer mechanism, the revelation of Bcl-2, Bcl-XL, and Bax which are genes related to apoptosis using the quantitative RT-PCR, change of mitochondria membrane permeability and membrane potential via flow cytometry, the cycle of cell mitosis, caspase cleavage and annexin V staining were examined. Results: 1. showed significant killing effect on stomach cancer cell than the control group with a time(6 hours later) and density dependent manner, which was statistical significance. 2. Extract of Curcuma longa L. showed suppressive effect on viability of stomach cancer cells that each test groups had more suppressive effects on viability of stomach cancer cells than the control group with a time(6 hours later), which was statistical significance.(p<0.05) 3. In the test about the revelation of genes related to apoptosis, the revelation of Bcl-2 and Bcl-XL decreased with a density manner which was statistical significance. but the revelation of Bax was not changed with statistical significance. 4. Extract of Curcuma longa L. caused apoptosis by decreasing the absorbance of mitochondria with statistical significance, and also induced apoptosis by decreasing the membrane potential of mitochondria. 5. Extract of Curcuma longa L. destructed the cell cycle of cell mitosos. 6. Cell apoptosis was induced by extract of Curcuma longa L. certificated by method of caspase cleavage and annexin V staining. Conclusion: This experiment showed that Curcuma longa L. has anti-tumor effect with statistical significance. This is in vitro experiment and basic experiment on Curcuma longa L.. We hope more progressive research on Curcuma longa L. will go on and its anti-tumor effects will be more practically identified.