• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7137-7141
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• 2013

Several studies have shown that nemo-like kinase (NLK) plays a vital role in apoptosis of cancer cells. The present research concerned effects and mechanisms of Taxol on NLK knockdown human laryngeal cancerHep-2 cell lines in vitro. Using RNAi, methyl-thiazoltetrazolium (MTT) assays, real-time RT-PCR, Western blotting and flow cytometry analysis, growth and the cell cycle progression of NLK knockdown Hep-2 cells and expression of downstream molecules were observed. Cell growth was obviously suppressed in the Taxol treated group (P<0.001, 48 hours). Cell numbers of combined Taxol-based chemotherapy with lentivirus mediated RNAi treatment group (Lv-shNLK+Taxol goup) were significantly different from NLK-specific siRNA lentivirus infected group (Lv-shNLK group) (p<0.001). Flow cytometry analysis revealed that Lv-shNLK+Taxol caused the G0/G1-phase DNA content to decrease from 44.1 to 3.33% (p<0.001) and the S-phase DNA content to increase from 38.4 to 82.0% (p<0.001), in comparison with the Lv-shNLK+Taxol group. Immunoblot analysis showed that knockdown of NLK led to significant reduction in the levels of cyclin D1, PCNA and PARP, whereas cyclin B1 was elevated in. Cell growth was also obviously suppressed in the Hep-2 cell line, knockdown of NLK making them more sensitive to Taxol treatment. NLK is expected to become a target of new laryngeal cancer gene therapies.

• The Journal of Internal Korean Medicine
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• v.33 no.2
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• pp.197-208
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• 2012

Objectives : Gleditsiae Spina has the effects of expelling toxins, draining pus, invigorating blood and resolving abscesses. Some clinicians apply the herb for patients suffering from cancer. However, its anti-cancer activities are not well understood. In the present study, anti-tumor agents from Gleditsiae Spina were purified. Methods : The viability of the PC-3 cell line was determined using MTT assay, and the induction of apoptosis by Gleditsiae Spina extract in PC-3 cells was measured by Annexin-V/propidium iodide double staining assay detected by flow cytometry. TLC and HPLC analysis were used to separate and identify the anti-cancer agents. Results : Treatment of the extract resulted in significant decreased cell viability of PC-3 cells in a dose- and time-dependent manner. Dose-dependent apoptotic cell death was also measured by flow cytometry analysis. The anti-cancer agents were successfully separated and identified by using TLC and HPLC analysis and the most potential agent among them was separated from EtOAC fraction. Conclusions : These results might be applied in developing new drugs from natural resources like Korean traditional medicine, and also support the clinical usefulness of herbal medicine.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.2
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• pp.290-296
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• 2002

Serum immunoglobulins (Igs) from Israeli carp were purified using affinity chromatography. Fish were immunized with purified mouse IgG, and the specific fish antibodies were purified from the immune serum on a mouse IgG-immobilized agarose gel. Rabbit anti-Israeli carp Igs (R $\alpha$ I. carp Igs) antibodies were produced following hyperimmunization with mouse IgG specific carp antibodies. SDS-PAGE analysis under reducing condition showed that Israeli carp Igs were composed of two $\mu$-like heavy chains with about 82 and 50 kd, respectively, and one light chain with about 25 kd. On immunoblotting analysis, however, R $\alpha$ I. carp Igs failed to react with the light chain. When both protein A and protein G-purified normal carp Ig were compared with mouse IgG-specific Israeli carp Ig, no significant structural differences among them were observed. To investigate if there is any homology between other fish Ig molecules, cross-reactivity of R $\alpha$ I. carp Igs against Ig molecules from 6 different fish sera and mouse control serum was checked on immunoblotting analysis. As a result, R $\alpha$ I. carp Igs responded to Israeli carp, common carp, and tilapia Ig molecules. In flow cytometry study, however, R $\alpha$ I. carp Igs appeared to recognize 42.0%, 35.8% and <5% of Israeli carp, common carp and tilapia $Ig^+$ head kidney cells, respectively. The result suggests the heterogeneity between receptor Igs on B-like lymphocytes and soluble Igs in serum. It is crucial to obtain pure fish Igs to produce reagent antibodies as tools for the study on their specific immune responses.

• Herbal Formula Science
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• v.23 no.2
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• pp.171-187
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• 2015

Objective This study aimed to evaluate the effects of Sagan-tang (SGT) on COPD mouse model. Methods The study was carried out by two ways (in vitro, in vivo). In vitro RAW264.7 cells (mouse macrophage) were used and analysed by flow cytometry, ELISA, Western blot. In vivo LPS and CSS challenged mice were used and its BALF had been analysed by cytospin image, FACS, ELISA, lung tissue by real-time PCR. Results In vitro, SGT maintained 80-100% rate of viablilty on 10 ~ 500 ㎍/㎖ concentration. In ELISA analysis with RAW264.7 cells, SGT significantly decreased NO over 30 ㎍/㎖. In flow cytometry, SGT 100 ㎍/㎖ dosage group displayed a tendency for decrease ROS. In Western blot analysis, SGT 100 ㎍/㎖ dosage group decreased NF-κB. In ELISA analysis, SGT significantly decreased TNF-α, IL-6 over 200 ㎍/㎖. In vivo SGT 200 ㎎/㎏ dosage group, application of SGT significantly decreased increase of neutrophils, TNF-α, IL-6 in BALF, muc5AC, TGF-β, TNF-α, expression of mRNA in lung tissue and histological lung injury. Conclusion This Study suggests usability of SGT for COPD patients by controlling lung tissue injury.

• The Journal of Korean Obstetrics and Gynecology
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• v.18 no.1
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• pp.181-191
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• 2005

Purpose : ${\beta}$-sitosterol is kind of phytosterols or plant which are structurally similar to cholesterol. This study was aimed to investigate the inhibitory effect of the ${\beta}$-sitosterol on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : We counted the number of death cells treated with indicated time of the ${\beta}$-sitosterol and investigated cell death rate by cell count assay. Furthermore, flow cytometry 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 the ${\beta}$-sitosterol $16{\mu}M$ was increased in a time dependent. 2) The result of flow cytometry analysis, subG1 phase arrest related cell apoptosis was investigated 16.97% in uterine leiomyoma cell treated with the ${\beta}$-sitosterol $16{\mu}M$ and showed the fashion of proportional time dependent. 3) The gene expression of p27, p21 related cell cycle was increased according to increasing time interval but cyclin E-CDK2 complex was decreased expression. 4) The character of apoptosis, DNA fragmentation was significantly observed on the time dependent. 5) The expression of pro-caspase 3 and PARP were decreased dependent on treatment with time dependent. Conclusion : This study showed that the ${\beta}$-sitosterol have the inhibitory effect on the proliferation of human uterine leiomyoma cell and the effect was related with apoptosis.

• Animal Bioscience
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• v.34 no.8
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• pp.1321-1330
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• 2021

Objective: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. Methods: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. Results: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. Conclusion: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

• Journal of Korean Society on Water Environment
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• v.40 no.4
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• pp.161-167
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• 2024

The demand for bottled water in South Korea is steadily increasing, but there are challenges regarding water sources and violations of water quality standards. Consumers struggle to identify products that do not meet these standards, highlighting the need for improved water management. This study aims to investigate the use of flow cytometry to identify microbial behavior in bottled water. Twelve different bottled water brands were selected for this study. A novel non-culture-based analysis method called total cell count via flow cytometry was utilized, which is not commonly used to assess drinking water quality. This method was compared to conventional culture-based methods for heterotrophic plate count and E. coli experiments, in order to introduce new indicators for hygiene management. Adenosine triphosphate analysis was also conducted to assess cell activity, and total organic carbon was measured to determine the presence of organic matter. The total cell counts varied among the different bottled water brands. The adenosine triphosphate levels ranged from 37.1ng/L to 221.7ng/L, while the total organic carbon ranged from 0.4 to 0.6 mg/L. Furthermore, E. coli was not detected in any of the bottled waters, and with the exception of two cases, the levels of heterotrophic bacteria did not exceed the drinking water standard of 100 CFU/mL. This study demonstrated a correlation between total cell count and heterotrophic plate count, suggesting that non-culture-based analysis could be valuable in promptly assessing microbial contamination, in contrast to the conventional methods that require approximately 48 hours for incubation.

• Development and Reproduction
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• v.2 no.1
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• pp.1-8
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• 1998

In mammalian ovary, major portion(>99%) of ovarian follicles undergo atresia. Recent studies have shown that this phenomenon is mediated via GC apoptosis. Ceramide, a product of sphingomyelin hydrolysis, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In the present study, we have examined the effect of ceramide on apoptotic cell death of GC in vitro. GCs were harvested by squeezing the antral follicles from the immature mice (3-4 weeks) and cultured in MEM medium with 10% fetal bovine serum. The cells were treated with various concentrations of ceramide (0 to 50 \mu M)and cultured up to 24 h.Cell death was determined by MTT cell viability assay and apoptosis was examined by acridine orange staining, in situ 3'-end labeling(TUNEL), and flow cytometry. Ceramid treatment induced apoptotic cell death of GC in a time- and a dose-dependent manner. Results of flow cytometric analysis showed that creamide-induced cell death was mostly confined to the $G_{0}$/$G_{1}$ cells. these results provide an evidence for ceramide as a lipid second messenger of apoptosis in mouse GC.

• Natural Product Sciences
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• v.10 no.1
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• pp.48-53
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• 2004

Two galloyl monosaccharides, 1,2,6-trigalloylglucose (1, TRgG) and 1,2,3,6- tetragalloylglucose (2, TEgG), were isolated from the stem-bark of Juglans mandshurica. Two galloylglucoses showed cytotoxic effects on human promyelocytic leukemia HL-60 cells. In order to elucidate their mechanism of action, we have investigated the flow cytometric analysis after Annexin V-FITC and PI staining, caspase-3 activity, and internucleosomal DNA fragmentation in HL-60 cells. HL-60 cells treated with both compounds 1 and 2 at 150 and $100\;{\mu}M$, respectively, led to a morphological features of apoptosis, such as plasma membrane blebbing and cell shrinkage. TRgG (1) and TEgG (2) increased the percentage of $FITC^+\;and\;FITC^+PI^+$ cells in flow cytometry after Annexin V-FITC and PI staining. The increase of apoptotic cells was preceded by the activation of caspase-3 reported to play a central role in apoptotic process and inducing internucleosomal DNA fragmentation. TEgG (2) showed to have stronger apoptosis inducing activity in HL-60 cell lines as compared with TRgG (1).

• The Journal of Pediatrics of Korean Medicine
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• v.20 no.1
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• pp.257-272
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• 2006

Objective : In this study, the ability of Oriental medicine Samultanggamibang(SMTG) to induce apoptosis was investigated in B16F10 melanoma cells. Method : Tetrazolium-based colorimetric assay was performed for cytotoxicity test. Several new assays for the basis of biochemical events associated with apoptosis such as DNA fragmentation by a flow cytometry, caspase-3 activation and PARP cleavage by Western blotting should be carried out potentially useful for the basis of biochemical events associated with apoptosis such as a flow cytometry and caspase-3 activation. Results : (1) The number of B16F10 melanoma cells was less than 30 % after exposure to 1 mg/ml SMTG for 48 h. SMTG increased cytotoxicity of B16F10 melanoma cells in a dose- and time-dependent manner. (2) The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 21 % at 24 h and 25 % at 48 h after treatment with 1 mg/ml SMTG. (3) SMTG-induced apoptosis was accompained by the activation of caspase-3 and the specific proteolytic cleavage of poly-ADP-ribose polymerase. (4) SMTG induces the activation of caspase-3 and the specific proteolytic cleavage of poly-ADP-ribose polymearse and eventually leads to apoptosis through c-Jun NH2-terminal protein kinase (JNK)-dependent manner in B16F10 melanoma cells. Conclusion : SMTG had a strong cytotoxic effect of B16F10 melanoma cells.