• Asian Pacific Journal of Cancer Prevention
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• 제15권12호
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• pp.5071-5076
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• 2014

Cholangiocarcinoma (CCA) is one of the aggressive cancers with a very poor prognosis. Several efforts have been made to identify and develop new agents for prevention and treatment of this deadly disease. In the present study, we examined the anticancer effect of luteolin on human CCA, KKU-M156 cells. Sulforhodamine B assays showed that luteolin had potent cytotoxicity on CCA cells with IC50 values of $10.5{\pm}5.0$ and $8.7{\pm}3.5{\mu}M$ at 24 and 48 h, respectively. Treatment with luteolin also caused a concentration-dependent decline in colony forming ability. Consistent with growth inhibitory effects, luteolin arrested cell cycle progression at the G2/M phase in a dose-dependent manner as assessed by flow cytometry analysis. Protein expression of cyclin A and Cdc25A was down-regulated after luteolin treatment, supporting the arrest of cells at the G2/M boundary. Besides evident G2/M arrest, luteolin induced apoptosis of KKU-M156 cells, demonstrated by a distinct sub-G1 apoptotic peak and fluorescent dye staining. A decrease in the level of anti-apoptotic Bcl-2 protein was implicated in luteolin-induced apoptosis. We further investigated the effect of luteolin on JAK/STAT3, which is an important pathway involved in the development of CCA. The results showed that interleukin-6 (IL-6)-induced JAK/STAT3 activation in KKU-M156 cells was suppressed by treatment with luteolin. Treatment with a specific JAK inhibitor, AG490, and luteolin diminished IL-6-stimulated CCA cell migration as assessed by wound healing assay. These data revealed anticancer activity of luteolin against CCA so the agent might have potential for CCA prevention and therapy.

• Advances in Traditional Medicine
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• 제1권1호
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• pp.45-54
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• 2000

Cannabidiol derivatives (1, 2 and 3), and 5-fluorouracil (4, 5-FU) were tested for their growth inhibitory effects against human oral epitheloid carcinoma cell lines (KB) using two different 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and sulforhodamine B protein (SRB) assay. These compounds showed a potent inhibitory activity in vitro in the micromolar range against KB cell lines. In general, the antitumor activity of these compounds (1, 2, 3 and 4) was in a dose-dependent over the micromolar concentration ranges from $1\;{\mu}M\;to\;100\;{\mu}M$. The comparison of $IC_{50}$ values of these compounds in tumor cell lines shows that their susceptibility to these compounds decreases in the following order: CBD > 5-FU > CBDME > CBDDE by the MTT assay and SRB assay. Cannabidiol derivatives (1, 2 and 3), and 5-FU were tested for their cytotoxic effects on NIH 3T3 fibroblasts using two different MTT assay and SRB assay. These compounds exhibited potent cytotoxic activities in vitro in the micromolar range against NIH 3T3 fibroblasts. In general, the cytotoxic activities of these compounds (1, 2, 3 and 4) were in a dose-dependent over the micromolar concentration range $1\;{\mu}M\;to\;100\;{\mu}M$. The comparison of $CD_{50}$ values of these compounds on NIH 3T3 fibroblasts shows that their susceptibility to these compounds decreases in the following order; CBD > 5-FU > CBDDE > CBDME by MTT assay, CBD > 5-FU > CBDME > CBDDE by SRB assay. These results suggest that cannabidiol (1, CBD) retains the most growth-inhibitory activity against KB cell lines.

• 동의생리병리학회지
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• 제17권5호
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• pp.1202-1207
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• 2003

In order to clarify the neuroprotective effect of Cornu Saigae Tataricae(CST) water extract on cultured mouse spinal motor neuron damaged by hydrogen peroxide (H₂O₂), MTT [3-(4,5-dimethylthiazole-2-yl)- 2,5-diphenyltetrazolium bromide] assay, LDH (Lactate Dehydrogenase) activity assay and SRB (Sulforhodamine B) assay were carried out after the cultured mouse spinal motor neuron were preincubated with various concentrations of CST water extract for 3 hours prior to exposure of hydrogen peroxide Cell viability of cultured mouse spinal motor neurons exposed to various concentrations of hydrogen peroxide for 6 hours was decreased in a dose-dependent manner. MTT50 values were 40 uM hydrogen peroxide. Cultured mouse spinal motor neurons in the medium containing various concentration of hydrogen peroxide for 6 hours showed increasing of LDH activity and decreasing of total protein synthesis. We know that hydrogen peroxide was toxic on cultured spinal motor neurons. Pretreatment of CST water extract for 3 hours following hydrogen peroxide prevented the hydrogen peroxide-induced neurotoxicity such as increasing of LDH activity and decreasing of total protein synthesis. These results suggest that hydrogen peroxide shows toxic effect on cultured spinal motor neurons and CST water extract is highly effective in protecting the neurotoxicity induced by hydrogen peroxide.

• Toxicological Research
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• 제20권1호
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• pp.37-42
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• 2004

본 연구는 Phellinus gilvus(PGE), Phellinus linteus (PLE) 그리고 Phellinus baumil(PBE)의 열수추출물에 대하여 SRB법과 MTT법으로 종양세포주(Sarcoma 180과 P388)를 이용하여 항암활성을 비교ㆍ평가하였다. 종양세포주는 PGE, PLE, PBE(7., 15, 30 ug/ml) 그리고 Doxorubicin(DOX)(0.001～10 mM)으로 처리되었다. 그 결과 DOX, PGE 그리고 PLE는 종양세포주에 대하여 농도의존적으로 억제하는 결과를 보였지만, PBEssm 30 ug/ml의 농도에서만 억제되는 항암활성을 보여주었다. 결론적으로 이 연구에서 사용된 PGE, PLE 및 PBE는 Sarcoma 180과 P388에 항암활성을 보여주었다. PLE는 sarcoma 180종양세포에 가장 효과가 뛰어났으나(p<0.05), SRB법에서는 PGE가 P388에 대해 가장 큰 항암활성을 나타내었다(p<0.05).

• 대한의생명과학회지
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• 제11권3호
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• pp.319-326
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• 2005

It is well known that oxidative stress of reactive oxygen species (ROS) may be a causative factor in the pathenogenesis of bone disorder on osteoblast or osteoclast. The purpose of this study was to evaluate the cytotoxicity of oxidative stress, protective effect of glutamate receptor antagoinst against ROS-induced osteotoxicity, secretion of tumor necrosis factor $(TNF)-\alpha$ and the expression of c-fos gene in the cultured rat osteoblasts and osteoclasts. Cell viability by MTS assay or !NT assay, activity of glutathione peroxidase (GPx), lipid peroxidation (LPO) activity, protein synthesis by sulforhodamine B (SRB) assay, alkaline phosphatase (ALP) activity, lactate dehydrogenase (LDH) activity, MTS assay for NMDA (N-methyl-D-aspartate) receptor antagonist or AMPA/kainate receptor antagonist, measurement for $TNF-\alpha$, and c-fos gene expression were performed after these cells were treated with or without various cocentrations of xanthine oxidase (XO), hypoxanthine (HX), D-2-amino-5-phosphonovaleric acid (APV), 7-chlorokynurenic acid (CKA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), respectively. In this study, XO/HX showed decreased cell viability and glutathione peroxidase (GPx) activity, but it showed increased LPO activity, $TNF-\alpha$ secretion and c-fos expression. APV and CKA incresed protein sythesis and ALP activity. While, CNQX or DNQX did not show any protective effect in LDH activity or cell viability. From these results, XO/HX showed cytotoxic effect in cultured rat osteoblast or osteoclast, and also NMDA receptor antagonist such as APV or CKA was effective in blocking XO/HX-induced osteotoxicity in these cultures.

• 생약학회지
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• 제51권2호
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• pp.93-99
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• 2020

This study was performed to find anti-inflammatory or antitumor compounds from the polar fraction obtained from the extract of Clinopodium chinense var. shibetchense (H. Lev) Koidz (Labiatae). Chromatography of the BuOH fraction yielded two flavonoid glycosides (compounds 1 and 2) and two saponins (compounds 3 and 4). On the basis of spectroscopic data, compounds 1 and 2 were identified to be ponciretin 7-O-α-L-rhamnopyranosyl-(1→6)-α-D-glucopyranoside (neoponcirin) and naringenin 7-O-α-L-rhamnopyranosyl-(1→6)-α-D-glucopyranoside (isonaringin). Compounds 3 and 4 were identified to be 3-O-{β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranosyl}-saikogenin F (buddlejasaponin IV) and 3-O-{β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranosyl}-21β-hydroxysaikogenin F (clinoposaponin XV). In addition, ursolic acid (5) was isolated and identified from the CHCl₃ fraction. Inducible nitric oxide synthase (iNOS) assay and sulforhodamine B (SRB) assay were performed to lead a potential anti-inflammatory or anti-tumor compounds from C. chinense var. shibetchense. Of the four compounds (1 - 4), compound 3 considerably inhibited cancer cell growth and NO production (IC₅₀s, 5.59 μM in iNOS assay and 6.62 - 14.88 μM in SRB assay).

• Journal of Microbiology and Biotechnology
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• 제26권11호
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• pp.1855-1862
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• 2016

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen, posing a high risk of disease to infants and immunocompromised individuals. In order to develop a quick, easy, and sensitive assay for detecting C. sakazakii, a rabbit anti-C. sakazakii immunoglobulin G (IgG) was developed using sonicated cell protein from C. sakazakii. The developed anti-C. sakazakii (IgG) was of good quality and purity, as well as species-specific. The developed rabbit anti-C. sakazakii IgG was attached to the surface of a sulforhodamine B-encapsulated liposome to form an immunoliposome. A test strip was then prepared by coating goat anti-rabbit IgG onto the control line and rabbit anti-C. sakazakii IgG onto the test line, respectively, of a plastic-backed nitrocellulose membrane. A purple color signal both on the test line and the control line indicated the presence of C. sakazakii in the sample, whereas purple color only on the control line indicated the absence of C. sakazakii in the sample. This immunochromatographic strip assay could produce results in 15 min with a limit of detection of $10^7CFU/ml$ in C. sakazakii culture. The immunochromatographic strip assay also showed very good specificity without cross-reactivity with other tested Cronobacter species. Based on these results, the developed immunochromatographic strip assay is efficient for the detection of C. sakazakii and has high potential for on-site detection.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.509-516
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• 2003

A model system for the immnunochemical detection of Escherichia coli O157:H7 using a combined immunomagnetic separation (IMS) and test-strip liposome immunoassay (LIA) procedure was developed. Immunomagnetic beads coated with anti-E. coli O157 IgG antibodies were used to separate the E. coli O157 (including the H7 serotype) from culture. Immunoliposomes, whose surface was conjugated to goat anti-E. coli O157:H7 IgG and which encapsulated the marker dye, sulforhodamine B, were used as a detection label. The test strip, onto which antibodies to goat IgG were immobilized, was the immunosensor capturing immunoliposomes that did not bind to E. coli O157:H7 on the immunomagnetic bead-E. coli O157:H7 complexes. In experiments, pure cell culture suspensions of $10^5 E.$ coli O157:H7 organisms per ml produced a measurable signal inhibition, whereas a weak yet detectable signal inhibition occurred with $10^3CFU/ml$. The inhibition signals increased, when the incubation time for IMS was extended to 90 min and higher IgG-tag density (0.4mol%) was used on the liposomes. With 0.2 and 0.4mol% IgG-tagged liposomes, the IMS-LIA procedure showed more improved signal inhibitions than those of a direct (no IMS) LIA. The combined assay, which measures the instantaneous signal from immunoliposomes, can be completed within 90 min, making it significantly faster than conventional plating methods and enzyme-linked immunosorbent assay (ELISA). Accordingly, it is quite feasible to use the combined immunoassay format of IMS and dye-loaded immunoliposomes for the detection of E. coli O157:H7.

• 대한치과교정학회지
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• 제27권3호
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• pp.503-513
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• 1997

본 연구는 산소 장력이 사람의 치주인대에서 유래된 세포의 활성과 세포외 기질 및 cytokine에 미치는 영향을 알아 보고자 저산소 및 과산소 조건에서 배양한 후 세포의 활성, 총 단백질 합성, 교원질 합성, 그리고 $IL-1{\beta}$, IL-6, $TNF-{\alpha}$를 측정하였다. 교정치료를 위해 발거한 제 1 소구치에서 사람의 치주인대 세포를 채취하여 $37^{\circ}C,\;5\%\;CO_2,\100\%$ 습도의 환경에서 배양한 후 5 내지 6 계대배양하여 실험에 사용하였다. Gaspack system에 $0.2{\mu}m$ Millipore filter를 부착하여 세균 감염을 막았고, 저산소군에는 $10\%\;O_2,\;5\%\;CO_2,\;85\%\;N_2$ 가스를, 과산소군에는 $90\%\;O_2,\;5\%\;CO_2,\;5\%\;N_2$ 가스를 연결하여 산소 장력에 변화를 주었으며 대조군에는 $5\%\;CO_2,\;95\%$ 공기를 공급하였다. $37^{\circ}C$ 에서 2, 4, 6일간 배양한 후에 세포활성을 측정하기 위해 tetrazolium(MTT) assay를 시행하였고 sulforhodamine B(SRB) assay를 이용하여 총 단백질 합성을 알아보았으며, 4-hydroxyproline 측정으로 교원질 합성을 측정해 보았다. 또한 효소면역측정법으로 $IL-1{\beta}$, IL-6, $TNF-{\alpha}$를 측정하여 다음과 같은 결과를 얻었다. 1. 저산소근에저 세포 활성과 총 단백질 합성은 대조군보다 다소 높거나 같은 정도이었다. 2. 과산소군에서 세포 활성은 대조군보다 낮았으며 총 단백질 합성도 다소 감소되었다. 3. 교원질 합성은 저산소군과 과산소군에서 대조군보다 유의하게 감소되었다가 배양기간이 길어짐에 따라 증가되어 대조군과 유사한 정도에 달했다. 4. 효소면역법 측정 결과 IL-6, $TNF-{\alpha},\;IL-1{\beta}$ 순으로 다량 검출되었다. 5. IL-6, $TNF-{\alpha},\;IL-1{\beta}$는 저산소군과 과산소군에서 배양기간이 길어짐에 따라 대조군에 비해 급격히 증가하였다. 5. IL-6, $TNF-{\alpha}$는 과산소 조건에서 배양 6일 후에는 대조군에 비해 유의하게 증가하였으며 이는 배양 2일 또는 4일 후보다도 유의하게 증가한 것이었다.

• 한국환경농학회지
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• 제16권2호
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• pp.149-155
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• 1997

Carbofuran에 의하여 손상된 NIH3T3 섬유모세포(mouse skin fibroblast)를 보상시킬 수 있는 물질을 개발하기 위한 기초자료를 제시하고자 carbofuran이 NIH3T3 섬유모세포에 미치는 세포독성을 검정하고, carbofuran의 독성에 대한 phenobarbital sodium(PB) 및 3-methylcholantherene(3-MC)의 보상효과를 비교${\cdot}$분석하였다. Carbofuran의 $IC_{50}$ 결정은 배양중인 NIH3T3 섬유모세포의 각 well당 1, 25, 50, $100{\mu}M$의 carbofuran을 첨가하여 48시간 배양한 후 MTT(Tetrazolium MTT), NR(Neutral red) 및 SBR(Sulforhodamine B protein)정량을 실시하여 이들에 대한 각각의 $IC_{50}$을 구하였으며, 여기에서 구한 carbofuran의 $IC_{50}$농도와 여러 농도의 PB 또는 3-MC를 배양액에 첨가하여 48시간 배양한 후 MTT, NR 및 SRB 정량을 실시하여 보상효과를 측정하고 광학현미경적 관찰을 실시하였다. Carbofuran의 세포독성 실험결과를 보면 MTT흡광도는 carbofuran의 농도증가에 따라 감소하였으며 $MTT_{50}$은 $60.7{\mu}M$이었고, NR흡광도는 $100{\mu}M$농도에서 급격히 감소하였으며, $NR_{50}$은 $82.5{\mu}M$이었고, SRB흡광도는 $50{\mu}M$농도에서 급격히 감소하였으며 $SRB_{50}$은 $87.0{\mu}M$로써 50%의 세포독성을 나타냈다. 보상효과 실험에서는 carbofuran IC50과 PB의 조합처리의 경우 MTT 정랑과 NR정량에서는 유사하게 PB $100{\mu}M$처리군에서부터 유의성있는 보상효과가 나타났으나, SRB정량에서는 보상효과가 인정되지 않았다. Carbofura $IC_{50}$과 3-MC의 조합처리의 경우 MTT정량은 3-MC $50{\mu}M$처리군에서부터, NR정량과 SRB정량의 경우는 동일하게 3-MC $100{\mu}M$처리군에서부터 유의성있는 보상효과가 나타났다. 세포의 광학현미경적 관찰소견의 경우에서도 carbofuran과 PB 또는 3-MC 조합처리 실험군 모두에서 세포가 회복되는 것을 관찰할 수 있었다. 이상의 결과에서 PB와 3-MC 모두 carbofuran의 세포독성을 감소시킬 수 있는 물질임을 알 수 있었다.