• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.293-299
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• 2000

Many methods have been developed for screening chemicals with hormonal activity. Using recombinant yeasts expressing either human estrogen receptor [Saccharomyces cerevisiae ER + LYS 8127 (YER)] or androgen receptor [S. cerevisiae AR + 8320 (YAR)], we evaluated the hormonal activities of several chemicals by induction of ${\beta}-galactosidase$ activity. The chemicals were $17{\beta}-estradiol$ (E2), testosterone (T), ${\rho}-nonylphenol$ (NP), bisphenol A (BPA), genistein (GEN), 2-bromopropane (2-BP), dibutyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and butylparaben (BP). To assess the estrogenicity of NP, the result of the in vitro recombinant yeast assay was compared with an E-screen assay using MCF-7 human breast cancer cells and an uterotrophid assay using ovariectomized mice. In the YER yeast cells, E2, NP, BPA, GEN, and BP exhibited estrogenicity in a doseresponse manner, while TCDD did not. All the chemicals tested, except T, did not show androgenicity in the YAR yeast cell. The sensitivity of the yeast (YER) assay system to the estrogenic effect of NP was similar to that of the E-screen assay. NP was also estrogenic in the uterotrophic assay. However, in terms of convenience and costs, the yeast assay was superior to the E-screen assay or uterotrophic assay. These results suggest that the recombinant yeast assay can be used as a rapid tool for detecting chemicals with hormonal activities.

• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.198-205
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• 2004

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Recent industrialized industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA. Some flavonoids such as genistein, daidzein, chrysin, naringenin and morin were also investigeted. These flavonoids decreased B(k)F infuced luciferase activity at low concentration. But, these flavonoids exhibited stimulatory effect at high concentration.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.213-217
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• 1998

This study was performed to observe cytotoxic effect of the pine needle extracts against cancer cell lines including human gastric carcinoma (KATOIII), human lung carcinoma (A549), human hepatocellular carcinoma (Hep3B) and human breast adenocarcinoma (MCF-7) using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and SRB (sulforhodamine B) method. The extracts were prepared by step-wise fractionation of ethanol extract of pine needles using diethylether, chloroform, ethylacetate, butanol and water. The growth of the cancer cells in medium containing pine needle extracts were significantly inhibited degree in proportion to the increase of the extract concentration. A significant shrinkage of Hep3B cells was observed when the cells were exposed into 0.5, 1 mg/mL of pinus rigida extract.

• Journal of the Korean Society of Food Science and Nutrition
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• v.30 no.5
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• pp.921-927
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• 2001

This study was performed to investigated the effects on the cytotoxicity and antigenotoxicity of Cordyceps militaris extracts on the human cancer cell lines. The ethanol extract and five fractions which were hexane, chloroform, ethylacetate, butanol and aqueous were screened for crytotoxicity on human lung carcinoma(A549). human breast adenocarcinoma (MCF-7) human epitheloid carcinoma(HeLa), human fibrosarcoma(HT1080) human hepatocellular carcinoma(Hep3B), human gastric carcinoma(KATOIII) and chronic myelogenous leukemia(K562) cell by SRB and MTT assays. The results showed that growth inhibition rates of the human cancer cell in the presence of Cordyceps militaris were inhibited with increasing concentration of the extract. The ethanol extract from Cordyceps militaris had strong inhibitory effects in1 mg/mL treatment by SRB assay , showing 89.4%, 85.7%, 72.9% and 65.5% inhibition in HT1080, HeLa, Hep3B and A549, respectively. The treatment of 1 mg/mL hexane fraction by SRB assay had the strongest cytotoxicity with 97.0% on HT1080 followed by MCF-7(92.9%) and HeLA(90.3%). The inhibition ration on KATOIII by MTT assay was much higher in the butanol (83.7%) and aqueous (80.4%) than in the ethanol extract (61.5%) And also, K562 showed similar tendency with KATOIII. The effects of Cordyceps militaris extracts on the frequencies of micronucleated polychromatic erythrocytes (MNPCEs) induced by N-methyl-N-nitro-N-nitrosoguanidime(MNNG) were investigated in the bone-marrow cells of ICR male mice. The amount of 10, 20, 40 and 80 mg/kg of each extract were administered to animals immediately after injection of MNNG, and the exposure time was 36 hours. Significant reductions(p<0.05) with 39.7%, 52.7%, 71.4% and 83.9% were observed in the frequencies of MNPCE when 10, 20, 40 and 80 mg/kg of the hexane fraction of Coryceps militarus extracts were given to the mice.

• Food Science of Animal Resources
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• v.32 no.5
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• pp.612-617
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• 2012

The antioxidant, antimicrobial, and cytotoxic activities of ovotransferrin were investigated in vitro. The antioxidant capacity of ovotransferrin was evaluated using the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging method, antimicrobial effects using the agar well diffusion method, and cytotoxicity using the 3-(4,5-dimethylthizol-2-yl)-2,5-diphenylatetetrazolium bromide (MTT) assay. The DPPH radical-scavenging capacity of ovotransferrin at 1 mg/mL level reached approximately 60% after 48 h of reaction. The antimicrobial effects of ovotransferrin against common food-borne pathogens, Staphylococcus aureus KCCM 32395, Bacillus cereus KCCM 40935, Listeria monocytogenes ATCC 15313, Escherichia coli O157:H7 ATCC 43895, and Helicobacter pylori HpKCTC 26695 were dose dependant. Gram-positive bacteria was more sensitive to ovotransferrin than gram-negative bacteria. Ovotransferrin showed stronger antimicrobial effect against L. monocytogenes than other gram-positive bacteria tested. The cytotoxicity of ovotransferrin was evaluated in human cancer cell lines, various tissue origins, including the larynx (Hep-2), stomach (AGS), lung (SK-MES-1), liver (HepG2), breast (MCF-7), cervix (HeLa), and colon (HT-29). Ovotransferrin displayed relatively high cytotoxicity (${\leq}60%$ inhibition effects) at 40 mg/mL. At lower concentrations (${\leq}10mg/mL$), however, ovotransferrin cytotoxic effects were not significant in all cancer cell lines tested. These results indicated that ovotransferrin has potential to be used as an antioxidant or antimicrobial agent in foods or a pharmaceutical agent against cancers.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.551-563
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• 2022

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50℃ and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30℃, 40℃, and 50℃, respectively. The enzyme reserved its activity at 30℃ and 37℃ up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC₅₀ of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC₅₀ values of 1.53 and 18 IU, respectively.

• Molecules and Cells
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• v.27 no.3
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• pp.351-357
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• 2009

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, $17{\beta}$-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER ${\alpha}$ as compared to $17{\beta}$-estradiol and genistein. Despite poor binding to ER ${\alpha}$, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER ${\alpha}$ at $Ser^{118}$. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

• Journal of the East Asian Society of Dietary Life
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• v.15 no.5
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• pp.542-548
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• 2005

In recent years, propolis has attracted much attention as an useful substance in medicine and functional food, even if it is known as a natural remedy in folk medicine since ancient times. propolis was registered as natural food since 1995 on Korean Food Act by Korean Food and Drug Administration(KFDA). The present study demonstrated the optimization of isolation of crude propolis by ethanol, and tumoricidal effect of pro polis. The optimal concentration of ethanol to separate a high quantity of propolis was $60\%$. The cytotoxic effect of ethanol extracted propolis against various cancer cell lines including murine lymphoma (Sarcoma-180), murine T-lymphoma (YAC-1), human breast carcinoma (MCF-7), human gastric carcinoma (KATOIII) and human hepatocellular carcinoma (Hep3B) and human lung adenocarcinoma (A-549) was observed using SRB and MIT assay. In order to investigate the curative activity by oral administration of propolis on tumor, ICR mice was subcutaneously implanted Sarcoma 180. In 300mg/kg and 600mg/kg propolis administered group, development of implanted tumors was inhibited by $40.9\%\;and\;67.9\%$ at 16th day, respectively. In the same dose of propolis administered group, development of implanted tumors was inhibited more strongly with dose dependent manner. Therefore, these data suggested propolis may show tumoricidal effects. In conclusion, these results indicate that propolis, one of the few natural remedies, can be used as functional food with tumoricidal effects.

• Journal of Life Science
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• v.13 no.6
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• pp.799-804
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• 2003

In order to verify the occurrence of an estrogenic compound in natural products, the estrogenic activity was measured using an in vitro detection system. For this system, human breast cancer cell line MCF7 was transfected using an estrogen responsive CAT reporter plasmid. Estrogenic activities of photosynthetic algae spirulina and sea lettuce were evaluated using this system. Estrogenic activities of a $500\mug/ml\; and\; 50 \mug/ml$ ethanol extracts of spirulina were as much as that of $10^{-8}$M standard solution (17$\beta$-estradiol) and activity of $5\mug/ml$ ethanol extract of spirulina was as much as that of $10^{-10}$ M standard solution. However, no significant estrogenic activity was observed using sea lettuce extract. Estrogenic activities of marine animals, such as star fish and shrimp, were also evaluated using this system, however, no significant estrogenic activity was observed in these extracts. In this result, it is confirmed that spirulina extract possesses estrogenic compound.

• Advances in Traditional Medicine
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• v.8 no.3
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• pp.228-235
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• 2008

This study was conducted to evaluate the estrogen activity of silkworm (Bombyx mori) pupa extracts and their fractions. Powdered samples of freeze-dried silkworm pupa were extracted at room temperature (RT), $40^{\circ}C$, $60^{\circ}C$, $80^{\circ}C$, and $100^{\circ}C$ in water (D.W), chloroform, ethyl acetate, and methanol for 6h and then filtered (0.45 um). The extracts were then freeze-dried. The estrogenic activity of these extracts was then investigated by competition binding assays using estrogen receptor ${\alpha}\;(ER{\alpha})$ and $ER{\beta}$, and by evaluating their effects on the proliferation of the human breast cancer cell line, MCF-7. Among the extracts evaluated, water extracts prepared at RT showed the highest binding affinity to $ER{\alpha}$ ($IC_{50}$, 1.76 ug/ml) and $ER{\beta}$ ($IC_{50}$, 0.07 ug/ml). In addition, MCF-7 cells that were treated with 62.5 ug/ml of the RT extract showed the greatest increase in proliferation (2-fold; 1291.79%) when compared to control cells (659.82%). Next, the water extract that was prepared at RT (sample 1) was dissolved in D.W. and further fractionated using a Dowex 50W - 8X ($H^+$) column. The flow-through and wash were then pooled together and freeze-dried (sample 2). The bound materials were then eluted with 20 mM NaCl, after which they were applied to a Dowex 1X2 - 200 ($Cl^-$) column and washed with D.W. to remove the sodium ions. The eluants were then freeze-dried (sample 3). Of these fractions, sample 2 showed the highest binding affinity to ER{\alpha} ($IC_{50}$, 1.44 ug/ml) and $ER{\beta}$ ($IC_{50}$, 1.18 ug/ml). In addition, MCF-7 cells that were treated with sample 2 (15.6 ug/ml) showed the largest increase in growth (1159.39%) when compared to control cells (525.26%). Taken together, these results suggest that the fraction of the RT water extract of silkworm pupa referred to as sample 2 may be useful as a phytoestrogen.