• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.6403-6407
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• 2012

Detoxifying enzymes are present in most epithelial cells of the human gastrointestinal tract where they protect against xenobiotics which may cause cancer. Induction of examples such as glutathione S-transferase (GST) and its thiol conjugate, glutathione (GSH) as well as NAD(P)H: quinoneoxidoreductase (NQO1) facilitate the excretion of carcinogens and thus preventing colon carcinogenesis. Pterostilbene, an analogue of resveratrol, has demonstrated numerous pharmacological activities linked with chemoprevention. This study was conducted to investigate the potential of pterostilbene as a chemopreventive agent using the HT-29 colon cancer cell line to study the modulation of GST and NQO1 activities as well as the GSH level. Initially, our group, established the optimum dose of 24 hours pterostilbene treatment using MTT assays. Then, effects of pterostilbene ($0-50{\mu}M$) on GST and NQO1 activity and GSH levels were determined using GST, NQO1 and Ellman assays, respectively. MTT assay of pterostilbene ($0-100{\mu}M$) showed no cytotoxicity toward the HT-29 cell line. Treatment increased GST activity in the cell line significantly (p<0.05) at 12.5 and $25.0{\mu}M$. In addition, treatment at $50{\mu}M$ increased the GSH level significantly (p<0.05). Pterostilbene also enhanced NQO1 activity significantly (p<0.05) at $12.5{\mu}M$ and $50{\mu}M$. Hence, pterostilbene is a potential chemopreventive agent capable of modulation of detoxifiying enzyme levels in HT-29 cells.

• Applied Biological Chemistry
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• 제44권1호
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• pp.1-6
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• 2001

본 연구에서는 Ttichoplusia ni 세포의 apoptosis 유도 및 억제 현상의 기초연구를 수행하였다. Apoptosis 유도제로 알려진 hygromycin B에 의한 세포 성장 저해는 $200\;{\mu}/ml$의 수준에서부터 나타났고, $400\;{\mu}/ml$ hygromycin B를 처리한 세포에서는 배양 후 2일부터 DNA가 분절되어지는 것을 확인할 수 있었다. 그러나 dexamethasone과 sodium butyrate를 첨가시 세포성장은 저해되었지만 DNA 분절현상이 보이지 않아 apoptosi의 유발여부를 확인할 수 없었다. 그리고 caspase 기능억제제의 apoptosis 지연효과를 보기 위해 $200\;{\mu}/ml$ hygromycin B로 apoptosis를 유발한 상태에서 Ac-DEVD-CHO를 첨가하여 세포성장을 비교해 본 결과 이 저해제에 의해 약 36%정도 apoptosis가 억제되었음을 확인하였다. N-acetylcysteine의 경우도 apoptosis지연 효과가 있었다. Bcl_계에 속하는 anti-apoptotic 유전자의 발현연구로서 apoptosis 저해 단백질인 bcl-2 유전자를 곤충세포에 형질전환시킨 후 이 단백질이 한시적으로 발현되는 것을 western blot분석법으로 확인하였으며 apoptosis가 지연된 곤충세포주의 개발이 가능하다는 결론을 보였다.

• Nutrition Research and Practice
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• 제9권6호
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• pp.586-591
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• 2015

BACKGROUND/OBJECTIVES: Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis. MATERIALS/METHODS: Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount$^{TM}$ cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate ($H_2DCF$-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction. RESULTS: Miconazole (3 and $10{\mu}M$) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole ($3{\mu}M$)-treated cells in a dose-dependent manner. Rhamnetin (1 and $3{\mu}M$) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation. CONCLUSIONS: Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels.

• Nutrition Research and Practice
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• 제2권4호
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• pp.322-325
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• 2008

The aim of present study was to investigate the effects of kaempferol on cellular proliferation and cell cycle arrest and explore the mechanism for these effects in human breast carcinoma MDA-MB-453 cells. Cells were treated with kaempferol at various concentrations (ranging from 1 to $200\;{\mu}M$) for 24 and 48 hrs. Kaempferol significantly inhibited cancer cell growth in cells exposed to 50 and $10\;{\mu}M$ of kaempferol and incubated for 24 and 48 hrs, respectively. Exposure to kaempferol resulted in cell cycle arrest at the G2/M phase. Of the G2/M-phase related proteins, kaempferol down-regulated CDK1 and cyclin A and B in cells exposed to kaempferol. In addition, small DNA fragments at the sub-G0 phase were increased by up to 23.12 and 31.90% at 10 and $50\;{\mu}M$ incubated for 24 and 48 hrs, respectively. The kaempferol-induced apoptosis was associated with the up-regulation of p53. In addition, the phosphorylation of p53 at the Ser-15 residue was observed with kaempferol. Kaempferol inhibits cell proliferation by disrupting the cell cycle, which is strongly associated with the induction of arrest at G2/M phase and may induce apoptosis via p53 phosphorylation in human breast carcinoma MDA-MB-453 cells.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.537-543
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• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• Toxicological Research
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• 제11권1호
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• pp.1-8
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• 1995

Fumonisins are a family of mycotoxins produced by the fungus Fusarium moniliforme which is a common contaminant in corn. Fumonisins are potent inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase), key enzymes in sphingolipid metabolism. The purpose of this study was to provide the evidence that the elevated levels of free sphingoid bases (primarily sphinganine) and depletion of complex sphingolipids were closely related to the inhibition of cell growth in LLC-$PK_1$ cells exposed to fumonisin $B_1$$(\leq 35 {\mu}M)$. Concentrations of fumonisin $B_1$ between 10 and $35 {\mu}M$ were known to inhibit cell growth without cytotoxicity in $LLC-PK_1$ cells (Yoo et al. Toxicol. Appl. Pharmacol. 114, 9-15, 1992). Cells exposed to 35$\mu M$ fumonisin B$_1$ for 48 and 72 hr developed a fibroblast-like (elongated and spindle-shaped) appearance and were less confluent than normal cells. At between 24 and 48 hr after exposure to fumonisin $B_1$ cells were beginning to show the inhibition of cell growth and at 72 hr the number of viable cells in fumonisin-treated cultures was about 50% of concurrent control cultures. During the 24 hr lag period preceding inhibition of cell growth, the free sphinganine levels in cells exposed to $35 {\mu}M$ fumonisin $B_1$ were highly elevated (approximately 230 fold higher than normal cells). The elevated levels of free sphinganine were $435\pm14$$pmoles/{10^6}$ cells at 48 hr and approximately TEX>$333\pm11$$pmoles/{10^6}$ cells in cells exposed to $35{\mu}M$ fumonisin$B_1$ at 72 hr, while the levels of free sphinganine in normal cells were less than 2$pmoles/{10^6}$ cells. Under the same condition, depletion of intracellular complex sphingolipids as a consequence of fumonisin inhibition of de novo sphingolipid biosynthesis and turnover pathway was appeared. Content of free sphingold bases in dividing cells was more elevated than in confluent cells at 24-48 hr after cells were exposed to $20{\mu}M$ fumonisin $B_1$. The dividing cells were showing the inhibition of cell growth at 48-72 hr and $20{\mu}M$ fumonisin $B_1$. The results of this study support the hypothesis that the inhibition of cell growth is very well related to the disruption of sphingolipid metabolism in $LLC-PK_1$ cells.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.457-462
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• 2002

Low Electromagnetic Field (EMF) intensity in the range of $1{\mu}T\;to\;10{\mu}T$(Tesla) was found to enhance the growth of CHO cells and the production of tPA in batch and perfusion cultivations. At $1{\mu}T\;intensity,\;1.3{\times}10^7$ viable cells/ml of maximum cell density and 80 mg/l of maximum tPA production were obtained in batch cultivation, compared to $2.8{\times}10^6$ viable cells/ml and 59 mg tPA/1 in unexposed case (control). A similar trend was observed in the perfusion process, where it was possible to obtain $1.2{\times}10^7$ viable cells/ml of maximum cell density and 81 mg tPA/l of maximum tPA production by more than 80 days of cultivation. However, there was not much difference between $1{\mu}T\;and\;10{\mu}T$ in perfusion cultivation, possibly due to better environmental growth conditions being maintained by continuous feeding of fresh medium into the reactor. On the contrary, both cell growth and tPA production were severely inhibited at higher than 1 mT intensity, showing no growth at 10 mT exposure. Specific growth rate was linearly correlated to specific tPA production rate at $1{\mu}T$EMF intensity, which represents a partially growth-related relationship. It was also found that a large amount of $Ca^2+$ was released at low EMF intensity, even though the cell growth was not much affected. Low EMF intensity significantly improved both cell growth and tPA production, and tPA production seemed to be more affected than the cell growth, possibly due to the changes of cell membrane characteristics. It can be concluded that the elaboration of EMF intensity less than $10{\mu}T$ could improve cell growth and tPA production, but mainly tPA secretion through batch or perfusion process in a bioreactor.

• Journal of Nutrition and Health
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• 제52권2호
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• pp.157-167
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• 2019

본 연구에서는 ice plant의 ethanol 추출물과 분획물의 항산화 및 대장암세포 성장억제 활성을 in vitro 수준에서 평가하는 것을 목적으로 하였다. Ethanol 추출물의 총 폴리페놀 함량 (3.7 mg GAE/g), 총 카로티노이드 함량 ($13.2{\mu}g/g$), DPPH 라디칼 소거활성 (21.0%), 철 환원력 (21.0%)보다 butanol 분획물의 총 폴리페놀 함량 (5.4 mg GAE/g), 총 카로티노이드 함량 ($86.6{\mu}g/g$), DPPH 라디칼 소거활성 (34.9%), 철 환원력 (80.8%)이 더 높았다. 또한 HCT116 대장암세포에서 세포 내 활성산소종 수준을 감소시키거나 세포 성장을 억제하는데 있어서 ethanol 추출물보다 butanol 분획물의 활성이 더 컸다. 대장암세포의 성장을 억제하는데 있어서 butanol 분획물이 ethanol 추출물보다 더 효과적이었던 것은 butanol 분획물의 apoptosis 유도활성이 ethanol 추출물의 활성보다 더 컸고 butanol 분획물만이 G2/M기억류활성을 나타냈기 때문인 것으로 생각된다. 앞으로 이와 같은 결과를 초래하는 주요 활성성분을 분리 동정하고 ice plant의 항산화 활성 및 대장암세포 성장억제 효과가 in vivo 수준에서 재현되는지 검증하며 이와 관련된 세부기전을 탐색하는 심도 있는 연구가 필요할 것으로 생각된다.

• 환경위생공학
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• 제20권3호
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• pp.10-16
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• 2005

Chestnut bark extract by methanol repressed the expression of tyrosinase gene of B16 mouse melanoma cell containing tyrosinase promoter. $10{\mu}g/ml{\ell}\;100{\mu}g/m{\ell}$, $1mg/m{\ell}$ of the extract repressed expression of tyrosinase gene about $38\%,\;47\%,\;and\;78\%$, respectively, compared with control. In the MTT assay, the same extract exhibited very low cytotoxicity at $1{\mu}g/m{\ell},\;10{\mu}g/m{\ell},\;100{\mu}g/m{\ell},\;and\;1mg/m{\ell}$, respectively. The fractions of Methylene chloride and ethyl acetate did not showed the repressive effect on the expression of tyrosinase gene, but the fraction of butyl alcohol repressed highly at $10{\mu}g/m{\ell}\;and\;100{\mu}g/m{\ell}$.

• Journal of Acupuncture Research
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• 제20권2호
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• pp.98-111
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• 2003

Objective : This study was designed to analyze the effects of bee venom and melittin on cell death in neuroblastoma cell line. Methods : MTT assay, morphologic method, DNA fragmentation, flow cytometry, immunocytochemistry analysis, RT-PCR and Western blot were performed. Results : The obtained results are summarized as follows: 1. The MTT assay demonstrated that neuroblastoma cell viability was significantly inhibitted dose-dependently by treatment with bee venom and melittin in comparison with control. 2. Cell culture demonstrated that control group proliferated highestly at he 5th day in comparison with the 4th day in bee venom and melittin group. And in bee venom and melitti group cell proliferation decreased 2.5 times than control group. 3. The morphologic study demonstrated that neuroblastoma cell showed apoptosis after treatment with bee venom and melittin for 6 hours using microscope. 4. The Flow cytometry demonstrated that apoptosis of neuroblastoma cell treated with bee venom and melittin was related with stop of cell cycle in stage of $G_0/G_1$. 5 .DNA fragmenation demonstrated that neuroblastoma cell treated with bee venom and melittin showed DNA ladder below 1 Kbp. 6. Immunocytochemistry assay demonstrated that Fos and MAPK which are related with cancer were down-regulated by treatment with bee venom and melittin. 7. RT-PCR analysis demonstrated that Fos and MAPK mRNA were transcripted. Fos was down-regulated form treatment with $5{\mu}g/ml$ bee venom and MAPK was down-regulated form $1{\mu}g/ml$ bee venom. 8. Western blot demonstrated that Fos was down-regulated from $1{\mu}g/ml$ bee venom whereas MAPK was expressed by $1{\mu}g/ml$ bee venom but down-regulated by $10{\mu}g/ml$ bee venom. Conclusions : We found that some cancer related genes ware down-regulated by treatment with bee venom and melittin. Further study is needed for investigating the anti-cancer effect of bee venom and melittin.