• International Journal of Industrial Entomology and Biomaterials
• /
• 제9권2호
• /
• pp.173-181
• /
• 2004

To delay the onset of apoptosis in the culture, transformed Tn 5B1-4 cells harboring anti-apoptotic genes, bcl-2 and baculovirus p35, have been established and analyzed for their anti-apoptotic ability in suspension culture using spinner flasks. In the suspension culture at agitation speeds of 100 rpm and 200 rpm, the cell growth of cell clone expressing Bcl-2 protein was much higher than other two clones and the maximum cell density of the clone was 6.0 ${\times}$ 10$^{6}$ cells/ml and 6.2 ${\times}$ 10$^{6}$ cells/ml at day three of the incubation. On the other hand, the cell growth of cell clone expressing baculovirus protein P35 was much higher than other two clones in suspension culture at agitation speed of 300 rpm and the maximum cell density of the clone was 6.1 ${\times}$ 10$^{6}$ cells/ml at day three of the incubation. Based on the pattern of genomic DNA laddering and the microscopic observation of apoptotic bodies, the more apoptotic bodies are induced in Tn 5B1-4 control cell clone at higher agitation speed. This result shows that the shear stress can be a main factor in inducing apoptosis in spinner flask culture. At low agitation speed, cell clone expressing Bcl-2 was more effective in delaying the onset of apoptosis than the cell clone expressing P35. On the other hand, at high agitation speed, cell clones expressing baculovirus P35 was more effective in delaying the onset of apoptosis than the cell clone expressing Bcl-2. Therefore, anti-apoptotic genes, bcl-2 and baculovirus p35, can playa distinct role depending on agitation speed in the suspension culture.

• The Korean Journal of Physiology and Pharmacology
• /
• 제20권6호
• /
• pp.595-603
• /
• 2016

Ribosomal S6 kinase is a family of serine/threonine protein kinases involved in the regulation of cell viability. There are two subfamilies of ribosomal s6 kinase, (p90rsk, p70rsk). Especially, p90rsk is known to be an important downstream kinase of p44/42 MAPK. We investigated the role of p90rsk on ethanol-induced cell proliferation of HepG2 cells. HepG2 cells were treated with 10~50 mM of ethanol with or without ERK and p90rsk inhibitors. Cell viability was measured by MTT assay. The expression of pERK1, NHE1 was measured by Western blots. The phosphorylation of p90rsk was measured by ELISA kits. The expression of Bcl-2 was measured by qRT-PCR. When the cells were treated with 10~30 mM of ethanol for 24 hour, it showed significant increase in cell viability versus control group. Besides, 10~30 mM of ethanol induced increased expression of pERK1, p-p90rsk, NHE1 and Bcl-2. Moreover treatment of p90rsk inhibitor attenuated the ethanol-induced increase in cell viability and NHE1 and Bcl-2 expression. In summary, these results suggest that p90rsk, a downstream kinase of ERK, plays a stimulatory role on ethanol-induced hepatocellular carcinoma progression by activating anti-apoptotic factor Bcl-2 and NHE1 known to regulate cell survival.

• Annals of Surgical Treatment and Research
• /
• 제95권5호
• /
• pp.240-248
• /
• 2018

Purpose: This study aimed to validate the synergistic effect of ABT-737 on docetaxel using MDA-MB-231, a triple negative breast cancer (TNBC) cell line overexpressing B-cell lymphoma-2 (Bcl-2). Methods: Western blot analysis was performed to assess expression levels of Bcl-2 family proteins and caspase-related molecules. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution was determined by flow cytometry analysis. Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) was used for pretreatment to assess the role of caspases. Results: Cell viability of MDA-MB-231 after combination treatment with ABT-737 and docetaxel was significantly lower than that after docetaxel or ABT-737 monotherapy based on MTT assay (both P < 0.001), with a combination index of 0.41. The proportion of sub-G1 population after combination treatment was significantly higher than that after docetaxel or ABT-737 monotherapy (P = 0.001, P = 0.003, respectively). Pretreatment with z-VAD-fmk completely restored cell viability of MDA-MB-231 from apoptotic cell death induced by combination therapy (P = 0.001). Although pro-caspase-8 or Bid did not show significant change in expression level, pro-casepase-9 showed significantly decreased expression after combination treatment. Cleaved caspase-3 showed increased expression while poly (ADP-ribose) polymerase cleavage was induced after combination treatment. However, hypoxia-inducible factor 1-alpha and aldehyde dehydrogenase 1 totally lost their expression after combination treatment. Conclusion: Combination of ABT-737 with docetaxel elicits synergistic therapeutic effect on MDA-MB-231, a TNBC cell line overexpressing Bcl-2, mainly by activating the intrinsic pathway of apoptosis. Therefore, adjunct of ABT-737 to docetaxel might be a new therapeutic option to overcome docetaxel resistance of TNBCs overexpressing Bcl-2.

• 대한예방한의학회지
• /
• 제16권1호
• /
• pp.71-80
• /
• 2012

Objective : The aim of this study is to evaluate anti-cancer effects of $Ampelopsisradix$ Extract (AE) on human lung cancer A549 cells. Method : The apoptotic activities and cell growth arrest activities of AE were measured using 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The molecules involved in apoptotic process were assessed by western blotting. Result : Treatment of AE potently reduced cell viability in a dose-dependent manner in A549 cells. AE (100-500 ${\mu}g/m{\ell}$) resulted in apoptosis via activation of caspase 9 following PARP cleavage in a time-and dose-dependent manner. The levels of Bax and Bad levels were increased by AE with a concomitant decrease of Bcl-xL. In addition, AE at the low dose (30 ${\mu}g/m{\ell}$) significantly inhibited cell growth in the presence of serum. Conclusion : AE has the potential as a therapeutic agent against lung cancer.

• Toxicological Research
• /
• 제18권2호
• /
• pp.183-190
• /
• 2002

The mechanism by which catechin-mediated cytotoxicity against tumor cells remains to be elusive. To elucidate the mechanical mights of anti-tumor effects, (-)epigallocatechin-gallate (EGCG) of catechin was applied to human prostate cancer DU 145 cells. Cell viability was measured by crystal violet staining. Cell lysates were wed to measure the catalytic activity of caspases by using fluorogenic peptide: Ac-DEVD-AMC for caspase-3 protease, Z-IETD-AFC for caspase-8 protease, Ac-LEHD-AFC for caspase-9 protease as substrates. The equal amounts of protein from cell lysate was separated on SDS-PAGE and analyzed by western blotting with anti-Fas antibody, anti-FasL antibody, anti-BCL2 antibody and anti-Bax antibody. (-)EGCG induced the death of DUl45 cells, which was revealed as apoptosis shown by DNA fragmentation. (-)EGCG induced the activation of caspase family cysteine proteases including caspase-3, -8 and -9 proteases in DU145 cells. Also, (-)EGCG increased the expression of Fas and Fas ligand (FasL) protein in DU145 colls. The expression level of BCL2 was decreased in (-)EGCG treated DU145 cells, whereas Bax protein was increased in a time-dependent manner. We suggest that (-)EGCG-induced apoptosis of DU145 cells is mediated by signaling pathway involving caspase family cysteine protease, mitochondrial BCL2-family protein and Fas/FasL.

• 대한한방부인과학회지
• /
• 제15권4호
• /
• pp.1-16
• /
• 2002

This work examines the effect of treatment with Gyukhachukeotang on the growth of uterine myomal cells. Comparisons of cell growth, MAP kinase activity and expression of bcl-2 (apoptosis-related gene) were made between the control and experimental samples. The results as fallows; 1. Any concentration of Gyukhachukeotang above 0.01% yielded growth inhibition. Concentrations of 5% and 10% stopped all cell growth, demonstrating the effectiveness of Gyukhachukeotang as a growth inhibitor on uterine myomal cells. 2. The MAP kinase activity in uterine myomal cells treated with Gyukhachukeotang was decreased to a high degree at the concentration of 10%, and some inhibition of activity was detected at a concentration of 5%. 3. The expression of bcl-2, a Cell Apoptosis-related gene, in uterine myoma cells treated with Gyukhachukeotang was gradually increased with increasing concentration of Gyukhachukeotang. These results indicate the ability of Gyukhachukeotang to control uterine myomal cell growth, with concurrent reduction of MAP kinase activity. Treatment with Gyukhachukeotang appears to trigger a normal apoptosis response, as indicated by increased bcl-2 expression. This observed increase in apoptosis indicates that Gyukhachukeotang is an appropriate prescription to treat uterine myomal cells.

• Molecular & Cellular Toxicology
• /
• 제2권1호
• /
• pp.15-20
• /
• 2006

Genistein is a potent, plant-derived isoflavone that displays estrogenic activity at low concentrations but inhibits proliferation at high amounts. However, the molecular mechanism of genistein is not completely understood. In the present study, the biphasic effects (estrogenic and antiestrogenic activity) of genistein on the growth of MCF-7 cells were identified. Genistein within a low range of concentration, $1-10\;{\mu}M$, stimulated proliferation, while $50-100\;{\mu}M$ caused apoptotic cell death. Additionally, genistein at a low concentration induced estrogen receptor (ER)-mediated gene expression and ER phosphorylation. When pre-treated with PD98059, an MEK inhibitor, ER-mediated gene expression and ER phosphorylation by genistein were noticeably increased. However, the increased gene expression and phosphorylation did not enhance cell proliferation. Moreover, it was observed that ER-mediated signaling performs an important role in the MAPK pathway. The proliferation and apoptosis in genistein-treated MCF-7 cells were partially dependent on the Bcl-2 level. The addition of IC1 182, 780, an estrogen receptor antagonist, inhibited Bcl-2 expression induced by genistein. This study suggests that there is a close relationship between Bcl-2 and the ER signaling pathways in MCF-7 cells.

• 한국재료학회:학술대회논문집
• /
• 한국재료학회 1999년도 추계학술발표강연 및 논문개요집
• /
• pp.142-142
• /
• 1999

• 한국재료학회:학술대회논문집
• /
• 한국재료학회 1997년도 한국재료학회 추계학술발표회
• /
• pp.104-104
• /
• 1997

• 생명과학회지
• /
• 제20권4호
• /
• pp.572-577
• /
• 2010

본 연구에서 60 주령된 쥐를 대상으로 8 주간 트레드밀 운동강도와 근섬유 유형에 따른 apoptosis 관련 인자를 연구하였다. 결론적으로 노후된 쥐의 apoptosis 관련 인자는 운동강도와 근육유형에 따라 차이가 났다. EDL이 Soleus보다 apoptosis인자의 변화가 운동강도에 의해 더 민감하게 나타났다. 또한 casepase-3의 발현은 고강도 운동에서 나타났으나 DNA 절편화는 나타나지 않았기에 실질적인 apoptosis는 발생되지 않았다. 이는 노후된 개체의 운동 수행방법으로 고강도 운동은 적합하지 않으며, 고강도 운동상태를 지속할 경우 근섬유의 apoptosis를 유도할 가능성이 있다는 것을 시사한다. 그러므로 추후 실험에서는 운동시간과 빈도에 의한 apoptosis의 time-course impact 관찰과 외재적 경로(caspase independent apoptosis)에서의 apoptosis 관련 단백질 변화에 대한 연구를 통하여 노화에 의한 근손실의 원인 규명이 요구된다.