Involvement of Phosphatidylinositol 3-kinase (PI3K) Pathway in Invasion and Motility H-ras-transformed MCF10A Cells

Shin, Il-Chung,Moon, Aree 德成女子大學校 藥學硏究所 2002 藥學論文誌 Vol.13 No.1

Many studies have identified the phosphatidylinositol 3-Kinase (PI3K) as a key regulator for various cellular functions including cell survival, growth and motility. We have previously shown that H-ras, but not N-ras, induces invasiveness and motility in human breast epithelial cells (MCF10A), while both H-ras and N-ras induce transformed phenotype. In the present study, we wished to investigate the functional role of PI3K pathway in H-ras-induced invasive phenotype and motility of MCF10A cells. Activation of PI3K in the parental, H-ras and N-ras MCF10A cells was examined by detecting phosphorylation of Akt a downstream molecule of PI3K, by Western blot analysis. Marked activation of Akt was detected not only in H-ras MCF10A cells but also in non-invasive/non-motile N-ras MCF10A cells at comparable levels. We then further investigated the functional significance of PI3K activation in invasion and motility by using known PI3k inhibitors, LY294002 and wortmannin. Treatment of LY 294002 and wortmannin significantly inhibited invasive phenotype and motility of H-ras MCF10A cells, suggesting that the activaation of PI3K pathway is not sufficient, but may be required for H-ras-induced invasion and motility. Prominent downregulation of MMP-2 and MMP-9 were observed in H-ras MCF10A cells treated with LY294002 in a dose-dependent manner. The results provide evidence that PI3K pathway is critical for H-ras-mediated upregulatio of MMPs in MCF10A cells, resulting in phenotypic conversion of non-invasive MCF10A cells to an invasive phenotype.

블루베리가 정상유선세포와 유방암세포의 ROS 축적과 세포사멸에 미치는 영향

이세나 ( Se Na Lee ),강금지 ( Keum Jee Kang ) 한국식품영양학회 2008 韓國食品營養學會誌 Vol.21 No.4

In an effort to elucidate the differential actions of blueberry(BB) in both normal and cancer cells, we utilized human breast cell lines to assess the accumulation of radical oxygen species(ROS) and ROS-associated apoptosis in both human normal breast epithelial(CMCF10A) and breast cancer(MCF7) cells. BB extract was added to the cultures at a final concentration of 20 μg/㎖ for 0(control), 6, 12, and 24 hr intervals. The MCF10A cells evidenced no marked ROS accumulation in the presence of BB, whereas the MCF7 cells evidenced clear ROS accumulation upon BB treatment from 12 hours forward. The number of dying or dead cells did not increase in the BB-treated MCF10A cell groups, whereas that number increased profoundly from 12 hr forward. Furthermore, the expression levels of certain stress-related, and pro- and anti-apoptotic gene products evidenced differential responses to BB treatment between the MCF10A and MCF7 cell groups. These results indicate that the components of BB extract differentiate cancer cells by not preventing ROS accumulation within cells and by inducing ROS-associated cell death in cancer cells. However, no marked ROS accumulation or induction of cell death was noted in the normal breast epithelial cells. The fact that BB extract exerted a differential effect on cancer cells opens further directions of research regarding the specific components that exert the differential BB-mediated effects in the selective prevention of normal cells and therapy for cancer tissues in the physiological body.

H-ras에 의하여 유도되는 침윤성 및 이동성에서 PI3K의 역할

신일청,문애리 德成女子大學校 藥學硏究所 2001 藥學論文誌 Vol.12 No.1

We have previously shown that H-ras, but N-ras, induces an invasiveness and cell motility in human breast epithelial cells (MCF10A), while both H-ras and N-ras induce transformed phenotype. It has been recently shown that phosphatidylinositol 3-kinase (PI3K) plays an important role on cell migration. In the present study, we wished to investigate the functional role of PI3K in H-ras-induced invasive phenotype and motility in MCF10A cells. The activation of PI3K was examined by detecting phosphorylation of Akt, a downstream molecule of PI3K, by Western blot analysis. We show that phosphorylated Akt level was upregulated both in H-ras MCF10A cells and N-ras MCF10A cells comparing to the parental MCF10A cells while the amount of total Akt was about the same in the parental, H-ras- and n-ras MCF10A cells. The results suggest that activation of PI3K is not sufficient for invasiveness and motility since PI3K is also activated in the N-ras MCF10A cells which have been shown to be non-invasion and non-motile. We then further investigated the functional significance of PI3K activation in invasion and motility by using the known PI3K inhibitors. LY294002 and wortmannin Treatment of LY294002 and wortmannin significantly reduced invasive phenotype and motility of H-ras MCF10A cells, suggesting that activation of PI3K is not sufficient, but may be required for H-ras-induced invasion and motility.

Capsaicin-Induced Apoptosis of H-Ras-Transformed Human Breast Epithelial Cells is Rac-Dependent via ROS Generation

Kim, Seon-Hoe,Moon, Aree The Pharmaceutical Society of Korea 2004 Archives of Pharmacal Research Vol.27 No.8

Many studies have focused on the anticarcinogenic, antimutagenic or chemopreventive activi-ties of capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) which is a major pungent ingredient in red pepper. We have previously shown that capsaicin selectively induces apoptosis in H-ras-transformed MCF10A human breast epithelial cells but not in their normal cell counter-parts (Int. J. Cancer, 103, 475-482,2003). In this study, we investigated the possible roles of reactive oxygen species (ROS) and Rac1 in capsaicin-induced apoptosis of H-ras MCF10A cells. Selective induction of ROS generation by capsaicin treatment was observed only in H-ras MCF10A cells. Pretreatment of H-ras MCF10A cells with an antioxidant N-acetylcysteine(NAC) significantly reversed capsaicin-induced growth inhibition, suggesting that ROS may mediate the apoptosis of H-ras-transformed cells induced by capsaicin. Rac1 was prominently activated by H-ras in MCF10A cells. Based on the studies using a wild type Rac1 and a domi-nant negative Rac1 constructs, we propose that Rac1 activity is critical for inhibitory effect of capsaicin on growth of H-ras-transformed MCF10A cells possibly through ROS generation.

Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by retinoic acids

CHOI, SANG-HO,KANG, HYUN-KYUNG,IM, EUN-OK,KIM, YOUNG JOON,BAE, YOUNG TAE,CHOI, YUNG HYUN,LEE, KYUNG HEE,CHUNG, HAE-YOUNG,CHANG, HEE-KYUNG,KIM, NAM DEUK 부산대학교 유전공학연구소 2000 분자생물학 연구보 Vol.16 No.-

The effects of retinoic acid (RA) and its analogs, all-trans RA, 9-cis RA and 13-cis RA, were investigated in human breast cancer MCF-7 cells and immortalized breast epithelial cell line MCF-10A. RA inhibited the telomerase activity of MCF-7 cells in a wide range of concentrations. RA at 10 μM also inhibited the growth of MCF-7 cells in a time-dependent manner. However, no significant growth inhibition was found between untreated control and RA-treated MCF-10A cells. Moreover, a marked inhibition of telomerase activity by RA was detected early in MCF-7 cells (after 24 h of RA treatment), which was preceded by a reduction of hTERT mRNA expression (after 12 h of RA treatment). However, MCF-10A cells showed a reduction of telomerase activity and down-regulation of hTERT after 4 days fo RA treatment. Simultaneous changes in hTERT mRNA expression and telomerase activity were found for MCF-10A cells. The expressions of hTR and hTEP1 telomerase component genes were not changed after RA treatment. These results indicate that the anti-breast cancer activity of RA could be mediated by its ability to down-regulate the expression of hTERT telomerase gene.

Hela 세포와 MCF10 세포 주에서 판코 빈혈 A 유전자 다량 발현

박우현 대한혈액학회 2006 Blood Research Vol.41 No.1

배경: 판코 빈혈(Fanconi anemia)은 열성으로 유전되는 질환으로, 임상적인 특징으로는 성장에 장애가 오며, 진행성 골수 이상과 암으로 대부분 사망하는 질환으로 알려져 있다. 특히 환자 세포에서 나타나는 주요 특징은 DNA crosslink인 mitomycin C (MMC)나 diepoxybutane에 대해 민감하게 반응을 한다. 방법: 본 실험을 하기 위해서 Hela 세포주와 MCF 10A 세포주를 사용하였으며, FANCA 단백을 발현하는 안정화 세포주를 만들기 위해서 retrovirus을 이용하였다. FANCA 단백을 발현하는 세포주의 선택을 위해서 puromycin을 사용하였으며, MMC에 대한 세포주들의 성장 억제 변화를 보기 위해서 MTT assay를 이용하였다. 결과: 성공적으로 FANCA 단백을 발현하는 HeLa 세포주와 MCF10A 세포주를 얻었으며, 이들 세포에 MMC을 처리하여 세포의 성장 억제를 분석한 결과, MMC 농도에 따라(0.01~10uM) HeLa 세포주와 FANCA를 발현시킨 HeLa 세포주는 세포성장의 차이에 아무런 변화를 보이지 않았다. MCF10A 세포주는 MMC 농도 0.01uM 부터 성적 억제가 관찰되었고(72 시간 배양), FANCA를 발현시킨 MCF10A 세포주는 MMC 농도(0.01~0.1uM)에서 성장이 확연히 덜 억제됨을 관찰할 수 있었다.

Capsaicin-Induced Apoptosis of H-Ras-Transformed Human Breast Epithelial Cells is Rac-Dependent via ROS Generation

Kim, Seonhoe,Moon, Aree 德成女子大學校 藥學硏究所 2004 藥學論文誌 Vol.15 No.1

Many studies have focused on the anticarcinogenic, antimutagenic or chemopreventive activities of capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) which is a major pungent ingredient in red pepper. We have previously shown that capsaicin selectively induces apoptosis in H-ras-transformed MCF10A human breast epithelial cells but not in their normal cell counter-parts (Int. J. Cancer, 103, 475-482, 2003). In this study, we investigated the possible roles of reactive oxygen species (ROS) and Racl in capsaicin-induced apoptosis of H-ras MCF10A cells. Selective induction of ROS generation by capsaicin treatment was observed only in H-ras MCF10A cells. Pretreatment of H-ras MCF10A cells with an antioxidant N-acetylcysteine (NAC) significantly reversed capsaicin-induced growth inhibition, suggesting that ROS may mediate the apoptosis of H-ras-transformed cells induced by capsaicin. Racl was prominently activated by H-ras in MCF10A cells. Based on the studies using a wild type Racl and a dominant negative Racl constructs, we propose that Racl activity is critical for inhibitory effect of capsaicin on growth of H-ras-transformed MCF10A cells possibly through ROS generation.

Analysis of the Cellular Stress Response in MCF10A Cells Exposed to Combined Radio Frequency Radiation

KIM, Han-Na,HAN, Na-Kyung,HONG, Mi-Na,CHI, Sung-Gil,LEE, Yun-Sil,KIM, Taehong,PACK, Jeong-Ki,CHOI, Hyung-Do,KIM, Nam,LEE, Jae-Seon Journal of Radiation Research Editorial Committee 2012 JOURNAL OF RADIATION RESEARCH Vol.53 No.2

<P>Exposure to environmental stressors can be measured by monitoring the cellular stress response in target cells. Here, we used the cellular stress response to investigate whether single or combined radio frequency (RF) radiation could induce stress response in human cells. Cellular stress responses in MCF10A human breast epithelial cells were characterized after exposure to 4 h of RF radiation [code division multiple access (CDMA) or CDMA plus wideband CDMA (WCDMA)] or 2 h RF radiation on 3 consecutive days. Specific absorption rate (SAR) was 4.0 W/kg for CDMA signal alone exposure and 2.0 W/kg each, 4.0 W/kg in total for combined CDMA plus WCDMA signals. Expression levels and phosphorylation states of specific heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs) were analyzed by Western blot. It was found that HSP27 and ERK1/2 phosphorylations are the most sensitive markers of the stress response in MCF10A cells exposed to heat shock or ionizing radiation. Using these markers, we demonstrated that neither one-time nor repeated single (CDMA alone) or combined (CDMA plus WCDMA) RF radiation exposure significantly altered HSP27 and ERK1/2 phosphorylations in MCF10A cells (<I>p</I> > 0.05). The lack of a statistically significant alteration in HSP27 and ERK1/2 phosphorylations suggests that single or combined RF radiation exposure did not elicit activation of HSP27 and ERK1/2 in MCF10A cells.</P>

In Vitro Bioassay for Transforming Growth Factor-$\beta$ Using XTT Method

Kim, Mi-Sung,Ahn, Seong-Min,Moon, Aree The Pharmaceutical Society of Korea 2002 Archives of Pharmacal Research Vol.25 No.6

Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-$\beta$, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-$\beta$ is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [$^3$H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascertain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-$\beta$ in a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-$\beta$ in three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-$\beta$, with a detection limit of as low as 10 pg/ml: Mv-1-Lu or H-ras MCF10A cells ($1{\times}10^5/well$) were incubated with TGF-$\beta$ at $37^{\circ}C$ in a humidified $CO_2$ incubator for 24 hr followed by XTT treatment and determination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-$\beta$.

Peroxiredoxin I and II inhibit H₂O₂-induced cell death in MCF-7 cell lines

Bae, Ji-Yeon,Ahn, Soo-Jung,Han, Wonshik,Noh, Dong-Young Wiley Subscription Services, Inc., A Wiley Company 2007 Journal of cellular biochemistry Vol.101 No.4

<P>Apoptosis is known to be induced by direct oxidative damage due to oxygen-free radicals or hydrogen peroxide or by their generation in cells by the actions of injurious agents. Together with glutathione peroxidase and catalase, peroxiredoxin (Prx) enzymes play an important role in eliminating peroxides generated during metabolism. We investigated the role of Prx enzymes during cellular response to oxidative stress. Using Prx isoforms-specific antibodies, we investigated the presence of Prx isoforms by immunoblot analysis in cell lysates of the MCF-7 breast cancer cell line. Treatment of MCF-7 with hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>) resulted in the dose-dependent expressions of Prx I and II at the protein and mRNA levels. To investigate the physiologic relevance of the Prx I and II expressions induced by H<SUB>2</SUB>O<SUB>2</SUB>, we compared the survivals of MCF10A normal breast cell line and MCF-7 breast cancer cell line following exposure to H<SUB>2</SUB>O<SUB>2.</SUB> The treatment of MCF10A with H<SUB>2</SUB>O<SUB>2</SUB> resulted in rapid cell death, whereas MCF-7 was resistant to H<SUB>2</SUB>O<SUB>2</SUB>. In addition, we found that Prx I and II transfection enabled MCF10A cells to resist H<SUB>2</SUB>O<SUB>2</SUB>-induced cell death. These findings suggest that Prx I and II have important functions as inhibitors of cell death during cellular response to oxidative stress. J. Cell. Biochem. 101: 1038–1045, 2007. © 2007 Wiley-Liss, Inc.</P>