인체유방암세포의 tight junction 기능 조절을 통한 genistein의 암세포 침윤 억제 효과 Anti-invasive Activity of Human Breast Carcinoma Cells by Genistein through Modulation of Tight Junction Function원문보기
Tight junctions (TJs)은 인접된 세포 사이의 전해질 및 거대분자 확산 조절에 관여하는 paracellular permeability의 장벽 역할을 한다. 본 연구에서는 MCF-7 및 MDA-MB-231 인체유방암세포에서 대두의 대표적인 생리활성물인 genistein에 의한 암세포의 침윤 억제에서 TJs의 견고성 및 투과성과의 연관성을 조사하였다. 본 연구의 결과에 의하면 genistein에 의한 유방암세포의 증식 억제, 암세포 이동성의 저하 및 침윤성의 억제는 TJs의 증가된 견고성과 연관이 있었으며, 이를 transepithelial electrical resistance의 증가 및 paracellular permeability의 감소로 확인하였다. Genistein은 두 유방암세포에서 TJs의 주요 조절 단백질로서 paracellular transport 조절에 중요한 역할을 하는 claudin-3 및 claudin-4의 발현을 억제시켰다. 그리고 genistein은 암세포의 전이 조절 관련 유전자들인 like growth factor-1 receptor 및 snail의 발현을 억제하였으며, thrombospondin-1 및 E-cadherin의 발현은 증가시켰다. 또한 small interfering RNA를 이용하여 genistein의 유방암세포의 침윤 억제에서 claudin-3단백질의 중요성을 확인하였다. 결론적으로 genistein이 TJs의 견고성 증가를 통하여 암세포의 침윤성을 억제할 수 있었으며, 이 과정에서 아마도 claudin 단백질의 발현 증가가 중요한 역할을 하고 있음을 알 수 있었다. 본 연구의 결과는 genistein이 종양 전이억제를 효과적으로 차단할 수 있음을 보여주는 것이다.
Tight junctions (TJs)은 인접된 세포 사이의 전해질 및 거대분자 확산 조절에 관여하는 paracellular permeability의 장벽 역할을 한다. 본 연구에서는 MCF-7 및 MDA-MB-231 인체유방암세포에서 대두의 대표적인 생리활성물인 genistein에 의한 암세포의 침윤 억제에서 TJs의 견고성 및 투과성과의 연관성을 조사하였다. 본 연구의 결과에 의하면 genistein에 의한 유방암세포의 증식 억제, 암세포 이동성의 저하 및 침윤성의 억제는 TJs의 증가된 견고성과 연관이 있었으며, 이를 transepithelial electrical resistance의 증가 및 paracellular permeability의 감소로 확인하였다. Genistein은 두 유방암세포에서 TJs의 주요 조절 단백질로서 paracellular transport 조절에 중요한 역할을 하는 claudin-3 및 claudin-4의 발현을 억제시켰다. 그리고 genistein은 암세포의 전이 조절 관련 유전자들인 like growth factor-1 receptor 및 snail의 발현을 억제하였으며, thrombospondin-1 및 E-cadherin의 발현은 증가시켰다. 또한 small interfering RNA를 이용하여 genistein의 유방암세포의 침윤 억제에서 claudin-3단백질의 중요성을 확인하였다. 결론적으로 genistein이 TJs의 견고성 증가를 통하여 암세포의 침윤성을 억제할 수 있었으며, 이 과정에서 아마도 claudin 단백질의 발현 증가가 중요한 역할을 하고 있음을 알 수 있었다. 본 연구의 결과는 genistein이 종양 전이억제를 효과적으로 차단할 수 있음을 보여주는 것이다.
Tight junctions (TJs) that act as paracellular permeability barriers play an essential role in regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. In this study, we investigated the correlation between the tightening of TJs, permeability and the invas...
Tight junctions (TJs) that act as paracellular permeability barriers play an essential role in regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. In this study, we investigated the correlation between the tightening of TJs, permeability and the invasive activity of genistein - a bioactive isoflavone of soybeans - in human breast carcinoma MCF-7 and MDA-MB-231 cells. The inhibitory effects of genistein on cell proliferation, motility and invasiveness were found to be associated with the increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance and a decrease in paracellular permeability. Additionally, the immunoblotting results indicated that genistein repressed the levels of the proteins that comprise the major components of TJ, claudin-3 and claudin-4, which play a key role in the control and selectivity of paracellular transport. Furthermore, genistein decreased the metastasis-related gene expressions of insulin like growth factor-1 receptor and snail, while concurrently increasing that of thrombospondin-1 and E-cadherin. In addition, we demonstrated that claudins play an important role in the anti-motility and invasiveness of genistein using claudin-3 small interfering RNA. Taken together, our results indicate a possible role for genistein as an inhibitor of cancer cell invasion through the tightening of TJs, which may counteract the up-regulation of claudins. In addition, our results indicate that this may be beneficial for the inhibition of tumor metastasis.
Tight junctions (TJs) that act as paracellular permeability barriers play an essential role in regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. In this study, we investigated the correlation between the tightening of TJs, permeability and the invasive activity of genistein - a bioactive isoflavone of soybeans - in human breast carcinoma MCF-7 and MDA-MB-231 cells. The inhibitory effects of genistein on cell proliferation, motility and invasiveness were found to be associated with the increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance and a decrease in paracellular permeability. Additionally, the immunoblotting results indicated that genistein repressed the levels of the proteins that comprise the major components of TJ, claudin-3 and claudin-4, which play a key role in the control and selectivity of paracellular transport. Furthermore, genistein decreased the metastasis-related gene expressions of insulin like growth factor-1 receptor and snail, while concurrently increasing that of thrombospondin-1 and E-cadherin. In addition, we demonstrated that claudins play an important role in the anti-motility and invasiveness of genistein using claudin-3 small interfering RNA. Taken together, our results indicate a possible role for genistein as an inhibitor of cancer cell invasion through the tightening of TJs, which may counteract the up-regulation of claudins. In addition, our results indicate that this may be beneficial for the inhibition of tumor metastasis.
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가설 설정
However, the identi-fication of genistein targets that mediate these effects on the regulation of metastasis is still necessary. Therefore, in this study, we investigated the relationship between the tighten-ing of TJZ permeability and the invasive activity of breast cancer cells treated with genistein. In addition, we examined the significance of claudin-3 and -4 in MCF-7 and MDA-MB- 231 cells.
제안 방법
Consequently, although we need further studies for con-formation of genistein roles in breast cancer cells the results from the experiments have three major impacts: First, we test a novel therapeutic paradigm in metastatic breast cancer, that isz the inhibition of metastasis by natural compound, genistein which increase TJ activity. The proof of concept of this novel approach provided the underpinnings for a new, powerful strategy to prevent cancer metastasis.
was performed. For this experiment, an assay was conducted to measure the rate of motility of the cells that filled an empty area of the tissue culture plate that was cre-ated by scratching a confluent monolayer of cells. Briefly, cells were grown to confluency on 30-mm cell culture dishes that were coated with rat tail collagen (20 jig/mlz BD Biosciences, Bedford, MA).
In this study to characterize the anti-metastatic activities of genistein as a tyrosine kinase inhibitor on breast cancer cells’ MCF-7 and MDA-MB 231 cells’ we tested genistein for the IGF signaling system, snail and TSP-1 as well as that of E-cadherin on two breast adenocarcinoma cells. The possi-bility that IGF-1 receptor signaling conveys invasive and metastatic capability in human tumors deserves broader in- vestigation, and prioritized development of pharmacological inhibitors may be warranted [12z27].
4 mm pore size, Costarz Corning, NY). The cells were then treated with genistein (Sigma) or vehicle (DMSO) for 48 hrz after which the average TER was determined using an EVOM Epithelial Tissue Voltohmmeter (World Precision Instruments, Sarasota, FL) to measure the TER of four different areas of each transwell.
The goal of this study was to characterize the biological effect of genistein on the invasion and metastasis of breast carcinoma cells using MCF-7 and MDA-MB 231 cells. Our study showed that treatment with genistein increased the TER and decreased the paracellular permeability in both cell lines in a dose-dependent manner, and this effect was asso- ciated with the inhibition of motility and invasiveness.
which increase TJ activity. The proof of concept of this novel approach provided the underpinnings for a new, powerful strategy to prevent cancer metastasis. Secondly, it spurs the clinical testing of genistein as a ther-apeutic to prevent breast cancer metastasis.
대상 데이터
MCF-7 and MDA-MB 231 cells were obtained from the American Type Culture Collection (Rockville, MD). MDA- MB-231 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRLZ Invitrogen, Carisbad, CA) and MCF-7 cells were cultured in RPMI-1640 (Gibco-BRL) with 10% FBSZ 2 mM L-glutamine, 1 mM so-dium pyruvate (Gibco-BRL), 10 |ig/ml insulin (Sigma Co” St.
데이터처리
of three in-dependent experiments. The significance was de-termined using a Student's t-test.*,p <0.
of three independent experiments. The significance was determined using a Student's f-test. * μ <0.
이론/모형
05 was employed. A two sided Student's t-test was used to test the differences between the means of control cells and drug-treated cells.
Transfection of synthetic RNA was then conducted using LipofectAMINE 2000 (Invitrogen, Carisbad, CA) according to the manu-facturer's protocol. Following transfection with siRNA, the cells were incubated for 24 hr followed by incubation at the indicated conditions.
성능/효과
Taken to-gether the results of these studies suggest that regulation of these gene expressions is closely related to the metastaic and invasive activity of cancer cells’ however, less is known about the impact the regulation of these protein expressions has on TJ complexes. In this study, the levels of E-cadherin and TSP-1 were markedly increased by genistein, while those of IGF-1R and snail were inhibited, and these effects occurred in a time-dependent fashion. Although the effects of treat-ment observed in this study were not specifically correlated with TJs function, they appear to be closely related to the dysregulation of the TER value and the permeability of TJs.
후속연구
Although further study is needed to determine the effects of genistein on the metastatic potential of breast cancer in vivo, the findings presented here indicate the pres-ence of a novel mechanism by which genistein, and possibly other phytochemicals, can prevent cancer and metastasis. In addition, the results of this study will also greatly enhance our understanding of the role of TJs and their composite proteins in breast cancer metastasis.
Although further study is needed to determine the effects of genistein on the metastatic potential of breast cancer in vivo, the findings presented here indicate the pres-ence of a novel mechanism by which genistein, and possibly other phytochemicals, can prevent cancer and metastasis. In addition, the results of this study will also greatly enhance our understanding of the role of TJs and their composite proteins in breast cancer metastasis.
The demonstration of the anti-metastatic ef-fects of genistein will also lead to the search for natural com-pound candiated anticancer activity drug that may have higher anti-metastatic activity. Thirdly, our studies will greatly enhance our understanding of the role of TJ and their composite proteins in breast cancer metastasis.
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