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Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7).

Dutta A, Sen T, Banerji A, Das S, Chatterjee A - J Oncol (2009)

Bottom Line: Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment.Conclusions.This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

View Article: PubMed Central - PubMed

Affiliation: Department of Receptor Biology & Tumor Metastasis, Chittaranjan National Cancer Institute, Kolkata 700 026, India.

ABSTRACT
Background. Vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We studied the effect of ATRA on MMP-2 in MCF-7, human breast cancer cells, and the probable signaling pathways which are affected by ATRA on regulating pro-MMP-2 activity and expression. Methods. Gelatin zymography, RT-PCR, ELISA, Western blot, Immunoprecipitation, and Cell adhesion assay are used. Results. Gelatin zymography showed that ATRA caused a dose-dependent inhibition of pro-MMP-2 activity. ATRA treatment downregulates the expression of MT1-MMP, EMMPRIN, FAK, NF-kB, and p-ERK. However, expression of E-cadherin, RAR, and CRABP increased upon ATRA treatment. Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment. Conclusions. The experimental findings clearly showed the inhibition of MMP-2 activity upon ATRA treatment. This inhibitory effect of ATRA on MMP-2 activity in human breast cancer cells (MCF-7) may result due to its inhibitory effect on MT1-MMP, EMMPRIN, and upregulation of TIMP-2. This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

No MeSH data available.


Related in: MedlinePlus

Western blot of MT1-MMP and EMMPRIN: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and presence of 30 μM ATRA (lane E) for 24 hours in SFCM. The cells were collected and were extracted in cell extraction buffer. 50 μg of protein from both control and ATRA-treated cell extracts were run on 7.5% SDS-PAGE and the proteins were transferred onto nitrocellulose membrane by western blot. The membrane was incubated with anti MT1-MMP (Figure 3(a)) and EMMPRIN (Figure 3(b)) antibody and then after washing membrane was incubated with respective alkaline phosphatase coupled secondary antibody. Bands were visualized using NBT-BCIP as substrate. Ig-G was used as internal control. The quantitative measurement of the western blot (Figures 3(a) and 3(b)) was performed by Image J Launcher (version 1.4.3.67). (C) is the amount of MT1-MMP and EMMPRIN expression in the control cells, respectively, and (E) is the amount of MT1-MMP and EMMPRIN expression of 30 μM ATRA-treated (for 24 hours) MCF-7 cells, respectively.
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fig3: Western blot of MT1-MMP and EMMPRIN: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and presence of 30 μM ATRA (lane E) for 24 hours in SFCM. The cells were collected and were extracted in cell extraction buffer. 50 μg of protein from both control and ATRA-treated cell extracts were run on 7.5% SDS-PAGE and the proteins were transferred onto nitrocellulose membrane by western blot. The membrane was incubated with anti MT1-MMP (Figure 3(a)) and EMMPRIN (Figure 3(b)) antibody and then after washing membrane was incubated with respective alkaline phosphatase coupled secondary antibody. Bands were visualized using NBT-BCIP as substrate. Ig-G was used as internal control. The quantitative measurement of the western blot (Figures 3(a) and 3(b)) was performed by Image J Launcher (version 1.4.3.67). (C) is the amount of MT1-MMP and EMMPRIN expression in the control cells, respectively, and (E) is the amount of MT1-MMP and EMMPRIN expression of 30 μM ATRA-treated (for 24 hours) MCF-7 cells, respectively.

Mentions: To study the status of pro-MMP-2 activation complex, we studied the membrane type 1 MMP (MT1-MMP) expression by western blot using anti-MT1-MMP polyclonal antibody. The result (Figure 3(a)) showed that 30 μM ATRA-treated (lane E) MCF-7 cells exhibit much lower expression of MT1-MMP compared to the untreated MCF-7 cells (lane C).


Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7).

Dutta A, Sen T, Banerji A, Das S, Chatterjee A - J Oncol (2009)

Western blot of MT1-MMP and EMMPRIN: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and presence of 30 μM ATRA (lane E) for 24 hours in SFCM. The cells were collected and were extracted in cell extraction buffer. 50 μg of protein from both control and ATRA-treated cell extracts were run on 7.5% SDS-PAGE and the proteins were transferred onto nitrocellulose membrane by western blot. The membrane was incubated with anti MT1-MMP (Figure 3(a)) and EMMPRIN (Figure 3(b)) antibody and then after washing membrane was incubated with respective alkaline phosphatase coupled secondary antibody. Bands were visualized using NBT-BCIP as substrate. Ig-G was used as internal control. The quantitative measurement of the western blot (Figures 3(a) and 3(b)) was performed by Image J Launcher (version 1.4.3.67). (C) is the amount of MT1-MMP and EMMPRIN expression in the control cells, respectively, and (E) is the amount of MT1-MMP and EMMPRIN expression of 30 μM ATRA-treated (for 24 hours) MCF-7 cells, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Western blot of MT1-MMP and EMMPRIN: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and presence of 30 μM ATRA (lane E) for 24 hours in SFCM. The cells were collected and were extracted in cell extraction buffer. 50 μg of protein from both control and ATRA-treated cell extracts were run on 7.5% SDS-PAGE and the proteins were transferred onto nitrocellulose membrane by western blot. The membrane was incubated with anti MT1-MMP (Figure 3(a)) and EMMPRIN (Figure 3(b)) antibody and then after washing membrane was incubated with respective alkaline phosphatase coupled secondary antibody. Bands were visualized using NBT-BCIP as substrate. Ig-G was used as internal control. The quantitative measurement of the western blot (Figures 3(a) and 3(b)) was performed by Image J Launcher (version 1.4.3.67). (C) is the amount of MT1-MMP and EMMPRIN expression in the control cells, respectively, and (E) is the amount of MT1-MMP and EMMPRIN expression of 30 μM ATRA-treated (for 24 hours) MCF-7 cells, respectively.
Mentions: To study the status of pro-MMP-2 activation complex, we studied the membrane type 1 MMP (MT1-MMP) expression by western blot using anti-MT1-MMP polyclonal antibody. The result (Figure 3(a)) showed that 30 μM ATRA-treated (lane E) MCF-7 cells exhibit much lower expression of MT1-MMP compared to the untreated MCF-7 cells (lane C).

Bottom Line: Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment.Conclusions.This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

View Article: PubMed Central - PubMed

Affiliation: Department of Receptor Biology & Tumor Metastasis, Chittaranjan National Cancer Institute, Kolkata 700 026, India.

ABSTRACT
Background. Vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We studied the effect of ATRA on MMP-2 in MCF-7, human breast cancer cells, and the probable signaling pathways which are affected by ATRA on regulating pro-MMP-2 activity and expression. Methods. Gelatin zymography, RT-PCR, ELISA, Western blot, Immunoprecipitation, and Cell adhesion assay are used. Results. Gelatin zymography showed that ATRA caused a dose-dependent inhibition of pro-MMP-2 activity. ATRA treatment downregulates the expression of MT1-MMP, EMMPRIN, FAK, NF-kB, and p-ERK. However, expression of E-cadherin, RAR, and CRABP increased upon ATRA treatment. Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment. Conclusions. The experimental findings clearly showed the inhibition of MMP-2 activity upon ATRA treatment. This inhibitory effect of ATRA on MMP-2 activity in human breast cancer cells (MCF-7) may result due to its inhibitory effect on MT1-MMP, EMMPRIN, and upregulation of TIMP-2. This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

No MeSH data available.


Related in: MedlinePlus