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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

(a) Cell Adhesion Assay:  MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (Control) and in presence of 30 μM ATRA for 24 hours in 10% MEM. Microtitre wells were coated with various concentrations of fibronectin and kept at 37°C for 1.5 hour. 1% BSA solution was used to block the nonspecific sites. Control and ATRA-treated cells were collected by trypsinization and 50,000 cells were added to each well. After 1 hour of incubation wells were washed thoroughly, number of bound cells was counted on a haemocytometer, and the % of cells adhered to the ligand was calculated. (b) RT-PCR of  α5 & β1: MCF-7 (300,000 cells/5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Two steps RT-PCR (Retroscript, Ambion) was done as before with equal amounts of total RNA using specific primers for α5 & β1. GAPDH was used as internal control. (c) ELISA of E-cadherin: ELISA of E-cadherin was performed with anti-E-cadherin antibody in control (C) and ATRA treated (E) MCF-7 cells. P = .01613  (P < .05) indicates that the difference in E-cadherin expression between control and ATRA treated MCF-7 cell is statistically significant.
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fig5: (a) Cell Adhesion Assay: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (Control) and in presence of 30 μM ATRA for 24 hours in 10% MEM. Microtitre wells were coated with various concentrations of fibronectin and kept at 37°C for 1.5 hour. 1% BSA solution was used to block the nonspecific sites. Control and ATRA-treated cells were collected by trypsinization and 50,000 cells were added to each well. After 1 hour of incubation wells were washed thoroughly, number of bound cells was counted on a haemocytometer, and the % of cells adhered to the ligand was calculated. (b) RT-PCR of α5 & β1: MCF-7 (300,000 cells/5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Two steps RT-PCR (Retroscript, Ambion) was done as before with equal amounts of total RNA using specific primers for α5 & β1. GAPDH was used as internal control. (c) ELISA of E-cadherin: ELISA of E-cadherin was performed with anti-E-cadherin antibody in control (C) and ATRA treated (E) MCF-7 cells. P = .01613 (P < .05) indicates that the difference in E-cadherin expression between control and ATRA treated MCF-7 cell is statistically significant.

Mentions: Figure 5(a) shows the effect of 30 μM ATRA for 24 hours on adhesion of MCF-7 cells to ECM protein (fibronectin). Binding of cells to fibronectin decreased significantly upon ATRA treatment for 24 hours whereas binding of cells to fibronectin remained almost unaltered for 12 hours ATRA-treated cells (result not shown).


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)

(a) Cell Adhesion Assay:  MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (Control) and in presence of 30 μM ATRA for 24 hours in 10% MEM. Microtitre wells were coated with various concentrations of fibronectin and kept at 37°C for 1.5 hour. 1% BSA solution was used to block the nonspecific sites. Control and ATRA-treated cells were collected by trypsinization and 50,000 cells were added to each well. After 1 hour of incubation wells were washed thoroughly, number of bound cells was counted on a haemocytometer, and the % of cells adhered to the ligand was calculated. (b) RT-PCR of  α5 & β1: MCF-7 (300,000 cells/5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Two steps RT-PCR (Retroscript, Ambion) was done as before with equal amounts of total RNA using specific primers for α5 & β1. GAPDH was used as internal control. (c) ELISA of E-cadherin: ELISA of E-cadherin was performed with anti-E-cadherin antibody in control (C) and ATRA treated (E) MCF-7 cells. P = .01613  (P < .05) indicates that the difference in E-cadherin expression between control and ATRA treated MCF-7 cell is statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712868&req=5

fig5: (a) Cell Adhesion Assay: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (Control) and in presence of 30 μM ATRA for 24 hours in 10% MEM. Microtitre wells were coated with various concentrations of fibronectin and kept at 37°C for 1.5 hour. 1% BSA solution was used to block the nonspecific sites. Control and ATRA-treated cells were collected by trypsinization and 50,000 cells were added to each well. After 1 hour of incubation wells were washed thoroughly, number of bound cells was counted on a haemocytometer, and the % of cells adhered to the ligand was calculated. (b) RT-PCR of α5 & β1: MCF-7 (300,000 cells/5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Two steps RT-PCR (Retroscript, Ambion) was done as before with equal amounts of total RNA using specific primers for α5 & β1. GAPDH was used as internal control. (c) ELISA of E-cadherin: ELISA of E-cadherin was performed with anti-E-cadherin antibody in control (C) and ATRA treated (E) MCF-7 cells. P = .01613 (P < .05) indicates that the difference in E-cadherin expression between control and ATRA treated MCF-7 cell is statistically significant.
Mentions: Figure 5(a) shows the effect of 30 μM ATRA for 24 hours on adhesion of MCF-7 cells to ECM protein (fibronectin). Binding of cells to fibronectin decreased significantly upon ATRA treatment for 24 hours whereas binding of cells to fibronectin remained almost unaltered for 12 hours ATRA-treated cells (result not shown).

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