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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

RT-PCR of MMP-2: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Cells were washed in PBS and total RNA were extracted (RNAqueous for PCR, Ambion). Two-step RT-PCR (Retroscript, Ambion) was done with equal amounts of total RNA using specific primers for MMP-2 PCR. 20 μL of each PCR products were run on a 2.5% agarose gel and bands visualised under UV. GAPDH primers were used to confirm equal loading. Documentation was done in Gel Doc (Image Master VDS, Pharmacia, Biotech).
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fig4: RT-PCR of MMP-2: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Cells were washed in PBS and total RNA were extracted (RNAqueous for PCR, Ambion). Two-step RT-PCR (Retroscript, Ambion) was done with equal amounts of total RNA using specific primers for MMP-2 PCR. 20 μL of each PCR products were run on a 2.5% agarose gel and bands visualised under UV. GAPDH primers were used to confirm equal loading. Documentation was done in Gel Doc (Image Master VDS, Pharmacia, Biotech).

Mentions: To analyze whether ATRA affects MMP-2 by regulating at transcription levels, we studied mRNA expression for MMP-2 by RT-PCR. Figure 4 demonstrates a reduction in MMP-2 mRNA expression after treating MCF-7 cells with 30 μM ATRA for 24 hours (lane E) comparing the control (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)

RT-PCR of MMP-2: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Cells were washed in PBS and total RNA were extracted (RNAqueous for PCR, Ambion). Two-step RT-PCR (Retroscript, Ambion) was done with equal amounts of total RNA using specific primers for MMP-2 PCR. 20 μL of each PCR products were run on a 2.5% agarose gel and bands visualised under UV. GAPDH primers were used to confirm equal loading. Documentation was done in Gel Doc (Image Master VDS, Pharmacia, Biotech).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712868&req=5

fig4: RT-PCR of MMP-2: MCF-7 (300,000 cells/1.5 mL) cells were grown in absence (lane C) and in presence of 30 μM ATRA (lane E) for 24 hours in SFCM. Cells were washed in PBS and total RNA were extracted (RNAqueous for PCR, Ambion). Two-step RT-PCR (Retroscript, Ambion) was done with equal amounts of total RNA using specific primers for MMP-2 PCR. 20 μL of each PCR products were run on a 2.5% agarose gel and bands visualised under UV. GAPDH primers were used to confirm equal loading. Documentation was done in Gel Doc (Image Master VDS, Pharmacia, Biotech).
Mentions: To analyze whether ATRA affects MMP-2 by regulating at transcription levels, we studied mRNA expression for MMP-2 by RT-PCR. Figure 4 demonstrates a reduction in MMP-2 mRNA expression after treating MCF-7 cells with 30 μM ATRA for 24 hours (lane E) comparing the control (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