All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules

Abstract

The 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 aimed to study the effect of ATRA on MMP-9 in MDA-MB-231, human breast cancer cells and the probable molecular mechanisms through which ATRA exerts its effect.

Results: Our experimental findings demonstrate that ATRA enters into the nucleus and regulates various signaling pathways viz. Integrin, FAK, ERK, PI-3K, NF-κB and also EGFR and down regulates pro-MMP-9 activity as well as its expression. As a result MDA-MB-231 cell migration on fibronectin medium gets retarded in presence of ATRA. ATRA up regulates TIMP-1 expression. Our study may help to understand the role of ATRA as a regulator of MMP-9 and the possible signaling pathways which are involved in this ATRA mediated down regulation of MMP-9.

Figure 1

Control and 48 h 20 µM ATRA treated (Treated) MDA-MB-231 cells were checked for viability with trypan blue assay.

Figure 2

(A) MDA-MB-231 (300,000 cells/1.5 ml) cells were grown in absence (lane Control) and in presence of 20 µM ATRA for 24 h (lane 20 µM ATRA 24 h) and for 48 h (lane 20 µM ATRA 48 h). SFCMs collected in all cases were subjected to gelatine zymography. Lane M is the marker lane, showing pro-MMP-9 and pro-MMP-2 activity in the SFCM of HT-1080 cells. (B) Total RNA was extracted from control (lane −ATRA) and 48 h 20 µM ATRA treated (lane +ATRA) MDA-MB-231 cells. Two-step RT-PCR was performed with MMP-9 and TIMP-1 primers. PCR products were run on 2% agarose gel and bands were visualized under UV. (C) MDA-MB-231 (300,000 cells/1.5 ml) cells were grown in absence (−ATRA) and in presence (+ATRA) of 20 µM ATRA for 48 h. SFCM was collected and total protein was extracted. 50 µl of SFCM as well as 50 µg protein from both control and ATRA treated cells were subjected to assay with ELSA using anti-MMP-9 antibody.

Figure 3

(A) SFCMs were collected from control and ATRA treated MDA-MB-231 cells, grown in presence and in absence of 1,000 ng/ml EGF for 8 h. Gelatin zymography was performed using sepharose 4B bead. Lane C denotes control MDA-MB-231 cells grown in absence of ATRA and EGF. Lane1 shows pro-MMP-9 activity of MDA-MB-231 cells grown in absence of ATRA, but in presence of EGF. Lane 2 represents pro-MMP-9 activity of ATRA treated MDA-MB-231 cells, grown in presence of EGF. (B) Total protein was extracted from both control (lane −ATRA) and ATRA treated (lane +ATRA) MDA-MB-231 cells. 100 µg of protein from each extract was subjected to western transfer on nitrocellulose membrane. Membrane was developed using anti-EGFR antibody, keeping actin as internal control.

Figure 4

(A) MDA-MB-231 cells grown in presence (Treated) and in absence (Control) of 20 µM ATRA were allowed to bind with different concentration of fibronectin (25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.125 µg/ml, 1.56 µg/ml) coated in 96 well plate. After 1.5 h incubation at 37°C wells were washed and cells were trypsinized. Numbers of bound cells were counted on a haemocytometer slide and % of adhesion was calculated. (B) MDA-MB-231 cells grown in presence (Treated) and in absence (Control) of 20 µM ATRA were allowed to bind with different concentration of vitronectin (5 µg/ml, 2.5 µg/ml, 1.25 µg/ml) coated in 96-well plate. After 1.5 h incubation at 37°C wells were washed and cells were trypsinized. Numbers of bound cells were counted on a haemocytometer slide and % of adhesion was calculated. (C) RT-PCR was performed in control (lane −ATRA) and ATRA treated (lane +ATRA) MDA-MB-231 cells with α5, β1, αv and β3 primer. GAPDH was used to confirm total RNA integrity and equal loading.

Figure 5

(A) Western blots were performed in MDA-MB-231 cells grown in absence (lane −ATRA) and in presence (lane +ATRA) of 20 µM ATRA for 48 h as described in methods. Membranes were developed using anti-ILK, anti-FAK, anti-NFκB and anti-VEGF primary antibody, keeping actin as internal control. (B) RT-PCR of FAK was performed in MDA-MB-231 cells grown without (lane −ATRA) or with (lane +ATRA) 20 µM ATRA. PCR products were run on 2% agarose gel to visualize the bands.

Figure 6

(A) Gelatin zymography was performed using the SFCM, collected from MDA-MB-231 cells grown in absence (lane C) and in presence of ERK (lane 1), PI-3K (lane 2), MEK (lane 3), p38 (lane 4) and both ERK & PI-3K (lane 5) inhibitors. (B) Western blots were performed in control (lane −ATRA) and ATRA treated (lane +ATRA) MDA-MB-231 cells as before. Membranes were developed with anti-ERK, anti-p-ERK, anti-PI-3K and anti-p-PI-3K antibodies. Actin was used to confirm equal loading.

Figure 7

(A) MDA-MB-231 cells were grown without (lane −ATRA) or with (lane +ATRA) ATRA for 48 h and total protein were extracted. Western blot was performed as before using anti-RAR and anti-e-cadherin antibodies. Actin was used as internal control. (B) ELISA of RAR and CRABP was performed as before in MDA-MB-231 cells, grown in presence (Treated) and in absence (Control) of ATRA for 48 h. (C) Immunocytochemical localization of CRABP was performed in MDA-MB-231 cells grown without (Control) or with (ATRA treated) 20 µM ATRA for 2, 3 and 4 h.

Figure 8

(A) MDA-MB-231 cells (300,000 cells/1.5 ml) grown in presence (lane 2) and in absence (lane 1) of 20 µM ATRA for 48 hrs. Both control and ATRA treated cells were then allowed to grow in presence of 20 µg/ml fibronectin for 2 h in SFCM. Lane C represents MDA-MB-231 cells grown in absence of fibronectin as well as ATRA. SFCMs were then subjected to gelatine zymography as before. Lane M is the marker lane, showing pro-MMP-9 and pro-MMP-2 activity in the SFCM, collected from HT-1080 cells. (B) Cell migration efficiency of Control and ATRA treated MDA-MB-231 cells were observed under inverted microscope by creating wounds in cell culture dishes. Cells were allowed to grow in presence of fibronectin ECM ligand to observe the efficacy of their migrations in SFCM.