All-Trans Retinoic Acid Downregulates Epidermal Growth Factor Receptor Mediated Regulation of Matrix Metalloproteinase-2 in B16F10 Murine Melanoma Cells

Abstract

Aims and Study Design: Epidermal growth factor receptor (EGFR) regulates a number of cellular processes, including cell motility, proliferation, differentiation and survival. EGFR mediated signal transduction via mitogen activated protein kinase (MAPK) modulates expression and activity of matrix metalloproteinases (MMPs). Elevated expression and activity of MMP-2 strongly correlates with increased tumour invasiveness. As numerous studies indicate that all-trans retinoic acid (ATRA) has considerable anti-tumorigenic potential, the effect of ATRA on EGFR mediated regulation of MMP-2 via MAPK was studied in this paper using the highly metastatic murine melanoma cell line B16F10 as a model. Methodology: B16F10 cells were cultured in the presence of ATRA (20 μM) for 6, 15 and 24 hrs. EGFR expression and phosphorylation and p38MAPK expression were assayed by Western blot. MMP-2 activity was assayed by gelatin zymography of culture supernatants. MMP-2 and TIMP-2 mRNA expression were assayed by RT-PCR. DNA fragmentation was used to assay the efficacy of ATRA in causing apoptosis. Results: Treatment of B16F10 murine melanoma cells with ATRA (20 µM) for 24 hrs led to an appreciable downregulation of EGFR phosphorylation and expression of EGFR and p38MAPK. Treatment of B16F10 cells with ATRA also inhibited MMP-2 activity and downregulated transcription of MMP-2 while transcription of TIMP-2 was upregulated. Treatment with ATRA did not show appreciable fragmentation of DNA. Conclusion: Downregulation of EGFR expression and phosphorylation and EGFR mediated signal transduction through p38 MAPK could lead to downregulation of MMP-2 transcription. Downregulation of MMP-2 transcription and upregulation of transcription of the MMP inhibitor TIMP-2 could result in loss of MMP-2 activity. Loss of MMP-2 activity would render cells less metastatic. Our findings indicate that treatment with ATRA can inhibit EGFR mediated regulation of MMP-2 activity in B16F10 murine melanoma cells. Such inhibition could have therapeutic potential in clinical management of tumours.