Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent

Abstract

The occurrence of a functional relationship between the release of metalloproteinases (MMPs) and the expression of cyclooxygenase (COX)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB), increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC) were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I-IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I-III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA), a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

Keywords: COX-2; MMP-9; Sirt1; angiogenesis; brain endothelial cells; inflammation; resveratrol.

Figure 1

Sirt1 gene expression profiling in grade I-IV brain tumour tissues. TissueScan™ cancer and normal tissue cDNA arrays from 43 clinical samples covering brain cancer four stages and normal brain tissues were used to analyze differential Sirt1 gene expression. Tissue cDNAs of each array are synthesized from high quality total RNAs of pathologist-verified tissues, normalized and validated with β-actin and provided with clinical information for 2 normal brain, 18 WHO grade I, 11 WHO grade II, 10 WHO grade III, and 2 WHO grade IV brain tumours. Abbreviation: NB, normal brain tissue.

Figure 2

Resveratrol dose-dependent inhibition of carcinogen-induced COX-2 protein and gene expression. (A) HBMEC were serum-starved in the presence of various concentrations of resveratrol and in combination with vehicle or 1 μM PMA for 18 hours. Lysates were isolated, electrophoresed via SDS-PAGE, and immunodetection of COX-2 and GAPDH performed as described in the Methods section (NS, non specific immunoreactivity). (B) Scanning densitometry of COX-2 expression was only performed in PMA-treated cells since no COX-2 was detectable in vehicle-treated HBMEC. Densitometric data of a representative blot is shown out of three independent experiments. (C) Total RNA isolation, RT-PCR, and qPCR were performed as described in the Methods section to assess COX-2 gene expression in the above-described conditions. (PMA = 1 μM; Resveratrol = 30 μM). Notes: Data are representative of three independent qPCR experiments. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance to the respective PMA treatment.

Figure 3

Resveratrol inhibition of carcinogen-induced MMP-9 gene expression and protein secretion. HBMEC were serum-starved in the presence of (A) various PMA concentrations for 18 hours, or (B) a combination of 1 μM PMA with increasing resveratrol concentrations. Conditioned media were then harvested and gelatin zymography was performed in order to detect PMA-induced proMMP-9 and hydrolytic activity as described in the Methods section. (C) Scanning densitometry was used to quantify the extent of proMMP-9 gelatinolytic activity in the combined PMA and resveratrol treatments. Data shown is representative of two independent experiments. (D) Total RNA isolation and qRT-PCR were performed as described in the Methods section to assess MMP-9 gene expression in the above-described conditions. (PMA = 1 μM; Resveratrol = 30 μM). Notes: Data are representative of three independent qPCR experiments. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance to the respective PMA treatment.

Figure 4

Carcinogen-induced IκB phosphorylation is inhibited by resveratrol. (A) HBMEC were serum-starved for 30 minutes in the presence or not of 30 μM resveratrol, then treated with 1 μM PMA for the indicated time. Lysates were isolated, electrophoresed via SDS-PAGE and immunodetection of phosphorylated IκB (P-IκB) and GAPDH proteins was performed as described in the Methods section. (B) Quantification was performed by scanning densitometry of the autoradiograms. Notes: Data were expressed as the percent of basal phospho-IκB/GAPDH ratios in vehicle (open circles) and resveratrol pre-treated cells (closed circles). Densitometric data of a representative blot out of three is shown.

Figure 5

Sirt1 contributes to the resveratrol inhibitory effect of PMA-induced IkB phosphorylation. HBMEC were transiently transfected with a scrambled siRNA sequence (Mock) or a siRNA designed to downregulate Sirt1 (siSirt1) as described in the Methods section. (A) Mock and siSirt1-transfected HBMEC were then serum-starved for 30 minutes in the presence or not of 50 μM resveratrol, then treated with 1 μM PMA for the indicated time. Lysates were isolated, electrophoresed via SDS-PAGE and immunodetection of phosphorylated IκB (P-IκB) and GAPDH proteins was performed as described in the Methods section. (B) Total RNA isolation, RT-PCR, and qPCR were performed as described in the Methods section to assess Sirt1 gene expression in the Mock and siSirt1-transfected HBMEC. (C) Quantification was performed by scanning densitometry of the autoradiograms obtained in (A). Notes: Data were expressed as the percent of basal phospho-IκB/GAPDH ratios in vehicle (open circles) and resveratrol pre-treated cells (closed circles). Densitometric data of a representative blot out of three is shown.

Figure 6

Sirt1-independent inhibition by resveratrol of PMA-induced COX-2 expression and of PMA-induced MMP-9 secretion. Mock or siSirt1-transfected HBMEC were serum-starved in the presence of 1 μM PMA, 30 μM resveratrol, or a combination of both agents for 18 hours. (A) Lysates were isolated, electrophoresed via SDS-PAGE, and immunodetection of COX-2 and GAPDH performed as described in the Methods section. (B) Conditioned media were also harvested and gelatin zymography was performed in order to detect PMA-induced proMMP-9 and hydrolytic activity as described in the Methods section.