Rottlerin inhibits ROS formation and prevents NFkappaB activation in MCF-7 and HT-29 cells

Abstract

Rottlerin, a polyphenol isolated from Mallotus Philippinensis, has been recently used as a selective inhibitor of PKC delta, although it can inhibit many kinases and has several biological effects. Among them, we recently found that Rottlerin inhibits the Nuclear Factor kappaB (NFkappaB), activated by either phorbol esters or H(2)O(2). Because of the redox sensitivity of NFkappaB and on the basis of Rottlerin antioxidant property, we hypothesized that Rottlerin could prevent NFkappaB activation acting as a free radicals scavenger, as other natural polyphenols. The current study confirms the antioxidant property of Rottlerin against the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in vitro and against oxidative stress induced by H(2)O(2) and by menadione in culture cells. We also demonstrate that Rottlerin prevents TNFalpha-dependent NFkappaB activation in MCF-7 cells and in HT-29 cells transfected with the NFkappaB-driven plasmid pBIIX-LUC, suggesting that Rottlerin can inhibit NFkappaB via several pathways and in several cell types.

Figure 1

Chemical structure of Rottlerin.

Figure 2

Rottlerin inhibits TNFα-induced NF-κB/DNA binding activities in MCF-7 and HT-29 cells. Cells were pretreated with Rottlerin (20 μM) or with Vitamin E (50 μM) with subsequent stimulation with TNFα (10 ng/mL) for 4 hr. Nuclear extracts (20 μg) were used to determine NF-B DNA binding activities by electrophoretic mobility shift assay (EMSA) using DIG-labled oligonucleotide containing NFκB element. The unlabeled NFκB consensus oligomer was used as the specific competitor and unlabeled NF-L6 consensus oligomer as the nonspecific competitor. Bottom panel shows the bands quantification of a representative blot. Complexes were visualized by chemiluminescence. Lanes: C, control; R, Rottlerin; T, TNFα; R+T; Rottlerin and TNFα; V, Vitamin E; V+T, Vitamin E and TNFα.

Figure 3

UV spectra of DPPH/Rottlerin solutions in absolute ethanol. (a) 20 μM DPPH ethanol solution (A); (b) spectrum of the mixture DPPH/Rottlerin obtained by mixing 2.5 ml of solution A with 0.5 ml of solution B (10 : 1 molar ratio); (c) 2.0 ml of solution A with 1 ml of solution B (4 : 1 molar ratio); (d) 1.5 ml of solution A with 1.5 ml of solution B (2 : 1 molar ratio); (e) spectrum of 10 μM Rottlerin ethanol solution (B).

Figure 4

EPR spectra of DPPH /Rottlerin solutions in absolute ethanol. (a) 20 μM DPPH ethanol solution (A); (b), (c), and (d) correspond to the (b), (c), and (d) solutions of Figure 2. All spectra were recorded in the same experimental conditions.

Figure 5

Rottlerin prevents both basal and induced intracellular production of H₂O₂. MCF-7 cells were pretreated with 5 and 20 μM Rottlerin for 1 hr and loaded by H₂O₂-specific fluorescent probe DHR-123. Cells were treated with menadione (100 μM) and hydrogen peroxide (100 μM) for 30 min and analyzed using flow cytometry.

Figure 6

Effect of Rottlerin on NFκB nuclear translocation and transcriptional activity in HT-29 transfected cells. HT-29 cells, stably transfected with the NFκB-driven plasmid pBIIX-LUC, were pretreated for 30 min with 5 and 20 μM Rottlerin and then treated with 10 ng/mL TNFα for 4 hr, before luciferase activity measurement. Data are the mean value ± SD of four independent experiments.