Identification of UV-protective activators of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) by combining a chemical library screen with computer-based virtual screening.

Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a master regulator of cellular antioxidant defense systems, and activation of this transcription factor is a promising strategy for protection of skin and other organs from environmental insults. To identify efficient Nrf2 activators in keratinocytes, we combined a chemical library screen with computer-based virtual screening. Among 14 novel Nrf2 activators, the most potent compound, a nitrophenyl derivative of 2-chloro-5-nitro-N-phenyl-benzamide, was characterized with regard to its molecular mechanism of action. This compound induced the expression of cytoprotective genes in keratinocytes isolated from wild-type but not from Nrf2-deficient mice. Most importantly, it showed low toxicity and protected primary human keratinocytes from UVB-induced cell death. Therefore, it represents a potential lead compound for the development of drugs for skin protection under stress conditions. Our study demonstrates that chemical library screening combined with advanced computational similarity searching is a powerful strategy for identification of bioactive compounds, and it points toward an innovative therapeutic approach against UVB-induced skin damage.

Hepatitis B virus induces expression of antioxidant response element-regulated genes by activation of Nrf2.

The expression of a variety of cytoprotective genes is regulated by short cis-acting elements in their promoters, called antioxidant response elements (AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related factor 2 (Nrf2). Human hepatitis B virus (HBV) induces a strong activation of Nrf2/ARE-regulated genes in vitro and in vivo. This is triggered by the HBV-regulatory proteins (HBx and LHBs) via c-Raf and MEK. The Nrf2/ARE-mediated induction of cytoprotective genes by HBV results in a better protection of HBV-positive cells against oxidative damage as compared with control cells. Furthermore, there is a significantly increased expression of the Nrf2/ARE-regulated proteasomal subunit PSMB5 in HBV-positive cells that is associated with a decreased level of the immunoproteasome subunit PSMB5i. In accordance with this finding, HBV-positive cells display a higher constitutive proteasome activity and a decreased activity of the immunoproteasome as compared with control cells even after interferon α/γ treatment. The HBV-dependent induction of Nrf2/ARE-regulated genes might ensure survival of the infected cell, shape the immune response to HBV, and thereby promote establishment of the infection.

STAT3 controls matrix metalloproteinase-1 expression in colon carcinoma cells by both direct and AP-1-mediated interaction with the MMP-1 promoter.

Aberrant activation of STAT3 in colorectal carcinoma (CRC) tissue is correlated with elevated expression of matrix metalloproteinase-1 (MMP-1). We analyzed transcriptional regulation of the human MMP-1 promoter in CRC cells by tyrosine phosphorylated (pY-) STAT3. One of six putative STAT binding elements within a 4.3 kb MMP-1 trancriptional promoter fragment showed a particular high affinity for STAT3 in vitro. However, the most profound regulatory influence on MMP-1 promoter activity resides in a proximal region relative to the transcriptional start, bearing a pair of putative binding sites for STAT3 and AP-1. Mutational analysis of the combined STAT3/AP-1 recognition element revealed that the integrity of the STAT3 binding site is necessary, but not sufficient for both DNA interaction and transcriptional regulation by activated STAT3. Instead, the adjacent AP-1 site was essential for pY-STAT3-mediated transcription on the MMP-1 promoter. DNA-protein binding assays provided strong evidence for complex formation of STAT3 and c-Jun governed by protein-protein contacts. We observed striking coincidence for concerted aberrant activation of both STAT3 and AP-1 in human colon cancer specimens. This finding supports the notion that the combination of inappropriate STAT3 and AP-1 activities drives elevated MMP-1 expression and tissue invasion in colorectal cancer and is of clinical relevance.

Enantiopure sulforaphane analogues with various substituents at the sulfinyl sulfur: asymmetric synthesis and biological activities.

A convergent and high-yielding approach for the asymmetric synthesis of sulforaphane 2 and four analogues differently substituted at the sulfinyl sulfur has been developed. The key step of the synthesis is the diastereoselective synthesis of sulfinate ester 23-S(S), using the DAG (diacetone-D-glucofuranose)-methodology. The biological activity of these compounds as inductors of phase II detoxifying enzyme has been studied by determining their ability to activate the cytoprotective transcription factor Nrf2.

Lung Adenocarcinomas and Lung Cancer Cell Lines Show Association of MMP-1 Expression With STAT3 Activation.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in the majority of lung cancer. This study aims at defining connections between STAT3 function and the malignant properties of non-small cell lung carcinoma (NSCLC) cells. To address possible mechanisms by which STAT3 influences invasiveness, the expression of matrix metalloproteinase-1 (MMP-1) was analyzed and correlated with the STAT3 activity status. Studies on both surgical biopsies and on lung cancer cell lines revealed a coincidence of STAT3 activation and strong expression of MMP-1. MMP-1 and tyrosine-phosphorylated activated STAT3 were found co-localized in cancer tissues, most pronounced in tumor fronts, and in particular in adenocarcinomas. STAT3 activity was constitutive, although to different degrees, in the lung cancer cell lines investigated. Three cell lines (BEN, KNS62, and A549) were identified in which STAT3 activitation was inducible by Interleukin-6 (IL-6). In A549 cells, STAT3 activity enhanced the level of MMP-1 mRNA and stimulated transcription from the MMP-1 promoter in IL-6-stimulated A549 cells. STAT3 specificity of this effect was confirmed by STAT3 knockdown through RNA interference. Our results link aberrant activity of STAT3 in lung cancer cells to malignant tumor progression through up-regulation of expression of invasiveness-associated MMPs.

Sulforaphane homologues: Enantiodivergent synthesis of both enantiomers, activation of the Nrf2 transcription factor and selective cytotoxic activity.

Reported is an enantiodivergent approach for the synthesis of both enantiomers of sulforaphane (SFN) homologues with different chain lengths between the sulfinyl sulfur and the isothiocyanate groups and different substituents on the sulfinyl sulfur. The homologues were designed in order to unravel the effect of all the diversity elements included in sulforaphane's structure. The key step of the approach is the diastereoselective synthesis of both sulfinate ester epimers at sulfur, using as single chiral auxiliary the sugar derived diacetone-d-glucose. The approach allows the first synthesis of both enantiomers of 5-methylsulfinylpentyl isothiocyanate, and the biologically important 6-methylsulfinylhexyl isothiocyanate (6-HITC) found in Japanese horseradish, wasabi (Wasabia japonica). The ability of the synthesized compounds as inductors of phase II detoxifying enzymes has been studied by determining their ability to activate the cytoprotective transcription factor Nrf2. The cytotoxic activity of all the synthesized compounds against human lung adenocarcinoma (A549) and foetal lung fibroblasts (MRC-5) is also reported.