Received for publication October 25, 2006.
Revised January 2, 2007.
Accepted for publication January 2, 2007.

BIFUNCTIONAL ALKYLATING AGENT-INDUCED p53 AND NONCLASSICAL NUCLEAR FACTOR-KAPPA B (NF-B) RESPONSES AND CELL DEATH ARE ALTERED BY CAFFEIC ACID PHENETHYL ESTER (CAPE): A potential role for antioxidant/electrophilic response element (ARE/EpRE) signaling

Abstract

Bifunctional alkylating agents (BFAs) such as mechlorethamine (nitrogen mustard; NM) and bis-(2-chloroethyl) sulfide (sulfur mustard; SM) covalently modify DNA and protein. The roles of NF-B and p53, transcription factors involved in inflammatory and cell death signaling, were examined in normal human epidermal keratinocytes (NHEKs) and immortalized HaCaT keratinocytes, a p53-mutated cell line, to delineate molecular mechanisms of action of BFAs. NHEKs and HaCaT cells exhibited classical NF-B signaling as degradation of IB occured within 5 min after exposure to tumor necrosis factor-alpha. However, exposure to BFAs induced nonclassical NF-B signaling as loss of IB was not observed until 2 or 6 hr in NHEKs or HaCaT cells, respectively. Exposure of a NF-B reporter gene-expressing HaCaT cell line to 12.5, 50, or 100 µM SM activated the reporter gene within 9 hr. Pretreatment with caffeic acid phenethyl ester (CAPE), a known inhibitor of NF-B signaling, significantly decreased BFA-induced reporter gene activity. A 1.5-hr pretreatment or 30-min post-exposure treatment with CAPE prevented BFA-induced loss of membrane integrity by 24 hr in HaCaT cells but not in NHEKs. CAPE disrupted BFA-induced phosphorylation of p53 and p90RSK in both cell lines. CAPE also increased Nrf2 and decreased AhR protein expression, both of which are involved in antioxidant/electrophilic response element (ARE/EpRE) signaling. Thus, disruption of p53/p90RSK-mediated NF-B signaling and activation of ARE/EpRE pathways may be effective strategies to delineate mechanisms of action of BFA-induced inflammation and cell death signaling in immortalized versus normal skin systems.

Key words: NF-kappaB, alkylating agents, antioxidant/electrophilic response element, caffeic acid phenethyl ester, mustards, p53