Bifunctional alkylating agent-induced p53 and nonclassical nuclear factor kappaB responses and cell death are altered by caffeic acid phenethyl ester: a potential role for antioxidant/electrophilic response-element signaling.

Bifunctional alkylating agents (BFA) such as mechlorethamine (nitrogen mustard) and bis-(2-chloroethyl) sulfide (sulfur mustard; SM) covalently modify DNA and protein. The roles of nuclear factor kappaB (NF-kappaB) and p53, transcription factors involved in inflammatory and cell death signaling, were examined in normal human epidermal keratinocytes (NHEK) and immortalized HaCaT keratinocytes, a p53-mutated cell line, to delineate molecular mechanisms of action of BFA. NHEK and HaCaT cells exhibited classical NF-kappaB signaling as degradation of inhibitor protein of NF-kappaBalpha (IkappaBalpha) occurred within 5 min after exposure to tumor necrosis factor-alpha. However, exposure to BFA induced nonclassical NF-kappaB signaling as loss of IkappaBalpha was not observed until 2 or 6 h in NHEK or HaCaT cells, respectively. Exposure of an NF-kappaB reporter gene-expressing HaCaT cell line to 12.5, 50, or 100 muM SM activated the reporter gene within 9 h. Pretreatment with caffeic acid phenethyl ester (CAPE), a known inhibitor of NF-kappaB signaling, significantly decreased BFA-induced reporter gene activity. A 1.5-h pretreatment or 30-min postexposure treatment with CAPE prevented BFA-induced loss of membrane integrity by 24 h in HaCaT cells but not in NHEK. CAPE disrupted BFA-induced phosphorylation of p53 and p90 ribosomal S6 kinase (p90RSK) in both cell lines. CAPE also increased nuclear factor E2-related factor 2 and decreased aryl hydrocarbon receptor protein expression, both of which are involved in antioxidant/electrophilic response element (ARE/EpRE) signaling. Thus, disruption of p53/p90RSK-mediated NF-kappaB 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.