**6.4 Pathways/mechanism that regulate Nrf2/ARE**

The major signaling pathways implicated in the modulation of ARE/Nrf2 activity include mitogen activated protein (MAP) kinases, phosphatidylinositol-3-kinase (PI3 kinase), and Protein kinase C (PKC). Activation of ERK signaling by sulforaphane and other agents was shown to be involved in ARE/Nrf2 induction (Yu et al., 1999), while p38 MAPK was found to negatively regulate ARE/Nrf2 in certain cell types (Keum et al., 2006; Yu et al., 2000a; Yu et al., 2000b). The PI3 kinase pathway has also been demonstrated to be a key component of ARE/Nrf2 regulation; induction of ARE-driven antioxidant genes by sulforaphane was abrogated by blocking PI3 kinase (Nakaso et al., 2003; Wang et al., 2008). Direct involvement of PKC in Nrf2 phosphorylation and ARE activation has also been established (Huang et al., 2000, 2002; Numazawa et al., 2003).

#### **6.5 Activation of the Nrf2/ARE pathway by Feverfew PFE**

Human epidermoid KB cells expressing the antioxidant response element promoter were treated with Feverfew PFE or with the well-known ARE-inducing agent Sulforaphane (Zhang et al., 1992) for a period of 24 hr. Treatment with Feverfew PFE led to 2-fold activation of the ARE promoter, which was at par with the induction mediated by Sulforaphane.

in various tissues and cell lines (Nguyen et al., 2004). As a result, the function of Nrf2 and its downstream target genes are vital for protection against cellular damage induced by oxidative stress or chemicals. Several studies have shown that Nrf2 knockout mice having decreased levels of phase II detoxification enzymes and antioxidant proteins are highly sensitive to cytotoxic electrophiles compared to their wild-type littermates (Lee and Johnson, 2004; Leung et al., 2003). The upregulation of protective detoxification and antioxidant genes by Nrf2/ARE pathway can synergistically increase the efficiency of our

The induction of several cytoprotective enzymes in response to reactive chemical entities or oxidative stress is regulated at the transcriptional level via activation of Nrf2. This transcriptional response is mediated by the ARE, and is activated in response to H2O2 (Purdom-Dickinson et al., 2007) and by chemical compounds with the capacity to either undergo redox cycling or be metabolically transformed to a reactive or electrophilic intermediate (Nguyen et al., 2003). Several electrophiles, including diethyl maleate, tertbutylhydroquinone, sulforaphane (Zhang et al., 1992) and curcumin (Balogun et al., 2003) have been shown to induce Nrf2-dependent transcriptional activation of downstream target genes. In addition, several phytochemicals, such as Sulforaphane obtained from cruciferous vegetables (Zhang et al., 1992), Resveratrol (Kode et al., 2008), and Celastrol from the medicinal plant Tripterygium wilfordii (Seo et al.) have also been shown to activate the antioxidant response element . Chemical compounds such as the isothiocyanates and diethyl maleate can directly react with sulfhydryl groups and do not require metabolism. They can mimic an oxidative insult by oxidizing cysteine residues and depleting reduced cellular glutathione (GSH). Elevated levels of reactive oxygen and other electrophilic species followed by a reduced antioxidant capacity can lead to the alteration of the cellular redox status and trigger the transcriptional response mediated by Nrf2/ARE (Nguyen et al., 2009).

The major signaling pathways implicated in the modulation of ARE/Nrf2 activity include mitogen activated protein (MAP) kinases, phosphatidylinositol-3-kinase (PI3 kinase), and Protein kinase C (PKC). Activation of ERK signaling by sulforaphane and other agents was shown to be involved in ARE/Nrf2 induction (Yu et al., 1999), while p38 MAPK was found to negatively regulate ARE/Nrf2 in certain cell types (Keum et al., 2006; Yu et al., 2000a; Yu et al., 2000b). The PI3 kinase pathway has also been demonstrated to be a key component of ARE/Nrf2 regulation; induction of ARE-driven antioxidant genes by sulforaphane was abrogated by blocking PI3 kinase (Nakaso et al., 2003; Wang et al., 2008). Direct involvement of PKC in Nrf2 phosphorylation and ARE activation has also been established (Huang et al.,

Human epidermoid KB cells expressing the antioxidant response element promoter were treated with Feverfew PFE or with the well-known ARE-inducing agent Sulforaphane (Zhang et al., 1992) for a period of 24 hr. Treatment with Feverfew PFE led to 2-fold activation of the ARE promoter, which was at par with the induction mediated by

**6.3 Activation of the Nrf2/ARE pathway by oxidative stress** 

**6.4 Pathways/mechanism that regulate Nrf2/ARE** 

**6.5 Activation of the Nrf2/ARE pathway by Feverfew PFE** 

2000, 2002; Numazawa et al., 2003).

Sulforaphane.

cellular defense system.

Fig. 4. Feverfew PFE induces activation of the ARE promoter.

Fig. 5. Feverfew PFE activates the Nrf2 transcription factor.

In order to analyze the effects of Feverfew PFE on the transcription factor, Nrf2, a TransAM ELISA was utilized. Primary human keratinocytes were treated with Feverfew PFE or Sulforaphane for 24 hr, following which the nuclear and cytoplasmic cell lysate fractions were separated. Translocation of Nrf2 was analyzed in the nuclear fractions. Feverfew PFE enhanced the nuclear translocation of Nrf2 by more than 70%.

The Botanical Extract Feverfew PFE Reduces

boosting the cellular antioxidant defense machinery.

reduce the damage to oxidatively challenged skin.

**9. Acknowledgements** 

**from DNA damage and DNA repair** 

intrinsic ROS formation.

DNA Damage and Induces DNA Repair Processes 541

agent, Psoralen both induce oxidative stress which can produce DNA damage (Martin et al., 2008). Feverfew PFE induced DNA repair activity in DNA damaged by Cisplatin or Psoralen. Thus Feverfew PFE can activate the endogenous nucleotide excision repair in human epidermal keratinocytes, which could repair DNA damaged by extrinsic or

In addition to the direct effects of Feverfew PFE on DNA repair enzymes, activation of the ARE/Nrf2 pathway by Feverfew might also play a crucial role in preventing DNA damage and augmenting DNA repair. Daily exposure to UV, smoke, and other external aggressors leads to the generation of high levels of reactive oxygen species in the body. In order to avoid the deleterious effects of reactive oxidative species, cells have developed different defensive mechanisms including antioxidant molecules such as glutathione, alphatocopherol, vitamins A and C, and enzymes such as superoxide dismutase (SOD) and catalase (Ames et al., 1993; Verjat et al., 2000). When the cellular balance between antioxidants and pro-oxidants is upset, free radicals lead to the formation of lesions in the DNA causing damage, which in most cases is endogenously repaired in the cells by several repair mechanisms. The Nrf2/ARE pathway induces production of a battery of downstream genes that mitigate damage from reactive oxygen species, and aid DNA repair (Villeneuve et al., 2009). It has been shown that activation of Nrf2 generates a glutathione gradient to protect skin from the damaging effects of UVB (Saw et al., Schafer et al.), and reduces UVAinduced apoptosis in fibroblasts (Hirota et al., 2005). Thus, Feverfew PFE may help protect the skin from DNA damage incurred by UV and other environmental aggressors by

**8. Summary and conclusions: The use of botanical extracts for protection** 

Skin is under continual assault from a variety of damaging environmental factors such as ultraviolet irradiation and atmospheric pollutants. As organisms age the cumulative damage exceeds the capacity of endogenous antioxidant defenses resulting in oxidative damage. Plants have adapted to chronic exposure to ultraviolet irradiation by producing phytochemicals which can mitigate reactive oxygen species and repair damaged DNA. Botanical extracts such as Feverfew PFE, containing naturally occurring antioxidants can replenish the depleted cutaneous stores and perhaps prevent oxidative stress. Feverfew has been shown to contain chlorogenic acid (Wu et al., 2007), which can activate the Nrf2-ARE pathway (Feng et al., 2005), increase DNA repair enzyme expression (Bernstein et al., 2007), and induce repair of DNA (Huang et al., 2008). Furthermore, chlorogenic acid might be one of several phytochemicals present in Feverfew that could protect from DNA damage and induce the DNA repair process. Through the ability to scavenge free radicals, preserve endogenous antioxidant levels, reduce DNA damage and induce repair of damaged DNA, Feverfew PFE may protect skin from numerous external aggressions encountered daily and

The authors would like to thank Katharine Martin, Neena Tierney, Peter Lyte, Dara Miller, Thierry Oddos, Karien Rodriguez and Dr. Balz Frei (Linus Pauling Institute,

Oregon State University) for research and discussions on Feverfew PFE.

The downstream functional effects of Nrf2/ARE activation by Feverfew PFE were analyzed by using an Oxidative Stress and Antioxidant Defense PCR array. Human epidermal skin equivalents treated with Feverfew PFE for 24 hr were analyzed using the PCR array. Feverfew PFE led to more than 2-fold increase in the expression of several genes involved in antioxidant defense and oxidative stress. These genes included Lactoperoxidase, an antioxidant-related gene which functions as a natural antibacterial agent, Glutathione Peroxidase which functions to protect the organism from oxidative damage by reducing free hydrogen peroxide to water, inducible Nitric Oxide Synthase-2 involved in superoxide metabolism, and G protein-coupled receptor-156 which is implicated in oxidative stress response.


Table 1. Fevefew PFE leads to induction of antioxidant defense genes.
