Antioxidant Activities and Anticancer Cell Proliferation Properties of Wild Strawberries

Fruit extracts from 17 to 18 representatives of three strawberry species IFragaria virginiana Mill., F. clz:Ioensi.s(L.) Mill., and J' xa,,a,za,ssa Duchesne ex Rozierl were tested for the ability to inhibit proliferation of A549 human lung epithelial cancer cells. The fruit extracts also were tested for activities against free radicals, (peroxyl radicals, h ydroxyl radicals, singlet oxygen, and superoxide radicals), the activities of antioxidant enzymes Iglutathione peroxidase (EC 1.11.1.9), superoxide disniutase (EC 1.15.1.1), guaiacol peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.1 1.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehvdroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2)1, and the activities of nonenzyme antioxidant components, ascorbic acid and glutathione. Correlations between the proliferation of cancer cells and these antioxidant activities ere calculated. At the species level, F. virginiana fruit extract inhibited the proliferation of A549 human lung epithelial cancer cells to a significantly greater extent (34% inhibition) than the extracts from fruit of either F. chiloensis (26%) or]-. xananassa (25%) (P < 0.0001). Extracts from fruit off. virginiana also had significantly greater antioxidant activities and higher activities of antioxidant enzymes and nonenzyme components than did extracts from the other two species. Among individual genotypes, there was a high positive correlation between antiproliferation of A549 cancer cells, antioxidant activities against free radicals, activities of antioxidant enzymes, and activities of nonenzyme components. Although all fruit extracts from all the strawberry genotypes inhibited proliferation of A594 cancer cells, fruit extracts from seven F. virginiana genotypes showed significantly greater antiproliferative effects than an' of the F. xananassa or F. chiloensis genotypes. These genotypes, CFRA 0982, JP 95-1-1, NC 95-19-1, RH 30, NC 96-48-1, .JP 95-9-6, and LH 50-4, may be especially useful in developing cultivars with greater anticancer potential. Reactive oxygen species, including peroxyl radicals (R00), hydroxyl radicals ( . OH), superoxide radicals (02), hydrogen peroxide (1-1 202), and singlet oxygen ('02), are generated as byproducts of normal metabolism. The accumulation of these reactive oxygen species or free radicals can cause oxidative damage to lipids, proteins, and nucleic acids, and thus promote cell death (Morel and Dangl, 1997; SatuéGracia et al., 1997). Physiological defenses against oxidative stress involve small-molecule antioxidants and antioxidant proteins (Frei et al., 1992). Antioxidants are compounds that can delay or inhibit the oxidation of lipids or other molecules by inhibiting oxidizing chain reactions, and they play an important role as a health-protecting factor (Velioglu et al., 1998). The antioxidant enzyme defense system consists of hundreds of different substances and mechanisms. Antioxidant enzymes have the capacity to lower the free radical burden and neutralize excess free radicals created by stress conditions. Antioxidant enzymes serveas catalysts that can affect one or Received for publication 26 Dec. 2006. Accepted for publication 7 May 2007. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture or the National Institute for Occupational Saléty and Health. We thank Mr. Sam Garrett for propagating the plants for testing, Mr. John Enns for establishing and maintaining the strawberry field and harvesting fruit, Sue Kim for technical assistance, and Drs, Peter Trivonen, James J. Luby, John Hartung, and the anonymous reviewers for their helpful comments. 'Corresponding author. E-mail; [email protected] . more of the three stages of free radical formation: initiation, propagation, and termination. Therefore it is possible that antioxidant enzymes can prevent cellular and tissue damage in the human body (Baldwin, 1996; Bode and Dong, 2000). Strawberries (Era garia L.) are good sources of natural antioxidants (Heinonen et al., 1989; Wang et al., 2005). Previous studies showed that strawberry extracts exhibited high enzymatic activity for oxygen detoxification (Wang et al., 2005) and a high level of antioxidant capacity against free radical species including R00, .OH, 02 , H202, and 02 (Wang and Jiao, 2000; Wang and Lin, 2000). The main antioxidant enzymes in strawberry fruit are superoxide dtsmutase (SOD), guaiacol peroxidase (G-POD), glutathione peroxidase (GSH-POD), ascorbate peroxidase (AsA-POD), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), with ascorbic acid (AsA) and glutathione (GSH) as important nonenzyme components. Strawberry extracts also exhibited chemopreventive and chemotherapeutic activities in vitro and in vivo (Canton et al., 2001; Meyers et al., 2003; Wang et al., 2005), inhibited proliferation of the human lung epithelial cancer cell line A549, and decreased tetradecanoylphorbol13-acetate (TPA)-induced neoplastic transformation of JB6 P mouse epidermal cells (Wang et al., 2005). Pretreatment of JB6 p mouse epidermal cells with strawberry extracts resulted in the inhibition of ultraviolet B (UVB)and TPA-induced protein-1 (AP-1), and nuclear factor-KB (NF-tcB) transactivation. Strawberry extracts J. AMER. Soc. HORT. So. 132(5):647-658. 2007. 647 also blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs) and UVB-induced phosphorylation of ERKs and c-June amino terminal kinases (JNKs) in JB6 P mouse epidermal cell culture (Wang et al., 2005). AP-1 and NF-KB are transcription factors associated with carcinogenesis (Bode and Dong, 2000). AP-1 is composed of homodirners or heterodimers of the JUN and fructo-oligosaccharide (FOS) families (Angel and Karin, 1991). Many stimuli induce the binding of AP-1 to the promoter region of various genes that govern cellular processes such as inflammation. proliferation, and apoptosis (Baldwin, 1996). Inhibition of AP-1 activity has been shown to lead to suppression of cell transformation (Dong et al.. 1997a). Some chemopreventive agents, including aspirin, tea polyphenols, and retinoic acid, have been reported to inhibit cell transformation and tumor promotion by suppressing AP-1 transactivation (Agadir ci al., 1999; Dong et al., 1997a, b). Nuclear factor-KB is a heterodimeric protein composed of different combinations of members of the Rel family of transcription factors. The NF-KB-regulated genes are part of the larger Rel/NF-KB family of transcription factors involved mainly in stress-induced, immune, and inflammatory responses. Nuclear factor-KB is also an important regulator in deciding cell fate, such as programmed cell death and proliferation control, and is critical in turnorigenesis (Baldwin. 1996). Reactive oxygen species (ROS) stimulate transcription by activating transcription factors such as AP-1 and NF-KB. Nuclear factor-KB and AP-1 signal transduction pathways are important in transformation and tumor promotion (Bode and Dong, 2000). TPA or UVB are carcinogens and can produce ROS and stimulate AP-1 and NF-,cB activity by activating mitogen-activated protein kinase (MAPK) signaling pathways such as the ERK 1/2, JNKs, p38, and MEKI/2 MAP kinases (Hou et al., 2004; Schulze-Osthoffet al., 1997). JB6 P mouse epidermal cells are sensitive to tumor promoter treatment and provide a cell culture-based model for studying the mechanism of tumor promotion (Hsu et al., 2000). Transformation-sensitive JB6 P cells are preferred for probing early events in oxidative stress-related signaling leading to carcinogenesis, and for identifying the molecular targets for chemoprevention (Dhar et al., 2002). Antioxidants and extracts of apple peel have been shown to inhibit AP-1 activity in JB6 cells (Ding et al., 2004; Dong et al., 1997a, b). Therefore, some component or components of strawberry extract may be highly effective as a chemopreventive agent that acts by targeting the downregulation of AP-1 and NF-KB activities, blocking MAPK signaling, and suppressing cancer cell proliferation and transformation. Although generally considered high, the levels of antioxidants and antioxidant capacity in strawberry extracts from whole fruit vary considerably among genotypes (Wang and Lin, 2000). This may he true partly because the cultivated strawberry (F. xananassa) is a hybrid of two very different wild species (Darrow, 1966). The paternal progenitor species, F. virginiana, is distributed throughout North America and was originally collected from the East Coast. The maternal progenitor species, F. chiloensis, is distributed on a thin strip of the western American coastline extending from the Aleutians through South America. Accessions of the progenitor species are valued by strawberry breeders as sources of novel traits, especially pest resistance and abiotic stress tolerance. Because strawberry is a relatively new crop, dating to the 1700s CHFf1ICALs. Ascorbate, chlorogenic acid, 13-carotene, histidine, H 202 (30% w/w), hydroxylamine hydrochloride. N,Ndimethyl-p-nitrosoani line, xanthine, xanthine oxide, ascorbate oxidase, dithiothreitol (DTT), oxidized form of GSH (GSSG), GSH, GR, guaiacol, j3-nicotinamide adenine dinucleotide (f3NADH, reduced form), f3-nicotinamide adenine dinucleotide phosphate (13-NADPH, reduced form), nitro blue tetrazolium (NBT), resveratrol (3,5,4'-trihydroxystilhene), bovine serum albumin, Chelex 100, FeSO4 , and MTT [3-(4, 5-diniethylthiazo!-2-yl)-2, 5,-diphenyl tetrazolium bromide] solution were purchased from Sigma Chemical Co. (St. Louis). 6-Hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox), and cL-tocopherol, and trichloroacetic acid were purchased from Aldrich (Milwaukee, WI). 2', 2' Azobis (2-amidinopropane) dihydrochloride was purchased from Wako Chemicals USA Inc. (Richmond, VA). STRAWBERRY GENOTYPES. Eighteen genotypes of three strawberry species (F. chiloensis, F. virginiana, and F. xananassa) (Table 1) were used in this study. Most of the accessions of wild strawberry, and one F. xananassa accession, were chosen because they are included in the core subset described by Hancock et al. (2002). Others were chosen based on past knowledge of their ability to thrive and produce fruit at Beltsville, MD. Three F. chiloensis accessions, 12 F. virginiana accessions, and three F. xananassa accessions were selected. The F. xananassa cultivar, Allstar. was selected for its relatively low antioxidant capacity, whereas 'Ovation' was selected for its relatively high antioxidant capacity among cultivars from the U.S. Department of Agriculture, Agricultural Research Station strawberry breeding program at Beltsville, MD (Lewers et al., 2004). (Darrow, 1966), as few as three baekcrosses can yield selections ofcultivar quality (J.F. Hancock, pers. comm.). Therefore, if thought to be of value in improving antioxidant capacity and health-promoting qualities, accessions from these progenitor species could be readily incorporated into a strawberry breeding program. Neither of the progenitor species has been evaluated for anticancer capability or activities of antioxidant enzymes and nonenzyme components. A core subset of the Fragaria collection maintained at the U.S. Department of Agriculture National Clonal Repository, Corvallis, OR, has been constructed to contain a group of native F. vi,giniana and F chiloensis thought to be of value to strawberry improvement. This core subset is being characterized tbr many horticultural traits useful to breeders (Hancock et al.. 2001a, b), including resistance to black root rot, common foliar diseases, and nematodes (Hancock et al., 2001b, 2002; Pinkerton and Finn, 2005). The objectives of this study are 1)10 identify wild strawberry genotypes with high anticancer and antioxidant activities for use in cultivar development, 2) to evaluate the anticancer capability and activities of antioxidant enzymes and nonenzyme components in representatives of the two progenitor species (F. virginiaiia and F. chiloensis) in comparison with representatives of the cultivated strawberry species (F. Xananassa), and 3) to determine which antioxidant activities are more closely correlated with anticancer activities among representatives of these three species. Materials and Methods 648 J. AMER. Soc. Howr. Sci. 132(5):647-658. 2007. Table 1. Fragaria (strawberry) cultivars, selections, and wild genotypes grown on the north farm of the Henry A. Wallace Agricultural Research Center at Beltsville, MD, and evaluated for inhibition of A594 human lung cancer cell proliferation, activities against free radicals, activities of antioxidant enzymes, and nonenzyme components. Taxon Plant introduction no. Common name Origin F. xananassa P1551406 Allstar Beltsville, MD 1 11 551929 CFRA 0638 Beltsville, MD P1634800 Ovation Beltsville, MD F. chiloensis P1 551735 CFRA 0368 near Juneau, AK