Blackberry Extracts Inhibit Activating Protein 1 Activation and Cell Transformation by Perturbing the Mitogenic Signaling Pathway Rentian Feng, Linda L. Bowman, Yongju Lu, Stephen S. Leonard, Xianglin Shi, Bing-Hua Jiang, Vince Castranova, Val Vallyathan, and Min Ding Abstract: Blackberries are natural rich sources of bio- flavonoids and phenolic compounds that are commonly known as potential chemopreventive agents. Here, we inves- tigated the effects of fresh blackberry extracts on prolifera- tion of cancer cells and neoplastic transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as well as the underlying mechanisms of signal transduction pathways. Using electron spin resonance, we found that blackberry ex- tract is an effective scavenger of free radicals, including hydroxyl and superoxide radicals. Blackberry extract inhib- ited the proliferation of a human lung cancer cell line, A549. Pretreatment of A549 cells with blackberry extract resulted in an inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation induced by ultraviolet B (UVB) irradiation. Black- berry extract decreased TPA-induced neoplastic transforma- tion of JB6 P + cells. Pretreatment of JB6 cells with black- berry extract resulted in the inhibition of both UVB- and TPA-induced AP-1 transactivation. Furthermore, blackberry extract also blocked UVB- or TPA-induced phosphorylation of ERKs and JNKs, but not p38 kinase. Overall, these results indicated that an extract from fresh blackberry may inhibit tumor promoter-induced carcinogenesis and associated cell signaling, and suggest that the chemopreventive effects of fresh blackberry may be through its antioxidant properties by blocking reactive oxygen species-mediated AP-1 and mitogen-activated protein kinase activation. Introduction Previous studies have demonstrated the chemopreventive effects of fruits and vegetables in animals and humans (1–3). Epidemiological investigations suggest that diet composition plays an important role in cancer risk control (4,5). Among the potential chemoprotective diets, growing attention has been dedicated to berry products, such as blackberry, straw- berry, cranberry, black raspberry, and blueberry (6–10). Al- though these berry products have been reported to possess anti-inflammatory and chemopreventive effects (6,9,10–14), little is known about the underlying mechanisms at the cellu- lar and molecular levels. The AP-1 signal transduction pathway is known to be an important molecular target of chemopreventive strategies. AP-1, a ubiquitous transcriptional activator, is composed of members of the Jun and Fos families that form homodimers or heterodimers and bind to a distinct DNA response ele- ment. Elevated AP-1 activities and its upstream regulators, mitogen-activated protein kinases (MAPK), are involved in many disease processes, such as inflammation, neoplastic transformation, tumor progression, metastasis, and angio- genesis (15–19). Reactive oxygen species (ROS) produced by cells exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV) irradiation can result in rapid acti- vation of MAPKs, ERKs, JNKs, and p38 (20). The develop- ment of an oxidant/antioxidant imbalance in preneoplasia may activate redox-sensitive transcription factor AP-1, which controls the expression of various genes implicated in inflammatory processes, cell differentiation, and stress re- sponses (19,21,22). Blockade of TPA-induced AP-1 activa- tion has been shown to inhibit neoplastic transformation (23). Inhibition of AP-1 activity in transformed JB6 RT101 cells causes reversion of the tumor phenotype (24). Further- more, the role of AP-1 transactivation in tumor promotion has been demonstrated using transgenic mice (25). There- fore, elevated AP-1 activity has been implicated as causal in transformation responses to tumor promoters. A potential chemopreventive strategy would be to block MAPK-AP-1 signal activation induced by environmental carcinogens us- ing natural antioxidants. Evidence for the involvement of oxidative stress in tumor promotion has accumulated in the past years. The antioxidant theory of cancer prevention has been widely documented (15). Early studies reported that natural antioxidants from vegetables or fruits could inhibit cancer cell growth and in- duce apoptosis (26–28). It has been proposed that the con- sumption of whole fruits may provide the antioxidant bal- NUTRITION AND CANCER, 50(1), 80–89 R. Feng, L. L. Bowman, Y. Lu, S. S. Leonard, X. Shi, V. Castranova, V. Vallyathan, and M. Ding are affiliated with Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505. B.-H. Jiang is affiliated with Mary Babb Randolph Cancer Center and the Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506.