Anthocyans and Chemoprevention: Evidence from Cellular

Chapter 26

Anthocyans and Chemoprevention: Evidence from Cellular Investigations

Downloaded by PURDUE UNIV on August 23, 2016 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch026

De-Xing Hou Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima City 890-0065, Japan

Anthocyans are polyphenols ring-based flavonoids, and widespread in fruits and vegetables with red-blue color. In recent years, we have used culture cells to investigate their chemopreventive effects. In mouse epidermal cells (JB6), a model for screening anticarcinogenic compounds, anthocyans inhibited cell transformation by targeting mitogen activated protein kinase (MAPK)-mediated activator protein-1 (AP-1) pathway. In mouse macrophage cells (RAW264), a model for identifying the anti-inflammation compounds, anthocyans suppressed LPS-evoked inflammation through targeting nuclear factor kappa Β (NF-κΒ), AP-1 and CCAAT/enhancer binding protein (C/EBPδ)-mediated cyclooxygenase-2 (COX2) expression. In human leukemia cells (HL-60), a model for testing antitumoral compounds, anthocyans induced apoptosis of cancer cells through the reactive oxygen species (ROS)/cJun NH -terminal kinase (JNK)-mediated mitochondrial dysfunction pathway. Our data provided the first molecular and cellular basis that anthocyans might have chemopreventive effects on several key steps involved in carcinogenesis. 2

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© 2008 American Chemical Society Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Anthocyans (in Greek anthos means flower, and kyanos means blue) are flavanols with polyphenols chemical structure. Anthocyan encompasses "anthocyanin" for the glycoside and "anthocyanidin" for the aglycon. The chemical structures are shown in Figure 1A (7, 2). Depending on their pH and the presence of chelating metal ions, they are intensely colored in blue, violet or red. Up to date, more than 400 anthocyanins have been found in nature (/, 2). On the other hand, the aglycon is a diphenylpropanoide-based polyphenolic ring structure, and is limited to a few structure variants. Only six kinds of anthocyanidins including delphinidin, cyanidin, petunidin, pelargonidin, peonidin and malvidin are common in fruits and vegetables (Figure IB) (7, 2).

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Figure. 1. Chemical structure of anthocyanins (A) and anthocyanidins (B) Anthocyans occur in very large amounts in some diets from colored crops, fruits and vegetables such as berries, purple sweet potatoes, red grapes and cabbages (7, 2). Servings of 200 g of aubergine or black grapes can provide up to 1500 mg anthocyans and servings of 100 g of berries up to 500 mg. Daily intake of anthocyans in humans has been estimated from several milligrams to grams (3), which depends on the nutrition customs. Mean dietary intake in Finland has been estimated to be 82 mg/day (3). The American diet can have much as 180215 mg/day (3). Recently, daily intake of several grams can be also obtained if an individual is consuming flavonoids supplements, such as bilberry, grape or elderberry extracts (4). There are some reports hinting at the potential anti-carcinogenicity of anthocyans. In a cohort of elderly individuals, who consumed large amounts of strawberries, the odds ratio for developing cancer at any site was 0.3, compared to subjects who refrained from high berry consumption (5). Consumption of colored fruits and vegetables has also been associated with a reduced risk of human breast cancer (6) and colorectal polyp recurrence (7). Anthocyancontaining foodstuffs have been linked with a decreased risk of coronary heart

Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


310 disease. They have been shown to possess beneficial effects in several parts of the organism (8), including the central nervous system and the eye, and have been suspected to account, at least in part, for the "French paradox", i.e. the decreased risk of cardiovascular disease despite a high-fat diet in individuals living in France. Accumulated data indicate anthocyans have considerable bioavailability (3), and biological activities including anti-oxidation, anti-mutagenicity, anti inflammatory, and anti-proliferation in some cancer cells (9, 10). These facts suggest that anthocyans might be potential chemoprevention agents, and that mechanisms on chemopreventive effects need to be considered at molecular level. Most of cancer chemoprevention-related compounds, such as components of tea or red wine, act to prevent tumor promotion by targeting signal transduction pathways to attenuate the expression of AP-1, and/or COX-2, or by inducing cell cycle arrest and apoptosis. Therefore, the preset study is to characterize the chemopreventive effects of anthocyans by targeting those wellaccepted cellular/molecular mechanisms that can at least partially explain the effectiveness of natural compounds as chemopreventive agents. The contents include that (i) anthocyans inhibit neoplastic transformation through the inhibition of AP-1 activation; (ii) anthocyans suppress inflammation by blocking COX-2 overexpression; and (iii) anthocyans inhibit proliferation/or growth of tumor cells by inducing apoptosis.

Results and Discussion Anti-carcinogenic Effects A mechanism-based model is important to investigate anti-carcinogenic effects of natural bioactive components. Mouse epidermal cells (JB6) provide a cell culture-based model for studying tumor promotion (77). In this cell line, tumor promoters such as TPA, EGF and TNF-a induce AP-1 activity and neoplastic transformation by activating MAPK including ERK, JNK or p38 kinase (77). The induced AP-1 activity and neoplastic transformation can be blocked by chemopreventive agents, such as retinoids (72), pyrrolidine dithiocarbamate (13), tea polyphenols (14), and glycoside compounds (75). Many of these inhibitors have been shown to be active not only in the JB6 transformation model but also in mouse skin tumor promotion in vivo. Thus, JB6 cells provide a validated model to screen cancer chemopreventive agents, and to elucidate their mechanisms at the molecular level. To investigate the anti-carcinogenic effects of anthocyans, four kinds of representative anthocyanins (Figure 2A) and six kinds of typical anthocyanidins (Figure 2B) were used to examine their effects on cell transformation in JB6



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cells. Within the concentration range of 5-20 juM, in which the cellular viability was not affect as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (data not shown). Of anthocyanins and anthocyanidins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring such as YGM3, Cy3Sam, Dp3Sam, delphinidin, cyanidin and petunidin (Figure 1) suppressed TPA-induced cell transformation in a dose-dependent manner (Figure 2), suggesting that the ortho-dihydroxyphenyl contributes to the inhibitory action. Molecular evidence revealed that 20 | i M of delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in ERK pathway at early times (2 h) and JNK signaling pathway at later times (12 h) (16). On the other hand, p38 kinase was not inhibited by 20 juM of delphinidin. Furthermore, 10 | i M of MAPK specific inhibitors (SP600125 for JNK and U0126 for MERK1/2) could specifically block the activation of c-Jun and ERK, and cell transformation. Thus, the active anthocyans might contribute to the inhibition of cell transformation by blocking activation of ERK and JNK pathway. On the other hand, TPA treatment in JB6 cells generated reactive oxygen species (ROS) that further promotes neoplastic transformation. We identified that TPA-induced superoxide anion contribute to AP-1 activation and cell transformation. Five | i M of delphinidin showed synergistic effect with 200U/ml of SOD to inhibit AP-1 activation and cell transformation (16). Our findings together with other reports suggest that the inhibitory effects of active anthocyans on AP-1 activation and cell transformation are due in part to their potent scavenging activity for superoxide radicals and in part to blocking ERK and JNK pathway. Both targets may be important in the cancer prevention activity of anthocyans (Figure 3). JB6 C141 cells (1 x 10 ) were exposed to 20 ng/mLTPA with or without 5-20 j^M of anthocyanins (A) or anthocyanidins (B) on soft agar medium. The cell colonies were scored with a computerized image analyzer after 14-day incubation in a 37°C, 5% C 0 incubator. The inhibitory efficiency of cell transformation is expressed as a percentage of the transformation frequency when the cells were treated with TPA alone. Each value represents the mean ± SD of 4-5 separate experiments. *P

Anthocyans and Chemoprevention: Evidence from Cellular

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