Vitexin-2-O-xyloside, raphasatin and (À)-epigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells Alessio Papi a,1 , Fulvia Farabegoli b,1 , Renato Iori c , Marina Orlandi a , Gina R. De Nicola c , Manuela Bagatta c , Donato Angelino d , Lorenzo Gennari d , Paolino Ninfali d,⇑ a Department of Experimental Evolutive Biology, University of Bologna, Bologna, Italy b Department of Experimental Pathology, University of Bologna, Bologna, Italy c Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali, Via di Corticella, 133, 40129 Bologna, Italy d Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Urbino (PU), Italy article info Article history: Received 1 August 2012 Received in revised form 19 October 2012 Accepted 20 November 2012 Available online 5 December 2012 Keywords: Colon carcinoma Vitexin-2-O-xyloside (–)-Epigallocatechin-3-gallate 4-Methylsulphanyl-3-butenyl isothiocyanate (raphasatin) Chemoprevention abstract Cytotoxic effects of the combination of the food components vitexin-2-O-xyloside (X), raphasatin (4- methylsulphanyl-3-butenyl isothiocyanates; G) and (–)-epigallocatechin-3-gallate (E) were investigated in colon (LoVo and CaCo-2) and breast (MDA-MB-231 and MCF-7) cancer cells. Breast cancer cells were more resistant than colon cells to X, G and E inhibition. On the contrary, marked synergistic effects among X, G and E on cell growth were found in both colon cancer cells. Further analysis revealed a G0/G1 arrest of the phase cell progression and apoptosis, linked to modulation of Bax, Bcl2, caspase-9 and poly(ADP-ribose) polymerase as well as Reactive Oxygen Species (ROS) generation in both colon cancer cells, whereas apoptosis and ROS were not significantly detected in normal human lymphocytes. We conclude that the X, G and E mixture might act by mitochondrial pathway activation of apoptosis, possibly elicited by ROS and the mixture may be effective in the chemoprevention of colon cancer. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Fruit and vegetables are rich sources of many bioactive com- pounds, which positively interact to decrease the risk of chronic diseases, including cancer (Surh, 2003). For this wide group of bio- active molecules, the term ‘‘chemopreventive phytochemicals’’ (CP) was coined (Sporn, 1980). Beyond the well-known radical scavenging activity, most of the CP interfere with the mechanisms that sustain cancer development such as: evasion of apoptosis, growth self-sufficiency, insensitivity to anti-growth signals, high replication rate and angiogenesis initiation (Lippman & Levin, 2005). It has been proposed that not only CP can prevent oncolog- ical events, but they also have a role in the treatment of neoplastic conditions (D’Incalci, Steward, & Gescher, 2005). CP are less spe- cific and have numerous molecular targets in relation to chemi- cally designed anti-neoplastic drugs (CDAD); however, CP operate in a narrow concentration range with relatively low toxic- ity (Davis, 2007). Moreover, pure CP, like polyphenols, glucosino- lates or carotenoids, can be combined together or with food extracts (Liu, 2004) and can positively increase the potential activ- ity of the mixture, in order to deregulate oncological events. Com- binations of natural molecules or extracts that effectively inhibit tumour development and progression are nowadays actively re- searched (Mertens-Talcott & Percival, 2005). Among the CP most frequently used in antitumour mixtures, catechins from green tea extracts and chiefly (–)-epigallocate- chin-3-gallate (E), showed relevant anticancer activity (Khan, Afaq, & Mukhtar, 2008; Yang, Wang, Lu, & Picinich, 2009) and optimal pharmacokinetic behaviour (D’Incalci et al., 2005). The combina- tion of soy phytochemicals and green tea extract was more effec- tive in inhibiting tumour angiogenesis than when either food was provided alone; similarly, soy phytochemicals and green tea synergistically inhibited tumour growth and metastasis in a mouse model of androgen sensitive human prostate cancer (Zhou, Yu, Mai, & Blackburn, 2004). Another class of CP used for anticancer cock- tails are the isothiocyanates (ITC), present in Cruciferous (Brassica- ceae) vegetables in the form of glucosinolates (GL) that are released following exposure of GL to the enzyme myrosinase (b-thiogluco- side glucohydrolase, EC. 3.2.1.147) (Barillari et al., 2008; Papi et al., 2008). One of these GL, named glucoraphasatin (GRH), re- leases a biologically important ITC, the 4-methylsulphanyl-3-bute- 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.112 ⇑ Corresponding author. Address: Department of Biomolecular Sciences, Univer- sity of Urbino ‘‘Carlo Bo’’, Via Saffi 2, 61029 Urbino (PU), Italy. Tel.: +39 0722 305288; fax: +39 0722 305324. E-mail address: paolino.ninfali@uniurb.it (P. Ninfali). URL: http://www.uniurb.it/orac/ (P. Ninfali). 1 These authors contributed equally to this work. Food Chemistry 138 (2013) 1521–1530 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem