In Vitro Antiproliferative Activity of Isothiocyanates and Nitriles Generated by Myrosinase-Mediated Hydrolysis of Glucosinolates from Seeds of Cruciferous Vegetables Claudio Nastruzzi,* ,†,‡ Rita Cortesi, † Elisabetta Esposito, † Enea Menegatti, † Onofrio Leoni, § Renato Iori, § and Sandro Palmieri § Dipartimento di Scienze Farmaceutiche, Universita ` di Ferrara, Ferrara, Italy, Istituto di Chimica e Tecnologia del Farmaco, Universita ` di Perugia, Via del Liceo, 06100 Perugia, Italy, and Istituto Sperimentale per le Colture Industriali MiRAAF, Bologna, Italy A comparison of the effect of isothiocyanates and nitriles derived from some glucosinolates, namely, epi-progoitrin, sinalbin, glucotropaeolin, glucocheirolin, and glucoraphenin, on human erythroleu- kemic in vitro cultured cells was studied. Many studies have in fact evidenced that a consumption of vegetable containing glucosinolates could reduce the development of colorectal cancer. In the experimental conditions used, the production of isothiocyanates and nitriles from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The obtained results demonstrated that in general nitriles are considerably less potent than the corresponding isothiocyanates in inhibiting cancer cell growth. Particularly, the isothiocyanates inhibitory activity on K562 cells growth is higher in the case of products derived from epi-progoitrin, glucotropaeolin, glucoraphenin, and glucocheirolin; while for nitriles the higher activity in inhibiting K562 cells growth is showed by sinalbin-derived product. Considering the antiproliferative activity found for isothiocyanates and nitriles, further studies will be aimed to the possible application of glucosinolate-derived products as chemopreventive cancer agents for the reduction of colorectal cancer. Keywords: Enzymes and enzyme reactions; antitumor compounds; antiproliferative agents INTRODUCTION The biological activity shown by glucosinolates (GLs) and their derived products, generally considered as detrimental compounds, has recently become more and more important due to their possible protective activity against intestine cancer (Lee et al., 1989; Manousos et al., 1983). GLs are characterized by a -D-thioglycosidic group and a variable side chain, generally represented by aliphatic, aromatic, or heteroaromatic residues. These compounds are contained in various quantities and ratios in the seeds, roots, stems, and leaves of some dicotyledonous angiosperms, the most important of which are typified by Cruciferae. In the intact cells, GLs are kept separate from the endogeneous enzyme myro- sinase, a -thioglucoside glucohydrolase able to catalyze the hydrolysis of GLs. When myrosinase and GLs react together after cell disruption, they result in the produc- tion of D-glucose and a series of different compounds, i.e., isothiocyanate, thiocyanate, and nitriles, depending on the substrate and the reaction conditions used, such as pH, temperature, and substrate structure (Gil and MacLeod, 1980). In this respect, it is important to emphasize that, at neutral pH and temperature above 25 °C, the formation of isothiocyanates is definitely favored; while using a protonation mechanism and low pH, the main degradation product is represented by nitriles (Gil and MacLeod, 1980). In our previous studies, we demonstrated that GL-derived products possess interesting antiproliferative activity toward tumoral cells (Nastruzzi et al., 1996; Leoni et al., 1997). The cytotoxic mechanism of these compounds is not completely understood, although it seems that these minor dietary constituents could be important inhibitors of carcinogenesis, acting by neutralization of a wide number of carcinogens or by the suppression of prolif- erative activity of neoplastic cells (Wattemberg, 1982). In the first case, GLs and derived products would prevent carcinogen molecules from reaching the target site or interacting with the reactive carcinogenic mol- ecules or activating important hepatic enzymes for the protection against several carcinogens, such as quinone reductase, glutathione S-transferase and UDP-glucoro- nosyl transferase (Zhang et al., 1992); in the second case, they may weaken the effects of genetic changes that occurred in the early stages of neoplastic transfor- mation (Nugon-Baudon et al., 1990). In addition, other studies demonstrated that some compounds, such as isothiocyanates, are able to inhibit protein synthesis and affect carbohydrate metabolism (Leblova, 1965). Taking into account these findings and those regard- ing our previous studies on the myrosinase-GL system (Leblova, 1965; Palmieri et al., 1986, 1987; Pessina et al., 1990; Visentin et al., 1992; Hochkoeppler and Palmieri, 1992; Lazzeri et al., 1993; Leoni et al., 1993), we studied the effect of some purified GLs enzymatic hydrolysis products (i.e., ITCs and nitriles) on in vitro cultured human K562 erythroleukemic cell line (Lozzio and Lozzio, 1975). This cell line is one of the best known in vitro experimental model systems mimicking eryth- roid differentiation when treated with suitable inducers * Corresponding author. Tel.:+39/075/5855158. Fax:+39/ 075/5847469. E-mail: nas@unipg.it. † Universita ` di Ferrara. ‡ Universita ` di Perugia. § Istituto Sperimentale per le Colture Industriali MiRAAF. 3572 J. Agric. Food Chem. 2000, 48, 3572-3575 10.1021/jf000191p CCC: $19.00 © 2000 American Chemical Society Published on Web 07/14/2000