Treatment of Metastatic Melanoma B16F10 by the Flavonoids Tangeretin, Rutin, and Diosmin CRISTINA MARTI ÄNEZ CONESA, † VICENTE VICENTE ORTEGA, † M. JOSEFA YA Ä N ˜ EZ GASCO Ä N, † MIGUEL ALCARAZ BAN ˜ OS, ‡ MANUEL CANTERAS JORDANA, # OBDULIO BENAVENTE-GARCI ÄA,* ,§ AND JULIA Ä N CASTILLO § Pathology, Radiology and Physical Medicine, and Biostatistics Departments, Faculty of Medicine, University of Murcia, 30100 Espinardo, Murcia, Spain, and Research and Development Department, Nutrafur-Furfural S.A., Camino Viejo de Pliego s/n, 80320 Alcantarilla, Murcia, Spain Melanoma is one of the most frequently metastasizing malignant neoplasias. This study examines an experimental model of pulmonary metastasis and the B16F10 cell subline, highly metastatic in the lung. Antimetastatic effects of the flavonoids tangeretin, rutin, and diosmin were analyzed, and at the same time an analysis of the metastatic activity of ethanol was performed, considered to be necessary because it is used as a vehicle for administering the flavonoids. Lentini’s model, which complements the macroscopic evaluation of nodule numbers by using a stereoscopic microscope and image analysis at the microscopic level, was used. The greatest reduction in the number of metastatic nodules (52%) was obtained with diosmin; similarly, the percentages of implantation, growth index, and invasion index (79.40, 67.44, and 45.23%, respectively), were all compared with those of the ethanol group, considered to be an effective control group. Rutin- and tangeretin-treated groups also showed reductions of the same index compared with the ethanol group. It would seem that structural factors would better explain these results and the antimetastatic activity of each flavonoid and the respective metabolites. KEYWORDS: Melanoma; B16F10; metastasis; flavonoid; ethanol; tangeretin; rutin; diosmin INTRODUCTION Melanoma is the fastest increasing cancer (3.3% per year growth between 1990 and 1996) among the Caucasian popula- tion of the United States (1) and is the 4th most common cancer in Australia and New Zealand and the 10th most common in the United States (2). It is calculated that ∼50000 new cases were diagnosed in the United States in 2001 and that at least 10000 people died as a result of metastasis (3). The study of new therapies for melanoma points to the frequent inadequacy of the actual treatments, its steadily growing incidence, and the gradually increasing mortality rates. It is one of the most rapidly metastasizing of malignant neoplasias; after the lymphatic glands, the lung is the most common organ to be attacked, with an incidence of between 12% (4) and 20% (5). Fidler’s classical studies dating from 1973 (6) demonstrated the predilection of melanomas to colonize different organs due to the presence of different characteristics: chemical, immunological, or through adhesion to the surface of tumoral cells (7). Flavonoids are polyphenols that have been shown, in both in vitro and in vivo test system models, to induce responses with the protective effects of diets rich in fruits and vegetables against degenerative conditions such as cardiovascular disease and cancer (8). Some of these flavonoids, especially rutin and diosmin, are commonly used as active constituents of several pharmaceutical products, mainly against cardiovascular diseases. Diosmin is also used as an antivaricose and vasoprotective agent, whereas rutin has been widely described as a powerful captor of free radicals and as anticarcinogenic (9). Both flavonoids may act in the different developmental stages of malignant tumors by protecting DNA against oxidative damage, inactivat- ing carcinogens, inhibiting the expression of mutagenic genes and enzymes responsible for activating procarcinogenic sub- stances, and activating the systems responsible for xenobiotics detoxification (10). In vitro, flavonoids have demonstrated their capacity to modify the activity of enzymatic systems in mammals (kinases, phospholipases, ATPase, lipooxygenases, cyclooxygenases, phosphorodiesterases, etc.), a correlation having been observed in some cases between the flavonoid structure and its enzymatic activity (11-13). More specifically, preliminary studies on melanoma lines using several flavonoids of a Citrus origin showed that the presence of the C2-C3 double bond on the C ring (14), * Author to whom correspondence should be addressed (telephone +34 968 892512; fax +34 968 890933; e-mail laboratorio@nutrafur.com). † Pathology Department. ‡ Radiology and Physical Medicine Department. # Biostatistics Department. § Nutrafur-Furfural S.A. J. Agric. Food Chem. 2005, 53, 6791-6797 6791 10.1021/jf058050g CCC: $30.25 © 2005 American Chemical Society Published on Web 07/23/2005