Antioxidant Extracts of African Medicinal Plants Induce Cell Cycle Arrest and Differentiation in B16F10 Melanoma Cells

956 INTERNATIONAL JOURNAL OF ONCOLOGY 43: 956-964, 2013 Antioxidant extracts of African medicinal plants induce cell cycle arrest and differentiation in B16F10 melanoma cells ANGELO GISMONDI1, LORENA CANUTI1, STEFANIA IMPEI1, GABRIELE DI MARCO1, MAURICE KENZO2, VITTORIO COLIZZI1 and ANTONELLA CANINI1 1Department of Biology, University of Rome ‘Tor Vergata’, I-00133 Rome, Italy; 2S/C 46 P.A. de Lingang Dschange, Cameroon Received April 26, 2013; Accepted June 9, 2013 DOI: 10.3892/ijo.2013.2001 Abstract. African ethnomedicine is essentially based on the dreams. These folkloric approaches are essentially based on the traditional use of vegetal extracts. Since these natural drugs use of plant extracts directly on wounds or as treatment by oral have shown health giving properties, in the present study we ingestion (3). Many studies have been performed to ascertain increased further the scientific basis supporting these data. We the scientific basis of African plant biological effects. In parti investigated the effects, on murine B16F10 melanoma cells, of cular, they have demonstrated how these vegetal compounds plant extracts that were directly obtained by a Cameroon ‘tradi- were characterized by a great amount of secondary metabolites tional healer’. After a preliminary study on the antioxidant with antiradical power (4-6). Environmental conditions highly functions of these compounds, already abundant in literature, influence the levels and the quality of these compounds that, Moringa oleifera Lam., Eremomastax speciosa (Hochst.) in nature, are synthesized only by plants, to protect themselves Cufod and Aframomum melegueta K. Schum extracts were from biotic and abiotic factors or to facilitate their propagation individually analyzed. We performed laboratory assessments (7). Since in the equatorial and tropical climates, UV exposure on these medicinal preparations in order to clearly demon- and temperatures are extreme, African plant phyto-complexes strate their antineoplastic features. All the treatments caused have been shown to be richer in antioxidant molecules than sub- in tumor cells a great reduction in growth and proliferation tropical and temperate ones (8). Alkaloids, tannins, steroids, rate, cell cycle arrest, increase of p53, p21WAF1/Cip1 and p27Kip1 terpenoids, terpenes, flavonoids, simple phenols, phenolic acids protein levels and induction of differentiation. These results, on and glycosides have been recognized as the principal metabo- the bioactivity and the biochemical characteristics of African lites whose powerful molecular activity has made African plant plant extracts, may increase the comprehension of indigenous extracts real alternative medicines, especially, on arthritis, flu, therapeutic practices and represent the first step for the indivi diabetes, psoriasis, tuberculosis, ulcers, infections, asthma, duation of new inexpensive and natural drugs able to prevent hypertension, central nervous system disorders, laryngitis, liver and contrast cancer onset. disorders, bronchitis and also cancer (2,9,10). After all, >50% of modern drugs are composed by molecules of vegetal origin Introduction because of their inexpensive production costs, high bioactive functions and low toxicity with respect to artificial chemical African natives infrequently visit public health services and substances (4). The present study was conducted with the drugs and therapies are usually too expensive for their standard aim to find vegetal substances able to represent efficient and of living. Therefore, they rely heavily on ethnomedicine and alternative substitutions to the actual tumor chemotherapeutics, ancient traditions, preserved from the past, in order to cure reported to have serious side-effects for patients (11). In this and prevent their pathologies (1,2). In African communities, study, different plant extracts, normally used in African local medicinal plant identification, processing and administration tradition as natural drugs, were characterized by their free to patients are commonly consigned to ‘traditional healers’ radical scavenging activity and total phenolic content. The most who receive relative guidelines by their ancestors during the antioxidant ones were also evaluated for their antiproliferative and differentiative effects on the B16F10 murine melanoma cell line. The knowledge of the medicinal plant properties on biological systems and the individuation of the molecular Correspondence to: Professor Antonella Canini, Department mechanisms that they can activate are essential aspects for the of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca identification and the development of new drugs as well as to Scientifica 1, Ι00133, Rome, Italy give scientific basis to African medical culture. E-mail: [email protected] Materials and methods Key words: Moringa oleifera Lam., Eremomastax speciosa (Hochst.) Cufod, Aframomum melegueta K. Schum, Cameroon ethnomedicine, Plant material and extract preparations. African plant mate- antineoplastic effects, B16F10 differentiation markers rials (Table I) were collected, by an indigenous ‘traditional GISMONDI et al: ANTINEOPLASTIC EFFECT OF AFRICAN PLANTS ON B16F10 CELL LINE 957 healers’, in the Cameroon forests (Central Africa) and then Flow cytometry analysis. Cells were washed twice in dried, under the sun, until they were completely dehydrated. phosphatebuffered saline and fixed for 30 min at 4˚C in cold Plant extracts were obtained in the laboratory according to methanol:acetone (4:1) solution. Then, cells have been treated, African tradition. Briefly, samples were ground with pestle, at room temperature, for 20 min with RNase A (100 µg/µl) mortar and liquid nitrogen and then boiled in bidistilled water and for further 20 min with propidium iodide (1 mg/ml), and (1 mg/ml) for 1 h. Extracts were filtered (0.22 µm) and finally analyzed by FACSCalibur instrument (Becton-Dickinson) stored at 20˚C. and the percentage of cells in the different cell cycle phases was measured by CellQuest software. Total phenolic content. Total phenolic content was assessed by FolinCiocalteau assay (12). Briefly, 9 ml of ddH2O and 1 ml of Western blotting. Cells were harvested, resuspended in RIPA Folin-Ciocalteau reagent (Sigma-Aldrich) were added to 1 ml lysis buffer, containing 1% protease inhibitor cocktail, and of each extract. After 5 min, the solution was supplemented centrifuged at 13,000 rpm for 30 min at 4˚C. Protein concen- with 10 ml of Na2CO3 7% (w/v) and 4 ml of ddH2O, vortexed tration was quantified by Bradford method (17), using bovine and incubated at room temperature for 1 h in the dark. Total serum albumin as standard. Proteins were separated on 12% phenolic concentration was detected by measuring the sample sodium dodecyl sulfate-polyacrylamide gel and transferred absorbance at 760 nm (UV-visible spectrophotometer Cary 50, onto Protran nitrocellulose membrane (Schleicher and Schuell). Bio Varian), with respect to a caffeic acid calibration curve Blots were incubated with the following primary antibodies: (20100 mg/l). Results are expressed as µg of caffeic acid rabbit polyclonal anti-β-actin (Sigma), mouse monoclonal equivalents per mg of dried sample (µg CAE/mg DW). anti-p53 (Santa Cruz), mouse monoclonal anti-p27Kip1 (BD Pharmingen), mouse monoclonal anti-p21WAF1/Cip1 (Sigma) and FRAP antiradical test. FRAP assay was performed as previ- goat polyclonal anti-MITF (microphthalmia-associated trans- ously reported (13). Plant extract (200 µl) was mixed with 1.8 ml cription factor, Santa Cruz). Finally, primary antibodies were FRAP reagent (10 mM TPTZ in 40 mM HCl, 20 mM FeCl3, revealed using horseradish peroxidaseconjugated antirabbit 0.3 M acetate buffer pH 3.6; 1:1:10 v/v/v) and placed in the dark, or anti-mouse or anti-goat antibodies (Sigma) and an ECL at 37˚C for 10 min. This test evaluated the capacity of natural chemiluminescence detection system (Pierce). Signal detection antioxidants to reduce the colorless Fe III - tripyridyltriazine and quantification was executed, respectively, by VersaDoc compound (Merck) to the blue-colored Fe II form, measuring Imaging System and Quantity One software (Bio-Rad). sample absorbance change at 593 nm. Ascorbic acid (AA in ddH2O) was used to obtain a standard solution (50500 µM). Tyrosinase activity and melanin content. In vitro L-3,4- Results are expressed as µg of ascorbic acid equivalents per mg dihydroxyphenylalanine (L-DOPA) oxidation by protein of dried sample (µg AA/mg DW). extracts was used as direct indicator of cell tyrosinase activity, as previously described (18). In summary, treated and untreated DPPH antioxidant assay. According to BrandWilliams et al cells were resuspended in 10 ml of lysis buffer (50 mM sodium (14), plant extract antioxidant activity was measured by the phosphate buffer pH 6.8, Triton X-100 1% and 0.1 mM phenyl- determination of its scavenging property against the stable methylsulfonyl fluoride) and frozen at 80˚C for 30 min. Then, free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, Merck). In cell extracts were centrifuged at 12000 rpm for 30 min at 4˚C. brief, the absorbance decrease, at 517 nm, of a 100 µM DPPH The supernatant (±8 ml) was mixed with 2 ml of L-DOPA methanolic solution was monitored, after 30 min of sample (2 mg/ml) and the absorbance at 492 nm of the solution was addition. The antiradical effect of the extract is reported as monitored, after incubation for 1 h at 37˚C, by the UVvisible IC50 (sample concentration causing 50% of DPPH activity spectrophotometer,

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