Introduction
African natives infrequently visit public health services and drugs and therapies are usually too expensive for their standard of living. Therefore, they rely heavily on ethnomedicine and ancient traditions, preserved from the past, in order to cure and prevent their pathologies (1,2). In African communities, medicinal plant identification, processing and administration to patients are commonly consigned to ‘traditional healers’ who receive relative guidelines by their ancestors during the dreams. These folkloric approaches are essentially based on the use of plant extracts directly on wounds or as treatment by oral ingestion (3). Many studies have been performed to ascertain the scientific basis of African plant biological effects. In particular, they have demonstrated how these vegetal compounds were characterized by a great amount of secondary metabolites with antiradical power (4–6). Environmental conditions highly influence the levels and the quality of these compounds that, in nature, are synthesized only by plants, to protect themselves from biotic and abiotic factors or to facilitate their propagation (7). Since in the equatorial and tropical climates, UV exposure and temperatures are extreme, African plant phyto-complexes have been shown to be richer in antioxidant molecules than sub-tropical and temperate ones (8). Alkaloids, tannins, steroids, terpenoids, terpenes, flavonoids, simple phenols, phenolic acids and glycosides have been recognized as the principal metabolites whose powerful molecular activity has made African plant extracts real alternative medicines, especially, on arthritis, flu, diabetes, psoriasis, tuberculosis, ulcers, infections, asthma, hypertension, central nervous system disorders, laryngitis, liver disorders, bronchitis and also cancer (2,9,10). After all, >50% of modern drugs are composed by molecules of vegetal origin because of their inexpensive production costs, high bioactive functions and low toxicity with respect to artificial chemical substances (4). The present study was conducted with the aim to find vegetal substances able to represent efficient and alternative substitutions to the actual tumor chemotherapeutics, reported to have serious side-effects for patients (11). In this study, different plant extracts, normally used in African local tradition as natural drugs, were characterized by their free radical scavenging activity and total phenolic content. The most 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 mechanisms that they can activate are essential aspects for the identification and the development of new drugs as well as to give scientific basis to African medical culture.
Materials and methods
Plant material and extract preparations
African plant materials (Table I) were collected, by an indigenous ‘traditional healers’, in the Cameroon forests (Central Africa) and then dried, under the sun, until they were completely dehydrated. Plant extracts were obtained in the laboratory according to African tradition. Briefly, samples were ground with pestle, mortar and liquid nitrogen and then boiled in bidistilled water (1 mg/ml) for 1 h. Extracts were filtered (0.22 μm) and finally stored at −20°C.
Total phenolic content
Total phenolic content was assessed by Folin-Ciocalteau assay (12). Briefly, 9 ml of ddH2O and 1 ml of Folin-Ciocalteau reagent (Sigma-Aldrich) were added to 1 ml of each extract. After 5 min, the solution was supplemented with 10 ml of Na2CO3 7% (w/v) and 4 ml of ddH2O, vortexed and incubated at room temperature for 1 h in the dark. Total phenolic concentration was detected by measuring the sample absorbance at 760 nm (UV-visible spectrophotometer Cary 50, Bio Varian), with respect to a caffeic acid calibration curve (20–100 mg/l). Results are expressed as μg of caffeic acid equivalents per mg of dried sample (μg CAE/mg DW).
FRAP antiradical test
FRAP assay was performed as previously reported (13). Plant extract (200 μl) was mixed with 1.8 ml FRAP reagent (10 mM TPTZ in 40 mM HCl, 20 mM FeCl3, 0.3 M acetate buffer pH 3.6; 1:1:10 v/v/v) and placed in the dark, at 37°C for 10 min. This test evaluated the capacity of natural antioxidants to reduce the colorless Fe III - tripyridyltriazine compound (Merck) to the blue-colored Fe II form, measuring sample absorbance change at 593 nm. Ascorbic acid (AA in ddH2O) was used to obtain a standard solution (50–500 μM). Results are expressed as μg of ascorbic acid equivalents per mg of dried sample (μg AA/mg DW).
DPPH antioxidant assay
According to Brand-Williams et al(14), plant extract antioxidant activity was measured by the determination of its scavenging property against the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, Merck). In brief, the absorbance decrease, at 517 nm, of a 100 μM DPPH methanolic solution was monitored, after 30 min of sample addition. The antiradical effect of the extract is reported as IC50 (sample concentration causing 50% of DPPH activity reduction with respect to the control).
Cell culture, treatments, proliferation assays and microscopic observations
Highly metastatic B16F10 murine melanoma cells were grown and propagated in Dulbecco’s modified Eagle’s medium (D-MEM), supplemented as reported in Gismondi et al(15), under standard culture conditions (16). To study African plant antiproliferative effects, melanoma cells were seeded in 35-mm dishes and treated with vegetal extracts (2 mg of plant dried weight per ml of cell culture media) for 24, 48 and 72 h (control cells were treated with phosphate-buffered saline). Other treatment concentrations were also tested (data not shown). Cell proliferation and treatment cytotoxicity were evaluated by counting cells, with a Neubauer modified chamber, after trypan blue staining (1%, w/v). In addition, cell growth, measured as function of mitochondrial activity, was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Sigma). Microscopic observations were also performed on cells by optical microscope (20X) (Nikon, TE2000-PFS).
Flow cytometry analysis
Cells were washed twice in phosphate-buffered saline and fixed for 30 min at 4°C in cold methanol:acetone (4:1) solution. Then, cells have been treated, at room temperature, for 20 min with RNase A (100 μg/μl) and for further 20 min with propidium iodide (1 mg/ml), and analyzed by FACSCalibur instrument (Becton-Dickinson) and the percentage of cells in the different cell cycle phases was measured by CellQuest software.
Western blotting
Cells were harvested, resuspended in RIPA lysis buffer, containing 1% protease inhibitor cocktail, and centrifuged at 13,000 rpm for 30 min at 4°C. Protein concentration was quantified by Bradford method (17), using bovine serum albumin as standard. Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel and transferred onto Protran nitrocellulose membrane (Schleicher and Schuell). Blots were incubated with the following primary antibodies: rabbit polyclonal anti-β-actin (Sigma), mouse monoclonal anti-p53 (Santa Cruz), mouse monoclonal anti-p27Kip1 (BD Pharmingen), mouse monoclonal anti-p21WAF1/Cip1 (Sigma) and goat polyclonal anti-MITF (microphthalmia-associated transcription factor, Santa Cruz). Finally, primary antibodies were revealed using horseradish peroxidase-conjugated anti-rabbit or anti-mouse or anti-goat antibodies (Sigma) and an ECL chemiluminescence detection system (Pierce). Signal detection and quantification was executed, respectively, by VersaDoc Imaging System and Quantity One software (Bio-Rad).
Tyrosinase activity and melanin content
In vitro L-3,4-dihydroxyphenylalanine (L-DOPA) oxidation by protein extracts was used as direct indicator of cell tyrosinase activity, as previously described (18). In summary, treated and untreated cells were resuspended in 10 ml of lysis buffer (50 mM sodium phosphate buffer pH 6.8, Triton X-100 1% and 0.1 mM phenylmethylsulfonyl fluoride) and frozen at −80°C for 30 min. Then, cell extracts were centrifuged at 12000 rpm for 30 min at 4°C. The supernatant (±8 ml) was mixed with 2 ml of L-DOPA (2 mg/ml) and the absorbance at 492 nm of the solution was monitored, after incubation for 1 h at 37°C, by the UV-visible spectrophotometer, Cary 50 (Bio Varian). Intracellular melanin quantification was measured and performed as suggested by Lotan and Lotan (19). Briefly, cells were harvested and lysed (Tris-HCl 50 mM pH 7.5, EDTA 2 mM, NaCl 150 mM, Triton X-100 1%, protease inhibitor 1%). Then samples were sonicated for 30 sec and centrifuged at 1400 rpm for 5 min. The supernatant was used for sample protein quantization (17). The pellet was washed twice with 1 ml of ethanol:diethyl ether (1:1) and finally resuspended in NH4OH 1 M at 37°C until it was completely dissolved. The melanin amount in the solution was determined by analyzing the absorbance value at 475 nm (UV-visible spectrophotometer Cary 50, Bio Varian). Results are expressed as μg melanin per mg of cell proteins.
Statistical analysis
All experiments were repeated in triplicate and the relative results are shown as the mean ± standard error of the mean (SEM) of the three independent measurements. Analysis of variance was conducted using one-way ANOVA test with SPSS (ver.19 ita) for Microsoft and the means were compared by Duncan tests. All p-values were <0.05 versus vehicle control-treated cells.
Results
Secondary metabolites and antioxidant properties
Fourteen African plants (Table I) were processed and subjected to aqueous extraction, as described in Materials and methods. The amount of phenolic compounds was measured in each extract by a spectrophotometric analysis (Fig. 1). ES, MN, MOC, MC and AM samples showed the highest aromatic secondary metabolite contents, respectively, 59.58, 55.83, 39.17, 33.33 and 32.92 μg CAE/mg DW. FRAP and DPPH assays were performed in order to determine the antiradical power of Cameroon plant extracts. The FRAP test (Fig. 2) allowed us to separate samples in three principal clusters, according to their antioxidant activity: the first group (including HC, PJ, EC, MNR, MNF, MNS and AP samples) revealed a radical scavenging property <5 μg AA/mg DW; the second cluster, made up of MC, ZO and MNC specimens, evidenced intermediate values (between 5 and 10 μg AA/mg DW) whilst the best antioxidant extracts (MOC, ES, MN and AM) exhibited a reducing activity >10 μg AA/mg DW. In DPPH assay (Fig. 3), MOC, MC, MN, ES, MNC, MNF and AM extracts were identified as the strongest antiradical solutions. In particular, they respectively, presented an IC50 value of 0.94, 0.80, 0.99, 0.36, 0.77, 0.61 and 0.55 μg extract per ml. In conclusion, in vitro tests showed MN, ES and AM were the most antioxidant samples.
Effects on cell growth and proliferation
B16F10 murine melanoma cells were treated for 24, 48 and 72 h with 2 mg/ml of MN, ES and AM extracts in order to analyze their effects on cell proliferation (Fig. 4). Proliferative tests were also performed with other treatment concentrations but data are not shown in this study because of irrelevance or the excessive effects. Cells incubated for 24 h with African samples did not show significant changes in cell growth, with respect to the control (CNT). On the other hand, treatments with MN, ES and AM for 48 h induced a reduction of cell proliferation, compared to control cells, of ~16, 32 and 39%, respectively. After 72 h of incubation, plant extracts caused the decrease of the number of cells of ~42 (MN), 47 (ES) and 61% (AM), with respect to the control. To determine if natural solutions could be toxic for cells, trypan blue exclusion test was also performed. As reported in Table II, treatments for 24 h did not cause any cell injury: in all cases, toxicity was <2% with respect to control cells. After 48 h, ES and AM solutions still showed slight cytotoxicity (<6%) whilst MN extract was ~12%, compared to control cells. Cells remained highly viable also after 72 h of contact with ES and AM extracts (only ~7% of toxicity was detected); in contrast, MN treatment induced the death of ~22% of cells, with respect to the control. Cell proliferation, after treatment with Cameroon plant extracts for 72 h, was further monitored by MTT assay (Fig. 5). MN, ES and AM treatments produced a decrease of cell growth of 42.7, 50.9 and 39.4%, respectively, compared to control cells.
Analysis of the cell cycle and related proteins
In order to verify if the previous reduction of B16F10 cell proliferation was associated with cell cycle modifications, FACS analysis was carried out. After treatment for 72 h with the different African preparations, very contrasting cell cycle profiles were revealed (Fig. 6), compared to the control (CNT). In particular, the amount of apoptotic cells, that was minimal in the control (2.6%), increased after MN treatment (21.2%) with respect to ES and AM, whose sub-G1 events were only 3.8 and 4.9%, respectively. As indicated in Fig. 7, with respect to the control, MN treatment produced an increase in G2/M phase (10.9%), ES solution induced an accumulation of cells (13.4%) between the S and G2/M peaks whilst AM extract clearly caused the arrest of the cell cycle in G1 phase (13.9%). Protein extraction was performed on B16F10 cells and the principal cell cycle regulators were detected by western blotting (Fig. 8). MN, ES and AM solutions enhanced p53 levels 50.8, 28.8 and 40.1%, in this order, with respect to the control. Similarly, p21WAF1/Cip1 content was highly increased following the treatments (>300% of the control). As a final point, p27Kip1 amount was investigated in cell protein extracts: the levels augmented of 29 and 25%, respectively, with ES and AM extracts and, remarkably, of 143% following MN treatment, with respect to control cells.
Differentiation induction
Differentiative properties of Cameroon plant extracts on B16F10 cell line, were also checked. After treatment for 72 h with MN, ES and AM extracts, with respect to the control, cells evidenced great morphological changes and decrease of cell density. Optical microscope images (Fig. 9) clearly showed how treated cells (especially MN and ES) had developed cytoplasmic dendritic protrusions and acquired a star shape. Melanin amount (Fig. 10) and tyrosinase activity (Fig. 11) were studied in cells after exposure to African preparations for 72 h. With respect to control cells, MN, ES and AM treatments, respectively, increased cellular pigment levels of 33.9, 68 and 59% and enzyme activity of 1.6-, 2.2- and 2.1-fold. In addition, MITF protein was detected in MN, ES and AM protein samples: respectively, western blot analysis revealed an increase of the transcription factor 1.6-, 1.8- and 1.9-fold, compared to control cells, as shown in Fig. 12).
Discussion
The detection and characterization of plant compounds have recently attracted research attention because of their impact on human health and economy. The great biological activity of these molecules and the low costs of their natural synthesis are largely substituting the production of the modern synthetic drugs (4). By contrast, in Africa the use of plant extracts as medicine is an actual and very common practice that hails from ancient rituals. Nevertheless, Africans have often attributed and associated plant therapeutic features to spiritualist agents (20). The aim of this study was to investigate the health giving properties of African plant extracts (Table I), collected from Cameroon forests, that indigenous peoples daily employ in ethnomedicine. We planned to identify the antineoplastic effects of plant sample preparations on murine cancer cells. We decided to carry out plant extracts by using hot water, even if a more organic and less polar solvent would have had a greater capacity in the extraction of plant compounds (21), in order to obtain solutions that would be similar to the vegetal preparations used by African natives. Oxidative and reducing processes are essential for cell survival but when their equilibrium is imbalanced cellular stability is altered. In particular, high levels of reactive oxygen species (ROS) have been demonstrated able to induce cell structural damage and apoptosis (22,23). Plant molecules, in general, have been recognized as strong antiradical compounds able to reduce cell oxidation: therefore, a correct diet, rich in vegetables and fruits, is considered an important factor for the prevention of several diseases, including cancer, by its ability to prevent and to rescue oxidative stress (24). Therefore, we begun this research by analyzing sample antioxidant activity. Secondary metabolites in plants can approximately range between 6.8 and 32.1 μg CAE/mg DW, although it highly depends on plant environmental and physiological conditions (25). In this study, ES and MN extracts were demonstrated to possess a total phenolic content that abundantly exceeded these values (Fig. 1). Moreover, the same plant species and AM sample also showed the strongest antioxidant properties (Figs. 2 and 3), with respect to the other African extracts and correlated literature data (26–28). Another important result was that M. oleifera (North region) leaves (MN) showed the most conspicuous quantity of free radical scavenging molecules with respect to the other plant districts of the same species (MNR, MNF, MNC and MNS samples). Over the past few decades, African plant features have been largely described in scientific publications (6,9,10). In other reports, M. oleifera has been demonstrated a rich source of ascorbic acid, oestrogenic substances, iron, calcium, phosphorus, copper, vitamins, riboflavin, nicotinic acid, folic acid, pyridoxine, β-carotene, proteins and essential amino acids (29). Its extract also exhibit antibacterial, antifungal, antihypertensive, diuretic, hepatoprotective and cholesterol lowering activities (30,31). Anticancer properties of this plant have been studied both in vivo on mice and more rarely in vitro on tumor cell lines (32,33). On the other hand, E. speciosa and A. melegueta species have shown antifungal, anti-inflammatory, antibacterical, gastro-protective and fertilizing effects (34–36). However, no report has been clearly focused on their antitumoral properties. Therefore, special core of this study was the investigation of the effects and the molecular mechanisms that MN, ES and AM preparations could activate and/or regulate on the melanoma B16–F10 murine cell line. We evaluated the effect of these solutions on cell viability. Trypan blue test (Table II) showed that ES and AM extracts had no toxic effects on cells, as expected because of their simple processing in water and the naturalness of their contents. Instead, surprisingly, MN treatment caused the death of ~22% of total cells, with respect to the control. Different plant extracts contain diverse metabolite profiles (7) and, probably, MN phyto-complex presented one or more compounds, absent in ES and AM, able to induce cell instability and death (33). However, after treatment for different times with MN, ES and AM extracts, cell proliferation (Fig. 4) and growth (Fig. 5) were greatly reduced. Cell cycle alteration and apoptosis inhibition are the principal characteristics of cancer cells (37); therefore, FACS analysis was performed to clarify if treated cells could undergo cell cycle modifications in order to justify the decrease of cell proliferation. As anticipated, samples produced different effects on the cell cycle: MN induced a G2/M phase arrest, ES associated the block of cells both in S phase and in G2/M and finally AM caused a G0–G1 stop (Fig. 7). Moreover, a large amount of apoptotic nuclei, detected by propidium iodide staining, was only observed in the sub-G1 area of MN specimen (Fig. 6); it confirmed further the results obtained in the exclusion test (Table II). It is well-known that p53 is a tumor suppressor protein: ~80% of cancer cells are characterized by alterations in p53 gene or activity. When DNA damage occurs in cells, p53 is activated and accumulated in the nucleus where it promotes the transcription of different genes involved in DNA repair, cell growth arrest and apoptosis thus preventing cancinogenesis (38). Of the p53 targets, the CIP/KIP p21 and p27 cyclin-dependent kinase (CDK) inhibitors are the most investigated genes because of their ability to induce cell cycle arrest (39). The study of these protein markers was complementary to cytofluorimetric analysis and essential for clarifying the basal molecular mechanisms that might have induced the inhibition of the proliferation. We demonstrated that all treatments (MN, ES and AM) enhanced p53, p21WAF1/Cip1 and p27Kip1 protein levels in B16F10 melanoma cells (Fig. 8). Probably, the African plant extracts produced in cells induction of p53 that consequently stimulated the p21WAF1/Cip1 and p27Kip1-dependent cell cycle arrest. Different cell cycle profiles (Fig. 7) could be explained by a different activity of p21WAF1/Cip1 and p27Kip1 in melanoma cells. Coqueret (39) reported that these two proteins inhibit all CDK complexes, without a specific restriction for a particular cell phase. Therefore, an increase in p21WAF1/Cip1 or p27Kip1 can be easily associated with both G0–G1 and S or G2-M phase block (40–42). A remarkable increase of p27Kip1 level was detected in an MN sample (Fig. 8). The overexpression of this protein might be the reason, or the consequence, of the activation of an apoptotic pathway in cells; in fact, it is proved that a great p27Kip1 increment triggers apoptosis in different cell lines (43,44). This preliminary hypothesis that should be further confirmed by other experiments, would explain the large amount of dead cells detected only after treatment with MN (Figs. 6 and 7). Alteration in cell morphology, with the development of dendritic protrusions, reduction of proliferation and activation of melanogenesis have been considered specific indicators of differentiation for melanoma cell lines (45,46). The induction of this process in B16F10, after treatment with Cameroon extracts, was firstly and clearly suggested by cell acquisition of typical cytoplasmic extensions (Fig. 9). In literature, it has been well documented that p53 is a transcriptional regulator in melanogenesis. Via p21WAF1/Cip1, it positively regulates the promoter of microphthalmia-associated transcription factor (MITF). The latter plays a central role in the expression of melanocyte-specific genes such as tyrosinase, the key enzyme of melanin synthesis (47). Melanin production (Fig. 10), tyrosinase activity (Fig. 11) and MITF levels (Fig. 12) were highly enhanced in all samples. These results rigorously and scientifically confirmed the antioxidant, antiproliferative and differentiative effects of the MN, ES and AM extracts that commonly Cameroon indigenous people adopt as food sources of medicinal compounds. By merging the knowledge of African tradition and laboratory evidences it might identify new nutraceutical products able to improve human health and to prevent cancer.