FARMACIA, 2017, Vol. 65, 1 ORIGINAL ARTICLE ANTICANCER ACTIVITY OF EUROPAEUS EXTRACT ON MELANOMA CELLS

BOGDAN SEVASTRE1, ORSOLYA SÁRPATAKI1, ROXANA STAN2*, MARIAN TAULESCU1, ALEXANDRA CRISTINA SEVASTRE-BERGHIAN2, NELI KINGA OLAH3,4, FLAVIA FURTUNA4, DANIELA HANGANU2, ADRIANA CORINA HANGAN2, MIHAI CENARIU1, IOANA BÂLDEA2

1University of Agricultural Science and Veterinary Medicine, 3-5 Calea Mănăștur Street, 400372, Cluj-Napoca, Romania 2“Iuliu Hațieganu” University of Medicine and Pharmacy, 8 Victor Babeș Street, 400012, Cluj-Napoca, Romania 3“Vasile Goldiș” West University, 94 Revoluției Street, 310025, Arad, Romania 4PlantExtrakt Ltd, Rădaia Baciu, Cluj, Romania

*corresponding author: [email protected] Manuscript received: November 2015

Abstract L. () (EE), known as spindle , is a commonly found all over European continent, used in traditional medicine for the treatment of ectoparasites. The present study investigates the in vitro anticancer effects of EE hydro-alcoholic extract prepared from fresh fruits. Qualitative and quantitative analysis of the EE extract content where made by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Cell Proliferation Assay and Anexin V-fluorescein isothiocyanate apoptosis detection where performed on both human melanoma cell line and normal human fibroblasts, in serial doses ranging from 0.05 µg/mL up to 50 µg/mL, at 6 and 24 hours exposure. Evodiamine, a potent anticancer compound was found in significant amounts. In vitro melanoma cells were inhibited in a concentration-dependent manner (p < 0.001), (IC50 = 6.76 µg/mL), and they were almost ten-fold more sensitive than fibroblasts (IC50 = 53.08 µg/mL). EE had a significant and selective antitumor activity.

Rezumat Euonymus europaeus L. (Celastraceae) (EE), este un arbust răspândit pe întregul continent european, folosit în medicina tradițională în tratamentul antiparazitar. Studiul de față investighează activitatea antitumorală a extractului hidroalcoolic de EE in vitro. Analiza cantitativă și calitativă a extractului de EE a fost realizat prin HPLC și spectrometrie de masă (HPLC- MS). Testele de proliferare celulară și de apoptoză (Anexin V - fluorescein isothiocyanate) au fost realizate pe culturi fibroblastice dermice umane normale și pe o linia celulară de melanom uman, în doze seriate de la 0,05 µg/mL până la 50 µg/mL la 6 și 24 de ore de expunere. A fost identificată evodiamina, un compus cu activitate puternică antitumorală. In vitro, proliferarea liniei de melanom a fost inhibată în manieră doză dependentă (p < 0,001), (IC50 = 6,76 pg/mL), și a fost aproape de zece ori mai sensibilă decât linia de fibroblaste (IC50 = 53,08 pg/mL). Activitatea antitumorală a EE a fost exercitată selectiv aupra celulelor tumorale.

Keywords: antitumor, Euonymus europaeus, evodiamine, cell viability

Introduction Eastern Asia, traditionally used for cancer treatment. There are several pathways responsible for its Despite some progress, cancer remains a major remarkable anticancer properties, but two of them research challenge. Research for new anticancer drugs are of main importance. First, it exhibits antitumor is a very active domain; natural products represent properties by inducing apoptosis via mitochondrial an important source of new drugs. Actually, more pathway [10, 19]. Second, EA inhibits tumour than 60% of the new therapeutic compounds are invasion, mainly by suppression of the matrix either isolated from natural products or incorporate metalloprotease-9 (MMP-9) activity [4, 9], effect synthetic compounds based on the chemical provided by dihydroxycinnamic acid (caffeic acid) structure of natural products [1, 5, 18]. [13]. The inhibition of MMP-2 and MMP-9 is mediated from the Celastraceae family have been used by NK-kappaB pathway [6]. for centuries in the field of traditional medicine, in Euonymus europaeus L., (Celastraceae) (EE) also South America, China, and Africa, for the treatment of known as the spindle tree, is the only European various diseases, including cancer [7, 21]. One of the representative of the Celastraceae family. The most investigated was (Thunb) dried fruits and are used against external Celastraceae (EA), a native from South- parasites as scabies, ticks and lice, while the seeds 56 FARMACIA, 2017, Vol. 65, 1 are emetic and purgative [2]. Relatively few phyto- Standardization of the tincture. The aspect of the chemical analysis on EE were performed, but they tincture was determined by observation of countenance, revealed active compounds including sesquiterpene colour and smell. The relative density was measured polyesters belonging to the alatol, 3-deoxymatol, using an Anton Paar 35 DMA digital densitometer. The 3,4-dideoxymaytol families, evonimate , result is the average of 3 independent determinations. dihydro-β-agarofuran polyesters [7], and lectins The dry residue was determined by drying at [17]. Evodiamine is a quinazolinecarboline 110ºC, for 3 hours. The result is the average of 3 initially isolated from Chinese herb Evodia rutaecarpa, independent determinations. Firstly, the ethanol represent a promising chemotherapeutic agent in the content was determined by distillation, and secondly multiple-drug resistant cancer cells [12]. Evodiamine by the relative density of the distillate solution has been shown to exhibit anticancer properties by correlated with the values from the alcoholometric various mechanisms as cell cycle arrest, induction tables. of apoptosis [19]. Determination of evodiamine content. The evodiamine In the present study, we investigated the anticancer content was determined by HPLC, using a method activity of EE. To differentiate the general cyto- provided by Phytolab and adapted. The determination toxicity from a selective antitumor effect, we tested was performed using a Varian ProStar HPLC system. the inhibitory potential on both normal fibroblastic We used a Phenomenex Luna C18 silicagel-C18 line and on melanoma cell line and then we column, 150 mm x 4.6 µm with a pre-column of investigated apoptosis in EE treated cultures. 5 mm x 4.6 µm, both having particles of 5 µm. As a mobile phase, a solvent gradient with phosphoric acid Materials and Methods pH = 2.5, water and acetonitrile, with a 1 mL/min flow rate were used. The detection was performed materials and extraction. Fresh fruits of EE, with a DAD detector at 228 nm. The UV-Vis harvested in September 2011, from a forest near spectra were recorded from 200 nm to 600 nm [22]. Cluj-Napoca, Romania, were used to prepare the 100 µL of EE extract from each concentration of hydro alcoholic extract. The harvest of fruits was standard evodiamine solution were injected. performed according to the Good Agricultural and Evodiamine was used as the standard. For the Collection Practices rules, from an unpolluted area, quantitative determination, a calibration curve with at a minimum of 3 km distance from any circulated concentration between 50 and 500 µg/mL was built. road. The fruits were botanically identified by The calibration curve’s equation is: Quality Control Laboratory of PlantExtrakt TC Ltd, A = 60110 x C – 478977 Rădaia, Cluj County, Romania. A voucher specimen and the correlation factor is: 0.9807. The was filed and kept in the company’s archives (B. identification was made based on the comparison no. 063811, CoA 4817/20.09.2011). between the retention time and UV-VIS spectra of The sampling of the vegetable material was performed compounds separated from EE extract and the according to the European Pharmacopoeia, and the standard evodiamine. The maximum absorption of botanic identification, according to the German evodiamine is at 228 nm. Homeopathic Pharmacopoeia. The moisture of the Cell culture. The assessment of antiproliferative fresh vegetal material was 70%. effect was performed both on normal human dermal The freshly cut was mixed with 90% vol. fibroblasts (HDFa - Invitrogen, Willow Creek, ethanol. The extraction ratio was 1 part fruit to 0.7 USA) and on a human radial growth phase (RGP) parts solvent. The extraction was performed at melanoma cell line (WM35). Fibroblasts were room temperature, by maceration and periodical cultivated in a Dulbecco's modified Eagle's medium mixing (minimum 10 min x 2 times a day x 10 (DMEM) supplemented with 5% fetal calf serum, days). After 10 days, the relative density and the 50 µg/mL gentamicin and 5 ng/mL amphotericin dry residue were evaluated. The mixture of plant (Biochrom AG, Berlin, Germany). Melanoma cells and solvent was subsequently pressed, left at (obtained from M. Herlyn, the Wistar Institute, maximum 30°C for 5 days and then filtered. The Philadelphia, PA, USA) [8] were maintained in resulting extract was the tincture used for the RPMI medium supplemented with 5% fetal calf further experiments. All preparation steps were serum, 50 µg/mL gentamicin and 5 ng/mL performed on an API-GMP certified production amphotericin (Biochrom AG, Berlin, Germany). flow at SC PlantExtrakt SRL in Rădaia, Cluj County, Both cultures were incubated in a humid atmosphere Romania, according to method 2a from the German at 37°C and 5% CO [16]. Homoeopathic Pharmacopoeia [20]. 2 Cell Proliferation Assay. The cell cytotoxicity assay Before use, the alcoholic solution was processed in was performed using the CellTiter 96® Aqueous a rotary evaporator at 40°C, until 3/4 of the content Non-Radioactive Cell Proliferation Assay (Promega evaporated, then refilled with sterile saline solution Corporation Madison, USA) as specified by the to initial volume for use on cell culture. manufacturer. The cells were seeded at a density of 57 FARMACIA, 2017, Vol. 65, 1 104/well in ELISA 96-well micro titration flat to Euonymus europaeus extract in concentrations of bottom plates and allowed to accommodate for 24 h 0.5 and 1 µg/mL for 6 and 24 hours, followed by in normal growth conditions. The cultures were FITC Annexin V and PI staining, according to the then exposed to EE extract (prepared as described manufacturer’s instructions. above) in increasing concentrations, ranging from The flow cytometric analyses were performed at 0.05 to 50 µg/mL for 6 and 24 hours. Each experiment room temperature with a BD FACSCanto II flow was carried out in triplicate. Cell cultures treated cytometer (Becton Dickinson & Company, Franklin only with medium where used as controls. Lakes, NJ, USA) equipped with two lasers as excitation After each individual time point, the E. europaeus sources: blue (488 nm, air cooled, 20 mW solid treated cells and their counterpart controls were state) and red (633 nm, 17 mW HeNe). The cytometer incubated for 2 hours with 20 µL of 3-(4,5-di- setup was performed using the BD Cytometer Setup & methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- Tracking beads (CS&T beads), while the compensation 2-(4-sulphenyl)-2H-tetrazolium MTS/phenazine was done using compensation beads (BD Comp- methosulfate (PMS) mixture in 100 µL culture Bead). The data was analysed using the BD FACSDiva medium [3]. The absorbance of each sample was Software (Becton Dickinson). A total number of read at 490 nm using an ELISA plate reader (Tecan, 10,000 events were recorded for each sample. The Männedorf, Switzerland). The viability was evaluated fluorescence spectrum of Annexin V and PI were based on the comparison with control cells. The detected using a 530/30 nm and a 575/26nm band- IC50 values representing the extract concentration pass (BP) filter, respectively. required to inhibit 50% of cell proliferation were Statistical analysis. All data are reported as the calculated from the calibration curve by linear mean ± SEM. The Gaussian distribution was regression using Microsoft Excel. checked using the Shapiro-Wilk normality test. Evaluation of apoptosis. The Annexin V-fluorescein Pearson’s correlation was used in order to assess the isothiocyanate (FITC) apoptosis detection kit was correlation between normally distributed variables; used in conjunction with the vital dye propidium this interpretation was made according to the Colton iodide (PI) (BD Biosciences, San Jose, CA, USA). scale. Statistical values and figures were obtained The induction of apoptosis was investigated by using GraphPad Prism version 5.0 for Windows, assessing positive cells (%) for Annexin V-FITC GraphPad Software, San Diego California, USA. and/or PI. The Annexin V-FTC positive cells were detected by green fluorescence, while the presence Results and Discussion of red fluorescence was the indicator for PI. The Standardization of the hydro alcoholic extract. The viable cells (showing no apoptosis) were identified obtained hydro alcoholic extract was a clear, as Annexin V (-)/PI (-); the apoptotic cells were brown-orange liquid. The quality parameters of the identified as Annexin V (+)/PI (-) (early apoptosis), extract were within the admissibility range of and Annexin V (+)/PI (+) (late apoptosis). The German Homeopathic Pharmacopoeia, as follows: differentiation among these three-cell-type populations relative density 0.953 (0.935 - 0.955), dry residue was made by flow cytometric detection. 5.76% (Min. 4%), ethanol content 45% vol. (40 - WM35 and fibroblast cells were seeded on Petri 50% vol.), digitoxin content of 0.0028% (Max. glass dishes (Nalgene, Nunc, USA) at a density of 0.01%). Evodiamine content was 0.404 g/mL [20]. 5 x 104/cm2 for 24 hours in medium, then exposed

Figure 1. HPLC chromatograms at 150 nm of standard evodiamine (A) and the compounds isolated from the ethanolic extract of Euonymus europaeus (B)

58 FARMACIA, 2017, Vol. 65, 1 Evodiamine presence in the EE was shown by UV-VIS spectra proved the presence of evodiamine HPLC chromatogram (Figure 1), while the recorded in the extract (Figure 2).

Figure 2. UV-Vis spectra of standard evodiamine (A) and the compound isolated from the ethanolic extract of Euonymus europaeus (B)

In vitro cytotoxicity. Human melanoma cell line normal fibroblasts, the IC50 at 6h was almost ten- (WM35) was compared to normal human dermal fold higher. The IC50 values for VM35 cells were fibroblasts for sensitivity to EE. The differences of 6.76 ± 1.08 µg/mL after 6 h and 5.89 ± 0.93 µg/mL absorption percentage among different concentrations after 24 h, while IC50 of the fibroblasts were 53.08 were statistically covered for both fibroblasts and ± 5.47 µg/mL and respectively 25.04 ± 3.73 µg/mL VM35 cells (p < 0.001). (Figure 3). The dose-response curves showed that cancer cells were, by far, more sensitive to EE treatment than

Figure 3. Comparative cytotoxicity of Euonymus europaeus extract on fibroblasts and VM35 cultures after 6 hours (A) and 24 hours (B) of exposure at a different dose versus untreated cells (mean ± SEM) (n = 3)

The fibroblasts inhibition showed a very different visible after 6 h, and enhanced after 24 h, a dose pattern, suffering a gradual dose-dependent and dependent reduction of cell concentration was time-dependent inhibition of viability, already clearly visible.

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Figure 4. Cell density at 0.5 µg/mL and 5 µg/mL

Apoptosis/necrosis assessment. To determine whether the growth inhibition is associated with cell death, Annexin V Propidium Iodide double staining of VM35 cells and fibroblasts followed by flow cytometric analysis were performed. As shown in Figure 4 in the upper left quadrant, the fluorescein isothiocyanate conjugate of Annexin V (FITC-A) staining in propidium iodide (PE-A) negative cells - early apoptotic cells - represented less than 0.5% of cells in all treatment conditions. This suggests that apoptosis was insignificant, therefore all cells positive for PE-A, positive or negative for FITC-A, confined in the right side of the panel, were considered as necrotic cells. In tumour cells cultures, the percentage of necrotic cells increased from 3.6% in control cells up to 18.6% at 1.0 µg/mL in 24 h. Fibroblasts were even more sensitive. The control cells showed 8.3% percentage of necrosis and 24 h later, the necrosis in the fibroblast subjected to a dose of 1.0 µg/mL, increased up to 72.1%. Apoptosis was measured using flow cytometric analysis. Figure 5 represents scatter plots of Annexin V-fluorescein isothiocyanate (FITC-A) (y - axis) versus vital dye propidium iodide (PE-A) labelling Figure 5. (x - axes). Lower left quadrants (absence of both Scatter plots of Annexin V-fluorescein markers) indicate viable cells; upper left quadrants isothiocyanate versus vital dye propidium iodide (FITC-A positive, PE-A negative) indicate the labelling apoptotic cells (early stage apoptosis). In the present study, the necrotic cells are represented by The present study investigates the underlying the right side of the panel (PE-A staining alone or mechanisms of tumoricidal properties of EE together with FITC-A). Fibroblasts and VM35 cells extract, already observed in the preliminary study were treated with Euonymus europaeus extract in [15]. The in vitro investigations were performed doses of 0.5 µg/mL and 1.0 µg/mL, after 6 hours using a human melanoma cell line VM35 and, in and 24 hours while untreated cells were used as order to check the specificity of inhibition against controls. tumour cells, we conducted parallel studies on a human fibroblasts cell line. As far as we know, no other studies focusing on tumouricidal properties of EE were ever published by other research groups. Despite the fact that no studies about EE are available, other Euonymus species, for example

60 FARMACIA, 2017, Vol. 65, 1 Euonymus alatus, already proved to have antitumor model - Ehrlich ascites carcinoma inoculated in properties. Euonymus alatus exhibits antitumour mice. This transplantable tumour model will give properties by induction of apoptosis via mitochondrial us also the possibility to investigate the side effect pathway [10] and inhibits tumour invasion by of tumour growth and the interaction between inducing the inhibition of matrix metalloprotease-9 tumour cells and the immune system. (MMP-9) activity [9]. Comparative studies on MMP-9 inhibition showed that Euonymus alatus Acknowledgement was among the most effective extracts providing an This study was supported by an internal research inhibitory activity of up to 90% [14]. Another grant of the University of Agricultural Science and antiproliferative mechanism was discovered for Veterinary Medicine Cluj Napoca, Romania. mammary and genital tumours, respectively the inhibition of aromatase activity. Aromatase is an Declaration of interests enzyme responsible for oestrogen synthesis. A large proportion of breast cancers express their own The authors report no declaration of interest. aromatase. Euonymus alatus was highly effective in inhibiting the intracellular aromatase in myometrial and References leiomyomal cells, in a dose-dependent manner [11]. 1. Alam M.N., Bristi N.J., Rafiquzzaman M., Review This is the first study showing the presence of on in vivo and in vitro methods evaluation of evodiamine in EE. Evodiamine was initially antioxidant activity. Saudi Pharmaceutical Journal, isolated from a Chinese herb named Evodia 2013; 21: 143-152. rutaecarpa and represents a promising chemo- 2. Aronson J.K., Meyler’s Side Effects of Herbal therapeutic agent in the multiple-drug resistant Medicines. Amsterdam, The Netherlands: Elsevier, cancer cells [12]. Therefore, in the present study, 2009; 94-95. the phytochemical investigation was focused on 3. 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