6- Artigo com foto viacount grafico Ethnopharmacology 2013 ATIV ANTICANCER

SAVE OFFLINE

Journal of Ethnopharmacology 149 (2013) 463–470

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Monoterpene bisindole alkaloids, from the African medicinal plant Tabernaemontana elegans, induce apoptosis in HCT116 human colon carcinoma cells Tayyab A. Mansoor a,b, Pedro M. Borralho a,c, Saikat Dewanjee a,b,1, Silva Mulhovo d, Cecília M.P. Rodrigues a,c, Maria-José U. Ferreira a,b,n a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal b Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal c Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal d Centro de Estudos Moçambicanos e de Etnociências, Universidade Pedagógica, Campus de Lhanguene, Av. de Moçambique, 21402161 Maputo, Mozambique

art ic l e i nf o

a b s t r a c t

Article history: Received 26 February 2013 Received in revised form 6 May 2013 Accepted 30 June 2013 Available online 17 July 2013

Ethnopharmacological relevance: Tabernaemontana elegans is a medicinal plant used in African traditional medicine to treat several ailments including cancer. The aims of the present study were to identify anticancer compounds, namely apoptosis inducers, from Tabernaemontana elegans, and hence to validate its usage in traditional medicine. Methods and materials: Six alkaloids, including four monomeric indole (1–3, and 6) and two bisindole (4 and 5) alkaloids, were isolated from the methanolic extract of Tabernaemontana elegans roots. The structures of these compounds were characterized by 1D and 2D NMR spectroscopic and mass spectrometric data. Compounds 1
6 along with compound 7, previously isolated from the leaves of the same species, were evaluated for in vitro cytotoxicity against HCT116 human colon carcinoma cells by the MTS metabolism assay. The cytotoxicity of the most promising compounds was corroborated by Guava-ViaCount flow cytometry assays. Selected compounds were next studied for apoptosis induction activity in HCT116 cells, by evaluation of nuclear morphology following Hoechst staining, and by caspase-3 like activity assays. Results: Among the tested compounds (1
7), the bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were found to be cytotoxic to HCT116 cells at 20 mM, with compound 5 being more cytotoxic than the positive control 5-Fluorouracil (5-FU), at a similar dose. In fact, even at 0.5 mM, compound 5 was more potent than 5-FU. Compounds 4 and 5 induced characteristic patterns of apoptosis in HCT116 cancer cells including, cell shrinkage, condensation, fragmentation of the nucleus, blebbing of the plasma membrane and chromatin condensation. Further, general caspase-3-like activity was increased in cells exposed to compounds 4 and 5, corroborating the nuclear morphology evaluation assays. Conclusions: Bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were characterized as potent apoptosis inducers in HCT116 human colon carcinoma cells and as possible lead/scaffolds for the development of anti-cancer drugs. This study substantiates the usage of Tabernaemontana elegans in traditional medicine to treat cancer. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Anti-cancer Apoptosis induction Monoterpene indole alkaloids HCT116 human colon carcinoma cells Tabernaemontana elegans

1. Introduction Apoptosis, also referred as ‘programmed cell death’, is a vital component of various biological processes of the body to get rid of abnormal and cancerous cells. Cell survival is maintained in the body by a balance between pro-apoptotic and anti-apoptotic stimuli. Deregulation of apoptosis disrupts the equilibrium between cell n

Corresponding author. Tel.: +351 21 7946470; fax: +351 21 7946470. E-mail address: [email protected] (M.J.U. Ferreira). 1 Present address: Department of Pharmaceutical Technology, Jadavpur University, Raja S C Mullick Road, Kolkata 700032, India. 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.06.051

growth and cell death, and is an important step in the development of cancer (Kinloch et al., 1999). This principle lies behind the search for novel molecules that can therapeutically activate apoptosis in cancer cells to eliminate them from body. Various classes of biologically active natural products, including several alkaloids, terpenoids, polyphenols, and saponins can induce apoptosis by interacting with important molecular targets such as DNA, microtubules, biomembranes and receptors (Taraphdar et al., 2001). The genus Tabernaemontana (Apocynaceae) has a wide distribution and plants belonging to this genus have been used in African traditional medicine to treat tumors and cancer (Graham et al., 2000). Tabernaemontana elegans Stapf. (Syn. Conopharyngia elegans

464

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

Stapf.) is an important member under this genus that occurs in tropical or subtropical regions including Indonesia, Malaysia, and Africa (Vanbeek et al., 1984). It is known in English as the "toad tree", due to the brown and wart like skin of its fruit. Folklore claims suggested that the root-bark is used for cancer treatment (Chhabra et al., 1987). Other usages include: root decoction is applied as a wash to wounds, and drunk for pulmonary diseases and chest pains (Watt and Breyer-Brandwijk, 1962; Arnold and Gulumian, 1984; Vanderheijden et al., 1986); treatment of heart diseases with the seeds, stem-bark and roots; and root decoction is also said to have aphrodisiac properties (Neuwinger, 1966). Tabernaemontana plants are characterized to produce various skeletal types of indole alkaloids, including iboga-type, aspidosperma-type, vobasinyl-ibogan-type and tetrakis-aspidosperma-type (Vanbeek et al., 1984, 1985). These alkaloids have shown a wide range of biological activities including cytotoxicity, reversal of vincristine-resistance, antimicrobial activity against Gram-positive bacteria, antifungal activity, and acetylcholinestrase inhibitory activity (Vanbeek, et al., 1985; Achenbach et al., 1997; Kam et al., 1998; Chaturvedula et al., 2003; Andrade et al., 2005). In our search for apoptosis inducing compounds from medicinal plants (Duarte et al. 2007; Mansoor et al. 2009b, 2011, 2012; Wesołowska et al. 2012), we report the isolation and identification of six compounds (1
6) from the MeOH extract of Tabernaemontana elegans roots. Our previous phytochemical studies of Tabernaemontana elegans leaves yielded several alkaloids, including compound 7 (Mansoor et al., 2009a, 2009b). Compounds 1
7 were evaluated for cytotoxicity/cell viability in HCT116 colon carcinoma cells. The most active compounds in cell viability assays were further subjected to

Guava-ViaCount flow cytometric assays and apoptosis induction activity by morphological evaluation of nuclear morphology following Hoechst staining and caspase-3 like activity assays.

2. Materials and methods 2.1. General experimental procedures NMR spectra were recorded on a Bruker ARX-400 NMR spectrometer (1H NMR 400 MHz; 13C NMR 100.61 MHz), using CDCl3 as solvent. Mass spectrometry was carried out on a Triple Quadrupole mass spectrometer (Micromass Quattro Micro API, Waters). Column chromatography was carried out on SiO2 (Merck 9385). TLC were performed on precoated SiO2 F254 plates (Merck 5554 and 5744), visualized under UV light and by spraying either with Dragendorff′s reagent or a solution of H2SO4–MeOH (1:1), followed by heating. 2.2. Plant material The roots of Tabernaemontana elegans were collected in Mozambique during February, 2011. Taxonomical identification was performed by the botanist Dr. Silva Mulhovo, Centro de Estudos Moçambicanos e de Etnociências, Universidade Pedagógica, Maputo, Mozambique. A voucher specimen (23/SM) has been deposited at the herbarium (LMA) of the Instituto de Investigação Agrária de Moçambique (IIAM), Maputo, Mozambique.

Fig. 1. Chemical structures of tabernaemontanine (1), dregamine (2), 16-epidregamine (3), tabernaelegantine C (4), tabernaelegantinine B (5), voacangine (6) and vobasine (7), isolated from Tabernaemontana elegans.

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

2.3. Test compounds These included tabernaemontanine (1), dregamine (2), 16epidregamine (3), tabernaelegantine C (4), tabernaelegantinine B (5), voacangine (6) and vobasine (7). The isolation and structural elucidation of compound 7 was previously reported (Mansoor et al., 2009b). The isolation of compounds 1
6 is described below. The purity of all the compounds (Fig. 1) was more than 95% based on HPLC analysis. We have included 5-fluorouracil (5-FU) as a positive control, since this cytotoxic agent has been used in the clinical treatment of colon cancer for several decades, and is a known inducer of apoptosis in colon cancer cells (Borralho et al., 2007, 2009; Hemaiswarya and Doble 2013). 2.4. Extraction and isolation The dried powdered roots (3.5 Kg) of Tabernaemontana elegans were macerated with MeOH (4  15 L) with continuous agitation. The MeOH extract (500 g, yield ≈14.3% w/w) was dissolved in Et2O (5 L) and extracted with 10% CH3COOH (3  6 L). The pH of the acid layer was adjusted to 9 by the addition of dilute NH4OH. The basic layer was successively extracted with CH2Cl2 (5  3 L) and EtOAc (5  3 L), yielding 90 and 2 g of CH2Cl2 and EtOAc soluble fractions, respectively. Dichloromethane soluble fraction (90 g) was subjected to silica gel column chromatography using mixtures of n-hexane-EtOAc and EtOAc-MeOH of increasing polarity, to yield fractions (A–K). Fraction A (0.75 g) was chromatographed with n-hexane-CH2Cl2 and CH2Cl23MeOH, to yield six sub-fractions (A1–6). Sub-fraction A1 was further chromatographed with n-hexane-CH2Cl2 and CH2Cl2-methanol to afford compound 6 (31 mg). Sub-fraction A3 was further chromatographed employing gradients of n-hexane–CH2Cl2 and CH2Cl23MeOH, yielding yellow crystals of compound 4 (27 mg). Fractions B (2.05 g) and D (17.45 g) were purified by direct crystallization method, using nhexane and few drops of ethyl acetate, to yield white amorphous solid of compound 2 (112 mg) and white crystals of compound 1 (643 mg), respectively. Fraction G (1.65 g) was eluted with n-hexane-CH2Cl2 and CH2Cl23MeOH of increasing polarity, to yield 8 sub-fractions (G1–8). The sub-fraction G2 (350 mg) was eluted by gradients of n-hexaneCH2Cl2 and CH2Cl23MeOH, yielding white crystals of compound 3 (4 mg). The sub-fraction G3 (590 mg) was eluted by gradients of nhexane–EtOAc and EtOAc–MeOH, and purified by RP silica column chromatography, using H2O–MeOH (60–100%) as eluent, to yield yellowish compound 5 (14 mg). 2.4.1. Tabernaelegantine C (4) 1 H NMR data (400 MHz; CDCl3) δ 7.68 (1H, br d, J¼ 8.0 Hz, H-9), 7.61 (2H, s, overlapped, 2NH), 7.27 (1H, d, J¼ 8.0 Hz, H-9′), 7.12 (1H, dt, J¼ 8.0, 1.0 Hz, H-10), 7.06 (1H, dt, J¼8.0, 1.0 Hz, H-11), 7.00 (1H, br d, J¼ 8.0 Hz, H-12), 6.84 (1H, d, J¼ 8.0 Hz, H-10′), 5.27 (1H, dd, J¼ 12.0, 3.0 Hz, H-3), 4.03 (1H, td, J¼9.1, 2.6 Hz, H-5), 3.97 (3H, s, OCH3–11′), 3.72 (3H, s, CO2CH3′), 3.34 (1H, m, H-21′), 3.32 (2H, m, H-6), 3.26 (1H, m, H-5′a), 3.10 (1H, m, H-5′b), 2.98 (1H, m, H-6′a), 2.92 (2H, overlapped, H-16, H-21a), 2.84 (1H, m, H-6′b), 2.74 (1H, m, H-14a), 2.71 (1H, m, H-15), 2.68 (1H, m, H-3′a), 2.58 (3H, s, N-CH3), 2.51 (3H, s, CO2CH3), 2.47 (1H, br d, J¼8.5 Hz, H-3′b), 2.41 (1H, br d, J¼12.5 Hz, H-21b), 1.90 (1H, m, H-14b), 1.75 (1H, br d, J¼14.0 Hz, H-17′a), 1.69 (1H, m, H-19a), 1.50 (1H, m, H-15′a), 1.47 (1H, m, H-19b), 1.43 (1H, m, H-14′), 1.34 (1H, m, H-20), 1.33 (2H, m, H-19′), 1.09 (1H, m, H-20′), 0.95 (3H, t, J¼7.6 Hz, CH3–18), 0.91 (1H, m, H-15′b), 0.82 (3H, t, J¼ 7.6 Hz, CH3–18′), 0.65 (1H, br d, J¼14.0 Hz, H-17′b); 13C NMR data (100 MHz; CDCl3) δ 174.9 (CO2CH3′), 172.6 (CO2CH3), 151.9 (C-11′), 136.7 (C-2), 136.1 (C-13), 136.0 (C-2′), 135.2 (C-13′), 129.5 (C-8), 124.4 (C-8′), 122.0 (C-11), 119.3 (C-10), 118.0 (C-9), 117.0 (C-9′), 114.7 (C-12′), 110.3 (C-7), 109.7 (C-12), 109.0 (C-7′), 104.8 (C-10′), 59.3 (C-5), 57.7 (C-21′), 56.8 (OCH3–11′), 54.6 (C-16′), 52.9 (C-5′), 52.3 (CO2CH3′), 51.1 (C-3′), 49.9

465

(CO2CH3), 46.8 (C-21), 43.9 (C-16), 43.1 (N-CH3), 42.8 (C-20), 38.9 (C20′), 36.7 (C-14), 35.2 (C-3), 35.0 (C-15), 34.6 (C-17′), 31.9 (C-15′), 27.0 (C-14′), 26.6 (C-19′), 25.7 (C-19), 22.1 (C-6′), 17.8 (C-6), 13.1 (C-18), 11.6 (C-18′); ESIMS m/z 707 [M+H]+. 2.4.2. Tabernaelegantinine B (5) 1 H NMR data (400 MHz; CDCl3) δ 7.65 (1H, br s, NH), 7.55 (2H, overlapped, NH, H-9), 7.06–7.04 (3H, overlapped, H-10, H-11, H-12), 6.85 (1H, s, H-9′), 6.80 (1H, s, H-12′), 5.05 (1H, br d, J¼12.0 Hz, H-3), 4.05 (1H, td, J¼9.1, 3.0 Hz, H-5), 3.95 (3H, s, OCH3–11′), 3.66 (3H, s, CO2CH3′), 3.45 (1H, m, H-21′), 3.34 (1H, m, H-6a), 3.32 (1H, m, H-3′), 3.15 (1H, m, H-6b), 3.09 (2H, m, H-5′), 2.85 (1H, m, H-14′), 2.73 (2H, overlapped, H-16, H-6′a), 2.67 (2H, m, CH2COCH3′), 2.65 (2H, overlapped, H-15, H-6′b), 2.65 (1H, m, H-6′b), 2.63 (3H, s, N-CH3), 2.44 (1H, m, H-21), 2.44 (3H, s, CO2CH3), 2.10 (3H, s, CH2COCH3′), 1.94 (1H, m, H-17′a), 1.87 (1H, m, H-17′b), 1.66 (2H, m, H-14), 1.34 (1H, m, H-20), 1.65 (1H, m, H-15′a), 1.22 (2H, m, H-19), 1.21 (1H, m, H-15′b), 1.21 (2H, m, H-19′), 1.20 (1H, m, H-20′), 0.94 (3H, t, J¼7.6 Hz, CH3–18), 0.85 (3H, t, J¼7.6 Hz, CH3-18′); 13C NMR data (100 MHz; CDCl3) δ 208.8 (CH2COCH3′), 175.7 (CO2CH3′), 171.8 (CO2CH3), 153.4 (C-11′), 137.9 (C13′), 135.9 (C-13), 135.1 (C-2), 134.7 (C-2′), 130.0 (C-8), 127.7 (C-10′), 122.4 (C-8′), 121.3 (C-11), 118.6 (C-10), 118.0 (C-9′), 117.3 (C-9), 110.9 (C7), 109.9 (C-7′), 109.8 (C-12), 92.5 (C-12′), 59.2 (C-5), 58.5 (C-21′), 55.8 (OCH3-11′), 54.8 (C-3′), 54.7 (C-16′), 52.5 (CO2CH3′), 51.3 (C-5′), 49.9 (C16), 49.8 (CO2CH3), 49.5 (C-21), 46.7 (CH2COCH3′), 43.9 (C-20), 42.4 (NCH3), 38.5 (C-20′), 37.6 (C-17′), 37.0 (C-3), 33.0 (C-14′), 31.8 (C-15), 31.0 (C-14), 30.8 (CH2COCH3′), 27.0 (C-15′), 26.7 (C-19′), 23.5 (C-19), 22.0 (C6′), 19.4 (C-6), 11.7 (C-18′), 11.4 (C-18); ESIMS m/z 763 [M+H]+. 2.5. Cell culture HCT116 human colon carcinoma cell line was obtained from European Collection of Cell Cultures (ECACC) (Porton Down, Salisbury, UK). HCT116 cells were grown in McCoy′s 5A supplemented with 10% fetal bovine serum, and 1% antibiotic/antimycotic (Invitrogen, Grand Island, NY, USA) and maintained at 37 1C in a humidified atmosphere of 5% CO2. Cells were seeded in 96-well plates at 5,000 cells/well for MTS assays; in 24-well plates at 75,000 cells/well for Guava ViaCount assay; in 40 mm dishes at 150,000 cells/dish and 300,000 cells/dish for morphological evaluation of apoptosis and caspase-3-like activity assays, respectively. 2.6. Compound exposure Stock solutions of 5–50 mM of test compounds (1
7) and positive control 5-FU (Sigma Chemical Co., St. Louis, MO, USA) were prepared in dimethyl sulfoxide (DMSO). Twenty-four hours after cell plating, media was removed and replaced with fresh media containing 5, 10, 20 or 50 μM of test compounds and 5-FU, or DMSO vehicle control, for the indicated exposure times. 2.7. Evaluation of cell viability and cytotoxicity After 48 h of cell incubation in the presence of each compound, and of positive and vehicle controls, cell viability was evaluated by the CellTiter 96s AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt (MTS). This method is routinely used to screen cancer cell sensitivity to commercially available, chemical synthesized or extracted test compounds (Borralho et al., 2009; Silva et al., 2012). In brief, this is a homogeneous, colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The CellTiter 96s AQueous Assay is composed of solutions of MTS and an electron coupling reagent, phenazinemethosulfate (PMS). MTS is

466

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

bioreduced by cells into a formazan product that is soluble in a tissue culture medium. The absorbance of the formazan product at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of the formazan product was measured in a Bio-Rad microplate reader Model 680 (Bio-Rad, Hercules, CA, USA) at 490 nm, as absorbance is directly proportional to the number of viable cells in culture. 2.8. Guava ViaCount assay ViaCount assay was used with the Guava easyCyte 5HT Flow cytometer (Guava Technologies, Inc., Hayward, CA, USA), to evaluate viable and dead cell populations on HCT116 cells exposed to compounds and controls. The ViaCount Assay distinguishes viable and non-viable cells based on differential permeability of two DNAbinding dyes in the Guava ViaCounts Reagent. The nuclear dye stains only nucleated cells, while the viability dye brightly stains dying cells. HCT116 cells were seeded in 24-well plates 75,000 cells/well. Twentyfour hours later, cells were exposed to compounds for 48 h. After treatment, cell culture supernatants were collected and adherent cells were detached with TrypLE (Invitrogen). Next, detached cells were pooled with cell culture supernatants and centrifuged for 5 min (650 g). Supernatants were discarded and the cells were resuspended in 2 ml phosphate buffered saline (PBS). Subsequently, 20 μl of cell

suspension were mixed with 180 μl of Guava ViaCount reagent, and incubated for 5 min at room temperature. Sample acquisition and data analysis were performed using the ViaCount software module. 2.9. Hoechst staining Hoechst labeling of cells was used to detect apoptotic nuclei by evaluation of nuclear morphology under fluorescence microscopy. In brief, cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4, for 10 min at 25 1C, incubated with Hoechst dye 33258 (Sigma Chemical Co.) at 5 μg/mL in PBS for 5 min, and next washed with PBS and mounted using PBS: glycerol (3:1, v/v). Fluorescent nuclei were scored and categorized according to the condensation and staining characteristics of chromatin. Normal nuclei showed non-condensed chromatin dispersed over the entire nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous to the nuclear membrane, as well as nuclear fragmentation of condensed chromatin. Three random microscopic fields per sample of approximately 100 nuclei were counted, and mean values were expressed as the percentage of apoptotic nuclei. 2.10. Caspase-3-like activity assay General caspase-3-like activity was determined in cytosolic protein extracts as previously described (Borralho et al., 2009; Mansoor et al., 2012). Briefly, after 16 h of compound exposure,

Fig. 2. Evaluation of compound effect on cell viability by MTS. Cell viability as assessed by the MTS assay after exposure of HCT116 colon carcinoma cells to: (A) 5, 10, 20, 50 mM of compounds (1
7) and 5-FU for 48 h; and (B) 0.5, 1.0, 2.5, 5, 10, 25, 50 mM of compounds (4 and 5) and vehicle control (DMSO). Results are expressed as the mean 7 SEM from control (DMSO), of at least three different experiments. †p o0.01 and *p o0.05 from DMSO vehicle control.

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

cells were harvested and homogenized in isolation buffer containing 10 mM Tris–HCl buffer, pH 7.6, 5 mM magnesium chloride, 1.5 mM potassium acetate, and 2 mM dithiothreitol. General caspase-3-like activity was determined by enzymatic cleavage of chromophore p-nitroanilide (pNA) from the substrate N-acetyl–Asp–Glu–Val–Asp-pNA (DEVD-pNA) (Sigma Chemical Co.). The proteolytic reaction was carried out in isolation buffer containing 50 μg of cytosolic protein and 50 μM of specific caspase substrate. The reaction mixtures were incubated at 37 1C for 1 h, and the formation of pNA was measured by monitoring A405 nm wavelength using a Bio-Rad microplate reader Model 680 (Bio-Rad), which is different from the UV maxima of 200–290 nm of tested compounds.

467

2.11. Statistical analysis All data were expressed as mean 7SEM from at least three independent experiments. Statistical analysis was performed using GRAPHPAD INSTAT version 3.00 (GraphPad Software, San Diego, CA, USA) for the Student′s t-test. Values of p o0.05 were considered significant.

3. Results and discussion The plants belonging to genus Tabernaemontana have been used in traditional medicine to treat cancer (Graham et al., 2000). These

Fig. 3. Evaluation of compound effect on cell viability by ViaCount. Cell populations obtained by Guava ViaCount flow cytometry after 48 h incubation of HCT116 colon carcinoma cells with compounds 4 and 5 (20 mM), 5 (0.5 mM), 5-FU (20 mM), or DMSO vehicle control. Results are expressed as the percentage (%) of viable or dead cells 7SEM of at least three different experiments. (A) Cell population dot plots. (B) Viable and dead cell populations. Results are expressed as mean 7SEM of at least three different experiments. *p o 0.05 and †p o 0.01 from DMSO vehicle control; and ‡ p o0.05 and §po 0.01 from 5-FU.

468

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

plants have been described to produce various classes of indole alkaloids with unusual structures as well as interesting bioactivities (Vanbeek et al., 1984, 1985). The phytochemical studies of the MeOH extract of Tabernaemontana elegans roots led to the isolation of four monoterpene indole (1–3 and 6) and two monoterpene bisindole (4 and 5) alkaloids. The structural identification of these compounds was carried out by comparison of their spectroscopic data with those reported in the literature (Bartlett et al., 1958; Bombardelli et al., 1976; Kutney et al., 1978). The isolation and characterization of the monoterpene indole alkaloid, vobasine (7) was previously reported from the MeOH extract of Tabernaemontana elegans leaves (Mansoor et al., 2009b). The alkaloids (1
7) (Fig. 1) were evaluated for anticancer and apoptosis induction activity in HCT116 human colon carcinoma cells.

The cytotoxic properties of compounds (1
7) were evaluated by the MTS assay in HCT116 human colon carcinoma cells. HCT116 cells were exposed to 5, 10, 20, and 50 μM of each compound for 48 h. Compounds 4 and 5 were more cytotoxic than the remainder compounds, reducing cell viability by 40% and 70%, respectively, at 20 μM concentrations (Fig. 2). Compounds 4 and 5 were further evaluated at lower concentrations (0.5, 1.0, and 2.5 mM). Compound 5 reduced cell viability up to 65% at 2.5 mM concentration. Furthermore, cell population analysis by flow cytometry using the ViaCount assay corroborated the cytotoxic behavior of compounds 4 and 5, demonstrating the induction of cell death following 48 h exposure to 20 μM concentrations (Fig. 3). Interestingly, compound 5 was the most cytotoxic, leading to  20% and 95% loss of cell viability at 0.5 and

Fig. 4. Evaluation of compound effect on apoptosis. Evaluation of changes in nuclear morphology by fluorescence microscopy of Hoechst stained nuclei, after 24 h incubation of HCT116 colon carcinoma cells with compounds 4 (20 mM) and 5 (0.5 mM), 20 mM 5-FU, or vehicle control (DMSO). (A) representative images of compound effect on apoptosis (400  magnification). (B) Quantification of apoptosis induced by compound exposure. Results are expressed as mean 7 SEM of at least three different experiments. *po 0.05 and †p o 0.01 from DMSO vehicle control; §p o0.05 from 5-FU.

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

Fig. 5. Evaluation of compound effect on caspase-3-like activity. Caspase-3-like activity after exposure of HCT116 colon carcinoma cells to compounds 4, 5, and 5-FU (20 mM) or DMSO vehicle control, for 16 h. Results are expressed as mean 7 SEM of at least three different experiments. *p o 0.05 from DMSO vehicle control and †p o0.05 from 5-FU.

20 μM, respectively. Similar results were obtained in the SW620 colon carcinoma cell line (data not shown). To determine the induction of apoptosis in HCT116 cells by compounds 4 and 5, we evaluated changes in nuclear morphology by fluorescence microcopy after Hoechst staining. Apoptotic nuclei are characterized by a distinct pattern of morphological changes, including cell shrinkage, condensation, fragmentation of the nucleus, blebbing of the plasma membrane and chromatin condensation (Galluzzi et al., 2009). HCT116 cells incubated with compound 4 at 20 μM for 24 h displayed 1.6-fold higher levels of apoptosis than the positive control 5-FU (Fig. 4A). Compound 5 at 0.5 μM (40x lower concentration) was a stronger inducer of morphologic changes characteristic of apoptosis in HCT116 cells, resulting in 2.7-fold increase in apoptotic cells as compared with vehicle control DMSO (Fig. 4B). At 20 μM concentration of compound 5, the assay was not possible since all cells were dead. Compounds 4 and 5 were further evaluated for general caspase-3-like activity (Galluzzi et al., 2009). Caspases are main executioners of the apoptotic process, belonging to a group of enzymes known as cysteine proteases and exist within the cell as inactive pro-forms or zymogens. These zymogens can be cleaved to form active enzymes following the induction of apoptosis. Induction of apoptosis via death receptors typically results in the activation of initiator caspases, such as caspase-8 or -10. In turn, these can then activate downstream effector caspases, such as caspase-3, -6 and -7. Effector caspases are responsible for the cleavage of key cellular proteins, such as cytoskeletal proteins, which lead to the typical morphological changes observed in cells undergoing apoptosis. In addition, induction of apoptosis is desired in the treatment of colorectal cancer, since it can be involved in the reduction of tumor growth (Borralho et al., 2007, 2009) and is a good indicator of drug cytotoxicity (Borralho et al., 2011, Aranha et al., 2007). Importantly, compound 5 displayed a 2.6-fold increase in caspase-3-like activity (Fig. 5). Compound 4 also showed 1.4-fold increase in caspase-3-like activity as compared to DMSO vehicle control (Fig. 5). These results corroborated the evaluation of nuclear morphology data after Hoechst staining and further demonstrated the ability of these compounds to induce large extent of apoptosis in HCT116 cells, although some degree of secondary necrosis may also be occurring in the culture system. An overview of the cell viability/cytotoxicity profiles of the compounds (1
7) reflected that the bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) displayed higher

469

levels of cytotoxicity than the other tested compounds (1
3, 6, and 7), which are monomeric indole alkaloids. It is also interesting to note here that compounds 1, 2 and 7 displayed significant levels of cytotoxicity as well as apoptosis induction activity toward HuH7 human hepatoma cancer cell line in our previous studies (Mansoor et al., 2009b), suggesting that these monoterepene bisindole alkaloids display differential cytotoxicity toward different cell lines. The data we report here on apoptosis induction for compounds 4 and 5 in colon cancer cells importantly expands the current knowledge on monoterepene bisindole alkaloids as anticancer agents based on colon cancer incidence and mortality (Jemal et al. 2010). Furthermore, tabernaelegantinine B (5) showed higher levels of cytotoxicity as well as apoptosis induction activity than tabernaelegantine C (4) in cell viability and apoptosis induction assays. Both structures are very similar, differing in the configuration at C-20 and linkage position of the two monoterpene indole units. In addition, tabernaelegantinine B (5) also has a different substitution at C-3′, having an extra –CH2COCH3 unit at this carbon whereas tabernaelegantine C (4) is unsubstituted at the same position (Fig. 1). These structural features seem to be responsible for stronger apoptosis induction activity of compound 5 as compared to tabernaelegantinine B (4), even at lower concentrations, as evaluated by Hoechst′s staining (Fig. 4). Monoterpene bisindole alkaloids such as vinblastine and vincristine are highly effective anti-cancer agents, currently used clinically against leukemia, Hodgkin′s lymphoma and other forms of cancer (Cragg and Newman 2005; Gurib-Fakim 2006). Similarly, the monoterpene indole alkaloid camptothecin (a topoisomerase inhibitor) and its analogues are routine anticancer agents (Lorence and Nessler 2004). These alkaloids are also potent apoptosis inducers in several other malignant cells, such as neuronal cancer cells, human epidermoid carcinoma cells, colon carcinoma cells, retinoblastoma cells, to name a few (Conway et al., 1998; Morris and Geller 1996; Chaturvedula et al., 2003; Kolomeichuk et al., 2008). Taken together, these results show that among the compounds evaluated in this study, monoterpene bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) can be considered as promising apoptosis inducers in HCT116 human colon carcinoma cells. Human colorectal cancer is one of the serious health problems in the western world. In fact, it is commonly acknowledged as one of the most prevalent cancers and is amongst the leading causes of cancer-related death (Jemal et al. 2010). In an anticancer therapeutic perspective, the use of apoptosis inducers 4 and 5 would be of extreme relevance, since apoptosis is often considered a ‘silent′ type of cell death, with virtual absence of inflammatory response. Thus, it is important to further characterize the mechanisms of action of tabernaelegantine C (4) and tabernaelegantinine B (5) in HCT116 cancer cells, as well as evaluate the selectivity toward tumor cells, before moving to in vivo studies of the anticancer potential of these compounds.

4. Conclusion In conclusion, our results demonstrate that the medicinal plant Tabernaemontana elegans possesses apoptosis-inducing compounds and therefore support the traditional use of this plant for treatment of tumors and cancer. The promising activity of tabernaelegantine C (4) and tabernaelegantinine B (5) in HCT116 cancer cells suggests their potential for possible anti-cancer drug scaffolds, warranting further study.

470

T.A. Mansoor et al. / Journal of Ethnopharmacology 149 (2013) 463–470

Acknowledgment This study was supported by the Portuguese Foundation for Science and Technology (FCT) through postdoctoral fellowships SFRH/BPD/30492/2006 and SFRH/BPD/70197/2010 and Projects PTDC/ QUI-QUI/099815/2008, PTDC/SAU-ORG/119842/2010 and PEst-OE/ SAU/UI4013/2011.

References Achenbach, H., Benirschke, M., Torrenegra, R., 1997. Alkaloids and other compounds from seeds of Tabernaemontana cymosa. Phytochemistry 45, 325–335. Andrade, M.T., Lima, J.A., Pinto, A.C., Rezende, C.M., Carvalho, M.P., Epifanio, R.A., 2005. Indole alkaloids from Tabernaemontana australis (Muell. Arg) Miers that inhibit acetylcholinesterase enzyme. Bioorganic Medicinal Chemistry 13, 4092–4095. Aranha, M.M., Borralho, P.M., Ravasco, P., Moreira da Silva, I.B., Correia, L., Fernandes, A., Camilo, M.E., Rodrigues, C.M., 2007. NF-kappaB and apoptosis in colorectal tumourigenesis. European Journal of Clinical Investigation 37, 416–424. Arnold, H.J., Gulumian, M., 1984. Pharmacopoeia of traditional medicine in Venda. Journal of Ethnophramacology 12, 35–74. Bartlett, M.F., Dickel, D.F., Taylor, W.I., 1958. The alkaloids of tabernanthe-iboga. 4. The structures of ibogamine, ibogaine, tabernanthine and voacangine. Journal of American Chemical Society 80, 126–136. Bombardelli, E., Bonati, A., Gabetta, B., Martinelli, E.M., Mustich, G., Danieli, B., 1976. Structures of tabernaelegantines A–D and tabernaelegantinines A and B, new indole alkaloids from Tabernaemontana elegans. Journal of Chemical Society, Perkin Transactions 1, 1432–1438. Borralho, P.M., Moreira da Silva, I.B., Aranha, M.M., Albuquerque, C., Nobre Leitão, C., Steer, C.J., Rodrigues, C.M., 2007. Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53. Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53. Biochimica et Biophysica Acta 1772, 40–47. Borralho, P.M., Kren, B.T., Castro, R.E., da Silva, I.B., Steer, C.J., Rodrigues, C.M., 2009. MicroRNA-143 reduces viability and increases sensitivity to 5-fluorouracil in HCT116 human colorectal cancer cells. FEBS Journal 276, 6689–6700. Borralho, P.M., Simões, A.E., Gomes, S.E., Lima, R.T., Carvalho, T., Ferreira, D.M., Vasconcelos, M.H., Castro, R.E., Rodrigues, C.M., 2011. miR-143 overexpression impairs growth of human colon carcinoma xenografts in mice with induction of apoptosis and inhibition of proliferation. PLoS One 6, e23787, http://dx.doi.org/ 10.1371/journal.pone.0023787. Chaturvedula, V.S.P., Sprague, S., Schilling, J.K., Kingston, D.G.I., 2003. New cytotoxic indole alkaloids from Tabernaemontana calcarea from the Madagascar Rainforest. Journal of Natural Products 66, 528–531. Chhabra, S.C., Mahunnah, R.L.A., Mshiu, E.N., 1987. Plants used in traditional medicine in eastern Tanzania. I. Pteridophytes and Angiosperms (Acanthaceae to Canellaceae). Journal of Ethnopharmacology 21, 253–277. Conway, R.M., Madigan, M.C., Billson, F.A., Penfold, P.L., 1998. Vincristine- and cisplatin-induced apoptosis in human retinoblastoma. Potentiation by sodium butyrate. European Journal of Cancer 34, 1741–1748. Cragg, G.M., Newman, D.J., 2005. Plants as a source of anti-cancer agents. Journal of Ethnopharmacology 100, 72–79. Duarte, N., Varga, A., Cherepnev, G., Radics, R., Molnár, J., Ferreira, M.J.U., 2007. Apoptosis induction and modulation of P-glycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids. Bioorganic and Medicinal Chemistry 15, 546–554. Galluzzi, L., Aaronson, S.A., Abrams, J., Alnemri, E.S., Andrews, D.W., Baehrecke, E.H., Bazan, N.G., Blagosklonny, M.V., 2009. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death and Differentiation 16, 1093–1107.

Graham, J.G., Quinn, M.L., Fabricant, D.S., Fransworth, N.R., 2000. Plants used against cancer—an extension of the work of Jonathan Hartwell. Journal of Ethnopharmacology 73, 347–377. Gurib-Fakim, A., 2006. Medicinal plants: traditions of yesterday and drugs of tomorrow. Molecular Aspects of Medicine 27, 1–93. Hemaiswarya, S., Doble, M., 2013. Combination of phenylpropanoids with 5fluorouracil as anti-cancer agents against human cervical cancer (HeLa) cell line. Phytomedicine 20, 151–158. Jemal, A., Siegel, R., Xu, J., Ward, E., 2010. Cancer Statistics 60, 277–300. Kam, T.S., Sim, K.M., Koyano, T., Toyoshima, M., Hayashi, M., Komiyama, K., 1998. Conodiparines A–D, new bisindoles from Tabernaemontana. Reversal of vincristine-resistance with cultured cells. Bioorganic and Medicinal Chemistry Letters 8, 1693–1696. Kinloch, R.A., Treherne, J.M., Furness, L.M., Hajimohamadereza, I., 1999. The pharmacology of apoptosis. Trends in Pharmacological Sciences 20, 35–42. Kolomeichuk, S.N., Bene, A., Upreti, M., Dennis, R.A., Lyle, C.S., Rajaskeran, M., Chambers, T.C., 2008. Induction of apoptosis by vinblastine via c-Jun autoamplification and p53-independent down-regulation of p21WAF1/CIP1. Molecular Pharmacology 73, 128–136. Kutney, J.P., Eigendorf, G.K., Matsue, H., Murai, A., Tanaka, K., Sung, W.L., Wada, K., Worth, B.R., 1978. Total synthesis of dregamine and epidregamine—general route to 2-acylindole alkaloids. Journal of American Chemical Society 100, 938–943. Lorence, A., Nessler, C.L., 2004. Molecules of interest—Camptothecin, over four decades of surprising findings. Phytochemistry 65, 2735–2749. Mansoor, T.A., Ramalhete, C., Molnar, J., Mulhovo, S., Ferreira, M.J.U., 2009a. Tabernines A—C, beta-carbolines from the leaves of Tabernaemontana elegans. Journal of Natural Products 72, 1147–1150. Mansoor, T.A., Ramalho, R.M., Mulhovo, S., Rodrigues, C.M., Ferreira, M.J.U., 2009b. Induction of apoptosis in HuH-7 cancer cells by monoterpene and β-carboline índole alkaloids isolated from the leaves of Tabernaemontana elegans. Bioorganic and Medicinal Chemistry Letters 19, 4255–4258. Mansoor, T.A., Ramalho, R.M., Luo, X., Ramalhete, C., Rodrigues, C.M., Ferreira, M.J. U., 2011. Isoflavones as apoptosis inducers in human hepatoma HuH-7 cells. Phytotherapy Research 25, 1819–1824. Mansoor, T.A., Ramalho, R.M., Rodrigues, C.M., Ferreira, M.J.U., 2012. Dibenzylbutane- and Butyrolactone-type lignans as apoptosis inducers in human hepatoma HuH-7 cells. Phytotherapy Research 26, 692–696. Morris, E.J., Geller, H.M., 1996. Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity. Journal of Cell Biology 134, 757–770. Neuwinger, N.D., 1966. African traditional medicine, 1st ed. Medpharm Scientific Publishers, Stuttgart. Silva, T.F., Martins, L.M., Guedes da Silva, M.F., Fernandes, A.R., Silva, A., Borralho, P. M., Santos, S., Rodrigues, C.M., Pombeiro, A.J., 2012. Cobalt complexes bearing scorpionate ligands: synthesis, characterization, cytotoxicity and DNA cleavage. Dalton Transactions 41, 12888–12897. Taraphdar, A.K., Roy, M., Bhattacharya, R.K., 2001. Natural products as inducers of apoptosis: implication for cancer therapy and prevention. Current Science 80, 1387–1396. Vanbeek, T.A., Verpoorte, R., Svendsen, A.B., Leeuwenberg, A.J.M., Bisset, N.G., 1984. Pharmacognostical studies of Tabernaemontana species. 8. Tabernaemontana L. (apocynaceae)—a review of its taxonomy, phytochemistry, ethnobotany and pharmacology. Journal of Ethnopharmacology 10, 1–156. Vanbeek, T.A., Verpoorte, R., Svendsen, A.B., 1985. Pharmacognostical studies of tabernaemontana species. 11. Antimicrobially active alkaloids from Tabernaemontana chippii. Journal of Natural Products 48, 400–423. Vanderheijden, R., Brouwer, R.L., Verpoorte, R., Vanbeek, T.A., Harkes, P.A.A., Svendsen, A.B., 1986. Pharmacognostical studies of Tabernaemontana species.16. Indole alkaloids from Tabernaemontana elegans. Planta Medica 2, 144–147. Watt, J.M., Breyer-Brandwijk, M.G., 1962. The medicinal and poisonous plants of southern and eastern Africa, 2nd ed Livingstone, London. Wesołowska, O., Wiśniewski, J., Sroda-Pomianek, K., Bielawska-Pohl, A., Paprocka, M., Duś, D., Duarte, N., Ferreira, M.J.U., Michalak, K., 2012. Multidrug resistance reversal and apoptosis induction in human colon cancer cells by some flavonoids present in citrus plants. Journal of Natural Products 75, 1896–1902.