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Vitexin-2-O-xyloside, raphasatin and (−)-epigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells
Food Chemistry 138 (2013) 1521–1530
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Vitexin-2-O-xyloside, raphasatin and ()-epigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells Alessio Papi a,1, Fulvia Farabegoli b,1, Renato Iori c, Marina Orlandi a, Gina R. De Nicola c, Manuela Bagatta c, Donato Angelino d, Lorenzo Gennari d, Paolino Ninfali d,⇑ a
Department of Experimental Evolutive Biology, University of Bologna, Bologna, Italy Department of Experimental Pathology, University of Bologna, Bologna, Italy Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali, Via di Corticella, 133, 40129 Bologna, Italy d Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Urbino (PU), Italy b c
a r t i c l e
i n f o
Article history: Received 1 August 2012 Received in revised form 19 October 2012 Accepted 20 November 2012 Available online 5 December 2012 Keywords: Colon carcinoma Vitexin-2-O-xyloside (–)-Epigallocatechin-3-gallate 4-Methylsulphanyl-3-butenyl isothiocyanate (raphasatin) Chemoprevention
a b s t r a c t Cytotoxic effects of the combination of the food components vitexin-2-O-xyloside (X), raphasatin (4methylsulphanyl-3-butenyl isothiocyanates; G) and (–)-epigallocatechin-3-gallate (E) were investigated in colon (LoVo and CaCo-2) and breast (MDA-MB-231 and MCF-7) cancer cells. Breast cancer cells were more resistant than colon cells to X, G and E inhibition. On the contrary, marked synergistic effects among X, G and E on cell growth were found in both colon cancer cells. Further analysis revealed a G0/G1 arrest of the phase cell progression and apoptosis, linked to modulation of Bax, Bcl2, caspase-9 and poly(ADP-ribose) polymerase as well as Reactive Oxygen Species (ROS) generation in both colon cancer cells, whereas apoptosis and ROS were not significantly detected in normal human lymphocytes. We conclude that the X, G and E mixture might act by mitochondrial pathway activation of apoptosis, possibly elicited by ROS and the mixture may be effective in the chemoprevention of colon cancer. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Fruit and vegetables are rich sources of many bioactive compounds, which positively interact to decrease the risk of chronic diseases, including cancer (Surh, 2003). For this wide group of bioactive molecules, the term ‘‘chemopreventive phytochemicals’’ (CP) was coined (Sporn, 1980). Beyond the well-known radical scavenging activity, most of the CP interfere with the mechanisms that sustain cancer development such as: evasion of apoptosis, growth self-sufficiency, insensitivity to anti-growth signals, high replication rate and angiogenesis initiation (Lippman & Levin, 2005). It has been proposed that not only CP can prevent oncological events, but they also have a role in the treatment of neoplastic conditions (D’Incalci, Steward, & Gescher, 2005). CP are less specific and have numerous molecular targets in relation to chemically designed anti-neoplastic drugs (CDAD); however, CP operate in a narrow concentration range with relatively low toxic-
⇑ Corresponding author. Address: Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Via Saffi 2, 61029 Urbino (PU), Italy. Tel.: +39 0722 305288; fax: +39 0722 305324. E-mail address: [email protected] (P. Ninfali). URL: http://www.uniurb.it/orac/ (P. Ninfali). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.112
ity (Davis, 2007). Moreover, pure CP, like polyphenols, glucosinolates or carotenoids, can be combined together or with food extracts (Liu, 2004) and can positively increase the potential activity of the mixture, in order to deregulate oncological events. Combinations of natural molecules or extracts that effectively inhibit tumour development and progression are nowadays actively researched (Mertens-Talcott & Percival, 2005). Among the CP most frequently used in antitumour mixtures, catechins from green tea extracts and chiefly (–)-epigallocatechin-3-gallate (E), showed relevant anticancer activity (Khan, Afaq, & Mukhtar, 2008; Yang, Wang, Lu, & Picinich, 2009) and optimal pharmacokinetic behaviour (D’Incalci et al., 2005). The combination of soy phytochemicals and green tea extract was more effective in inhibiting tumour angiogenesis than when either food was provided alone; similarly, soy phytochemicals and green tea synergistically inhibited tumour growth and metastasis in a mouse model of androgen sensitive human prostate cancer (Zhou, Yu, Mai, & Blackburn, 2004). Another class of CP used for anticancer cocktails are the isothiocyanates (ITC), present in Cruciferous (Brassicaceae) vegetables in the form of glucosinolates (GL) that are released following exposure of GL to the enzyme myrosinase (b-thioglucoside glucohydrolase, EC. 3.2.1.147) (Barillari et al., 2008; Papi et al., 2008). One of these GL, named glucoraphasatin (GRH), releases a biologically important ITC, the 4-methylsulphanyl-3-bute-
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nyl isothiocyanate (G) in the presence of the myrosinase. G is known to have notable antioxidant and apoptotic activities (Papi et al., 2008). Another emerging group of anti-proliferative and pro-apoptotic flavonoids, never used in anticancer mixtures, are the vitexin-glycosides. Among them, the vitexin 2-O-xyloside (X), present in Swiss chard leaves (Gil, Ferreres, & Tomàs-Barberàn, 1998) and seeds (Gennari et al., 2011) was shown to be very active as a cytotoxic agent against different human cancer cell lines (Gennari et al., 2011; Ninfali et al., 2007). The molecules X, G and E are structurally different, as they belong to distinct classes of phytochemicals: C-glycosyl-flavones (X), glucosinolates-ITC (G) and flavan-3-ols (E). Therefore, these molecules, when used in combinations, may exhibit complementary synergistic effects in their cytotoxic activity on cancer cell lines. To demonstrate synergy, it is necessary that the effect of the compound mixture is superior to the sum of the effects caused by each compound individually. If the effect of the mixture corresponds to the sum of the effects of the individual phytochemicals, the interaction is additive; whereas, it is antagonistic when the measured effect is less than the sum of the individual components. In this study, the three molecules X, G and E were evaluated, individually and in combination, with respect to their cytotoxicity on colon-rectal and breast carcinoma cell lines. We were particularly interested on identifying synergistic interactions, to support the hypothesis that the intake of specific food bioactive components may offer remarkable advantages for cancer prevention.
2. Materials and methods 2.1. Materials Plant source. Raphanus sativus L. var. major (Daikon) seeds, cultivar 0P38 (Brassicaceae) and Beet green seeds (Chenopodiaceae) were supplied by SUBA SEEDS COMPANY, Longiano (FO). Glucoraphasatin (GRH; MW: 435.5 Da) was isolated from 7-day-old freeze-dried R. sativus L. sprouts, by two sequential steps: ion exchange and size exclusion chromatography, as reported (Barillari et al., 2005). GRH purity was close to 95%, as estimated by HPLC analysis of the desul-
pho-derivative (ISO 9167-1 method). GRH (4.6 mg/ml) was dissolved in water and stored at 20 °C. The relative ITC, 4methylsulphanyl-3-butenyl isothiocyanate (G), was produced via myrosinase (b-thioglucoside glucohydrolase, EC.3.2.1.147) catalysed hydrolysis, performed by an ‘‘in situ’’ method (Nastruzzi et al., 1986) (Fig. 1). In every treatment, 5 lL of myrosinase (30 IU/ ml; 60 IU/mg protein) isolated from seeds of Sinapis alba L., was added to the cell culture medium, which contained increasing concentrations of GRH. One myrosinase unit was defined as the amount of enzyme able to hydrolyse 1 lmol sinigrin per min at pH 6.5 and 37 °C. Vitexin-2-O-xyloside (X) was extracted and purified from seeds of Beta vulgaris cicla by Sephadex LH-20 and Davisil C18 column chromatography, as reported (Gennari et al., 2011). X (MW: 564 Da) was identified by HPLC–MS, analysing the m/z of molecular ion and the m/z signal generated by the loss of xylose, in positive ESI mode. X (10 mg/ml) was dissolved in 60% ethanol and diluted for use in cell culture medium at the desired concentrations. (–)-Epigallocatechin-3-gallate (E) (MW: 458 Da) was purchased from Sigma Chem. Co, USA, dissolved in water at 10 mg/ml and diluted for use in cell culture medium at the desired concentrations. 2.2. Cell culture LoVo and Caco-2 colon cancer cell lines and MCF-7 and MDAMB-231 breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI 1640 1 (Lovo and CaCo-2), E-MEM (MCF-7) or D-MEM (MDA-MB-231) supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, 50 U/ml penicillin, 50 lg/ml streptomycin and grown at 37 °C in a humidified atmosphere with 5% CO2. Media and supplements were purchased from Sigma Chem. Co., USA. 2.3. Lymphocytes isolation Human lymphocytes were isolated from peripheral blood of a healthy donor and grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 50 g/ml streptomycin and 50 U/ ml penicillin and grown at 37 °C in a humidified atmosphere with 5% CO2.
Fig. 1. Molecular structures of compounds X, G and E. Compound 1, was purified from Rhapanus sativus L. var. major (Daikon) sprouts (Brassicaceae); compound 2 was released from 1 by myrosinase catalysed hydrolysis and indicated as G; compound 3, vitexin-2-O-xyloside (X), was prepared from Beta vulgaris cicla (Swiss chard) seeds (Chenopodiaceae); compound 4, ()-epigallocatechin-3-gallate (E), was purchased as pure compound.
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2.4. Cell viability assay
2.7. Annexin V/propidium iodide apoptosis detection assay
Cell viability was evaluated by the Sulphorhodamine (SRB) assay. The SRB assay is based on the ability of SRB dye to bind basic amino acid residues. The amount of dye incorporated by the cells indicates the number of living cells. Cells (104/well) were plated in 96-well plates in triplicate and treated with E, X and G for 24, 48 and 72 h. At the end of treatment, the cell culture medium was removed and fresh medium was added (50 lL/well). Cells were fixed using 25 lL/well of 50% aqueous trichloroacetic acid (TCA) for 1 h at 4 °C, rinsed with water several times and incubated for 30 min with 50 lL/well SRB solution (0.4%) (Sigma, MO, USA). After rinsing with 1% acetic acid and solubilising in 10 mM Tris for 5 min, the absorbance was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. The viability results were expressed as a percentage of living cells in treated vs control (untreated) wells.
The cells were treated for 24 h with different doses of X, G and E (40, 5 and 10 lg/ml, respectively) and analysed for Annexin-V-FITC positive staining by flow cytometry using Bender-Medsystem (Austria) apoptosis kit. Briefly, cells were treated by trypsin, centrifuged and stained with Annexin-V-FITC/Propidium Iodide (PI) and analysed using flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA). Unstained cells were classified as alive; cells only stained for Annexin V were ‘‘early apoptotic’’; cells positive for both Annexin V and PI were ‘‘late apoptotic’’; cells only stained for PI were classified as dead cells or debris. Results were expressed as % of cells in the indicated condition.
2.5. Synergy evaluation To identify specific combinations of phytochemicals that exhibit synergistic interactions, compounds were tested individually and then mixed in pairs and triplets in the cell culture medium and the cytotoxicity evaluated by the SRB assay. In total, 20 combinations were tested and the observed cell viability values were recorded. The measured cell viability (V%) was converted into % of growth inhibitory effect (GIE) by the calculation: % GIE = (100 V%) and this value was taken as an indication of the cytotoxic effect. The experimentally measured cytotoxicity ‘‘Measured Cytotoxicity’’ in each mixture was divided by the ‘‘Expected Cytotoxicity’’, which is the mathematical sum of the cytotoxicity derived from the individual phytochemicals. This mathematical approach belongs to the category of the summation of effects, where the molecules X, G and E behave as mutually nonexclusive effectors (Chou, 2006). If measured values were significantly higher than expected values, i.e. Measured/Expected Ratio > 1.0 (p < 0.05), and a synergistic effect was considered to have occurred in the mixture. Consequently, Measured/Expected Ratio < 1.0 (p < 0.05) indicated an antagonistic effect; Measured/Expected Ratio = 1.0 (p < 0.05) indicated an additive interaction (Fuhrman, Volkova, Rosenblat, & Aviram, 2000).
2.6. Reactive Oxygen Species (ROS) assay Generation of ROS in colon cancer cells was monitored by the conversion of 20 ,70 -dichlorodihydrofluorescein diacetate (DCFHDA) to highly fluorescent dichlorofluorescein (DCF). DCFH-DA is transformed by cellular esterase into the reduced non-fluorescent form DCFH, which, in the presence of ROS, is oxidised to highly fluorescent DCF. Cells were grown on coverslips (2 104/well) in 24-well plates and treated for 4 h with X, G and E individually at 40, 5 and 10 lg/ml, respectively, and in the mixture at the same concentrations. After washing with PBS, the cells were incubated at room temperature in the dark for 20 min with 10 lM DCF-DA (Sigma, MO, USA) dissolved in PBS. As a positive control, cells treated with 1 lM H2O2 were included in each experiment. After incubation, the cells were rinsed three times with PBS and mounted with a solution of Hoescht (10 lg/ml) and 1,4-diazabicyclooctane (DABCO). The samples were observed at a Nikon epifluorescence microscope. Cells showing a bright and intense fluorescence were counted as positive, whereas cells having no or low fluorescence were counted as negative. At least three fields for each sample were analysed and 100–200 cells for sample were counted. Results were expressed as % DCF fluorescent positive cells vs the control.
2.8. Cell cycle analysis Cells treated for 24 h with different doses of X, G and E (40, 5 and 10 lg/ml, respectively) were fixed with ice cold 70% ethanol in PBS, treated with 10 lg/ml RNase A, stained with 50 lg/ml PI, and analysed by flow cytometry for DNA synthesis and cell cycle status (FACSCalibur, Becton Dickinson, San Jose, CA, USA). 2.9. Apoptosis detection by DAPI staining Cells were grown on coverslips. After 24 h of treatment the cells were fixed with 4% paraformaldehyde and thereafter permeabilised with Triton X-100 (0.1% in PBS). Cells were stained with 2.5 lg/ml DNA dye, 40 ,6-diamidine-2-phenylindole (DAPI) in PBS, for 30 min at 20 °C and analysed by fluorescence microscopy. At 20-fold magnification cells with condensed or fragmented nuclei were counted on adjacent four fields of each coverslip for a total of 160–180 cells. The percentage of cells with chromatin condensation and nuclear fragmentation was determined in two samples for each treatment and compared with controls. 2.10. Immunocytochemistry of caspase-3 Cells were grown on coverslips and after treatment they were fixed with 4% paraformaldehyde and treated with Triton X-100 (0.1%) in PBS and saturated in PBS–BSA 4% for 30 min. Then, cells were incubated with anti-caspase-3-FITC-conjugated antibody for 1 h at room temperature. After washings, slides were mounted in glycerol-PBS 1 medium containing 30 mg/ml DABCO. Evaluation of antibody specificity was also carried out by omitting the primary antibody. Cells were observed using fluorescence microscope and the percentage of caspase-3 positive cells was calculated by counting at least 200 cells in different microscope fields. 2.11. Western blot To determine BAX, BCL2, caspase-9 and poly(ADP-ribose) polymerase (PARP-1) protein levels, the cells were plated and treated with X, G and E individually and in the mixture. After treatment, cells were detached and collected by centrifugation at 300g, and the pellets were suspended in lysis buffer (20 mM Tris–HCl, pH 7.5, 0.5 mM EDTA, 0.5% Triton X-100, 5 lM Na3VO4) and sonicated on ice, in the presence of protease inhibitors. Proteins (50 lg) from cell lysate were size fractioned in 12% SDS–polyacrylamide gel, and transferred to Hybond TM-C Extra membranes (GE Healthcare, UK) by standard protocols. After blocking, the following antibodies were used according to supplier recommended concentrations: anti-caspase-9 (Sigma, USA), anti-PARP-1 (S. Cruz, USA), anti-Bax (Applied Biosystem, USA), anti-Bcl2 (Sigma, USA). The primary antibodies were diluted 1:500 in TBS-5% milk and the respective peroxidase-conjugated secondary antibody was diluted 1:1000. The proteins were detected by luminol (GE Healthcare, UK). Bands
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were quantified by using densitometric images analysis software (Image Master VDS, Pharmacia Biotech, Sweden). Protein loading was controlled by actin (1:1000) (Sigma, USA) detection after stripping of membranes with stripping solution (Thermo Scientific, USA). Values were normalised against the actin control and statistically evaluated. 2.12. Statistical analysis Experiments were replicated twice with three samples analysed per replicate (n = 6). Results were expressed as mean ± SD of six obtained values. Statistic significance was assessed by ANOVA followed by Bonferroni’s multiple comparison test or two-tail Student’s t-test with standard deviation (SD), as appropriate, using PRISM 5.1 (Graph pad Software, La Jolla, CA, USA). The level for accepted statistical significance was p < 0.05.
3. Results Cytotoxic effects of the individual compounds X, G and E (Fig. 1) were first investigated on the four types of cancer cell lines and the number of living cells in culture was evaluated. The IC50 values of X, G and E on the four cell lines are shown in Table 1. All cancer cell lines were markedly sensitive to the cytotoxic effect of G, produced in situ via myrosinase reaction. This enzyme was shown to be ineffective when added to the cells in the absence of G (data not shown). The average IC50 of G in all cell lines was 26 ± 5 lg/ml. The CaCo-2 cancer cell line was markedly sensitive to E, but moderately sensitive to X; whereas LoVo cancer cell line was moderately sensitive to both E and X. On the contrary, MCF-7 and MDA-MB-231 were found to be relatively resistant to both X and E, as deduced by the elevated IC50 values (Table 1). Individual compounds X, G and E and 20 different combinations were tested on all cell lines at 24, 48, and 72 h of incubation. On both colon cancer cell lines, estimation of the cell viability showed that some pairs and triplets of the CP induced marked cytotoxicity at all incubation times (Supplementary Fig. 1 for CaCo-2; Supplementary Fig. 2 for LoVo). To identify specific combinations of X, G and E that exhibited synergistic interactions, we evaluated measured/expected cytotoxicity ratios. Fig. 2 shows these ratios of the Caco-2 (Fig. 2A) and LoVo (Fig. 2B) cancer cell lines. The histograms provide an immediate picture of the CP interaction type, with reference to the horizontal baseline corresponding to the ratio = 1, i.e. additive effect. For instance, in the CaCo-2 cell line (Fig. 2A), eight pairs and four triplets showed synergistic interactions, being all other interactions additive. In the LoVo cell line (Fig. 2B) all triplets gave synergy, whereas only four pairs, out of 12, showed synergistic effects. As no one combination of X, G and E was synergistically effective in MCF-7 and MDA-MB-231 breast cancer cell lines (data not shown), these cells were not considered as a suitable subjects
for further investigations on the cell cycle, which was only restricted to LoVo and CaCo-2 cells. On the basis of the results obtained, we pinpointed one combination of X, G and E eliciting synergistic cytotoxicity and we treated LoVo and CaCo-2 colon cancer cell lines, first individually and then with the mixture, in order to detect fluctuations of specific biomarkers of the cell cycle and apoptosis. Fig. 3 shows the effects of X, G and E on the cell cycle progression by the percentages of cells in the G0/G1, S and G2 phases in both colon cancer cell lines. In the CaCo-2 cell line (Fig. 3A), an increase in the G0/G1 population was induced in the presence of G individually, whereas X and E were not significantly effective. The combination of X, G and E increased the G0/G1 phase (p < 0.05) and markedly decreased the cells in both G2 (p < 0.01) and S phase (p < 0.05) (Fig. 3A). In the LoVo cell line (Fig. 3B), the mixture of X, G and E resulted in a decrease of the cell number in G2 and S phases, although G0/G1 phase was not significantly different from the control. Hence, the combined CP hampered the progression of the cycle towards the G2 phase in both colon cancer cells lines. These results suggested that a number of cells underwent apoptosis. We therefore evaluated the effect of X, G and E treatment on the extent of the apoptosis by fluorescence microscopy analysis of nuclear morphology after DAPI staining and by flow cytometry, after Annexin V/PI co-staining. Fig. 4 shows the results obtained by means of the two techniques. The DAPI staining provided evidence of a statistically significant increase in the number of apoptotic nuclei in both CaCo-2 (Fig. 4A) and LoVo (Fig. 4B) cell lines after the CP mixture treatment. Notably, the resulting effect of the mixture was greater than that provided by the individual compounds. We also compared the effect of X, G and E treatment on normal human lymphocytes from peripheral blood. Lymphocytes, stained by DAPI, did not show any significant increase in the number of apoptotic nuclei after exposure to the CP, either individually or in the mixture (Fig. 4C). Apoptosis investigation by Annexin V/PI staining showed that in the CaCo-2 cell line (Fig. 4D), the percentage of apoptotic Annexin V/PI positive cells increased three folds and the percentage of necrotic PI positive cells increases two folds after the X, G and E combined treatment vs control. In the LoVo cancer cell lines (Fig. 4E), late apoptotic Annexin V/PI positive and PI positive necrotic cells increased after mixture treatment and the difference vs control or G and X individual agent treatments was significant (Fig. 4E). In order to further investigate the occurrence of apoptosis, as well as to obtain an insight in the apoptotic pathways involved, we evaluated the effect of X, G and E treatment on Bax, Bcl2, PARP-1, caspase-3 and caspase-9 protein expression. The rationale for the choice of these proteins can be briefly summarised as follows. Apoptosis/survival is regulated by the relative balance of pro-apoptotic BAX and anti-apoptotic BCL family members and their heterodimers. PARP is the enzyme which contributes to the DNA repair after oxidative breaks. This enzyme is cleaved during
Table 1 Values of IC50 for vitexin-2-O-xyloside, 4-methylsulphanyl-3-butenyl isothiocyanate and ()-epigallocatechin-3-gallate in human breast and colon cancer cell lines after 72 h incubation. Cancer cell lines
CaCo-2 LoVo MCF-7 MDA-MB-231
IC50 (lg/ml) Vitexin-2-O-xyloside (X)
4-Methylsulphanyl-3-butenyl ITC (G)
()-Epigallocatechin-3-gallate (E)
120 ± 9 158 ± 13 350 ± 48 1200 ± 66
16 ± 4 36 ± 5 31 ± 4 21 ± 6
21 ± 3 135 ± 16 350 ± 47 1980 ± 94
The data represent the average median ± SD of two replicate experiments with three samples analysed per replicate (n = 6). For the dose–response curves five concentrations of each compound were used: X (30, 50, 80, 100, 120 lg/ml), G (5, 10, 15, 30, 50 lg/ml) and E (10, 20, 30, 40, 50 lg/ml). Colon cancer cell lines: CaCo-2 and LoVo. Breast cancer cell lines: MCF-7 and MDA-MB-231.
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Fig. 2. Synergistic combinations of X, G and E in CaCo-2 (A) and LoVo (B) colon cancer cell lines. Values are shown as measured/expected cytotoxicity ratios after 72 h incubation using the following concentrations: X = 20 lg/ml (35 lM); 2X = 40 lg/ml (70 lM); G = 5 lg/ml (11 lM); 3G = 15 lg/ml (33 lM); E = 10 lg/ml (22 lM); 2E = 20 lg/ml (44 lM). Data are expressed as mean ± SD of two replicate experiments with three samples analysed per replicate (n = 6). Statistically significant differences from the ratio = 1 (additive effect) are indicated with: a, p < 0.05; b, p < 0.01; c, p < 0.001; n.s., not significant.
Fig. 3. Effects of X, G and E on cycle progression and chromatin condensation. Flow cytometry analysis of X, G and E on cell cycle progression of CaCo-2 (A) and LoVo (B) colon cancer cell lines after 24 h exposure to X (40 lg/ml), G (5 lg/ml) and E (10 lg/ml) and their mixture at the same concentrations. Histograms are representative of the percentage of cells in G0/G1, S and G2 phases after treatment with the phytochemicals, compared with controls (CTR), 100% dotted line. Data are presented as the mean ± SD of two replicate experiments with three samples analysed per replicate. ANOVA; n = 6. Statistically significant differences from the CTR are indicated with a, p < 0.05; b, p < 0.01; n.s., not significant.
apoptosis into two fragments, one of which is a fragment of 89 kDa, called PARP-1. Detection of PARP-1 is widely used in apoptotic cells as it is an excellent marker of the activation of caspase-3. Caspase3 is activated by caspase-9, following a change of mitochondria permeability, release of cytochrome c and formation of the apoptosome. Fig. 5 shows the results of this molecular analysis for CaCo-2 (A, left column) and LoVo (B, right column). In the CaCo-2 cell line, the combination of X, G and E caused significant up-regulation of Bax protein expression (p < 0.05), down-regulation of Bcl2 protein (p < 0.05), significant increase of PARP-1 (p < 0.05) and caspase-9 (p < 0.05) expression (Fig. 5A, left column). The LoVo cancer cell line, exposed to the mixture of X, G and E, significantly up-regulated Bax expression, down-regulated expression of Bcl2, significantly increased activated caspase-9 and PARP-1 expression (Fig. 5B, right column). The mixture X, G and E was always found
to be effective in modifying the expression of the above proteins, although the same does not hold true for the individual compounds. Furthermore, an increased expression of cleaved caspase-3 in both cancer cell lines (Fig. 5C and D) treated with the individual CP (p < 0.05), was found; this expression further increased after the mixture treatment (p < 0.01) with respect to the individual agents. Overall, the data of Fig. 5 demonstrate that, in both colon cancer cell lines, the X, G and E mixture provided a marked increase in biomarkers of the mitochondrial apoptotic pathways, although not all the agents were individually able to exert such effects. We hypothesised that ROS generation might be the primary event able to trigger the underlying mitochondrial apoptotic mechanism. Fig. 6 shows the level of ROS, generated by exposure to X, G and E individually as well as to the mixture, in both colon carcinoma cell lines and in normal human lymphocytes. X, G and E,
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Fig. 4. Effects of X, G and E on lymphocytes, CaCo-2 and LoVo colon cancer cell lines for evaluating apoptosis by DAPI staining and flow cytometry. For the DAPI staining, CaCo-2 (A), LoVo (B) colon cancer cell lines and human resting lymphocytes (C) were treated 24 h with X (40 lg/ml), G (5 lg/ml) and E (10 lg/ml) and their mixture at the same concentrations were then fixed and stained with DAPI. Nuclear morphology was analysed and cells showing fragmented nuclei were counted as apoptotic and values were expressed as % apoptosis. For flow cytometry analysis, CaCo-2 (D) and LoVo (E) cancer cells were treated as above. Data were expressed as % cells in the indicated cell phase (mean ± SD). Two replicate experiments with three samples analysed each replicate were performed in both techniques. Significant statistical differences among treated samples and control were assessed: ANOVA, n = 6; a, p < 0.05; b, p < 0.01; n.s., not significant.
neither individually nor in the mixture, were able to significantly increase the ROS generation in normal lymphocytes (Fig. 6A). In contrast, a statistically significant increase in ROS generation was found in both carcinoma cell lines, after treatment with the mixture. ROS increase was fourfold greater in treated than in untreated CaCo-2 cell line (Fig. 6B) and threefold in the LoVo cancer cell line (Fig. 6C). Individually, G only was able to provide a significant increase in ROS generation in both colon cancer cell lines (Fig. 6B and C). Fig. 7 summarises the possible sequential events able to account for the synergistic cytotoxicity of our CP mixture. 4. Discussion The present study investigates the synergistic cytotoxic effects of the food derived phytochemicals X, G and E in four cancer cell lines. Our results showed that G, individually, was the most effective cytotoxic compound in all cancer cell lines. To the cytotoxic effect of G certainly contributed to the fact that this isothiocyanate was stoichiometrically released from the glucoraphasatin, by myrosinase activity, and added to the cell culture medium (Abdull Razis, De Nicola, Pagnotta, Iori, & Ioannides, 2012). G is a potent
inducer of rat hepatic phase II enzymes and a potential chemopreventive agent. Twenty different combinations of X, G and E, in couples and triplets, were tested for cytotoxicity on breast cancer cell lines, which were quite resistant and none of the mixtures showed synergistic cytotoxic effect. On the contrary, against LoVo and CaCo-2 colon cancer cells, X, G and E interacted synergistically in many pairs and triplets, thus generating powerful cytotoxic mixtures. The different responsiveness to X, G and E combinations, between colon and breast cancer cell lines, depends on the genetic diversity of the two types of neoplastic cells. They can in fact metabolise the CP at a different rate, due to the expression of the detoxificating enzymes or the diverse efficacy of the multidrug resistance system in the exclusion of the CP (Fimognari, Turrini, Ferruzzi, Lenzi, & Hrelia, 2012; Hemalswarya & Doble, 2006). The CaCo-2 cell line was found to be more responsive to the effect of the CP mixtures than the LoVo cell line. In the first instance, this result can be explained by the fact that CaCo-2 cell line is representative of a more differentiated type of colon carcinoma, whereas LoVo cells derive from an advanced stage of colon carcinoma and represent a very aggressive type of neoplasm. Regarding to the concentrations of the CP, chosen for testing synergy, we can infer that the G and E concentrations, used on
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Fig. 5. Detection of apoptosis in X, G and E treated CaCo-2 and LoVo cell lines by Western blot analysis and immuno-cytochemistry. For Western blot analysis (A and B columns), cells were treated with: (1) control; (2) X (40 lg/ml); (3) G (5 lg/ml); (4) E (10 lg/ml) and (5) X + G + E at the same concentrations for 24 h. Bcl2, Bax, cleaved caspase-9 and PARP protein levels, were assessed in cell lysate of CaCo-2 (left column A) and LoVo (right column B) colon cancer cell lines. Densitometric data were expressed as percentage of protein expression (mean ± SD) in treated samples with respect to the control (1). Two replicate experiments with three samples analysed per replicate were performed. ANOVA, n = 6; a, p < 0.05; n.s., not significant. For immunocytochemistry of caspase-3 (C and D histograms), CaCo-2 (C) and LoVo (D) colon cancer cell lines were exposed to the phytochemicals individually and in the mixture, as above, fixed, then incubated with anti-cleaved caspase-3-FITC antibody, photographed and counted. Data are expressed as % cleaved caspase-3 activation (mean ± SD) in treated samples with respect to control (CTR). Two replicate experiments with three samples analysed per replicate were performed. Statistical significant differences among treated samples and control were assessed: ANOVA, n = 6; a, p < 0.05; b, p < 0.01; n.s., not significant.
Caco-2 and LoVo cells, were a little bit higher than the plasmatic concentrations observed during ‘‘in vivo’’ experiments (Boreddy, Pramanik, & Srivastava, 2011; Khan et al., 2008). Similar studies are lacking for X, as its anti-cancer bioactivity was only recently
discovered (Gennari et al., 2011). To detect synergism, we used only pure molecules in order to control the working concentrations and avoid effects due to unknown components, as it happens when whole extracts are used. The compound E was commercially
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Fig. 7. Putative molecular mechanism of the cytotoxic effect of X, G and E mixture. The phytochemicals X, G and E increase ROS production in cancer cells. ROS trigger the mitochondrial apoptosis pathway through caspase-9 activation, which induces cleaved caspase-3 and PARP activation.
Fig. 6. ROS detection in lymphocytes and colon cancer cell lines. Lymphocytes (A), CaCo-2 (B) and Lovo (C) cells were treated with X (40 lg/ml), G (5 lg/ml) and E (10 lg/ml) and their mixture at the same concentrations for 4 h and DCF-DA analysis was performed. Cells showing bright fluorescence were considered positive and counted. Results were expressed as % DCF fluorescent cells in treated samples and control (CTR). Data are mean % values ± SD of twice replicate experiments, with three samples analysed for each replicate. Statistical significant differences among treated samples and control were assessed: ANOVA, n = 6; a, p < 0.05; b, p < 0.01; n.s., not significant of treated samples vs CTR. ⁄Individual agent treatments significantly different with p < 0.05 vs the mixture (X + G + E).
available, but X and GRH, the precursor of G, were purified inhouse with lab-scale procedures and characterised by NMR and mass spectrometry analysis, as they are not yet commercially available. The ability to purify CP by chromatographic equipment is an important resource and lab-scale purification protocols may be easily scaled up to obtain consistent amounts of CP to be combined in mixtures. The molecular mechanism underlying the cytotoxic effect of the mixture of X, G and E was based on arrest of the cell cycle in the G1
phase and apoptosis, possibly originating from the mitochondrial pathway. The present results suggest that an activation of this pathway, caused by ROS, occurred in both CaCo-2 and LoVo cancer cell lines, as we observed remarkable ROS production in both colon cancer cells after treatment. On the contrary, the resting lymphocytes did not show any significant increase in ROS production and apoptosis. The sequential cytotoxic events due to ROS production were found to be linked to deregulation of the proteins of the BCL2 family, activation of caspase-9 and -3, cleavage of the repairing enzyme PARP and occurrence of apoptosis. An interesting aspect of this work is related to the dual behaviour of CP. How can a pro-oxidant action be generated in tumour cells by compounds considered to be strong antioxidants? These opposite effects may be related to the concentration of the agents utilised to treat the cells. In fact, several antioxidant CP follow a hormetic mechanism (Son, Camandola, & Mattson, 2008), in the sense that at low concentrations CP can effectively work as antioxidants but, at higher concentrations, the beneficial antioxidant effect is reversed and the CP become cytotoxic. Cytotoxicity derives from a rapid and dose dependent inhibition of the enzymes (phases I and II) involved in xenobiotic metabolism, that cause mitochondrial damage and consequently ROS production (Fimognari et al., 2012; Hemalswarya & Doble, 2006). In this report, cytotoxicity and ROS production occurred at the applied X, G and E concentrations in colon cancer cells, but not in normal lymphocytes. The explanation of this behaviour lies in the biology of the tumour cell, which is, in many aspects, similar to the stem cell (Andrews, Przyborski, & Thomson, 2001). The malignant cells up-regulate multiple signalling pathways to promote proliferation, inhibit apoptosis, to finally migrate and invade the tissues. This performance is supported energetically by high oxygen consumption and metabolic rate (Cairns, Harris, & Mak, 2011). In this context, interacting with enzymes involved in oxidative reactions, CP enlarge the production of ROS, deregulated several molecular targets, leading to a marked derangement in many cell signalling pathways, thus amplifying the consequent genotoxic cell damage (Forester & Lambert, 2011). In this study, we demonstrated that the compounds G and E were clearly able to trigger
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the ROS production in cells. A pro-oxidant effect of G was already demonstrated to occur from 5 to 10 lM in cancer (Valgimigli & Iori, 2009) and normal stem cells (Zanichelli et al., 2012). In the present study, G was used at 5 lg/ml, i.e. 11 lM, concentration and therefore it fell into the pro-oxidant range. E was found to arrest lung cancer cells growth in vitro by pro-oxidant activity (Li et al., 2010) and it was demonstrated to trigger apoptosis in various types of cancer cells both in vitro and in vivo, but not in normal cells (Forester & Lambert, 2011). X is a novel flavone-C-glycoside and its pro-oxidant activity has never been studied in detail, but its aglycone, apigenin, was found to be able to initiate cell death in prostate cancer cells by ROS generation (Shukla & Gupta, 2010). We cannot exclude that a few part of cytotoxic effects in colon cancer cells, that we observed, might be related to E dimers, superoxide radical and hydrogen peroxide that are formed in cell culture medium as a consequence of the high instability of E molecule. Some of the reported biological activities of E are in fact considered H2O2-dependent, as a consequence of radicals spontaneously generated in vitro by E auto-oxidation (Long, Hoi, & Halliwell, 2010; Yang et al., 2006). However, the moderate cytotoxicity we observed in colon cancer cells after individual E treatment do not support the hypothesis that this is the most important mechanism leading to cell death after mixture treatment. As far as the synergism among the CP is concerned, the effect could likely be due to the fact that the CP contemporaneously reach different targets of the same signalling pathway, controlling cell cycle and apoptosis, thus accelerating the overall process (D’Incalci et al., 2005). An increase of cell permeability of one CP mediated by the others, the so called ‘‘permissive role’’ or alternatively, the inhibition performed by one of the CP on the multi-drug resistance system, which reduced the rate of exclusion of the other two CP from the cells, may contribute to the synergism. In conclusion, our results show that X, G and E added in the mixture synergistically induced cytotoxicity mediated by apoptosis in colon cancer cells. A ROS oxidative insult was exhibited by the X, G and E mixture to a greater extent than after individual treatment. Either individually or in the mixture X, G and E have no cytotoxic effect in normal human lymphocytes. These findings provide the fundamentals for further in vivo validation of the combination of X, G and E as a beneficial CP mixture, potentially able to decrease cancer cell growth and to be better investigated in experimental animal models.
Conflict of interest None declared.
Acknowledgements This study was supported by Pallotti Legacy for Cancer Research, Cornelia Pallotti Fund for Cancer Research and RFO (Orientated Research Fund). The authors wish to thank SUBA SEEDS COMPANY, Longiano (FO) for providing Beta vulgaris cicla and Raphanus sativus seeds and Dr. Rebecca L. Morris and Dr. Whitney N. Ajie for their support during the preparation of the English manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2012. 11.112.
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Vitexin-2-O-xyloside, raphasatin and ()-epigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells Alessio Papi a,1, Fulvia Farabegoli b,1, Renato Iori c, Marina Orlandi a, Gina R. De Nicola c, Manuela Bagatta c, Donato Angelino d, Lorenzo Gennari d, Paolino Ninfali d,⇑ a
Department of Experimental Evolutive Biology, University of Bologna, Bologna, Italy Department of Experimental Pathology, University of Bologna, Bologna, Italy Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali, Via di Corticella, 133, 40129 Bologna, Italy d Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Urbino (PU), Italy b c
a r t i c l e
i n f o
Article history: Received 1 August 2012 Received in revised form 19 October 2012 Accepted 20 November 2012 Available online 5 December 2012 Keywords: Colon carcinoma Vitexin-2-O-xyloside (–)-Epigallocatechin-3-gallate 4-Methylsulphanyl-3-butenyl isothiocyanate (raphasatin) Chemoprevention
a b s t r a c t Cytotoxic effects of the combination of the food components vitexin-2-O-xyloside (X), raphasatin (4methylsulphanyl-3-butenyl isothiocyanates; G) and (–)-epigallocatechin-3-gallate (E) were investigated in colon (LoVo and CaCo-2) and breast (MDA-MB-231 and MCF-7) cancer cells. Breast cancer cells were more resistant than colon cells to X, G and E inhibition. On the contrary, marked synergistic effects among X, G and E on cell growth were found in both colon cancer cells. Further analysis revealed a G0/G1 arrest of the phase cell progression and apoptosis, linked to modulation of Bax, Bcl2, caspase-9 and poly(ADP-ribose) polymerase as well as Reactive Oxygen Species (ROS) generation in both colon cancer cells, whereas apoptosis and ROS were not significantly detected in normal human lymphocytes. We conclude that the X, G and E mixture might act by mitochondrial pathway activation of apoptosis, possibly elicited by ROS and the mixture may be effective in the chemoprevention of colon cancer. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Fruit and vegetables are rich sources of many bioactive compounds, which positively interact to decrease the risk of chronic diseases, including cancer (Surh, 2003). For this wide group of bioactive molecules, the term ‘‘chemopreventive phytochemicals’’ (CP) was coined (Sporn, 1980). Beyond the well-known radical scavenging activity, most of the CP interfere with the mechanisms that sustain cancer development such as: evasion of apoptosis, growth self-sufficiency, insensitivity to anti-growth signals, high replication rate and angiogenesis initiation (Lippman & Levin, 2005). It has been proposed that not only CP can prevent oncological events, but they also have a role in the treatment of neoplastic conditions (D’Incalci, Steward, & Gescher, 2005). CP are less specific and have numerous molecular targets in relation to chemically designed anti-neoplastic drugs (CDAD); however, CP operate in a narrow concentration range with relatively low toxic-
⇑ Corresponding author. Address: Department of Biomolecular Sciences, University of Urbino ‘‘Carlo Bo’’, Via Saffi 2, 61029 Urbino (PU), Italy. Tel.: +39 0722 305288; fax: +39 0722 305324. E-mail address: [email protected] (P. Ninfali). URL: http://www.uniurb.it/orac/ (P. Ninfali). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.112
ity (Davis, 2007). Moreover, pure CP, like polyphenols, glucosinolates or carotenoids, can be combined together or with food extracts (Liu, 2004) and can positively increase the potential activity of the mixture, in order to deregulate oncological events. Combinations of natural molecules or extracts that effectively inhibit tumour development and progression are nowadays actively researched (Mertens-Talcott & Percival, 2005). Among the CP most frequently used in antitumour mixtures, catechins from green tea extracts and chiefly (–)-epigallocatechin-3-gallate (E), showed relevant anticancer activity (Khan, Afaq, & Mukhtar, 2008; Yang, Wang, Lu, & Picinich, 2009) and optimal pharmacokinetic behaviour (D’Incalci et al., 2005). The combination of soy phytochemicals and green tea extract was more effective in inhibiting tumour angiogenesis than when either food was provided alone; similarly, soy phytochemicals and green tea synergistically inhibited tumour growth and metastasis in a mouse model of androgen sensitive human prostate cancer (Zhou, Yu, Mai, & Blackburn, 2004). Another class of CP used for anticancer cocktails are the isothiocyanates (ITC), present in Cruciferous (Brassicaceae) vegetables in the form of glucosinolates (GL) that are released following exposure of GL to the enzyme myrosinase (b-thioglucoside glucohydrolase, EC. 3.2.1.147) (Barillari et al., 2008; Papi et al., 2008). One of these GL, named glucoraphasatin (GRH), releases a biologically important ITC, the 4-methylsulphanyl-3-bute-
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nyl isothiocyanate (G) in the presence of the myrosinase. G is known to have notable antioxidant and apoptotic activities (Papi et al., 2008). Another emerging group of anti-proliferative and pro-apoptotic flavonoids, never used in anticancer mixtures, are the vitexin-glycosides. Among them, the vitexin 2-O-xyloside (X), present in Swiss chard leaves (Gil, Ferreres, & Tomàs-Barberàn, 1998) and seeds (Gennari et al., 2011) was shown to be very active as a cytotoxic agent against different human cancer cell lines (Gennari et al., 2011; Ninfali et al., 2007). The molecules X, G and E are structurally different, as they belong to distinct classes of phytochemicals: C-glycosyl-flavones (X), glucosinolates-ITC (G) and flavan-3-ols (E). Therefore, these molecules, when used in combinations, may exhibit complementary synergistic effects in their cytotoxic activity on cancer cell lines. To demonstrate synergy, it is necessary that the effect of the compound mixture is superior to the sum of the effects caused by each compound individually. If the effect of the mixture corresponds to the sum of the effects of the individual phytochemicals, the interaction is additive; whereas, it is antagonistic when the measured effect is less than the sum of the individual components. In this study, the three molecules X, G and E were evaluated, individually and in combination, with respect to their cytotoxicity on colon-rectal and breast carcinoma cell lines. We were particularly interested on identifying synergistic interactions, to support the hypothesis that the intake of specific food bioactive components may offer remarkable advantages for cancer prevention.
2. Materials and methods 2.1. Materials Plant source. Raphanus sativus L. var. major (Daikon) seeds, cultivar 0P38 (Brassicaceae) and Beet green seeds (Chenopodiaceae) were supplied by SUBA SEEDS COMPANY, Longiano (FO). Glucoraphasatin (GRH; MW: 435.5 Da) was isolated from 7-day-old freeze-dried R. sativus L. sprouts, by two sequential steps: ion exchange and size exclusion chromatography, as reported (Barillari et al., 2005). GRH purity was close to 95%, as estimated by HPLC analysis of the desul-
pho-derivative (ISO 9167-1 method). GRH (4.6 mg/ml) was dissolved in water and stored at 20 °C. The relative ITC, 4methylsulphanyl-3-butenyl isothiocyanate (G), was produced via myrosinase (b-thioglucoside glucohydrolase, EC.3.2.1.147) catalysed hydrolysis, performed by an ‘‘in situ’’ method (Nastruzzi et al., 1986) (Fig. 1). In every treatment, 5 lL of myrosinase (30 IU/ ml; 60 IU/mg protein) isolated from seeds of Sinapis alba L., was added to the cell culture medium, which contained increasing concentrations of GRH. One myrosinase unit was defined as the amount of enzyme able to hydrolyse 1 lmol sinigrin per min at pH 6.5 and 37 °C. Vitexin-2-O-xyloside (X) was extracted and purified from seeds of Beta vulgaris cicla by Sephadex LH-20 and Davisil C18 column chromatography, as reported (Gennari et al., 2011). X (MW: 564 Da) was identified by HPLC–MS, analysing the m/z of molecular ion and the m/z signal generated by the loss of xylose, in positive ESI mode. X (10 mg/ml) was dissolved in 60% ethanol and diluted for use in cell culture medium at the desired concentrations. (–)-Epigallocatechin-3-gallate (E) (MW: 458 Da) was purchased from Sigma Chem. Co, USA, dissolved in water at 10 mg/ml and diluted for use in cell culture medium at the desired concentrations. 2.2. Cell culture LoVo and Caco-2 colon cancer cell lines and MCF-7 and MDAMB-231 breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in RPMI 1640 1 (Lovo and CaCo-2), E-MEM (MCF-7) or D-MEM (MDA-MB-231) supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, 50 U/ml penicillin, 50 lg/ml streptomycin and grown at 37 °C in a humidified atmosphere with 5% CO2. Media and supplements were purchased from Sigma Chem. Co., USA. 2.3. Lymphocytes isolation Human lymphocytes were isolated from peripheral blood of a healthy donor and grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 50 g/ml streptomycin and 50 U/ ml penicillin and grown at 37 °C in a humidified atmosphere with 5% CO2.
Fig. 1. Molecular structures of compounds X, G and E. Compound 1, was purified from Rhapanus sativus L. var. major (Daikon) sprouts (Brassicaceae); compound 2 was released from 1 by myrosinase catalysed hydrolysis and indicated as G; compound 3, vitexin-2-O-xyloside (X), was prepared from Beta vulgaris cicla (Swiss chard) seeds (Chenopodiaceae); compound 4, ()-epigallocatechin-3-gallate (E), was purchased as pure compound.
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2.4. Cell viability assay
2.7. Annexin V/propidium iodide apoptosis detection assay
Cell viability was evaluated by the Sulphorhodamine (SRB) assay. The SRB assay is based on the ability of SRB dye to bind basic amino acid residues. The amount of dye incorporated by the cells indicates the number of living cells. Cells (104/well) were plated in 96-well plates in triplicate and treated with E, X and G for 24, 48 and 72 h. At the end of treatment, the cell culture medium was removed and fresh medium was added (50 lL/well). Cells were fixed using 25 lL/well of 50% aqueous trichloroacetic acid (TCA) for 1 h at 4 °C, rinsed with water several times and incubated for 30 min with 50 lL/well SRB solution (0.4%) (Sigma, MO, USA). After rinsing with 1% acetic acid and solubilising in 10 mM Tris for 5 min, the absorbance was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. The viability results were expressed as a percentage of living cells in treated vs control (untreated) wells.
The cells were treated for 24 h with different doses of X, G and E (40, 5 and 10 lg/ml, respectively) and analysed for Annexin-V-FITC positive staining by flow cytometry using Bender-Medsystem (Austria) apoptosis kit. Briefly, cells were treated by trypsin, centrifuged and stained with Annexin-V-FITC/Propidium Iodide (PI) and analysed using flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA). Unstained cells were classified as alive; cells only stained for Annexin V were ‘‘early apoptotic’’; cells positive for both Annexin V and PI were ‘‘late apoptotic’’; cells only stained for PI were classified as dead cells or debris. Results were expressed as % of cells in the indicated condition.
2.5. Synergy evaluation To identify specific combinations of phytochemicals that exhibit synergistic interactions, compounds were tested individually and then mixed in pairs and triplets in the cell culture medium and the cytotoxicity evaluated by the SRB assay. In total, 20 combinations were tested and the observed cell viability values were recorded. The measured cell viability (V%) was converted into % of growth inhibitory effect (GIE) by the calculation: % GIE = (100 V%) and this value was taken as an indication of the cytotoxic effect. The experimentally measured cytotoxicity ‘‘Measured Cytotoxicity’’ in each mixture was divided by the ‘‘Expected Cytotoxicity’’, which is the mathematical sum of the cytotoxicity derived from the individual phytochemicals. This mathematical approach belongs to the category of the summation of effects, where the molecules X, G and E behave as mutually nonexclusive effectors (Chou, 2006). If measured values were significantly higher than expected values, i.e. Measured/Expected Ratio > 1.0 (p < 0.05), and a synergistic effect was considered to have occurred in the mixture. Consequently, Measured/Expected Ratio < 1.0 (p < 0.05) indicated an antagonistic effect; Measured/Expected Ratio = 1.0 (p < 0.05) indicated an additive interaction (Fuhrman, Volkova, Rosenblat, & Aviram, 2000).
2.6. Reactive Oxygen Species (ROS) assay Generation of ROS in colon cancer cells was monitored by the conversion of 20 ,70 -dichlorodihydrofluorescein diacetate (DCFHDA) to highly fluorescent dichlorofluorescein (DCF). DCFH-DA is transformed by cellular esterase into the reduced non-fluorescent form DCFH, which, in the presence of ROS, is oxidised to highly fluorescent DCF. Cells were grown on coverslips (2 104/well) in 24-well plates and treated for 4 h with X, G and E individually at 40, 5 and 10 lg/ml, respectively, and in the mixture at the same concentrations. After washing with PBS, the cells were incubated at room temperature in the dark for 20 min with 10 lM DCF-DA (Sigma, MO, USA) dissolved in PBS. As a positive control, cells treated with 1 lM H2O2 were included in each experiment. After incubation, the cells were rinsed three times with PBS and mounted with a solution of Hoescht (10 lg/ml) and 1,4-diazabicyclooctane (DABCO). The samples were observed at a Nikon epifluorescence microscope. Cells showing a bright and intense fluorescence were counted as positive, whereas cells having no or low fluorescence were counted as negative. At least three fields for each sample were analysed and 100–200 cells for sample were counted. Results were expressed as % DCF fluorescent positive cells vs the control.
2.8. Cell cycle analysis Cells treated for 24 h with different doses of X, G and E (40, 5 and 10 lg/ml, respectively) were fixed with ice cold 70% ethanol in PBS, treated with 10 lg/ml RNase A, stained with 50 lg/ml PI, and analysed by flow cytometry for DNA synthesis and cell cycle status (FACSCalibur, Becton Dickinson, San Jose, CA, USA). 2.9. Apoptosis detection by DAPI staining Cells were grown on coverslips. After 24 h of treatment the cells were fixed with 4% paraformaldehyde and thereafter permeabilised with Triton X-100 (0.1% in PBS). Cells were stained with 2.5 lg/ml DNA dye, 40 ,6-diamidine-2-phenylindole (DAPI) in PBS, for 30 min at 20 °C and analysed by fluorescence microscopy. At 20-fold magnification cells with condensed or fragmented nuclei were counted on adjacent four fields of each coverslip for a total of 160–180 cells. The percentage of cells with chromatin condensation and nuclear fragmentation was determined in two samples for each treatment and compared with controls. 2.10. Immunocytochemistry of caspase-3 Cells were grown on coverslips and after treatment they were fixed with 4% paraformaldehyde and treated with Triton X-100 (0.1%) in PBS and saturated in PBS–BSA 4% for 30 min. Then, cells were incubated with anti-caspase-3-FITC-conjugated antibody for 1 h at room temperature. After washings, slides were mounted in glycerol-PBS 1 medium containing 30 mg/ml DABCO. Evaluation of antibody specificity was also carried out by omitting the primary antibody. Cells were observed using fluorescence microscope and the percentage of caspase-3 positive cells was calculated by counting at least 200 cells in different microscope fields. 2.11. Western blot To determine BAX, BCL2, caspase-9 and poly(ADP-ribose) polymerase (PARP-1) protein levels, the cells were plated and treated with X, G and E individually and in the mixture. After treatment, cells were detached and collected by centrifugation at 300g, and the pellets were suspended in lysis buffer (20 mM Tris–HCl, pH 7.5, 0.5 mM EDTA, 0.5% Triton X-100, 5 lM Na3VO4) and sonicated on ice, in the presence of protease inhibitors. Proteins (50 lg) from cell lysate were size fractioned in 12% SDS–polyacrylamide gel, and transferred to Hybond TM-C Extra membranes (GE Healthcare, UK) by standard protocols. After blocking, the following antibodies were used according to supplier recommended concentrations: anti-caspase-9 (Sigma, USA), anti-PARP-1 (S. Cruz, USA), anti-Bax (Applied Biosystem, USA), anti-Bcl2 (Sigma, USA). The primary antibodies were diluted 1:500 in TBS-5% milk and the respective peroxidase-conjugated secondary antibody was diluted 1:1000. The proteins were detected by luminol (GE Healthcare, UK). Bands
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were quantified by using densitometric images analysis software (Image Master VDS, Pharmacia Biotech, Sweden). Protein loading was controlled by actin (1:1000) (Sigma, USA) detection after stripping of membranes with stripping solution (Thermo Scientific, USA). Values were normalised against the actin control and statistically evaluated. 2.12. Statistical analysis Experiments were replicated twice with three samples analysed per replicate (n = 6). Results were expressed as mean ± SD of six obtained values. Statistic significance was assessed by ANOVA followed by Bonferroni’s multiple comparison test or two-tail Student’s t-test with standard deviation (SD), as appropriate, using PRISM 5.1 (Graph pad Software, La Jolla, CA, USA). The level for accepted statistical significance was p < 0.05.
3. Results Cytotoxic effects of the individual compounds X, G and E (Fig. 1) were first investigated on the four types of cancer cell lines and the number of living cells in culture was evaluated. The IC50 values of X, G and E on the four cell lines are shown in Table 1. All cancer cell lines were markedly sensitive to the cytotoxic effect of G, produced in situ via myrosinase reaction. This enzyme was shown to be ineffective when added to the cells in the absence of G (data not shown). The average IC50 of G in all cell lines was 26 ± 5 lg/ml. The CaCo-2 cancer cell line was markedly sensitive to E, but moderately sensitive to X; whereas LoVo cancer cell line was moderately sensitive to both E and X. On the contrary, MCF-7 and MDA-MB-231 were found to be relatively resistant to both X and E, as deduced by the elevated IC50 values (Table 1). Individual compounds X, G and E and 20 different combinations were tested on all cell lines at 24, 48, and 72 h of incubation. On both colon cancer cell lines, estimation of the cell viability showed that some pairs and triplets of the CP induced marked cytotoxicity at all incubation times (Supplementary Fig. 1 for CaCo-2; Supplementary Fig. 2 for LoVo). To identify specific combinations of X, G and E that exhibited synergistic interactions, we evaluated measured/expected cytotoxicity ratios. Fig. 2 shows these ratios of the Caco-2 (Fig. 2A) and LoVo (Fig. 2B) cancer cell lines. The histograms provide an immediate picture of the CP interaction type, with reference to the horizontal baseline corresponding to the ratio = 1, i.e. additive effect. For instance, in the CaCo-2 cell line (Fig. 2A), eight pairs and four triplets showed synergistic interactions, being all other interactions additive. In the LoVo cell line (Fig. 2B) all triplets gave synergy, whereas only four pairs, out of 12, showed synergistic effects. As no one combination of X, G and E was synergistically effective in MCF-7 and MDA-MB-231 breast cancer cell lines (data not shown), these cells were not considered as a suitable subjects
for further investigations on the cell cycle, which was only restricted to LoVo and CaCo-2 cells. On the basis of the results obtained, we pinpointed one combination of X, G and E eliciting synergistic cytotoxicity and we treated LoVo and CaCo-2 colon cancer cell lines, first individually and then with the mixture, in order to detect fluctuations of specific biomarkers of the cell cycle and apoptosis. Fig. 3 shows the effects of X, G and E on the cell cycle progression by the percentages of cells in the G0/G1, S and G2 phases in both colon cancer cell lines. In the CaCo-2 cell line (Fig. 3A), an increase in the G0/G1 population was induced in the presence of G individually, whereas X and E were not significantly effective. The combination of X, G and E increased the G0/G1 phase (p < 0.05) and markedly decreased the cells in both G2 (p < 0.01) and S phase (p < 0.05) (Fig. 3A). In the LoVo cell line (Fig. 3B), the mixture of X, G and E resulted in a decrease of the cell number in G2 and S phases, although G0/G1 phase was not significantly different from the control. Hence, the combined CP hampered the progression of the cycle towards the G2 phase in both colon cancer cells lines. These results suggested that a number of cells underwent apoptosis. We therefore evaluated the effect of X, G and E treatment on the extent of the apoptosis by fluorescence microscopy analysis of nuclear morphology after DAPI staining and by flow cytometry, after Annexin V/PI co-staining. Fig. 4 shows the results obtained by means of the two techniques. The DAPI staining provided evidence of a statistically significant increase in the number of apoptotic nuclei in both CaCo-2 (Fig. 4A) and LoVo (Fig. 4B) cell lines after the CP mixture treatment. Notably, the resulting effect of the mixture was greater than that provided by the individual compounds. We also compared the effect of X, G and E treatment on normal human lymphocytes from peripheral blood. Lymphocytes, stained by DAPI, did not show any significant increase in the number of apoptotic nuclei after exposure to the CP, either individually or in the mixture (Fig. 4C). Apoptosis investigation by Annexin V/PI staining showed that in the CaCo-2 cell line (Fig. 4D), the percentage of apoptotic Annexin V/PI positive cells increased three folds and the percentage of necrotic PI positive cells increases two folds after the X, G and E combined treatment vs control. In the LoVo cancer cell lines (Fig. 4E), late apoptotic Annexin V/PI positive and PI positive necrotic cells increased after mixture treatment and the difference vs control or G and X individual agent treatments was significant (Fig. 4E). In order to further investigate the occurrence of apoptosis, as well as to obtain an insight in the apoptotic pathways involved, we evaluated the effect of X, G and E treatment on Bax, Bcl2, PARP-1, caspase-3 and caspase-9 protein expression. The rationale for the choice of these proteins can be briefly summarised as follows. Apoptosis/survival is regulated by the relative balance of pro-apoptotic BAX and anti-apoptotic BCL family members and their heterodimers. PARP is the enzyme which contributes to the DNA repair after oxidative breaks. This enzyme is cleaved during
Table 1 Values of IC50 for vitexin-2-O-xyloside, 4-methylsulphanyl-3-butenyl isothiocyanate and ()-epigallocatechin-3-gallate in human breast and colon cancer cell lines after 72 h incubation. Cancer cell lines
CaCo-2 LoVo MCF-7 MDA-MB-231
IC50 (lg/ml) Vitexin-2-O-xyloside (X)
4-Methylsulphanyl-3-butenyl ITC (G)
()-Epigallocatechin-3-gallate (E)
120 ± 9 158 ± 13 350 ± 48 1200 ± 66
16 ± 4 36 ± 5 31 ± 4 21 ± 6
21 ± 3 135 ± 16 350 ± 47 1980 ± 94
The data represent the average median ± SD of two replicate experiments with three samples analysed per replicate (n = 6). For the dose–response curves five concentrations of each compound were used: X (30, 50, 80, 100, 120 lg/ml), G (5, 10, 15, 30, 50 lg/ml) and E (10, 20, 30, 40, 50 lg/ml). Colon cancer cell lines: CaCo-2 and LoVo. Breast cancer cell lines: MCF-7 and MDA-MB-231.
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Fig. 2. Synergistic combinations of X, G and E in CaCo-2 (A) and LoVo (B) colon cancer cell lines. Values are shown as measured/expected cytotoxicity ratios after 72 h incubation using the following concentrations: X = 20 lg/ml (35 lM); 2X = 40 lg/ml (70 lM); G = 5 lg/ml (11 lM); 3G = 15 lg/ml (33 lM); E = 10 lg/ml (22 lM); 2E = 20 lg/ml (44 lM). Data are expressed as mean ± SD of two replicate experiments with three samples analysed per replicate (n = 6). Statistically significant differences from the ratio = 1 (additive effect) are indicated with: a, p < 0.05; b, p < 0.01; c, p < 0.001; n.s., not significant.
Fig. 3. Effects of X, G and E on cycle progression and chromatin condensation. Flow cytometry analysis of X, G and E on cell cycle progression of CaCo-2 (A) and LoVo (B) colon cancer cell lines after 24 h exposure to X (40 lg/ml), G (5 lg/ml) and E (10 lg/ml) and their mixture at the same concentrations. Histograms are representative of the percentage of cells in G0/G1, S and G2 phases after treatment with the phytochemicals, compared with controls (CTR), 100% dotted line. Data are presented as the mean ± SD of two replicate experiments with three samples analysed per replicate. ANOVA; n = 6. Statistically significant differences from the CTR are indicated with a, p < 0.05; b, p < 0.01; n.s., not significant.
apoptosis into two fragments, one of which is a fragment of 89 kDa, called PARP-1. Detection of PARP-1 is widely used in apoptotic cells as it is an excellent marker of the activation of caspase-3. Caspase3 is activated by caspase-9, following a change of mitochondria permeability, release of cytochrome c and formation of the apoptosome. Fig. 5 shows the results of this molecular analysis for CaCo-2 (A, left column) and LoVo (B, right column). In the CaCo-2 cell line, the combination of X, G and E caused significant up-regulation of Bax protein expression (p < 0.05), down-regulation of Bcl2 protein (p < 0.05), significant increase of PARP-1 (p < 0.05) and caspase-9 (p < 0.05) expression (Fig. 5A, left column). The LoVo cancer cell line, exposed to the mixture of X, G and E, significantly up-regulated Bax expression, down-regulated expression of Bcl2, significantly increased activated caspase-9 and PARP-1 expression (Fig. 5B, right column). The mixture X, G and E was always found
to be effective in modifying the expression of the above proteins, although the same does not hold true for the individual compounds. Furthermore, an increased expression of cleaved caspase-3 in both cancer cell lines (Fig. 5C and D) treated with the individual CP (p < 0.05), was found; this expression further increased after the mixture treatment (p < 0.01) with respect to the individual agents. Overall, the data of Fig. 5 demonstrate that, in both colon cancer cell lines, the X, G and E mixture provided a marked increase in biomarkers of the mitochondrial apoptotic pathways, although not all the agents were individually able to exert such effects. We hypothesised that ROS generation might be the primary event able to trigger the underlying mitochondrial apoptotic mechanism. Fig. 6 shows the level of ROS, generated by exposure to X, G and E individually as well as to the mixture, in both colon carcinoma cell lines and in normal human lymphocytes. X, G and E,
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Fig. 4. Effects of X, G and E on lymphocytes, CaCo-2 and LoVo colon cancer cell lines for evaluating apoptosis by DAPI staining and flow cytometry. For the DAPI staining, CaCo-2 (A), LoVo (B) colon cancer cell lines and human resting lymphocytes (C) were treated 24 h with X (40 lg/ml), G (5 lg/ml) and E (10 lg/ml) and their mixture at the same concentrations were then fixed and stained with DAPI. Nuclear morphology was analysed and cells showing fragmented nuclei were counted as apoptotic and values were expressed as % apoptosis. For flow cytometry analysis, CaCo-2 (D) and LoVo (E) cancer cells were treated as above. Data were expressed as % cells in the indicated cell phase (mean ± SD). Two replicate experiments with three samples analysed each replicate were performed in both techniques. Significant statistical differences among treated samples and control were assessed: ANOVA, n = 6; a, p < 0.05; b, p < 0.01; n.s., not significant.
neither individually nor in the mixture, were able to significantly increase the ROS generation in normal lymphocytes (Fig. 6A). In contrast, a statistically significant increase in ROS generation was found in both carcinoma cell lines, after treatment with the mixture. ROS increase was fourfold greater in treated than in untreated CaCo-2 cell line (Fig. 6B) and threefold in the LoVo cancer cell line (Fig. 6C). Individually, G only was able to provide a significant increase in ROS generation in both colon cancer cell lines (Fig. 6B and C). Fig. 7 summarises the possible sequential events able to account for the synergistic cytotoxicity of our CP mixture. 4. Discussion The present study investigates the synergistic cytotoxic effects of the food derived phytochemicals X, G and E in four cancer cell lines. Our results showed that G, individually, was the most effective cytotoxic compound in all cancer cell lines. To the cytotoxic effect of G certainly contributed to the fact that this isothiocyanate was stoichiometrically released from the glucoraphasatin, by myrosinase activity, and added to the cell culture medium (Abdull Razis, De Nicola, Pagnotta, Iori, & Ioannides, 2012). G is a potent
inducer of rat hepatic phase II enzymes and a potential chemopreventive agent. Twenty different combinations of X, G and E, in couples and triplets, were tested for cytotoxicity on breast cancer cell lines, which were quite resistant and none of the mixtures showed synergistic cytotoxic effect. On the contrary, against LoVo and CaCo-2 colon cancer cells, X, G and E interacted synergistically in many pairs and triplets, thus generating powerful cytotoxic mixtures. The different responsiveness to X, G and E combinations, between colon and breast cancer cell lines, depends on the genetic diversity of the two types of neoplastic cells. They can in fact metabolise the CP at a different rate, due to the expression of the detoxificating enzymes or the diverse efficacy of the multidrug resistance system in the exclusion of the CP (Fimognari, Turrini, Ferruzzi, Lenzi, & Hrelia, 2012; Hemalswarya & Doble, 2006). The CaCo-2 cell line was found to be more responsive to the effect of the CP mixtures than the LoVo cell line. In the first instance, this result can be explained by the fact that CaCo-2 cell line is representative of a more differentiated type of colon carcinoma, whereas LoVo cells derive from an advanced stage of colon carcinoma and represent a very aggressive type of neoplasm. Regarding to the concentrations of the CP, chosen for testing synergy, we can infer that the G and E concentrations, used on
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Fig. 5. Detection of apoptosis in X, G and E treated CaCo-2 and LoVo cell lines by Western blot analysis and immuno-cytochemistry. For Western blot analysis (A and B columns), cells were treated with: (1) control; (2) X (40 lg/ml); (3) G (5 lg/ml); (4) E (10 lg/ml) and (5) X + G + E at the same concentrations for 24 h. Bcl2, Bax, cleaved caspase-9 and PARP protein levels, were assessed in cell lysate of CaCo-2 (left column A) and LoVo (right column B) colon cancer cell lines. Densitometric data were expressed as percentage of protein expression (mean ± SD) in treated samples with respect to the control (1). Two replicate experiments with three samples analysed per replicate were performed. ANOVA, n = 6; a, p < 0.05; n.s., not significant. For immunocytochemistry of caspase-3 (C and D histograms), CaCo-2 (C) and LoVo (D) colon cancer cell lines were exposed to the phytochemicals individually and in the mixture, as above, fixed, then incubated with anti-cleaved caspase-3-FITC antibody, photographed and counted. Data are expressed as % cleaved caspase-3 activation (mean ± SD) in treated samples with respect to control (CTR). Two replicate experiments with three samples analysed per replicate were performed. Statistical significant differences among treated samples and control were assessed: ANOVA, n = 6; a, p < 0.05; b, p < 0.01; n.s., not significant.
Caco-2 and LoVo cells, were a little bit higher than the plasmatic concentrations observed during ‘‘in vivo’’ experiments (Boreddy, Pramanik, & Srivastava, 2011; Khan et al., 2008). Similar studies are lacking for X, as its anti-cancer bioactivity was only recently
discovered (Gennari et al., 2011). To detect synergism, we used only pure molecules in order to control the working concentrations and avoid effects due to unknown components, as it happens when whole extracts are used. The compound E was commercially
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Fig. 7. Putative molecular mechanism of the cytotoxic effect of X, G and E mixture. The phytochemicals X, G and E increase ROS production in cancer cells. ROS trigger the mitochondrial apoptosis pathway through caspase-9 activation, which induces cleaved caspase-3 and PARP activation.
Fig. 6. ROS detection in lymphocytes and colon cancer cell lines. Lymphocytes (A), CaCo-2 (B) and Lovo (C) cells were treated with X (40 lg/ml), G (5 lg/ml) and E (10 lg/ml) and their mixture at the same concentrations for 4 h and DCF-DA analysis was performed. Cells showing bright fluorescence were considered positive and counted. Results were expressed as % DCF fluorescent cells in treated samples and control (CTR). Data are mean % values ± SD of twice replicate experiments, with three samples analysed for each replicate. Statistical significant differences among treated samples and control were assessed: ANOVA, n = 6; a, p < 0.05; b, p < 0.01; n.s., not significant of treated samples vs CTR. ⁄Individual agent treatments significantly different with p < 0.05 vs the mixture (X + G + E).
available, but X and GRH, the precursor of G, were purified inhouse with lab-scale procedures and characterised by NMR and mass spectrometry analysis, as they are not yet commercially available. The ability to purify CP by chromatographic equipment is an important resource and lab-scale purification protocols may be easily scaled up to obtain consistent amounts of CP to be combined in mixtures. The molecular mechanism underlying the cytotoxic effect of the mixture of X, G and E was based on arrest of the cell cycle in the G1
phase and apoptosis, possibly originating from the mitochondrial pathway. The present results suggest that an activation of this pathway, caused by ROS, occurred in both CaCo-2 and LoVo cancer cell lines, as we observed remarkable ROS production in both colon cancer cells after treatment. On the contrary, the resting lymphocytes did not show any significant increase in ROS production and apoptosis. The sequential cytotoxic events due to ROS production were found to be linked to deregulation of the proteins of the BCL2 family, activation of caspase-9 and -3, cleavage of the repairing enzyme PARP and occurrence of apoptosis. An interesting aspect of this work is related to the dual behaviour of CP. How can a pro-oxidant action be generated in tumour cells by compounds considered to be strong antioxidants? These opposite effects may be related to the concentration of the agents utilised to treat the cells. In fact, several antioxidant CP follow a hormetic mechanism (Son, Camandola, & Mattson, 2008), in the sense that at low concentrations CP can effectively work as antioxidants but, at higher concentrations, the beneficial antioxidant effect is reversed and the CP become cytotoxic. Cytotoxicity derives from a rapid and dose dependent inhibition of the enzymes (phases I and II) involved in xenobiotic metabolism, that cause mitochondrial damage and consequently ROS production (Fimognari et al., 2012; Hemalswarya & Doble, 2006). In this report, cytotoxicity and ROS production occurred at the applied X, G and E concentrations in colon cancer cells, but not in normal lymphocytes. The explanation of this behaviour lies in the biology of the tumour cell, which is, in many aspects, similar to the stem cell (Andrews, Przyborski, & Thomson, 2001). The malignant cells up-regulate multiple signalling pathways to promote proliferation, inhibit apoptosis, to finally migrate and invade the tissues. This performance is supported energetically by high oxygen consumption and metabolic rate (Cairns, Harris, & Mak, 2011). In this context, interacting with enzymes involved in oxidative reactions, CP enlarge the production of ROS, deregulated several molecular targets, leading to a marked derangement in many cell signalling pathways, thus amplifying the consequent genotoxic cell damage (Forester & Lambert, 2011). In this study, we demonstrated that the compounds G and E were clearly able to trigger
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the ROS production in cells. A pro-oxidant effect of G was already demonstrated to occur from 5 to 10 lM in cancer (Valgimigli & Iori, 2009) and normal stem cells (Zanichelli et al., 2012). In the present study, G was used at 5 lg/ml, i.e. 11 lM, concentration and therefore it fell into the pro-oxidant range. E was found to arrest lung cancer cells growth in vitro by pro-oxidant activity (Li et al., 2010) and it was demonstrated to trigger apoptosis in various types of cancer cells both in vitro and in vivo, but not in normal cells (Forester & Lambert, 2011). X is a novel flavone-C-glycoside and its pro-oxidant activity has never been studied in detail, but its aglycone, apigenin, was found to be able to initiate cell death in prostate cancer cells by ROS generation (Shukla & Gupta, 2010). We cannot exclude that a few part of cytotoxic effects in colon cancer cells, that we observed, might be related to E dimers, superoxide radical and hydrogen peroxide that are formed in cell culture medium as a consequence of the high instability of E molecule. Some of the reported biological activities of E are in fact considered H2O2-dependent, as a consequence of radicals spontaneously generated in vitro by E auto-oxidation (Long, Hoi, & Halliwell, 2010; Yang et al., 2006). However, the moderate cytotoxicity we observed in colon cancer cells after individual E treatment do not support the hypothesis that this is the most important mechanism leading to cell death after mixture treatment. As far as the synergism among the CP is concerned, the effect could likely be due to the fact that the CP contemporaneously reach different targets of the same signalling pathway, controlling cell cycle and apoptosis, thus accelerating the overall process (D’Incalci et al., 2005). An increase of cell permeability of one CP mediated by the others, the so called ‘‘permissive role’’ or alternatively, the inhibition performed by one of the CP on the multi-drug resistance system, which reduced the rate of exclusion of the other two CP from the cells, may contribute to the synergism. In conclusion, our results show that X, G and E added in the mixture synergistically induced cytotoxicity mediated by apoptosis in colon cancer cells. A ROS oxidative insult was exhibited by the X, G and E mixture to a greater extent than after individual treatment. Either individually or in the mixture X, G and E have no cytotoxic effect in normal human lymphocytes. These findings provide the fundamentals for further in vivo validation of the combination of X, G and E as a beneficial CP mixture, potentially able to decrease cancer cell growth and to be better investigated in experimental animal models.
Conflict of interest None declared.
Acknowledgements This study was supported by Pallotti Legacy for Cancer Research, Cornelia Pallotti Fund for Cancer Research and RFO (Orientated Research Fund). The authors wish to thank SUBA SEEDS COMPANY, Longiano (FO) for providing Beta vulgaris cicla and Raphanus sativus seeds and Dr. Rebecca L. Morris and Dr. Whitney N. Ajie for their support during the preparation of the English manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2012. 11.112.
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