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Antitumoural activity of viniferin-enriched extracts from Vitis vinifera L.cell culturesL. Giovannellia, M. Innocentib, A.R. Santamariac, E. Bigaglia, G.Pasquac & N. Mulinacciba Division Pharmacology and Toxicology, Department ofNEUROFARBA, V. le G. Pieraccini, 6, 50139 Florence, Italyb Division Pharmaceutical and Nutraceutical Sciences, Departmentof NEUROFARBA, CeRA (Multidisciplinary Centre of Research onFood Sciences), Via U. Schiff6, 50019 Sesto Fiorentino, Florence,Italyc Department of Environmental Biology, “Sapienza” University,Piazz. le Aldo Moro, 5, 00185 Rome, ItalyPublished online: 20 Jun 2014.

To cite this article: L. Giovannelli, M. Innocenti, A.R. Santamaria, E. Bigagli, G. Pasqua & N.Mulinacci (2014) Antitumoural activity of viniferin-enriched extracts from Vitis vinifera L. cellcultures, Natural Product Research: Formerly Natural Product Letters, 28:22, 2006-2016, DOI:10.1080/14786419.2014.924935

To link to this article: http://dx.doi.org/10.1080/14786419.2014.924935

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Antitumoural activity of viniferin-enriched extracts from Vitis vinifera L.cell cultures

L. Giovannellia, M. Innocentib, A.R. Santamariac, E. Bigaglia, G. Pasquac* and N. Mulinaccib

aDivision Pharmacology and Toxicology, Department of NEUROFARBA, V. le G. Pieraccini 6, 50139Florence, Italy; bDivision Pharmaceutical and Nutraceutical Sciences, Department of NEUROFARBA,CeRA (Multidisciplinary Centre of Research on Food Sciences), Via U. Schiff 6, 50019 Sesto Fiorentino,Florence, Italy; cDepartment of Environmental Biology, “Sapienza” University, Piazz. le Aldo Moro 5,00185 Rome, Italy

(Received 3 April 2014; final version received 13 May 2014)

The aim of this work was to evaluate the effect of stilbenes from different cultivars ofVitis vinifera on tumour proliferation. Extracts were obtained from elicited V. viniferacell cultures and characterised by HPLC/DAD/MS. Cell growth was evaluated in fourhuman cancer cell lines and in normal human fibroblasts. The cells were exposed to theextracts or to trans-resveratrol, used as reference molecule, for 48 h, at 1–10mMconcentrations of total stilbenoids. All the extracts exhibited antiproliferative activity,mediated by modulation of the cell cycle and induction of cytotoxicity in cancer but notin normal cell lines, and positively correlated with the content in dimeric stilbenoids.The Alphonse Lavallee extract was the most active, and the obtained stilbenoid fractionresulted 8–10 times more active than trans-resveratrol. Extracts from V. vinifera cellcultures could represent new sources of active stilbenoid compounds to be furtherassayed in in vivo studies for their antitumoural properties.

Keywords: stilbenes; viniferins; trans-resveratrol; breast cancer cell lines;hepatocellular carcinoma cells

1. Introduction

During the last 10 years much attention has been devoted to the potential beneficial effects on

human health of trans-resveratrol (3,40,5-triidrossistilbene), a phytoalexin produced during

defence reactions in stressed plants. Resveratrol attracted initial interest because of its

cardioprotective activity, and was later on shown to have chemopreventive properties first on

skin tumours and then on other types of tumour, such as breast, skin, gastric, colon, oesophageal,

prostate and pancreatic cancer, as well as leukaemia (Shukla & Singh 2011). The in vitro and

in vivo evidences and the initial clinical studies indicate a potential for a nutrapharmacological

use of trans-resveratrol in a variety of chronic diseases, ranging from diabetes and obesity to

chronic inflammatory diseases and cancer (Chachay et al. 2011).

Although trans-resveratrol is well absorbed, it is also rapidly metabolised upon oral

assumption in man (Wenzel & Somoza 2005), so that after 30min the plasma concentration of

free trans-resveratrol is virtually zero, whereas the serum half-life of total trans-resveratrol

metabolites is ,9.2 h (Walle et al. 2004). As a consequence of this very low bioavailability,

rather high doses (1–5 g) are currently used in clinical trials, whose long-term effects are not yet

defined. Thus, the search for analogues or mixtures of stilbenoids endowed with higher

bioavailability and/or potency appears of interest.

q 2014 Taylor & Francis

*Corresponding author. Email: gabriella.pasqua@uniroma1.it

Natural Product Research, 2014

Vol. 28, No. 22, 2006–2016, http://dx.doi.org/10.1080/14786419.2014.924935

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Vitis vinifera (Decendit et al. 2002) is the most relevant source of resveratrol in the human

diet, although wine contains modest amounts (few mg/L) of this phenol (Gambuti et al. 2004;

Vitrac et al. 2005). Along with trans-resveratrol, plants contain other stilbenoid compounds,

derived from resveratrol oxidation by endogenous peroxidases, such as resveratrol

dehydrodimers (1- and d-viniferin), trimers ((a-viniferin), tetramers (b-viniferin) and oligomers

(g-viniferin). Similar to trans-resveratrol, viniferins are produced by plants upon fungal

infections and other types of stress conditions (Pezet et al. 2003). They have been identified in

leaves and cell cultures from V. vinifera (Pezet et al. 2003), in grapes (Gonzalez-Barrio et al.

2006) and in some wines in amounts varying from 1 to a few mg/L (Baderschneider &

Winterhalter 2000; Vitrac et al. 2005).

Compared with the wide literature on trans-resveratrol biological effects, the studies on

viniferins and other resveratrol derivatives are scarce. However, it appears from the available

data that at least some of the biological activities of trans-resveratrol are also displayed by

other stilbenoids. Viniferins have exhibited antioxidant (Privat et al. 2002; Ha et al. 2009)

and hepatoprotective properties (Oshima et al. 1995). The mono-oxydrilated metabolite of

trans-resveratrol, piceatannol, has exhibited anti-tumour activity (Potter et al. 2002). 1-

Viniferin has been reported to be more potent than trans-resveratrol in inhibiting the

proliferation of multiple myeloma cells (Barjot et al. 2007), and human cytochrome P450

enzymes, particularly CYP1A1, CYP1B1 and CYP2B6 (Piver et al. 2003). Oligostilbenes

also exhibit anti-inflammatory properties (Ha et al. 2009; Choi et al. 2011). Moreover, some

studies suggest possible synergistic activities of resveratrol and its oligomeric derivatives in

reducing cancer cell growth and inflammation (Billard et al. 2002; Colin et al. 2008; Wang

et al. 2011).

Plant cell cultures allow a continuous and controlled production of active metabolites,

independent of season and climate conditions. Furthermore, in cell cultures the levels of target

molecules can be modulated by modifying exogenous (culture media composition, light,

temperature, precursor molecule availability) and/or endogenous factors (expression of specific

biosynthetic pathways genes) (Rao & Ravishankar 2002).

Recent studies on V. vinifera L. highlighted that cell cultures from the cvs Gamay Freaux and

Cabernet Sauvignon are able to produce glycosylated stilbenes after a suitable elicitation

treatment (Decendit et al. 2002; Larronde et al. 2005). trans- and cis-Resveratrol were isolated

from cell cultures prepared from the leaves of cv. Barbera. upon elicitation with methyl

jasmonate, sodium orthovanadate and jasmonic acid (Tassoni et al. 2005). In Red Globe and

Michele Palieri cvs subjected to elicitation with methyl jasmonate, an increased biosynthesis of

stilbenoids, mainly trans-piceid and e-viniferin, has also been shown, which was also cultivar-

dependent (Santamaria et al. 2010). The same research group has monitored stilbenoid

production in elicited cell cultures from Malvasia (MAL) and Italia (ITA) cvs, showing the

presence of significant amounts of stilbene monomers and dimers, with a prevalence of

viniferins (Mulinacci et al. 2010; Santamaria et al. 2011). Recently, it has been reported that

trans-resveratrol obtained from grapevine cultures (Monastrell cv.) inhibited the proliferation of

lymphocytic and monocytic leukaemia cell lines (Fernandez-Perez et al. 2012). Thus, V. vinifera

cell cultures appear to be a feasible system to produce trans-resveratrol and stilbenoid

derivatives such as viniferins.

The aim of this study was to use elicited cell cultures of V. vinifera to obtain extracts rich in

viniferins and to evaluate their in vitro effects on cancer cell growth. For this purpose, the

extracts were tested on three different human breast cancer cell lines and one human

hepatocellular carcinoma cell line. Pure trans-resveratrol was used as a positive control. The

toxicity on non-tumour cells was also evaluated using primary human fibroblasts.

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2. Results and discussion

2.1 Extract preparation and characterisation

Our objective was to prepare characterised extracts enriched in stilbenes, mainly viniferins,

applying the purification procedure summarised in Figure S1. The first step was performed on

Alphonse Lavallee (A. Lavallee) cv. cell cultures because of the higher biomass productivity.

The extractive mixture (ethanol/acidic water) in a 7:3 v/v ratio was first applied on elicited dried

cells, and the obtained hydroalcoholic extract was purified by a liquid/liquid extraction with

ethyl acetate. The stilbene content was subsequently increased applying this latter extract on a

C18 cartridge and removing the polar interferents by water wash. The final methanol eluate (AL-

1 extract) after the HPLC/DAD control was tested for antitumoural activity towards human

cancer cell lines. As this procedure was time consuming and not suitable for a possible scaling up

of the process, a modification was applied, substituting the ethanol/water extractive mixture with

ethanol only (Figure S1) in the first step. The elimination of water allowed to get less polar

interferents in the ethanol extracts, thus avoiding the need for further purification steps by solid-

phase extraction (SPE-cartridge). Finally, to increase the stilbene content in the final extracts, a

liquid/liquid extraction with ethylacetate was carried out as done earlier for the AL-1 sample

(Figure S1). This simplified method allowed to obtain in a shorter time the AL-2, MAL and ITA

extracts, with a stilbenoid content comparable to AL-1. The identification of the main stilbenoids

in the extracts was done with the help of mass spectra in negative ionisation mode according to a

previous work (Mulinacci et al. 2010). Based on the HPLC profiles (shown in Figure S2), the

stilbene content was evaluated in mg/g of dried extract (Table 1). As expected, the stilbenoid

component of the extracts consisted mainly of resveratrol, piceid, 1-viniferin and d-viniferin.The presence of high amounts of d-viniferin in these extracts is worth noticing, as a peculiar

aspect of samples derived from V. vinifera cell cultures elicited by methyl jasmonate

(Santamaria et al. 2011). The ITA extract displayed the highest content of total stilbenoids, due

to the considerable amount of viniferins, especially d-viniferin; the MAL extract was richer in

trans-resveratrol but the viniferins were anyway the main components. The AL-1 and AL-2

extracts, both from A. Lavallee cv., revealed similar HPLC profiles as expected, with a total

amount of stilbenes ranging from 12.12mg/g (AL-2) to 18.02mg/g (AL-1). In all the samples,

d-viniferin was the main dihydrodimer, but the ratio between 1 and d-viniferin and between

stilbene monomers and dimers was different among the samples. The peculiar composition can

be a key to partially explain the different antitumour potency exhibited by the extracts (see later).

The amount of total stilbenoids in each extract was calculated based on the molecular weight and

Table 1. Concentrations of the different stilbenes in the investigated extracts.

mg/g dried extract

Stilbenes AL-1 AL-2 ITA MAL

Piceid (mw 390) 1.29 0.89 2.63 3.75trans-Resveratrol (mw 228) 1.68 0.35 1.78 2.98Resv der. (mw 228) 0.891-Viniferin (mw 454) 4.91 4.53 7.28 5.38Viniferin der. (mw 454) 1.97 1.75 2.95 0.00d-viniferin (mw 454) 6.88 4.59 16.38 7.42Viniferin der. (mw 454) 0.41 1.08Total 17.14 12.12 31.03 21.50Extract (mmol/g) 0.041 0.028 0.073 0.057Ratio t-resv/total stilbenes 0.098 0.029 0.057 0.139Ratio dimers/monomers 4.77 8.76 6.04 1.82

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relative amount of the single compounds in each extract (see Table 1), and turned out to be 0.042

and 0.028mmol/g extract for AL-1 and AL-2, respectively, 0.073 for MAL and 0.057mmol/g

extract for ITA. No detectable amount of stilbenoid compounds was found in the AL-2 ‘blank’

extract, prepared from non-elicited cultures.

The first evidence on trans-resveratrol ability to block the three stages of carcinogenesis

(initiation, promotion and progression) was provided by the seminal work of Jang et al. (1997).

The mechanism of this activity is probably complex, related to the antioxidant action, the

protection from DNA damage, the ability to inhibit phase I and to stimulate phase II xenobiotic

metabolism, the ability to inhibit cell proliferation, to modify the cell cycle and to induce

apoptosis in tumour cells (Cucciolla et al. 2007; Chachay et al. 2011). Although no information

is available on their bioavailability and metabolic fate, the resveratrol dehydrodimers 1 and d-viniferins have the potential to share numerous biological effects attributed to trans-resveratrol.

Plant cell cultures can be elicited to produce significant amounts of stilbenoid compounds

including oligomeric resveratrol derivatives, and extracts rich in these compounds can be

prepared from these cultures. The advantages of using complex extracts compared with pure

compounds are the reduced cost of the preparation and the possibility of different and more

useful biological activities in the mixture compared with single compounds. Thus, the

four viniferin-rich extracts from V. vinifera cell cultures and the blank extract were

tested for antitumour activity on human cell lines, and their effects compared with that of pure

trans-resveratrol. The in vitro doses were expressed as mmol of total stilbenoids in the culture

medium.

2.2 Antiproliferative activity and cytotoxicity

Figure 1 shows the effect of trans-resveratrol and V. vinifera cell culture extracts on the

proliferation of the human breast cancer cell lines HCC1954, HCC1500 and MCF7. The latter

appears to be more resistant to trans-resveratrol, being inhibited about 30% with 100mM. The

other two cell lines were already significantly inhibited (about 60% inhibition) at 25 (HCC1500)

and 50mM (HCC1954). The AL-1 and AL-2 extracts were the most effective on HCC1500 and

HCC1954 cell lines, inducing an almost complete growth inhibition (280% or more) at 5mM(total stilbenoids) and being significantly effective (about 250% growth) at 1mM. In MCF7

cells, the AL-1 extract yielded a smaller antiproliferative effect than AL-2. The MAL and ITA

extracts were in general less effective, reaching from 20% (MCF7) to about 50% growth

inhibition (HCC1500 and HCC1954) at the highest concentration. The growth of non-tumoural,

normal fibroblast was not strongly affected by the extracts: the most potent, AL-1 and AL-2,

induced about 30% inhibition at 5mM concentration (Figure 1). The net effect of the stilbenoid

component was calculated for the AL-2 extract, using the corresponding control extract devoid

of stilbenoids (AL-2 blank, see Methods). To do this, the growth inhibitory effect of the AL-2

blank was subtracted from that of the AL-2 extract for MCF7 and HCC1500 breast cancer cell

lines and for hepatocellular carcinoma HEPG2 cells (Figure 2). It can be seen that the AL-2

blank (i.e. the non-stilbenoid component of the AL-2 extract) also exerted an inhibitory action on

tumour cell growth, which was stronger in breast cancer than in hepatocellular carcinoma cells.

The stilbenoid content (net effect) resulted to be responsible for about 50% of the total effect of

the extract in breast cancer cells and for about 75% in HEPG2 cells. In all the tested breast cancer

cell lines, the growth inhibition induced specifically by the stilbenoids contained in the AL-2

extract at 5mM was about 80%, whereas in HEPG2 cells it was about 50%. In order to clarify

whether the observed effects on cell growth were due to modulation of the cell cycle and/or

induction of cell death, we focused on the AL-2 extract and carried out the analysis of the effects

of this extract on the cell cycle of one line of breast cancer, HCC1954, and on HEPG2 cells.

Upon 48 h exposure to the concentration of 5mM total stilbenoids, an accumulation of cells in S

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phase was observed in HCC1954 (from 22.2% to 50.7%, p , 0.01) but not in HEPG2 cells (from

31.5% to 23.8%, non-significant). The percentage of HCC1954 cells in the G0/G1 phase was

reduced by this treatment (Figure S3, upper panel). The AL-2 blank extract induced a lesser but

significant S phase increase in HCC1954 cells. A significant increase in G2/M phase was also

found in HCC1954 cells. The analysis of cell death was performed measuring the release of LDH

in the medium, evaluated as the percentage of total LDH in the sample. The results of these

experiments show that AL-2 induced an increase in cell death both in HEPG2 (þ40%) and in

HCC1954 (about fourfold), and that the AL-2 blank extract was also able to induce cell death in

HCC1954, although to a lesser extent (þ78%), but not in HEPG2 cells (Figure S3, middle

panel). These data are in agreement with the less potent effect exerted both by AL-2 and AL-2

blank extract in HEPG2 as compared with breast cancer cell lines. None of the employed

extracts exerted toxic effects on normal non-cancer cells, as shown in Figure S3 (lower panel),

where the measured LDH release from MRC5 human fibroblasts upon 48-h treatment with the

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Figure 1. Antiproliferative effect of trans-resveratrol (upper panel) and four extracts from V. vinifera cellcultures (48 h exposure) on three breast cancer cell lines (HCC1954, MCF7 and HCC1500). Viabilitymeasured with the MTS method is expressed as % of viable cells compared with control cells (treated with0.4% DMSO) (mean ^ SE, n ¼ 4). For the extracts, the concentration refers to the total stilbene content.

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different extracts is shown. It can be observed that none of the extracts, including the blank and

trans-resveratrol, increased this release over the control level.

All the extracts exhibited antiproliferative activity on both breast and liver tumour cell lines.

It is important to notice that altogether the stilbenoids in the extract exhibited antiproliferative

activity in the micromolar concentration range, whereas trans-resveratrol was one order of

magnitude less active. Furthermore, the extracts from A. Lavallee cv., particularly AL-2, were

more active than those from MAL or ITA, even if both the total stilbene and the total viniferin

content of these latter samples was higher compared with AL-2. These results indicate that

the relative content of the different stilbenes can play a relevant role in modulating the

antiproliferative activity of the extracts. We found that the antiproliferative activity of

the extracts was negatively associated with the trans-resveratrol/total stilbenoids ratio, but

positively associated with the dimer/monomer ratio: the AL-2 extract, the most active, displayed

the highest dimer/monomer ratio (about 9) compared with the other samples (Table 1).

Considering these observations along with the fact that in our experimental conditions pure

trans-resveratrol was about 10 times less potent than the total stilbenoids in the extracts, the

existence of interactions between dimeric and monomeric stilbenoids can be hypothesised, with

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Figure 2. Antiproliferative effect (48 h exposure) of A. Lavallee extract 2 (AL-2) from V. vinifera culturesand of the corresponding blank extract (AL-2 blank), devoid of the stilbenoid component, in MCF7 andHCC1500 breast cancer cells (upper and lower left panel) and in hepatocellular carcinoma HEPG2 cells(upper right panel). Lower right panel: net effect of the stilbenoid compounds contained in AL-2. The neteffect was calculated for each cell line by subtracting from the dose–response curve for the whole extractthe corresponding curve obtained with AL-2 blank extract (see Methods). Viability measured with the MTSmethod is expressed as% of viable cells compared with control cells (treated with 0.4%DMSO) (mean^ SE,n ¼ 4). The concentration refers to the total stilbene content in each extract.

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the dimeric component possibly acting synergistically with the monomeric one. In agreement

with these observations, Colin et al. (2008) have found that a grapevine shoot extract (vineatrol),

containing mainly trans-resveratrol and 1-viniferin, exhibits higher antiproliferative activity

towards human hepatocellular carcinoma cells than the single compounds. Wang et al. (2011)

reported synergistic anti-inflammatory effects of trans-resveratrol and some oligostilbenoids,

among which 1-viniferin, in an in vivo model of lipopolysaccharide-induced arthritis.

To evaluate the possible role of other co-present compounds derived from the culture

medium and from the constitutive components of Vitis cultured cells, we tested the activity of an

AL-2 blank extract prepared from non-elicited cultures and thus containing undetectable amount

of stilbenoids. The specific (‘net’) stilbenoid effect of AL-2 was then calculated by subtracting

from the dose–response curves of the whole extract the curves obtained with the blank extract.

The results showed that the stilbenoid component was responsible for 50–75% of the observed

antiproliferative activity. We do not exclude that extract components other than the stilbenoids

might exert the observed effects. However, the use of specific elicitors that selectively stimulate

the stilbene biosynthesis, the analytical controls by the coupled DAD and MS detector to obtain

a widespread control on the other co-present molecules and the use of a blank derived from the

same A. Lavallee cell cultures confirmed that the stilbenoid fraction plays a major role in the

induced growth inhibition. Furthermore, it is important to notice that the extract doses that were

effective on tumour cell lines did not exert significant cytotoxic or cytostatic activity on normal

human fibroblasts.

Regarding the mechanisms involved in the antitumoural activity, in general single stilbenes,

such as trans-resveratrol, inhibit proliferation at lower doses and induce apoptosis at higher

doses (Fernandez-Perez et al. 2012). Vineatrol and resveratrol exhibited antiproliferative and

cytotoxic action in cultures of leukaemic cells, whereas 1-viniferin alone was only slightly

effective (Billard et al. 2002). These authors also showed that the tested compounds only slightly

affected the survival of normal peripheral blood mononuclear cells. In human colon cancer cell

lines, resveratrol- and vineatrol-induced accumulation of cells in early S phase of the cell cycle,

and again 1-viniferin did not demonstrate any significant activity when tested alone (Colin et al.

2009). Our data on cell cycle modulation indicate that the most active extract, AL-2, did induce a

strong accumulation in S phase, similar to that reported for resveratrol, but also a significant

increase in G2/M phase in breast cancer cells. Barjot et al. (2007) showed a G2/M phase

increase, along with caspase-mediated apoptosis induction, upon 1-viniferin exposure of

multiple myeloma cells. Thus, it is possible that the modifications of the cell cycle observed by

us are due to a combination of effects of the different stilbenoids in the extract. Changes in cell

cycle were accompanied by extensive cell death, again in agreement with previously reported

data on stilbenes (Fernandez-Perez et al. 2012). Moreover, the effect of the AL-2 extract was

cell-dependent, as breast cancer cells showed higher sensitivity than HEPG2 cells.

3. Experimental

3.1 Plant cell cultures of V. vinifera L

Cell cultures of MAL, ITA and A. Lavallee cultivars were obtained by culturing stem and tendril

explants excised from the plants. The explants were cut into 1 cm pieces and cultured in 1%

(w/v) agarised B5 medium (Gamborg et al. 1968) supplemented with naphthaleneacetic acid

0.2mgL21, kinetin 1mgL21, sucrose 20 g L21 and agar 9.8 g L21. The explants were

maintained in continuous dark at 26 ^ 18C. Subcultures of the callus were prepared every

20 days. To obtain extracts rich in stilbenoids, cell cultures were treated with methyl jasmonate

(25mM dissolved in 100% ethanol) at day 10 of culture, which corresponded to the midpoint of

the exponential growth phase. Instead, the not-elicited control cells, used to prepare the blank

extract, were obtained by adding to the medium ethanol without methyl jasmonate. Both elicited

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and control cells were harvested 6 days after the treatment by vacuum filtration, weighed and

stored at 2208C until extraction.

3.2 Extraction of the stilbenes from V. vinifera L. cell cultures

Preliminary procedure. The extraction of the stilbenoids has been carried out from lyophilised cell

cultures obtained from different V. vinifera cultivars. A hydro-alcoholic extraction with ethanol/

acidified H2O (pH 3.2 by HCOOH) 7:3 v/v and three successive liquid/liquid fractionations with

ethylacetate were applied to A. Lavallee cv. (Figure S1). Subsequently, the dried ethylacetate

fraction was re-suspended in water and applied on a C18 cartridge (Strata Phenomenex, Le Pecq

Cedex, France). After removing potentially interfering polar compounds by washing with water,

the compounds of interest were eluted with methanol. The extract obtained with this procedure

(AL-1) was subjected to HPLC/DAD/MS analysis and tested for antitumour activity.

Optimised extractive procedure. The first extraction from lyophilised cell cultures was done

using only ethanol, and followed by ethylacetate fractionation (Figure S1) to obtain three

extracts: AL-2 from A. Lavallee cv., MAL from MAL cv. and ITA from ITA cv. The same

procedure was also applied to A. Lavallee untreated cell cultures to obtain the blank extract

(AL-2-blank) which served as a blank control to be compared with AL-2 extract.

3.3 Analyses by HPLC/DAD/MS of the extracts from V. vinifera cell cultures

The analyses of the extracts fromV. vinifera cell cultureswere carried out, according to our previous

works (Mulinacci et al. 2010; Santamaria et al. 2011), using an HP 1100L liquid chromatograph

equippedwith aDADandmanagedbyanHP9000workstation (all fromAgilentTechnologies, Palo

Alto,CA,USA). The elutionmethodwas amulti-step linear solvent gradient, changing from20% to

44%of solvent Bwithin 25min, then to 100%ofB in 3min. Solvent Awas acidic H2O (pH 3.2with

HCOOH) and solvent B was CH3CN, both HPLC grade. Total time of analysis was 28min;

equilibration time 10min, flow rate 0.4mL min21. The columnwas a SynergymaxRP-12 150mm

£ 3mm i.d., 4mmmaintained at 278Cwith a pre-column containing the same phase (Phenomenex,

Castel Maggiore, Bologna, Italy).

3.4 Quantitative determination of stilbenes in the extracts

trans-Resveratrol, piceid and the other stilbenes were quantified by a five-point calibration curve

at 307 nm (r 2 ¼ 0.9999) using pure trans-resveratrol as external standard (Extrasynthese,

Genay, France) from 0 to 1.98mg. A multiplicative correction factor of 2 was applied to express

the concentration of the viniferins and other dimers of resveratrol.

3.5 Cell cultures and treatments

MCF7 (human breast cancer cell line), HEPG2 (human liver hepatocellular carcinoma cell

line) and MRC5 (normal human fibroblast cell line derived from foetal lung tissue) cells

were cultured in high-glucose DMEM (BioWhittaker, Lonza, Milan, Italy), supplemented with

10% foetal bovine serum (FCS, BioWhittaker, Lonza, Milan, Italy) and penicillin–streptomycin

(Sigma-Aldrich, Milan, Italy). HCC1500 and HCC1954 (human breast cancer cell lines) were

cultured in high-glucose RPMI-1640 medium (BioWhittaker, Lonza, Milan, Italy), with 10%

FCS and penicillin–streptomycin as mentioned earlier, with the addition of Na-pyruvate (1mM)

and HEPES buffer (10mM). trans-Resveratrol (Shaanxi Sciphar Biotechnology, Xi’an, China)

and the four extracts were dissolved in DMSO and then diluted to the final concentration in the

culture medium. DMSO concentration in the culture medium never exceeded 0.4%. The cultures

were treated for 48 h.

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3.6 Cell proliferation and death assay

Cell viability and proliferation under the various experimental conditions were evaluated with

the MTS spectro-photometric method (Cell Titer 96wNon-Radioactive Cell Proliferation Assay

Kit, Promega Italia, Milan, Italy). Cell viability was expressed as % of viable cells compared

with the control (0.4% DMSO for 48 h). Cell death was assessed measuring LDH release in the

culture medium and in the corresponding attached cells (Cytotoxicity Detection Kit-LDH,

Roche Italia, Monza, Italy). LDH release in the medium was expressed as % of total LDH per

each experimental point.

3.7 Cell cycle distribution analysis by flow cytometry

HEPG2 andHCC1954 cell lines were seeded in 12-well microtitre plates and treatedwith AL-2 and

AL-2 blank extract at a concentration corresponding to 5mM total stilbenoids in the AL-2 extract,

and analysed after 48 h incubation.After treatment, 1 £ 106 cells/mLwerefixedby addition of 2mL

of cold ethanol-phosphate buffered saline (PBS) (70–30%) for 30min at 48C. Prior to staining, cellswere centrifuged (250g at RT for 10min) and re-suspended in 800mL of PBS. Cells were then

treated with 100mL of RNase (1mg/mL) and 100mL propidium iodide (PI, 400mg/mL) (Sigma-

Aldrich) for 30min at 378C in the dark. The fluorescence of stained cells was analysed in a

Fluorescence Activated Cell Sorting flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA)

equippedwith a 5-Watt argon ion laser. Thefluorescence ofPI-stained nucleiwas excited at 488 nm,

and histograms of the number of cells versus linear integrated red fluorescence were recorded for

50,000 nuclei/sample. DNA histograms were analysed using theMultiCycle DNA content and cell

cycle analysis software (Phoenix Flow Systems, San Diego, CA, USA).

3.8 Statistical analysis

All data are given as the mean^ SE of a minimum of three experiments. Data were analysed by

ANOVA and p values #0.05 were considered statistically significant.

4. Conclusions

The present data show that the stilbenoids contained in the extracts from V. vinifera cell cultures

display a significant antiproliferative activity in vitro on cell lines from different human tumours,

and that this activity is mediated by modulation of the cell cycle and induction of cytotoxicity in

cancer but not in normal cell lines. This activity appears to be related to the relative amount of

stilbenoids in the extract, and in particular to a high dimer/monomer ratio, and is exerted at doses

that are much lower than those previously used with trans-resveratrol alone. Importantly, these

doses of extract are not toxic towards normal non-tumour cells. Finally, grapevine cell cultures

represent a reliable system to produce biologically active extracts.

Supplementary material

Supplementary material relating to this article is available online, alongside Figures S1–S3.

Acknowledgements

The authors are very grateful to Dr. Manuela Balzi of the Department of Biomedical Experimental andClinical Sciences, University of Florence, for helpful discussion.

Funding

This study was supported by the University of Florence. The authors thank the Ente Cassa di Risparmio diFirenze for supplying part of the instrumentation used for this research.

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