Antiproliferative Activity of Violaxanthin Isolated from Bioguided Fractionation of Dunaliella tertiolecta Extracts

Dunaliella tertiolecta (DT) was chemically investigated to isolate molecules inhibiting cancer cell proliferation and inducing apoptosis in vitro. The potency to inhibit cell growth was used for the bio-guided fractionation and isolation of active compounds using chromatographic techniques. The DT dichloromethane extract exhibited a strong anti-proliferative activity on MCF-7 and LNCaP cells, and was further fractionated and sub-fractionated by RP-HPLC. High resolution mass spectrometry and spectrophotometric analysis unequivocally identified violaxanthin as the most antiproliferative molecule present in DT DCM extract. Violaxanthin purified from DT induced MCF-7 dose-dependent growth inhibition in continuous and discontinuous treatments, at concentrations as low as 0.1 μg·mL−1 (0.17 μM). Phosphatidylserine exposure, typical of early apoptosis, was observed after 48 h treatment at 8 μg·mL−1 (13.3 μM) but no DNA fragmentation, characteristic of late apoptosis steps, could be detected even after 72 h treatment at 40 μg·mL−1 (66.7 μM). Taken together, our results demonstrate the strong antiproliferative activity of violaxanthin on one human mammary cancer cell line, and suggest that studying the pharmacology of violaxanthin and pharmacomodulated derivatives on cancer cells may allow potent antiproliferative drugs to be obtained.


Introduction
Despite significant progress in prevention, diagnosis, and development during the last 25 years, cancer still represents the second cause of mortality in developed countries, after cardiovascular diseases. Discovering new drugs that are more active, more selective, and less toxic, limiting deleterious side effects and tumor multidrug resistance, will obviously be a challenge for the 21st century. The isolation of potent anticancer molecules from the marine environment has generated interest in many groups to purify original compounds, understand their biological activity, and also identify the pharmacological targets of molecules previously known for their ecological function. Extensive screening of marine microalgae has led to the isolation and chemical determination of over 15,000 compounds, including fatty acids, sterols, phenolic compounds, terpenes, enzymes, polysaccharides, alkaloids, toxins and pigments [1]. The pharmacology of most of these molecules remains to be clearly established. Bio-guided fractionation of microalgae extracts, followed by studies on human cells, has demonstrated that many pigments, beyond their ecological function as light harvesting molecules, also act as potent bioactive compounds on cancer cells and may have great potential in the prevention and treatment of cancers [2]. In particular, carotenoids have received increasing attention because of the decreased incidence of cancers associated with their consumption in fruits and vegetables [3,4]. Marine microalgae contain up to 0.2% of carotenoids (w:w dry weight) and may thus be of high interest as functional food to prevent cancer, or as a source of pure carotenoids [5][6][7]. A large number of studies have demonstrated that purified carotenoids (β,β-carotene, β,α-carotene, lutein, zeaxanthin, lycopene, fucoxanthin, astaxanthin, neoxanthin) exert a direct antiproliferative activity on cancer cells grown in vitro and induce their apoptosis [8][9][10][11][12][13][14][15]. The molecular mechanisms ruling this cytotoxicity remain to be clearly established as a large variety of pharmacological effectors regulating cell proliferation, differentiation and apoptosis are affected by carotenoids.
As part of our ongoing activity dedicated to the research and pharmacomodulation of natural anticancer compounds, we screened extracts from various microalgae species, in order to purify and identify antiproliferative molecules. We report here the bioassay-guided isolation of violaxanthin as the major antiproliferative pigment in the dichloromethane extract of the Chlorophyceae Dunaliella tertiolecta. Violaxanthin exerted a potent antiproliferative activity on MCF-7 breast cancer cells, and induced biochemical changes typical of early apoptosis.

Antiproliferative Activity of Microalgae Extracts
For each extract, the concentration inhibiting 50% of cell growth (Growth Inhibition 50%; GI 50 ) was determined (Table 1). DCM and EtOH DT extracts inhibited MCF-7 growth with equivalent potency and at low concentrations (GI 50 ≈ 60 μg·mL −1 ). The DT DCM extract also inhibited LNCaP growth, with a GI 50 close to the value determined on MCF-7 (GI 50 = 60.9 μg·mL −1 ). No extract inhibited MDA-MB-231 growth. The DT DCM extract, active both on MCF-7 and LNCaP cells, was selected to purify antiproliferative molecules by fractionation.  Identification of the 11 major peaks present on the chromatogram was not performed at this step as we hypothesized that some fractions may not be active on cancer cells. The antiproliferative activity of each fraction was studied on the MCF-7 cell line as it was the most sensitive to the starting DCM extract, and grew faster than LNCaP. Table 2 presents the antiproliferative activity of the four DT DCM fractions and the four F1 sub-fractions on MCF-7.  Table 2). The GI 50 of F 2 , F 3 and F 4 were superior to 100 μg· mL −1 ( Table 2), indicating that they did not contain potent antiproliferative molecules. F 1.2 , F 1.3 and F 1.4 strongly inhibited MCF-7 growth, with GI 50 values of 20.5, 18.9 and 11.7 μg·mL −1 , respectively ( Table 2). The GI 50 values of these three sub-fractions were in the range of that of the F 1 fraction, and confirmed that the three sub-fractions contained active molecules ( Table 2). The GI 50 of F 1.1 was greater than 40 μg·mL −1 . Figure 2 presents the GI 50 (μg·mL −1 ) measured on MCF-7 with the starting DT DCM extract, the F 1 fraction and the F 1.4 subfraction.  The GI 50 decreased with purification steps, indicating that the antiproliferative activity measured in the initial crude extract was not due to a synergistic action between several molecules in the mixture.

Effect of the F 1.4 Sub-Fraction on MCF-7 Growth
The antiproliferative activity of the most active sub-fraction, F 1.4 , was assessed on MCF-7 continuously exposed for 72 h to increasing concentrations in the cell culture medium. F 1.4 inhibited MCF-7 growth at a concentration as low as 0.1 μg·mL −1 and in a dose-dependent manner from 0.1 to 40 μg·mL −1 (Figure 3). A concentration of 40 μg·mL −1 was necessary to observe a cytostatic activity on MCF-7 ( Figure 3). MCF-7 cells were also exposed for 72 h to various concentrations of F 1.4 in the cell culture medium, before changing the medium to a fresh control cell culture medium ( Figure 4). At all tested concentrations, the growth rate increased when the culture medium containing F 1.4 was replaced with fresh culture medium, demonstrating that F 1.4 exerted a strong antiproliferative effect, without however killing all cells, in the range of the pharmacological concentrations studied.

Characterization of the Antiproliferative Molecule Contained in F 1.4
Analytical RP-HPLC at 435 nm of F 1.4 demonstrated that 95% of this fraction corresponded to a single molecule M eluting at t = 17.326 min ( Figure 5). Iterative semi-preparative RP-HPLC allowed the collection of 0.910 mg of F 1.4 from 1500 mg freeze-dried DT cells, indicating that M represented 0.0576% (w:w) of the freeze-dried microalgae content, considering the extraction yield to be 100%. The absorption spectrum of M was characteristic of a carotenoid pigment and presented maximal absorption peaks at 417.2, 441.5 and 471.9 nm, with a band III:II ratio of 96% ( Figure 5). These values were compared to data from reference spectra [16] which suggested that M was most probably violaxanthin, as its maximal absorption wavelengths and band III:II ratio were very close to the values measured with standard violaxanthin in ethanol. HRMS analysis unambiguously confirmed that M corresponded to violaxanthin (Table 3; Figure 6C).    Only a small proportion of control cells ( Figure 7A) were identified as apoptotic (red arrows) or early necrotic (bright green spots). Violaxanthin 8 and 20 μg·mL −1 (Figures 7B and 7C, respectively) evoked MCF-7 apoptosis as indicated by the important increase in the number of cells binding annexin V without being labeled by BOBO-1. Only a few cells were identified as late necrotic after the violaxanthin treatment (green arrows).

Violaxanthin Does Not Evoke MCF-7 DNA Fragmentation
Violaxanthin doses evoking phosphatidylserine translocation in MCF-7 cells did not induce DNA fragmentation, even after a 72 h treatment ( Figure 8B). A higher dose of violaxanthin was tested, in the range of rational pharmacological doses (40 μg·mL −1 ) (66.4 µM), but no DNA fragmentation was observed even after 72 h incubation (Figure 8).

Microalgae Culture, Collection and Storage
Microalgae were grown at IFREMER PBA, Nantes, France. DT strain CCMP364 (CCMP, USA) was cultivated in 10 L flasks under continuous illumination at an average light intensity of 180 µmol· m −2 s −1 . Growth was performed at 20 °C , in pH unregulated batch culture, in Walne (Conway) medium diluted in 0.22 µm sterile-filtered natural seawater. The DT cell suspension was harvested at the end of the exponential growth phase, and cells were separated from culture medium by soft centrifugation (4000 g, 20 min, 10 °C ). Cells were frozen at −20 °C , sent to laboratory LIENSs, La Rochelle and freeze-dried at −55 °C and P < 1 hPa, on a freeze-dryer equipped with a HetoLyoPro 3000 condenser and Heto cooling trap (Therma Electron Corporation, France).

Successive Extractions in Dichloromethane, Ethanol and Water
In order to extract most microalgal organic molecules, on a wide range of polarity, successive extractions were performed in dichloromethane (DCM), ethanol (EtOH) and ultrapure water. A 1 g sample of freeze-dried microalgae powder was first extracted for 2 h in 100 mL DCM (1% w/v), at room temperature, under continuous shaking and in the dark. The mixture was filtered through a PVDF0.22 μm membrane, and the DCM extract was evaporated to dryness in dark vials (45 °C , vacuum). The insoluble residue was collected, dried and successively extracted in ethanol and ultrapure water, in the same conditions, except that the water extract was collected by centrifugation (8000 g, 10 min, 4 °C ) and filtered through a nitrocellulose membrane. The EtOH extract was evaporated to dryness in dark vials (45 °C, vacuum) and the aqueous extract was freeze-dried.

Cell Viability Assay
The algal extracts and related fractions were evaporated to dryness and stock solutions were prepared in DMSO before being diluted in the cell culture medium. The final DMSO concentration was lower than 1% and tested as a negative control. Cell viability was studied using the MTT assay.

Phosphatidylserines Translocation and DNA Staining
Ten thousand cells were grown for 24 h on epifluorescence live cell array slides (Nunc, Dutscher, France) and treated for 72 h with control culture medium or microalgae extracts diluted in cell culture medium. Phosphatidylserines translocation onto the outer side of the plasma membrane of early apoptotic cells was detected using Annexin-V-Alexa 568 fluorochrome (Roche, France). Since necrotic cells also expose phosphatidylserines because of the loss of membrane integrity, necrotic cells were distinguished from apoptotic cells by BOBO-1 labeling. BOBO-1 is a DNA-binding fluorochrome, excluded from living and apoptotic cells. Cells were incubated for 15 min at 20 °C with the labeling mix solution, and observed using a Leica epifluorescence microscope, equipped with an I3 epifluorescence filter block (blue excitation 450-490 nm) and a numeric camera. Early necrotic cells, having lost their cytoplasmic membrane integrity but with a round shaped nucleus, were labeled by BOBO-1 only and appeared as round green spots. Early apopotic cells, having exposed phosphatidylserines but with no cytoplasmic membrane damages, appeared as red spots. Late necrotic cells exhibited a red and green co-staining and a shrunk nucleus.

DNA Fragmentation
DNA was extracted and purified from cancer cells using a combination of two kits (Macherey-Nagel). In a first step, 10 6 MCF-7 cells were lysed at 70 °C for 30 min in a lysis buffer (112 µL buffer T1, 25 µL proteinase K and 112 µL buffer B3 from the "Genomic DNA from Tissue" Kit). Then, DNA was purified using 360 µL of BB buffer ("Circulating DNA from Plasma" kit), which allows the purification of high and low molecular weight DNA fragments. DNA samples (5 µg) from control cells, cells treated with violaxanthin or cell treated with a control apoptosis inducer, DIM (3,3′-Diindolylmetane), were separated by electrophoresis on a 1.5% agarose/Tris-borate-EDTA (TBE) gel, stained with ethidium bromide and compared to standards ranging from 100 to 3000 bp (Fermentas) using a UV transilluminator.

High Resolution Mass Spectrometry (HRMS)
Accurate molecular weight of the bioactive molecules contained in DT extracts was determined by HRMS at the "Centre Ré gional de Mesures Physiques de l'Ouest", University of Rennes 1, France. The mass spectrometer was a Bruker MicrO-Tof-Q 2, equipped with an ESI source, and samples were dissolved in CH 2 Cl 2 :CH 3 OH (90:10).

Chemicals and Standards
All solvents used in this study were HPLC grade. Standard pigments were obtained from Chromadex, France. Ultra-pure water was obtained using a Milli-Q system (Millipore, France).

Conclusions
Breast cancer is a major cause of mortality worldwide, and the development of new drugs is necessary to reduce mortality and limit tumor resistance to chemotherapy. This study unequivocally demonstrates the strong antiproliferative activity of violaxanthin on MCF-7 human mammary cancer cells grown in vitro and suggests that violaxanthin and derivatives obtained by pharmacomodulation should be studied as possible new drugs to cure breast cancer. Violaxanthin was already identified as possibly involved in the strong antiproliferative and pro-apoptotic activity of Chlorella ellipsoidea extracts on HCT116 human colon cancer cells [19]. Further studies should be undertaken to define the pharmacological mechanisms involved in its antiproliferative activity in human cancer cells. In this study, violaxanthin only represented 0.0576% (w:w) of the total freeze-dried DT content, with a theoretical extraction yield of 100%. Thus, it seems unrealistic to consider that violaxanthin could be purified from DT for therapeutic applications, even if physiological studies in Dunaliella or Chlorella may be carried out to increase production yields. Moreover, there is no clear evidence that epoxycarotenoids, despite their abundance in dietary fruits and vegetables, are absorbed per os by humans [20]. Recent studies indicate that epoxycarotenoids are metabolized before being absorbed per os in mice [21], suggesting that consumption of marine microalgae as functional food to obtain epoxycarotenoids for a putative cancer prevention or treatment is most probably less beneficial. It is, however, interesting to note that the oral administration of high doses of fucoxanthin in mice does not evoke acute or chronic toxicity, except the risk of hypercholesterolemia [22]. Additional studies will be necessary to get a clear understanding of epoxycarotenoids pharmacology and to consider their possible use to inhibit cancer cells growth in vivo.