Brown Algae Himantothallus grandifolius ( Desmarestiales , Phaeophyceae ) Suppresses Proliferation and Promotes Apoptosis-Mediated Cell Death in Tumor Cells

Marine algae have been used as food since ancient times and today are consumed as a regular part of the diet. In this study, we hypothesized that H. grandifolius phlorotannins-enriched extract exerts cytotoxic selective effects against tumor cell lines promoting cell death trough apoptosis mechanisms. The aim of the present study is to characterize chemically and investigate the selective cytotoxic effect of the H. grandifolius extract on epithelial tumour cell lines (A375, A549, Hep-2, HeLa) compared to non-malignant cell line (Hek-293), and evaluate possible molecular mechanisms involved in the programmed cell death pathway. High-resolution directly-infusion mass spectrometry (HR-DIMS) analysis with electrospray ionization (ESI) was performed in positive and negative mode. Cytotoxicity was evaluated through colorimetric assay and morphological alteraCorresponding author.


Introduction
Marine Natural Products (MNP) offer a rich chemical content of bioactive molecules that have become an important source of pharmacologically active metabolites [1].The Antarctic Peninsula presents a wide variety of MNP, mainly marine algae.The western region of the Peninsula exhibits abundant macroalgae biomass, about 44% are endemic [2].It is possible to classify macroalgae into three groups according to predominant photosynthetic pigments, storage products, and cell wall components: Green algae, Red algae, and Brown algae [3].
Marine algae have been used as food since ancient times, and today are consumed as a regular part of the diet, especially in Asian countries [4]- [6].A derivate from the cyanobacteria group known as spirulina has been used as food taken its high amount of protein, among them the phycocyanin, and essential nutrients such as carotenoids, vitamins, and minerals [7].Lipid extract from these group of algae also were able to exert antiinflammatory properties [8].Macroalgae are currently used in the diet and may provide human beings with beneficial fatty acid once used as nutrient sources in food products [9].The brown algae (class Phaeophyceae) are applied in treatment of several disorders in traditional medicine.Sargassum spp., belonging to family Sargassaceae, is a kind of edible brown algae that has been used as food and medicine commonly found in tropical and sub-tropical countries [10] [11].
The brown macroalgae Himantothallus grandifolius (A & E Gepp) Zinova is an eukaryotic organism typically found in marine environment with geographical distribution in the Antarctic islands and subantarctic regions, being the prevalent species in regions such as the Potter Cove [12].A recent study found the percentage distribution of Antarctic algae, and within the class Phaeophyceae, H. grandifolius has shown increased colonization and is, therefore, classified as endemic [13].However, because it's geographical localization, which implies in uncommon sun irradiation and cold weather, it is expected to be found active compounds as secondary metabolites.Iken et al. (2007) determined significant amounts of phlorotannins in H. grandifolius, but few studies have investigated their possible biological activity to date [2].
In this study, we hypothesized that H. grandifolius phlorotannins-enriched extract exerts cytotoxic selective effects against tumor cell lines promoting cell death trough apoptosis mechanisms.Algae have been reported to generate beneficial effects on health, thus our findings can help to increase the knowledge in the human nutrition field and life sciences research.The aim of the present study is to characterize chemically and investigate the selective cytotoxic effect of the H. grandifolius extract on epithelial tumour cell lines (A375, A549, Hep-2, HeLa) compared to non-malignant cell line (Hek-293), and evaluate possible molecular mechanisms involved in the programmed cell death pathway.

Himantothallus grandifolius Extract (HgExt)
Himantothallus grandifolius was collected in Punta Plaza (62˚5'34.82"S× 58˚24'15.27"W).The air dried material was triturated to obtain a uniform sample.0.5 g of the sample was extracted with 15 mL of acetone: water (1:1) for 15 min assisted by an ultrasound bath at 55 Hz.The liquid phase was separated and evaporated under vacuum.The dry extract was dissolved in ethanol 20% and filter through a 0.2 µm.The extract was further diluted with cell culture medium.The final ethanol concentration used was 1% of total volume of the medium in all treatments, including the negative control group.

Chemical Analysis
The dry extract was dissolved in a solution of 50% (v/v) chromatographic grade acetonitrile (Tedia, Fairfield, OH, USA), 50% (v/v) deionized water and 0.1% formic acid.The solutions were infused directly individually or with HPLC (Shymadzu ® ) assistance into the ESI source by means of a syringe pump (Harvard Apparatus) at a flow rate of 10 μL•min −1 .ESI(+)-MS and tandem ESI(+)-MS/MS were acquired using a hybrid high-resolution and high accuracy (5 μL/L) microTOF-Q II (Q-TOF) mass spectrometer (Bruker Scientific ® ) under the following conditions: capillary and cone voltages were set to +3500 V and +40 V, respectively, with a de-solvation temperature of 100˚C.For ESI(+)-MS/MS, the energy for the collision induced dissociations (CID) was optimized for each component.Diagnostic ions in fraction were identified by combinations of isotopic ratio, exact mass and fragmentation pathway in its ESI(+)-MS/MS and ESI(-)-MS/MS.For data acquisition and processing, TOF control (Bruker Scientific) was used.The data were collected in the m/z range of 70 -1000 at the speed of two scans per second, providing the resolution of 50,000 (FWHM) at m/z 200.No important ions were observed below m/z 100 or above m/z 800, therefore ESI(+)-MS data is shown in the m/z 100 -800 range.

Cell Viability Assay
Cytotoxicity was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) method.Therefore cells were seeded in 96-well plates at a density of 2 × 10 4 cells/cm 2 .After 24 h incubation, the cells were treated with increasing concentrations of HgExt (100 -500 µg/mL) for 24, 48 and 72 h.Ethanol 1% was added to medium as a negative control.MTT solution (0.4 mg/mL) was added after treatment and incubated for 2 h, followed by dimethyl sulfoxide incubation.The optical density (OD) was measured at 570 nm.The cell viability rate (%) was calculated as follows: (OD of the treated group/OD control group) × 100.The results represent the averages of 3 independent experiments performed in triplicate.

Morphological Characterization and Detection of Cell Apoptosis
Cells were seeded into 24-well plates containing coverslips.After 24 h incubation, the cells were treated with HgExt extract for 48 h at IC 50 (µg/mL), and stained with Accustain ® Giemsa Stain (Sigma-Aldrich ® ) according standard giemsa procedure established by the supplier.The cell morphological changes of treated and nontreated cells (negative control) were observed and photographed under a light microscope at ×200 (Olympus BX43).After treatment, the cells were incubated with 2.5 µg/mL of Annexin V (abcam ® -ab14196) primary antibody diluted in biding buffer (Hepes 10 mM, NaCl 150 mM, KCl 5 mM, MgCl 2 1.8 mM, CaCl 2 pH = 7,4) for 30 minutes.They were washed once with binding buffer an then incubate with 2.5 µg/mL Dylight ® 488 (ab-cam ® -ab115637) secondary antibody.The cells in the late apoptosis or necrosis were staining with propidium iodate (PI) (Sigma-Aldrich ® -P4170).Briefly, they were fixed with formalin 4% for 15 minutes and then mounted with antifade mounting media.The apoptosis was analyzed by fluorescent microscopy at ×400 (Olympus BX43).The percentage of cells undergoing early (annexin V positive) or late (annexin V + PI positive) apoptosis was estimated by counting 200 cells or twenty fields.

Statistical Analysis
All the experiments were repeated at least three times and the results were expressed as mean ± standard deviation.The statistical differences were analyzed using one-way ANOVA followed by a Tukey post hoc.Values of p < 0.05 were considered significant.

Chemical Characterization of H. grandifolius Macroalgae Extract
Considering the sample complexity, it is necessary a robust and sensitive tool with good resolution to generate chemical information.To date, there are no complete studies about the chemical composition for Antarctic algae as H. grandifolius.Here, high-resolution directly-infusion mass spectrometry (HR-DIMS) analysis with electrospray ionization (ESI) was performed, which has been an important device to characterize and identify complex natural metabolites.The instrument accurate mass measurement gives the exact mass, the isotopic ratio and the of parent fragmentation pathway.The extract was tested in positive and negative mode.According to this information, the elemental composition was defined and is shown in Table 1.Since the widely accepted accuracy threshold for confirmation of elemental compositions was established as 5 ppm [18], we have considered compounds bellow this threshold, providing highly reliable identification of the target compounds.
In negative mode the results indicate presence of alcohols because these compounds suffer easy ionization.D-Mannitol, D-Glucitol, Galactitol (FW C 6 H 14 O 6 , m/z 182.0730), all previously isolated by marine algae, are the probable compounds presented here [19].In positive mode, several compounds have been identified in its molecular formula and are listed in Table 1.The full mass spectrum is shown in Figure 1(a), with representative compounds with peaks showing m/z between 370 and 430.Highlighted is the isotopic ratio, which was used for molecular formula definition.
On the other hand, important information can be obtained from fragmentation pathway.For example, the mass loses with m/z 177.0327 (394.0917-217.0590) and 32.9720 (264.0797-231.1077),demonstrates the presence of glycosides pattern and sulfur, respectively.In mode MS-2 the compound with m/z 426.0601 revealed the fragmentation with m/z 243.0467 (C 14 H 11 O 2 S diff.5.24 ppm), which corroborates this information.The occurrence of sulfated glycosides from Antarctic algae had already been reported by Berteau et al. [20].Important information was observed by the peaks with m/z 700 and 400 fragmentation, which can be defined as chlorogenic acid (C 16 H 18 O 9 , m/z 355.0977) in Figure 1(b).The presence of this compound in algae has been determined by Onofrejova et al. [21].Nevertheless, the structural elucidation of some compounds by its MS spectrum is not trivial and for complete deduction more studies are required.In this way, the isolation of the compounds and chemical structures information will be done by other spectroscopic methods.

Effect of H. grandifolius Extract on Cell Cytotoxicity
For cytotoxic analysis MTT assay was performed based on the conversion of MTT into formazan crystals by living cells.This assay is widely used for measuring cytotoxic potential of drugs on cell lines or primary patient cells.Figure 2 shows the cytotoxic effect of HgExt in the viability of A375 (A), A549 (B), HeLa (C) and Hep-2 (D) cells.For all tumor lines analyzed, cell viability was decreased in a concentration and time-dependent manner.H. grandifolius macroalgae extract was most effective on 72 h treatment compared to 24 h and 48 h.The half maximal inhibitory concentration (IC 50 ) obtained are listed in Table 2. HeLa cells were more susceptible to HgExt than other cells lines, followed by Hep2, A375 and A549 at all different incubation times of treatment.Non-tumor cell line Hek-293 was not sensitive to the extract, presenting 100% of survival at the highest HgExt concentration tested (500 µg/mL).The results presented in Figure 3 are related to 48 h treatment and demonstrated that all tumor cell lines responded to the treatment with HgExt in a similar way, suggesting a common extract mechanism of action.
Table 1.High-resolution directly-infusion mass spectrometry (HR-DIMS) analysis with electrospray ionization (ESI) was performed in positive and negative mode.Diagnostic ions in fraction were identified by combinations of isotopic ratio, exact mass and fragmentation pathway.The data were collected in the m/z range of 70 -1000, providing the resolution of 50,000 (FWHM) at m/z 200.No important ions were observed below m/z 100 or above m/z 800.Table 2. Experiments were repeated in triplicates and results were expressed as mean ± standard deviation.The statistical differences were analyzed using one-way ANOVA followed by a Tukey post hoc.Values of p < 0.05 were considered significant.Different superscript letters indicate significant differences between treatment time (hours) of in each cell line.Different superscript symbols indicate significant differences between cell lines for each time analyzed.

Effect of H. grandifolius Extract on the Morphology of Cells and Induction of Apoptosis
Detachment and shrinkage of cell structure could be observed in all lines tested.Morphologically changes of cells were more evident after 48 h of HgExt at IC 50 (µg/mL) compared to the negative control.Signaling to death at early stages of apoptosis was a major event in cell treated with HgExt compared to the untreated control group (Figure 4).Here, early apoptosis (Annexin V positive) presented significant higher expression compared to late apoptosis events (Annexin V and PI positive) for all cells investigated (Figures 4(a)-(d)).

Discussion
Himantothallus grandifolius extract obtained in this study present a novel and complex chemical characterization and important cytotoxic effect against tumor cell lines.To date this is the first report that investigates a crude extract obtained from brown algae showing an important biological activity.It is known that cancer is the second major cause of death worldwide, with increasing death rate every year [22].Thus, focusing on new drugs  of natural products that contribute to prevention strategies and treatment are very important [23].Many vitamins from seaweed are essential due to its biochemical functions and antioxidant activity and also as health benefits such as decreasing of blood pressure, prevention of cardiovascular diseases, and reducing the risk of cancer [24].
(a) ( Between 1998 and 2006, 592 marine natural products were investigated for its activity in pharmacology studies [1].Cytotoxic studies using total extract obtained from a variety of algae have demonstrated that brown algae are a potential source of compounds presenting biological activities on tumor cells [15] [16] [25].The results here presented with acetone extraction at different treatment times (24 h, 48 h, 72 h) showed IC 50 values ranging from 111.43 µg/mL to 528.03 µg/mL for the tumor cells lines analyzed.Other studies based on tumor activity of algae extracts on cancer cells reported similar IC 50 values.Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier (brown algae) methanol extract against HT-29 and Caco-2 colon adenocarcinoma cells lines IC 50 showed greater IC 50 (>1000 μg/mL) [25].A study with red algae Gracilaria tenuistipitata C.F. Chang & B.M. Xia using methanol extraction against Ca9-22 oral cancer cells presented IC 50 326 µg/mL [26], very close to the ones here reported.A green algae Ulva lactuca Linnaeus (as U. fasciata) extracted with ethanol showed 50% growth inhibition for HCT 116 colon cancer cells at concentration of 200 μg/ml [27].
The selective induction of cell death is very important in chemotherapy to decreases possible side effects [23].The HgExt showed no cytotoxic effects to Hek-293 cell line at all concentrations and incubation time tested.The differences of cytotoxicity from tumor and non-tumor lines are related to the ultrasound assisted extraction method, optimized to yield higher amounts of polyphenols (data not shown).Phlorotannins purified from others brown algae such as Ecklonia sp. has shown cytocompatible effects to human fetal lung fibroblasts cell line (MRC-5) [28] and macrophage-like form cell (THP-1 cell) [15].A recent report using dieckol, an isolated compound from Ecklonia stolonifera Okamura algae extracts, also presented more selective effects to Hek-293 compared to a tumor cell line Hep3B [29].
Here, the chlorogenic acid (CHL) was defined as a molecule found in the extract obtained from the algae H. grandifolius.CHL has been attributed to have chemopreventive properties, modulating several mechanisms involved in the carcinogenesis process.CHL was able to inhibit metalloproteinases MMP-9 [30], microsomal glucose-6-phosphate translocase of U-87 of brain tumor-derived glioma cells [31] and inhibit proliferation of A549 human cancer cells due to the inhibitory effects on TPA-or NF-B, Activator Protein-1, and MAPK signaling [32].Recently, the chlorogenic acid was shown to be able to inhibit tumor progression by changing gene expression involved in immune pathways [33].
The cytotoxic effects here observed also promoted changes in the morphology of cells and induction of cell death.Kerr & Wyllie (1972) were the first authors that described such morphological features [34].It is known that changes in cell morphology are related to physiological and pathological modifications by a variety of environmental stimuli.Apoptosis, autophagy and necrosis are the three main forms of cell death, easily distinguished by their morphological peculiarities [35].Giemsa staining analyzes cell morphology using a simple colorimetric technique that gives a good high-contrast polychromatic stain.The typical morphology of apoptosis could be observed by Giemsa staining in all tumor cell lines here investigated treated with HgExt (IC 50 for 48 h).Apoptosis is a programmed form of cell death observed in tissues during development and maintains tissue homeostasis through elimination of excessive or injured cells [35] [36].Recent studies have shown that several bioactive compounds obtained from algae induced tumor cell death by apoptotic mechanism [14] [16] [37].However, our study is pioneer in demonstrating these morphological changes through in situ analysis induced by the extract obtained from H. grandifolius.
The exposure of phosphatidylserine (PS) at the outer leaflet of the plasma membrane serves a physiological role in the recognition and subsequent removal of the dying cell by means of phagocytosis [38].PS exposure during apoptosis seems to be a phenomenon during programmed cell death, not limited to mammalian cells, but also occurring in insect and plant cells [39].The annexin V distribution observed in the tumor cells here investigated revealed that apoptosis was induced in a significant manner in all cell lines treated with the HgExt in comparison with the control group.The early apoptosis (Annexin V positive) event was significantly expressed compared to late apoptosis/necrosis (Annexin V + PI positive).Similar studies using apoptotic parameters revealed apoptotic mechanisms with total methanolic extract of red algae Gracilaria tenuistipitata [26], and isolated compounds like sargaquinoic acid from the brown algae Sargassum incisifolium (Turner) C. Agardh (as Sargassum heterophyllum) [40], and low-molecular-weight fucoidan obtained from Cladosiphon navae-caledoniae Kylin [16] [41].It is known that tumor resistance to apoptotic cell death is a hallmark of cancer and that election of drugs which promote and induce apoptosis are desired.Many therapeutic agents for cancer, such as cisplatin, paclitaxel, isothiocyanate and adriamycin, have been reported to eliminate tumor cells by inducing apoptotic cell death [42] [43].

Conclusion
In conclusion, MNP have been only recently exploited and investigated as molecules that present promising antitumor activities.Algae have been reported to generate beneficial effects on health, thus we believe that our findings can help to increase the knowledge in the human nutrition field and life sciences research.It is essential to identify bioactive molecules in different extract, isolating compounds and determining whether the cytotoxic activity enhances with the isolated molecules or if it is a synergistic activity between compounds.Here, selective antitumor activity of extract taken from the alga H. grandifolius was able to suppress proliferation and promote apoptosis-mediated cell death with induction of initial stages of apoptosis in different cell lines.Given the chemical complexity of the extract and the lack of material found in the literature for this sort of brown macroalgae, more studies are necessary for better understanding what molecules are bioactive and which pathways are involved in programmed cell death.

Figure 1 .
Figure 1.Spectra from dry extract dissolved in chromatographic grade solution.The solutions were infused directly, individually or with HPLC assistance into the ESI source by means of a syringe pump.A-Full mass spectra in positive mode, several compounds have been identified in its molecular formula with representative compounds with peaks showing m/z between 370 and 430.Highlighted is the isotopic ratio, which was used for molecular formula definition.B-Peaks with m/z 700 and 400 fragmentation can be defined as chlorogenic acid (C 16 H 18 O 9 , m/z 355.0977).

Figure 3 .
Figure 3. Cells treated for 48 h with negative control and HgExt at IC 50 (µg/mL).Living (none fixed) cells are represented at first lines and stained fixed cell with Giemsa at second lines.A: A375 cell line.B: A549 cell line.C: HeLa cell line.D: Hep-2 cell line.First columns are control groups and second columns are represented as treated groups.Photomicrographs were taken under light microscopy (200×).

Figure 4 .
Figure 4. Percent of cells immunostained in early (Annexin V positive) or late apoptosis (Annexin V positive + PI positive) events.Results were obtained from three independent experiments expressed in median ± SD.A: A375 cell line.B: A549 cell line.C: HeLa cell line.D: Hep-2 cell line.