The marine environment has been recognized as a rich source of bioactive metabolites with various biological and pharmacological activities. The chemical complexity and biological diversity of the marine-derived compounds is enormous, so that bioprospecting of marine organisms represents today a major tool for the discovery of new therapeutic agents and drug candidates [1
Cnidarians polyps and jellyfish have increasingly become an attractive source of physiologically active compounds. Extracts from different species were reported to exert hemolytic [9
], insecticidal [10
], cardiovascular [11
], antioxidant [12
], anti-microbial [13
], and cytotoxic [9
] effects. Strong fibrinogenolytic factors have been recently found in the moon jellyfish Aurelia aurita
tentacle extract [14
]. Partially purified venom from the mauve stinger jellyfish Pelagia noctiluca
displayed potent anti-tumoral properties against U87 cells [15
]. Collagen hydrolysate from edible jellyfish Rhopilema esculentum
exerted antioxidant and protective effects on mice skin subjected to photo aging induced by UV irradiation [16
]. Collagen from giant jellyfish Nemopilema nomurai
has been shown to exert immunostimulatory effect on hybridoma cell line HB4C5 and human peripheral blood lymphocytes [17
Marine jellyfish are currently recognized as subject to worldwide proliferations in coastal areas, becoming a crucial ecological and societal issue in recent decades [19
]. Occurrences of jellyfish outbreaks increasingly interfere with human economic and recreational activities, such as bathing, fishery, tourism, etc.
, as well as the public health [20
] and coincide with human proliferations and man-induced environmental perturbations [21
]. Jellyfish have been reported to clog fishing nets, spoil commercial catches, cause serious damage to aquaculture, clog the cooling systems of coastal power plants, and sting or even kill tourist swimmers [22
]. Conversely, under an opposite perspective, the large amount of jellyfish biomass could be considered as a valuable source of bioactive compounds including bioactive peptides, collagen and gelatin, oligosaccharides, fatty acids, enzymes, calcium, water-soluble minerals, and biopolymers. The identified various biological activities, including antioxidant activity, make them a potentially valuable material for food, cosmetic, and biomedical industries, such as has been proposed for seafood processing by-products [30
The scyphozoan Cotylorhiza tuberculata
(Macrì, 1778) [31
], the most common rhizostome jellyfish in the Mediterranean Sea, is subject to summer population outbreaks. High jellyfish abundances can be found in enclosed bays such as Vlyho Bay in the Ionian Island of Lefkada-Greece [32
] and in coastal lagoons, such as the Mar Menor in the western Mediterranean Sea where annual blooms have been observed since 1995 [33
]. To protect human leisure activities from high C. tuberculata
abundance in Mar Menor, local maritime authorities now constantly implement management plans by use of fishing vessels for removal of large jellyfish biomasses or by use of protective coastal nets to create safe bathing areas [34
]. In 2002 and 2003, approximately five thousands tons of C. tuberculata
were yearly removed during summers months [35
An important biological aspect, linked to the success of C. tuberculata
populations, is the occurrence of endosymbiotic, unicellular dinoflagellates, usually referred as zooxanthellae. As for several other rhizostome jellyfish species and other cnidarians [32
], C. tuberculata
hosts in its endodermal tissue a dense population of the dinoflagellate Symbiodinium
]. The endosymbiotic association occurs very early in the life cycle of C. tuberculata
] and the occurrence of zooxanthellae in the polyp stage is required to activate the process of medusa formation (strobilation) of C. tuberculata
]. Apparently, the importance of autotrophy provided by zooxanthellae seems to be lower than heterotrophic uptakes for jellyfish growth and survival [39
]. Nevertheless, we observed that unfed polyps maintained for two years under natural daylight conditions and laboratory room temperature in closed aquarium system, did not show any sign of ageing or reduction in size. The molecular regulation and maintenance of the symbiotic relationships between the microalgae and their animal hosts is still largely unknown, but algal-derived metabolites, resulting from photosynthetic framework and secondary metabolism play crucial roles in homeostatic mechanisms [41
]. In Symbiodinium
dinoflagellates, the majority of the photosynthetic pigments are associated to light-harvesting pigment-protein complexes [43
]. These photosynthetic pigments are believed to be central to photoprotection in jellyfish-associated Symbiodinium
, by stabilization of the chloroplast thylakoids, and vital to the prevention or quenching of ROS in the host [45
]. Another key area of metabolic activity within the symbiosis is the free fatty acid synthesis and translocation within the dinoflagellate symbionts and their cnidarian host. Indeed, lipids are the main energy stores in cnidarians and the primary products derived from photosynthetically fixed carbon and translocated from the dinoflagellate symbionts to the host [47
]. Total lipids from the symbiosis (triacylglycerols, wax esters, phospholipids and free fatty acids), account for 10%–46% of the cnidarian tissue dry weight [48
]. Production and accumulation of long-chain, highly unsaturated fatty acids (HUFA), n
-3 and n
-6, such as DHA [22:6 (n
-6)], is well documented [49
]. Beyond the significance of essential FAs in symbiosis regulation, the importance of n
-3 and n
-6 derived essential FAs, such as DHA, to supplement animal diets is well known [51
]. Essential fatty acids (EFA) are PUFAs involved in biological processes, but not synthesized by animal cells. These fatty acids represent a necessary component of animal diets, since they are used as starting points for building longer chains of fatty acids and their deficiency may lead to severe damage to the organism. These FAs are produced by the symbiotic microalgae and, in some cases, in large amounts [53
]. Therefore, the relationship with the symbiotic dinoflagellate S. microadriaticum
makes the jellyfish C. tuberculata
a heterogeneous and complex biomass consortium rich in diverse and potentially bioactive compounds.
The chemical, biological and ecological diversity of marine metabolites has largely contributed to the discovery of potent compounds with strong antitumor activities [54
]. However, the structural diversity of the marine compounds, from simple linear peptides to complex macrocyclic polyethers, represents one of the first difficulties in new drug discovery from marine natural products. Recent advances in sophisticated technologies for the isolation and characterization of marine natural products and the development of high-throughput screening methods have substantially increased the rate of discovery of various compounds of biomedical application. High-throughput screening, combinatorial chemistry and, most recently, in silico
virtual screening techniques partly supported successful attempts to identify new drug candidates. However, the classical approach to recognize bioactive compounds through analysis of their specific biologic activity remains highly effective, especially when associated to the above-mentioned technological approaches.
One of the mechanisms underlying the multistage carcinogenesis process is the homeostatic regulation disorder related to abnormal gap junction intercellular communications (GJIC).
GJ are communicating junctions that are present in most cells in animal tissue. The GJ channels are formed by four-pass transmembrane proteins sharing a similar function and overall structure but encoded from different gene families, pannexins and connexins in chordates, and pannexins and innexins in invertebrates. The molecular architecture of GJIC consists of a couple of hexameric structures in the membrane of two adjacent cells, docking each other and forming a channel with a central pore, which allows the diffusion of metabolites, ions, small signaling molecules, and mediate electrical synapses [55
]. Gap junctions are grouped in plaques at the cell plasma membrane surface allowing molecules smaller than about 1–2 kDa to pass directly from the cytoplasm of one cell to the cytoplasm of another adjacent cell. Traditionally, the function of GJ proteins was associated to the formation of membrane channels; however, recent studies revealed additional functions, including sensing of the extracellular environment, cell-cell adhesion, facilitation of cell migration, and modulation of endocrine, pain, signal transduction and apoptotic pathways [58
The mechanisms controlling GJ functionality are strictly regulated [60
] at multiple levels, ranging from gene transcription to gap junction trafficking and degradation [59
]. Connexin phosphorylation is involved both in changing single channel conductance and in protein trafficking to the cell surface and degradation. The regulation of GJIC by connexin phosphorylation is quite complex, as the outcome of this post translational modification is both connexin- and kinase specific [61
GJIC has been speculated to be a necessary, if not sufficient, biological function of metazoan cells for the regulation of growth control, differentiation and apoptosis of normal progenitor cells, able to regulate both tissue homeostasis and the triggering of intra-cellular signal transduction mechanisms. Normal, contact-inhibited cells have functional GJIC, while most, if not all, tumor cells have dysfunctional homologous or heterologous GJIC [65
]. Cancer cells are characterized by the lack of growth control, inability to terminally differentiate or apoptose under normal conditions and have extended or immortalized life spans. Cancer cells either have no connexin expression or have expressed connexins but no functional GJIC; as a consequence, cancer cells lose the ability to respond appropriately to extra-cellular stimuli [66
]. In this context it would seem that GJIC is the ultimate down-stream cell function that must be maintained to prevent cancer [67
]. The classical model of carcinogenesis begins with an initiation
step, when the exposure to a carcinogen results in an irreversible genetic change within a single cell. The reversible down regulation of GJIC plays a role during the promotion
phase of carcinogenesis and the presence of GJIC is closely linked to the suppression of tumorigenic phenotypes [65
]. In normal tissues, GJIC is necessary for apoptosis and by blocking apoptosis with chemicals, it is possible to promote initiated premalignant cells or by increasing apoptosis by increasing GJIC, one could prevent tumorigenesis [68
]. Increasing evidences indicated that prevention of GJIC during the promotion
phase or the up-regulation of GJIC in tumorigenic cells represents a mechanistic-based strategy for chemoprevention or chemotherapy [69
]. A consistent observation is that many chemopreventive compounds such as phytochemicals [70
], antitumor-promoting agents and anticancer drugs can reverse the down-regulation of GJIC [65
As the inhibition of GJIC is considered an in vitro
biomarker of tumor promotion, the GJIC enhancement, through several processes, could be regarded as anti-tumor mechanism. The GJIC modulation can be proposed as an antitumor mechanism underlying the action of several natural bioactive compounds [70
Several bioactive marine compounds may affect GJIC. For instance, the depsipeptides isolated from the sponge Geodia corticostylifera
have antiproliferative activity on breast cancer cells and increases the size of GJ plaques in HTC-Cx43-GFP cells [77
]. Astaxanthin and canthaxanthin, xanthophylls synthesized by microalgae and yeast, with demonstrated cancer preventive effect in animal studies, were able to modulate GJIC, inducing changes in the phosphorylation state of connexin 43 proteins [78
Conversely, similarly to several tumor promoter chemicals, the potential tumor promoting effects of several marine cyanobacteria seems related to their ability to inhibit GJIC [80
From a computational analysis of naturally occurring marine compounds, based on protein-ligand interactions, many compounds have shown selective interaction towards gap junction and cell adhesive communication proteins resulting as ligand against the specific target molecules [81
]. Despite the limits of this approach, many molecules reported for other interesting bioactivities, such as the anti-inflammatory sesterterpene manoalide [82
] were identified as good ligands of the GJ connexin 43 and connexin 26 proteins. Dihydroxrytetrahydrofurans, some stereoisomers of which had been recognized as metabolites of the marine brown alga Notheia anomala
], ascosalipyrrolidinone A [86
], an antimicrobial alkaloid isolated from the endophytic obligate marine fungus Ascochyta salicorniae
, and solenolide A, a briariane diterpene lactone from the cnidarian octocoral Solenopodium excavatum
], they all resulted as ligands specifically against GJ protein targets, connexin 43 and connexin 26 [81
The present study aimed to isolate and identify potentially bioactive compound(s) occurring in the tissues of C. tuberculata jellyfish. Here the extraction, fractioning and biological activity assessment of different fractions of a jellyfish hydroalcoholic extract are described. The effects of the extract fractions on cancer (MCF-7) and non cancer (HEKa) cells were evaluated by cell-based assays allowing the quantification of cytotoxicity of jellyfish-derived compounds and their effect on the GJIC functionality as target of anti-tumor promoting activity.
3. Experimental Section
3.2. Jellyfish Samples
Live jellyfish specimens of C. tuberculata were collected in south Adriatic and Ionian Sea (Otranto and Santa Caterina, Lecce, Italy) in September 2010 and 2011. After biometric measurement (weight and diameter), samples were immediately frozen in liquid nitrogen and stored at −80 °C until lyophilization. Large specimens were cut and quarter radial sectors of the jellyfish were used as homogeneous portion representative of the whole animal. Frozen jellyfish were freeze-dried for 4 days at −55 °C in a chamber pressure of 0.110 mbar in a freeze dryer (Freezone 4.5L Dry System, Labconco Co. Thermo Scientific, Kansas City, MO, USA). After lyophilization, the dry weight was recorded and the samples were stored at −20 °C until use.
3.3. Preparation and Fractionation of the Jellyfish Extract
Four grams of lyophilized jellyfish sample were subjected to hydro alcoholic extraction by stirring in 16 volumes (w/v) of 80% ethanol, overnight at 4 °C. Samples were then centrifuged at 9000× g for 30 min, at 4 °C, and the supernatants indicated as Total Extract (TE) were used for biochemical assays. TE was concentrated by vacuum rotary evaporator (Buchi), at low temperature and protected from light, in order to evaporate the ethanol, and then frozen in liquid nitrogen and lyophilized.
Dried extract was weighed and 50 mg were solubilized in 1 mL of acetonitrile/water 1:1 (v/v) at 4 °C, in order to separate proteins. After gentle shacking, the suspension was left on ice in order to facilitate the phase separation. Three phase were obtained (Figure 2
) indicated as Upper Phase (UP), Intermediate Phase (IP) and Lower Phase (LP). Protein concentration, antioxidant activity and total phenol content were evaluated in the TE and in UP, IP, and LP fractions.
3.4. Protein Concentration
Protein content in TE, UP, IP and LP was estimated by modified Bradford assay [139
] using bovine serum albumin (BSA) as a standard for curve construction.
3.5. Determination of Phenol Content
The total content of phenols was determined by a modified Folin-Ciocalteau colorimetric method. The test solutions containing 100 μL of sample were mixed with 500 μL of Folin-Ciocalteu’s phenol reagent and with 500 μL of 7.5% sodium carbonate (Na2CO3). After 2 h, at room temperature in the dark, the absorbance was spectrophotometrically measured at 760 nm. The calibration curve was plotted versus concentrations of gallic acid ranging from 25 to 200 μg/mL, used as standard. The results were expressed as gallic acid equivalents per gram of dry extract.
3.6. In Vitro Antioxidant Activity Analysis
The total antioxidant activity was determined spectrophotometrically by using the Trolox Equivalent Antioxidant Capacity (TEAC) method, as described in Leone et al.
], using the radical cation ABTS•+
and Trolox as standard. Ten microliters of the jellyfish extract or fractions were assayed into 1 mL of the reaction mixture and the absorbance decrease was measured at 734 nm. Solutions of 80% ethanol, acetonitrile or acetonitrile/water were used as control. A Trolox calibration curve in a range of 2.5–20 μM was prepared under the same conditions of the samples. The antioxidant capacity of the samples was calculated, on the basis of the inhibition exerted by standard Trolox concentrations at 734 nm, inhibition time being fixed at 6 min. Results were expressed as nmol of Trolox equivalents per gram of sample or per mg of contained proteins.
3.7. Fatty Acid Profiles Determination
Fatty acid methyl esters (FAME) were obtained using BF3
according to Szczesna-Antczak et al.
] with some modifications. Upper phase (1 mL) was saponified at 90 °C for 20 min with 0.5 M KOH in methanol (3 mL). Forty-nine micrograms of the internal standard (methyl tricosanoate) were added before saponification. The fatty acids were methylated by adding 14% BF3 in MeOH (2 mL) and heating at 90 °C for 10 min. After cooling, hexane (1 mL) was added and vigorously stirred for 30 s before the addition of 1 mL of sodium chloride solution (0.6%). The esterified sample was left at 4 °C for better phase separation. After collecting the supernatant, another 1.0 mL of hexane was added and stirred. The supernatant was collected and added to the previous fraction. The sample was concentrated to a final volume of 1.0 mL for GC-MS analysis.
3.8. HPLC Analysis of the UP Fraction
The pigments in upper phase were separated according to Guaratini et al.
] using an Agilent 1100 HPLC. Analyses were carried out as described by Fraser et al.
], with slight modifications. Isoprenoids were separated using a reverse-phase C30 column (5 μm, 250 × 4.6 mm) (YMC Inc., Wilmington, NC, USA) with mobile phases consisting of methanol (A), 0.2% ammonium acetate aqueous solution/methanol (20/80 v/v) (B), and terz-methyl butyl ether (C). The isocratic elution was as follows: 0 min, 95% A and 5% B; 0 to 12 min, 80% A, 5% B, and 15% C; 12 to 42 min, 30% A, 5% B, and 65% C; 42 to 60 min, 30% A, 5% B, and 65% C; 60 to 62 min, 95% A, and 5% B. The column was re-equilibrated for 10 min between runs. The flow rate was 1.0 mL/min, and the column temperature was maintained at 25 °C. The injection volume was 10 μL. Absorbance was registered by diode array at wavelengths of 450 and 475 nm for carotenoids and 657 nm for chlorophylls. Isoprenoids were identified by comparing their retention times and UV-visible spectra to authentic standards peridinin, lutein in ethanol and chlorophylls.
3.9. GC-MS Analysis
The analyses were performed on a GC-MS system, Shimadzu GC-17A version 3.0 (Shimadzu Co., Kyoto, Japan), with MS QP5050A according to Talà et al.
]. Compounds were separated on DB-5 capillary column having 30 m length, 0.25 mm ID and 0.25 μm thickness. The GC parameters were as follows: the column temperature was 80 °C at the injection then programmed at 10 °C/min to 150 °C, at 5 °C/min to 250 °C and maintained at 250 °C for 15 min. Split injection was conducted with a split ratio of 50:1, the flow-rate was 1.0 mL/min, carrier gas used was 99.999% pure helium, the injector temperature was 250 °C and the column inlet pressure was 74 kPa. The MS detection conditions were as follows: 250 °C interface temperature; ionization mode, EI+
; electron energy, 70 eV; scanning method of acquisition, ranging from 30 to 450, for mass/charge (m
) optimization. Spectrum data were collected at 0.5 s intervals. Solvent cut time was set at 2 min and 45 min retention time enough for all fatty acids separation all. Compounds were identified by using online NIST-library spectra and published MS data. Moreover, FAME mix (C8
) and PUFA No. 3 (from menhaden oil) authentic standards were used to confirm MS data.
3.10. Protein Analysis by SDS-PAGE
Different component proteins in the total extract and fractions (TE, IP and LP) and their molecular weights were assessed by electrophoresis (SDS-PAGE) on 12% separating gel with a 4% stacking gel. The Precision Plus Protein Dual Color Standard (Bio-Rad, Hertfordshire, UK) was used as molecular weight marker. The protein bands were stained with 0.25% Coomassie brilliant blue R250 in 10% acetic acid and 50% methanol for 20 min. The gel was then detained with 10% acetic acid and 30% methanol overnight.
3.11. Cell Cultures
Breast cancer cell line, MCF-7, containing the estrogenic receptor, was obtained from the European Collection of Cell Cultures (ECACC, London, UK). Cell line was routinely grown in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine (GLN), 50 U/mL penicillin G, 50 μg/mL streptomycin (all from GIBCO) in 75 cm2 plastic flasks (Corning, NY, USA) at 37 °C in a 5% CO2 humidified atmosphere. Cells were passaged at 70%–80% confluence, about twice a week by trypsinization.
HEKa cells (Invitrogen) were cultured in Epilife Medium supplemented with HKGS, 50 U/mL of penicillin G and 50 μg/mL of streptomycin (all from GIBCO). Cells were plated at density of 150,000 cell/mL in 75 cm2 for HEKa and 250,000 cell/mL for MCF7 plastic flasks and incubated at 37 °C in a 5% CO2 humidified atmosphere and trypsinized when reached 80%–90% confluences.
3.12. Cell Viability and Treatments
Cell viability was assayed by MTS assay, as indirect measure of viable cell number, and by Trypan blue dye exclusion associated to automated cell counting (Countess®
Automated Cell Counter, Invitrogen Carlsbad, CA, USA), as suitable method to asses the real number of live and dead cells, in order to overcoming the possible inaccuracy of MTS assay in presence of supplements or new bioactive chemicals [144
3.12.1. MTS Assay
MCF-7 cells were seeded in flat bottomed 96-well plates (Corning, NY, USA) at 25 × 104 cells/well in 200 μL of RPMI medium supplemented with FBS, GLN and AA and HEKa were seeded at 25 × 104 cells/well in 200 μL supplemented Epilife Medium, and allowed to attach for 24 h at 37 °C in a 5% CO2 humidified atmosphere. Subsequently, the culture medium was replaced with complete medium containing the extract fractions at the following protein concentrations: UP, ranging from 0.0005 to 0.015 μg/μL and IP and LP from 0.005 to 0.08 μg/μL. For negative controls, the test compounds were replaced with solvent only (0.1% acetonitrile) or medium only, controls were included on each plate. Each treatment was performed in quadruplicate. The cells were then incubated for 16 h at 37 °C and 5% CO2 and assayed for vitality by MTS assay. Twenty microliters of CellTiter 96® Aqueous One Solution Reagent (Promega, Madison, WI, USA) were added to each well according to the manufacturer’s instructions. After 1 h in culture the cell viability was determined as assessment of metabolic activity by measuring the absorbance at 490 nm using a 3550 Ultra Microplate Reader (Biorad Instruments) (Bio-Rad, Hertfordshire, UK). The assay was performed in quadruplicate for three independent experiments and the results were expressed as a percentage of control.
3.12.2. Trypan Blue Dye Exclusion Assay
Cell viability was determined by Trypan blue (Invitrogen™, Carlsbad, CA, USA) dye exclusion in order to evaluate the cytotoxic effect induced by extract treatment. HEKa cells were seeded at a density of 6 × 105 cells and MCF-7 were seeded at 106 cells in 35 mm plate in 2 mL of the relative complete medium and treatments were performed when cells reached the 90% of confluence. Treatments and controls were performed as for MTS assay. At the end of treatment, cultures were trypsinized and a sample of cell suspension (20 μL) was mixed with Trypan blue (20 μL; 0.4% dye solution). A Cell Countess system (Invitrogen) was used to count the number of viable and non-viable cells and the percentage viability was calculated. Three independent experiments were performed in triplicate for each treatment and controls with vehicles and medium only.
3.13. Estimation of GJIC by Scrape-Loading Dye Transfer (SL/DT) Assay
GJIC was assessed using the SL/DT technique described by El-Fouly [146
]. Cells were seeded at 6 × 105
cells/plate (HEKa) and at 106
cells/plate (MCF-7) in 35 mm cell culture dishes, and incubated at 37 °C and 5% humidified atmosphere. Only cells at 95% confluence were used for the experiments herein, they were incubated with the test compounds (UP, IP and LP fractions) at the two lower tested concentrations in the vitality tests. The incubation with test substances was 30 min, 2 h or 4 h. Controls with only medium or vehicles were included in each experiment. After treatment, the MCF-7 and HEKa cells were rinsed carefully with PBS without Ca2+
, and then scraped and incubated with 0.5 mL of 0.1% (w/v) Lucifer Yellow CH (Molecular Probes, Invitrogen), for 3 min. The cells were then washed with PBS and fixed with 4% paraformaldehyde. The distance that Lucifer Yellow had travelled through gap junctions was observed with a laser scanning confocal microscope (Carl Zeiss, Munchen, Germany) and the number of fluorescent cells (cell in communication via gap junctions) was compared with the controls. Six random images were recorded for each plate and means were considered. Three independent experiments for each cell type were performed in triplicate for each treatment and for controls with the vehicles and medium only.
3.14. Laser Scanning Confocal Microscopy
Fresh jellyfish tissues containing symbiont cells were imaged using a Zeiss LSM Pascal confocal microscope excited using a 488 nm (argon) and 543 nm (He–Ne) lasers. Emissions were collected using two detection channels within 505–530 nm (virtual green) and over 560 nm (virtual red) emission. Following spectral mapping of innate cell fluorescence, online profiling was used to image a consistent number of clustered cells in tissues from different jellyfish anatomical portions. The offline tools of confocal microscopy were used to measure the cell and subcellular dimensions.
3.15. Statistical Analysis
Statistical analysis was performed using a Student’s t-test and an analysis of variance (ANOVA) test to analyse any differences in the measured endpoints observed between controls and the treatment groups. In the GJIC assays, these tests were used to analyse fluorescent cell average values. The mean and standard deviation (SD) were calculated for all data.