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original scientific article	DOI 10.19233/ASHN.2017.15
received: 2017-07-13
ANTITUMORAL ACTIVITY IN INKS OF SEPIA OFFICINALIS AND OCTOPUS VULGARIS (CEPHALOPODA) FROM THE NORTHERN TUNISIAN COAST
(CENTRAL MEDITERRANEAN SEA)
Emna SOUFI-KECHAOU & Ichrak SARIYA
Department of Animal Sciences, Halieutics and Food Technology, National Institute of Agronomy of Tunis, 43 avenue Charles Nicolle,
1082-Tunis-Mahrajene,Tunisia e-mail: esoufi@gmail.com
Amine BEZAA, Neziha MARRAKCHI & Mohammed EL AYEB
Laboratory of Venoms and Toxins, Pasteur Institute of Tunis. 13, place Pasteur, B.P. 74. 1002Tunis-Belvedere, Tunisia
abstract
In the present study, authors investigate the antitumor effects of peptides from the ink of two cephalopod species: the common cuttlefish, Sepia officinalis (Linnaeus, 1758), and the common octopus, Octopus vulgaris (Cuvier, 1797), from specimens sampled in the northern Tunisian coasts (central Mediterranean). The results indicate that the crude ink show anti-adhesion properties of the IGR39 cells depending on the Extra Cellular Matrixes (ECM) tested. The partially purified fractions with a molecular weight inferior to 10 kDa for Sepia officinalis (F.nf10) and the superior to 10 kDa for Octopus vulgaris (Fsup10) revealed anti-invasion, anti-migration and anti-adhesive activities on the U87 glioma cell lines, with a dose-dependent response. No antiproliferative activity was found for both of the partially purified fractions and the MTT assay showed toxicity effect only for high ink fraction concentrations.
Key words: Cephalopoda, Sepia ink, Octopus ink, antitumor, enzymatic hydrolysis, oligopeptide, Tunisia, central
Mediterranean
attivitA antitumorale di inchiostri di sepia officinalis e octopus
vulgaris (cefalopoda) provenienti dalla costa settentrionale della
tunisia (mediterraneo centrale)
SINTESI
Nel presente studio gli autori analizzano gli effetti antitumorali dei peptidi ricavati dall'inchiostro di due specie di cefalopodi: la seppia comune, Sepia officinalis (Linnaeus, 1758), e il polpo comune, Octopus vulgaris (Cuvier, 1797), provenienti da campioni prelevati lungo la costa settentrionale della Tunisia (Mediterraneo centrale). I risultati indicano che l'inchiostro grezzo mostra proprieta di anti-adesione delle cellule IGR39, in dipendenza delle matrici extra cellulari (ECM) analizzate. Le frazioni parzialmente purificate, con un peso molecolare inferiore a 10 kDa per Sepia officinalis (Finf10), e superiore a 10 kDa per Octopus vulgaris (Fsup10), hanno evidenziato attivita anti-in-vasione, anti-migrazione e anti-adesivita sulle linee cellulari degli gliomi U87, con una risposta dose-dipendente. Nessuna attivita antiproliferativa e stata trovata per entrambe le frazioni parzialmente purificate, e il dosaggio MTT ha dimostrato l'effetto tossicita solo per elevate concentrazioni di frazioni di inchiostro.
Parole chiave: Cefalopoda, inchiostro di seppia, inchiostro di polpo, antitumorale, idrolisi enzimatica,
oligopeptide, Tunisia, Mediterraneo centrale
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INTRODUCTION
In recent years, researchers have focused on identifying novel natural products as anticancer drugs. Anticancer peptides have characteristics of multi-function, high sensitivity and stability (Leng et a/., 2005; Simmons et a/., 2005).
Molluscan species, such as sea hares show a wide range of uses in pharmacology as they produce bioactive metabolites used in the treatment of cancerous tumors (Chakraborty & Ghosh, 2010). Peptides as antitumor drugs can improve immune response, inhibit the tumor angiogenesis and metastasis of tumor cells, directly eradicate tumor cells and induce the apoptosis of tumor cells and stop the cell cycle (Shen et a/., 2000; Aneiros & Garateix, 2004; Zheng et a/., 2007). The most studied, Do/astatins is a family of cytotoxic peptides isolated from the mollusk Do//abe//a auricu/aria, where the linear pentapeptide Do/astatin-10 and the depsipeptide Do/as-tatin 15 have had the most promising antiproliferative activity reported (Pettit et a/., 1995; Garteiz et a/., 1998; Pettit et a/., 1998). The pentapeptide Dolastatin-1 0 is characterized by four of the residues being structurally unique but with many side effects. Also, the Keenamide A is a cytotoxic cyclic hexapeptide isolated from the mollusk P/eurobranchus forska/ii, elicits antitumor activity via unknown mechanisms. This compound exhibited significant activity against the P388, A549, MEL-20 and HT-29 tumor cell lines (Wesson et a/., 1996). Strong anticancer peptides were also found from Meretix meretrix with IC of 10 pg-mL-1 (Liu & Qiu, 2004).
Cephalopoda ink had been used in the treatment of hemostasis for centuries in Chinese traditional medicine (Zhong et a/., 2009). As early as 1982, it was reported that Sepia ink could regulate gastric juice secretion and had antiulceration activity (Andersen & Roepstorff, 1982). Researchers in Japan found that the peptidogly-can extracted from Sepia ink had higher antitumor activity than the other fractions (Takaya et a/., 1996, 1997). Other research works reported antitumor activity of cephalopoda ink (Naraoka et a/., 2000; Palumbo et a/., 2000). For example, it has been reported that Sepia ink has antitumor activity against Meth-A fibrosarcoma in BALB/c mice and its fraction containing peptidoglycan showed higher antitumor activity than the other fractions (Tetsushi et a/., 2000; Mayer et a/., 2010). Nowadays, none of the currently available anticancer drugs acts solely on carcinoma cells. Anticancer drugs are usually extremely toxic and kill both malignant and normal cells. However, despite its wide spectrum of clinical uses, they are known to cause several adverse effects. These limits on the use of chemotherapeutic agents thus constrain their use in effective therapy.
Protein hydrolysates formed by the enzymatic digestion of aquatic and marine by-products are an important source of bioactive peptides. Purified peptides from these sources show cytotoxic effect on several human
cancer cell lines such as HeLa, AGS, and DLD-1 (Wang et a/., 2010). These characteristics imply that the use of peptides from marine sources has a great potential for the prevention and treatment of cancer, and that they might also be useful as molecular models in anticancer drug research.
In this paper, the inks from Sepia officina/is and Octopus vu/garis had been used for in vitro antitumor activities. These two marine species have been chosen because of their widespread geographical distribution in Tunisia and also because of the popularity of this seafood. The cephalopoda ink, which is the natural substance released for defence purpuses against predators is composed mainly of melanin and proteoglycans (Shen et a/., 2007; Mayer et a/., 2013). It is produced by the ink gland, a by-product of marine-product processing, generated after gutting procedures. The objective of this research work was the characterization and the evaluation of anticancer potential from the ink of S. officina/is and O. vu/garis through the antiproliferative effect, inhibition on invasion, migration of tumor cells, as well as cytotoxicity. A characterization of the nature the peptide has also been carried out after an enzymatic hydrolysis process.
MATERIAL AND METHODS Biological material
Like all the cephalopoda species, S. officina/is and O. vu/garis (Fig. 1) are positioned in a high level in the marine food web and are carnivorous since egg hatchling S. officina/is is a demersal and neritic species present in the infra and circalittoral zones, on sandy or muddy-sandy bottoms and phanerogam meadows, from the coast up to 150 meters. The cuttlefish specimens are present in the coastal waters from April till October. In the winter, they migrate to deeper zones searching for abundant food and more adequate temperatures.
O. vu/garis is a benthic, neritic species occurring from the coastline to the outer edge of the continental shelf (in depths from 0 to 200 m), where it is found in a variety of habitats, such as rocks, coral reefs, and grass beds. Throughout its distribution range, this species is known to undertake limited seasonal migrations, usually overwintering in deeper waters and occurring in shallower waters during summer. In the western Mediterranean, large mature or maturing individuals migrate inshore in early spring, followed later on by smaller, immature individuals. These two groups begin their retreat into deeper waters by August/September and November/ December respectively.
Specimens of S. officina/is and O. vu/garis were captured off Bizerte coasts (Fig. 1), (North of Tunisia, Mediterranean Sea) by deep-sea trawling for the Cuttlefish and by traditional coast-fishing for Octopus, during April 2010. The specimens were then transported to the
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Fig. 1: Location of the capture sites (FAO fisheries department 2005) of the biological material. A. Sepia officinalis and B. Octopus vulgaris.
Sl. 1: Lokalitete, kjer je bil nabran biološki material (FAO fisheries department 2005). A. Sepia officinalis in B. Octopus vulgaris.
laboratory packed in ice and the ink gland was extracted. In order to avoid biochemical variation due to the physiological state of the animals, inks were homogenized by triturator and stored at -20°C before use.
Ink extraction and preparation
At a first time, the crude cuttlefish or Octopus inks was dissolved in PBS buffer (500 mL). After centrifuga-tion at 1000 g during 10 min., the supernatants were collected for the antitumoral tests on tumor cell lines deriving from human melanoma IGR39. The sediment is stored at -80°C for further analyses. In a second time, the crude cuttlefish or Octopus inks were homogenized with acetone at -30°C (4V), according to the method of Takaya et al. (1994). The supernatant was collected after centrifugation at 1000 g during 15 min. and lyophilised. The sediment was stored at -80°C for further analyses. The lyophilised extracts were dissolved in a 0.1 M Tris-
HCl solution (PH = 6.8) (40 v) for 72 hours at 4°C and then centrifuged at 11.000 g during 30 min (Mikro 200 R, Hettich Zentrifuger), in Amicon centrifuging cells (YM10) in order to separate the ink extracts according to their molecular weight. Two fractions were obtained for each cephalopoda species: the Fsup 10 molecular compounds with a molecular weight higher then 10kDa and another fraction denoted Finf with the molecular weight inferior to 10 kDa. For Cuttlefish, as well as Octopus, these two fractions were lyophilised under vacuum (LABCONCO, 2.5 (Plus) Freezone). Finally, the freeze-dried fractions, F.nf 10 and the Fsup 10 of Sepia and Octopus were dissolved in PBS (v/v) according to the method of Naraoka et al. (2000). Aliquots were stored at -80°C.
Enzymatic hydrolysis
The Pepsin (EC 3.4.23.1) was provided by DSM. The enzymatic hydrolysis conditions were: a temperature of
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45°C, pH2, an E/S ratio of 0.1% and an hydrolysis time of 10 hours. The reaction was stopped by heating the solution to 85°C to inactivate the enzyme. The resulting hydrolysate was centrifuged at 20,000 g for 20 min.
Isolation and purification of anticancer peptide
Sepia ink hydrolysates were fractionated into a high and low molecular weight fractions and by ultrafiltration at 4°C by PM-10 membrane (MWCO = 3000 Da) and kept for use in gel filtration. Prior to use, the membrane was washed with 10 mL of distilled water. The ultrafil-trate was filtered again through a Millipore membrane filter (0.45 pm) and applied to a (2.8 cm x 90 cm) column saturated in Sephadex G-25 resin. The Sepha-dex G-25 column was eluted with distilled water and fractions were collected every 4 minutes with a fraction collector. The absorbance was measured at 280 nm. The hydrolysate was fractionated into five fractions by gel filtration chromatography. Each fraction was tested for anticancer activities.
The fraction showing the highest anticancer activity was further purified using reverse-phase HPLC on a Primesphere 10 C column (10 mm x 250 mm) with a linear gradient of 1 acetonitrile (0-50% for 20min) containing 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 mL-min . The absorbance of the eluent was monitored at 280 nm. Active peak representing anticancer activity was pooled and freeze-dried immediately for a future analysis of the bioactive fraction.
The yield of Sepia ink oligopeptides
The fraction provided by the G-25 gel chromatog-raphy was concentrated in vacuum under 25°C and concentrated liquid was dried under 70°C by vacuum freeze-drying. Then the powder of Sepia ink oligopep-tides was collected and weighed.
Cell line and cell culture
Human glioma cell lines U87 and melanoma cell lines IGR 39 were used for anticancer activity tests with non purified ink fractions. The human prostate cancer cell lines PC-3 were used later for the purified and isolated active peptide. The cell lines were grown at 37°C in a 5% CO2, 95% air humidified atmosphere, in MEM medium for U87 and DMEM-Ham's F12 medium for IGR39. The medium was supplemented with 10% FCS + 5% heat inactivated horse serum to which streptomycin (100 pg/ mL) and penicillin (100 U/mL) had been added. The cells grown in flasks were washed with PBS, trypsinized (3 mL of trypsine-EDTA, 500 pg/ml), centrifuged at 800 rpm for 5 min and dissolved in fresh culture medium at 104 cells/90 pL. Subsequently, a 90 pL volume of suspension cells was added to each well of a 96-well microplate and incubated at 37°C for 24 hours.
Cell migration and invasion assay in vitro
The assays were achieved in Boyden Chambers (Bec-ton Dickinson). A 24-well transwell (Corning, NY, USA) was used to evaluate the motility and invasive ability of U87 and IGR29 cells in vitro. The upper surface of polycarbonate filters with 8 pm pores (0,3 cm2) was coated with 5 mg of Matrigel, fibrinogen (Sigma-Aldrich) at 50 pg/mL of PBS and incubated during 2 hours at 37°C . The lower chambers were filled with the medium (MEM/ BSA 0,1%) (Sigma-Aldrich) (500 pl in the lower well, and 200 pl in the upper one). The glioma cells U87 were pre-incubated with different doses of the molecular fractions Finf 10 and the Fsup 10 of Sepia and Octopus or 1% BSA (negative control) for 24 hours at 37 °C in a CO2 incubator and then washed with PBS, detached with the versene (Gibco) and resuspended in serum-free MEM. A suspension of cells (2 x105 cells/200 mL) was placed in the upper chambers. After 5 h of incubation at 37°C under optimal conditions, the supernatant was completely removed and the upper and lower faces of the membranes are washed with PBS. Cells on the upper surface of the filter (that did not migrate) were completely removed by wiping them with a cotton swab. Cells that invaded the Matrigel and were fixed during 10 min with glutaraldehyde 1% and then stained with crystal violet at 0.5 % for 30 min. The cellular migration was quantified by counting the number of cells that migrated using a microscope (Leica) with 5 mm2 fields at a magnification of x 400, or by measuring the absorbance at 560 nm after solubilization of the colorant in SDS 1%.
Antiproliferative activity assay
This assay aims to evaluate the effect of the molecular fractions, Finf 10 and the Fsup 10 of Sepia and Octopus on the multiplication of tumor cells. U87 cells are incubated in the wells of a microplate (5 x 103 cells/ well) in 50 pl MEM/10% FCS (Fœtal Calf Serum/ 5% horse serum. After one hour incubation, the medium is renewed in presence of the fractions to test. Each day, 3 wells are washed with PBS and the cells are fixed with glutaraldehyde 1% then fixed with PBS. At the end of the week, the cells are stained with crystal violet 0.1% and quantified by measuring the absorbance at 560 nm.
Cytotoxicity assay
The MTT assay (Mosmann, 1983) allows the evaluation of the effect of the molecular fractions F. ..„and
inf 10
the Fsu 10 on the cell viability of cancer cells U87. The peptidic ink fractions from Cuttlefish and Octopus, at a concentration of 10 mg/mL was prepared in PBS 0.1 M (pH 7.4), and diluted 10-fold in cell culture medium containing the cells. The microplate was then incubated at 37 °C for 24, 48 and 72 h, changing the culture medium every 24 h and adding the ink fractions at a
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final concentration of 1 mg/mL. At the end of every incubation period, 15 pL of 5 mg/mL tetrazolium salt (MTT) solution was added to each well, and the plate was incubated for 3 h. To stop succinate-tetrazolium reductase activity and solubilize formazan crystals, 200 pL of dimethyl sulfoxide (DMSO) was then added to each well and kept at 37 °C for 1 h. Absorbance was read on a plate reader at 570 nm.
Cellular adhesion assay
The aim of this assay was to analyze the ability of ink fractions to inhibit the adhesion of tumor cells IGR39 on the proteins of the extracellular matrix (ECM). Four proteins were assyed: fibrinogen, fibronectin, collagen type I and polylysin. The substrates of adhesion were prepared by coating the wells of a microplate (Nunc) by 50 pL of a proteic solution : fibrinogen at 50 pg/mL, la polylysine (PL) at 20 pg/mL, the collagen type I (Coll I) at 10pg/mL and fibronectine (Fn) at 5pg/mL and incubating it during 2 hours at 37°C. The wells are then saturated with 50 pL PBS/BSA 0.5 % during 1 hour at 37°C. During saturation, the cells were detached by PBS/EDTA and washed twice with adhesion buffer (MEM, NaHCO3 1,2 g/l, HEPES 15 mM pH 7.3 and BSA 0,2%). For the assay of the ink fractions, the cells were pre-incubated during 30 min at room temperature with stirring and then deposited in the wells where 50 pL cellular suspension (106 cells/ mL adhesion buffer) were added and incubated during 1 hour at 37°C. After incubation, the non adherent cells are eliminated by washing with an adhesion buffer. The adherent cells are fixed with glutaraldehyde 1% during 10 min at room temperature and are washed twice with distilled water and stained during 30 min by 100 pL of a crystal violet solution at 0.5%. Cellular adhesion was quantified by measuring the absorbance at 560 using a microplate reader (£960 Metertech). All the data were analyzed by the software of SPSS.
RESULTS AND DISCUSSION
Effect of the crude ink on the adhesion of IGR23 to the proteins of the ECM
Cephalopoda ink had been studied for its antimicrobial and antiviral activities, but also for its toxicity for some cell lines (Derby et a/., 2007). To this context, we investigated the antitumor potential of S. officinalis and O. vu/garis inks. At a first time, the crude inks were diluted in PBS and then centrifuged. We evaluated the anti-adhesive effect of the supernatants on IGR39 cell lines deriving from human melanoma, on 3 different ECM: fibrinogen, fibronectin and collagen type I. The results showed that cuttlefish ink - at a concentration of 5,28 pg/ml - significantly (p < 0.05) inhibits the adhesion to fibrinogen, with an inhibition percentage of 60%. This inhibition was 25% on fibronectin. There
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Fg	Fn	Coll I
Extracellular matrixes (ECM)
Fig. 2: Effect of crude Sepia and Octopus ink on the adhesion of the melanoma IGR 39 cells to fibrinogen, fibronectin and collagen type I.
Sl. 2: Učinek surovega črnila sipe in hobotnice na lepljenje celic melanoma IGR 39 na fibrinogen, fibronectin in kolagen tipa I.
was no inhibition of the adhesion of the IGR 39 cells on the collagen type I (Fig. 2). Concerning Octopus ink, we noticed that the adhesion of IGR 39 cells on fibronectin is significantly (p < 0.05) decreased by 40% (concentration of 8,75 pg/mL), but there was no inhibition for fibrinogen and collagen type I.
Effect of ink fractions on the adhesion of tumor cells U87 to the proteins of the ECM
After the fractionation of the inks in Amicon cells, two fractions were obtained for each cephalopoda specie, the Fsup 10 (MW > 10 kDa) and the Finf 10 (MW < 10 kDa). With a concentration of 30 pg/mL, the cuttlefish Fsup 10 poorly inhibits the adhesion of U87 cells on fibrinogen (Fig. 3.A), whereas the Finf fraction inhibits cell adhesion on fibrinogen in a dose-dependent manner, with an IC50 of 25pg/ml (Fig. 3.B). In the same way, Octopus Fsup 10 inhibits cell adhesion with an IC50 of 75 pg/mL (Fig. 4.A). However, the fraction Finf 10 assayed at dose 100pg/ mL did not show a significant antitumor effect at the level of 5%. (Fig. 4.B). The adhesion assays of the U87 cells on Polylysine-L showed an inhibition that did not exceed 20 % with cuttlefish Finf 10 and Octopus Fsup 10. At that point we cannot conclude yet that these inhibitions are integrin-dependent (Fig. 5.A and B).
Effect of ink fractions on the migration of U87 cells
The cellular migration plays a very important role in the metastatic dissemination and requires cellular adhesion to the proteins of the extracellular matrixes (ECM). Because the extracts of the active fractions, F .,,. of Se-
inf 10
pia ink and Fsup 10 of Octopus ink exhibited an inhibiting potential of the adhesion of U-87 tumoral cells, we have essayed the effect of these extracts on their migration.
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Fig. 3: Effect of the Sepia ink fractions on the adhesion of the glioma U87 cells on fibrinogen. A: Fsup 10 (30^g/ ml), B: Amount of Finf 1 fraction.
Sl. 3: Učinek frakcije surovega črnila sipe na lepljenje celic glioma U87 na fibrinogen. A: Fsup 10 (30^g/ml), B: Količina Finf 1 frakcije.
Fig. 4: Effect of the Octopus ink fractions on the adhesion of the glioma U87 cells on fibrinogen. A: F.nf 10 (100^g/mL); B: Amount of Fsup 10 fraction. Sl. 4: Učinek frakcije surovega črnila hobotnice na lepljenje celic glioma U87 na fibrinogen. A: Fsup 10 (30^g/ml), B: Količina Fsup 10 frakcije.
Fig. 5: Dose-response effect of ink fractions on the adhesion of U87 cells to Polylysin-L. A: Sepia Fsu 10, B:
Octopus F;nf 10
Sl. 5: Učinek frakcij črnila na lepljenje U87 celic na polilizin-L v odvisnosti od doziranja. A: Sepia Fsu 10, B:
Octopus F;nf 10.
The U87 cells treated with increasing concentrations of cuttlefish F.nf10 or Octopus Fsup 10 stopped the migration of the cells and their adhesion to fibrinogen, with a dose-dependence (IC50 =15pg/ml) for Sepia F.nf 10 and (IC50 = 40pg/ml) for Octopus Fsup 10 (Fig. 6 and Fig. 7).
Antiproliferative effect of ink fractions on the U87 cells
Because the ink fractions Sepia Finf 10 and Octopus Fsu io showed interesting antitumor activities (inhibits adh esion and migration of U87), their antiproliferative potential was assayed. Our results showed that the ink fractions did not show any significant inhibition of cell proliferation during 4 days (Fig. 8 A and B).
Isolation and purification of the Sepia ink peptide
Because the peptidic fraction inferior to 10 kDa of Sepia ink (Finf 10) is the one who was the most active, and because biologically active peptides have low molecular weight in general, we decided to concentrate our study on Sepia ink anticancer peptides. After ultrafiltration using a 3000 Da MWCO membrane the permeates of Pepsin hydrolysates were loaded on a gel filtration column (Sephadex G-25). Five anticancer peptide fractions with the highest activity were collected. The purity was
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Fig. 6: Effect of the Sepia Finf 1 ink fraction on the U87 Fig. 7: Effect of the Octopus Fsu 1 ink fraction on the
cell migration and morphological changes. (I): Microscopic observation. A-D: Cells were incubated with F inf 10 Sepia ink fractions (0, 10, 20, 30 pg/mL) during 5 hours at 37°C. (II): U87 cellular migration (%) according to the concentration of Sepia ink fraction Finf10 (0, 10, 20, 30 pg/mL).
Sl. 6: Učinek frakcije črnila sipe (F.nf 10) na migracijo celic U87 in morfološke spremembe. (I): Mikroskopska opazovanja. A-D: Celice inkubirane s frakcijami F.nf 1 črnila sipe (0, 10, 20, 30 pg/mL) v peturnem obdobju pri 37°C. (II): U87 celična migracija (%) glede na koncentracijo frakcij črnila sipe F.nf10 (0, 10, 20,30 pg/mL).
o	'	sup 10
U87 cell migration and morphological changes. (I): Microscopic observation. A-D: Cells were incubated with Fsup 10 Octopus ink fractions (0, 25, 50, 100 pg/mL) during 5 hours at 37°C. (II): U87 cellular migration (%) according to the concentration of Octopus ink fraction Fsup 10 (0, 25, 50, 100 pg/mL).
Sl. 7: Učinek frakcije črnila hobotnice (Fsup 10) na migracijo celic U87 in morfološke spremembe. (I): Mikroskopska opazovanja. A-D: Celice inkubirane s frakcijami Fsup 10 črnila sipe (0, 10, 20, 30 pg/mL) v peturnem obdobju pri 37°C. (II): U87 celična migracija (%) glede na koncentracijo frakcij črnila hobotnice Fsu 10 (0, 25, 50, 100 pg/mL).
then detected by HPLC. The sample was divided into five peaks and the peak 2 had the highest anticancer activity. After a further reverse-phase HPLC, the second peak had a molecular weight of 340.6. After freeze drying, 1.74 g Sepia ink oligopeptide was obtained from 100 g Sepia ink. So the yield was 1.74 %.
Effect of Sepia ink oligopeptides on cell viability
The PC-3 cells (human prostate cancer cell lines) were treated with 2-10 pg-mL of Sepia ink oligopep-tides for 24-72 h. PC-3 cells displayed dose-dependent decreases in viability, detectable as early as 24 h. At 24 h, the threshold concentration which caused a decrease in PC-3 cell viability was 1.89 pg-mL (89 % of control, P > 0.05). The Sepia ink oligopeptide concentration that produced the maximal effect was 10 pg/mL (26% of control, P < 0.05), and the half inhibitory concentration (IC ) was 7.45 pg-mL .
5At 48 h, the threshold concentration was 2,2 pg-mL-1 (33 % of control, P < 0.05), the maximal effect was 10 pg-mL-1 (0.24% of control, P < 0.05), and the IC50 was 1.23 pg-mL-1. At 72 h, the threshold concentration was again 2 pg-mL-1 (43 % of control, P < 0.05). The maximal effect was 10 pg-mL-1 (0% of control, P < 0.05), and the IC50 was 1.64 mg-mL-1. The threshold concentration at 24, 48 and 72 h were the same (2 pg-mL-1), even though the level of the significance increased from day 1 to days 2 and 3. The IC50 decreased from 7.45 pg-mL-1 at 24 h to
1.23 pg-mL-1 at 48 h. These results suggest that Sepia ink oligopeptides had a dose-dependent deleterious effect on PC-3 cell viability.
Biologically active antitumor compounds have been isolated from different marine sources. Recently research has been focused on peptides from marine animal sources, since they have been found as secondary metabolites from sponges, ascidians, tunicates, and mollusks. The structural characteristics of these peptides include various unusual amino acid residues which may be responsible for their bioactivity. However, many side effects had been observed in clinical trials and the complexity and low yield of chemical synthesis, together with low water solubility, have been significant obstacles to broader clinical evaluation, triggering the development of analog compounds (De Arruda et al., 1995; Pitot et al., 1999; Tamura et al., 2007).
Even if the bioactive peptides from marine mollusks had been well documented, there had been a few publications on anticancer peptides from cephalopoda, specifically the species O. vulgaris and S. officinalis ink wastes.
Interestingly in our study, the crude Sepia and Octopus inks assayed on tumor cells IGR39, showed a selective inhibition according to the cellular matrix used. In order to refine this investigation, we adopted an acetone fractioning of the ink and interested particularly to the supernatant. The two fractions obtained
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-Mean CT -Mean S (1pg/mL) Mean S (5pg/mL) -Mean S (10pg/mL)
4
if3 Is 182
1
Mean CT
Mean P (10^g/mL) Mean P (15^g/mL) Mean P (25^g/mL)
Fig. 8: Anti-proliferative effect of the active fractions on
U87 cells. (A): Sepia Ff 10, (B): Octopus Fsup 10.
Sl. 8: Anti-proliferativni učinek aktivnih frakcij na U87
celice. A): Sepia Fnf o (B): Octopus Fsup o
Finf 10 (MW< 10kDa) and Fsup 10 (MW> 10kDa) were then assayed in vitro on glioma cell lines U87. The in anti-adhesive activities of the ink fractions belonging to the two cephalopoda species are not comparable. Sepia Finf 10 and Octopus Fsup 10 inhibit the adhesion of U87 cells on fibrinogen, according to their concentrations (dose-dependent) with an IC50 = 25pg/mL for cuttlefish ink fraction and 75pg/mL for Octopus. However, both of the ink extracts slightly inhibit the non-specific adhesion of U87 cells on Polylysin-L. This result suggests that the fractions would own an inhibition mechanism through one or more membrane receptors. It is also important to mention that the anti-adhesive effect requires a high concentration of the Octopus Fsup 10 (~50pg/mL), unlike Sepia Finf 10 fraction (~10 pg/mL). We can thus emit the hypothesis that these ink fractions may contain antagonistic activities. According to the literature, the antitumor effect is correlated to a synergy between different chemical ink compounds. This action is related to the
tyrosinase activity and peptidoglycans (Naraoka et al., 2000). We also showed that with concentrations of 10 pg/mL of Sepia ink fraction, the cell migration is reduced and is completely stopped with a concentration of 30 pg/ml. However, concerning the Octopus ink fraction, we observe an inhibition of cell migration starting from a concentration of 25 pg/mL. This inhibition is complete at 100 pg/mL. Somehow, there was no inhibition of cell proliferation. Our results are in concordance with the research work on squid (Ommastrephes bartrami) ink where the authors did not detect evident antiprolifertive activity on tumor cells Hep G2, but induces a suppression of cell invasion and cell migration, according to the concentrations of ink fractions (Chen et al., 2010). The cytotoxicity assays of the F.nf 10 and Fsup 10 of Sepia and Octopus during 5 hours showed that these fractions are toxic only at very high concentrations. It had previously been reported that the tyrosinase (MW=94 kDa) is responsible of the toxic effect of cephalopoda ink (Prota et al., 1981, Palumbo et al., 1985, 1994, Takaya et al., 1994, Naraoka et al., 2000, 2003). At this point, we can only hypothesize that the cytotoxicity of the Octopus Fsup10 is also due to the enzymatic effect of tyrosinase, but this is to be confirmed.
The Sepia ink oligopeptides extracted using the protease Pepsin also inhibited the growth of PC-3 cells. In U-87 cells, Sepia ink oligopeptides caused a linear decrease of cell viability in a dose-dependent manner. However, the mechanism of the anticancer activity is unclear. Therefore, further studies are needed to identify the mechanism of the potent antitumor activity.
Finally we can deduce that the fractions F.nf 10 et Fsup 10, respectively from S. officinalis and O. vulgaris, do not have antiproliferative but are responsible of antiadhe-sive and anti-migration activity. However, we still have to investigate whether these antitumor activities are due to one or more chemical components and to determine their chemical nature and molecular mechanisms that are implied. The results of our study also demonstrated the effect of Sepia ink oligopeptides on growth inhibition and could be a potentially useful adjunct in the treatment of cancer. Hence, since the cephalopod species S. officinalis and O. vulgaris are easily accessible Tunisian marine resources, their ink protein wastes are attractive as a protein source for the future industrial production of functional peptides.
A
is O 2 o
B
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protitumorska aktivnost črnila pri sipi sepia officinalis in hobotnici octopus vulgaris (cephalopoda) iz severne tunizijske obale (osrednje
sredozemsko morje)
POVZETEK
Avtorji poročajo o protitumorskih učinkih peptidov iz črnila dveh glavonožcev in sicer sipe, Sepia officinalis (Linnaeus, 1758), in hobotnice, Octopus vulgaris (Cuvier, 1797), dobljenih na primerkih, ujetih ob severnotunizijskih obalah (osrednje Sredozemsko morje). Rezultati prikazujejo, da učinkovine iz surovega črnila kažejo protiadhezijsko aktivnost na celice IGR39 v odvisnosti od testiranih izvenceličnih matriksov. Delno prečiščena frakcija z molekulsko maso, manjšo od 10 kDa pri vrsti Sepia officinalis (F.nf10) in višjo od 10 kDa pri vrsti Octopus vulgaris (Fsup10) sta pokazali koncentracijsko odvisno protiinvazivno, protimigracijsko in protiadhezivno aktivnost na celičnih linijah glioma U87. Delno prečiščene frakcije niso pokazale nobenih protiproliferativnih aktivnosti, MTT protokol pa je pokazal toksični učinek le v primeru visoke koncentracije frakcije črnila.
Ključne besede: Cephalopoda, črnilo sipe, črnilo hobotnice, protitumorska aktivnost, encimatska hidroliza,
oligopeptidi, Tunizija, osrednje Sredozemsko morje
REFERENCES
Andersen, S.O. & P. Roepstorff (1982): Sclerotiza-tion of insect cuticle: An unsaturated derivative of N-acetyldopaminc and its role in sclerotization. J. Insect Biochem, 12(3), 269-276.
Aneiros, A. & A. Garateix (2004): Bioactive peptides from marine sources: Pharmacological properties and isolation procedures. J. Chromatogr. B: Biomed. Sci. App., 803, 41-53.
Chakraborty, S. & U. Ghosh (2010): Oceans: a store of house of drugs - A review. J. Pharm. Res., 3, 12931296.
Chen, S., J. Wang, C. Xue, H. Li, B.Sun, Y. Xue & W. Chai (2010): Sulfation of a squid ink polysaccharide and its inhibitory effect on tumor cell Metastasis. Carbohydr. Polym., 81, 560-566.
De Arruda, M., C.A. Cocchiaro, C.M. Nelson, C.M. Grinnell, B. Janssen, A. Haupt & T. Barlozzari (2008):
1995LU1 03793 (NSC D-669356): A synthetic peptide that interacts with microtubules and inhibits mitosis. Cancer Res, 55, 3085-3092.
Fearon, E.R. & B. Vogelstein (1990): A genetic model for colorectal tumorigenesis. Cell, 61, 759-767.
Garteiz, D.A., T. Madden, D.E. Beck, W.R. Huie, K.T. McManus, J.L. Abbruzzese, W. Chen & R.A. Newman (1998): Quantitation of dolastatin-10 using HPLC/elec-trospray ionization mass spectrometry: application in a phase I clinical trial. Cancer Chemother. Pharmacol., 41, 299-306.
Leng, B.O., D.L. Xiao & X.C. Qing (2010): Inhibitory effects of anticancer peptide from Mercenaria on the
73
ANNALES ■ Ser. hist. nat. ■ 27 ■ 2017 ■ 2
Emna SOUFI-KECHAOU et al.: ANTITUMORAL ACTIVITY IN INKS OF SEPIA OFFICINALIS AND OCTOPUS VULGARIS (CEPHALOPODA) FROM ..., 125-74
BGC-823 cells and several enzymes. FEBS Letters, 579 (5), 1187-1190.
Liu, X.D., L. Qiu & Q. Wu (2004): Studies on the physiological activity of a natural peptide from clam (Meretrix meretrix Linnaeus). J. Xiamen Univ. (Nat. Sci.), 43 (4), 432-435.
Luesch, H., R.E. Moore & V.J. Paul (2001): Isolation of dolastatin 10 analogue from the marine cyanobacte-rium symploca species VP642 and total stereochemistry and biological evaluation of its analogue symplostatin 1. Nat.Prod., 64(7), 907-910.
Mayer, A.M.S., K.B. Glaser, C. Cuevas, R.S. Jacobs, W. Kem, R.D. Little, J.M. Mcintosh, D.J. Newman, B.C. Potts & D.E. Shuster (2010): The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends in Pharmacological Sciences, 31, 255-265.
Mayer, A.M.S., A.D. Rodriguez, O. Taglialatela-Sca-fati & N. Fusetani (2013): Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-inflammatory, Antiprotozoal, Antituberculosis and Antiviral Activities; affecting the Immune and Nervous System, and other Miscellaneous Mechanisms of Action. Mar. Drugs, 11, 2510-2573.
Monique, W.J., B. Den & N. Bastiaan (2005): Pharmaceutical development of a parenteral lyophilised formulation of the investigational anticancer agent ES-285.HCl. PDA J. Pharm. Sci. Technol., 59(4), 246-257.
Mosmann, T. (1983): Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55.
Naraoka, T., H.S. Chung, H. Uchisawa, J. Sasaki & H. Matsue (2000): Tyrosinase activity in antitumor compounds of squid ink. Food Sci. Technol. Res., 6, 171-175.
Naraoka, T., H. Uchisawa, H. Mori, H. Matsue, S. Chiba & A. Kimura (2003): Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus. Eur. J. Biochem., 270, 4026-4038.
Palumbo, A., A. Poli, A. Di Cosmo & M. D'Ischia (2000): N-Methyl-D-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric oxide/c GMP signal transduction pathway. J. Biol. Chem., 275, 16885-16890.
Pettit, G.R., J.K. Srirangam, , J. Barkoczy, M.D. Williams, K.P. Durkin, M.R. Boyd, R. Bai, E. Hamel, J.M. Schmidt & J.C. Chapuis (1995): Antineoplastic agents 337. Synthesis of dolastatin 10 structural modifications. Anticancer Drug Des., 10, 529-544.
Pettit, G.R., E.J. Flahive, M.R. Boyd, R. Bai, E. Hamel, R.K. Pettit & J.M. Schmidt (1998): Antineoplastic agents 360. Synthesis and cancer cell growth inhibitory studies of dolastatin 15 structural modifications. Anticancer Drug Des., 13, 47-66.
Pitot, H.C., E.A. McElroy, J.M. Reid, A.J. Windebank, J.A. Sloan, C. Erlichman, P.G. Bagniewski, D.L. Walker, J. Rubin, R.M. Goldberg & al. (1999): Phase I trial of dolastatin-10 (NSC 376128) in patients with advanced solid tumors. Clin. Cancer Res., 5, 525-531.
Sanjay, G., C.M. Alain & M. Monica (2009): A phase I study of eribulin mesylate (E7389), a mechanistically novel inhibitor of microtubule dynamics, in patients with advanced solid malignancies. J. Clin. Cancer Res., 15(12), 4207-4212.
Shen, G., R. Layer & R. McCabe (2000): Conopep-tides: From deadly venoms to novel therapeutics. Drug Discovery Today, 5, 98-106.
Shen, C., J. Xie & K. Xu (2007): The components of cuttlefish (Sepiella maindroni de Rocheburns) oil. Food Chem., 102, 210-214.
Simmons, T.L., E. Andrianasolo, K. McPhail, & al. (2005): Marine natural products as anticancer drugs. Mol. Cancer Ther., 4(2), 333-342.
Susan, A, A. Brooke, J. Hannah, N. Lomax-Browne, M. Tracey, J. Carter, F. Chloe, C.E. Kinch, M.S. Debbie & I. Hall (2010): Molecular interactions in cancer cell metastasis. Acta Histochem., 112, 3-25.
Takaya, Y., H. Uchisawa & F. Narumi (1996): Illexins A, B and C from Squid ink should have a branched structure. Biochem. Biophys. Res. Commun., 226(2), 335-338.
Takaya, Y., H. Uchisawa & K. Hanamatsu (1997):
Novel fucose-rich glycosaminoglycans from squid ink bearing repeating unit of trisaccharide structure(-6Gal-NAca1-3GlcAp1 3Fuca-1) . Biochem. Biophys. Res. Commun., 198(2), 560-567.
Tamura, K., K. Nakagawa, T. Kurata, T. Satoh, T. Nogami, K. Takeda, S. Mitsuoka, N. Yoshimura, S. Ku-doh, S. Negoro & al. (2007): Phase I study of TZT-1027, a novel synthetic dolastatin 10 derivative and inhibitor of tubulin polymerization, which was administered to patients with advanced solid tumors on days 1 and 8 in 3-week courses. Cancer Chemother. and Pharmacol., 60, 285-293.
Tetsushi, N., C. Hun-Sik & U. Hidemitsu (2000):
Tyrosinase activity in antitumor compounds of Squid ink. Food Sci.Technol. Res., 6(3), 171-175.
Wang, Y., H. He, G. Wang, H. Wu, B. Zhou, X. Chen & Y. Zhang (2010): Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and immunostimulating effects in BALB/c Mice. Mar. Drugs, 8, 255-268.
Wesson, K. & M. Hamann (1996): Keenamide, A, a bioactive cyclic peptide from the marine mollusk Pleu-robranchus forskalii. J. Nat. Prod., 59, 629-631.
Zeng, M.J., D.M. Zhang & J.H. Chen (2007):
Research advances of antitumor peptides. Chinese J. Biochem. Pharmacol., 28(2), 139-141
Zhong, J.P., G. Wang & J.H. Shang (2009): Protective effects of Squid ink extract towards hemopoietic injuries induced by cyclo- phosphamine. Mar. Drugs, 7(1), 9-18.
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