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Research Paper
Journal of Pharmacy And Pharmacology
Cytotoxic effects on tumour cell lines of fatty acids from the marine sponge Scopalina ruetzleri Renata Biegelmeyera, Rafael Schröderb, Douglas F. Ramboa, Roger R. Drescha, E. Paige Stoutc, João L.F. Carrarod, Beatriz Mothese, José C.F. Moreirab, Tadeusz F. Molinskic, Mário L.C. da Frota Juniorb and Amélia T. Henriquesa a Laboratório de Farmacognosia, Faculdade de Farmácia, bCentro de Estudos em Estresse Oxidativo, Dpto. Bioquímica, Universidade Federal do Rio Grande do Sul (UFRGS), eFundação Zoobotânica, Museu de Ciências Naturais, Porto Alegre, RS, dMuseu Nacional, Departamento de Invertebrados, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil and cDepartment of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA, USA
Keywords cancer; lipid peroxidation; marine sponge; PUFAs; Scopalina ruetzleri Correspondence Renata Biegelmeyer, Laboratório de Farmacognosia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752, 90.610.000, Porto Alegre, RS, Brazil. E-mail: [email protected] Received May 29, 2014 Accepted November 16, 2014 doi: 10.1111/jphp.12366
Abstract Objectives Marine sponges are among the most promising sources of chemically diversified fatty acids (FAs). In addition, several studies have shown the effect of polyunsaturated FAs on cancer therapy. This research carried out a biological and chemical evaluation of the sponge Scopalina ruetzleri collected on the South Brazilian coastline. Methods Bioassay-guided fractionation of S. ruetzleri was performed in human glioma (U87) and neuroblastoma (SH-SY5Y) cell lines, and the in-vitro effects on free radicals were evaluated. Key findings The ethyl acetate fraction of S. ruetzleri showed promising cytotoxic effects in cancer cell lines, with IC50 < 20 μg/ml. Fingerprint 1H Nuclear Magnetic Resonance (NMR) analysis showed that this fraction is mainly constituted of FAs. Through FA methyl ester analysis, it was possible to identify 32 FAs. In addition, some minor unusual FAs for the marine biosphere were identified. The results of conjugated dienes method showed that FAs fraction, at concentrations above 50 μg/ml, has a pro-oxidant effect, indicating that lipid peroxidation may be partially responsible for the mechanism of cytotoxicity on cancer cells. Conclusion This work also contributes to studies that focus on the application of FAs on cancer therapy as a new adjuvant to radio or chemotherapy, or as a chemotherapeutic agent.
Introduction The marine environment has been screened as a potential source of biologically active compounds. Of all the marine organisms, sponges represent one of the most promising sources of chemically diversified fatty acids (FAs), which sometimes have unprecedented or unique structures.[1] The study of the FAs composition of marine organisms is an important contribution to chemotaxonomic biomarkers, given that every species or genus has its own characteristic composition of FAs. Furthermore, polyunsaturated fatty acids (PUFAs) are involved in many physiological roles, and many biological activities have been reported for this class of compounds.[2] Several studies have shown the effect of PUFAs and their metabolites against cancer. Also, it is important to highlight 746
the consensus in the literature that the cytotoxic and/or anti-proliferative action of PUFAs is selective to tumour cells, as normal cells are affected to a much smaller degree, or even unaffected. The mechanism of action of PUFAs is related to oxidative stress, it’s metabolism to active eicosanoids and to changes in membrane composition. Therefore, PUFAs are now viewed as an important adjuvant to radio or chemotherapy, and also as a possible chemotherapeutic agent.[1,3] Of the various forms of cancer, brain tumours are a serious and life-threatening condition. They can destroy and compress normal brain tissue, causing damage that is often disabling and sometimes fatal.[4] In addition, the severity of this kind of tumour has been associated with
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metastatic processes.[5] Gliomas are considered the most devastating primary tumours of the brain, representing 30% of all cases.[6] On the other hand, neuroblastoma is the most common extracranial solid cancer in childhood, observed mainly in children under 1 year of age.[7] Although the Brazilian coastline is extensive, there have been few reports on the screening of Brazilian sponge species for anticancer activity.[8] Scopalina ruetzleri is a marine sponge that deserves special attention, mainly because it has fine and delicate tissue, but despite this, it has high potential to defend the organism. An epibiotic profile was observed for this sponge; that is, it often has the potential to live on other marine sponge host species that interact in the marine environment, and these sponges do not have the ability to cover its delicate tissue.[9] At this moment, only four metabolites were isolated from S. ruetzleri collected in Harrington Sound, Bermuda.[10–12] The first compound isolated was an unusual lipid, 17Ztetracosenyl 1-glycerol ether.[10] More recently, organic extracts of S. ruetzleri have shown cytotoxic activity against human breast cancer cells[13] and antifungal effects against Candida albicans.[14] These data open up very interesting perspectives for the study of this sponge. In this context, the aim of this research was to carry out a biological and chemical evaluation of the marine sponge S. ruetzleri, collected on the South Brazilian coast. We evaluated the cytotoxic effect of fractions of S. ruetzleri in human glioma (U87) and neuroblastoma (SH-SY5Y) cell lines, and also the in-vitro effects on redox properties. For the chemical characterization of bioactive fraction, fatty acid methyl ester (FAMEs) analysis was performed. An ex-vivo method was also used to evaluate the effects on lipid peroxidation. In this paper, we emphasize the assessment of cytotoxicity of a differentiated set of FAs that is characteristic of natural products from marine sponges. This article will help towards a better understanding of the application of FAs as a potential adjuvant to kill cancer cell lines.
Materials and Methods Reagents MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) and Trolox were purchased from Sigma-Aldrich (St Louis, MO, USA). Methanol, ethyl acetate (EtOAc) and hexane (high-performance liquid chromatography (HPLC) grade) were obtained from Tedia (Fairfield, TX, USA).
Sponge collection The marine sponge Scopalina ruetzleri was collected by hand at a depth around −15 m, in locations along the
Cytotoxic fatty acids from S. ruetzleri
coastline of the state of Santa Catarina, Brazil. A specimen of the sponge was deposited at the Museu de Ciências Naturais (Museum of Natural Sciences), Porifera collection of the Fundação Zoobotânica do Rio Grande do Sul, Brazil.
Preparation of extracts and fractions The marine sponge sample was frozen and lyophilized. Extracts were prepared using animal sample (m = 75.1 g – dry weight) in the Ultra-Turrax system (Marconi Ltda, Piracicaba, SP, Brazil) with methanol. The raw methanol extract was partitioned with other solvents to obtain the initial aqueous, hexane and EtOAc fractions, with a yield of 18.72%, 3.92% and 0.46%, respectively. First, the volume of the methanol extracts was reduced. Next, 10% water was added to the methanolic extract. The methanol-aqueous suspension was partitioned against hexane, and then methanol was evaporated using a rotary evaporator. The remaining aqueous suspension was partitioned against EtOAc.
Cell cultures The human glioma (U87) and human neuroblastoma (SHSY5Y) cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Glioma cells were grown and maintained in low glucose Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Carlsbad, CA, USA), containing 0.1% fungizone, 100 U/l gentamicin and supplemented with 10% fetal bovine serum and neuroblastoma cells in a mixture 1:1 of Ham’s F12 and DMEM supplemented with 10% heat-inactivated FBS, 2 mM of glutamine, 0.28 μg/μl of gentamicin and 250 μg of amphotericin B. The cells were kept at 37°C in a humidified atmosphere with 5% CO2. The cell media were replaced every 3 days, and all the treatments were performed when cell confluence reached 70–80%.
Treatments Fractions of S. ruetzleri were dissolved in dimethylsulfoxide (DMSO) to obtain final concentrations of 50 mg/ml (w/v). Successive dilutions were made with DMEM medium to obtain final concentrations ranging from 1.0 to 100 μg/ml. The cultures were treated with sponge extracts and fractions for 24 h. DMSO (0.25% final concentration) was proven not to affect the experiments. Control cultures were performed in the same way, but without the fractions.
Assessment of glioma and neuroblastoma cell viability Cell viability was determined by the reduction of 3-(4,5 dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product by viable cells. After the
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treatments, the medium was discarded and a new medium containing 0.5 mg/ml MTT was added to the wells. The cells were incubated for 45 min at 37°C in a humidified atmosphere with 5% CO2. At the end of incubation, this medium was then discarded, and DMSO was added and left for 30 min to solubilize the formazan crystals. Absorbance was measured at 550 nm (test) and 690 nm (reference) in a SoftMax Pro Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The half maximal inhibitory concentration values (IC50) were estimated from a semilog plot of fractions and extract concentrations versus percentage inhibition of tumour cell line growth.
Evaluation of antioxidant activity using the total reactive antioxidant potential method The total reactive antioxidant potential (TRAP) is widely employed to estimate the antioxidant capacity of samples in vitro. This method is based on the quenching of luminolenhanced chemiluminescence (CL) derived from the thermolysis of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) as the free radical source.[15] The stock solution was prepared with AAPH (10 mm). Luminol (8 nm) in a glycine buffer (0.1 m; pH 8.6) was added. The system was left to stabilize under constant light intensity (2 h) before the first reading. After the stabilization, 20 μl of each sample, Trolox or system (glycine buffer) were placed in a 96-well plate and the stock solution was added to obtain a final volume of 200 μl. The CL produced by the free radical reaction was quantified in a liquid scintillator counter (Wallac 1409, Perkin Elmer, Boston, MA, USA; with 10 s defined as the count time, with a total of 3000 s). The samples (concentration of 50 mg/ml) were diluted with glycine buffer to reach the final concentrations (1, 10, 25, 50 and 100 μg/ml) and the final percentage of DMSO (0.25%) was proven not to affect the system. Trolox was prepared with glycine buffer. The results were expressed as the plotting percentage of counts per minute (% cpm) versus time (s) and area under the curve (AUC). Trolox equivalent antioxidant capacity (TEAC) was calculated using the standard curve, which was obtained by plotting the concentration of Trolox and the AUC (between 0.05 and 0.4 μm Trolox). Conjugated dienes The conjugated dienes method was performed according to Recknagel and Glende[16] with some modifications. A rat liver homogenate was prepared (0.16 g/ml) in saline buffer and incubated with samples or controls for 60 min. Trolox (200 nm in 0.1 m glycine buffer, pH 8.6) was used as antioxidant agent, and hydrogen peroxide (20 mm) as positive control. The residue of chloroform-free lipid was dissolved in 2 ml of cyclohexane and the optical density (1 cm light 748
path) was recorded at 233 and 242 nm, as described for Corongiu and Banni.[17] The results were expressed as % absorbance in comparison with Trolox. The sample blank was performed by incubating the liver homogenate with the sample, without hydrogen peroxide. Nuclear Magnetic Resonance (NMR) analysis NMR fingerprint analysis of S. ruetzleri EtOAc fraction was recorded on a Jeol spectrometer (Jeol, Pleasanton, CA, USA) equipped with a two channel 1H,19F(15N,31) inversedetect probe operating at 500 MHz. The 1H spectrum is referenced to residual solvent signal for CDCl3 (1H, δ 7.26 ppm). Fatty acid methyl ester transesterification FAMEs were obtained according a procedure developed by MIDI Inc. (Newark DE, USA). Approximately 5 mg of EtOAc fraction was placed in a round-bottomed tube with a screw-cap, and 1 ml of a solution 3.75 m NaOH in MeOH/ H2O solution was added and vigorously vortexed for 5–10 s. The tube was then placed in a hot water bath (100°C) for 30 min. Two millilitres of the esterification solution (6 N HCl/MeOH) were added to the tube and briefly vortexed. After vortexing, the tube was returned to the hot water bath (100°C) for 10 min and immediately cooled to room temperature. The extraction of the FAMEs was performed with a solution of (1:1, v/v) hexane : methyl tert-butyl ether. This extract was mixed with 1.2% NaOH in H2O and then the organic phase was injected into a gas chromatography (GC). Fatty acid methyl esters analysis FAMEs analysis was performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Wilmington, DE, USA) equipped with a flame ionization detector and connected with MIS Sherlock (MIDI, Inc.) and Agilent ChemStation software. FAMEs were separated on an Agilent Ultra 2 column, 25 m × 0.2 mm phenyl methyl siliconefused silica capillary. The temperature programme ramp was from 170°C to 270°C at 5°C/min. The temperature was then increased to 300°C for 2 min to clean the column. Hydrogen was the carrier gas, and nitrogen was the ‘make up’ gas.
Statistical analysis The in-vitro and ex-vivo procedures were carried out with n = 3, whereas cell culture experiments were conducted with n = 6 in two independent experiments. Data were expressed as mean ± standard error of the mean (SEM). The results were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. In all cases,
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 746–753
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Cytotoxic fatty acids from S. ruetzleri
toma cell lines. A significant drop in glioma and neuroblastoma cell viability was observed after treatment for 24 h with EtOAc fraction of S. ruetzleri. However, the hexane and aqueous fractions did not appear to affect the viability of the cancer cells. The cytotoxic effect of EtOAc fraction was slightly higher for neuroblastoma cells, with an IC50 around 10 μg/ml, compared with the effects on glioma cells (IC50 = 18.35 μg/ml). Redox properties of marine sponge fractions were evaluated using a method based on the quenching of luminolenhanced CL of AAPH and were compared with the antioxidant standard Trolox (water-soluble vitamin E analogue). Significant dose-dependent antioxidant effects of S. ruetzleri fractions are shown in Figures 1–2. The EtOAc fraction (10 μg/ml) and hexane fraction (50 μg/ml) showed an AUC comparable with Trolox (Figure 1), but a slightly different CL profile, because these fractions do not have the ability to block the production of free radical to basal levels, as Trolox does (Figure 2). The aqueous fraction of
differences were considered statistically significant if P < 0.05. Data analyses were performed using the GraphPad software version 5.00 (San Diego, CA, USA).
Results Table 1 shows the antitumor results of S. ruetzleri fractions in the U87 human glioma and SH-SY5Y human neuroblas-
Table 1 In-vitro growth inhibitory activity against human glioma and neuroblastoma cell lines and antioxidant effect expressed as Trolox equivalent antioxidant capacity (TEAC) of S. ruetzleri fractions IC50 (μg/ml)a
S. ruetzleri fractions
U87
SH-SY5Y
TEAC (μM Trolox/g marine sponge)
Ethyl acetate Aqueous Hexane
18.35 ± 1.03 na na
10.51 ± 1.12 na na
21.17 4.32 5.46
na, not active. aAll values given as mean ± standard error of the mean.
(a)
400000
(b)
(c) *
AUC
300000 200000
*
*
*
*
*
* *
*
*
* 100000
*
*
0 Trolox 1 10 25 50 100 Trolox 1 10 25 50 100 Trolox 1 10 25 50 100 Concentration (µg/ml)
Chemiluminescence (%cpm)
Figure 1 Total reactive antioxidant potential (TRAP) from marine sponge S. ruetzleri: ethyl acetate (EtOAc) fraction (a) hexane fraction (b) and aqueous fraction (c). The effect of different concentrations of fractions on free radical-induced chemiluminescence (CL) was measured as area under curve (AUC). Trolox (0.05 μg/ml) was used as standard antioxidant. Bars represent mean ± standard error of the mean. *P < 0.05 indicates a significant difference compared to control (one-way analysis of variance followed by Tukey’s test).
150 10 µg/ml EtOAc Fraction 50 µg/ml Aqueous Fraction
100
50 µg/ml Hexane Fraction Trolox 200 nM 50
0 0
1000
2000
3000
Time (min) Figure 2 Chemiluminescence (CL) intensity (% cpm) measured after the addition of S. ruetzleri fractions. The CL profile of samples is shown for the concentration that exhibited similar area under curve (AUC) to the standard Trolox. EtOAc, ethyl acetate. © 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 746–753
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Table 2
Renata Biegelmeyer et al.
FA composition of ethyl acetate fraction of marine sponge S. ruetzleri, expressed as % of total FAs
Name of FAMEs (IUPAC name)
Retention time (min)
% of FAMEs
Dodecanoic acid Tetradecanoic acid (3Z,6Z,9Z,12Z)-3,6,9,12-Pentadecatetraenoic acid 13-Methyltetradecanoic acid 12-Methyltetradecanoic acid Pentadecanoic acid 14-Methylpentadecanoic acid 13-Methylpentadecanoic acid (9Z)-9-Hexadecenoic acida Hexadecanoic acidb 10-Methylhexadecanoic acid (9Z)-13-Methyl-9-Hexadecenoic acid 15-Methylhexadecanoic acid 14-Methylhexadecanoic acid cis-9,10-Methylene-Hexadecanoic acid (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid (9Z,12Z)-9,12-Octadecadienoic acid (9Z)-9-Octadecenoic acid (11Z)-11-Octadecenoic acid Octadecanoic acid (11Z)-10-Methyl-11-Octadecenoic acid 1,1-Dimethoxyoctadecane 17-Methyloctadecanoic acid Nonadecanoic (5Z,8Z,11Z,14Z)-5,8,11,14-Icosatetraenoic acidc (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-Icosapentaenoic acidd (11Z,14Z,17Z)-11,14,17-Icosatrienoic acid 18-Methylnonadecanoic acid (11Z,14Z)-11,14-Icosadienoic acid (11Z)-11-Icosenoic acid cis-13,14-Methylene-Nonadecanoic acid (4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-Docosahexaenoic acide ΣSFA ΣMUFA ΣPUFA ΣTUFA
12.00 13.99 14.49 14.62 14.71 15.00 15.62 15.74 15.82 15.99 16.41 16.57 16.62 16.72 16.89 17.58 17.72 17.77 17.82 17.99 18.08 18.46 18.63 18.99 19.40 19.47 19.56 19.63 19.73 19.81 19.96 21.32
0.4 2.31 19.15 1.12 0.25 0.49 1.09 1.21 8.85 8.1 0.65 1.07 0.87 1.15 0.37 1.39 1.38 1.63 4.82 6.93 0.3 0.33 0.51 0.33 10.9 17.25 0.56 0.46 1.16 0.65 3.2 1.1 29.77 17.32 52.89 70.21
Other names: apalmitoleic acid; bpalmitic acid; carachidonic acid (AA); deicosapentaenoic acid (EPA); edocosahexaenoic acid (DHA). FA, fatty acid; FAME, fatty acid methyl ester; IUPAC, International Union of Pure and Applied Chemistry; MUFA, monounsaturated fatty acid; PU, polyunsaturated fatty acids; SFA, saturated fatty acid; TUFA, Total Unsaturated Fatty Acid.
S. ruetzleri presented an interesting CL profile: it initially led to increased free radical levels, which dropped a few seconds later (Figure 2). On the other hand, the TEAC allows comparison of the antioxidant activity of marine sponge fractions. The results of TEAC are summarized in Table 1. Analysing these results, it can be demonstrated that of the fractions tested, the EtOAc fraction was the one that exerted the highest antioxidant capacity, with a TEAC value of around 20 μg/g of marine sponge. The bioassay-guided fractionation showed the EtOAc fraction to be a promising source of bioactive metabolites. Based on NMR proton analysis, EtOAc fraction of S. ruetzleri is mainly constituted of FAs (Figure S1). A total of 32 FAs were identified by GC–FAME analysis (Table 2), most of these consisting of PUFAs, representing 52.89%, 750
followed by 29.77% saturated fatty acids (SFAs) and 17.32% of monounsaturated fatty acids (MUFAs). The major compounds were PUFAs: (3Z,6Z,9Z,12Z)-3,6,9,12pentadecatetraenoic acid (19.15%), eicosapentaenoic acid (EPA; 17.25%) and arachidonic acid (AA; 10.9%). The main MUFAs and SFAs were palmitoleic acid (8.85%) and palmitic acid (8.1%), respectively. As minor compounds, several long-chain branched FAs were identified. In addition, some minor compounds were identified that are unusual for marine organisms (Figure 3): ciclopropane FAs (1–2), methoxylated (3) and also two long-chain branched unsaturated FAs (4–5). To evaluate the cytotoxic mechanism of FAs, the EtOAc fraction was assayed using the conjugated dienes method. The EtOAc fraction demonstrated an antioxidant and
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 746–753
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Cytotoxic fatty acids from S. ruetzleri
O C
13
(1)
19
OH O
16
C
9
(2) OH
O
CH3 (3) CH3 O
18
O
9
C
CH3
(4) OH
17 13 11
O C
10
(5) OH
CH3 18
#
#
50
nM 2 10 0 n M 0 µg / 50 m µg l 25 /m µg l 10 /m µg l / 1 ml µg /m l
0
* 100
#
#
#
50
0
x lo Tr o
x
2O 2
H
lo Tr o
Concentration (µg/ml)
*
nM 2 10 0 n 0 M µg 50 /m µg l 25 /m µg l 10 /m µg l / 1 ml µg /m l
100
#
20 0
*
150
2O 2
*#
(b)
H
150
% absorbance in comparison with Trolox (242 nM)
(a)
20 0
% absorbance in comparison with Trolox (233 nM)
Figure 3 Unusual fatty acids identified for S. ruetzleri: cis-13,14-Methylene-Nonadecanoic acid (1); cis-9,10-Methylene-Hexadecanoic acid (2); 1,1Dimethoxyoctadecane (3); (9Z)-13-Methyl-9-Hexadecenoic acid (4); (11Z)-10-Methyl-11-Octadecenoic acid (5).
Concentration (µg/ml)
Figure 4 Conjugated dienes from marine sponge S. ruetzleri: 233 nm (a) and 242 nm (b). The effect of different concentrations of ethyl acetate (EtOAc) fraction on lipid peroxidation was measured as % absorbance in comparison with Trolox effects (0.05 μg/ml). Bars represent mean ± standard error of the mean. *P < 0.05 indicates a significant difference compared with Trolox. #P < 0.05 indicates a significant difference compared to H2O2 (one-way analysis of variance followed by Tukey’s test).
pro-oxidant profile (Figure 4a–b). At concentrations below 25 μg/ml, a protective effect of lipid peroxidation was observed, similar to Trolox. On the other hand, the EtOAc fraction at concentrations above 50 μg/ml, showed increased lipid peroxidation, because the effect was comparable or higher than that of H2O2.
Discussion Considering that S. ruetzleri has delicate tissue, and in view of its epibiotic profile,[9] its potential for chemical defence is evident. The cytotoxic activity of S. ruetzleri was also previously reported by Prado et al.,[13] although this study only
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investigated crude extracts of S. ruetzleri. The methanol extract showed a cytotoxic effect against the human breast tumour cell line (T4TD) and had strong effects on microtubule organization and cell cycle progression.[14] Our results demonstrated significant cytotoxic effects on glioma and neuroblastoma human cell lines and also lower IC50 due to the bioassay-guided fractionation of methanol extract of S. ruetzleri. Prior to the present study, chemical analysis had only been performed only for S. ruetzleri collected in Harrington Sound, Bermuda. Vanwagenen et al.[12] isolated a phosphorylated hydantoin and another structurally related compound. Cardellina II et al.[11] isolated an indolic metabolite, and the first compound identified for this sponge was an unusual lipid, 17Z-Tetracosenyl 1-Glycerol Ether.[10] To the best of our knowledge, our study is the first report on the chemical analysis of S. ruetzleri collected in the Brazilian coastline; on the other hand, the presence of lipid compounds found in our study is in agreement with the initial report for this sponge species. The ability to induce a cytotoxic effect by FAs varies depending on the number of double bonds, and on the carbon chain length. Generally, saturated and monounsaturated FAs are least able to induce an effect than PUFAs. AA (20:4 n-6), EPA (20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) are the most widely reported FAs, with strong effects on cancer therapy.[2,3,18] This could explain the potential cytotoxic activity observed for EtOAc fraction of S. ruetzleri, consisting mainly of PUFAs, especially AA and EPA. There is increasing evidence that oxidative processes play an important part in the initiation and progression of cancer.[19] Strategies that modulate the cellular redox potential are being applied to kill cancer cells,[20] and several studies have demonstrated that lipid peroxidation is the major mechanism of FAs, especially PUFAs action on cancer cells.[3,18] The ability to lose a hydrogen atom from the PUFA double bond is responsible for initiating lipid peroxidation due to the production of reactive species that can propagate further reactions, and also act on the macromolecules causing the damage.[3] Here, we found that EtOAc fraction of S. ruetzleri has the potential to modulate free radicals, because we observed in-vitro and ex-vivo antioxidant potential and also ex-vivo pro-oxidant effect in higher concentrations, indicating that lipid peroxidation is partially responsible for the cytotoxic effect of EtOAc fraction of S. ruetzleri in neuroblastoma and glioma human cell lines. The inhibition of lipid peroxidation by EtOAc fraction in low concentrations can be linked to antioxidant potential of
References 1. Pereira DM et al. Fatty acids in marine organisms: in the pursuit of bioactive 752
PUFAs. Some authors support that the antioxidant potential of PUFAs is related to the inhibition of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase, which is one of the major responsible for production of reactive oxygen species (ROS) in endothelial. Moreover, PUFAs can act as a ‘skin’ trapping free radicals and becoming oxidized.[21,22] However, this oxidation produce lipid peroxides, which can explain the results observed in Figure 4: in higher concentrations, an excess of peroxides can increase lipid peroxidation and in lower concentrations can protect. Lipid peroxidation evaluation of FAs is generally carried out using the thiobarbituric acid reactive method (TBARS).[3] However, the application of the conjugate dienes method brings some important advantages in relation to TBARS: (1) it is able to measure early lipid peroxidation reaction events, whereas TBARs measures end products; (2) greater sensitivity; (3) the fact that it is an ex-vivo experiment.[23] In conclusion, this work sheds new light on the bioactive potential and chemical analysis of the marine sponge Scopalina ruetzleri collected along the Brazilian south coast. We performed a bioassay-guided fractionation of this sponge for anticancer activity. The results demonstrate a potential cytotoxic effect in human glioma and neuroblastoma cell lines for the EtOAc fraction of this marine sponge. Chemical analysis of this active fraction led to the identification of 32 FAs, consisting mainly of PUFAs. The majority of FAs identified are commonly found in marine sponges; however, some minor compounds were also identified that are unusual for the marine biosphere. Moreover, our findings suggest that lipid peroxidation may be partially responsible for the mechanism of the EtOAc fraction on cancer cells. This work therefore shows the importance of bioassayguided studies on marine sponges using fractions, focusing on the active metabolite. In particular, it contributes to studies focusing on the application of FAs in cancer therapy, as a new adjuvant to radio- or chemotherapy or as a chemotherapeutic agent.
Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.
Funding This work was supported by CNPq and CAPES.
agents. Curr Pharm Anal 2011; 7: 108– 119. 2. Bergé J-P, Barnathan G. Fatty acids from lipids of marine organisms: mole-
cular biodiversity, roles as biomarkers, biologically active compounds, and economical aspects. Adv Biochem Eng Biotechnol 2005; 96: 49–125.
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3. Diggle CP. In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects? Prog Lipid Res 2002; 41: 240–253. 4. American Cancer Society. Information and resources for cancer. 2013. http://www.cancer.org/Cancer/index (Accessed December 13, 2013). 5. DeAngelis LM, Posner MD. Brain metastases. In: Kufe DW et al., eds. Holland–Frei Cancer Medicine. Hamilton, ON: BC Decker, 2004: 1227–1231. 6. Binello E, Germano IM. Targeting glioma stem cells: a novel framework for brain tumors. Cancer Sci 2011; 102: 1958–1966. 7. Park JR et al. Neuroblastoma: biology, prognosis, and treatment. Hematol Oncol Clin North Am 2010; 24: 65–86. 8. Frota Junior MLC et al. Current status on natural products with antitumor activity from Brazilian marine sponges. Curr Pharm Biotechnol 2012; 13: 235–244. 9. Engel E, Pawlik JR. Interactions among Florida sponges. I. Reef habitats. Mar Ecol Prog Ser 2005; 303: 133– 144. 10. Cardellina JH II et al. 17Z-tetracosenyl 1-glycerol ether from the sponges Cinachyra alloclada and Ulosa ruetzleri. Lipids 1983; 18: 107–110. 11. Cardellina JH II et al. Plant growth regulatory indoles from the sponges
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12.
13.
14.
15.
16.
17.
18.
19.
Dysidea etheria and Ulosa ruetzleri. J Nat Prod 1986; 49: 1065–1067. Vanwagenen BC et al. Ulosantoin, a potent insecticide from the sponge UIosa ruetzleri. J Org Chem 1993; 58: 335–337. Prado MP et al. Effects of marine organisms extracts on microtubule integrity and cell cycle in cultured cells. J Exp Mar Biol Ecol 2004; 313: 125–137. Galeano E, Martínez A. Antimicrobial activity of marine sponges from Urabá Gulf, Colombian Caribbean region. J Mycol Med 2007; 17: 21–24. Dresch MTK et al. Optimization and validation of an alternative method to evaluate total reactive antioxidant potential (TRAP). Anal Biochem 2009; 385: 107–114. Recknagel RO, Glende EA. Spectrophotometric detection of lipid conjugated dienes. Methods Enzymol 1984; 105: 331–337. Corongiu FP, Banni S. Detection of conjugated dienes by second derivative ultraviolet spectrophotometry. Methods Enzymol 1994; 233: 303–310. Hossain Z et al. Growth inhibition and induction of apoptosis of colon cancer cell lines by applying marine phospholipid. Nutr Cancer 2009; 61: 123–130. Trachootham D et al. Targeting cancer cells by ROS-mediated mechanisms: a
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20.
21.
22.
23.
radical therapeutic approach? Nat Rev Drug Discov 2009; 8: 579–591. Booy EP et al. The immune system, involvement in neurodegenerative diseases, ageing and cancer. Curr Med Chem Anti Inflamm Anti Allergy Agents 2005; 4: 349–352. Massaro M et al. The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKC epsilon inhibition. Proc Natl Acad Sci U S A 2006; 103: 15184– 15189. Doriane R et al. Polyunsaturated fatty acids as antioxidants. Pharmacol Res 2008; 57: 451–455. Vasankari T et al. Measurement of serum lipid peroxidation during exercise using three different methods: diene conjugation, thiobarbituric acid reactive material and fluorescent chromolipids. Clin Chim Acta 1995; 234: 63–69.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. S. ruetzleri 500 MHz).
1
H NMR spectra of EtOAc fraction (CDCl3,
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Research Paper
Journal of Pharmacy And Pharmacology
Cytotoxic effects on tumour cell lines of fatty acids from the marine sponge Scopalina ruetzleri Renata Biegelmeyera, Rafael Schröderb, Douglas F. Ramboa, Roger R. Drescha, E. Paige Stoutc, João L.F. Carrarod, Beatriz Mothese, José C.F. Moreirab, Tadeusz F. Molinskic, Mário L.C. da Frota Juniorb and Amélia T. Henriquesa a Laboratório de Farmacognosia, Faculdade de Farmácia, bCentro de Estudos em Estresse Oxidativo, Dpto. Bioquímica, Universidade Federal do Rio Grande do Sul (UFRGS), eFundação Zoobotânica, Museu de Ciências Naturais, Porto Alegre, RS, dMuseu Nacional, Departamento de Invertebrados, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil and cDepartment of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA, USA
Keywords cancer; lipid peroxidation; marine sponge; PUFAs; Scopalina ruetzleri Correspondence Renata Biegelmeyer, Laboratório de Farmacognosia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga 2752, 90.610.000, Porto Alegre, RS, Brazil. E-mail: [email protected] Received May 29, 2014 Accepted November 16, 2014 doi: 10.1111/jphp.12366
Abstract Objectives Marine sponges are among the most promising sources of chemically diversified fatty acids (FAs). In addition, several studies have shown the effect of polyunsaturated FAs on cancer therapy. This research carried out a biological and chemical evaluation of the sponge Scopalina ruetzleri collected on the South Brazilian coastline. Methods Bioassay-guided fractionation of S. ruetzleri was performed in human glioma (U87) and neuroblastoma (SH-SY5Y) cell lines, and the in-vitro effects on free radicals were evaluated. Key findings The ethyl acetate fraction of S. ruetzleri showed promising cytotoxic effects in cancer cell lines, with IC50 < 20 μg/ml. Fingerprint 1H Nuclear Magnetic Resonance (NMR) analysis showed that this fraction is mainly constituted of FAs. Through FA methyl ester analysis, it was possible to identify 32 FAs. In addition, some minor unusual FAs for the marine biosphere were identified. The results of conjugated dienes method showed that FAs fraction, at concentrations above 50 μg/ml, has a pro-oxidant effect, indicating that lipid peroxidation may be partially responsible for the mechanism of cytotoxicity on cancer cells. Conclusion This work also contributes to studies that focus on the application of FAs on cancer therapy as a new adjuvant to radio or chemotherapy, or as a chemotherapeutic agent.
Introduction The marine environment has been screened as a potential source of biologically active compounds. Of all the marine organisms, sponges represent one of the most promising sources of chemically diversified fatty acids (FAs), which sometimes have unprecedented or unique structures.[1] The study of the FAs composition of marine organisms is an important contribution to chemotaxonomic biomarkers, given that every species or genus has its own characteristic composition of FAs. Furthermore, polyunsaturated fatty acids (PUFAs) are involved in many physiological roles, and many biological activities have been reported for this class of compounds.[2] Several studies have shown the effect of PUFAs and their metabolites against cancer. Also, it is important to highlight 746
the consensus in the literature that the cytotoxic and/or anti-proliferative action of PUFAs is selective to tumour cells, as normal cells are affected to a much smaller degree, or even unaffected. The mechanism of action of PUFAs is related to oxidative stress, it’s metabolism to active eicosanoids and to changes in membrane composition. Therefore, PUFAs are now viewed as an important adjuvant to radio or chemotherapy, and also as a possible chemotherapeutic agent.[1,3] Of the various forms of cancer, brain tumours are a serious and life-threatening condition. They can destroy and compress normal brain tissue, causing damage that is often disabling and sometimes fatal.[4] In addition, the severity of this kind of tumour has been associated with
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metastatic processes.[5] Gliomas are considered the most devastating primary tumours of the brain, representing 30% of all cases.[6] On the other hand, neuroblastoma is the most common extracranial solid cancer in childhood, observed mainly in children under 1 year of age.[7] Although the Brazilian coastline is extensive, there have been few reports on the screening of Brazilian sponge species for anticancer activity.[8] Scopalina ruetzleri is a marine sponge that deserves special attention, mainly because it has fine and delicate tissue, but despite this, it has high potential to defend the organism. An epibiotic profile was observed for this sponge; that is, it often has the potential to live on other marine sponge host species that interact in the marine environment, and these sponges do not have the ability to cover its delicate tissue.[9] At this moment, only four metabolites were isolated from S. ruetzleri collected in Harrington Sound, Bermuda.[10–12] The first compound isolated was an unusual lipid, 17Ztetracosenyl 1-glycerol ether.[10] More recently, organic extracts of S. ruetzleri have shown cytotoxic activity against human breast cancer cells[13] and antifungal effects against Candida albicans.[14] These data open up very interesting perspectives for the study of this sponge. In this context, the aim of this research was to carry out a biological and chemical evaluation of the marine sponge S. ruetzleri, collected on the South Brazilian coast. We evaluated the cytotoxic effect of fractions of S. ruetzleri in human glioma (U87) and neuroblastoma (SH-SY5Y) cell lines, and also the in-vitro effects on redox properties. For the chemical characterization of bioactive fraction, fatty acid methyl ester (FAMEs) analysis was performed. An ex-vivo method was also used to evaluate the effects on lipid peroxidation. In this paper, we emphasize the assessment of cytotoxicity of a differentiated set of FAs that is characteristic of natural products from marine sponges. This article will help towards a better understanding of the application of FAs as a potential adjuvant to kill cancer cell lines.
Materials and Methods Reagents MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide) and Trolox were purchased from Sigma-Aldrich (St Louis, MO, USA). Methanol, ethyl acetate (EtOAc) and hexane (high-performance liquid chromatography (HPLC) grade) were obtained from Tedia (Fairfield, TX, USA).
Sponge collection The marine sponge Scopalina ruetzleri was collected by hand at a depth around −15 m, in locations along the
Cytotoxic fatty acids from S. ruetzleri
coastline of the state of Santa Catarina, Brazil. A specimen of the sponge was deposited at the Museu de Ciências Naturais (Museum of Natural Sciences), Porifera collection of the Fundação Zoobotânica do Rio Grande do Sul, Brazil.
Preparation of extracts and fractions The marine sponge sample was frozen and lyophilized. Extracts were prepared using animal sample (m = 75.1 g – dry weight) in the Ultra-Turrax system (Marconi Ltda, Piracicaba, SP, Brazil) with methanol. The raw methanol extract was partitioned with other solvents to obtain the initial aqueous, hexane and EtOAc fractions, with a yield of 18.72%, 3.92% and 0.46%, respectively. First, the volume of the methanol extracts was reduced. Next, 10% water was added to the methanolic extract. The methanol-aqueous suspension was partitioned against hexane, and then methanol was evaporated using a rotary evaporator. The remaining aqueous suspension was partitioned against EtOAc.
Cell cultures The human glioma (U87) and human neuroblastoma (SHSY5Y) cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Glioma cells were grown and maintained in low glucose Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Carlsbad, CA, USA), containing 0.1% fungizone, 100 U/l gentamicin and supplemented with 10% fetal bovine serum and neuroblastoma cells in a mixture 1:1 of Ham’s F12 and DMEM supplemented with 10% heat-inactivated FBS, 2 mM of glutamine, 0.28 μg/μl of gentamicin and 250 μg of amphotericin B. The cells were kept at 37°C in a humidified atmosphere with 5% CO2. The cell media were replaced every 3 days, and all the treatments were performed when cell confluence reached 70–80%.
Treatments Fractions of S. ruetzleri were dissolved in dimethylsulfoxide (DMSO) to obtain final concentrations of 50 mg/ml (w/v). Successive dilutions were made with DMEM medium to obtain final concentrations ranging from 1.0 to 100 μg/ml. The cultures were treated with sponge extracts and fractions for 24 h. DMSO (0.25% final concentration) was proven not to affect the experiments. Control cultures were performed in the same way, but without the fractions.
Assessment of glioma and neuroblastoma cell viability Cell viability was determined by the reduction of 3-(4,5 dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product by viable cells. After the
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treatments, the medium was discarded and a new medium containing 0.5 mg/ml MTT was added to the wells. The cells were incubated for 45 min at 37°C in a humidified atmosphere with 5% CO2. At the end of incubation, this medium was then discarded, and DMSO was added and left for 30 min to solubilize the formazan crystals. Absorbance was measured at 550 nm (test) and 690 nm (reference) in a SoftMax Pro Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The half maximal inhibitory concentration values (IC50) were estimated from a semilog plot of fractions and extract concentrations versus percentage inhibition of tumour cell line growth.
Evaluation of antioxidant activity using the total reactive antioxidant potential method The total reactive antioxidant potential (TRAP) is widely employed to estimate the antioxidant capacity of samples in vitro. This method is based on the quenching of luminolenhanced chemiluminescence (CL) derived from the thermolysis of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) as the free radical source.[15] The stock solution was prepared with AAPH (10 mm). Luminol (8 nm) in a glycine buffer (0.1 m; pH 8.6) was added. The system was left to stabilize under constant light intensity (2 h) before the first reading. After the stabilization, 20 μl of each sample, Trolox or system (glycine buffer) were placed in a 96-well plate and the stock solution was added to obtain a final volume of 200 μl. The CL produced by the free radical reaction was quantified in a liquid scintillator counter (Wallac 1409, Perkin Elmer, Boston, MA, USA; with 10 s defined as the count time, with a total of 3000 s). The samples (concentration of 50 mg/ml) were diluted with glycine buffer to reach the final concentrations (1, 10, 25, 50 and 100 μg/ml) and the final percentage of DMSO (0.25%) was proven not to affect the system. Trolox was prepared with glycine buffer. The results were expressed as the plotting percentage of counts per minute (% cpm) versus time (s) and area under the curve (AUC). Trolox equivalent antioxidant capacity (TEAC) was calculated using the standard curve, which was obtained by plotting the concentration of Trolox and the AUC (between 0.05 and 0.4 μm Trolox). Conjugated dienes The conjugated dienes method was performed according to Recknagel and Glende[16] with some modifications. A rat liver homogenate was prepared (0.16 g/ml) in saline buffer and incubated with samples or controls for 60 min. Trolox (200 nm in 0.1 m glycine buffer, pH 8.6) was used as antioxidant agent, and hydrogen peroxide (20 mm) as positive control. The residue of chloroform-free lipid was dissolved in 2 ml of cyclohexane and the optical density (1 cm light 748
path) was recorded at 233 and 242 nm, as described for Corongiu and Banni.[17] The results were expressed as % absorbance in comparison with Trolox. The sample blank was performed by incubating the liver homogenate with the sample, without hydrogen peroxide. Nuclear Magnetic Resonance (NMR) analysis NMR fingerprint analysis of S. ruetzleri EtOAc fraction was recorded on a Jeol spectrometer (Jeol, Pleasanton, CA, USA) equipped with a two channel 1H,19F(15N,31) inversedetect probe operating at 500 MHz. The 1H spectrum is referenced to residual solvent signal for CDCl3 (1H, δ 7.26 ppm). Fatty acid methyl ester transesterification FAMEs were obtained according a procedure developed by MIDI Inc. (Newark DE, USA). Approximately 5 mg of EtOAc fraction was placed in a round-bottomed tube with a screw-cap, and 1 ml of a solution 3.75 m NaOH in MeOH/ H2O solution was added and vigorously vortexed for 5–10 s. The tube was then placed in a hot water bath (100°C) for 30 min. Two millilitres of the esterification solution (6 N HCl/MeOH) were added to the tube and briefly vortexed. After vortexing, the tube was returned to the hot water bath (100°C) for 10 min and immediately cooled to room temperature. The extraction of the FAMEs was performed with a solution of (1:1, v/v) hexane : methyl tert-butyl ether. This extract was mixed with 1.2% NaOH in H2O and then the organic phase was injected into a gas chromatography (GC). Fatty acid methyl esters analysis FAMEs analysis was performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Wilmington, DE, USA) equipped with a flame ionization detector and connected with MIS Sherlock (MIDI, Inc.) and Agilent ChemStation software. FAMEs were separated on an Agilent Ultra 2 column, 25 m × 0.2 mm phenyl methyl siliconefused silica capillary. The temperature programme ramp was from 170°C to 270°C at 5°C/min. The temperature was then increased to 300°C for 2 min to clean the column. Hydrogen was the carrier gas, and nitrogen was the ‘make up’ gas.
Statistical analysis The in-vitro and ex-vivo procedures were carried out with n = 3, whereas cell culture experiments were conducted with n = 6 in two independent experiments. Data were expressed as mean ± standard error of the mean (SEM). The results were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. In all cases,
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toma cell lines. A significant drop in glioma and neuroblastoma cell viability was observed after treatment for 24 h with EtOAc fraction of S. ruetzleri. However, the hexane and aqueous fractions did not appear to affect the viability of the cancer cells. The cytotoxic effect of EtOAc fraction was slightly higher for neuroblastoma cells, with an IC50 around 10 μg/ml, compared with the effects on glioma cells (IC50 = 18.35 μg/ml). Redox properties of marine sponge fractions were evaluated using a method based on the quenching of luminolenhanced CL of AAPH and were compared with the antioxidant standard Trolox (water-soluble vitamin E analogue). Significant dose-dependent antioxidant effects of S. ruetzleri fractions are shown in Figures 1–2. The EtOAc fraction (10 μg/ml) and hexane fraction (50 μg/ml) showed an AUC comparable with Trolox (Figure 1), but a slightly different CL profile, because these fractions do not have the ability to block the production of free radical to basal levels, as Trolox does (Figure 2). The aqueous fraction of
differences were considered statistically significant if P < 0.05. Data analyses were performed using the GraphPad software version 5.00 (San Diego, CA, USA).
Results Table 1 shows the antitumor results of S. ruetzleri fractions in the U87 human glioma and SH-SY5Y human neuroblas-
Table 1 In-vitro growth inhibitory activity against human glioma and neuroblastoma cell lines and antioxidant effect expressed as Trolox equivalent antioxidant capacity (TEAC) of S. ruetzleri fractions IC50 (μg/ml)a
S. ruetzleri fractions
U87
SH-SY5Y
TEAC (μM Trolox/g marine sponge)
Ethyl acetate Aqueous Hexane
18.35 ± 1.03 na na
10.51 ± 1.12 na na
21.17 4.32 5.46
na, not active. aAll values given as mean ± standard error of the mean.
(a)
400000
(b)
(c) *
AUC
300000 200000
*
*
*
*
*
* *
*
*
* 100000
*
*
0 Trolox 1 10 25 50 100 Trolox 1 10 25 50 100 Trolox 1 10 25 50 100 Concentration (µg/ml)
Chemiluminescence (%cpm)
Figure 1 Total reactive antioxidant potential (TRAP) from marine sponge S. ruetzleri: ethyl acetate (EtOAc) fraction (a) hexane fraction (b) and aqueous fraction (c). The effect of different concentrations of fractions on free radical-induced chemiluminescence (CL) was measured as area under curve (AUC). Trolox (0.05 μg/ml) was used as standard antioxidant. Bars represent mean ± standard error of the mean. *P < 0.05 indicates a significant difference compared to control (one-way analysis of variance followed by Tukey’s test).
150 10 µg/ml EtOAc Fraction 50 µg/ml Aqueous Fraction
100
50 µg/ml Hexane Fraction Trolox 200 nM 50
0 0
1000
2000
3000
Time (min) Figure 2 Chemiluminescence (CL) intensity (% cpm) measured after the addition of S. ruetzleri fractions. The CL profile of samples is shown for the concentration that exhibited similar area under curve (AUC) to the standard Trolox. EtOAc, ethyl acetate. © 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 746–753
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Table 2
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FA composition of ethyl acetate fraction of marine sponge S. ruetzleri, expressed as % of total FAs
Name of FAMEs (IUPAC name)
Retention time (min)
% of FAMEs
Dodecanoic acid Tetradecanoic acid (3Z,6Z,9Z,12Z)-3,6,9,12-Pentadecatetraenoic acid 13-Methyltetradecanoic acid 12-Methyltetradecanoic acid Pentadecanoic acid 14-Methylpentadecanoic acid 13-Methylpentadecanoic acid (9Z)-9-Hexadecenoic acida Hexadecanoic acidb 10-Methylhexadecanoic acid (9Z)-13-Methyl-9-Hexadecenoic acid 15-Methylhexadecanoic acid 14-Methylhexadecanoic acid cis-9,10-Methylene-Hexadecanoic acid (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid (9Z,12Z)-9,12-Octadecadienoic acid (9Z)-9-Octadecenoic acid (11Z)-11-Octadecenoic acid Octadecanoic acid (11Z)-10-Methyl-11-Octadecenoic acid 1,1-Dimethoxyoctadecane 17-Methyloctadecanoic acid Nonadecanoic (5Z,8Z,11Z,14Z)-5,8,11,14-Icosatetraenoic acidc (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-Icosapentaenoic acidd (11Z,14Z,17Z)-11,14,17-Icosatrienoic acid 18-Methylnonadecanoic acid (11Z,14Z)-11,14-Icosadienoic acid (11Z)-11-Icosenoic acid cis-13,14-Methylene-Nonadecanoic acid (4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-Docosahexaenoic acide ΣSFA ΣMUFA ΣPUFA ΣTUFA
12.00 13.99 14.49 14.62 14.71 15.00 15.62 15.74 15.82 15.99 16.41 16.57 16.62 16.72 16.89 17.58 17.72 17.77 17.82 17.99 18.08 18.46 18.63 18.99 19.40 19.47 19.56 19.63 19.73 19.81 19.96 21.32
0.4 2.31 19.15 1.12 0.25 0.49 1.09 1.21 8.85 8.1 0.65 1.07 0.87 1.15 0.37 1.39 1.38 1.63 4.82 6.93 0.3 0.33 0.51 0.33 10.9 17.25 0.56 0.46 1.16 0.65 3.2 1.1 29.77 17.32 52.89 70.21
Other names: apalmitoleic acid; bpalmitic acid; carachidonic acid (AA); deicosapentaenoic acid (EPA); edocosahexaenoic acid (DHA). FA, fatty acid; FAME, fatty acid methyl ester; IUPAC, International Union of Pure and Applied Chemistry; MUFA, monounsaturated fatty acid; PU, polyunsaturated fatty acids; SFA, saturated fatty acid; TUFA, Total Unsaturated Fatty Acid.
S. ruetzleri presented an interesting CL profile: it initially led to increased free radical levels, which dropped a few seconds later (Figure 2). On the other hand, the TEAC allows comparison of the antioxidant activity of marine sponge fractions. The results of TEAC are summarized in Table 1. Analysing these results, it can be demonstrated that of the fractions tested, the EtOAc fraction was the one that exerted the highest antioxidant capacity, with a TEAC value of around 20 μg/g of marine sponge. The bioassay-guided fractionation showed the EtOAc fraction to be a promising source of bioactive metabolites. Based on NMR proton analysis, EtOAc fraction of S. ruetzleri is mainly constituted of FAs (Figure S1). A total of 32 FAs were identified by GC–FAME analysis (Table 2), most of these consisting of PUFAs, representing 52.89%, 750
followed by 29.77% saturated fatty acids (SFAs) and 17.32% of monounsaturated fatty acids (MUFAs). The major compounds were PUFAs: (3Z,6Z,9Z,12Z)-3,6,9,12pentadecatetraenoic acid (19.15%), eicosapentaenoic acid (EPA; 17.25%) and arachidonic acid (AA; 10.9%). The main MUFAs and SFAs were palmitoleic acid (8.85%) and palmitic acid (8.1%), respectively. As minor compounds, several long-chain branched FAs were identified. In addition, some minor compounds were identified that are unusual for marine organisms (Figure 3): ciclopropane FAs (1–2), methoxylated (3) and also two long-chain branched unsaturated FAs (4–5). To evaluate the cytotoxic mechanism of FAs, the EtOAc fraction was assayed using the conjugated dienes method. The EtOAc fraction demonstrated an antioxidant and
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Cytotoxic fatty acids from S. ruetzleri
O C
13
(1)
19
OH O
16
C
9
(2) OH
O
CH3 (3) CH3 O
18
O
9
C
CH3
(4) OH
17 13 11
O C
10
(5) OH
CH3 18
#
#
50
nM 2 10 0 n M 0 µg / 50 m µg l 25 /m µg l 10 /m µg l / 1 ml µg /m l
0
* 100
#
#
#
50
0
x lo Tr o
x
2O 2
H
lo Tr o
Concentration (µg/ml)
*
nM 2 10 0 n 0 M µg 50 /m µg l 25 /m µg l 10 /m µg l / 1 ml µg /m l
100
#
20 0
*
150
2O 2
*#
(b)
H
150
% absorbance in comparison with Trolox (242 nM)
(a)
20 0
% absorbance in comparison with Trolox (233 nM)
Figure 3 Unusual fatty acids identified for S. ruetzleri: cis-13,14-Methylene-Nonadecanoic acid (1); cis-9,10-Methylene-Hexadecanoic acid (2); 1,1Dimethoxyoctadecane (3); (9Z)-13-Methyl-9-Hexadecenoic acid (4); (11Z)-10-Methyl-11-Octadecenoic acid (5).
Concentration (µg/ml)
Figure 4 Conjugated dienes from marine sponge S. ruetzleri: 233 nm (a) and 242 nm (b). The effect of different concentrations of ethyl acetate (EtOAc) fraction on lipid peroxidation was measured as % absorbance in comparison with Trolox effects (0.05 μg/ml). Bars represent mean ± standard error of the mean. *P < 0.05 indicates a significant difference compared with Trolox. #P < 0.05 indicates a significant difference compared to H2O2 (one-way analysis of variance followed by Tukey’s test).
pro-oxidant profile (Figure 4a–b). At concentrations below 25 μg/ml, a protective effect of lipid peroxidation was observed, similar to Trolox. On the other hand, the EtOAc fraction at concentrations above 50 μg/ml, showed increased lipid peroxidation, because the effect was comparable or higher than that of H2O2.
Discussion Considering that S. ruetzleri has delicate tissue, and in view of its epibiotic profile,[9] its potential for chemical defence is evident. The cytotoxic activity of S. ruetzleri was also previously reported by Prado et al.,[13] although this study only
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investigated crude extracts of S. ruetzleri. The methanol extract showed a cytotoxic effect against the human breast tumour cell line (T4TD) and had strong effects on microtubule organization and cell cycle progression.[14] Our results demonstrated significant cytotoxic effects on glioma and neuroblastoma human cell lines and also lower IC50 due to the bioassay-guided fractionation of methanol extract of S. ruetzleri. Prior to the present study, chemical analysis had only been performed only for S. ruetzleri collected in Harrington Sound, Bermuda. Vanwagenen et al.[12] isolated a phosphorylated hydantoin and another structurally related compound. Cardellina II et al.[11] isolated an indolic metabolite, and the first compound identified for this sponge was an unusual lipid, 17Z-Tetracosenyl 1-Glycerol Ether.[10] To the best of our knowledge, our study is the first report on the chemical analysis of S. ruetzleri collected in the Brazilian coastline; on the other hand, the presence of lipid compounds found in our study is in agreement with the initial report for this sponge species. The ability to induce a cytotoxic effect by FAs varies depending on the number of double bonds, and on the carbon chain length. Generally, saturated and monounsaturated FAs are least able to induce an effect than PUFAs. AA (20:4 n-6), EPA (20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) are the most widely reported FAs, with strong effects on cancer therapy.[2,3,18] This could explain the potential cytotoxic activity observed for EtOAc fraction of S. ruetzleri, consisting mainly of PUFAs, especially AA and EPA. There is increasing evidence that oxidative processes play an important part in the initiation and progression of cancer.[19] Strategies that modulate the cellular redox potential are being applied to kill cancer cells,[20] and several studies have demonstrated that lipid peroxidation is the major mechanism of FAs, especially PUFAs action on cancer cells.[3,18] The ability to lose a hydrogen atom from the PUFA double bond is responsible for initiating lipid peroxidation due to the production of reactive species that can propagate further reactions, and also act on the macromolecules causing the damage.[3] Here, we found that EtOAc fraction of S. ruetzleri has the potential to modulate free radicals, because we observed in-vitro and ex-vivo antioxidant potential and also ex-vivo pro-oxidant effect in higher concentrations, indicating that lipid peroxidation is partially responsible for the cytotoxic effect of EtOAc fraction of S. ruetzleri in neuroblastoma and glioma human cell lines. The inhibition of lipid peroxidation by EtOAc fraction in low concentrations can be linked to antioxidant potential of
References 1. Pereira DM et al. Fatty acids in marine organisms: in the pursuit of bioactive 752
PUFAs. Some authors support that the antioxidant potential of PUFAs is related to the inhibition of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase, which is one of the major responsible for production of reactive oxygen species (ROS) in endothelial. Moreover, PUFAs can act as a ‘skin’ trapping free radicals and becoming oxidized.[21,22] However, this oxidation produce lipid peroxides, which can explain the results observed in Figure 4: in higher concentrations, an excess of peroxides can increase lipid peroxidation and in lower concentrations can protect. Lipid peroxidation evaluation of FAs is generally carried out using the thiobarbituric acid reactive method (TBARS).[3] However, the application of the conjugate dienes method brings some important advantages in relation to TBARS: (1) it is able to measure early lipid peroxidation reaction events, whereas TBARs measures end products; (2) greater sensitivity; (3) the fact that it is an ex-vivo experiment.[23] In conclusion, this work sheds new light on the bioactive potential and chemical analysis of the marine sponge Scopalina ruetzleri collected along the Brazilian south coast. We performed a bioassay-guided fractionation of this sponge for anticancer activity. The results demonstrate a potential cytotoxic effect in human glioma and neuroblastoma cell lines for the EtOAc fraction of this marine sponge. Chemical analysis of this active fraction led to the identification of 32 FAs, consisting mainly of PUFAs. The majority of FAs identified are commonly found in marine sponges; however, some minor compounds were also identified that are unusual for the marine biosphere. Moreover, our findings suggest that lipid peroxidation may be partially responsible for the mechanism of the EtOAc fraction on cancer cells. This work therefore shows the importance of bioassayguided studies on marine sponges using fractions, focusing on the active metabolite. In particular, it contributes to studies focusing on the application of FAs in cancer therapy, as a new adjuvant to radio- or chemotherapy or as a chemotherapeutic agent.
Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.
Funding This work was supported by CNPq and CAPES.
agents. Curr Pharm Anal 2011; 7: 108– 119. 2. Bergé J-P, Barnathan G. Fatty acids from lipids of marine organisms: mole-
cular biodiversity, roles as biomarkers, biologically active compounds, and economical aspects. Adv Biochem Eng Biotechnol 2005; 96: 49–125.
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 746–753
Renata Biegelmeyer et al.
3. Diggle CP. In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects? Prog Lipid Res 2002; 41: 240–253. 4. American Cancer Society. Information and resources for cancer. 2013. http://www.cancer.org/Cancer/index (Accessed December 13, 2013). 5. DeAngelis LM, Posner MD. Brain metastases. In: Kufe DW et al., eds. Holland–Frei Cancer Medicine. Hamilton, ON: BC Decker, 2004: 1227–1231. 6. Binello E, Germano IM. Targeting glioma stem cells: a novel framework for brain tumors. Cancer Sci 2011; 102: 1958–1966. 7. Park JR et al. Neuroblastoma: biology, prognosis, and treatment. Hematol Oncol Clin North Am 2010; 24: 65–86. 8. Frota Junior MLC et al. Current status on natural products with antitumor activity from Brazilian marine sponges. Curr Pharm Biotechnol 2012; 13: 235–244. 9. Engel E, Pawlik JR. Interactions among Florida sponges. I. Reef habitats. Mar Ecol Prog Ser 2005; 303: 133– 144. 10. Cardellina JH II et al. 17Z-tetracosenyl 1-glycerol ether from the sponges Cinachyra alloclada and Ulosa ruetzleri. Lipids 1983; 18: 107–110. 11. Cardellina JH II et al. Plant growth regulatory indoles from the sponges
Cytotoxic fatty acids from S. ruetzleri
12.
13.
14.
15.
16.
17.
18.
19.
Dysidea etheria and Ulosa ruetzleri. J Nat Prod 1986; 49: 1065–1067. Vanwagenen BC et al. Ulosantoin, a potent insecticide from the sponge UIosa ruetzleri. J Org Chem 1993; 58: 335–337. Prado MP et al. Effects of marine organisms extracts on microtubule integrity and cell cycle in cultured cells. J Exp Mar Biol Ecol 2004; 313: 125–137. Galeano E, Martínez A. Antimicrobial activity of marine sponges from Urabá Gulf, Colombian Caribbean region. J Mycol Med 2007; 17: 21–24. Dresch MTK et al. Optimization and validation of an alternative method to evaluate total reactive antioxidant potential (TRAP). Anal Biochem 2009; 385: 107–114. Recknagel RO, Glende EA. Spectrophotometric detection of lipid conjugated dienes. Methods Enzymol 1984; 105: 331–337. Corongiu FP, Banni S. Detection of conjugated dienes by second derivative ultraviolet spectrophotometry. Methods Enzymol 1994; 233: 303–310. Hossain Z et al. Growth inhibition and induction of apoptosis of colon cancer cell lines by applying marine phospholipid. Nutr Cancer 2009; 61: 123–130. Trachootham D et al. Targeting cancer cells by ROS-mediated mechanisms: a
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 746–753
20.
21.
22.
23.
radical therapeutic approach? Nat Rev Drug Discov 2009; 8: 579–591. Booy EP et al. The immune system, involvement in neurodegenerative diseases, ageing and cancer. Curr Med Chem Anti Inflamm Anti Allergy Agents 2005; 4: 349–352. Massaro M et al. The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKC epsilon inhibition. Proc Natl Acad Sci U S A 2006; 103: 15184– 15189. Doriane R et al. Polyunsaturated fatty acids as antioxidants. Pharmacol Res 2008; 57: 451–455. Vasankari T et al. Measurement of serum lipid peroxidation during exercise using three different methods: diene conjugation, thiobarbituric acid reactive material and fluorescent chromolipids. Clin Chim Acta 1995; 234: 63–69.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. S. ruetzleri 500 MHz).
1
H NMR spectra of EtOAc fraction (CDCl3,
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