Toxicon 40 (2002) 885891

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Antibacterial activity against resistant bacteria and cytotoxicity of four alkaloid toxins isolated from the marine sponge Arenosclera brasiliensis
Yohandra R. Torres a, Roberto G.S. Berlinck a,*, Gislene G.F. Nascimento b, Sergio C. Fortier c, Claudia Pessoa c, Manoel O. de Moraes c
Instituto de Qumica de Sao Carlos, Universidade de Sao Paulo, CP 780, CEP 13560-970 Sao Carlos, SP, Brazil Faculdade de Ciencias da Saude, Universidade Metodista de Piracicaba, Rodovia do Acucar Km 156, 13400-901 Piracicaba, SP, Brazil c Laboratorio de Oncologia Experimental, Universidade Federal do Ceara, CP 3219, Porangabussu, CEP 60431-970 Fortaleza, CE, Brazil
b a

Received 11 September 2001; accepted 6 December 2001

Abstract Arenosclerins AC and haliclonacyclamine E, new tetracyclic alkylpiperidine alkaloids isolated from the marine sponge Arenosclera brasiliensis, were subjected to antimicrobial and cytotoxic bioassays. Fourteen samples of microorganisms were used: Candida albicans, Staphylococcus aureus, Escherichia coli, and 12 antibiotic-resistant bacteria isolated from hospital environment. The minimum inhibitory concentration activity of each alkaloid was determined. The four compounds displayed antibacterial activity, but no antifungal activity against C. albicans. Haliclonacyclamine E and arenosclerins A and C were active against a larger number of bacteria strains than arenosclerin B. However, arenosclerins B and C presented more potent antibacterial activity. The alkaloids displayed inhibitory activity against both Gram positive and Gram negative bacteria. Cytotoxicity bioassays using the MTT method showed that these compounds present cytotoxic activity against human HL60 (leukemia), L929 (brosarcoma), B16 (melanoma) and U138 (colon) cancer cell lines at concentrations between 1.5 and 7.0 mg/ml. The results obtained indicated that A. brasiliensis alkaloids have a potent toxic activity. The broad cytotoxic and antimicrobial activities presented by A. brasiliensis alkaloids suggest a defensive role of arenosclerins and haliclonacyclamine E against microbial infection and/or the action of potential predators at the sponge's natural habitat. q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Marine sponge; Arenosclera brasiliensis; Alkaloids; Antibiotic-resistance; Cytotoxicity; Staphylococcus aureus; Brazil

1. Introduction Sponges are the most primitive invertebrates, and represent an important constitutive group of the coral reef fauna (Van Soest, 1994). These animals are frequently exposed to intense predation and/or tissue infection by microorganisms (Faulkner et al., 2000; Newbold et al., 1999). However, despite being sessile and soft bodied, sponges appear to be predated only by selected groups of marine animals, such as turtles (Meylan, 1988), a few sh species (Wulff, 1995; Dunlap and Pawlik, 1998), nudibranchs (Karuso, 1988;
* Corresponding author. Tel.: 155-16-2739954; fax: 155-162739982. E-mail address: rberlinck@iqsc.sc.usp.br (R.G.S. Berlinck).

Faulkner, 1992; Proksch, 1994), sea urchins (Birenheide et al., 1993) and sea stars (Waddell and Pawlik, 2000b). While sponge tissue and skeleton constituents appear to have little or no activity against potential predators (Chanas and Pawlik, 1995; Chanas and Pawlik, 1996; Pawlik et al., 1995), sponge secondary metabolites clearly present a defensive role against predation (Pawlik et al., 1995; Assmann et al., 2000; Kubanek et al., 2000; Lindel et al., 2000; Wilson et al., 1999; Waddell and Pawlik, 2000a). Additionally, marine sponge crude extracts present a high incidence of antibacterial activity against terrestrial pathogenic bacteria (Amade et al., 1982,1987; McCaffrey and Endeau, 1985; Uriz et al., 1992; Muricy et al., 1993; Becerro et al., 1994), but a low incidence of antibacterial activity against marine bacteria (Amade et al., 1982,1987; Newbold

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Fig. 1. Structures of Arenosclera brasiliensis alkaloids haliclonacyclamine E and arenosclerins AC.

et al., 1999). Only a few cases of sponge infection by exogenous microorganisms are known, presumably because of its production and/or accumulation of compounds that have antimicrobial activity (Amade et al., 1987; Newbold et al., 1999; Hentschel et al., 2001). Up to 800 antibiotic compounds have been isolated from marine sponges, a number that corroborates assumptions that sponges appear to defend themselves against infections by producing and/or accumulating secondary metabolites. Our group has been interested in the search for new biologically active secondary metabolites from marine sponges (see Torres et al., 2000 and references mentioned therein). During our continuing program with this aim, we have observed that the crude extract of the sponge Arenosclera brasiliensis (Haplosclerida, Demospongiae) displayed strong antimicrobial activity against common bacteria (Muricy et al., 1993) and cytotoxicity against cancer cells. A subsequent chemical investigation of the A. brasiliensis crude extract led to the isolation and structure determination of four new alkaloids (Fig. 1), arenosclerins AC and haliclonacyclamine E (Torres et al., 2000). Therefore, we suspected that the alkaloids isolated from A. brasiliensis may be responsible for the biological activities observed for the crude extract. Herein, we report the antibacterial activity of arenosclerins AC and haliclonacyclamine E against a panel of sensitive and antibiotic-resistant microorganisms, as well as their cytotoxic activity against four different cancer cell lines.

were obtained during a previous work (Torres et al., 2000). Samples were stored in a freezer at 24 8C until needed. 2.2. Microbial samples Fourteen samples of microorganisms were used (Table 1): one sample of yeast Candida albicans (organism number 4 in Table 1); two sensitive bacteria: Staphylococcus aureus ATCC 6538 (number 1) and Escherichia coli ATCC 25922 (number 2); as well as 11 antibiotic-resistant bacteria isolated in hospital environment: one Pseudomonas aeruginosa (number 3) and 10 strains of methicillin-resistant Staphylococcus aureus (MRSA, numbers 11135). The bacteria resistance panel is presented in Table 1. 2.3. Antimicrobial assay The bacteria were grown in Brain Heart Infusion liquid medium at 37 8C. After 6 h of growth, each microorganism was inoculated on the surface of MuellerHinton agar plates at a concentration of 10 6 cells/ml. Subsequently, previously sterilized lter paper discs (6 mm in diameter) were impregnated with the A. brasiliensis alkaloids (20 ml of a 400 500 mg/ml solution of each alkaloid) and were placed on the surface of each inoculated plate (agar diffusion method). The plates were incubated at 37 8C for 24 h. Zones of growth inhibition greater than 7 mm were considered susceptible to A. brasiliensis alkaloids. The alkaloids were subsequently tested to determine the minimal inhibitory concentration (MIC) for each bacterial sample. Seven bacterial samples (numbered 13, 11, 18, 36, 135) were grown in nutrient broth for 6 h. Afterwards, 100 ml of 10 6 cells/ml were inoculated in tubes with Nutrient Broth supplemented with different concentrations (5, 10,

2. Materials and methods 2.1. Alkaloids isolation from Arenosclera brasiliensis Arenosclerins AC and haliclonacyclamine E (Fig. 1)

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Table 1 Panel of bacteria resistance to antibiotics. Microorganism #1: Staphylococcus aureus ATCC 6538; microorganism #2: Escherichia coli ATCC 25922; microorganism #3: antibiotic-resistant Pseudomonas aeruginosa; microorganism #4: Candida albicans; microorganisms #11, 18, 35, 36, 61, 63, 68, 108, 115, 135: 10 methicillin-resistant strains of Staphylococcus aureus. Antibiotics: AMAmikacin; APAmpicillin; ASB Sulbactam/Ampicillin; ATMAztreonam; CFCephalothin; CFRCefpirome; CRCarbenicillin; CAZCeftazedime; CFOCefoxitin; CPMCefepime; CLIClindamicin; COChloramphenicol; CROCeftriaxone; CXMCefuroxime; CTXCefotaxime; EI Erytromycin; GNGentamicin; IPMImipenen; KNKanamycin; LNLincomycin, OXOxacillin; MUPMupirocine; NANalidixic Acid; NETNetilmicin; NORNoroxacin; NTNitrofurantoin; PNPenicillin; PPPiperacillin; RFRifampicin; SFSulfonamide; SFTSulfamethoxazole; TBTobramycin; TTTetracycline Microrganisms 1 2 3 4 11 18 35 36 61 63 68 108 115 135 Drugs Susceptible to antibiotics Susceptible to antibiotics AM AP CR TB SFT AN Susceptible to antibiotics AM AP CF PN RIF CLI AP CF CTX SFT CPM ASB AP CF CFO AP CF CTX CXM MUP OX AP CF CTX AP CF CTX MUP OX AM AP CF ASB CXM MUP AP CF CTX CXM OX AP CTX CFO CPM ASB CXM AP CF CTX CXM MUP OX

CF NET CTX IPM CFO CXM EI CFO CFO CFO CFO OX CFO CAZ OX CFO

CTX NOR CFO ATM CAZ MUP PN CAZ CAZ CAZ CAZ CAZ CRO CAZ

CFO CAZ SFT CRO OX CLI CRO CRO CRO CRO CRO CO CRO

CFR CRO NET GM ATM EI PN PN TT NOR TT KN

CRO CO CPM KM ASB PN ATM RIF TB EI TB NOR

CO TT ASB TB CXM RIF SFT CLI PN PN EI PN

GN GN CXM NOR MUP CLI CPM ATM RIF RIF PN CLI

KN KN MUP EI OX ATM ASB SFT CLI CLI RIF ATM

PP TB OX PN SFT CXM CPM ATM ATM CLI SFT

NET NOR CLI CPM MUP ASB SFT SFT ATM ASB

TT EI ATM ASB OX CXM CPM ASB SFT

50, 100, 200, 300, 400 and 500 mg/ml) of the substances. After 24 h at 37 8C, the MIC of each sample was determined by measuring the optical density in a spectrophotometer (620 nm), comparing the sample readout with the readout of control samples, which have not been inoculated with alkaloids solutions in the nutrient broth. 2.4. Cytotoxicity bioassays For HL-60 (leukemia), L929 (brosarcoma), B16 (melanoma) and U138 (colon) cancer cell lines, microassay for cytotoxicity was performed using the MTT method (Mosmann, 1983), by the following procedure. Adherent cancer cell lines (L929, B16 and U138) at the concentration of 0.3 10 6 cells/ml and suspended cells (HL-60) at 0.5 10 6 cells/ml were seeded in 96 well microplates. The adherent cells were incubated during 24 h in order to allow cell attachment. The alkaloid solutions were added to the cell cultures at concentrations of 0.3925 mg/ml, and the cells were incubated for three days. The MTT solution was added 3 h before the end of the incubation time. Cell

survival was evaluated with a multiwell scanning spectrophotometer at 540 nm.

3. Results 3.1. Antimicrobial activity of haliclonacyclamine E and arenosclerins AC The antimicrobial activity of arenosclerins AC (24) and haliclonacyclamine E (1) is presented in Table 2. The results indicated that the four compounds display antibacterial activity (inhibition zone between 7 and 13 mm) against S. aureus, P. aeruginosa and MRSA, but they have no effect on bacterium (E. coli) or yeast (C. albicans). Haliclonacyclamine E (1), arenosclerin A (2) and arenosclerin C (4) present antimicrobial activity over all samples of microorganisms tested except against E. coli and C. albicans, while arenosclerin B inhibited the growth of only ve microorganism strains (S. aureus, P. aeruginosa and three antibiotic-resistant strains of S. aureus). Due to the similar biological activity observed for A. brasiliensis

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Y.R. Torres et al. / Toxicon 40 (2002) 885891 Table 3 MIC of haliclonacyclamine E (1) and arenosclerins A (2), B (3) and C (4) against fungi, Gram positive and Gram negative bacteria. Substances: (1) haliclonacyclamine E; (2) arenosclerin A; (3) arenosclerin B; (4) arenosclerin C; (1) susceptibility (inhibition zone of 713 mm); (2) absence of susceptibility; bacterial strains: 1 S. aureus (ATCC 6538); 2 E. coli (ATCC 25922); 3 P. aeruginosa; 11, 18, 36, 135 diverse oxacillin-resistant S. aureus. Experiments were performed with n 2; each in duplicate Microorganisms Substances (concentration in mg/ml) (1) (2) 1 2 1 2 1 1 1 1 1 1 1 1 1 1 (3) 1 2 1 2 1 1 2 2 2 2 2 2 2 1 (4) 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 (Gram 1) 2 (Gram 2) 3 (Gram 2) 11 (Gram 1) 18 (Gram 1) 36 (Gram 1) 135 (Gram 1) 100 400 200 50 10 50 50 (2) 112 400 400 100 10 100 50 (3) 100 100 5 100 5 100 100 (4) 25 100 50 100 5 100 50

Table 2 Antimicrobial activity of haliclonacyclamine E (1) and arenosclerins AC (24). Substances: (1) haliclonacyclamine E; (2) arenosclerin A; (3) arenosclerin B; (4) arenosclerin C; (1) susceptibility (inhibition zone of 713 mm); (2) absence of susceptibility; bacterial strains: 1 S. aureus (ATCC 6538); 2 E. coli (ATCC 25922); 3 P. aeruginosa; 4 C. albicans; 11, 18, 35, 36, 61, 63, 68, 108, 115, 135 diverse oxacillin-resistant S. aureus. Each assay was performed with 6 mm lter paper discs impregnated with 20 ml of a 400 500 mg/ml solution of each alkaloid (approximately 810 mg per disc). Experiments were performed with n 2; each in duplicate Microrganisms Substances (1) 1 2 3 4 11 18 35 36 61 63 68 108 115 135 1 2 1 2 1 1 1 1 1 1 1 1 1 1

4. Discussion Marine sponges are an outstanding source of unusual biologically active natural products, which may be considered as potential new drug leads useful for the treatment of human diseases (Fusetani, 2000). In particular, marine sponges of the order Haplosclerida have an array of alkylpyridine and alkylpiperidine alkaloids, and most of these compounds present pronounced biological activities (Andersen et al., 1996; Almeida et al., 1997; Sepcic, 2000). In the present work, the evaluation of the antibacterial activity of A. brasiliensis alkaloids demonstrated that these compounds inhibited the growth of several bacteria, including 11 antibiotic-resistant strains. Haliclonacyclamine E (1), arenosclerin A (2) and arenosclerin C (4) inhibited the growth of all antibiotic-resistant S. aureus tested, while arenosclerin B (3) inhibited the growth of three of the antibiotic-resistant S. aureus strains. These results indicate that the different stereochemistry of (3) at the bis-piperidine ring system plays a signicant role in the antibiotic activity, since this is the only difference between the alkaloids (2), (3) and (4). Additionally, although haliclonacyclamine E (1) and arenosclerin A (2) differ in the presence of a hydroxyl group, their MIC on the strains tested are quite similar, suggesting that such structural change plays no signicant role in the antibacterial activity. However, the change of the stereochemistry of the bis-piperidine ring system appear to have a major inuence in the antibacterial activity, because the MIC of both arenosclerins B (3) and C (4) are much lower than those observed for haliclonacyclamine E (1) and arenosclerin A (2). Haliclonacyclamine E (1) and arenosclerins AC (24) presented a similar range of cytotoxicity, irrespective of the cancer cell line tested (Fig. 2 and Table 4). The cytotoxicity of all alkaloids against HL-60, L929, B16 and U138 cancer

alkaloids and the small quantity of material available, only seven microorganisms were used in the determination minimum inhibitory concentration (MIC; see Table 3) of the antibacterial activity. Gram negative bacteria was less susceptible to the antimicrobial alkaloids (MIC 50400 mg/ml range) than Gram positive bacteria (MIC 5112 mg/ml range). Arenosclerins B (3) and C (4) display a much higher antibacterial activity against P. aeruginosa than (1) and (2). The antibacterial activity of (3) and (4) is also pronounced against non-resistant S. aureus (strain 1) and oxacillin-resistant S. aureus (strain 18). 3.2. Cytotoxic activity against cancer cell lines The cytotoxic activity of haliclonacyclamine E (1) and arenosclerins AC (24) is presented in Table 4 and in the graphs of Fig. 2. All the A. brasiliensis alkaloids display cytotoxic activity at a range of 1.57.1 mg/ml when tested against HL-60 (leukemia), L929 (brosarcoma), B16 (melanoma) and U138 (colon) cancer cell lines. Haliclonacyclamine E is slightly less active than the arenosclerin alkaloids, but this difference lies in the experimental error and, therefore, is not signicative. Since the alkaloids were also active against normal cells in the same concentration range (data not shown), the overall cytotoxic results suggested that these alkaloids present a broad cytotoxic activity rather than a particular mode-of-action.

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Table 4 Growth-inhibitory effects of arenosclerins AC and haliclonacyclamine E on leukemia HL-60, brosarcoma L929, melanoma B16 and colon U138 cancer cell lines Compound IC50 a (mg/ml, mean ^ SE) HL60 Haliclonacyclamine E (1) Arenosclerin A (2) Arenosclerin B (3) Arenosclerin C (4)
a

B16 1.82 ^ 0.14 1.77 ^ 0.09 1.76 ^ 0.13 1.71 ^ 0.19

U138 6.06 ^ 1.03 3.83 ^ 0.24 3.62 ^ 0.30 3.60 ^ 0.18

L929 3.89 ^ 0.27 2.34 ^ 0.34 2.24 ^ 0.26 2.17 ^ 0.22

4.23 ^ 0.79 4.31 ^ 0.75 4.07 ^ 0.59 3.65 ^ 0.65

Concentration promoting 50% inhibition of cell growth. Experiments with n 1; each in triplicate.

Fig. 2. Cytotoxicity of haliclonacyclamine E (1), arenosclerin A (2), arenosclerin B (3) and arenosclerin C (4) against leukemia HL-60 (A), brosarcoma L929 (B), melanoma B16 (C) and colon U138 (D) cancer cell lines. Doses are expressed in mg/ml.

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cell lines is almost identical. Therefore, the stereochemistry of the bis-piperidine ring system and the presence of the hydroxyl group appear to play only a negligible role to differentiate the cytotoxic activity of A. brasiliensis alkaloids. The antibiotic and cytotoxic activities observed for the A. brasiliensis alkaloids are in agreement with previous reports on the biological activity of Haplosclerida alkaloids. Potent cytotoxic (against P388 cells), antibiotic (Bacillus subtilis) and antifungal (C. albicans and Trichophyton mentagrophytes) activities have been observed for haliclonacyclamines A (IC50 of 0.8 mg/ml), B (IC50 of 0.6 mg/ml) and C (IC50 of 0.7 mg/ml), closely related bis-piperidine alkaloids isolated from an Australian sponge of the genus Haliclona (Charan et al., 1996; Clark et al., 1998). Halicyclamine B isolated from Xestospongia sp. displayed marginal antimicrobial activity against E. coli and B. subtilis, no antifungal activity against C. albicans and moderate cytotoxicity against murine mouse cancer cells PO3 and M17 (Harrison et al., 1996). Halicyclamine A from another Haliclona sp. inhibited the activity of inosine monophosphate dehydrogenase (Jaspars et al., 1994). Other biogenetically related, but structurally distinct, Haplosclerida alkaloids present diverse and potent biological activities (Andersen et al., 1996; Almeida et al., 1997; Sepcic, 2000). The common biogenetic origin of such potently bioactive alkaloids strongly suggests that these compounds are sponge toxins that may have a protective role against microbial infection and/or tissue consumption by predators. A criterious investigation demonstrated that although the sponge Haliclona sp. has a symbiotic association with the dinoagellate Symbiodinium microadriaticum, Haliclona sp. is the true producer of the haliclonacyclamine alkaloids (Garson et al., 1998). These compounds also presented antifeedant activity against shes, general toxicity against the coral Acropora sp. and antifouling activity against larvae of the ascidian Herdmania curvata (Clark, 2000). Therefore, it seems likely that alkylpiperidine and related alkaloids appear to be, at least in part, responsible for Haplosclerid sponges ecological success in their natural environment. Finally, it is worth mentioning that the antibacterial activity of A. brasiliensis alkaloids against antibiotic-resistant bacteria points to a more detailed study of their mode-ofaction. The growing incidence of pathogenic antibioticresistant bacteria is a subject of concern to the medical and scientic community, because the therapeutic resources available for the treatment of many infections are running out due to the increasing ineffectiveness of antibiotics. This fact becomes even more disturbing as the population of hospitalized immunodecient patients increases with the multi-resistant bacterial strains, for which there are often no therapeutic options, whatsoever. In particular, methicillinresistant S. aureus (MRSA) and oxacillin-resistant S. aureus (ORSA) strains have extended worldwide, and the increase in their predominance is a matter of public health. The problem of microbial resistance is growing and the outlook

for the use of antimicrobial drugs in the future is still uncertain. Therefore, actions must be taken to reduce this problem by controlling the use of antibiotics, developing research to better understand the genetic mechanisms of resistance, and continuing studies toward the discovery of new effective antibiotics, either synthetic or natural. The strong antibiotic activity of arenosclerins AC and haliclonacyclamine E suggest that these compounds may be regarded as potentially useful new drug leads. Acknowledgements Financial support was provided by a FAPESP thematic grant 96/04316-5 and the American Society Research Starter Grant (1998), which are gratefully acknowledged. YRT also thanks to FAPESP for a PhD fellowship (97/03907-2). References
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