Search for cytotoxic agents in multiple Laurencia complex seaweed species (Ceramiales, Rhodophyta) harvested from the Atlantic Ocean with emphasis on the Brazilian State of Espírito Santo

Search for cytotoxic agents in multiple Laurencia complex seaweed species (Ceramiales, Rhodophyta) harvested from the Atlantic Ocean with emphasis on the Brazilian State of Espírito Santo

Erika M. Stein^I,II; Daniel X. Andreguetti^I; Cleidiane S. Rocha^I; Mutue T. Fujii^II,III; Mauricio S. Baptista^I; Pio Colepicolo^I; Guilherme L. Indig^IV

^IDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil

^IIDepartamento de Botânica, Instituto de Biociências, Departamento de Botânica, Universidade de São Paulo, Brazil

^IIIInstituto de Botânica, Secretaria do Meio Ambiente, Brazil

^IVDepartment of Chemistry and Biochemistry, University of Wisconsin, USA

^{Correspondence}

ABSTRACT

The development of new anti-cancer drugs of algal origin represents one of the least explored frontiers in medicinal chemistry. In this regard, the diversity of micro- and macroalgae found in Brazilian coastal waters can be viewed as a largely untapped natural resource. In this report, we describe a comparative study on the cytotoxic properties of extracts obtained from the Laurencia complex: Laurencia aldingensis, L. catarinensis, L. dendroidea, L. intricata, L. translucida, L. sp, and Palisada flagellifera. All of these species were collected in the coastal waters of the State of Espírito Santo, Brazil. Four out of the twelve samples initially investigated were found to show significant levels of toxicity towards a model tumor cell line (human uterine sarcoma, MES-SA). The highest levels of cytotoxicity were typically associated with non-polar (hexane) algal extracts, while the lowest levels of cytotoxicity were found with the corresponding polar (methanol) extracts. In this report, we also describe a biological model currently in development that will not only facilitate the search for new anti-cancer drug candidates of algal origin, but also permit the identification of compounds capable of inducing the destruction of multi-drug resistant tumors with greater efficiency than the pharmaceuticals currently in clinical use.

Keywords: macroalgae, Laurencia complex, anti-cancer activity, MES-SA, MES-SA/Dx5, Hela

Introduction

Out of the more than 25,000 known species of algae, only about fifteen are currently grown on a large-scale for biotechnological applications (Raja et al., 2008). Algae are considered to be a largely untapped natural resource and investigations involving them are one of the least explored frontiers of biotechnology and natural products chemistry (Pinto et al. 2000; Cardozo et al., 2007; Chakabourty et al., 2009). Significant advances in the discovery and development of new anti-cancer drugs of algal origin have been recently reported in the literature. Newly identified algal drug candidates include representatives of the terpene, steroid, and polyketide families (Mayer & Gustafson, 2008). Drug-induced over-expression of the multi-drug efflux pump P-glycoprotein (P-gp) is thought to be one of the leading causes of chemotherapy failure in clinical oncology (Higgins, 2007; Dawson & Locker, 2006; Lage, 2008; Perez-Thomas, 2006). Because P-gp can remove many unrelated chemotherapeutic agents from the target cells, including agents to which the tumor had not been previously exposed (Higgins, 2007; Dawson & Locker, 2006), these cells are referred to as multi-drug resistant (MDR) mutants.

One of the most gratifying findings that can arise from any drug screening program is the identification of new drug candidates that are capable of eliminating MDR tumor cells more efficiently than the drugs currently in clinical use. The discovery of new, more benign inhibitors of such drug efflux pumps can also be facilitated by the biological model currently in development in our laboratories.

In this report, we describe our initial results on the search for novel anti-cancer agents in marine algae found on the coast of the Brazilian State of Espírito Santo. We also outline a biological model currently in development to guide our screening efforts. This model aids in the identification of potential new anti-cancer candidates and, at the same time, of those that should be less susceptible to the action of promiscuous therapy-induced drug-efflux pumps such as P-gp.

Materials and Methods

Reagents and cell culture media

McCoy's 5A modified medium, Dulbecco's modified Eagle's medium (DMEM), doxorubicin hydrochloride, fungizone (amphotericin B), penicillin, streptomycin, thiazolyl blue tetrazolium bromide (MTT), ethylenediamine tetraacetic acid (EDTA; disodium salt), trypsin, and Trypan Blue were obtained from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) from Vitrocell Embriolife (Campinas, Brazil). NaCl from Synth (Diadema, Brazil), sodium bicarbonate from Merck (Darmstadt, Germany), D-glucose from Becto Chemical (São Paulo, Brazil), KCl from Cetus (São Paulo, Brazil), Dulbecco's phosphate buffered saline (DPBS) from Gibco BRL (Grand Island, USA), and dimethylsulfoxide (DMSO) from Vetec Química Fina Ltda. (Duque de Caxias, Brazil) were all of high-purity grade and used as received. Water was distilled, deionized and filtered prior to use (Millipore Milli-Q system; resistivity, 18 MΩ cm).

Instruments and methods

Stock solutions of doxorubicin were prepared in water and their respective concentrations determined by absorption spectroscopy on a Shimadzu (Kyoto, Japan) Model UV-1650 PC spectrophotometer employing a molar extinction coefficient of 11,500 M^-1 cm^-1 at 480 nm (Zeman et al., 1998).

Algal extracts were prepared via initial sonication (30 min) of approximately 30 g of the respective powdered and dried biomass with 300 mL of hexane. After standing overnight, the mixture was filtered and the solid mass subjected to two more cycles of extraction with hexane. The hexane-extracted biomass was then further processed by performing three extraction cycles with chloroform followed by three cycles of methanol extraction. The solvent was evaporated from the filtrates on a rotary evaporator to obtain three distinct classes of algal extracts, namely the hexane extract, the chloroform extract, and the methanol extract. In selected experiments, we extracted the original algal dry powder sample only with methanol; these extracts are referred to here as "crude" methanol extracts. Stock solutions of the final algal extracts were prepared in either DMSO or ethanol and subsequently diluted in the cell growth media in order to maintain the final concentration of organic solvent in the medium below 0.5% (by volume). The species of algae investigated are listed in Table 1.

The trypan blue dye exclusion assay (Perry et al., 1997) was routinely employed to assess the quality of our original cell cultures; in all experiments performed with cell cultures, cell viability was at least 95%. All experiments were carried out with cells in the exponential growth phase. Cell viability assays were carried out using the MTT assay (Mosmann, 1983; Garcia-Peres et al., 2010). For this purpose, 96-well flat-bottomed microtiter plates were seeded at a cell density of 5,000 cells per well (100 μL of growth medium per well) and the cells allowed to attach and grow for 24 h. The sub-culturing protocols used in this study were always in accord with the respective ATCC recommendations. The cells were subsequently exposed to growth medium containing either the standard drug (doxorubicin) or the algal extracts for a period of time that typically varied from 24 to 72 h prior to MTT analysis. The MTT assays were performed on a Model Infinite M200 multi-well plate reader from Tecan (Männedorf, Switzerland). Cells exposed to the growth medium alone or to the growth medium plus the drug/extract vehicle (DMSO or ethanol) served as the controls. Under our experimental conditions, no significant cytotoxicity was observed for the vehicle (organic solvent used to prepare the stock solutions). Cell mortality was expressed as the viability of cells treated with the drug or extract relative to that of the corresponding controls.

Cell Lines

The MES-SA human uterine sarcoma cell line (ATCC CRL-1976™) and the corresponding doxorubicin-resistant mutant (MES-SA/Dx5, ATCC CRL-1977™) were obtained from the American Type Culture Collection (ATCC; Manassas, VA). These cells were grown in modified McCoy's 5A medium supplemented with 10% FBS, amphotericin B (0.63 μg/mL), penicillin (100 units/mL) and streptomycin (100 μg/mL). The medium used to grow the MES-SA/Dx5 mutants also contained doxorubicin (0.5 μM), as required for the maintenance of the drug-resistance phenotype (Harker, 1983; Harker & Sikic, 1985; Larroque-Lombard et al., 2010). HeLa (CCL-2™) human cervix adenocarcinoma cells were kindly provided by Dr. Mari Cleide Sogayar (Departamento de Bioquímica, Universidade de São Paulo) and grown in DMEM medium, supplemented as described above for the McCoy's medium used to grow the MES-SA cells. Cells were grown at 37 ºC in a humidified atmosphere of 5% CO₂ in air.

Results and Discussion

In this initial screening, we explored the extent to which the chemical agents extracted from a variety of Laurencia complex seaweed species exhibited cytotoxicity effects toward our primary model tumor cell line (MES-SA). For this purpose, we initially exposed cultures of MES-SA cells to mixtures containing substantial amounts of the algal extracts (i.e., 500 μg of algal extract/mL of cell culture media), subsequently measuring the cell survival rates after 24 h of continuous exposure to the extract. Table 2 shows that substantial cytotoxicity was associated with some, but not all, of these algal extracts. The methanol extracts obtained by our sequential extraction protocol (i.e., hexane/chloroform/methanol) showed the lowest levels of toxicity, even lower than that of the crude methanol extracts obtained via direct methanol extraction of the original algae dry powder samples. The somewhat higher toxicity of the crude methanol extracts relative to the sequential methanol extracts is presumably the result of the presence of less polar chemical entities in the former that are absent in the latter extract because of removal during the prior extractions with the less polar solvents. Indeed, the hexane extracts of Laurencia catarinensis Cord.-Mar. & M.T. Fujii, L. dendroidea J. Agardh, and L. translucida M.T. Fujii & Cord.-Mar all show high levels of cytotoxicity. Likewise, the chloroform extract of L. dendroidea also exhibits a high degree of cytotoxicity, but the corresponding extract of L. aldingensis does not (Table 2).

The overall trends observed in Table 2 were further confirmed by a subsequent experiment employing the highly cytotoxic hexane extracts of L. dendroidea and L. translucida and the methanol extract of L. translucida. In this second study, MES-SA cells were exposed for 72 h to variable concentrations of the extracts and cell viability again measured by the MTT assay. Figure 1 confirms that the methanol extract of L. translucida is virtually non-toxic, while the corresponding hexane extract of the same alga is highly toxic, with an apparent IC50 of about 16 μg/mL. The IC50 associated with the hexane extract of L. dendroidea was ca. 5-fold higher (i.e., 91 μg/mL) than that of the hexane extract of L. translucida. For comparative purposes, an analogous experiment carried out with doxorubicin (a classical and potent anti-cancer drug) gave an ID50 of 0.05 μM (i.e., 0.03 μg/mL) for MES-SA cells (Figure 2).

The use of MES-SA cells for this screening was motivated in part by the fact that these cells grow quickly (doubling time of ca. 24 h), show desirable morphology, are large enough to facilitate counting/analysis of drug-induced mechanisms of damage via microscopic analysis, are highly susceptible to a large variety of anti-cancer drugs (i.e., relatively easy to perform in vitro assays of mortality), and are also easy to manipulate and maintain (Larroque-Lombard, 2010; Lacerda et al., 2005; Belostotsky et al., 2011). The susceptibility of MES-SA cells to doxorubicin is analogous to that observed for HeLa cells (compare panels A and B in Figure 2). HeLa cells have been extensively used in pre-clinical studies and, for this reason, the inclusion of this cell line in selected screening experiments may facilitate the comparison of the potency associated with putative new drug candidates with that of previous candidates studied with HeLa cells. On the other hand, the MES-SA line has a feature that is quite attractive in terms of the development of novel screening protocols: the existence of a commercially-available MES-SA mutant (namely the MES-SA/Dx5 line) that is also easy to work with and maintain and shows the classical multi-drug resistance phenotype (i.e., over-expression of the drug efflux pump, P-gp). Therefore, the inclusion of this mutant cell line in any screening protocol permits the identification not only of novel anti-cancer agents capable of inducing the mortality of MDR tumor cells, but also of novel inhibitors of P-gp. For comparative purposes, the ID50 for doxorubicin was 0.05 μM for the wild type cell line (MES-SA), but almost two orders of magnitude higher (i.e., 3.4 μM; see Figure 2, panel A) for the MDR mutant (MES-SA/Dx5). Analogous differences in the response of these cell lines to doxorubicin have been reported by others authors (Larroque-Lombard et al., 2010).

Conclusions

Our initial studies indicate that four of the twelve extracts of the Laurencia complex seaweeds currently under investigation exhibit significant cytotoxicity towards MES-SA cells. We are cautiously optimistic with respect to these preliminary findings and further investigations designed to isolate and identify novel and potent anti-cancer drug candidates from these extracts are already in progress. The biological model under development here is currently being used for a wider systematic screening of algal species present in waters along the Brazilian seashore. This should expand our knowledge of the presence of cytotoxic compounds in Brazilian algal species and may identify novel candidate compounds for the more effective treatment of multi-drug resistant tumors (Higgins, 2007; Dawson & Locker, 2006; Lage, 2008; Perez-Tomas, 2006). In addition, we envision that our biological model should also facilitate the search for novel chemical inhibitors of P-gp that show positive synergism with classical anti-cancer drugs. Thus, any enhancement in MES-SA/Dx5 cell mortality (e.g., at ID20) in the presence of doxorubicin plus a non-toxic extract relative to that of doxorubicin alone would suggest the possible presence of P-gp inhibitors or other agents (e.g., pro-apoptotic factors) in the extract. Hence, in our future screening studies, even algal extracts that do not show cytotoxic properties will be tested for synergy with the standard drug doxorubicin.

Acknowledgment

This work was supported by the Brazilian research funding agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo a Pesquisa do Estado de São Paulo and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Ministério da Saúde, Ministério de Ciência e Tecnologia and CNPq-INCT-Redoxoma.

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Received 15 Dec 2010

Accepted 5 Jan 2011

Publication Dates

• Publication in this collection
  22 Apr 2011
• Date of issue
  Apr 2011

History

• Received
  15 Dec 2010
• Accepted
  05 Jan 2011