Sulforhodamine B (SRB) is an aminoxanthene dye bearing two sulfonic groups, with a strong pink color. The SRB assay for cell proliferation inhibition testing relies on the ability of this molecule to bind to basic amino acids residues in acidic conditions and dissociate at basic pH. This binding is stoichiometric and thus, the amount of dye extracted from stained cells is directly proportional to the cell mass. For this reason, this test has been widely used for the evaluation of inhibition of cell proliferation [12].

In the work herein, meroditerpenoids from S. flabelliforme were tested for their ability to inhibit cell proliferation using a panel of human (Caco-2 and SH-SY5Y) and non-human (RBL-2H3 and RAW.267) cancer cell lines, as well as V79 non-cancer cells.

All cancer cells were affected by exposure to the compounds, although the several cell lines exhibited different sensitivities (Figures 2–6). Among the cell lines assayed, RAW.267 was one of the most affected and showed a significant inhibition of cell proliferation (Figure 2), with epitaondiol, epitaondiol monoacetate and stypotriol triacetate displaying nearly 100% inhibition at all tested concentrations. Epitaondiol diacetate, 14-ketostypodiol diacetate and stypodiol also displayed a concentration-dependant activity, although to a lesser extent (Figure 2). In fact, in general, epitaondiol, epitaondiol monoacetate and stypotriol triacetate were the most toxic compounds to all cell lines (Figures 2–6).

SH-SY5Y, a thrice-cloned neuroblastoma, originally from SK-N-SH, was the cell line displaying the highest susceptibility to the meroditerpenoids (Figure 3), on a par with RAW.267. Overall, with the exception of epitaondiol diacetate, all compounds exhibited nearly 100% inhibition of cell proliferation at the highest tested dose. IC₅₀ of 12.2 μM and 14 μM were found for epitaondiol and stypotriol triacetate, respectively, which were the most active compounds (Figure 3a). The positive control used in this study, vincristine, yielded an IC₅₀ of 0.03 μM. The neurotoxicity of meroditerpenoids has been observed before, but only 2β,3α-epitaondiol, an isomer of epitaondiol, was studied using a mouse cell line, neuro2a. With this mouse cell line, Sabry and colleagues [13] observed the effect of 2β,3α-epitaondiol and other meroditerpenoids and a LD₅₀ between 2 and 11 μM was found [13]. To the best of our knowledge, this is the first time that the human neurotoxicity of these compounds is addressed. This cell line is particularly prone to damage caused by oxidative stress [14,15].

For Caco-2 cell line all compounds showed a concentration-dependent inhibitory effect, with stypotriol triacetate being the most active, followed by epitaondiol monoacetate and epitaondiol (Figure 4). A similar trend was found regarding RBL-2H3 cell line (Figure 5).

Regardless of the effect against cancer cells, cytotoxic compounds are interesting for human chemotherapy only when their toxicity is higher for cancer cells than for non-cancer ones. For this reason we tested the ability of the meroditerpenoids to inhibit cell proliferation of the non-cancer cell line V79 (Figure 6). Epitaondiol diacetate, 14-ketostypodiol diacetate and stypodiol revealed to have little or no effect on non-cancer V79 cell line, while against the cancer cell lines these same compounds displayed a selective activity (Figures 2–5). This selectivity against specific cancer cell lines is one of the requirements for the development of new drugs in order to lower the extent of side-effects. 14-Ketostypodiol diacetate has already proved to inhibit microtubules assembly and cell proliferation in DU-145 human prostatic cancer cells [16].

The results obtained in the present work point to the possible use of this class of compounds in a context of human chemotherapy against cancer. Nevertheless, further studies are required in order to understand the mechanism of action of these compounds.

Compounds exhibiting antimicrobial properties for concentrations lower than 64 μg/mL are accepted as having notable antimicrobial activity [17], while those showing activity at concentrations below 10 μg/mL are considered as “clinically significant” [17,18].

Epitaondiol monoacetate, stypotriol triacetate and stypodiol showed some antimicrobial capacity, with the first displaying the major effect against gram-positive and gram-negative bacteria (MIC ≥ 114 μg/mL) (Table 1). E. faecalis appeared to be the most sensitive species. As for the remaining compounds, all bacteria were found to be resistant under the tested concentrations (Table 1). Thus, the compounds studied can hardly be considered good antimicrobial candidates.

The brown alga Stypopodium flabelliforme was obtained from the underwater environment near Easter Island, Pacific Ocean. Isolation and characterization procedures were previously reported by us [19–21]. Generally, fresh algae were repeatedly extracted with dichloromethane. The extract was concentrated under reduced pressure and applied to a Sephadex LH-20 column. Because of the instability of the compounds, polar fractions were immediately acetylated with pyridine/acetic anhydride in order to avoid oxidation. Purification was done by thin layer chromatography (TLC) and column chromatography (CC) using grade silica gel. Complete spectroscopic characterization, including ¹H NMR, ¹³C NMR and UV data has been published before for stypotriol triacetate, epitaondiol diacetate, epitaondiol monoacetate and epitaondiol [21], as well as for 14-ketostypodiol diacetate [20]. Stypodiol was obtained by hydrolysis of stypodiol diacetate previously isolated by the team [19]. Briefly, stypodiol diacetate (30 mg) was refluxed in basic aluminum oxide (1 mg) in benzene (30 mL) for 12 h [21]. Benzene was removed under reduced pressure and the residue dissolved in water (10 mL), extracted with dichloromethane (3 × 20 mL), dried, and evaporated to yield a solid (20 mg).

Stypodiol. ¹H NMR (400 MHz, CDCl₃) δ 6.42 (1H, brs), 6.40 (1H, brs), 3.25 (1H, dd, J = 11.6, 4.8), 3.21 (1H, d, J = 16.4), 2.75 (1H, d, J = 16.4), 2.18 (3H, s), 0.94 (6H, s), 0.86 (3H, s), 0.77 (3H, s), 0.67 (1H, d, J = 6.3). In agreement with literature [22].

Peak purity of all compounds was checked by HPLC analysis, using an HPLC unit (Gilson) and a Spherisorb ODS2 column (4.6 × 250 mm, 5 μm particle size). The eluent used was acetonitrile, at a flow rate of 0.9 mL/min. Detection was achieved with a Gilson diode array detector. Spectral data from all peaks were accumulated in the range of 200–400 nm, and chromatograms were recorded at 280 nm for all compounds, excepting sytpodiol, which was determined at 305 nm. The data were processed on a Unipoint Software system (Gilson Medical Electronics, Villiers le Bel, France). All tested compounds were >99% pure, excepting 14-ketostypodiol diacetate (>95%) and stypodiol (>80%).

Sulforhodamine B (SRB), dimethylsulfoxide (DMSO) and vincristine were from Sigma-Aldrich (Steinheim, Germany). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS), human transferrin, fungizone and penicillin were from GIBCO (Invitrogen, UK). Tris-base, acetic acid and trichloroacetic acid were from Merck (Germany). Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) media were purchased from Liofilchem (Teramo, Italy). The water was treated in a Milli-Q water purification system (Millipore, Bedford, MA).

Caco-2, V79, SH-SY5Y and RAW.267 cells were a kind gift from Professor Fernando Remião from the Laboratory of Toxicology, Faculty of Pharmacy, Porto University, Portugal. RBL-2H3 cells were obtained from ATTC. All cells were maintained in DMEM with 10% FBS and 1% penicillin, excepting RBL-2H3, which were cultivated in 15% FBS. V79 and Caco-2 cells were additionally added non-essential amino acids and, in the case of Caco-2, human-transferrin and fungizone. All cells were grown in an incubator at 37 °C and 5% CO₂.

Stock solutions of all compounds in DMSO were prepared and diluted in media for testing. Cells were allowed to attach for 24 h, after which they were exposed to the compounds.

The method by Houghton et al. [23], with modifications [24], was followed. Cells were plated with a density of 2 × 10⁴ (V79, RBL-2H3, Caco-2 and RAW.267) and 1.5 × 10⁴ (SH-SY5Y) cells/well and allowed to attach for 24 h at 37 °C, with 5% CO₂. On the following day the medium was removed and cells were gently washed with warm PBS. Compounds in media were added and the plates were incubated for 48 h. The anticancer compound vincristine was used as a positive control.

After the incubation period media was removed, 100 μL of cold 40% trichloroacetic acid was added and plates were maintained at 4 °C for 60 min. Plates were then washed 5 times with tap water and allowed to dry. Afterwards, 50 μL of 0.4% SRB in 1% acetic acid were added and plates were incubated for 30 min.

After the incubation period plates were quickly washed with 1% acetic acid, in order to remove unbound dye, and allowed to dry. 100 μL of tris-base were added and after 10 min the plates were shaken.

Absorbance at 492 nm was determined in a multi-plate reader. OD values were plotted against concentration. Three independent assays were conducted, each one of them in triplicate. Values of inhibition of cell proliferation were expressed against a control of 0.1% DMSO.

Comparisons of data from different groups versus control were performed by one-way ANOVA test, using GraphPad Prism 5. A p value lower than 0.05 was considered statistically significant.