Cytotoxic and Antimicrobial Activity of Pseudopterosins and seco-Pseudopterosins Isolated from the Octocoral Pseudopterogorgia elisabethae of San Andrés and Providencia Islands (Southwest Caribbean Sea)

To expand the potential of pseudopterosins and seco-pseudopterosins isolated from the octocoral Pseudopterogorgia elisabethae of San Andrés and Providencia islands (southwest Caribbean Sea), we report the anti-microbial profile against four pathogenic microorganisms (Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans) and report a more complete cytotoxic profile against five human cells lines (HeLa, PC-3, HCT116, MCF-7 and BJ) for the compounds PsG, PsP, PsQ, PsS, PsT, PsU, 3-O-acetyl-PsU, seco-PsJ, seco-PsK and IMNGD. For the cytotoxic profiles, all compounds evaluated showed moderate and non-selective activity against both tumor and normal cell lines, where PsQ and PsG were the most active compounds (GI50 values between 5.8 μM to 12.0 μM). With respect to their anti-microbial activity the compounds showed good and selective activity against the Gram-positive bacteria, while they did not show activity against the Gram-negative bacterium or yeast. PsU, PsQ, PsS, seco-PsK and PsG were the most active compounds (IC50 2.9–4.5 μM) against S. aureus and PsG, PsU and seco-PsK showed good activity (IC50 3.1–3.8 μM) against E. faecalis, comparable to the reference drug vancomycin (4.2 μM).

In regards to their anti-inflammatory activity, our experiments showed that fractions enriched with pseudopterosins (PsG, PsK, PsP, PsQ, PsS, PsT and PsU) and seco-pseudopterosins (seco-PsJ and seco-PsK) were able to inhibit the inflammation in a 12-O-tetradecanoyl-phorbol-acetate (TPA)-induced edema assay, with results comparable to those shown by indomethacin used as the control standard [21]. Additionally, in in vitro experiments, PsQ, PsS, PsT, PsU and IMNGD showed good activity for the inhibition of MPO release; PsP, PsT and IMNGD inhibit NO release [21] and PsR inhibited thromboxane B2 (TXB 2 ) and the superoxide anion (O 2 − ) [11].
While there has been considerable effort towards characterizing the biological activity of pseudopterosins isolated from octocoral samples collected in the northern Caribbean, much is unknown about the bioactivity of such compounds isolated from octocoral collected from the Islands of San André s and Providencia. For this reason, and with a goal of expanding the potential of these compounds, here we report for the first time the antimicrobial profile against four pathogen microorganisms (S. aureus, E. faecalis, Pseudomonas aeruginosa and Candida albicans) and report a more complete cytotoxic profile against five human cells lines (HeLa, PC-3, HCT116, MCF-7 and BJ) for the compounds: PsG, PsP, PsQ, PsS, PsT, PsU, 3-O-acetyl-PsU, seco-PsJ, seco-PsK and IMNGD ( Figure 1).

Results and Discussion
In this study, PsG, PsP, PsQ, PsS, PsT, PsU, 3-O-acetyl-PsU, seco-PsJ, seco-PsK and IMNGD were isolated ( Figure 1) by flash chromatography and HPLC and identified by spectroscopic means. The structures of all compounds were previously reported by us [7,10], and preliminary assessment of their cytotoxic (Table 1) and antimicrobial ( Table 2) properties is presented here.

Cytotoxic Activity
The cytotoxic activity of the pseudopterosins (PsG, PsP, PsQ, PsS, PsT, PsU and 3-O-acetyl-PsU), seco-pseudopterosins (seco-PsJ and seco-PsK) and IMNGD against five cell lines: HeLa (cervical cancer), PC-3 (prostate cancer), HCT116 (colorectal cancer), MCF-7 (breast cancer) and BJ (fibroblasts) was investigated using the MTT reduction colorimetric assay [22]. Although no compound showed significant activity, according to the GI 50 cut off value of 10 nM for pure compounds suggested by Bugelski et al. [23], all compounds evaluated showed moderate to weak (GI 50 5.8-83.9 µM) and non-selective activity against both tumor and normal cell lines as shown in Table 1.
While none of these compound showed comparable activity to the reference drug staurosporine (GI 50 13.6-105.6 nM) against the four tumor cell lines, PsQ and PsG were the most active compounds (GI 50 values between 5.8 µM to 12.0 µM) and IMNGD showed a moderate activity with GI 50 values of 9.7-19.9 µg/mL. These results are comparable with those previously reported by Rodriguez et al. [11], who determined the GI 50 values for PsQ, PsU and PsV in the NCI-H460-cell line. Results for PsQ showed a GI 50 between 1.7-5.8 μM. In the same screen, PsU and PsV were generally much less toxic (GI 50 20-100 μM). For the normal cell line BJ (Table 1)  The selectivity of the cytotoxic activity of the compounds, measured as the differential effect on growth of different types of cell lines, was made by comparing the effect of the compounds on inhibiting cell growth in both normal and tumor cells. The results showed no selectivity of compounds between the lines used, and all compounds induced reduction in cell survival to a similar magnitude in all lines. Preliminary conclusions regarding structure-activity relationships can be drawn from an examination of the cytotoxic activity. The position of glycosylation on the terpene skeleton appears to affect the inhibitory activity profile as, for example, PsG (glycosylated in C-9 with fucopyranose) is more active than PsP (glycosylated in C-10 with fucopyranose). Further, the type of sugar moiety also influences the activity as, for example, PsP which is glycosylated with fucopyranose is more active than PsT which is glycosylated with arabinopyranose. Likewise, PsQ (C-4' mono-acetylated fucose as sugar moiety) is more active than PsU (C-4' mono-acetylated arabinose as sugar moiety) and seco-PsK (non-acetylated fucose as sugar moiety) is more active than seco-PsJ (mono-acetylated arabinose as sugar moiety).

Antimicrobial Activity
The antimicrobial activity of the pseudopterosins, seco-pseudopterosins and IMNGD was investigated against one Gram-negative bacterium (P. aeruginosa), two Gram-positive bacteria (S. aureus and E. faecalis) and one yeast (C. albicans), using a microdilution method [24]. The majority of compounds evaluated showed a good IC 50 (2.9-7.64 µM) and all showed a selective activity against both Gram-positive bacteria tested (Table 2), while they did not show activity against the Gram-negative bacterium or the yeast.
PsU, PsQ, PsS, seco-PsK, PsG were the most active compounds (IC 50 2.3-4.5 µM) against S. aureus. The IMNGD showed good activity with IC 50 of 2.3 µg/mL. For E. faecalis, PsG, PsU and seco-PsK showed better activity (Table 2) compared to the reference drug vancomycin (4.2 µM) while, seco-PsJ and PsT exhibited similar activity. The IMNGD showed good activity, with an IC 50 of 3.5 µg/mL. These results are comparable with the activity previously reported for PsA-E, PsK, PsX and PsY isolated from specimens collected in the north Caribbean Sea [5], which were reported to have minimum inhibitory concentration (MIC) values between 4.2 and 8.8 µM.
The selectivity observed in our study is comparable to that reported by Ata et al., [5] who reported that other pseudopterosins (PsA-E, PsK, PsX and PsY) inhibit the growth of Gram-positive bacteria (S. pyogenes, S. aureus,and E. faecalis), while they were inactive against Gram-negative bacteria (E. coli and P. aeruginosa). This finding can be related to the fact that many Gram-negative bacteria are resistant to toxic agents in the environment, due to the barrier of lipopolysaccharides on their outer membrane [25]. Examination of the data in Table 2 suggests the following structure-activity relationships. Firstly, PsG (glycosylated in C-9 with fucopyranose) is more active than PsP (glycosylated in C-10 with fucopyranose). Secondly, PsT glycosylated with arabinopyranose is more active than PsP, which is glycosylated with fucopyranose. Likewise, PsU (mono-acetylated arabinose as sugar moiety) is more active than PsQ and PsS (mono-acetylated fucose as sugar moiety). However, this behavior initially observed in pseudopterosins (amphilectane skeleton) is not retained when the results are compared with seco-psedopterosins (serrulatane skeleton), where, the seco-PsK (glycosylated with fucoyranose) showed more activity than seco-PsJ (glycosylated with arabinopyranose). Clearly, additional work is required to fully understand the structure-activity relationships for this family of terpenes.
From the above discussion, one can note that the pseudopterosins, seco-pseudopterosins and IMNGD isolated from P. elisabethae collected at the Islands of San André s and Providencia (southwest Caribbean Sea) have a similar drug-like potential as the related compounds isolated from specimens collected in the north Caribbean Sea (the Bahamas, Bermuda and the Florida Keys). Further, due to their wide applicability in commercial additives, in the cosmetic industry, it might be important to consider P. elisabethae collected in Colombian waters as an alternative source of such compounds. As has been shown, the anti-inflammatory, anti-microbial and cytotoxic activity of this family of compounds isolated from P. elisabethae collected at different locations at the Caribbean Sea are quite similar, regardless of a small number of differences in their structure (type of sugar moiety, position of glycosylation and stereochemistry).

Chemicals and Reagents
The following substances were purchased from Sigma-Aldrich (

Cell Lines
The cell lines HeLa (cervical cancer), PC-3 (prostate cancer), HCT116 (colorectal cancer), MCF-7 (breast cancer) and normal human cell line BJ (skin fibroblasts) were used and maintained in DMEM with 5% of FBS and gentamicin 50 μg/mL. Cultures were held in 75 cm 2 culture flasks at 37 °C , 5% CO 2 and 100% relative humidity, changing media at least twice a week.

Cytotoxic Assay
Cells were harvested, counted, and transferred into 96 well plates and incubated for 24 h prior to the addition of the test compounds. Test sample solutions (10 µL) at the desired dilutions were added to the wells containing the cells and incubated for 48 h. Microtitration colorimetric method of MTT reduction was used to determine surviving cells at the end of the treatment period [22].
A solution of MTT (0.5 mg/mL) was prepared in PBS and 100 µL of this solution was added to each well after the removal of 100 µL of treatment solution. The plates were incubated at 37 °C for 4 h. The solution in each well, containing media, unmetabolized MTT, and dead cells, was removed by suction and 100 µL of DMSO was added to each well. The DMSO was mixed by pipetting and optical density was recorded using a BioTek Synergy HT microplate reader to measure the absorbance at 570 nm. Percentages of cell survival relative to vehicle control wells (wells containing only cells and DMSO) were calculated.
Stock solutions (5 mg/mL) were prepared by dissolving pure compounds in DMSO and storing at 4 °C. Serial dilutions with culture media were prepared just prior to addition to test plates. The concentration of DMSO in wells was consistent across dilutions at 0.38% v/v. Staurosporine was used as the positive control and vehicle (DMSO + media) as blank. Control and test compounds (PsG, PsP, PsQ, PsS, PsT, PsU, 3-O-Ac-PsU, seco-PsJ, seco-PsK and IMNGD) were assayed in duplicate for each concentration and replicated for each cell line at concentrations of 5000, 1000, 500, 100, 50, 10, 5 and 1 nM for staurosporine and 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, and 20.0 µg/mL for compounds.

Microbial Strains and Culture Media
Compounds isolated from P. elisabethae were tested against a panel of microorganisms including the bacteria Staphylococcus aureus ATCC 375, Enterococcus faecalis ATCC 10741, Pseudomonas aeruginosa ATCC 14210 and the yeast Candida albicans ATCC 14035. Bacterial strains were cultured in Luria-Bertani Broth (LB) and C. albicans was cultured in Sabouraud dextrose broth (SD), for 18 h at 37 °C and 220 rpm in a humidified incubator.

Antimicrobial Assay
The antimicrobial activity was determined by a microdilution method (IC 50 ) in 96-well microtiter plates using LB media for bacteria and SD media for the yeast seeded liquid media [24]. In all cases, a pre-inoculated dilution was made with fresh broth. A volume of 180 L of each microorganism suspension was inoculated in each well (10 5 cells/mL) and mixed with 20 µL of treatment solutions. Stock solutions were prepared by dissolving pure compounds and controls in 20% DMSO and storing at 4 °C. Penicillin G (S. aureus), vancomycin (E. faecalis), gentamicin (P. aeruginosa) and nystatin (C. albicans) were used as positive controls and vehicle (DMSO + media) as blank. The final DMSO concentration used for dissolving the extracts was less than 20%. Controls and test compounds (PsG, PsP, PsQ, PsS, PsT, PsU, 3-O-Ac-PsU, seco-PsJ, seco-PsK and IMNGD) were assayed in duplicate for each of the following concentrations: 64.0, 32.0, 16.0, 4.0, 1.0, 0.25 and 0.06 µg/mL.
Microplates were incubated for 24 h at 37 °C and optical density was recorded using a BioTek Synergy HT microplate reader to measure the absorbance at 600 nm. For each experiment, correction for background absorbance was made by subtracting the value for OD 600 after the compounds were added (time 0 h). Percentages of microorganism survival relative to vehicle control wells (wells containing only microorganism and DMSO) were calculated. The IC 50 of the controls were also determined in parallel experiments in order to control for the sensitivity of the standard test organisms.

Statistical Analysis
Data were normalized and analyzed using non-linear regression to elucidate IC 50 values from sigmoidal dose-response curves. Comparison between cell lines and microorganisms used extra sum-of-squares F test to evaluate IC 50 between data sets. P < 0.05 was considered as indicative of significance using GraphPad Software, Prism V. 5.0.

Conclusions
All results presented here contribute to the demonstration that the compounds isolated from P. elisabethae (SW Caribbean) are promising molecules with potentially useful antimicrobial activity profiles. This confirms that this marine organism has great value as a source of lead compounds with pharmaceutical applications. Specifically, PsU, PsQ, PsS, seco-PsK and PsG are the most promising anti-bacterial agents. For this reason, it will be interesting to continue with molecular targeted high-throughput screens in order to understand their mechanism of action.