Antiproliferative Scalarane-Based Metabolites from the Red Sea Sponge Hyrtios erectus

Two new sesterterpenes analogs, namely, 12-acetoxy,16-epi-hyrtiolide (1) and 12β-acetoxy,16β-methoxy,20α-hydroxy-17-scalaren-19,20-olide (2), containing a scalarane-based framework along with seven previously reported scalarane-type sesterterpenes (3–9) have been isolated from the sponge Hyrtios erectus (order Dictyoceratida) collected from the Red Sea, Egypt. The structures of the isolated compounds were elucidated on the basis of their spectroscopic data and comparison with reported NMR data. Compounds 1–9 exhibited considerable antiproliferative activity against breast adenocarcinoma (MCF-7), colorectal carcinoma (HCT-116) and hepatocellular carcinoma cells (HepG2). Compounds 3, 5 and 9 were selected for subsequent investigations regarding their mechanism of cell death induction (differential apoptosis/necrosis assessment) and their influence on cell cycle distribution.

Relative configuration at C-12, C-16 and C-20 was detected and confirmed based on their coupling constants and NOESY correlations (Supplementary Materials, Figure S12). On the basis of the coupling constants, the diaxial coupling of H-12 (δ H 4.88; dd, J = 10.8 and 3.6 Hz) with H-11 and NOESY cross-peaks with α oriented H-9 and H-14 indicate its α-configuration ( Figure 5). Relative configuration at C-12, C-16 and C-20 was detected and confirmed based on their coupling constants and NOESY correlations (Supplementary Materials, Figure S12). On the basis of the coupling constants, the diaxial coupling of H-12 (δH 4.88; dd, J = 10.8 and 3.6 Hz) with H-11 and NOESY cross-peaks with α oriented H-9 and H-14 indicate its α-configuration ( Figure 5).  Similarly, the diaxial coupling of H-16 (δ H 4.08; dd, J = 9.0 and 6.6 Hz) with H-15 indicates the α-configuration of H-16 which was confirmed by NOESY cross-peaks with the α oriented H-14 ( Figure 5). Finally, cross-peaks between H-20 and β-OMe in NOESY indicate its β-configuration ( Figure 5). SRB-U assay was used to assess the antiproliferative effects of compounds 1-9 (Table 3)   In MCF-7 breast cancer cells, compound 5 and compound 9 showed the most potent cytotoxic profile with IC 50 s of 1.1 µM, and 3.3 µM, respectively. Compound 6 was the weakest against MCF-7 cells with IC 50 s higher than 100 µM. Other compounds (1, 2, 3, 4, 7 and 8) showed considerable cytotoxic profile with IC 50 s ranging from 12.7 µM to 40.3 µM (Table 3).
In HepG2 liver cancer cells, compounds 3, 5 and 9 showed relatively potent cytotoxic effect with IC 50 s of 9.6 µM, 1.1 µM and 1.7 µM, respectively. Compound 6 possessed weak cytotoxicity against HepG2 cells with IC 50 s higher than 100 µM. Other compounds (1, 2, 4, 7 and 8) showed moderate cytotoxicity with IC 50 s ranging from 15.5 µM to 42.5 µM. Accordingly, compounds 3, 5 and 9 were selected for subsequent investigations regarding their mechanism of cell death induction (differential apoptosis/necrosis assessment) and their influence on cell cycle distribution.

Programmed Cell Death Induced by Compounds 3, 5 and 9 against HCT-116 Cells
Annexin V-FITC/PI staining coupled with flowcytometry was used to assess proportion of cells undergoing necrosis or cells undergoing programmed cell death (apoptosis). HCT-116 was treated with 5 µM of compounds 3, 5 and 9 for only 24 h and apoptosis/necrosis cell death was assessed. Compound 5 significantly increased total cell death by 20 fold compared to control. Cell death induced by compound 5 is attributed mainly to apoptosis induction; and a much lesser extent is attributed to necrosis ( Figure 6A,C,E). Compound 9 significantly induced total cell death by 1.2 fold compared to control cells. Similarly, compound 9 mainly induced cell killing effect via the activation of programmed cell death rather than non-specific necrosis cell death ( Figure 6A,D,E). Compound 3 was the weakest among the other selected compounds; it only induced more apoptosis with reciprocal less necrosis compared to control cells. However, total cell death induced by compound 3 was not significant from control untreated cells ( Figure 6A,B,E). blue exclusion assay was used to confirm percent of cells with lost membrane integrity (necrosis and late apoptosis populations). After cell exposure for 72 h, compound 3 and compound 9 induced moderate membrane integrity damage indicated by 5.1% ± 1.3% and 9.5% ± 2.1% positive trypan blue cells, respectively. On the other hand, compound 5 induced profound membrane integrity damage with 67.6% ± 4.2% trypan blue positive cells (Supplementary Table S1). Figure 6 Effect of potentially active compounds on cell death profile against HCT-116 cells.  Cell cycle distribution using DNA flow cytometry was used to investigate the influence of compounds 3, 5 and 9 on the proliferation profile of tumor cells. Cells were exposed to compounds 3, 5 and 9 (1 µ M) for 24 h and cell cycle phases were assessed as mentioned in the experimental section. All tested compounds (3, 5 and 9) exerted significant anti-proliferative effect against HCT-116 cells appeared as increased cell population in G0/G1-phase from 53% ± 1.0% to 59.8% ± 0.2%, 68.4% ± 1.7% and 71.5% ± 0.5% respectively. The increased non proliferating cell population in compounds 3, 5 and 9 was accompanied by reciprocal decrease in cells in S-phase from 35.4% ± 1.7% to 31.8% ± 0.2%, 21.8% ± 1.2% and 18.7% ± 1.1% respectively. Additionally, only compound 3 treatment resulted in decreasing G2/M cells from 11.6% ± 0.9% to 8.4% ± 0.1% after 24 h ( Figure 7A-E). In addition, compounds 5 and 9 significantly increased cells in Pre-G phase (apoptotic cells) from 1.0% ± 0.1% to 65.1% ± 1.0% and 4.8% ± 0.1% respectively ( Figure 7F). It can be concluded that the potential anticancer activity of compound 3 might be solely attributed to antiproliferative effect while compounds 5 and 9 possess mixed cytotoxic and antiproliferative activities. After exposure of HCT-116 cells to the pre-determined IC 50 's of selected compounds, trypan blue exclusion assay was used to confirm percent of cells with lost membrane integrity (necrosis and late apoptosis populations). After cell exposure for 72 h, compound 3 and compound 9 induced moderate membrane integrity damage indicated by 5.1%˘1.3% and 9.5%˘2.1% positive trypan blue cells, respectively. On the other hand, compound 5 induced profound membrane integrity damage with 67.6%˘4.2% trypan blue positive cells (Supplementary Table S1). Figure 6 Effect of potentially active compounds on cell death profile against HCT-116 cells.

Influence of Compounds 3, 5 and 9 on Cell Cycle Distribution of HCT-116 Cells
Cell cycle distribution using DNA flow cytometry was used to investigate the influence of compounds 3, 5 and 9 on the proliferation profile of tumor cells. Cells were exposed to compounds 3, 5 and 9 (1 µM) for 24 h and cell cycle phases were assessed as mentioned in the experimental section. All tested compounds (3, 5 and 9) exerted significant anti-proliferative effect against HCT-116 cells appeared as increased cell population in G 0 /G 1 -phase from 53%˘1.0% to 59.8%˘0.2%, 68.4%˘1.7% and 71.5%˘0.5% respectively. The increased non proliferating cell population in compounds 3, 5 and 9 was accompanied by reciprocal decrease in cells in S-phase from 35.4%˘1.7% to 31.8%˘0.2%, 21.8%˘1.2% and 18.7%˘1.1% respectively. Additionally, only compound 3 treatment resulted in decreasing G 2 /M cells from 11.6%˘0.9% to 8.4%˘0.1% after 24 h ( Figure 7A-E). In addition, compounds 5 and 9 significantly increased cells in Pre-G phase (apoptotic cells) from 1.0%˘0.1% to 65.1%˘1.0% and 4.8%˘0.1% respectively ( Figure 7F). It can be concluded that the potential anticancer activity of compound 3 might be solely attributed to antiproliferative effect while compounds 5 and 9 possess mixed cytotoxic and antiproliferative activities. Further molecular studies Mar. Drugs 2016, 14, 130 9 of 14 on compounds 5 and 9 to assess their modes of action are highly recommended. Due to the low yield of compounds from marine origin on the top of their non-renewability [40], it might be difficult to harvest sufficient material for use in a clinical setting. However, synthetic chemists are highly urged to use this nucleus as a lead compound in the field of anti-cancer drug discovery [41][42][43].
Mar . Drugs 2016, 14, 130 9 of 14 studies on compounds 5 and 9 to assess their modes of action are highly recommended. Due to the low yield of compounds from marine origin on the top of their non-renewability [40], it might be difficult to harvest sufficient material for use in a clinical setting. However, synthetic chemists are highly urged to use this nucleus as a lead compound in the field of anti-cancer drug discovery [41][42][43]. Figure 7 presents the effects of potentially active compounds on cell cycle distribution of HCT-116 cells.
(E) Cell cycle distribution (F) Pre-G phase Figure 7. Effect of Compounds 3, 5 and 9 on the cell cycle distribution of HCT-116 cells. The cells were exposed to Compounds 3 (B), 5 (C), and 9 (D) for 24 h and compared to control cells (A). Cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted (E) as percentage of total events. Sub-G cell population was taken as representative of total cell death and was plotted as percent of total events (F). Data are presented as mean ± SD; n = 3. *: significantly different from control group.

General Experimental Procedures
Optical rotation was measured on the automatic high-speed laboratory polarimeter P3000 (A.KRUSS Optronic Gmbh, Hamburg, Germany). UV spectra were measured on a Hitachi 300 Spectrophotometer (Hitachi High-Technologies Corporation, Kyoto, Japan). High-resolution ESIMS data were recorded with an Ultra-High Resolution (UHR) TOF spectrometer (Impact, Bruker, Bremen, Germany). NMR spectra were obtained in CDCl3 on a Bruker Avance DRX 600-MHz spectrometer (Bruker) at 600-MHz for 1 H NMR and 150 MHz for 13

Biological Materials
Specimen of the marine sponge, Hyrtios erectus (Keller, 1889) (Figure 1) was collected from Sharm el-Sheikh, Red Sea Egypt, using scuba diving at a depth of 11 m and 17 m. The sponge material was immediately frozen after collection and kept at´20˝C until investigation. The sponge was kindly identified by Dr. R. van Soest (Institute of Systematic Population Biology, Amsterdam University, The Netherlands) as Hyrtios erectus (class: Demospongiae, order: Dictyoceratida, family: Thorectidae). A voucher specimen is kept in the collections of the Zoological Museum of the University of Amsterdam, under ZMAPOR19761 registration number.

Purification of Compounds 1-9
The sponge materials (0.91 kg, wet wt.) were cut into small pieces and were extracted three times at room temperature with MeOH (3ˆ2 L). The combined extracts were concentrated under reduced pressure to afford the organic crude extract (86 g). The concentrated total extract was subjected to silica gel column using VLC (vacuum liquid chromatography) stepwise gradient elution (n-hexane-CHCl 3 -MeOH) to obtain fractions 1-9. Fraction 4 (n-hexane-CHCl3, 1:3) was subjected to heat-inactivated fetal bovine serum. Cells were propagated in a humidified incubator at 37˝C with 5% (v/v) CO 2 atmosphere.

Trypan-Blue Exclusion Assay
Viability of cells was confirmed prior to seeding using trypan-blue exclusion assay. Briefly, exponentially growing cells were detached using trypsin/EDTA solution. Aliquots of live cell suspensions were exposed to trypan blue solution (0.4%) and percent of trypan blue positive cells was determined. Cell suspensions were not used with viability less than 95%.

Antiproliferative Assessment
The antiproliferative activities of the compounds 1-9 on breast adenocarcinoma (MCF-7), colorectal carcinoma (HCT-116) and hepatocellular carcinoma cells (HepG2) were evaluated using the sulforhodamine B (SRB) assay as previously described [44]. Briefly, exponentially growing cells were collected using 0.25% Trypsin-EDTA and plated in 96-well plates at 1000-2000 cells/well. Cells were exposed to serial concentrations of test compounds for 72 h and subsequently fixed with TCA (10%) for 1 h at 4˝C. After washing trice, cells were exposed to 0.4% SRB solution for 10 min in dark place and subsequently washed with 1% glacial acetic acid. After drying overnight, Tris-HCl was used to dissolve the SRB-stained cells and color intensity was measured at 540 nm.
The dose response curve of compounds was analyzed using E max model (Equation (1)).

% cell viability " p100´Rqˆˆ1´r
where (R) is the residual unaffected fraction (the resistance fraction), (D) is the drug concentration used, (K d ) or IC 50 is the drug concentration that produces a 50% reduction of the maximum inhibition rate and (m) is a Hill-type coefficient. IC 50 was defined as the drug concentration required to reduce absorbance to 50% of that of the control (i.e., K d = absolute IC 50 when R = 0 and E max = 100´R) [45].

Apoptosis Assessment Using Annexin V-FITC Staining Coupled with Flowcytometry
To assess the potential of selected active compounds in inducing programmed cell death, apoptosis and necrosis cell populations were determined using Annexin V-FITC apoptosis detection kit (Abcam Inc., Cambridge Science Park, Cambridge, UK). Briefly, HCT-116 cells were treated with compounds 3, 5 and 9 for 24 h and collected by trypsinization, washed twice with ice-cold PBS, and re-suspended in 0.5 mL of annexin V-FITC/PI solution for 30 min in dark according to manufacturer protocol. After staining at room temperature, cells were injected through ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed for FITC and PI fluorescent signals using FL1 and FL2 signal detector, respectively (λ ex/em 488/530 nm for FITC and λ ex/em 535/617 nm for PI). For each sample, 12,000 events were acquired and positive FITC and/or PI cells were quantified by quadrant analysis and calculated using ACEA NovoExpress™ software version 1.1.0 (ACEA Biosciences Inc.).

Analysis of Cell Cycle Distribution
To assess the effect of selected active compounds on cell cycle distribution, HCT-116 cells were treated with compounds 3, 5 and 9 for 24 h. After treatment, cells were collected by trypsinization; washed twice with ice-cold PBS and re-suspended in 0.5 mL of PBS. Two milliliters of 70% ice-cold ethanol was added gently while vortexing. Cells were kept in ethanol solution at 4˝C for 1 h for fixation. Upon analysis, fixed cells were washed and re-suspended in 1 mL of PBS containing 50 µg/mL RNAase A and 10 µg/mL propidium iodide (PI). After 20 min incubation in dark place at room temperature, cells were analyzed for DNA contents by FACS-VantageTM (Becton Dickinson Immunocytometry Systems). For each sample, 10,000 events were acquired. Cell cycle distribution was calculated using ACEA NovoExpress™ software.

Statistical Analysis
Data are presented as mean˘SEM using GraphPad prism™ software version 5.00 (GraphPad software Inc., La Jolla, CA, USA) for windows version 5.00. Analysis of variance (ANOVA) with Bonferroni post hoc test was used for testing the significance using SPSS ® for windows, version 17.0.0. p < 0.05 was taken as a cut off value for significance.