EP (methyl (2S)-2-[[(2Z)-2-(3-hydroxy-5-oxo-4-phenylfuran-2-ylidene)-2-phenylacetyl] amino]-4-methylpentanoate) was isolated from Acarospora schleicheri A. Massal, and collected in altitudinal gradients in Enquelga-Isluga (19°14′S, 68°47′W) in Chile´s alpine zones. In each site, at least ten thalli were randomly collected from rock surfaces. Voucher specimens were deposited in the Lichen Herbarium of the School of Chemistry and Pharmacy, Universidad de Valparaíso. The collection of the specimens was authorized by the National Forest Corporation (CONAF).
Thalli were cleaned and washed with distilled water, and dried at 60 °C. EP was extracted in acetone at room temperature (20 °C ± 2°) for 48 and 24 h successively. The extract was then purified chromatographically using a silica gel Merck 60 G (0.032–0.063 nm) column eluted with a mixture of hexane and ethyl-acetate with increasing polarity. The fractions were monitored by thin-layer chromatography (TLC) using silica gel Merck 60 F254 plates. The blots were developed using a H2SO4 spray reagent and UV light (254/365 nm).
RMN spectroscopic analysis was utilized. The 1H- and 13C-NMR spectra were recorded in CDCl3 solutions and are referenced to the residual peaks of CHCl3 at δ = 7.26 ppm and δ = 77.00 ppm for 1H and 13C on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1H and 100.6 MHz for 13C.
Optical rotation was measured with a sodium lamp (λ = 589 nm, D line) on a Atago AP-300 digital polarimeter equipped with 1 dm cells at 23 °C.
The human breast adenocarcinoma cell line MCF-7 (American Type Culture Collection, (ATCC® HTB-22™), Rockville, MD, USA) and the human epithelial kidney HEK293 cells (ATCC® CRL-11268™) were grown in DMEM (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, PAA Laboratories GmbH, Linz, Austria), 2 mM glutamine, 10 U/L penicillin and 100 μg/mL streptomycin (Thermo Fischer Scientific, Waltham, MA USA). The cells were cultured in an incubator (Thermo Forma) with a 5% CO2 humidified atmosphere.
Cell proliferation assay
Cells were seeded into 96-well cell culture plates at a density of 5 × 103 cells/well. After 24 h incubation, cells were exposed for 48 h to 14, 28, 42, 56, 70, 84 and 98 μM EP in dimethyl sulfoxide (DMSO). Equivalent concentrations of DMSO vehicle, corresponding to the different dilutions of the test metabolite, and cells without treatment were included as negative controls. Cell proliferation inhibition by 1.3 μM tamoxifen (TMX) was used as positive control. Cell proliferation was determined with sulforhodamine-B (SRB, Sigma Aldrich, St Louis, MI) assay . At the end of the culture period, proteins were precipitated with 50% w/v trichloroacetic acid and cells were stained with 50 μL of SRB (0.4% w/v in 1% v/v acetic acid). Finally, 200 μL 10 mM tris(hydroxymethyl) aminomethane (TRIS) were added to each well and absorbance was read at 540 nm using a microplate reader (Merck Sensident Scan).
DNA fragmentation assay
Detection of DNA fragmentation as indicator of apoptosis was performed by the in Situ Cell Death Detection Kit (TUNEL Kit, Roche Applied Science, Manheim, Germany) . MCF-7 cells were grown on silanized slides until 40% confluence. Then, the cells were treated for 12 h with 28 μM EP in DMSO, and 50 μM TMX, DMSO, and untreated cells as positive, vehicle and negative controls, respectively. At the end of the exposure, cells were washed five times with phosphate saline buffer (PBS) and fixed 20 min with 2% p-formaldehyde at 4 °C. After washing them five times with PBS, apoptosis was determined following manufacturer’s instructions, adding 4’,6-diamidino-2-phenylindole (DAPI) to stain the nuclei and using a technical negative control with a slide to which no terminal deoxynucleotidyl transferase (TdT) was added. Images were visualized with an Olympus BX 51 fluorescence microscope provided with a U-MWU2 Olympus filter.
Cell cycle analysis by flow cytometry
This protocol was performed by adapting the report of Riccardi and Nicoletti . For this, 20 × 104 cells were seeded in cell culture flasks and incubated for 24 h, after which they were treated for 48 h with 28 μM EP, 1.3 μM TMX, DMSO, and untreated cells were used as positive, vehicle, and negative controls, respectively. Cells were then trypsinized, aliquoted into flow cytometry tubes (1 × 106 cells/tube) and centrifuged 5 min at 240g. The pellet was resuspended into 500 μL PBS, and kept in ethanol at 4 °C until its analysis, at which time cells were centrifuged for 5 min at 240g, pellets were washed with 1 mL 10% p/v albumin in PBS, centrifuged for 5 min at 240g, and resuspended with 500 μL Krisham solution (1.12 mg/mL sodium citrate, 0.046 mg/mL propidium iodide, 0.01% v/v triton X-100 and 0.01 mg/mL RNAase A) for DNA staining. DNA fluorescence was detected with a flow cytometer [Coulter (R) Epics (R)]. For cell counting, a minimum of 3500 events were recorded for each treatment. Fluorescence intensity histograms versus event numbers were recorded.
Mitochondrial oxygen reactive species assay
The mitoSOX red fluorescent probe was utilized for the assessment of mitochondrial derived oxygen reactive species . The assay was performed seeding 5000 MCF-7 cells on a 96-wells culture plate. After 24 h, the cells were exposed to 1 µM doxorubicin (positive control), 28 μM EP or 1% DMSO containing cell culture media for 48 h. Then, the cells were washed twice with PBS and exposed to mytoSOX for 10 min; next, they were washed twice again with PBS and the fluorescence detected at 510/595 nm of excitation and emission, respectively, with a Varioscan™LUX multimode plate reader (Thermoscientific). Next, the protein content was determined by using the sulforhodamine B (SRB) assay. For this, the cells were fixed using cold 1% acetic acid in methanol and then exposed to SRB 0.5% w/v for 1 h at 37 °C. After removing the SRB, the wells were gently washed with 1% acetic acid, then the plate was dried and the fixed dye solubilized by adding 200 μL of 10 mM tris pH 10. The absorbance was read with the Varioscan™LUX multimode plate reader (Thermoscientific) at 580 nm. The results were expressed as relative fluorescence units normalized by the protein content of each well.
Testing was performed according to standard S. typhimurium reverse mutation assay (Ames test) procedures [1, 21]. In brief, four different histidine-deficient (his−) S. typhimurium strains TA98, TA100, TA102 and TA 104 were used. For metabolic activation, S9 fraction was obtained from the supernatant of post-mitochondrial mouse liver fraction exposed to sodium phenobarbital and β-naphtoflavone . E was dissolved in DMSO and tested at 0.125, 12.5 and 125 μg/plate. Each strain was incubated in the presence of E at 37 °C for 48 h with and without metabolic activation. Spontaneous reversions and mutations in response to DMSO and known mutagens (4-nitroquinoline-N-oxide for TA98, methylmethanesulfonate for TA 100 and TA102 and methylglyoxal for TA104) were also determined for negative and positive controls, respectively. Each treatment was performed in triplicate.
For proliferation assays, 42 independent experiments were conducted. The percentage of inhibition for E was expressed as π (%iEX) with the following statistical hypothesis: H0: π (%iEX) = 0 and H1: π (%iEX) > 0. The results of the assay gave an estimation of π (%iEX) which was called p (%iARX), and a function of this sample proportion allowed to analyze the hypothesis. The Stata software was used  to calculate the signification probability, which allowed to reject H0 when it was below 0.05.