In vitro experiments

Human MCF-7, HepG2 and Fibroblasts cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum, 4 mmol L⁻¹ of L-glutamine and a mixture of antibiotics (5 mg mL⁻¹ of streptomycin and 5 mg mL⁻¹ penicillin). Cells were incubated at 37 °C in humidified atmosphere containing 5% CO₂. After cells reached ~ 80% of confluence, cells were harvested and seeded in a 96 well plate or 12 well plate, according to the experimental protocol.

The WST-1 viability assay is a colorimetric assay for the quantification of cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells producing a colorimetric product, formazan (CELLPRO-Roche, Mannheim, Germany). Briefly, 5 × 10³ of MCF-7 cells/well were added in 96 wells plate. Cells were incubated with vehicle (0) or fruti (31.2, 62.5, 125 and 250 µM) dissolved in 0.1% DMSO/DMEM for 24 h. Then, the medium was replaced by new medium to which 10 µL of WST-1was added and the cells were incubated for 2 h at 37 °C in humidified atmosphere 5% CO₂. Additionally, fibroblasts were also incubated with fruti to evaluate the cytotoxicity of fruti in non-transformed cells. Viability was measured by spectrophotometer at 450 and 690 nm, after 1 min shaking the plate to equilibrate the intracellular formazan. The results were expressed as cell viability (%). In addition, to evaluate the necroptosis pathway, the same procedure described above was performed, including the incubation of cells with 50 µM necrostatin-1 (Nec-1) (Sigma-Aldrich, St. Louis, Missouri, EUA), an inhibitor of receptor-associated kinase 1 (RIPK1).

To perform reduced glutathione (GSH) measurement, MCF-7 cells were seeded in 96 wells plate until reaching 80% of confluence. Vehicle (0) or fruti (12.5, 25, 50 and 100 µM) was incubated for 0.5, 2, 16 and 24 h to better evaluate if alterations in quinone concentration over a period of time would deplete this peptide. After the incubation, cells were scratched in the presence of iced cold 10% perchloric acid and centrifuged for 10 min at 14,000×g 4 °C. To increase the pH to 8, 3.0 M K₂PO₄ was added in the supernatant and samples were immediately frozen in liquid nitrogen. After 30 min samples were thawn and incubated with work solution (DTNB and NADPH) diluted in GSH buffer (0.1 M Na₂HPO₄.2H₂O, 0.1 M NaH₂PO₄.2H₂O and 0.05 M Na₂EDTA.2H₂O) in a 96 wells plate. The reaction was started by addition of glutathione reductase (GR) and the absorbance was monitored at 412 nm at 37 °C on a Clariostar (BMG LabTech, Cary, USA) analyzer. The results were interpolated with GSH standards and were expressed in nmol GSH/well³⁸.

Additionally, to measure total reactive oxygen species (ROS), MCF-7 cells were seeded in a black 96 wells plate. After 24 h cells were briefly washed with DMEM without phenol red and supplemented with 1.9 g L⁻¹ NaHCO₃, 5 mM glucose and 0.1% fetal bovine serum. 5 µM DCFH-DA was added to monitor ROS production following addition of fruti concentrations (25, 50, 75 and 100 µM), dissolved in 0.1% DMSO/DMEM (-) phenol red. The fluorescence was monitored on a Clariostar analyzer at 488/20, 520/20 nm at 37 °C, 5% CO₂. Results were expressed as DCFH-DA fluorescence (AUC/sec)³⁹.

Human MCF-7, HepG2 and Fibroblast cells were seeded in 12 wells plate. After reaching confluence, cells were incubated with vehicle (0) or fruti at 10, 25, 50, 75 or 100 µM for 24 h (concentrations previously chosen by WST1 assay screening). Total RNA was extracted from cultured cells using Trizol reagent (Sigma-Aldrich, St. Louis, Missouri, USA) and measured by Nanodrop 100 (Thermo Scientific, Waltham, Massachusetts, USA). Complementary DNA was synthesized from 2 µg total RNA with an oligo-dT, random hexamers primers and deoxythymidine oligomer, Ribolock RNAse inhibitor and RevertAid reverse transcriptase. Real-time polymerase chain reaction was performed in a Lightcycler apparatus (Roche, Mannheim, Germany) using the sensiFAST SYBR No-ROX kit (Bioline, London, UK). Initial fluorescent values were calculated by LinRegPCR3 (version 2013.0; Academic Medical Center, Amsterdam, The Netherlands). Primer sequences were described in Supplementary Table ^S2. The most stable reference genes were calculated using geNorm⁴⁰. Transcript levels were normalized to housekeeping genes ratio GAPDH and Cyclophilin (CyP) for MCF-7 and Fibroblasts cells, and the genes ratio HPRT and Actin for HepG2 cells.

MCF-7 cells were seeded in a 12 wells plate until reaching confluence. Then the cells were incubated with vehicle (0) or fruti at 10, 25 and 50 µM for 24 h (concentrations previously chosen by WST1 assay screening), harvested in iced cold PBS and homogenized in RIPA buffer freshly supplemented with PhosSTOP phosphatase inhibitor (Roche, Mannheim, Germany) and 5 mM EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany). The samples were centrifuged for 10 min at 4 °C and the supernatant was used for protein measurement by the bicinchonic acid assay. Forty micrograms of protein were electrophoresed on 8–10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidine difluoride (PVDF) membrane by semidry blotting. The membrane was blocked overnight in 5% nonfat milk/TBST followed of overnight incubation with primary antibody in a cold room at 4 °C. The PVDF membrane was washed 3 times with TBST and incubated with secondary antibody with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G antibody (BioRad, California, USA) for 1 h at room temperature. All antibodies were diluted in 1% nonfat milk/TBST. The lists of antibodies and dilutions used were described in Supplementary Table ^S3. The bands in the membrane were detected using chemiluminescence reagents (100 mM Tris HCl pH 8.5, 1.25 nM luminol 0.2 mM p-coumarin and freshly added 3 mM H₂O₂) by ImageQuant LAS 4000 (GE Healthcare Life Sciences, Pittsburgh, USA) and compared with endogenous GAPDH protein.

As human MCF-7 cells do not express caspase 3 protein⁴¹, the caspase 3/7 activity assay was performed seeding HepG2 cells in a 96 wells plate. The cells were incubated with vehicle (0) or fruti at 10, 25, 50, 75, 100 and 125 µM for 16, 24, 48 and 72 h to better detect the effect of different fruti concentrations on caspase 3/7 activity over a time-course. Caspase 3/7 activity solution was added according to the manufacture instructions (SensoLyte, AnaSpec EGT Group, Fremont, USA). The fluorescence was monitored at 485–490/520–535 nm on a Clariostar analyzer at 37 °C. Results were expresses as relative fluorescence.