Melanoma inhibition by anthocyanins is associated with the reduction of oxidative stress biomarkers and changes in mitochondrial membrane potential

Zoriţa Diaconeasa1, Huseyin Ayvaz2, Dumitriţa Ruginǎ1, Loredana Leopold1, Andreea Stǎnilǎ1, Carmen Socaciu1, Flaviu Tăbăran1, Lavinia Luput3, Diana Carla Mada4, Adela Pintea1, Andrew Jefferson5

1University of Agricultural Science and Veterinary Medicine, Cluj-Napoca 400372, Romania;

2Canakkale Onsekiz Mart University, Canakkale 17020, Turkey;

3Babeș-Bolyai University, Cluj-Napoca 400084, Romania

4Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca 400006 Romania

5Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

*Corresponding author. E-mail: , Telephone: +40 741962857

Supplementary data

Reagents All chemicals were purchased from Sigma-Aldrich (Germany), unless otherwise specified.

Samples Nine berries, blackberries (Rubus nigra), blackcurrants (Ribes nigrum), blackthorns (Prunus spinosa), blueberries (Vaccinium myrtlius), chokeberries (Aronia melanocarpa), cranberries (Vaccinium macrocarpon), mulberries (Morus nigra), dwarf elderberries (Sambucus ebulus) and red grapes (Vitis Vinifera Moldova), were harvested in 2016 from an area located in Cluj-Napoca, Romania (Coordinate: 46°57′23″N 23°46′50″E46°57′23″N 23°46′50″E). Samples were then washed 3 times with water and kept at - 20°C until further analysis.

HPLC-PDA-ESI/MS Analysis of Anthocyanins HPLC analysis was performed on a Shimadzu HPLC-PDA system equipped with a binary pump delivery system LC-20 AT (Prominence), a degasser DGU-20 A3 (Prominence), a diode-array SPD-M20 and a UV-VIS detector following a previously published protocol [8]. Quantification of anthocyanins was done using Cy-3-O-glu, as a standard. The chromatograms were recorded at 520 nm and anthocyanins were identified by HPLC-ESI/MS [8].

Cell Proliferation Assay

For all cell lines, 8×103 cells/well were seeded in 96-well plates for 24h. This was replaced by fresh media containing AREs, in concentrations ranging from 0 to 400 μg/mL, for another 24h. The media from each experimental well was then replaced with MTT reagent (0.5 mg/mL) for 2h, discarded and the formazan crystals formed dissolved in DMSO. The solubilized formazan formed in viable cells was measured at 550 and 630 nm (for sample and background, respectively) using the microplate reader (HT BioTek Synergy, BioTek Instruments, Winooski, VT, USA).

Measurement of LDH Release Level

LDH release assay was performed according to kit instructions (Pierce LDH Cytotoxicity Assay Kit, Rockford, lL 61105 USA) and the cells were treated as described for the MTT assay. The absorbance at 490 nm and 680nm respectively was measured using the microplate reader (BioTek Instruments, Winooski, VT, USA) and the results were expressed as percentage variation of LDH release normalized to control.

Assessment of Malondialdehyde level

In order to evaluate the levels of lipid peroxidation as malondialdehyde (MDA) was quantified by HPLC using a calibration curve done with hydolyzed 1,1,3,3-Tetraethoxypropane (Sigma-Aldrich, Berlin, Germany). Cell lysates were deproteinized with HClO4, then centrifuged at 4500g for 5 min and the supernatants injected into the HPLC system [18]. A RP-18 column (5 µm) (Supelco, Bellefonte, PA, USA) was used for MDA quantification on an isocratic elution system with 30 mM KH2PO4/MeOH (65:35, v/v) at a flow rate of 0.5 mL/min. Detection was performed at 254 nm and data were expressed as µM MDA and were normalized to the protein concentration of cell lysates.

Measurement of Mitocondrial Membrane Potential

3x104cells each of cell line were spotted onto glass slides and allowed to adhere for 24h at 37°C with 5% CO2. The medium was then replaced with complete DMEM medium containing 500 nM MitoTracker reagent, and the cultures were incubated for 20 min, then washed with 1x PBS, and placed in 4% paraformaldehyde for 20 min. The slides were washed with 1x PBS and incubated for 30 min with Phalloidin Alexa 488. After this step, the slides were washed with 1x PBS, and DRAQ5 (5 μM) was added for a further 5 min, being finally scanned on an Axio Observer Z1 Inverted Microscope.

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Tabel 1. Data about the chemical structure and content of individual anthocyanins identified in analyzed berries samples

Peak no / Molecular ion
[M-H]+ (m/z) / Fragment ion
(m/z) / Tentative identification / Individual anthocyanins content
(mg /100 g FW) / Peak no / Molecular ion
[M-H]+
(m/z) / Fragment ion
(m/z) / Tentative
identification / Individual anthocyanins content
(mg /100 g FW) / Total
anthocyanins
(mg /100 g FW)
Blackberry
1 / 449 / 287 / Cy-3-O-glu / 108.8 / 4 / 535 / 287 / Cy-3-O-mal-glu / 30.6
2 / 419 / 287 / Cy-3-O-ara / 40.3 / 5 / 593 / 287 / Cy-3-O-di-glu / 70.0 / 310.2
3 / 595 / 449/287 / Mal deriv / 60.6
Blackthorn
1 / 449 / 287 / Cya-3-O-glu / 310.1 / 3 / 463 / 301 / Peo-3-O-glu / 360.3 / 1110.0
2 / 595 / 287 / Cy-3-O-rut / 203.2 / 4 / 609 / 301 / Peo-3-O-rut / 236.4
Chokeberry
1 / 449 / 287 / Cy-3-O-gal / 550.2 / 3 / 463 / 301 / Cy-3-O-ara / 199.6 / 799.3
2 / 449 / 287 / Cy-3-O-glu / 22.1 / 4 / 609 / 301 / Cy-3-O-xyl / 27.4
Dwarf elderberry
1 / 449 / 287 / Cy-3-O-gal / 309.4 / 3 / 565 / 433/271 / Pel-3-O-sam / 5.1 / 320.2
2 / 581 / 449/287 / Cy-3-O-sam / 5.7
Blackcurrant
1 / 465 / 303 / Delph-3-O-glu / 22.5 / 3 / 449 / 287 / Cy-3-O-glu / 9.9 / 121.0
2 / 300 / 283/252 / Delp-3-O-rut / 48.0 / 4 / 595 / 287 / Cy-3-O-rut / 40.6
Blueberry
1 / 465 / 303 / Delp-3-O-gal / 26.2 / 7 / 419 / 287 / Cy-3-O-ara / 6.5 / 146.5
2 / 465 / 303 / Delp-3-O-glu / 4.26 / 8 / 463 / 301 / Peo-3-O-gal / 4.9
3 / 449 / 287 / Cy-3-O-gal / 8.5 / 9 / 449 / 317 / Pet-3-O-ara / 25.3
4 / 435 / 303 / Delp-3-O-ara / 9.5 / 10 / 493 / 331 / Mal-3-O-gal / 19.5
5 / 449 / 287 / Cy-3-O-glu / 3.7 / 11 / 465 / 303 / Mal-3-O-glu / 5.8
6 / 479 / 317 / Pet-3-O-gal / 15.9 / 12 / 465 / 303 / Mal-3-O-ara / 16.3
Cranberry
1 / 449 / 287 / Cy-3-O-gal / 96.9 / 5 / 463 / 301 / Peo-3-O-gal / 92.8 / 362.1
2 / 419 / 286 / Cy-3-O-ara / 66.3 / 6 / 624 / 463 / Mal-3-O-glu vinylcathecol / 9.9
3 / 449 / 316 / Pet-3-O-ara / 79.4 / 7 / 535 / 331 / Mal-6-acetyl-3-gal / 3.9
4 / 493 / 332 / Mal-3-O-gal / 12.9
Mulberry
1 / 449 / 287 / Cy-3-O-glu / 65.3 / 3 / 625 / 317 / Pet-3-(6-O-p-coumaryl-glu) / 11.0 / 96.7
2 / 595 / 287 / Cy-3-O-rut / 20.4
Red grapes
1 / 465 / 303 / Del-3-O-glu / 4.80 / 4 / 479 / 317 / Pet-3-O-glu / 63.5 / 257.7
2 / 449 / 287 / Cy-3-O-glu / 3.82 / 5 / 433 / 271 / Pel-3-O-glu / 176.5
3 / 493 / 331 / Mal-3-O-glu / 9.05

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