| |

Received: 30 December 2020    Revised: 20 April 2021    Accepted: 24 April 2021

DOI: 10.1111/jfbc.13760

ORIGINAL ARTICLE

Antiproliferative potential of Andean Berry (Vaccinium

meridionale Swartz) juice in combination with Aspirin in human
SW480 colon adenocarcinoma cells

Sandra S. Arango-­Varela1  | Ivan Luzardo-­Ocampo2,3  | Camilo Reyes-­Dieck4 |

Elhadi M. Yahia5  | Maria E. Maldonado-­Celis4

1
Biomedical Research and Innovation Group
(GI2B), Instituto Tecnológico Metropolitano Abstract
(ITM), Medellín, Colombia Andean Berry (Vaccinium meridionale Sw.) is a South American fruit rich in phyto-
2
Instituto de Neurobiología, Universidad
chemicals with promising anti-­cancer properties as co-­adjuvants to nonsteroidal anti-­
Nacional Autónoma de México, Juriquilla,
Mexico inflammatory drugs such as Aspirin. This study aimed to evaluate the antiproliferative
3
Research and Graduate Program in potential of Andean Berry Juice (ABJ) in combination with Aspirin in human SW480
Food Science, Universidad Autónoma de
Querétaro, Queretaro, Mexico
colon adenocarcinoma cells. ABJ primarily contained 3,4-­dihydroxybenzoic and chlo-
4
Escuela de Nutrición y Dietética, rogenic acids. The combined treatment of ABJ (IC50: 30.0 ± 0.11%) and Aspirin (IC50:
Universidad de Antiquia, Medellín, Colombia 20.0 ± 0.57) exhibited a higher (p < .01) antiproliferative effect than each counter-
5
Facultad de Ciencias Naturales, Universidad
part. Moreover the same mixture displayed a lower reduced glutathione/oxidized
Autónoma de Querétaro, Queretaro, Mexico
glutathione ratio (GSH/GSSG) than the untreated cells. ABJ-­A spirin combination
Correspondence
induced late apoptosis stage without stimulating mitochondrial depolarization and
Maria E. Maldonado-­Celis, Escuela de
Nutrición y Dietética, Universidad de prompted phosphatidylserine relocalization. These results emphasize the antiprolif-
Antiquia, Ciudadela de Robledo Cra. 75 #65-­
erative potential of bioactive compounds from ABJ and Aspirin combinations.
87, Medellín, Colombia.
Email: maria.maldonado@udea.edu.co Practical applications
Funding information Natural products such as Andean Berry (V. meridionale Sw.) juice (ABJ) contains antiox-
Departamento Administrativo de Ciencia, idant polyphenols that could reduce the need to use non-­steroidal anti-­inflammatory
Tecnología e Innovación, Grant/Award
Number: 7851 and FP44842-­211-­2018; drugs, currently employed in cancer treatment, to prevent its side effects. The high
Dirección General de Asuntos del Personal abundance of polyphenols from this underutilized berry could stimulate the stand-
Académico, Universidad Nacional Autónoma
de México, Grant/Award Number: 5267 ardization of its production and industrial exploitation to be transformed into suita-
ble food products delivering natural bioactive compounds with potential anti-­cancer
effects in vitro.

KEYWORDS

antiproliferative, apoptosis, Aspirin, colorectal cancer, Vaccinium meridionale Swartz

Abbreviations: ABJ, Andean berry (Vaccinium meridionale Sw.) juice; C3G, cyanidin-­3-­glucoside; CE, (+)-­c atechin equivalents; COX, cyclooxygenase; CRC, colorectal cancer; DCFH-­DA,
2,7-­dichlorodihydrofluorescein diacetate; DiOC6, 3,3′-­dihexyloxacarbocyanine iodide; DMEM, Dulbecco’s modified eagle medium; DPPH, 2,2-­diphenyl-­1-­picrylhydrazyl; DTT,
dithiothreitol; EGFR, epidermal growth factor receptor; FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalents; GSH, reduced glutathione; GSSG, oxidized glutathione;
HPLC-­DAD, high-­performance liquid chromatography; IC 50, half-­inhibitory concentration; ITS, insulin-­transferring-­selenium medium; LRU, luminescence relative units; MAPK,
mitogen-­activated protein kinase; n.d., not detected; NEM, N-­ethylmaleimide; NF-­κ B, Nuclear factor κB; NMBA, N-­nitrosomethylbenzylamine; NSAIDs, Nonsteroidal anti-­inflammatory
drugs; OD, optical density; PI, propidium iodide; PS, phosphatidylserine; RFU, relative fluorescence units; ROS, reactive oxygen species; RT, retention time; SOD, superoxide dismutase;
SRB, sulphorhodamine B; TA, total monomeric anthocyanins; TCA, trichloroacetic acid; TE, trolox equivalents; TEAC, trolox equivalent antioxidant capacity; TF, total flavonoids; TPC,
total phenolic compounds; VEGF, vascular endothelial growth factor; Wnt, wingless-­related integration site; ΔΨm, mitochondrial membrane potential.

J Food Biochem. 2021;00:e13760. wileyonlinelibrary.com/journal/jfbc |

© 2021 Wiley Periodicals LLC.     1 of 16
https://doi.org/10.1111/jfbc.13760
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2 of 16       ARANGO-­VARELA et al.
1 |  I NTRO D U C TI O N
Colorectal cancer (CRC) is a high-­impact disease globally, with an
estimated incidence of 1.8 million new cases (GLOBOCAN,  2020).
CRC ranked third in terms of incidence and second in terms of mor-
tality among cancer conditions (Bray et al., 2018). The sporadic CRC
carcinogenesis pathway typically includes a process that takes 10–­
20 years and ends progressively to adenocarcinoma and metastatic
phenotype (Greystoke & Mullamitha,  2012). CRC prevention and
further treatment could be considered using chemopreventive strat-
egies focusing on adenoma formation or its transformation to carci-
noma (López et al., 2014).
Epidemiological studies have shown that the intake of nonsteroi-
dal anti-­inflammatory drugs (NSAIDs) such as Aspirin could reduce
CRC incidence (Tsioulias et  al.,  2015). NSAIDs could act hindering
prostaglandin-­catalyzers such as cyclooxygenase (COX) enzymes,
involved in increased cancer cells’ proliferation (Thun et al., 2012).
For instance, NSAIDs exhibit antiproliferative effects by inhibiting
the sirtuin-­1 (SIRT1) deacetylase activity, stimulate acetylation and
activity of p53 in vitro and in vivo, and increase the activity of p21 in
vitro (Dell’Omo et al., 2019).
Another important chemopreventive strategy of CRC is the di-
etary phytochemicals, which can delay, stop, or reverse carcinogen-
esis. Many of these substances have been identified in fruits and
vegetables (Baena Ruiz & Salinas Hernández,  2016). For example,
phenolic compounds from berries have displayed anti-­carcinogenic
effects protecting against oxidative damage, suppressing inflamma-
tion, and inhibiting cell proliferation by modulating cell cycles and
apoptosis (Afrin et al., 2016).
Andean Berry (Vaccinium meridionale Swartz), or “mortiño” as lo-
cally known, is a native fruit from the west forest zone of the Andean
region (Abreu et al., 2014). Research studies on V. meridionale Swartz
have shown the presence of several bioaccessible and antioxidant phy-
tochemicals (Agudelo, Luzardo-­Ocampo, et al., 2018), with antiprolif-
erative and pro-­apoptotic effects on CRC cell lines in vitro (Agudelo
et  al.,  2017; Agudelo, Luzardo-­Ocampo, et  al.,  2018; Maldonado-­
Celis et al., 2014). Andean Berry, in combination with Aspirin, has
shown a coadjuvant anti-­inflammatory effect in vitro (Arango-­Varela
et  al.,  2020), and natural products-­drugs combination could allow to
use a lower dosage to reduce NSAIDs’ side effects (González-­Vallinas
et al., 2013; Landis-­piwowar & Iyer, 2014). Therefore, the objective of
this study was to evaluate the antiproliferative potential of Andean
Berry (Vaccinium meridionale Sw.) juice combined with Aspirin in
SW480 cells. As far as we know, this is the first report of these effects
of ABJ combined with Aspirin in such a cancer cell line.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Andean Berry juice preparation
Ripe Andean Berry (V. meridionale Swartz) fruits were harvested
in El Retiro (Antioquia, Colombia) at an altitude of 2,175 meters
above sea level (6º8′6″ N and 75º25′3″ W), and mean tempera-
ture of 16°C. The samples were identified in the Universidad
de Antioquia’s Herbarium (Registro Nacional de Colecciones
Biológicas #027). The juice was prepared as described by Franco
Tobón et al. (2016). Briefly, the harvested fruit was processed in a
blender (2,500 rpm, 2 min) and freeze-­dried (50ºC, 0.43 ± 0.50 mm
Hg pressure). The obtained powder was mixed with sterile water
(12% wt/vol). Sugar was added at 9% wt/vol. ABJ was sonicated
for 3 hr (42 kHz, 135 W) and filtered through a 0.22 µm pore size
membrane to reduce the particle size. Finally, it was stored at
−20°C and protected from light.
2.2 | Chemical characterization
Undiluted ABJ (100% prepared juice or 56.78  mg freeze-­dried
ABJ/mL) was used for the chemical characterization. Aspirin was as-
sayed at the same concentration used for cell culture (25 mM). For
the ABJ + Aspirin mixture, both were analyzed as to their initial con-
centrations (100% prepared juice + 25 mM Aspirin).
2.2.1 | Total phenolic compounds (TPC)
TPC were measured using a modified Folin-­Ciocalteau method,
suitable for a 96-­well microplate originally described by Singleton
and Rossi (1965). Briefly, an aliquot of ABJ (100% juice or 56.78 mg
freeze-­dried ABJ/ml) or gallic acid (6.25–­100 μg/mL) was mixed
with 250 μl distilled water and 125 μl of the Folin-­Ciocalteu rea-
gent (47,641, Sigma-­Aldrich, St. Louis, MO, US). The samples were
sonicated for 5 min at room temperature (25 ± 1ºC) in a sonicator
(Branson Sonicator B3510, Ultrasonic Corporation, Boston, MA,
USA). Afterward, 625 μl of Na2CO3 (222,321, Sigma-­Aldrich) were
added, vortexed (3 s), and incubated for 2 hr at room temperature.
The mixture was added to 96-­well plates (250 μl/well) in tripli-
cates. TPC were determined in a microplate reader (MRX, Dynex
Technologies, Chantilly, VA, US) at 760 nm. A calibration curve was
constructed using gallic acid as the standard (G7384, Sigma-­Aldrich),
and the results were presented as milligrams of gallic acid equiva-
lents per 100  ml of sample (mg GAE/100  ml). Three independent
experiments were carried out.
2.2.2 | Total flavonoids (TF)
The TF content was quantified as described by Yahia et al. (2017).
Mixtures of 1,250 μl distilled water, 75 μl of 5% NaNO2 (237,213,
Sigma-­Aldrich), 150 μl of 10% AlCl3 (237,051, Sigma-­Aldrich), 500 μl
of 1  M NaOH (S8045, Sigma-­Aldrich), and 525 μl of ABJ samples
were prepared. After 30  min, the Optical Density (OD) was meas-
ured at 310 nm in a microplate reader (MRX, Dynex Technologies).
Results were presented as milligrams of (+)-­catechin equivalents per
100 ml (mg CE/100 ml).
ARANGO-­VARELA et al.
2.2.3 | Total monomeric anthocyanins (TA)
A pH differential method was used (Giusti & Wrolstad,  2001).
Briefly, ABJ samples (100% juice or 56.78  mg freeze-­dried ABJ/
ml) or its combination with Aspirin (25  mM) were diluted using
2 buffers (pH 1.0, 0.025  M KCl; pH 4.5, 0.40  M sodium acetate
buffer), reaching a final reaction volume of 200 μl (final concentra-
tion: 60% ABJ or 34.10 mg freeze-­dried ABJ/ml). The pH from the
Aspirin, ABJ and their combination after dissolution in the buff-
ers was recorded using a potentiometer (Hanna HI 2,550, Hanna
Instruments, Woonsocket, RI, US). The OD at 530 and 700 nm was
measured in a spectrophotometer (D%-­65, Beckman Instruments
Inc., Fullerton, CA, US) using pH buffers (1.0 and 4.5). The total
monomeric anthocyanins (TA) were calculated using the formula:
TA (mg/100 ml) = ΔΑ × MW × 100/Ɛ, where: ΔA = [(A 530 − A700)
pH 1.0 − (A 530 − A700) pH 4.5], with molar extinction coefficient
(Ɛ) for cyanidin–­3–­glucoside (C-­3-­G) of 26,900 L/mol·cm−1, and a
molecular weight (MW) of 449.20 g/mol.
2.2.4 | High-­performance liquid chromatography
coupled to diode-­array-­detection (HPLC-­DAD) analysis
The identification of the compounds present in the extract was
conducted following the procedure described by Corrales-­Bernal
et al. (2016) with slight modifications. Twenty microliters of the ABJ
samples were automatically injected into an HPLC system (HP 1,100
Series, Hewlett-­Packard/Agilent Technologies, Santa Clara, CA, US)
coupled to a DAD (Dionex UltiMate 3,000, Thermo Fisher Scientific,
Waltham, MA, US). An RP18 C18 X-­Terra Column (250  ×  4.6  mm,
5 µm of spherical particle size) was used (Agilent Technologies) with
two mobile phases: formic acid (1%, A) and acetonitrile:formic acid
(2%, B) with an elution gradient of 2%–­100% (B) for 60 min. The flow
rate was 0.5 ml/min, and the injection volume was 20 μl. Compounds
were detected at 280 nm, and their quantification was carried out
using calibration curves from prepared standards (Sigma-­Aldrich,
St. Louis, MO, US) including p-­coumaric (C9008), chlorogenic
(C3878), ferulic (Y0001013), sinapic (D7927); 3,4-­dihydroxybenzoic
(36,333), vanillic (94,770), syringic (S6881), caffeic (C0624), cinnamic
(1,133,933), gentisic (85,707), 2-­hydroxycinnamic (H22809), and
trans-­cinnamic acids (C80857). (+)-­C atechin (C1251), epicatechin
(E4018), kaempferol (60,010), myricetin (70,050), and naringenin
(N5893).
2.3 | Antioxidant capacity
For the antioxidant capacity assays, ABJ was evaluated undi-
luted (100% prepared juice or 56.78 mg freeze-­dried ABJ/ml), and
Aspirin was assayed using the same concentration as for the cell
culture tests (25 mM). For the ABJ + Aspirin mixture, both were
used in their initial concentrations (100% prepared juice + 25 mM
Aspirin).
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2.3.1 | 2,2-­diphenyl-­1-­picrylhydrazyl (DPPH) assay
The free radical 2,2-­diphenyl-­1-­picrylhydrazyl (DPPH) assay was
conducted following the method reported by Moore et al. (2011).
Aliquots of 20 µl of the extract (100% ABJ or 56.78 mg freeze-­dried
ABJ/ml) and 280 µl of DPPH/methanol (DPPH: D9132, 0.15 mM;
methanol: 34,860, Sigma-­A ldrich) were incubated for 30 min in the
dark. The OD was then measured at 490 nm in a microplate reader
(MRX, Dynex Technologies). A calibration curve was plotted
using Trolox as a standard (0–­8 00 μM) (238,813, Sigma-­A ldrich).
The results were reported in Trolox equivalent antioxidant ca-
pacity (TEAC) as μmol of Trolox equivalents per ml sample (μmol
TE/ml). A single concentration was used to assess the antioxidant
capacity by the DPPH method. For the calculation of TEAC, an
absorbance versus concentration curve of Trolox antiradical ac-
tivity (% ARA) was mathematically determined (linear curve). The
antiradical activity was calculated as follows: [(Absorbance con-
trol − Absorbance sample)/(Absorbance control)] × 100. All %ARA
values of the samples were calculated and then mathematically
transformed to TEAC.
2.3.2 | Ferric reducing antioxidant power
(FRAP) assay
The Ferric Reducing Antioxidant Power (FRAP) assay was carried
out following the method reported by Yahia et al. (2017). Aliquots
of 280 μl of FRAP reagent (MAK369, Sigma-­A ldrich) and 20 μl of
ABJ extract were added to each well of the 96-­well plate. The
plates were incubated for 30 min in the dark and read at 630 nm
in a microplate reader (MRX, Dynex Technologies). Trolox was
used as a standard for the calibration curve (0–­8 00 μM), and re-
sults were expressed as μmol TE/ml sample. A single concentra-
tion was used to evaluate the antioxidant capacity by the FRAP
assay.
2.4 | Cell culture assays
2.4.1 | Cell culture
Human SW 480 (European Collection of Animal Cell Culture, ECACC
87,092,801) colon adenocarcinoma cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM, 11,995,065, Gibco, Fisher
Scientific, Hampton, NH, US) supplemented with 25  mM glucose,
2  mM L-­Glutamine, 10% vol/vol horse serum (16,050, HS Gibco,
Invitrogen, Carlsbad, USA), 100 UI/mL penicillin and 100  µg/ml
streptomycin (11,548,876, Gibco), and 1% non-­essential amino
acids (12,084,947, Gibco, Invitrogen, Carlsbad, CA, US). The cells
were incubated at 37°C and 5% CO2. Insulin-­Transferrin-­Selenium
(ITS) supplement (15,383,661, Corning, Fisher Scientific) was used
mixed with DMEM supplemented with 3% horse serum (11,540,636,
Gibco).
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4 of 16       ARANGO-­VARELA et al.
2.4.2 | Experimental design
For this experiment, ABJ concentrations equivalents to 0, 10, 20, 30,
and 40% vol/vol were used. Concentrations were expressed in the
volume of ABJ as this is a complex mixture where calculations of the
molarity of compounds cannot be easily conducted (Kisitu,  2019).
However, to ensure reproducibility, considering that the original ABJ
formulation includes 30 g of freeze-­dried juice, 28.35 g of sucrose,
and 500 ml water (Agudelo, Luzardo-­Ocampo, et al., 2018), ABJ con-
centrations for the cell treatments were equivalents to 5.67, 11.35,
17.03, 22.71, and 28.39 mg/ml, respectively. For preparing the ABJ-­
cell culture medium dilutions, ABJ was directly diluted and filtered
through sterile 0.45 and 0.22 μm Acrodisc® syringe filters.
For the Aspirin (70264–­013, Bayer, Leverkusen, Germany) treat-
ments, concentrations of 0, 5, 10, 15, 20, 30, and 40 mM were evalu-
ated. These concentrations were selected based on previous studies
with RAW 264.7 murine macrophages (Arango-­Varela et al., 2020). A
30% v/v ABJ (17.03 mg/ml fruit) and 20 mM Aspirin were chosen for
the mixture, considering the obtained IC50 values from each treat-
ment. For preparing the Aspirin dilutions, Aspirin was mixed with
cell culture medium, sonicated for 15  min in a Branson Sonicator
(Ultrasonic Corporation, US) at room temperature, and the solution
was filtered through sterile 0.45 and 0.22 μm Acrodisc® syringe
filters.
2.4.3 | Cell viability assay
The cell viability was determined with the Sulphorhodamine B
(SRB) colorimetric assay. This assay relies on SRB binding to pro-
tein components of cells previously fixed to culture plates (Vichai
& Kirtikara, 2006). Cultured cells exhibiting cell viability >95% were
used. The cells (2 × 10 4 cells/well) were seeded in 96-­well plates using
ITS-­added DMEM medium (3% horse serum). After 24 hr-­incubation,
the cells were treated for the following 24 hr with ABJ (10%, 20%,
30%, 40% vol/vol) and Aspirin (0, 5, 10, 15, 20, 30, 40  mM) (250
μL/well) by replacing the complete media with the treatments. The
cells were then fixed for 1  hr after treatment with 100 µl at 50%
trichloroacetic acid (TCA, 11,964,064, Fisher Scientific) at 4°C.
The plate was washed for TCA removal, and cells were stained for
30 min with 100 µl 0.4% SRB solution (11,520,666, Invitrogen, Fisher
Scientific). The unbound dye was rinsed with 1% vol/vol acetic acid.
Afterward, 200 µl of 10  mM Tris solution (pH 10.5) was added to
every well, and the plate was incubated under stirring for 30  min.
The OD value was measured at 560  nm in a microplate reader
(GlomaxTM, Promega Co., Madison, WI, US). The cell viability (%)
was calculated using the equation: (ODt/ODc) × 100, where ODt is
the OD value of treated cells, and ODc is the OD value of the control
cells. A linear regression on this data was used to determine the half
inhibitory concentration (IC50).
The interaction index between both substances (ABJ and
Aspirin) was calculated following the proposed equation of Tallarida
(2016): b/Bi + a/Ai, where a and b are the smallest dose of each drug
to produce a desirable effect, while Ai and Bi are the largest doses of
each drug to get a desirable effect. If interaction index = 1 the effect
was considered additive, <1 synergistic, and >1 antagonistic. For the
smallest doses, a and b were selected as the doses of ABJ and Aspirin
producing at least 80% inhibition of SW480 cell viability. For the
largest doses (Ai and Bi ), ABJ and Aspirin concentrations producing
a maximum 80% cell viability inhibition of RAW 264.7 macrophages
were selected (Arango-­Varela et  al.,  2020), considering that these
cells have been used to evaluate cytotoxicity (Clemens et al., 2012;
Hala Ragab et al., 2016). Results from the interaction index are pre-
sented in Supplementary Table S1.
2.4.4 | Intracellular detection of reactive oxygen
species (ROS)
The 2,7-­ dichlorodihydrofluorescein diacetate (DCFH-­DA) is a fluo-
rescence probe that is deacetylated and oxidized to form the fluo-
rescent compound DCF due to ROS-­induced deacetylase enzyme
activation, serving as indirect marker of ROS. The cells (1  × 10 4
cells/well) were seeded on a 96-­well plate using phenol-­red free
ITS-­added DMEM medium (3% horse serum) for 24  hr. The cells
were then treated with ABJ (10, 20, 30, 40% vol/vol), Aspirin (10,
15, 20  mM), and the mixture (IC50 ABJ: 30% vol/vol; IC50 Aspirin:
20 mM, in 1:1 proportion) (250 ml/well) by replacing the media with
the treatments and incubated for 24 hr. The medium was rinsed off,
and a solution of 100 µl of 8 µM DCFH-­DA (D6883, Sigma-­Aldrich)
with PBS (806,552, Merck, Sigma-­Aldrich) was added for 60  min.
Fluorescence was measured at excitation/emission 520/485  nm
wavelengths in a fluorescence plate reader (Varioskan, Thermo
Fisher Scientific, Waltham, MA, US). Data were expressed in rela-
tive fluorescence units (RFU). The interaction index for ROS was
calculated as indicated in Section 2.4.2. The results are presented in
Supplementary Table S1. Hydrogen peroxide (40 mM) was used as a
positive control for ROS production.
2.4.5 | Intracellular oxidative stress analysis
Intracellular oxidative stress of treated cells was evaluated follow-
ing the manufacturer’s instructions of the commercially available
kit GSH/GSSG-­Glo™ (V6911, Promega), which assesses the pro-
portion of reduced glutathione (GSH) versus oxidized glutathione
(GSSG). Both GSH and GSSG exist in healthy cells, mostly as GSH.
A change in the GSH and GSSG levels is an indicator of oxidative
stress evaluation. Lower GSH levels are indicative of diminished
antioxidant capacity. The cells (1 × 10 4 cell/well) were seeded on a
96-­well plate using ITS-­added DMEM medium (3% horse serum) for
24 hr. Then, the cells were treated with ABJ (10%, 20%, 30%, 40%
vol/vol), Aspirin (10, 15, 20 mM) and the mixture (IC50 ABJ: 30% vol/
vol; IC50 Aspirin: 20 mM, in 1:1 proportion) (250 ml/well) by replacing
the incubation media with the treatments and incubated for 24 hr.
The medium was rinsed off, and a 25 µl GSSG passive lysis buffer
ARANGO-­VARELA et al. |
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solution (0.50 µl Luciferin-­NT; 0.25 µl N-­ethylmaleimide or NEM, 5 Annexin-­V binding buffer (10  mM HEPES, 0.14  M NaCl, 2.5  mM
µl Passive lysis buffer 5×, 19.25 µl distilled water) was added to each CaCl2; pH 7.4) and 4 µl Annexin-­V (Annexin-­V–­FLUOS staining kit,
well for 5 min under stirring at room temperature (RT). Then, 25 µl Sigma Aldrich). Cells were incubated for 15 min at 4°C in the dark.
of glutathione S-­transferase solution (0.70 µl dithiothreitol or DTT The flow cytometer (FACSCantoTM II, BD Biosciences) was used to
10 mM, 1.50 µl Glutathione-­S-­Transferase, and 22.80 µl Glutathione analyze 10.000 events. The interaction index for PS relocalization
Reaction Buffer) was added to each well for 60  min at room tem- was calculated as indicated in Section  2.4.2. The results are pre-
perature. Finally, 50 µl of Luciferin Detection Reagent per well were sented in Supplementary Table S1.
added for 15  min at room temperature. Luminescence was meas-
ured in a microplate reader, and results were reported as lumines-
cence relative units (LRU) from the GSH/GSSG ratio, calculated as 2.5 | Statistical analysis
[(LRUTotal Glutathione − LRUGSSG treated cells)/(LRU GSSG treated cells/2)]. The
interaction index for GSH/GSSG ratio was calculated as indicated in Unless indicated, all experiments were conducted with at least three
Section 2.4.2. The results are presented in Supplementary Table S1. independent experiments in triplicates. Data analysis was carried
Hydrogen peroxide (40 mM) was used as a positive control for intra- out using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego,
cellular oxidative stress. CA). A one-­way ANOVA and post hoc Tukey-­Kramer’s test were con-
ducted for multiple comparison tests. Significance was established
at p < .01.
2.4.6 | Analysis of mitochondrial membrane potential
(ΔΨm) with flow cytometry
3 | R E S U LT S
Positively charged fluorescence probes can be accumulated inside
mitochondria inversely related to ΔΨm, following the Nernst equa- 3.1 | Chemical characterization and antioxidant
tion. Hence, more polarized mitochondria (negatively charged core) capacity of ABJ and ABJ+Aspirin
will accumulate more fluorescence dye (Ronot & Leverve,  2011).
Changes in the mitochondrial membrane permeability were meas- The total phenolic content and the antioxidant capacity of the
ured using 3,3′-­dihexyloxacarbocyanine iodide (DiOC6). The cells ABJ+Aspirin mixture suggested that Aspirin did not influence the
(1  × 106 cells/well) were seeded in 6-­well plates for 24  hr. Then, detection of total phenolic compounds nor the FRAP quantifica-
the cells were treated with IC50 ABJ (30% vol/vol), IC50 Aspirin tions based on the absence of differences (p  > .01) (Table  1). The
(20  mM), and their combination (1:1 proportion) (250  ml/well) by Aspirin-­added samples showed lower TF and DPPH (−19.48 ± 0.60,
replacing the incubation media (ITS-­added DMEM media) with the and −34.28 ± 1.31%, respectively) but exhibited a higher TA content
treatments for 24 hr. The cells were then collected using trypsin 1X (32.67 ± 0.50%) than ABJ, indicating the major contribution of an-
and incubated with 50 µM DiOC6 (D273, Thermo Fisher Scientific) thocyanins to the DPPH inhibition.
and 10 µl of Propidium Iodide (PI) (P4170, Sigma-­Aldrich) (1 mg/ml)
for 30  min in the dark at room temperature (25  ±  1ºC). Flow cy-
tometry assay (FACSCantoTM II, BD Biosciences, Franklin Lakes, NJ, TA B L E 1   Total polyphenolic content and antioxidant capacity of
US) analyzed 10,000 events per sample at excitation and emission ABJ and its combination with 25 mM Aspirin
detection wavelengths with green filters (530/515  nm). Hydrogen
ABJ +Aspirin
peroxide (40 mM) was used as a positive control for mitochondrial Component ABJ (25 mM)
polarization.
Total polyphenolic composition
TPC (mg GAE/100 ml) 3,570 ± 260a 3,260 ± 46a
a
TF (mg CE/100 ml) 2,310 ± 20 1,860 ± 30 b
2.4.7 | Apoptosis detection by phosphatidylserine
TA (mg. C3G/100 L) 129 ± 20 b 172 ± 20a
(PS) relocalization
Antioxidant capacity (TEAC, μmol TE/ml sample)

The plasmatic cell membrane polarity changes were evaluated FRAP 127.60 ± 4.20a 112.20 ± 9.60a
a
throughout its binding to Annexin V, allowing the detection of the DPPH 35.43 ± 7.10 23.29 ± 9.30 b
exposed PS in the external membrane. This phospholipidic com- Note: The results are the mean ± SD of three independent experiments
ponent is usually located in the internal plasmatic membrane, but in triplicates. Rows not sharing the same letter are significantly different
pro-­apoptotic processes stimulate the catalytic activity of flippase, (p < .01) by Student’s t-­test.
Abbreviations: ABJ, Andean Berry juice; CE, (+)-­c atechin equivalents;
exposing PS to the external membrane. Hence, Annexin V bind-
C3G, cyanidin-­3-­glucoside; DPPH, 2,2-­diphenyl-­1-­picryl-­hydrazyl;
ing suggests PS relocalization (Demchenko,  2013). Briefly, the cell FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalents;
culture, treatments, and sample collection were done as previously TE, trolox equivalent; TA, total monomeric anthocyanins; TF, total
described (section 2.4.3). The obtained cells were mixed with 1 ml flavonoids; TPC, total phenolic compounds.
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6 of 16       ARANGO-­VARELA et al.
Based on the presented chromatogram peaks from Supplementary
Figure  S1, six out of 17 phenolics were identified, being ABJ the
sample with the highest content of 3,4-­hydroxybenzoic acid,
2-­hydroxycinnamic acid, and kaempferol (Table 2). Since p-­Coumaric
acid was only detected in the mixture, it could be inferred a potential
impact of Aspirin on TF and TA contents.
3.2 | Antiproliferative effect of ABJ,
Aspirin, and their combination in SW480 cells
Results from the antiproliferative effect of the treatments on SW480
cells are presented in Figure 1. The results depict the effect of ABJ
concentrations (Figure  1a) and its IC50 calculation (Figure  1b); the
Aspirin effect on the same parameter (Figure 1c) and its IC50 calcula-
tion (Figure 1d); and the combined effect ABJ-­A spirin on the cell via-
bility (Figure 1e). The ABJ doses above 20% showed antiproliferative
potential (cell viability <80%), while Aspirin doses displayed the same
dose-­response trend in concentrations >5 mM. Since the ABJ-­IC50
was calculated at 30 ± 0.11% (17.04 ± 0.06 mg freeze-­dried ABJ/mL)
using a linear curve (Supplementary Table S2), the concentration was
equivalent to 243.62  ±  0.48  μg/100  ml 3,4-­hydroxybenzoic acid,
408.87 ± 0.43 μg/100 ml chlorogenic acid, 81.22 ± 0.89 μg/100 ml
p-­coumaric acid, 0.13  ± 0.01 μg/100  ml 2-­hydroxycinnamic acid,
0.70  ± 0.01 μg/100  ml naringenin, and 1.11  ± 0.01 μg/100 ml
kaempferol. The evaluation of the calculated ABJ-­IC50 showed cell
viabilities close to 50% (ABJ: 51.90  ±  0.56% or 29.47  mg freeze-­
dried ABJ/mL, Aspirin: 42.70  ±  0.78%). However, for the ABJ-­IC50
+ Aspirin-­IC50 treatment, cell viability was 32.90 ± 8.66%. The in-
teraction index between ABJ and Aspirin (Supplementary Table S1)
showed a synergistic effect of both substances contributing to de-
creasing this parameter.
3.3 | Antioxidant effect of treatments on
SW480 cells
Significant increases (p  < .01) in the ROS production were found
at ABJ concentrations ≥20% vol/vol and Aspirin concentrations
RT
Phenolic compound (min) ABJ
3,4-­dihydroxybenzoic acid 32.20 812.06 ± 1.59a
Chlorogenic acid 34.30 1,362.91 ± 1.43a
p-­coumaric acid 43.80 nd.
2-­hydroxycinnamic acid 48.80 0.44 ± 0.00a
a
Narigenin 56.40 2.35 ± 0.01
Kaempferol 58.80 3.71 ± 0.04
Note: Compounds were reported in μg equivalents of each phenolic/100 ml ABJ. The results are
the mean ± SD of three independent experiments in triplicates. Rows not sharing the same letter
are significantly different (p <.01) by Student’s t-­test.
Abbrevitions: ABJ, Andean Berry Juice; nd., not detected; RT, retention time.
>15 mM (Figure 2a). The mixture had the same ROS production as
the 20 mM Aspirin, but ABJ-­IC50 exhibited higher ROS levels (~ 50%
higher) (Figure 2b).
ABJ (p  < .01) reduced the intracellular oxidative stress (GSH/
GSSG) levels, whereas the 30 ± 0.11% (17.04 ± 0.06 mg freeze-­dried
ABJ/mL) concentration was the most effective one (Figure 3a). Only
15-­ and 20-­mM Aspirin showed a lower ratio, while the mixture was
not different (p  > .01) than the individual substances (IC50 Aspirin
or IC50 ABJ) (Figure 3c). In both ROS and GSH/GSSG levels, as the
Aspirin-­ABJ mixture did not show differences (p  > .01) compared
to the individual counterparts, the antagonistic interaction index
calculated for both effects (2.05 and 1.46 for ROS and GSH/GSSG,
respectively) could explain this situation (Supplementary Table S1).
3.4 | Impact of the treatments on the mitochondrial
membrane potential (ΔΨm) and apoptosis from
SW480 cells
Figure 4a shows representative pictures of the effect of the treatments
on both mitochondrial membrane potential and apoptosis/necrosis on
SW480 cells after double staining with DiOC6/PI, while their quantifica-
tion is shown in Figure 4b. Flow cytometry using the fluorescent probe
DiOC6 was performed to evaluate the influence of the treatments
on changes of mitochondrial membrane potential (ΔΨm) since reduc-
tions of DiOC6 are associated with lower ΔΨm (Kataoka et al., 2005).
Moreover, DNA staining with PI allowed the study of losses on the
cell membrane integrity. The combined DiOC6/PI staining permits
the identification of dying cells, discriminating non-­apoptotic/necrotic
cells, low ΔΨm, and low membrane integrity (Q1: DiOC6−/PI+); late
apoptosis, high ΔΨm, and low membrane integrity (Q2: DiOC6+/PI−);
high ΔΨm and proper membrane integrity (Q3: DiOC6+/PI−); and early
apoptosis, low ΔΨm, and proper membrane integrity (Q4: DiOC6−/PI−)
(Pacor et al., 2015). Compared to the untreated cells or negative control,
ABJ (IC50: 30 ± 0.11% vol/vol or 17.03 ± 0.06 mg freeze-­dried ABJ/mL)
Aspirin (IC50: 20 ± 0.56 mM), and their combination (1:1 IC50 ABJ and
IC50 Aspirin) showed 30.10 ± 2.37%, 18.50 ± 0.88%, and 81.5 ± 0.64%
positive late apoptotic SW480 cells after staining in Q2 (DiOC6+/PI+),
while the positive control (H2O2, 40  mM) increased the number of
TA B L E 2   Identification and
ABJ +aspirin
quantification of some individual phenolic
(50 mM)
compounds by HPLC-­DAD
537.44 ± 3.73b
1,640.82 ± 265.40a
225.15 ± 0.08b
0.27 ± 0.01b
2.33 ± 0.05a
nd.
ARANGO-­VARELA et al. |
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F I G U R E 1   Impact of ABJ and Aspirin on SW480 cell viability. (a) Cell viability and (b) calculated IC50 after several ABJ treatments (0%–­
40%). (c) Cell viability and (d) calculated IC50 after several Aspirin treatments (0–­4 0 mM); (e) Cell viability from the ABJ+Aspirin mixture.
The results were expressed as mean ± SD of three independent experiments in triplicates. Different letters are significantly different
(p < .01) by Tukey-­Kramer’s test. ABJ: Andean Berry (Vaccinium meridionale Swartz); IC50: Half inhibitory concentration (equivalent
to 243.62 ± 0.48 μg/100 ml 3,4-­hydroxybenzoic acid, 408.87 ± 0.43 μg/100 ml chlorogenic acid, 81.22 ± 0.89 μg/100 ml p-­coumaric
acid, 0.13 ± 0.01 μg/100 ml 2-­hydroxycinnamic acid, 0.70 ± 0.01 μg/100 ml naringenin, and 1.11 ± 0.01 μg/100 ml kaempferol). ABJ
concentrations are equivalents to 5.67, 11.35, 17.03, 22.71, and 28.39 mg of freeze-­dried ABJ/ml, respectively for 10, 20, 30, and 40%
vol/vol ABJ
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8 of 16       ARANGO-­VARELA et al.

F I G U R E 2   Effect of ABJ and Aspirin on the intracellular reactive oxygen species (ROS) production of SW480 cells. Impact of ROS after
several concentrations of (a) ABJ and Aspirin; and (b) ABJ + Aspirin mixture. The results were expressed as mean ± SD of three independent
experiments in triplicates. Different letters are significantly different (p < .01) by Tukey-­Kramer’s test. ABJ, Andean Berry (Vaccinium
meridionale Swartz); H2O2, hydrogen peroxide (40 mM); RFU, relative fluorescence units. ABJ concentrations are equivalents to 5.67, 11.35,
and 17.03 mg of freeze-­dried ABJ/ml, respectively for 10%, 20%, and 30% vol/vol ABJ

Q2-­positive cells. Hence, ABJ and Aspirin mixtures augmented late ap-
optotic cells together with enhanced mitochondrial depolarization.
3.5 | Effect of ABJ, Aspirin, and their mixture on
phosphatidylserine relocalization in SW480 cells
median values, agreeing with the abovementioned results (Figure  4),
indicating the ABJ-­Aspirin mixture was the most pro-­apoptotic treat-
ment. Particularly for this outcome, the interaction index revealed a
synergistic effect from both treatments, potentially associated with a
3.99 ± 0.44 and 2.49 ± 0.58-­fold increase in the median of externalized
PS-­positive cells, compared to Aspirin and ABJ, respectively.

PS relocalization due to its binding with Annexin V was evaluated in

Figure 5. Based on the representative histogram from the flow cytome- 4 | D I S CU S S I O N
try analysis (Figure 5a), the quantification of the median of externalized
PS-­positive cells revealed no differences between the untreated con- This study aimed to evaluate the antiproliferative effect of ABJ and
trol and Aspirin (20 mM). However, the ABJ-­Aspirin showed the highest Aspirin in SW480 cells. Aspirin has been largely used as NSAID in
ARANGO-­VARELA et al. |
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F I G U R E 3   Influence of treatments on the intracellular oxidative stress of SW480 cells. GSH/GSSG ratio after treatment with (a) ABJ juice
and Aspirin; (b) Aspirin; and (c) Mixture. The results were expressed as mean ± SD of three independent experiments in triplicates. Different
letters are significantly different (p < .01) by Tukey-­Kramer’s test. ABJ, Andean Berry (Vaccinium meridionale Swartz) juice; GSH, reduced
glutathione; GSSG, Oxidized glutathione; H2O2, hydrogen peroxide (40 mM). ABJ concentrations are equivalents to 5.67, 11.35, and 17.03 of
freeze-­dried ABJ mg/ml, respectively for 10%, 20%, and 30% ABJ

colorectal cancer treatment as its daily consumption has been linked
to reduced deaths from several common cancers in randomized clini-
cal trials (Pan, Huang, et al., 2018). However, its known side effects,
such as gastrointestinal bleeding and peptic ulcers (van Kruijsdijk
et  al.,  2015), is still an open question stimulating research aiming
to reduce its concentration. In this sense, phytochemical-­rich prod-
ucts such as ABJ could be used as co-­adjuvant in cancer preven-
tion (Mokhtari et  al.,  2017). Hence, plant-­derived phytochemicals
act modulating the tumor environment at an epigenetic level, po-
tentially reversing adverse epigenetic mutations, inhibiting tumo-
rigenesis progression, preventing the metastatic process, or making
cancer cells more sensitive to radio and chemotherapy (Pistollato
et  al.,  2015). Particularly for the relationship between Aspirin and
polyphenol-­rich diets, more research is still needed since clinical tri-
als have suggested mixed results in colon cancer prevention (Pan,
Huang, et al., 2018). Moreover it has been observed that the combi-
nation of two treatments could provide a therapeutic effect that is
generally better than a single component (Huang et al., 2019).
The chemical characterization of ABJ showed TPC values higher
than those previously reported (2.64 ± 0.25 fold), whereas TF were
lower (5.20 ± 1.2 fold) (Arango-­Varela et al., 2020). This could be ex-
plained by the high heterogeneity at which V. meridionale is cultivated
since TF and TPC are secondary metabolites produced in response to
hydric or thermal stress (Cheynier et al., 2013; Ligarreto et al., 2011).
Besides, the ABJ’s processing conditions could impact its polyphe-
nolic content as it has been reported that rising temperatures from
60 to 100°C can increase the polyphenol content (up to 724.24 mg
GAE/L), FRAP and ORAC values (up to 978.51  mg ascorbic acid/L
and 14.17 mM Trolox, respectively), while the total anthocyanin con-
tent decrease at temperatures >80°C (Zapata et al., 2019). There are
no reports on TA for ABJ, but it has been reported values of 73 mg
C3G/L for V. corymbosum (Zapata et  al.,  2020) and 182  mg C3G/L
for V. ashei (Zhang et al., 2019). Augmentation in the TA content in
the ABJ + Aspirin mixture could be associated with the acidic pH of
Aspirin (Ediriweera et al., 2018), enhancing TA extraction and stabil-
ity (Cesa et al., 2017). Moreover acidity improves the transformation
of flavanones into stable anthocyanins, favoring their accumulation
(Oyama et al., 2019). In this experiment, once ABJ was prepared and
mixed with Aspirin, pH decreased from 3.06 ± 0.42 to 2.61 ± 0.03,
potentially confirming these outcomes.
The heterogeneity in the chemical composition in berries could
be optimized to produce high-­quality products with the amount of
required phytochemicals to prevent the risk of developing colorectal
cancer. Plant genotyping strategies for recognizing specific genetic
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10 of 16       ARANGO-­VARELA et al.

F I G U R E 4   (a) Representative images of the analysis of mitochondrial membrane potential (ΔΨm) of SW480 cells by flow cytometry after
treatments of ABJ, Aspirin, and their mixture; (b) Quantification of positive cells (%) for each quartile. Hydrogen peroxide (H2O2, 40 mM)
was used as positive control while the negative control was untreated SW480 cells. The mixture was 30% ABJ + 20 mM Aspirin. The ABJ
concentration (30 ± 0.11% vol/vol) equals to 17.03 ± 0.06 mg of freeze-­dried ABJ/ml. For the quartiles, Q1 (DiOC6−/PI+): dying cells
(nonapoptotic/necrotic), low ΔΨm, and low membrane integrity; Q2 (DiOC6+/PI−): Late apoptosis, high ΔΨm, and low membrane integrity;
Q3 (DiOC6+/PI−): High ΔΨm and good membrane integrity; Q4 (DiOC6−/PI−): low ΔΨm, and good membrane integrity. ABJ, Andean Berry
(Vaccinium meridionale Swartz) juice; DiOC6, 3,3′-­dihexyloxacarbocyanine iodide; PI, propidium iodide

resources, genetic improvement, and transgenic approaches could Regarding the antioxidant capacity, the ABJ + Aspirin solution
be used to reach the intended nutritional quality in berries (Mazzoni contained lower TF but higher TA, suggesting the major contri-
et al., 2016). bution of flavonoids to the observed effect. The abundance of
ARANGO-­VARELA et al. |
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F I G U R E 5   Phosphatidylserine (PS) relocalization in SW480 cells after ABJ and Aspirin treatments. (a) Representative histograms; (b)
Median of positive cells to PS externalization. Results from B are expressed as mean ± SD of three independent experiments in triplicates.
Different letters are significantly different (p < .01) by Tukey-­Kramer’s test. ABJ, Andean Berry (Vaccinium meridionale Swartz) juice. The
control corresponded to untreated SW480 cells. The ABJ concentration (30 ± 0.11% vol/vol) equals to 17.03 ± 0.06 of freeze-­dried ABJ mg/
ml

flavonoids in the samples is beneficial as these phytochemicals
have been linked to extensive anti-­inflammatory, antiprolifera-
tive, anti-­m etastatic, and anti-­angiogenic properties in a wide
range of cancer cells (Pistollato et  al.,  2015). The DPPH results
are close to those previously informed for ABJ and ABJ + Aspirin
(49.75 and 51.27 μmol TE/ml sample) (Arango-­Varela et al., 2020).
Although no reports for ABJ+Aspirin were found, Quintero-­
Quiroz et  al.  (2019) reported lower values for a V. meridionale
nectar (0.10  μmol TE/mLl of sample), but the amount of freeze-­
dried ABJ was much lower than this study (36.9 mg/ml), and the
sample was diluted more than 50 times. ABJ DPPH values were
within those reported for V. floribundum (47.90  μM TE/g) (Vasco
et  al.,  2009) and higher than those reported for V. meridionale
exposed at several pH values (pH: 2.79–­7.40, ~10–­11 μM Trolox
equivalents) (Zapata et al., 2019). For the FRAP method, the values
from Table 1 agree with other reports for the same fruit (87.0 μmol
TE/g) (Garzón et al., 2010), but higher than FRAP values have been
informed for local genotypes of V. corymbosum and V. uliginosum
(3.07–­17.80 μmol TE/g) (Kraujalytė et al., 2015). For both antioxi-
dant capacity assays, the evaluation of a single concentration was
carried out as a reference value to compare with other reports to
estimate changes in these values and track potential differences
in the biological properties from several V. meridionale batches.
As such, the obtained DPPH values are similar to those reported
by Arango-­Varela et  al.  (2020) for ABJ, ensuring that a potential
comparison could be conducted based on the antioxidant prop-
erties of ABJ.
Compounds such as 3,4-­hydroxybenzoic and chlorogenic acids
were the most abundant phenolics. It has not been reported the
presence of the 3,4-­dihydroxybenzoic acid in V. meridionale fruit,
but it has been found in cranberry (V. macrocarpon) as a flavonoids’
metabolite (Baron et al., 2020). The absence of p-­Coumaric acid in
the ABJ-­A spirin mixture could be explained by the susceptibility
of this compound to low pH values (highest absorbance at pH 4.5–­
7.0) (Benvidi et  al.,  2019). Several phenolics have been informed
for V. meridionale Sw. and ABJ. For instance, Garzón et  al.  (2010)
and Agudelo, Luzardo-­Ocampo, et al. (2018) identified chlorogenic
acid or chlorogenic acid derivatives (86.1 and 153.99 μg/g, respec-
tively) as the most abundant phenolics and a lower abundance of
caffeic acid and derivatives, gallic acid, and caffeoyl methyl quinate.
However, Arango-­Varela et al. (2020) reported gallic acid, rutin, and
ellagic acid as the most concentrated phenolics (650.75, 109.96, and
69.73 μg/g, respectively) in ABJ.
The observed antiproliferative effect cannot be attributed to a
nutrient deprival as other experiments evaluating ABJ mixtures with
DMEM (0%, 10%, 20%, 30%, 40%, and 50% vol/vol) did not impact
(p  < .05) cell viability (>90%) of RAW 264.7 macrophages (Arango-­
Varela et  al.,  2020). Our viability results from ABJ-­IC50 were higher
than those informed by Agudelo et al. (2017) for the same cell line (19%
vol/vol). There are few reports on the antiproliferative effect of ABJ,
but phenolic compounds isolated from Rabbiteye blueberries (V. ashei)
such as ursolic acid, β-­amyrin, and β-­Sitosterol (3-­O-­glucopyranoside)
were associated with antiproliferative effects on HCT116 cells (IC50:
2.13, 3.12, and 4.86  μg/ml, respectively) (Neto,  2007). Enriched an-
thocyanin extracts from V. myrtillus have also displayed antiprolifer-
ative effects on other colorectal cancer cells, such as HT29 (7% at
200 mg/ml) and HCT116 (34% at 4 mg/ml) (Katsube et al., 2003; Zhao
et al., 2004). It has been hypothesized that anthocyanins are the most
important compounds linked to the antiproliferative effect of V. me-
ridionale, but the synergistic activity of phenolic acids and flavonoids
should not be discarded (Maldonado Celis et al., 2017). Indeed, consid-
erable evidence has suggested that the combined effects produced by
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12 of 16       ARANGO-­VARELA et al.
plant-­derived extracts and targeted treatments can alter the biological
activity of a mixture, and only some components offer a synergistic
effect. In contrast, some of them could have no effect on the expected
outcome (Caesar & Cech, 2019). Although no individual isolated poly-
phenols from ABJ were evaluated in this research to validate the
observed biological effects, most anticancer properties of vegetable
and fruit-­derived extracts are a consequence of combining these com-
pounds (Brglez Mojzer et al., 2016).
Aspirin has shown antiproliferative effects on SW480 cells (IC50:
3  mM) (Dibra et  al.,  2010) and exhibited reduced cell viability on
SW480, HCT116, and HCT116-­chr3 cells (1, 2.5, 5, and 10 mM con-
centrations after 48 and 72 hr) (Goel et al., 2003). Reports have also
suggested the involvement of Aspirin in the downregulation of pro-
teins such as Sp1, Sp3, and Sp4, associated with cell growth promotion
(activating the epidermal growth factor receptor or EGFR and cyclin
D1), survival (Bcl-­2 and survivin), angiogenesis (vascular endothelial
growth factor or VEGF) and inflammation (NF-­κB) (Pathi et al., 2012).
ROS are by-­p roducts of cell metabolism and play critical roles
in cell homeostasis, inducing oxidative stress on DNA, protein,
and lipids, promoting gene instability and tumorigenesis (Costa
et  al.,  2014). ROS could also induce cancer cell proliferation par-
ticipating in metabolic signaling mechanisms (Liou & Storz, 2010).
Increases in the ROS production derived from the ABJ-­A spirin
mixture suggest losses in their pro-­oxidant potential, an effect
that has also been reported for LPS-­challenged RAW 264.7 mac-
rophages treated with 7.79% v/v ABJ and increasing Aspirin con-
centrations (2.5, 5.0, 7.5, and 10 mM) (Arango-­Varela et al., 2020).
Aspirin protects cells from hydroxyl radical groups (OH−) at a
faster rate (reaction rate constant: 3.6 × 1010 M−1 s−1) than notori-
ous antioxidants such as ascorbate, glutathione, and cysteine, by
inhibiting NF-­κ B activation by pro-­inflammatory inductors (lipo-
polysaccharide, silica, or TNF-­α) (Shi et al., 1999). For example, en-
dothelial cells treated with Aspirin (0.5, 1.0, and 2.5 mM) for 1 hr
exhibited ROS reduction after previous treatment with oxidized
low-­d ensity lipoprotein (Chen et  al.,  2012). High Aspirin concen-
trations (up to 50  mM) were assayed in vivo showing significant
inhibition of COX-­2 , potentially activated by the ROS-­induced
NF-­κ B stimulatory activity (M. E. Lee et al., 2012). Particularly for
cancer cells, ROS are increased due to overexpression of transfer-
rin and copper receptors (Lee et al., 2013), potentially involved in
oxygen-­reactive labile redox complexes between ABJ and metal
ions (León-­G onzález et al., 2015).
GSH is the most abundant antioxidant on living organisms,
being found at elevated levels in cancer cells (Bansal & Celeste
Simon,  2018). Hence, reducing GSH to produce GSSG is an inter-
esting strategy to reduce tumor growth, promoting ROS imbalance
(Ortega et al., 2011). Results obtained from the impact of the treat-
ments in the GSH/GSSG ratio are in agreement with the ROS ob-
servations since ROS could react with antioxidant enzymes such
as superoxide dismutase (SOD) in association with catalase or glu-
tathione peroxidase (GPX) to oxidize GSH (Aquilano et  al.,  2014).
Agudelo et al. (2017) informed about the reducing effect of ABJ on
the GSH/GSSG ratio on SW480 cells, associated with an oxidative
stress-­induced pro-­apoptotic effect. Moreover the antioxidant po-
tential of ABJ was previously examined by our research group in a
clinical study involving healthy men and women (18–­60 years) with
dietary factors for colorectal cancer (Agudelo, Ceballos, et al., 2018).
In this study, ABJ consumption (250 ml for 14 days) showed increased
plasmatic antioxidant capacity (p > .01) and significant 8-­isoprostane
reductions (p  < .01), compared to the basal state of participants.
Although the IL-­6 and TNF-­a reduction were not significant, the ob-
served antioxidant potential could be linked to significant findings
of IL-­1β reductions, attributed to ABJ phenolics such as gallic acid
(Arango-­Varela et al., 2020; Luzardo-­Ocampo et al., 2020).
Mitochondria from cancer cells exhibit structural and functional
differences compared to non-­malignant cells, such as the membrane
potential (ΔΨm), dependent on chemical and electrical components
from the electron transporter chain (Zhang et  al.,  2015). Changes
in ΔΨm are linked to Krebs’ cycle alterations and ATP production,
leading to permeability alterations and release of death cell inducer
proteins to the intermembrane space (Waterhouse et  al.,  2002).
The potential pro-­apoptotic effects observed in SW480 after ABJ
treatments could be explained by the involvement of anthocy-
anins and phenolics in these mechanisms (Joshi & Goyal,  2011).
Phenolics such as 3-­hydroxybenzoic acid have shown cell death in-
duction by increasing caspase-­3 and ROS production and the num-
ber of cells from the sub-­G 0/G1 cell cycle populations in HCT116
and HCT15 cells (Anantharaju et al., 2017). Reports have indicated
that 2-­hydroxycinnamic acid has exhibited antiproliferative ef-
fects, cell cycle regulation, and apoptosis modulation in colorectal
cancer cells (Rocha et  al.,  2012). More recently, it was reported
the effectiveness of 3,4-­dihydroxybenzoic acid as a flavonoid me-
tabolite in the inhibition of cancer cell proliferation (HCT116, HT-­
29, Caco-­2, and MDA-­MB-­231 cells) involving a SLC5A8 transport
and CDK-­dependent mechanism (Sankaranarayanan et  al.,  2019).
Chlorogenic acid has promising inhibitory effects on colorectal can-
cer cells reducing ROS production and modulating the NF-­κB, ac-
tivator protein 1 (AP-­1), and the mitogen-­activated protein kinase
(MAPK) pathways (Xu et  al.,  2013). Aspirin has been associated
with growth inhibition and induction of apoptosis in colon cancer
cells (RKO, SW480, HT-­29, and HCT116 cells), showing VEGF and
Wnt/b-­catenin inhibition (Borthwick et al., 2006; Bos et al., 2006).
Mixtures of Aspirin and strawberry displayed high PCNA reductions
and slight impact decreasing Cyclin D1, Cyclin A2, and CDK4 in rats
with N-­nitrosomethylbenzylamine (NMBA)-­induced esophageal
tumors in vivo, compared to NMBA only-­treated rats (Pan, Peiffer,
et al., 2018). It is important to note that additional analyses involving
evaluating the impact of treatments on caspase 3/7 activity or the
modulation of target pro-­apoptotic proteins would indicate a more
straightforward intrinsic or extrinsic pro-­apoptotic pathway.
5 | CO N C LU S I O N S
In conclusion, the results suggested that the combination of ABJ with
Aspirin increases the cytotoxic effect on SW480 cells compared to
ARANGO-­VARELA et al.
separate ABJ or Aspirin treatments. ABJ favored the ROS produc-
tion and reduced the GSH/GSSG ratio, indicating an intracellular
oxidative environment. Moreover, the combined ABJ + Aspirin
treatments exhibited loss of mitochondrial membrane and increased
phosphatidylserine relocalization. These effects could be attributed
to the joint activity between the rich polyphenolic composition of
ABJ and Aspirin, highlighting the potential use of natural compounds
and synthetic molecules as potential co-­adjuvants delivering anti-
proliferative effects in vitro.
AC K N OW L E D G E M E N T S
Author Sandra S. Arango appreciate the granted Ph.D. scholarship from
Departamento Administrativo de Ciencia, Tecnología e Innovación
–­ COLCIENCIAS (grant number: 7851) and Instituto Tecnológico
Metropolitano (ITM), Medellín, Colombia. Author Ivan Luzardo-­
Ocampo acknowledges Programa de Becas Posdoctorales de la UNAM
(DGAPA-­CTIC) for his postdoctoral scholarship [grant number: 5267].
The support given by COLCIENCIAS through the Program Ecosistema
Científico Cod. FP44842-­211-­2018 [project number: 58580].
C O N FL I C T O F I N T E R E S T
The authors declared that they have no conflict of interest.
AU T H O R C O N T R I B U T I O N S
Conceptualization; Data curation; Formal analysis; Funding acquisi-
tion; Investigation; Methodology; Resources; Writing-­
original draft;
Writing-­review & editing: Sandra S. Arango-­Varela. Formal analysis;
Investigation; Methodology; Validation; Writing-­original draft; Writing-­
review & editing: Ivan Luzardo-­Ocampo. Formal analysis; Investigation;
Methodology; Writing-­
review & editing: Camilo Reyes-­Dieck.
Conceptualization; Formal analysis; Funding acquisition; Methodology;
Project administration; Supervision; Validation; Writing-­review & edit-
ing: Elhadi M. Yahia. Conceptualization; Formal analysis; Funding acqui-
sition; Project administration; Supervision; Validation; Writing-­review &
editing: Maria Elena Maldonado-­Celis.
ORCID
Sandra S. Arango-­Varela  https://orcid.
org/0000-0003-2422-3439
Ivan Luzardo-­Ocampo  https://orcid.org/0000-0002-8033-3520
Elhadi M. Yahia  https://orcid.org/0000-0002-3557-8975
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