Invest Clin 63(1): 7 - 18, 2022 https://doi.org/10.54817/IC.v63n1a01
Corresponding author. Yenddy Carrero. Universidad Técnica de Ambato. Facultad Ciencias de la Salud. C/ Salva-
dor entre México y Colombia, Ingahurco. Ambato 180150, Ecuador. Tel: + 59 3 33730268 ext. 5239.
E-mail: yenddycarrero@yahoo.es
Arracacia xanthorrhiza Bancr compounds
decrease β-actin, hypoxia-inducible factor-1
and nitric oxide production in HeLa cells.
Yenddy Carrero1,2, Jenny Moya2, Michael Acosta2 and Jesús Mosquera-Sulbarán3
1Universidad Técnica de Ambato. Facultad Ciencias de la Salud. Ambato, Ecuador.
2Universidad Técnica de Ambato. Facultad de Ciencias de la Salud. Laboratorio
de Biología Molecular y Celular. Ambato, Ecuador.
3Instituto de Investigaciones Clínicas “Dr. Américo Negrette”. Facultad de Medicina,
Universidad del Zulia, Maracaibo, Venezuela.
Key words: actin; nitric oxide; HIF-1; Arracacia xanthorrhiza Bancr; Hela cells.
Abstract. The treatment of cancer patients with anti-cancer drugs is often
accompanied by the presence of undesirable side effects. The use of natural
plant derivatives alone, or in conjunction with existing anti-neoplastic drugs,
has been suggested to obtain better results and decrease these side effects.
Nitric oxide (NO•), the hypoxia-inducible factor-1 (HIF-1), and decreased con-
centration of actin play important roles in cancer progression. The beneficial
effects of polyphenols in various organ disorders including cancer has been re-
ported. The aim of this study was to determine the effect of Arracacia xanthor-
rhiza Bancr extracts, white (WAXB) and red (RAXB) variants (compounds rich
in polyphenols) on the concentrations of β-actin, NO• and HIF-1 in Hela cells
cultures, to uncover possible anti-neoplastic effects. Extracts from the plant
leaves were added to Hela cell cultures at a concentration of 10-3 mg/mL, and
after 24 hours of culture, the concentrations of β-actin, NO• and HIF-1 were de-
termined by immunohistochemical, biochemical and western blot assays. Both
extracts reduced the concentrations of β-actin, NO• and HIF-1 (p<0.001), sim-
ilar to the methotrexate effect. These results suggest an antineoplastic effect
of the studied plant extracts and highlight the possibility of their use in the
treatment of neoplasms.
8 Carrero et al.
Investigación Clínica 63(1): 2022
Los componentes de la Arracacia xanthorrhiza Bancr
disminuyen las producciones de la actina β, del factor-1
inducible por la hipoxia y del óxido nítrico en células Hela.
Invest Clin 2022; 63 (1): 7 – 18
Palabras clave: actina; óxido nítrico; HIF-1; Arracacia xanthorrhiza Bancr; células Hela.
Resumen. El tratamiento de pacientes con cáncer utilizando drogas-
antineoplásicas presenta problemas relacionados con efectos colaterales inde-
seables. Se ha sugerido el uso de derivados de plantas naturales solas, o en
combinación con drogas antineoplásicas existentes para obtener mejores re-
sultados y disminuir los efectos colaterales. Así mismo, se ha reportado que
el óxido nítrico (NO•), el factor-1 inducible por hipoxia (HIF-1) y la disminu-
ción de la expresión de la actina tienen un papel en la progresión del cáncer.
También se ha reportado los efectos beneficiosos de lo polifenoles en varios
desordenes orgánicos, incluyendo el cáncer. El objetivo de este estudio fue de-
terminar los efectos de los extractos procedentes de la Arracacia xanthorrhiza
Bancr blanca (AXBB) y la variedad roja (AXBR) (compuestos ricos en polifeno-
les) en las concentraciones de la actina-beta, el NO• y el HIF-1 en cultivo de
células Hela, para destacar sus posibles efectos antineoplásicos. A los cultivos
de células Hela se les agregaron los extractos de las hojas de AXBB o AXBR
(10-3 mg/mL, concentración final) y después de 24 horas de cultivo se deter-
minaron las concentraciones de la actina-beta, el NO• y el HIF-1 por métodos
inmunohistoquímicos, bioquímicos y western blot. Ambos extractos disminuye-
ron las concentraciones de la actina-beta, el NO• y el HIF-1 (p<0,001) de una
manera similar al efecto del metotrexato. Estos resultados sugieren un efecto
antineoplásico de estos extractos y destacan la posibilidad de ser usados para el
tratamiento de las neoplasias.
Received: 13-08-2021 Accepted: 03-09-2021
INTRODUCTION
Cellular motility is the basis for can-
cer cell invasion and metastasis. Migration
involves an intricate actin branched net-
work. Regulation of cell migration is nec-
essary for development, wound healing,
and immune responses, whereas aberrant
and uncontrolled cell motility is a feature
in cancer cells 1. Therefore, compounds
capable of inducing degradation of actin,
could be important in the production of
anti-cancer drugs by decreasing the actin-
dependent cell motility. In addition to the
alterations of actin, nitric oxide (NO•), a
vasodilator, has been shown to have a role
in the regulation of cancer progression and
metastasis 2; effects that probably are medi-
ated by angiogenesis, apoptosis and cellu-
lar motility 3,4. In addition, NO• increases
the hypoxia-inducible factor-1 (HIF-1), a
transcription factor that enhances several
hypoxia-inducible genes and induces cancer
metastasis 5. Previous studies have shown
Arracacia xanthorrhiza Bancr and Hela cells 9
Vol. 63(1): 7 - 18, 2022
that methotrexate is capable of inducing
apoptosis 6, an important cytotoxic mecha-
nism of anti-cancer drugs. It is well known
that apoptosis is associated with alterations
of the reorganization and concentration of
actin 7, 8. Therefore, drugs that target ac-
tin, NO•, and HIF-1may be important in the
treatment of cancer. Thus, the aim of this
study was determining the effect of two eco-
variants of Arracacia xanthorrhiza Bancr
extracts, white (WAXB) and red (RAXB)
variants on the concentrations of β-actin,
NO• and HIF-1 in Hela cells cultures (uter-
ine cervix cancer cell line) and compared
their effects with the effect of methotrex-
ate, a well-known anti-cancer drug.
MATERIALS AND METHODS
Source of plants
Plants were obtained from the follow-
ing localities: Red Arracacia xanthorrhiza
Bancr (RAXB) from the province of Tungu-
rahua, Quinchicoto (Municipality Tisaleo),
Ecuador (Latitude: 1° 23’ 54” S; Longitude:
78° 24’ 41” W; Altitude: 3,260 meters above
sea level). White Arracacia xanthorrhiza
Bancr (WAXB) from El Triunfo (Municipal-
ity Baños), Ecuador (Latitude: 1°10’ 18” S;
Longitude: 78°32’ 33” W; Altitude: 1,683
meters above sea level). Plant samples used
for this research have been authorized by
the Ecuadorian government under the
framework of the United Nations Conven-
tion on Biodiversity.
Extract procedure
Fresh plant leaves were collected and
dried in an oven at 40°C for 24 hours until
a constant weight was obtained. The speci-
mens were stored at room temperature in
the dark prior to their extraction and subse-
quent testing. Stock solutions of RAXB and
WAXB were prepared by dissolving 60 g of
leaves in ethanol (20%) at 80°C for one hour.
Ethanol was extracted by a rotary evaporator
and active compounds remained in water.
Extracts were grinded into a powder (VirTis
Bench Top, SP Scientific, NY, USA), and then
dissolved in PBS and filtered (0.22 µm). Fil-
tered stock solutions (0.411 mg/mL) were
stored at -80°C until use. Total phenolic
content (TPC) and antioxidant activity (AA)
assays in microplates were determined. An
intra-laboratory validation was performed 9
of the Folin-Ciocalteu microplate method to
measure TPC and the 2,2-diphenyl-1-picryl-
hydrazyl (DPPH) microplate method to mea-
sure AA.
Cell culture
The human cervical cell line (HeLa) was
obtained from the American Type Culture
Collection (CCL-2). Cells were maintained
in DMEM media, supplemented with 10% fe-
tal bovine serum, 100 U/mL penicillin, and
100U/mL streptomycin. The cells were then
incubated at 37°C with 5% CO2 saturation.
Cells were used in the linear phase of growth
with a passage number of 5 to 7.
Viability assay
The cytotoxic effect of WAXB and RAXB
on HeLa cells was evaluated by MTT assay ac-
cording to the manufacturer’s instructions
(Thermo Fisher, MA, USA). The cells were
seeded at 1x104 cells per well in a 96-well
plate and incubated at 37°C for 24 h. Cells
were treated with increasing doses of WAXB
or RAXB by 2-fold dilution, starting with
10–1mg/mL until 10–11mg/mL. Cells were
treated for 24h. The MTT assay results were
obtained using a spectrophotometer plate
reader (Victor X3; Perkin Elmer, USA) at
570 nm. Half maximal inhibitory concentra-
tion (IC50: 10–3 mg/mL) was obtained by no
lineal regression analysis (Graphpad Prism
7.0 Software Inc., San Diego, CA, USA). All
experiments were performed in triplicate to
evaluate half-maximal inhibitory concentra-
tion for both plant extracts against the HeLa
cell line.
Western Blot
Untreated and WAXB or RAXB- treated
Hela cell cultures (10–3 mg/mL) and metho-
10 Carrero et al.
Investigación Clínica 63(1): 2022
trexate-treated cultures (10 µM), were lysed
with RIPA buffer for 30 min on ice. Then, cells
were centrifuged at 1200 rpm for 15 min at
4°C, and the supernatant was collected. The
protein concentration was measured by the
Bradford protein assay. Similar amount of
protein from each sample was separated by
SDS-PAGE and transferred onto PVDF mem-
branes. The membranes were blocked with
7% skimmed milk for 2 h at room tempera-
ture and incubated overnight at 4°C with a
primary anti- β actin antibody (Santa Cruz
Biotechnology Inc, Texas, USA). After wash-
ing three times with TBST(10 mM Tris-HCL,
pH 7.4, 150 mM NaCl, 0.05% Tween 20), the
membranes were incubated with the appro-
priate horseradish peroxidase-conjugated
secondary antibody (1:5000 dilution: Santa
Cruz Biotechnology Inc, Texas, USA). Ex-
periments were performed in triplicate and
at least three independent experiments were
done. Controls represent untreated HeLa
cultures.
β-actin determination
HeLa cells were seeded on coverslips at
5x105 cells per well. After 48 h of culture,
cells were treated with WAXB, RAXB (10–3
mg/mL) or methotrexate (10 µM) for addi-
tional 24 h. Cells were fixed and permeabi-
lized by a paraformaldehyde solution (4%, 15
min) and cold acetone (15 min), respectively.
Then, cells were reacted with a monoclonal
antibody against β-actin (β-Actin (C4): sc-
47778 conjugated Alexa Fluor®647; Santa
Cruz Biotechnology Inc, Texas, USA) for 30
min at 37°C. Thereafter, cells on coverslips
were analyzed through a fluorescence mi-
croscopy (Leica-DMi8, Wetzlar, Germany).
Experiments were performed in triplicate
and at least three independent experiments
were done. DAPI staining was used to iden-
tify nuclei. Results were expressed as fluo-
rescence intensity units (502/530 nm) per
x 630 field. Controls represent untreated
HeLa cultures.
Nitric oxide determination
Nitric oxide was determined by detec-
tion of nitrite formed by the spontaneous
oxidation of NO• using a Griess Reagent Kit
(G-7921: Molecular Probe Inc. Eugene, USA)
following the manufacture’s indications. Re-
sults are expressed as µM per mg of protein.
Hypoxia-inducible factor-1 determination
HIF-1 was determined in WAXB, RAXB
(10–3 mg/mL) treated Hela cell cultures us-
ing Image-iT™ Hypoxia Reagents (Invitro-
gen, Termo Fisher Scientific, Eugene, USA)
following the manufacture’s indications.
Cells on coverslips were analyzed through
a fluorescence microscopy (Leica-DMi8,
Wetzlar, Germany). Experiments were per-
formed in triplicate and at least three inde-
pendent experiments were done. DAPI stain-
ing was used to identify nuclei. Results were
expressed as fluorescence intensity units
(502/530 nm) per x 630 field. Controls rep-
resent untreated HeLa cultures.
Statistical analysis
Experiments were in triplicate and per-
formed at least three times. Results were ex-
pressed as the means ± standard deviation.
Statistical analysis was performed using one
way ANOVA and Bonferroni´s posttest us-
ing Graph Pad Prism software (version 7.0;
Graph Pad Software Inc., San Diego, CA,
USA). Statistical significance is expressed as
p<0.05.
RESULTS
The antioxidant activity of WAXB and
RAXB and their phenolic content are ob-
served in Table 1. The cytotoxicity of both
extract plants was determined by the MTT
assay. The MTT assay indicated that both
WAXB and RAXB have a half maximal inhib-
itory concentration (IC50) of 10–3 mg/mL
(Fig. 1). Western blot analysis showed de-
creased thickness and intensity of the actin
Arracacia xanthorrhiza Bancr and Hela cells 11
Vol. 63(1): 7 - 18, 2022
Table 1
Phenolic content and antioxidant activity of Arracacia xanthorrhiza Bancr
(white carrot and red carrot) extracts.
WABX RABX
Phenolic content
(mg Eq AG/g powder) 78.6 ± 0.002 59.7 ± 0.001
Antioxidant activity
(µg Eq Trolox/g powder) 0.595 ± 0.03 0.395 ± 0.02
(µmol Eq Trolox/100g powder) 237.7 ± 0.03 157.8 ± 0.02
WABX: White Arracacia xanthorrhiza Bancr; RAXB: Red Arracacia xanthorrhiza Bancr.
Fig. 1. Viability assay in Hela cell cultures. White (WABX) and red (RABX) Arracacia xanthorrhiza Bancr
leaves cytotoxic effect on HeLa cells was evaluated by MTT assay. Cells were seeded at 1x104 per well
and treated with increased doses of WAXB (A) and RAXB (B) from 10–11mg/mL to 10–1mg/mL. Expe-
riments were performed in triplicate to evaluate half-maximal inhibitory concentration (IC50: 10–3
mg/mL) for plant extracts.