Archives of Medical Research 51 (2020) 65e75

ORIGINAL ARTICLE
In Vitro Employment of Recombinant Taenia solium Calreticulin as a Novel
Strategy Against Breast and Ovarian Cancer Stem-like Cells
Alejandro Schcolnik-Cabrera,a,1 Mandy Ju
arez,a,1 Bernardo Oldak,b,c Mayra Cruz-Rivera,b Ana Flisser,b
Alfonso Due~nas-Gonz
alez,a,d Vinnitsa Buzoianu-Anguiano,e Sandra Orozco-Suarez,e and
Fela Mendlovicb,c
a
Divisi
on de Investigaci
on B
asica, Instituto Nacional de Cancerolog
ıa, Ciudad de M
exico, Mexico
b
Departamento de Microbiolog
ıa y Parasitolog
ıa, Facultad de Medicina, Universidad Nacional Aut
onoma de M
exico, Ciudad de M
exico, Mexico
c
Facultad de Ciencias de la Salud, Universidad An
ahuac M
exico Norte, Huixquilucan, Estado de M
exico, Mexico
d
Unidad de Investigaci

on Biom
edica en C
ancer, Instituto de Investigaciones Biom
edicas de la Universidad Nacional Aut
onoma de M
exico/Instituto Nacional
de Cancerolog
ıa, Ciudad de M
exico, Mexico
e
Unidad de Investigaci
on M
edica en Enfermedades Neurol
ogicas, Hospital de Especialidades CMN Siglo XXI, Ciudad de M
exico, Mexico
Received for publication June 7, 2019; accepted December 3, 2019 (ARCMED_2019_476).

Background and Aims. Calreticulin is a chaperone and master regulator of intracellular

calcium homeostasis. Several additional functions have been discovered. Human and
parasite calreticulin have been shown to suppress mammary tumor growth in vivo. Here,
we explored the capacity of recombinant Taenia solium calreticulin (rTsCRT) to modu-
late cancer cell growth in vitro.
Methods. We used different concentrations of rTsCRT to treat cancer cell lines and
analyzed viability and colony formation capacity. We also tested the combination of
the IC20 or IC50 doses of rTsCRT and of the chemotherapeutic drug 5-fluorouracil on
MCF7 and SKOV3 cell lines. As a control, the non-tumorigenic cell line MCF10-A
was employed. The effect of the drug combinations was also assessed in cancer stem-
like cells. Additionally, scavenger receptor ligands were employed to identify the role
of this receptor in the rTsCRT anti-tumoral effect.
Results. rTsCRT has a dose-dependent in vitro anti-tumoral effect, being SKOV3 the
most sensitive cell line followed by MCF7. When rTsCRT/5-fluorouracil were used,
MCF7 and SKOV3 showed a 60% reduction in cell viability; colony formation capacity
was also diminished. Treatment of cancer stem-like cells from MCF7 showed a higher
reduction in cell viability, while those from SKOV3 were more sensitive to colony disag-
gregation. Finally, pharmacological inhibition of the scavenger receptor, abrogated the
reduction in viability induced by rTsCRT in both the parental and stem-like cells.
Conclusion. Our data suggest that rTsCRT alone or in combination with 5-fluorouracil
inhibits the growth of breast and ovarian cancer cell lines through its interaction with
scavenger receptors. Ó 2020 Published by Elsevier Inc. on behalf of IMSS.
Key Words: Taenia solium, Calreticulin, Cancer stem-like cells, Drug synergism, Cancer.

Introduction
Cancer is a major health problem worldwide. It was esti-
1
These authors contributed equally for this work. mated that during 2018, only in the United States, more
Address reprint requests to: Fela Mendlovic, Departamento de Micro- than 1.7 million people acquired some type of neoplastic
biolog
ıa y Parasitolog
ıa, Facultad de Medicina, Universidad Nacional disease, and close to 600,000 persons died due to cancer
Aut
onoma de M
exico, Col. Universidad Nacional Aut
onoma de M
exico,
(1). Tumors share high histopathological diversity and
Ciudad Universitaria, Alcald
ıa Coyoac
an, Avenida Universidad 3000,
04510 Ciudad de M
exico, Mexico; Phone: (þ52) 55 5623 2466; FAX: contain regions delimited by various degrees of differentia-
(þ52) 55 56232382.; E-mail: [email protected] tion, proliferation, vascularity, inflammation, and/or

0188-4409/$ - see front matter. Copyright Ó 2020 Published by Elsevier Inc. on behalf of IMSS.
https://doi.org/10.1016/j.arcmed.2019.12.003
66 Schcolnik-Cabrera et al./ Archives of Medical Research 51 (2020) 65e75

invasiveness (2). Cancer stem-like cells (CSC) are found receptor class A and the F class scavenger receptor SREC1
within the heterogeneous population of cells that comprise (24,25). The SRF is a diverse group of evolutionally
the tumor and several signaling pathways are recognized to conserved membrane-bound proteins that bind a heteroge-
play essential roles in their regulatory capacity. Specific neous number of polyanionic ligands, modified low-
signaling pathways mediate various stem cell properties, density lipoproteins, extracellular matrix proteins and path-
such as self-renewal, cell fate decisions, survival, prolifera- ogen products (26). Furthermore, members of the SRF are
tion, and differentiation (3e5), and many of these crucial involved in cell adhesion and in the regulation of tumor
pathways are dysregulated in cancer, leading CSC to be development, progression and immunomodulation in
resistant to conventional therapies and ultimately promot- response to cancer (27). For instance, in ovarian carcinoma,
ing tumor growth and recurrence (6). overexpression of scavenger receptor A3 correlates with tu-
Chemotherapy, along with radiotherapy and surgical mor progression and recurrence (28,29). Also, signaling
resection, are the common treatment options for cancer. pathways of LOX-1, a class E scavenger receptor, has been
While cancer drug treatment started in the 1940s with nitro- implicated in breast cancer cell line growth, adhesion and
gen mustards (7), now targeted biological therapies and invasion (30).
drug repurposing are areas of research with high investment We previously cloned the CRT from the helminth Taenia
that aim to find new options for treating cancer cells. One of solium and expressed it as a functional recombinant cal-
the most recognized chemotherapy drugs in use is 5- cium binding protein (rTsCRT) (31). In this study, we
fluorouracil (5-FU), a drug commonly used to treat solid explored the capacity of rTsCRT to modulate cancer cell
neoplasia including breast, ovarian, and gastrointestinal growth in vitro by using multiple malignant cell lines from
cancers (8,9). However, one side effect of 5-FU is cardio- different tissue origins, and particularly evaluated the effect
toxicity; its incidence varies between 0e35% depending of rTsCRT on the viability of CSC from ovarian and breast
on the dose, cardiac comorbidities and the schedule of cancer cell lines, with a possible synergistic effect when
chemotherapy (9,10). This fact, along with the discovery used together with 5-FU. We hypothesized that due to its
that loss of p53 function in cancer may diminish cellular affinity for some members of the SRF, rTsCRT binds to
sensitivity to 5-FU, has promoted the employment of 5- scavenger receptors and precludes tumor cell viability.
FU in combination with other agents to improve response
and to reduce the associated side effects (10).
In addition to drug repurposing, the identification of new Materials and Methods
molecules for the treatment of cancer is much needed. Cal-
reticulin (CRT) is a 46 KDa multifunctional highly Epithelial Cell Cultures
conserved calcium-binding protein, commonly found in The cancer cell lines HeLa, MCF7, SW480, PC3, MDA-
the endoplasmic reticulum of every nucleated cell except MB-231 and SKOV3 from cervical, breast, colon, prostate,
in yeast, that acts as a quality control chaperone with spec- breast and ovarian adenocarcinomas, respectively, as well
ificity towards glycoprotein molecules due to its lectin-like as the non-tumorigenic breast epithelial cell line MCF10-
properties (11,12). Although the main role of CRT is to A, were obtained from ATCC (Manassas, VA, USA). All
control intracellular Ca2þ homeostasis, it also participates cell lines were cultured at 37 C in a humidified atmosphere
in the assembly and surface expression of MHC class I mol- containing 5% CO2. All cells were grown in complete me-
ecules for antigen presentation to CD8þ T cells, in cell dium, which was composed of Dulbecco’s modified Eagle’s
motility, secretion pathways, gene transcription regulation, medium and F12 medium (DMEM-F12) supplemented
protein synthesis, cellular adhesion, and apoptosis (13e16). with 10% fetal bovine serum (FBS) and 1% antibiotic-
Several years ago, human calreticulin (HuCRT) and vaso- antimycotic solution (all from Invitrogen Life Technolo-
statin, a fragment that comprises the N terminal of the pro- gies, Carlsbad, CA, USA). The complete medium for
tein, were shown to have anti-tumor effects by inhibiting MCF10-A was composed of DMEM-F12 supplemented
angiogenesis (17,18). CRT is also found in parasites, both with 5% horse serum (Invitrogen, CA, USA), 20 mg/mL hu-
protozoa and helminths, and is present in their excretion/ man epidermal growth factor (Sigma-Aldrich, MO, USA),
secretion products (19e21). Indeed, the native, as well as 0.5 mg/mL hydrocortisone (Stem Cell Technologies, Van-
the recombinant form of the CRT from the protozoan para- couver, CA), 10 mg/mL insulin (Eli Lilly, IN, USA) and
site Trypanosoma cruzi (TcCRT), was shown to have 1% antibiotic/antimycotic solution.
in vivo anti-mammary tumor capacity and to be more effi-
cient than HuCRT in its anti-angiogenic effect and in inhib-
CSC Cultures
iting endothelial cell migration and differentiation, and thus
suppressing tumor growth (22,23). Ovospheres from SKOV3 origin were grown in low-
CRT has been shown to interact with some members of adherence flasks (Corning, ME, USA) under stem-cell con-
the scavenger receptor family (SRF), that include scavenger ditions: serum-free DMEM-F12 supplemented with 4 mg/
 Taenia solium Calreticulin as an Anticancer Agent 67

mL insulin, 20 ng/mL hEGF, 1 ng/mL hydrocortisone Synergism Evaluation

(Stem Cell Technologies, Vancouver, CA) and 1X B27 Sup-
Increasing IC doses of rTsCRT (IC20, IC30, IC40, and IC50)
plement (Gibco, NY, USA). Mammospheres from MCF7
were combined with their respective increasing IC doses of
origin were grown in MammoCult Basal Medium (Stem
5-FU (IC20, IC30, IC40, and IC50). The resulting mixes of
Cell Technologies, Vancouver, CA) supplemented with
rTsCRT and 5-FU (IC20/IC20e50, IC30/IC20e50, IC40/
10% MammoCult Proliferation supplement (Stem Cell
IC20e50, and IC50/IC20e50) were employed for viability
Technologies, Vancouver, CA), 4 mg/mL heparin (Sigma-
curve assays. Cells were seeded into 12 well microtiter
Aldrich, MO, USA) and 0.48 mg/mL hydrocortisone.
plates at 3  104 cells/well densities in 1 mL complete
DMEM-F12. After a pre-incubation period of 24 h, cells
Expression and Purification of Recombinant Taenia were treated for 72 h with the different rTsCRT/5-FU com-
solium Calreticulin (rTsCRT) binations and cellular viability was assessed as previously
described. The synergistic interaction was determined using
The full-coding region of the mature recombinant Taenia
the combination index method from the formula of Chou
solium CRT (rTsCRT) without the signal peptide was cloned,
and Talalay with the Calcusyn software (Biosoft) (33).
expressed and the resulting protein was purified as previ-
ously described (32). Briefly, Escherichia coli cultures were
induced with 0.75 mM isopropylthio-galactoside (IPTG) to Clonogenic Assays
express rTsCRT. The induced bacteria were resuspended in SKOV3, MCF7 and MCF10-A cells were treated with the
25 mM Tris buffer pH 7.4, centrifuged and sonicated. combination of rTsCRT and 5-FU at IC20/IC20, IC50/IC50,
rTsCRT was submitted to 10% SDS-PAGE gel electropho- or the control, which was composed of complete DMEM-
resis and the resulting 53 KDa band was excised and F12 containing the vehicle for either rTsCRT (25 mM Tris,
electro-eluted using an electro-eluter model 422 (BioRad pH 7.4) or 5-FU (ethanol at a final volume of 0.1%) at the
Hercules, CA, USA). Endotoxins were measured as reported same concentration as the higher dose for each treatment,
elsewhere (32). Purified rTsCRT was filtered using a 0.2 mM during 72 h. Then, 1000 cells were recovered from each
filter, and kept at 70 C until use. condition by using a 0.5% trypsin-0.1% EDTA solution
and were plated in 6 well microtiter plates (Corning, ME,
Viability Curves USA). Cells were allowed to grow during 14 d without
treatment, with complete media removal and addition of
Cells were grown in 12-well microtiter plates (Corning, ME, fresh complete media every 48 h. After the 14 d period, col-
USA) at a density of 3  104 cells/mL in complete DMEM- onies (defined by the presence of O50 cells grouped
F12. After a pre-incubation period of 24 h, cells were treated together) were fixed in a methanol and acetic acid solution
during 72 h with increasing concentrations of either rTsCRT, (3:1 v/v), stained with crystal violet and counted using the
ranging from 0.1e1 mM, or 5-fluorouracil (5-FU, Sigma- ImageJ software (2.0 version). ImageJ software has a plu-
Aldrich, MO, USA, F6627) ranging from 0.1e3 mM. Fresh gin that allows the user to count colonies in an automatized
complete medium containing each compound was changed manner. This counter works with 8 bit images, and it auto-
every 24 h and after the 72 h period, cells were detached with matically counts the number of colonies according to the
a 0.5% trypsin-1% EDTA solution (Invitrogen, CA, USA) values of size and circularity previously entered by the user,
for cell counting using the trypan blue exclusion assay. which depends on the colonies observed in control cultures
Recovered cells were centrifuged (5 min at 1200 rpm) and of each cell line.
the remaining cellular pellet was resuspended in 1 mL com-
plete DMEM-F12. Cells were gently mixed at a 1:1 ratio
CSC Sorting with CD44þ/CD24- Markers
with trypan blue stain solution (Life Technologies) and cell
viability was evaluated using a TC10Ô Automated Cell MCF7 and SKOV3 cell lines were seeded in 75 cm2 culture
Counter (Biorad). The cytotoxic effect of each treatment flasks and incubated in complete medium to achieve 80%
were expressed as a percentage of cell viability relative to confluence. Cells were then harvested with a 0.5%
control cells, which were treated with the vehicle of either trypsin-1% EDTA solution and detached cells were washed
rTsCRT (Tris 25 mM pH 7.4) or 5-FU (ethanol at final vol- twice with PBS and resuspended in 1 mL wash buffer
ume of 0.1%) at the same concentration as the higher dose (PBS þ 3% FBS). Cells (5  104) were incubated with
for each treatment. The data of the dose-response curves at fluorophore-conjugated monoclonal antibodies against hu-
different concentrations per compound were plotted using man CD44-FITC (BD Biosciences, 555742) and human
the SigmaPlot software 10.0 to generate the inhibitory con- CD24-PE (BioLegend, Inc, 311106) or against their respec-
centrations (IC)20e50. The IC20eIC50 values for rTsCRT or tive isotype controls (FITC-IgG2bk, BD Biosciences,
5-FU represent the concentration of each compound that 555742; PE-IgG2ak, BD Biosciences, 555574). Antibodies
achieved 20e50% growth inhibition as compared to the were used at concentrations of 2 mg/1  106 cells and were
control. incubated at 4 C in the dark for 40 min, with two posterior
68 Schcolnik-Cabrera et al./ Archives of Medical Research 51 (2020) 65e75

additional washing steps. The cells were then sorted with a Dunn’s post hoc test. A p value of !0.05 was considered
BD FACSAria TM II (BD Biosciences) and evaluated using statistically significant.
the BD FACSDiva version 6.1.3 (BD Biosciences). CD44þ/
CD24‒ sorted cells, representing the CSC population from
both MCF7 and SKOV3 cell lines, were recovered and Results
seeded in low-adherence flasks under stem-cell conditions
during 15 d at 37 C in humidified atmosphere with 5% Calreticulin and 5-FU Induce Dose-dependent Cellular
CO2 for further viability assays. Viability Reduction in HeLa, MCF7, SW480 and SKOV3
cell Lines
Employment of the Scavenger Receptor Inhibitors We explored the anti-tumoral activity of rTsCRT on
Polyinosinic Acid and Fucoidan in Parental Cells and different cancer cell lines. First, we screened 6 human
CSC Cultures tumorigenic cell lines, MDA-MB-231 and MCF7 of breast,
Parental and CSC forms of MCF7 and SKOV3 were SKOV3 of ovarian, HeLa of cervix, SW480 of colon and
cultured as stated before. Cells were pre-treated for PC3 of prostate adenocarcinomas. We used increasing
30 min with either polyinosinic acid potassium salt (poly- doses of rTsCRT during 72 h and found that MDA-MB-
I, 10 mg/mL, Sigma-Aldrich, MO, USA) or fucoidan 231 and PC3 cell lines were resistant to rTsCRT treatment,
(150 mg/mL, Sigma-Aldrich, MO, USA), according to as the cellular viability was only slightly diminished even at
(34,35), alone or in combination with the IC50 of rTsCRT. the highest dose (data not show). We found that SKOV3
When rTsCRT was used, it was added right away after was the most sensitive cell line to rTsCRT treatment, fol-
the incubation period of 30 min into the cell culture me- lowed by the MCF7 cell line (Figure 1A). Next, we evalu-
dium, for treatment during a 24 h period. This procedure ated the viability response to the chemotherapeutic drug
was repeated thrice with replacement of fresh complete me- 5-FU on the same four malignant cell lines and observed
dium in each occasion for a final period of 72 h. The inhib- a dose-dependent viability response to this treatment. Inter-
itory effect of both compounds on rTsCRT activity was estingly, both SKOV3 and MCF7 were the most resistant
evaluated as the percentage of cell viability relative to con- cell lines to 5-FU (Figure 1B). Table 1 shows the IC20e50
trol cells by employing trypan blue, as described above. values for rTsCRT (A) and 5-FU (B) obtained from the sur-
vival curves. Due to the higher sensitivity of SKOV3 to
rTsCRT and resistance to 5-FU, this cell line was selected
Confocal Microscopy Evaluation of MCF7 and SKOV3
to evaluate the combinatory effect of rTsCRT with 5-FU
Parental Cells
and to analyze a possible synergistic role using the different
MCF7 or SKOV3 parental cells (1  104) were seeded in IC values obtained from SigmaPlot. The data were intro-
culture slides (BD Falcon, MA, USA) and allowed to attach duced in the CalcuSyn software to determine rTsCRT-5-
to the slides for 24 h. Cells were treated for an additional FU interactions and the combination index values were
24 h as follows: control (rTsCRT vehicle), rTsCRT, fucoi- generated. All the evaluated combinations demonstrated
dan, poly-I, fucoidan þ rTsCRT, or poly-I þ rTsCRT. Then, pharmacological interactions around the nearly additive
cells were fixed with cold acetone (J.T. Baker, 9006-03) range (0.9e1.2), and no combination followed a clearly
during 5 min and were washed twice with sterile PBS. Next, synergistic or antagonistic interaction (data not shown).
cells were dyed with a solution of 5 mL Hoescht stain (Mo- Since SKOV3 and MCF7 were the most sensitive cell lines
lecular Probes, Life Technologies) in 5 mL PBS during to rTsCRT and the most resistant to 5-FU, we decided to
7 min and two additional washing steps with PBS were per- use them for further analysis.
formed. Slides were mounted with Vectashield (Vector Lab)
and images were obtained with a Nikon Ti Eclipse inverted The Combination of rTsCRT and 5-FU Reduces Cell
confocal microscope equipped with an A1 imaging system Viability and Clonogenic Potential in Both MCF7 and
(both controlled by the proprietary software NIS Elements SKOV3 Cell Lines
v.4.5.0). The images were acquired and analyzed using NIS
elements V.4.50. We compared the lowest (IC20/IC20) and the highest (IC50/
IC50) dose combinations of rTsCRT and 5-FU in both
MCF7 and SKOV3 cell lines. Results show that the IC20/
Statistical Analysis
IC20 and the IC50/IC50 doses significantly reduced the
Three independent experiments were performed in tripli- cellular viability of treated SKOV3 and MCF7 parental cell
cate and data were expressed as means  SD. Data were lines. Clonogenic potential was inhibited by both drug com-
analyzed by using GraphPad Prism V6 software (GraphPad binations in SKOV3, but in MCF7 only the IC50/IC50 com-
Software Inc., La Jolla, CA, USA). Significant differences bination showed a significant effect. Cellular viability of
among the test groups were determined using the the MCF10-A cell line used as control of an immortalized
Kruskal-Wallis one-way analysis of variance followed by non-tumorigenic human cell line, was only moderately
 Taenia solium Calreticulin as an Anticancer Agent 69

A rTsCRT B 5-FU
*** *** **** ****
* ** ** **** Control
*** **** *** ** ** ** Control
*** 0.1 μM *** ** 0.1 μM
100 0.3 μM 100
1 μM
0.5 μM 3 μM
75 75
% Viability

% Viabiity
0.7 μM
1 μM
50 50

25 25

0 0
0

0
V3

V3
a

F7

F7
48

48
eL

eL
C

C
O

O
SW

SW
H

H
M

M
SK

SK
Figure 1. Inhibitory effect of rTsCRT and 5-FU in different cancer cell lines. Effect of rTsCRT treatment (0.1, 0.3, 0.5, 0.7 and 1 mM) on cellular viability of
HeLa, MCF7, SW480 and SKOV3 cell lines (A). Effect of 5-FU treatment (0.1, 1 and 3 mM) on cell viability of HeLa, MCF7, SW480 and SKOV3 cell lines
(B). All cell lines were treated for 72 h. Data represent results from triplicate cultures of 3 independent experiments. Kruskal-Wallis: *p !0.05; **p !0.01;
***p !0.001; ****p !0.0001.

affected when the IC50/IC50 concentration was used, but not effect on colony formation for both combinations are shown
with the IC20/IC20 dose, suggesting that non-tumorigenic in Figure 3C and F.
cells are resistant to the same compound concentrations
(Figure 2A and B).
The Blockade of Scavenger Receptors Prevents rTsCRT-
mediated Antitumor Effects on Epithelial and CSC-
enriched Populations of MCF7 and SKOV3 Cell Lines
The Combination of rTsCRT and 5-FU Reduces Cellular In order to figure out whether the anti-tumoral effect of
Viability and Colony Formation Ability in CSC-enriched rTsCRT is mediated by the scavenger receptor family
Populations (SRF), we employed the scavenger receptor ligands fucoi-
Since it has been reported that both MCF7 and SKOV3 dan or poly-I on both the epithelial and CSC-enriched pop-
have CD44þ/CD24‒ subpopulations that show CSC proper- ulations of MCF7 and SKOV3. We found that pre-treatment
ties, known as mammospheres and ovospheres, respectively with either fucoidan or poly-I of MCF7 and SKOV3
(3,36), we decided to test the effect of the IC20/20 and IC50/ parental cells inhibits the anti-tumoral effects of rTsCRT,
þ ‒ as compared to cells treated with rTsCRT alone. A slight
50 doses on these cells. We sorted the CD44 /CD24 pop-
ulation, and treated the CSC-enriched population with the reduction in cellular viability was observed with fucoidan
compound combinations during 72 h to evaluate the per- and poly-I treatment that was not significant when
centage of cell viability and the number of colonies formed compared to the control (Figure 4A and B). For the
in both cell lines. In mammospheres, regarding cell MCF7 cell line, the effect of the pre-incubation with
viability, treatment with the IC20/IC20 and IC50/IC50 doses poly-I or fucoidan on cellular viability was similar to that
resulted in a significant reduction of sphere viability as observed when rTsCRT was added after the addition of
compared to the control. Furthermore, a significant differ- the SRF inhibitors (Figure 4A). However, fucoidan did
ence between the IC20/IC20 and IC50/IC50 combinations
was seen; mammospheres treated with the IC50/IC50 doses Table 1. rTsCRT (A) and 5-FU (B) inhibitory concentrations (IC)20e50
exhibited a O75% reduction in viability (Figure 3A). In in HeLa, MCF7, SW480 and SKOV3 cell lines
addition, we observed a significant reduction in the number
A. Cell line IC20 (mM) IC30 (mM) IC40 (mM) IC50 (mM)
of colonies formed as compared to the control with both
drug combinations (Figure 3B). In ovospheres, there was HeLa 0.05 0.16 0.35 0.65
a significant decrease in the cellular viability and in the MCF7 0.14 0.25 0.39 0.58
number of colonies formed with the employment of both SW480 0.23 0.36 0.49 0.63
SKOV3 0.23 0.32 0.42 0.51
combinations, as compared to the respective controls. There
was no significant difference between the IC20/IC20 and B. Cell line IC20 (mM) IC30 (mM) IC40 (mM) IC50 (mM)
IC50/IC50 combinations. In contrast to mammospheres, in
ovospheres there was a higher effect on the number of HeLa 0.05 0.08 0.12 0.18
MCF7 0.14 0.45 0.98 1.77
generated colonies than on cellular viability (Figure 3D SW480 0.04 0.06 0.09 0.11
and E). Representative micrographs of the cell culture
70 Schcolnik-Cabrera et al./ Archives of Medical Research 51 (2020) 65e75

Figure 2. Combinatory effect of the IC20/IC20 and IC50/IC50 doses of rTsCRT and 5-FU on cell viability and clonogenicity of MCF10-A, MCF7 and SKOV3.
Effect of IC20/IC20 and IC50/IC50 combinations on cell viability of MCF10-A, MCF7 and SKOV3 cell lines (A). Effect of IC20/IC20 and IC50/IC50 combi-
nations on clonogenicity of MCF10-A, MCF7 and SKOV3 cell lines at the end of the 14 d period of the clonogenic assays (B). Data represent results from
triplicate cultures of 3 independent experiments. Kruskal-Wallis: *p !0.05; **p !0.01; ****p !0.0001.

not fully inhibit the anti-tumoral effect of rTsCRT in the least partially, by the SRF. However, we did not observe a
SKOV3 cell line (Figure 4B). Interestingly, in the SKOV3 synergistic effect of 5-FU and rTsCRT.
cell line, treatment with rTsCRT resulted in an apparent 5-FU is a widely used chemotherapeutic agent that
loss of the cell-to-cell interactions observed in the control; mostly acts on dividing cells at the S phase, where it inserts
the cells became round and showed decreased spreading altered uracil residues into newly formed RNA, blocking
(Figure 4C). splicing and translation of the sequence. The effect is regu-
Pre-treatment of mammospheres with poly-I or fucoidan lated by the integrity of sequence repairing mechanisms of
inhibited the anti-tumoral effect of rTsCRT on cell the cell (9). 5-FU incorporation into RNA also results in
viability; a slight increase in the number of cells was loss of clonogenic survival of MCF7 cells (37). Due to
observed with pre-treatment with poly-I in the presence the potential cardiotoxicity and low response rate, 5-FU is
of rTsCRT. Fucoidan or poly-I treatment showed a slight usually used in combination with other anti-tumoral drugs.
decrease in cell viability that was not statistically signifi- Fucoidan, that acts through the SRF, has been used in com-
cant (Figure 5A). However, treatment with fucoidan and bination with 5-FU. A reduced toxicity of the chemo-
rTsCRT, as well as with fucoidan alone, but not with therapy and longer duration of clinical effectiveness in
poly-I, resulted in a higher reduction in colony formation cancer patients was reported (38). Thus, we decided to
ability as compared to rTsCRT (Figure 5B). Figure 5C analyze the combinatorial effect of 5-FU and rTsCRT, since
shows representative micrographs of the different treat- CRT from different species has been shown to act through
ments on colony formation. On the other hand, treatment the SRF. Our findings show that rTsCRT had no synergistic
of ovospheres with both inhibitors prior to the addition of anti-tumoral effect in combination with 5-FU on reducing
rTsCRT, resulted in the inhibition of rTsCRT anti-tumoral cell viability in the two cell lines studied. However, treat-
effect, both on cell viability and colony formation potential. ment with IC50/IC50 of rTsCRT þ 5-FU had a nearly addi-
A slight increase in the number of cells and colonies was tive effect that killed 60% of the MCF7 and SKOV3 cells
observed with pre-treatment with fucoidan and poly-I in and reduced the proliferative capacity of the surviving pop-
the presence of rTsCRT, which was not statistically signif- ulation, as evidenced by the clonogenic assays. Studies
icant when compared to the control (Figure 5De5F). involving treatment with 5-FU alone or in combination
have yielded contradictory results both in vivo and
in vitro (8,39e41). Results depend on the particular type
and stage of cancer, whether the study was performed
Discussion
in vitro or in vivo, as well as on the demonstrated sensitivity
In this work we demonstrated that rTsCRT, the recombinant or chemoresistance of the cell line or tumor.
CRT from the helminth Taenia solium, has a concentration- CSC have gained attention in recent years. Moreover,
dependent anti-tumoral effect on cancer cell lines from cer- in vitro treatment of CSC has been proposed as a promising
vix (HeLa), ovarian (SKOV3), breast (MCF7) and colon strategy for anti-neoplastic drug treatment (4).
(SW480) in vitro. SKOV3 was the most sensitive cell line, CD44þCD24‒ CSC cells have the ability to self-renew
followed by MCF7. rTsCRT treatment resulted in reduced and form new tumors with a relatively low quantity of cells,
viability and clonogenic capacity of MCF7 and SKOV3 and are thought to be responsible for metastasis and relapse
parental cells and affected both viability and colony forma- (3). A common cause of relapse in cancer therapy is drug
tion ability of CSC obtained from the same cell lines. More- resistance. Recent studies demonstrate that CSC are at least
over, we showed that the anti-tumor effect was mediated, at partly responsible for this phenomenon, since these cells
 Taenia solium Calreticulin as an Anticancer Agent 71

Figure 3. Effect of the IC20/IC20 and IC50/IC50 doses of rTsCRT and 5-FU on CSC from MCF7 and SKOV3 cell lines. Effect of the compound combinations
on cellular viability (A) and on colony formation (B) of mammospheres. Effect of the same compound combinations on cellular viability (D) and on colony
formation (E) of ovospheres. Representative micrographs of mammospheres (C) and ovospheres (F). For both C and F, cells were treated with either control
media (a), IC20/IC20 (b), or IC50/IC50 (c) doses. Data represent results from triplicate cultures of 3 independent experiments. Kruskal-Wallis: *p !0.05;
**p !0.01; ***p !0.001; ****p !0.0001.

suffer a process of dedifferentiation which alters genes was upregulated in breast cancer samples compared to
implicated in nucleic acid repairing processes, thus modi- non-cancerous breast tissue and adjacent normal tissue. In
fying the effects of antimetabolites such as 5-FU (42,43). addition, they used the MCF7 cell line with a transfected
5-FU treatment of the breast cancer cell line MDA-MB- form of the SRB-1 (without the intracellular domain),
460 during 6 generations changed the quantity and colony which inhibited proliferation compared to control cells
formation capacity of CSC, and resulted in fluctuations in (46). Moreover, knockdown or pharmacological inhibition
the expression of drug resistance and anti-apoptotic genes of SRB-1 reduced cellular proliferation and cell migration
(44). Our studies using the IC20/IC20 and IC50/IC50 of in vitro and reduced tumor growth in vivo, suggesting that
rTsCRT and 5-FU, resulted in a decrease in the viability SRB-1 is involved in promoting breast cancer metastasis
and colony formation capacity of mammospheres and ovo- and proliferation (47,48). Since CRT from different species
spheres. The combination with rTsCRT could help prevent has been shown to bind to the SRF, and because such bind-
the drug resistance effects observed during treatment with ing is inhibited by SRF ligands such as fucoidan, carra-
5-FU. Thus, the analysis of the effect of rTsCRT in geenan and poly-I (24,25), we hypothesized that the anti-
conjunction with 5-FU on drug resistance and anti- tumoral effect of rTsCRT is SRF-mediated. We showed that
apoptotic gene expression should be addressed, as well as the anti-tumoral effect of rTsCRT in MCF7 and SKOV3
the capacity of rTsCRT to reduce the required doses for parental cell lines, as well as mammospheres and ovo-
chemotherapy by 5-FU or other drugs to help dampen spheres, is abrogated by fucoidan and poly-I, suggesting
possible cytotoxic effects. that rTsCRT acts through the SRF.
The involvement of scavenger receptors in the anti- Fucoidan and poly-I alone did not affect cellular
tumoral effect of rTsCRT was evaluated using fucoidan viability of the parental cell lines MCF7 and SKOV3, as
and poly-I, which are competitive polyanionic ligands of well as of mammospheres. On the other hand, poly-I
the SRF. Although most studies on scavenger receptors reduced the viability of ovospheres, with no significant ef-
have focused on macrophages, these receptors are also ex- fect by fucoidan. This effect could be explained by the
pressed in smooth muscle, endothelial and epithelial cells. different binding specificities and affinities of the SRF li-
Different members of the SRF have been implicated as reg- gands. Poly-I is a single-stranded RNA homologue that
ulators of tumor development, progression and immunomo- forms a quadruplex structure that interacts with the collag-
dulation in response to cancer (27e29,45). For example, enous domain, whereas fucoidan is a sulfated fucose poly-
SR class B member 1 (SRB-1) has been reported in saccharide obtained from different species of brown algae
different cancer cells. Cao et al. demonstrated that SRB-1 (49e53).
72 Schcolnik-Cabrera et al./ Archives of Medical Research 51 (2020) 65e75

Figure 4. Fucoidan and poly-I protect against rTsCRT-induced reduction in cellular viability in parental forms of MCF7 and SKOV3 cell lines. Cellular
viability of the parental forms of either MCF7 (A) and SKOV3 (B) was evaluated after the addition of control media, rTsCRT, fucoidan, poly-I,
fucoidan þ rTsCRT or poly-I þ rTsCRT. Representative confocal micrographs showing the morphological changes in SKOV3 (C) after the treatment with
control media (a), rTsCRT (b), fucoidan þ rTsCRT (c) or poly-I þ rTsCRT (d). Fucoidan (fuco). Data represent results from triplicate cultures of 3 inde-
pendent experiments. Kruskal-Wallis: *p !0.05; ***p !0.001; ****p !0.0001.

Fucoidans have been shown to have different biological vesiculosus differ in their ability to inhibit CYP450. In
functions, including anti-viral, anti-oxidant, anti-coagulant the same study, a limited growth inhibitory activity by both
and anti-tumoral activities (54). Diverse SRF ligands fucoidans was observed in 8 cell lines, including MCF7 and
induce differential effects on the target cell. For example, SKOV3 (55). These data are in agreement with our results
fucoidan induces the pro-inflammatory cytokines TNF-a showing that fucoidan from Fucus vesiculosus alone did not
and IL-1, whereas OxLDL, another SRF ligand, only in- affect cellular viability in parental cell lines, as well as
duces TNF-a but no IL-1 production, activating different mammospheres and ovospheres. However, reports in the
signaling pathways (53). Furthermore, fucoidan, as well literature are controversial. Banafa et al. (56) showed that
as other polysaccharides, show different bioactivities de- fucoidan induces apoptosis and cell arrest, but the species
pending on the biopharmaceutical and biochemical struc- from which the fucoidan was extracted was not specified.
tures that depend on the species, extraction and The differences in the treatment outcome could also be
purification methods, as well as the tumor type (54). For due to the lower fucoidan concentration used in our studies.
example, fucoidans from Undaria pinnatifida and Fucus Thus, reaching conclusions on the relation between the
 Taenia solium Calreticulin as an Anticancer Agent 73

Figure 5. Fucoidan and poly-I protect against rTsCRT-induced reduction in cellular viability in CSC of MCF7 and SKOV3 cell lines. Cellular viability and
colony percentage of either mammospheres (A, B) and ovospheres (D, E) were evaluated after the addition of control media, rTsCRT, fucoidan, poly-I,
fucoidan þ rTsCRT, or poly-I þ rTsCRT. Representative micrographs showing the morphological changes on mammospheres (C) and ovospheres (F) after
the treatment with control media (a), rTsCRT (b), fucoidan (c), poly-I (d), fucoidan þ rTsCRT (e), or poly-I þ rTsCRT (f). Fucoidan (fuco). Data represent
results from triplicate cultures of 3 independent experiments. Kruskal-Wallis: *p !0.05; **p !0.01; ***p !0.001; ****p !0.0001.

specific bioactivities of the fucoidans from different sources SRF-induced disaggregation by fucoidan. Accordingly, a
and their structural characteristics have been difficult (57). recent study demonstrated the importance of the SRF-
Interestingly, while cell viability was not hampered after mediated endocytosis of functionalized silica particles in
the treatment with either fucoidan or in combination with mammospheres. Moreover, SRF in mammospheres are
rTsCRT, we found a significant reduction in the colony for- up-regulated as compared to their MCF7 more differenti-
mation capacity of mammospheres in the presence of fucoi- ated parental counterparts (34). Nevertheless, the specific
dan. These results are consistent with studies showing that pathways of the anti-tumoral effects of fucoidan have not
although fucoidan has anti-tumoral effects by inducing been fully characterized (60).
apoptosis in cancer cells in vivo, in vitro fucoidan alone The rTsCRT and fucoidan treatment changed the
did not cause apoptosis but inhibited colony formation morphology of the parental cancer cells, and this was
(58). Alternatively, fucoidan has been shown to decrease particularly evident in the SKOV3. Cell-cell interactions
the extracellular matrix-associated protein expression. Spe- were inhibited by rTsCRT treatment, and this effect was
cifically, it has been reported that fucoidan down-regulates abolished by the SRF ligands, suggesting the involvement
fibronectin, vimentin, a-SMA and the COL1A1 protein of rTsCRT in the adhesion properties of the ovarian cancer
levels on leiomyoma cells (59). Since some of these pro- cell line SKOV3. Accordingly, members of the SRF have
teins are important for intercellular junctions, their loss been shown to mediate cell adhesion in macrophages by
could be responsible for the decrease in the number and activating actin polymerization and formation of focal
size of mammosphere colonies. Further studies analyzing adhesion complexes; this effect is inhibited by fucoidan
the extracellular matrix protein levels in our model are (61,62). In addition, fucoidan binds to fibronectin, disrupt-
warranted. ing cell-cell interactions (63). Alternatively, rTsCRT could
The reduction in the colony formation capacity of mam- interact with thrombospondin-1 on the cancer cell surface
mospheres was not observed in the ovospheres. These re- and mediate focal adhesion disassembly, which is essential
sults suggest that mammospheres are more susceptible to for cell migration and tumor metastasis. Residues 19e36 in
74 Schcolnik-Cabrera et al./ Archives of Medical Research 51 (2020) 65e75

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Conflict of Interest an early stage of death modulates the clearance by macrophages of
The authors declare that they have no conflict of interest. apoptotic cells. Front Immunol 2017;8:1034.
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Acknowledgments 18. Pike SE, Yao L, Jones KD, et al. Vasostatin, a calreticulin fragment,
Alejandro Schcolnik-Cabrera is a student belonging to the Plan de inhibits angiogenesis and suppresses tumor growth. J Exp Med 1998;
Estudios Combinados en Medicina (PECEM), UNAM. Mandy 188:2349e2356.
19. Queiroz RM, Ricart CA, Machado MO, et al. Insight into the exopro-
Ju
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teome of the tissue-derived trypomastigote form of trypanosoma cru-
qu
ımicas, UNAM. Bernardo Oldak is a student belonging to the
zi. Front Chem 2016;4:42.
Maestr
ıa en Ciencias M
edicas at the Universidad An
ahuac M
exico 20. Guillou F, Roger E, Mone Y, et al. Excretory-secretory proteome of
Norte. We would like to thank Vadim P
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Laboratorio Nacional de Microscop
ıa Avanzada, CMN Siglo XXI, of Biomphalaria glabrata. Mol Biochem Parasitol 2007;155:45e56.
for his kind support with the use of the confocal microscope. 21. Mendlovic F, Cruz-Rivera M, Avila G, et al. Cytokine, antibody and
Funding: This work was supported by the Department of proliferative cellular responses elicited by Taenia solium calreticulin
Microbiology and Parasitology of the Faculty of Medicine at the upon experimental infection in hamsters. PLoS One 2015;10:e0121321.
Universidad Nacional Aut
onoma de M
exico (UNAM), M
exico. 22. Lopez NC, Valck C, Ramirez G, et al. Antiangiogenic and antitumor
effects of Trypanosoma cruzi Calreticulin. PLoS Negl Trop Dis 2010;
4:e730.
23. Ramirez-Toloza G, Abello P, Ferreira A. Is the antitumor property of
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