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Citation: Guillamón, E.; Mut-Salud, N.;
Rodríguez-Sojo, M.J.; Ruiz-Malagón,
A.J.; Cuberos-Escobar, A.; Martínez-
Férez, A.; Rodríguez-Nogales, A.;
Gálvez, J.; Baños, A. In Vitro
Antitumor and Anti-Inflammatory
Activities of Allium-Derived
Compounds Propyl Propane
Thiosulfonate (PTSO) and Propyl
Propane Thiosulfinate (PTS).
Nutrients 2023,15, 1363. https://
doi.org/10.3390/nu15061363
Academic Editor: Rosaria
Maddalena Ruggeri
Received: 3 February 2023
Revised: 7 March 2023
Accepted: 8 March 2023
Published: 11 March 2023
Copyright: © 2023 by the authors.
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nutrients
Article
In Vitro Antitumor and Anti-Inflammatory Activities of
Allium-Derived Compounds Propyl Propane Thiosulfonate
(PTSO) and Propyl Propane Thiosulfinate (PTS)
Enrique Guillamón1, Nuria Mut-Salud 1, María Jesús Rodríguez-Sojo 2, Antonio Jesús Ruiz-Malagón2,
Antonio Cuberos-Escobar 3, Antonio Martínez-Férez 4, Alba Rodríguez-Nogales 2, * , Julio Gálvez 2, 5, *
and Alberto Baños 1
1DMC Research Center, 18620 Granada, Spain
2
Department of Pharmacology, Center for Biomedical Research (CIBM), Instituto de Investigacion Biosanitaria
de Granada (ibs.GRANADA), University of Granada, 18071 Granada, Spain
3Primary Care, Andalusian Health Service District Malaga-Guadalhorce, 29004 Malaga, Spain
4
Chemical Engineering Department, University of Granada, Avenida Fuentenueva s/n, 18071 Granada, Spain
5CIBER de Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, 28029 Madrid, Spain
*Correspondence: albarn@ugr.es (A.R.-N.); jgalvez@ugr.es (J.G.)
Abstract:
Increasing rates of cancer incidence and the side-effects of current chemotherapeutic
treatments have led to the research on novel anticancer products based on dietary compounds.
The use of Allium metabolites and extracts has been proposed to reduce the proliferation of tumor
cells by several mechanisms. In this study, we have shown the
in vitro
anti-proliferative and anti-
inflammatory effect of two onion-derived metabolites propyl propane thiosulfinate (PTS) and propyl
propane thiosulfonate (PTSO) on several human tumor lines (MCF-7, T-84, A-549, HT-29, Panc-1,
Jurkat, PC-3, SW-837, and T1-73). We observed that this effect was related to their ability to induce
apoptosis regulated by oxidative stress. In addition, both compounds were also able to reduce the
levels of some pro-inflammatory cytokines, such as IL-8, IL-6, and IL-17. Therefore, PTS and PTSO
may have a promising role in cancer prevention and/or treatment.
Keywords: antitumor; anti-inflammatory; Allium; organosulfur compounds
1. Introduction
Despite the fact that some recent research has shown that certain risk factors can
ease the appearance of some types of cancer, the reasons why some people develop these
processes while others do not remain unknown. Among these risk factors, the exposure to
chemicals and radiation, age, genetics, lifestyle, or underlying chronic diseases, such as
inflammatory disorders, seem to have a prominent role, representing major signaling cues
driving the activation of cancer in humans [
1
]. After cardiovascular diseases, cancer remains
the second leading cause of death around the world, with 19.3 million new cases and almost
10.0 million deaths in 2020 [
2
]. This scenario is projected to be amplified drastically soon;
hence, continued searching for more effective chemoprevention and treatment therapies is
clearly needed to increase surveillance and to lower the treatment cost for cancer care.
Cancer is caused by the proliferation and progression of abnormal cells. Exposure to
carcinogens causes DNA damage and mutations at the cellular level due to the failure in
DNA repair mechanisms. The proliferation of the damaged cells causes inflammation of
the cells and tissues, finally leading to a tumor formation [
3
]. One of the mutual traits of
cancer is the quick formation of aberrant cells that expand beyond their normal borders,
affecting neighboring sections of the body and migrating to other tissues. This process is
known as metastasis and is the chief reason for death due to cancer [4].
The advent of modern drug therapies has undeniably improved cancer patients’ cares
and lives. However, advanced metastasized cancers remain untreatable, and conventional
Nutrients 2023,15, 1363. https://doi.org/10.3390/nu15061363 https://www.mdpi.com/journal/nutrients
Nutrients 2023,15, 1363 2 of 17
treatment methods, such as chemotherapy, radiation therapy, or immunotherapy, have
major side effects and toxicities [
5
]. Therefore, the identification and development of
novel anticancer products based on natural substances with fewer adverse effects have
gained attention in recent years [
6
]. For example, there is a growing interest in the use
of natural immunostimulants in combination with the common therapeutic modalities in
the treatment of cancer. The supplementation with these products pretends to improve
the immune response against tumors and reduce the suppression effect produced by the
chemotherapy [7].
The immune system exhibits the chief function in the defense against infected pathogens
and harmful antigens, including tumor cells [
8
]. Although the induction of an acute im-
mune response plays a cardinal aspect in the detection of and combating tumor cells, it is
widely described that the shifting into chronic inflammation, as in case of chronic inflam-
matory bowel diseases, may also increase the incidence of cancer generation due to the
excessive production of inflammatory mediators such as cytokines, reactive oxygen species
(ROS), and growth factors. Supporting this, it is well known that chronic inflammation
triggers different epigenetic mechanisms that shape the tumor microenvironment, affecting
the cell plasticity, differentiation, and polarization of immune cells, promoting the release
of ROS and cytokine production [
9
,
10
]. In fact, elevated levels of ROS in association with
an impaired redox balance are common features of cancer progression and resistance to
treatments [
11
]. In addition, ROS production causes DNA damage, which also contributes
to cancer development. It has been estimated that 25% of cancer-causing factors are related
to chronic inflammation [
3
]. Accordingly, the current targets of the treatments include
mediators-associated inflammatory pathways and/or oxidant-generating enzymes, as the
evaluation of different anti-inflammatory drugs in several clinical trials has revealed that
they can play dual roles in inflammation and tumorigenesis. However, some severe prob-
lems related to a long-term use of these drugs have been identified [
3
,
9
]. Consequently, the
search for natural compounds with antiproliferative and/or anti-inflammatory properties,
which provides a safe use profile, constitutes a line of work of great interest [
12
–
15
]. Further-
more, the potential synergism of conventional drugs with natural compounds introduces a
new aspect to fight cancer, which involves a promising approach to improve the effective-
ness of treatment while minimizing the adverse effects associated with chemotherapy [
16
].
A large number of studies point out that suitable dietary patterns may help to prevent
cancer or inhibit tumor development in cancer patients [
17
]. For instance, some plants are
rich in bioactive compounds that possess anticancer and immunomodulatory activities
with a low risk of cytotoxicity and side effects. These biologically active plant metabolites
are known as “phytochemicals” [
18
,
19
]. Although phytochemicals are not associated with
nutritional functions, they play a key role as responsible compounds for multiple health
benefits. Epidemiological studies, as well as
in vivo
,
in vitro
, and clinical trials, have shown
the ability of a variety of dietary compounds to reduce the risk of different chronic diseases,
including cancer. Many phytochemicals have been reported for their immunomodulatory
activities and their uses in treatment of and combating several types of cancer through
different mechanisms: the enhancement of the activity of the enzymes involved in the inac-
tivation of carcinogens, the suppression of the growth of cancer cells, or affecting metabolic
processes [
20
,
21
]. Some examples of phytochemicals with anticancer and immunostim-
ulant properties include curcumin from turmeric, epigallocatechin-3-gallate from green
tea, resveratrol from grapes, sulforaphane from broccoli, glucosinolates from cruciferous
vegetables, or gingerol from ginger [
22
]. Among these dietary substances, those derived
from alliaceous plants such as garlic, onion, or leek stand out. In general, vegetables
from the Allium genus contain different reputed bioactive molecules including flavonoids,
oligosaccharides, amino acids, selenium, and organosulfur compounds (OSCs) [18,19].
The beneficial properties of OSCs obtained from Allium spp., such as antimicrobial,
anti-inflammatory, antidiabetic, antioxidant, and immunomodulatory, among others, have
been broadly reported [
22
–
26
]. In addition, some of these compounds, mainly allylic
derivatives from garlic, have been described to display a direct antitumoral effect [
11
,
26
] or
Nutrients 2023,15, 1363 3 of 17
used as adjuvants in chemotherapy treatment, enhancing the activity of drugs or reducing
its side effects [
27
–
29
]. For instance, the anticancer potential of allicin (e.g., diallyl thiosul-
finate), the most important OSC from garlic, has been recently reviewed [
30
], revealing
that it suppresses the growth of different types of tumors. However, this compound is
very unstable, and even at optimal processing and storage conditions, easily lead to the
spontaneous decomposition of secondary OSCs such as diallyl disulfide (DADS). Sev-
eral experimental studies have demonstrated that DADS also exhibits anti-tumor activity
against many lines of tumor cells, including hematological cancers (leukemia, lymphoma),
lung cancer, prostate cancer, or colorectal cancer (CRC) [
31
]. However, DADS has caused
appreciable allergic reactions and toxicity, affecting normal cells too. Thus, the use of these
compounds in the prevention and treatment of cancer is limited presently [32].
In contrast, despite the fact that epidemiological studies have confirmed that regular
onion consumption reduces the incidence of various forms of cancer as well as other
diseases associated with oxidative stress [
33
], much less is known about the biological
activities of OSCs derived from onion. In particular, propyl propane thiosulfinate (PTS),
the saturated analogue to allicin, and its oxidized derivative propyl propane thiosulfonate
(PTSO) (Figure 1), also present antimicrobial and immunomodulatory activities [
34
,
35
],
but they are highlighted for their higher stability compared to the OSCs derived from
garlic. Therefore, as a first contribution to deep in the potential of PTS and PTSO in
the chemotherapy treatment of cancer, the present study aims to characterize the
in vitro
antiproliferative and anti-inflammatory properties of both compounds and analyze some
of their mechanisms of action.
Nutrients 2023, 15, x FOR PEER REVIEW 3 of 19
substances, those derived from alliaceous plants such as garlic, onion, or leek stand out.
In general, vegetables from the Allium genus contain different reputed bioactive molecules
including flavonoids, oligosaccharides, amino acids, selenium, and organosulfur com-
pounds (OSCs) [18,19].
The beneficial properties of OSCs obtained from Allium spp., such as antimicrobial,
anti-inflammatory, antidiabetic, antioxidant, and immunomodulatory, among others,
have been broadly reported [22–26]. In addition, some of these compounds, mainly allylic
derivatives from garlic, have been described to display a direct antitumoral effect [11,26]
or used as adjuvants in chemotherapy treatment, enhancing the activity of drugs or re-
ducing its side effects [27–29]. For instance, the anticancer potential of allicin (e.g., diallyl
thiosulfinate), the most important OSC from garlic, has been recently reviewed [30], re-
vealing that it suppresses the growth of different types of tumors. However, this com-
pound is very unstable, and even at optimal processing and storage conditions, easily lead
to the spontaneous decomposition of secondary OSCs such as diallyl disulfide (DADS).
Several experimental studies have demonstrated that DADS also exhibits anti-tumor ac-
tivity against many lines of tumor cells, including hematological cancers (leukemia, lym-
phoma), lung cancer, prostate cancer, or colorectal cancer (CRC) [31]. However, DADS
has caused appreciable allergic reactions and toxicity, affecting normal cells too. Thus, the
use of these compounds in the prevention and treatment of cancer is limited presently
[32].
In contrast, despite the fact that epidemiological studies have confirmed that regular
onion consumption reduces the incidence of various forms of cancer as well as other dis-
eases associated with oxidative stress [33], much less is known about the biological activ-
ities of OSCs derived from onion. In particular, propyl propane thiosulfinate (PTS), the
saturated analogue to allicin, and its oxidized derivative propyl propane thiosulfonate (PTSO)
(Figure 1), also present antimicrobial and immunomodulatory activities [34,35], but they
are highlighted for their higher stability compared to the OSCs derived from garlic. There-
fore, as a first contribution to deep in the potential of PTS and PTSO in the chemotherapy
treatment of cancer, the present study aims to characterize the in vitro antiproliferative
and anti-inflammatory properties of both compounds and analyze some of their mecha-
nisms of action.
Figure 1. Chemical structure of PTS and PTSO.
2. Materials and Methods
2.1. Chemicals and Reagents
PTSO and PTS (93.5% purity) were chemically isolated and provided by DOMCA
S.A.U. (Granada, Spain). Both compounds were previously dissolved in dimethyl sulfox-
ide (DMSO). From these solutions, the different dilutions to be tested were prepared using
Dulbecco’s Modified Eagle Medium (DMEM) without fetal bovine serum (FBS) or antibi-
otics. All reagents were purchased from Sigma-Aldrich Química S.L (Madrid, Spain), un-
less otherwise stated.
2.2. Cell Lines and Culture
MCF-7 (a human breast adenocarcinoma line; ECACC 86012803), T-84 (a human co-
lon carcinoma line; ECACC 88021101), A-549 (a human lung carcinoma line; ECACC
86012804), HT-29 (a human colon adenocarcinoma line; ECACC 91072201), Panc-1 (a hu-
man pancreatic cancer line; ECACC 87092802), Jurkat E6.1 (a human leukemia line;
Figure 1. Chemical structure of PTS and PTSO.
2. Materials and Methods
2.1. Chemicals and Reagents
PTSO and PTS (93.5% purity) were chemically isolated and provided by DOMCA
S.A.U. (Granada, Spain). Both compounds were previously dissolved in dimethyl sulfoxide
(DMSO). From these solutions, the different dilutions to be tested were prepared using Dul-
becco’s Modified Eagle Medium (DMEM) without fetal bovine serum (FBS) or antibiotics.
All reagents were purchased from Sigma-Aldrich Química S.L (Madrid, Spain), unless
otherwise stated.
2.2. Cell Lines and Culture
MCF-7 (a human breast adenocarcinoma line; ECACC 86012803), T-84 (a human colon
carcinoma line; ECACC 88021101), A-549 (a human lung carcinoma line; ECACC 86012804),
HT-29 (a human colon adenocarcinoma line; ECACC 91072201), Panc-1 (a human pancreatic
cancer line; ECACC 87092802), Jurkat E6.1 (a human leukemia line; ECACC 88042803), PC-3
(a human prostate adenocarcinoma line; ECACC 90112714), and SW-837 (a human rectum
adenocarcinoma line; ECACC 91031104) were obtained from the Cell Cultures Unit of the
University of Granada (Spain). T1-73 (a human osteosarcoma line; CRL-7943) and hMSCs
(human adipose-derived multipotent mesenchymal cells; PCS-500-01) were supplied by the
American Type Culture Collection (ATCC). PBMCs (peripheral blood mononuclear cells)
were obtained from blood samples of healthy volunteers and provided by the biobank of
“Sistema Sanitario Público de Andalucía (SPPA)”. All cell lines were cultured in darkness
at 37
◦
C, with a humidified atmosphere of 5% CO
2
, using DMEM supplemented with 10%
FBS, 10 mL/L of penicillin–streptomycin 100
×
, and 2 mM of L-glutamine, except hMSCs
Nutrients 2023,15, 1363 4 of 17
that were supplemented with 20% FBS and without antibiotics, and PBMCs that were
cultured with RPMI-1460 medium supplemented with 10% FBS.
2.3. In Vitro Antiproliferative Assays
In order to calculate the half-maximal inhibitory concentration (IC
50
) values of PTS
and PTSO, adherent cells (MCF-7, T-84, A-549, HT-29, Panc-1, SW-837, PC-3, and T1-73)
were seeded in sterile 96-well plates (Thermo Fisher Scientific, Denmark) at a high density
(1.4
×
10
4
cells/well) and incubated at 37
◦
C with 5% CO
2
for 24 h to allow for cell
adhesion. In non-adherent cells (Jurkat and PBMCs), the induction was conducted directly.
Increasing concentrations of PTS and PTSO (1–250
µ
M) were added in the corresponding
wells and incubated for 72 h at 37
◦
C with 5% CO
2
. The effect of both compounds on
adherent cell lines was evaluated using a colorimetric technique with Sulforhodamine-B
(SRB) [
36
]. Non-adherent cell lines were quantified by the MTT assay [
37
]. The optical
density values of adherent and non-adherent cells were determined by colorimetry at
490 nm using a microplate reader (Multiskan EX, Thermo Electron Corporation). The
assessment of absorbance was obtained using the SkanIt RE 5.0 for Windows v.2.6 (Thermo
Labsystems, USA) and a regression analysis for each cell line using Statgraphics 18 software
(Statistical Graphics Corp, 2000, Warrenton, VA, USA) was conducted. The IC
50
values
were calculated from the semi-logarithmic dose–response curve by linear interpolation.
Finally, the therapeutic index (TI) of each compound was determined to determine the
margin of safety of PTS and PTSO when used as an antiproliferative. TI was calculated by
establishing the ratio between the IC
50
values obtained in non-tumoral cells and in a tumor
cell line. For each cell line, the assays were performed in duplicate.
2.4. Oxidative Stress Assays
MCF-7 and T-84 cells were seeded in 96 well-plates at a high density in sextuplicate.
At 24 h, the cells were induced with increasing concentrations of PTS and PTSO, with
or without 5mM of NAC (N-Acetyl-Cystein) for 1 h pre-induction. After 72 h, the cell
viability was evaluated using the SRB method. The IC
50
values were calculated from the
semilogarithmic dose–response curve by linear interpolation. Assays were performed in
duplicate.
The production of intracellular ROS was detected by fluorescence microscopy. MCF-7
cells were seeded at 5
×
10
3
cells/well for 24 h on a
µ
-Slide 8 well high glass bottom
(Ref.80807, Ibidi, Gräfelfing, Germany). Then, the cells were preincubated in the presence
or absence of 5mM of NAC for 1 h and treated with PTS or PTSO for 24 h at doses of IC50.
Subsequently, the cells were incubated with 2,7-dichlorofluorescein diacetate (DCFH-DA)
(10
µ
M) in darkness at 37
◦
C for 30 min. Fluorescent images were taken with a confocal
laser scanning microscope Confocal Leica TCS-SP5 (Leica, Munich, Germany) at 25
×
of
magnification and 1.5×of zoom.
2.5. Apoptosis Assays
The cell viability was determined by flow cytometry using the Annexin V-FITC kit
(Trevigen, Gaithersburg, MD, USA). MCF-7 and T-84 cells were seeded at a high density
(2 ×105cells/cm2)
in 6-well plates. After 24 h, the cells were induced with PTS and PTSO
for 48 h at the IC
50
concentration for each cell line. The cells were detached with the TrypLE
Express Enzyme (ThermoFisher Scientific, Waltham, MA, USA), washed with PBS, and
collected by centrifugation at 300
×
gfor 10 min. Then, the cells were washed again and
incubated with annexin-V FITC and propidium iodide (PI) in an annexin-V binding buffer
for 15 min. After incubation, the cells were diluted with the binding buffer and examined
immediately in a FACScan flow cytometer, using FlowJo (v.7.6.5, Tree Star, Inc., Ashland,
OR, USA). This assay was performed in duplicate.
Nutrients 2023,15, 1363 5 of 17
2.6. In Vitro Anti-Inflammatory Assays
The HT-29 and T-84 cells were seeded at a high density (1.4
×
10
4
cells/well) in 96-well
plates for 24 h. Then, the supernatants were discarded and the compound dissolved in the
supplemented medium was added. After 1 h of incubation with PTS or PTSO, 1
µ
g/mL of
lipopolysaccharide from Salmonella enterica serotype typhimurium (LPS) was added, and
the plates were incubated for 24 h at 37
◦
C and 5% CO
2
. All the concentrations tested were
performed in sextuplicate. After induction, the supernatants were collected, centrifuged at
1000
×
gfor 10 min, and stored at
−
80
◦
C. Finally, the IL-8, IL-6, and IL-17 determination
was carried out by an ELISA using cytokines commercial kits (Invitrogen-ThermoFisher
Scientific, Bethlehem, PA, USA). The assays were performed in duplicate.
2.7. Statistical Analysis
The results of absorbance in cytotoxicity assays and IC
50
were evaluated using an
analysis of variance (ANOVA), using the statistical software SPSS 11.5 (IBM, New York,
NY, USA). All the results were expressed as mean
±
standard deviations (SD). Figures
and statistical analysis for apoptosis assays and anti-inflammatory assays were generated
with GraphPad prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA) using a
one-way ANOVA test supplemented with Tukey’s post hoc. Differences were considered
statistically significant when p< 0.05. The relative fluorescence intensity was quantified
using the software Image J (v. 1.53t).
3. Results
3.1. In Vitro Antiproliferative Effects of PTS and PTSO
The antiproliferative activity of PTS and PTSO was evaluated in all the cell lines
described above. Both compounds inhibited cellular proliferation in a dose–response
manner with a different efficacy against the cell lines used (Table 1). The results revealed
that the IC
50
values from PTSO were higher than for PTS, except in MCF-7, Jurkat, SW-
837, and Panc-1 (Table 1). Therefore, these findings indicated a different but remarkable
antitumor effectiveness of PTS and PTSO (Table 1).
Table 1.
Antiproliferative activity of PTS and PTSO against MCF-7, T-84, A-549, HT-29, Panc-1, Jurkat,
SW-837, PC-3, T1-73, and PBMCs 72 h post-induction. Different letters differ significantly (p< 0.05).
Cell Line IC50 PTS (µM) IC50 PTSO (µM)
MCF-7 (human breast adenocarcinoma) 17.7 ±1.9 a6.9 ±0.7 b
T-84 (human colorectal carcinoma line) 18.2 ±2.2 a37.3 ±0.8 c
A-549 (human lung adenocarcinoma line) 10.4 ±1.2 d38.6 ±1.1 c
HT-29 (human grade II colorectal adenocarcinoma line) 15.6 ±2.5 a50.8 ±3.1 e
Panc-1 (human pancreatic carcinoma line) 34.5 ±3.7 c33.8 ±4.2 c
Jurkat (human tumor T lymphocytes line) 15.7 ±1.4 a10.6 ±1.3 d
SW-837 (human rectum adenocarcinoma tumor line) 150.8 ±2.4 f132.8 ±1.7 g
PC-3 (human prostate adenocarcinoma tumor line) 128.5 ±2.3 g198.7 ±3.5 h
T1-73 (human osteosarcoma tumor line) 76.4 ±3.2 i98.2 ±2.2 j
PBMCs (Peripheral blood mononuclear cells) 229.2 ±3.7 h,k 248.5 ±3.6 k
In order to determine the
in vitro
TI of PTS and PTSO, their effect by performing
cultures with PBMCs was studied under the conditions described in Section 2.2. In this cell
line, the results obtained show an IC
50
value of 229.2
µ
M for PTS and 248.5
µ
M for PTSO.
Therefore, the TIs for PTSO and PTS were 12.9 and 36, respectively, taking as reference the
MCF-7 line, and 14.6 and 23.4 considering the Jurkat line.
To confirm the harmlessness of PTS and PTSO in healthy cells, their effect was also
tested on hMSCs. It was found that both compounds hardly produced toxicity on these
cells as very high concentrations are required to affect cell viability (Figure 2). Moreover,
certain concentrations of these compounds even were able to induce the proliferation of
Nutrients 2023,15, 1363 6 of 17
hMSCs. Specifically, induction with 10
µ
M of PTS or PTSO increased the population by
17% and almost 11%, respectively, compared to the control. At concentrations greater than
20 µM, it was noted that PTSO was generally less harmful on hMSCs than PTS.
Nutrients 2023, 15, x FOR PEER REVIEW 6 of 19
PBMCs (Peripheral blood mononuclear cells) 229.2 ± 3.7
h,k
248.5 ± 3.6
k
In order to determine the in vitro TI of PTS and PTSO, their effect by performing
cultures with PBMCs was studied under the conditions described in Section 2.2. In this
cell line, the results obtained show an IC
50
value of 229.2 μM for PTS and 248.5 μM for
PTSO. Therefore, the TIs for PTSO and PTS were 12.9 and 36, respectively, taking as ref-
erence the MCF-7 line, and 14.6 and 23.4 considering the Jurkat line.
To confirm the harmlessness of PTS and PTSO in healthy cells, their effect was also
tested on hMSCs. It was found that both compounds hardly produced toxicity on these
cells as very high concentrations are required to affect cell viability (Figure 2). Moreover,
certain concentrations of these compounds even were able to induce the proliferation of
hMSCs. Specifically, induction with 10 μM of PTS or PTSO increased the population by
17% and almost 11%, respectively, compared to the control. At concentrations greater than
20 μM, it was noted that PTSO was generally less harmful on hMSCs than PTS.
Figure 2. Effects of increasing concentrations of PTS and PTSO on hMSCs viability three days post-
induction. The histogram depicts means ± SD of six determinations. * p < 0.05; ** p < 0.01; ns = non-
significant.
3.2. Oxidative Stress Assays
In order to determine if the mechanism of action of PTS and PTSO was related to ROS
production, their cytotoxicity was tested in MCF-7 and T-84 cells in the presence or ab-
sence of NAC 5 mM. In MCF-7 cells, the IC
50
of PTS and PTSO in the presence of NAC
barely affected the cell viability, reducing the population by 10% compared to controls
without NAC (Figure 3A). Thus, the presence of NAC decreased the anti-proliferative ef-
fect of both compounds, increasing by 40% the cell population. In T-84 cells, the IC
50
of
PTS and PTSO in the presence of NAC were able to reduce the population by 20% and
15%, respectively, increasing the cell viability by 33–34% compared to the controls without
NAC. From these results, it may be concluded that NAC protects tumor cells from the
activity of PTS and PTSO.
Figure 2.
Effects of increasing concentrations of PTS and PTSO on hMSCs viability three days
post-induction. The histogram depicts means
±
SD of six determinations. * p< 0.05; ** p< 0.01;
ns = non-significant.
3.2. Oxidative Stress Assays
In order to determine if the mechanism of action of PTS and PTSO was related to ROS
production, their cytotoxicity was tested in MCF-7 and T-84 cells in the presence or absence
of NAC 5 mM. In MCF-7 cells, the IC
50
of PTS and PTSO in the presence of NAC barely
affected the cell viability, reducing the population by 10% compared to controls without
NAC (Figure 3A). Thus, the presence of NAC decreased the anti-proliferative effect of both
compounds, increasing by 40% the cell population. In T-84 cells, the IC
50
of PTS and PTSO
in the presence of NAC were able to reduce the population by 20% and 15%, respectively,
increasing the cell viability by 33–34% compared to the controls without NAC. From these
results, it may be concluded that NAC protects tumor cells from the activity of PTS and
PTSO.
These results were also confirmed by the determination of intracellular ROS by a
confocal microscope using DCFH-DA in the MCF-7 line. Thus, the cells were treated with
PTS and PTSO at IC
50
concentrations, in the absence or presence of NAC 5 mM. As it
can be observed in Figure 3B. NAC incubation reduced the high fluorescence of tumor
control cells. Remarkably, the incubation for 24 h only with PTS or PTSO in MCF-7 cells
diminished considerably the cell population in the absence of the antioxidant, and so did
the fluorescence (Figure 3B). On the contrary, when MCF-7 cells were incubated with PTS or
PTSO and previously supplemented with NAC, the findings revealed a higher fluorescence
because the population had barely been affected (Figure 3B).
3.3. Study of Apoptosis
Cultures of MCF-7 and T-84 cells were incubated with the IC
50
concentrations of PTS
and PTSO for 48 h. The apoptosis induction was assessed by an annexin V FITC assay using
flow cytometry. Figure 4A shows the different apoptotic stages in both cell lines when
they were incubated with both compounds. Specifically, the percentage of the different
apoptotic stages was quantified, and the results showed that in MCF-7 cells, the fraction of
early apoptosis increased from 12.7% to 20.2% in cultures treated with PTS, and to 17.3%
with those treated with PTSO. The number of late apoptotic cells also increased in treated
cells, from 1.6% to 10.7% with PTS and to 5.9% with PTSO (Figure 4B). In T-84 cells, the
percentages of early and late apoptotic cells were also higher in treated cells compared to
the control for both compounds, although the induction of apoptosis was more evident
with PTS, increasing from 0.9% to 7.4% of early apoptotic and 0.3% to 2.8% of late apoptotic
cells (Figure 4B).
Nutrients 2023,15, 1363 7 of 17
Nutrients 2023, 15, x FOR PEER REVIEW 7 of 19
(A)
MCF-7 cells T-84 cells
(B)
Control
Control with NAC
PTS 20 μM
PTS 20 μM with NAC
PTSO 20 μM
PTSO 20 μM with NAC
Figure 3. Cont.
Nutrients 2023,15, 1363 8 of 17
Nutrients 2023, 15, x FOR PEER REVIEW 8 of 19
Figure 3. (A) Cytotoxicity of PTS and PTSO on MCF-7 and T-84 cells in absence or presence of NAC
5 mM. The concentration of PTS and PTSO was equal to IC50 and all cells were induced for 72 h. The
histogram depicts means ± SD of six determinations. **** = p < 0.0001. (B). Effect of PTS and PTSO
on ROS production detected by DCFH-DA staining (magnification 25× and zoom 1.5×) in MCF-7
cells. The relative fluorescence intensity was expressed as the mean ± SD. Different letter between
columns indicates significant differences at p < 0.05.
These results were also confirmed by the determination of intracellular ROS by a
confocal microscope using DCFH-DA in the MCF-7 line. Thus, the cells were treated with
PTS and PTSO at IC50 concentrations, in the absence or presence of NAC 5 mM. As it can
be observed in Figure 3B. NAC incubation reduced the high fluorescence of tumor control
cells. Remarkably, the incubation for 24 h only with PTS or PTSO in MCF-7 cells dimin-
ished considerably the cell population in the absence of the antioxidant, and so did the
fluorescence (Figure 3B). On the contrary, when MCF-7 cells were incubated with PTS or
PTSO and previously supplemented with NAC, the findings revealed a higher fluores-
cence because the population had barely been affected (Figure 3B).
3.3. Study of Apoptosis
Cultures of MCF-7 and T-84 cells were incubated with the IC50 concentrations of PTS
and PTSO for 48 h. The apoptosis induction was assessed by an annexin V FITC assay
using flow cytometry. Figure 4A shows the different apoptotic stages in both cell lines
when they were incubated with both compounds. Specifically, the percentage of the dif-
ferent apoptotic stages was quantified, and the results showed that in MCF-7 cells, the
fraction of early apoptosis increased from 12.7% to 20.2% in cultures treated with PTS, and
to 17.3% with those treated with PTSO. The number of late apoptotic cells also increased
in treated cells, from 1.6% to 10.7% with PTS and to 5.9% with PTSO (Figure 4B). In T-84
cells, the percentages of early and late apoptotic cells were also higher in treated cells
compared to the control for both compounds, although the induction of apoptosis was
more evident with PTS, increasing from 0.9% to 7.4% of early apoptotic and 0.3% to 2.8%
of late apoptotic cells (Figure 4B).
0
20
40
60
80
100
Fluorescence intensity/mm2
b
a
b
c
d
e
PTS PTSO-
NAC
PTS PTSO-
Figure 3.
(
A
) Cytotoxicity of PTS and PTSO on MCF-7 and T-84 cells in absence or presence of NAC
5 mM. The concentration of PTS and PTSO was equal to IC
50
and all cells were induced for 72 h. The
histogram depicts means
±
SD of six determinations. **** = p< 0.0001. (
B
). Effect of PTS and PTSO
on ROS production detected by DCFH-DA staining (magnification 25
×
and zoom 1.5
×
) in MCF-7
cells. The relative fluorescence intensity was expressed as the mean
±
SD. Different letter between
columns indicates significant differences at p< 0.05.
Nutrients 2023, 15, x FOR PEER REVIEW 9 of 19
(A)
(B)
(C)
Figure 4. Analysis of apoptosis in MCF-7 (A) and T-84 (B) cell viability with and without PTS or
PTSO for 48 h, revealed with annexin V-FITC/PI staining by cytometry. The graphics show the per-
centages of early apoptotic, late apoptotic, and necrotic cells after induction with PTS and PTSO at
a concentration of IC50 in MCF-7 cells. (C) Histograms of MCF-7 and T-84 cells. Control = untreated
control cells
.
The experiment was repeated independently three times yielding similar results. **** =
p < 0.0001; *** = p < 0.001.
Figure 4. Cont.
Nutrients 2023,15, 1363 9 of 17
Nutrients 2023, 15, x FOR PEER REVIEW 9 of 19
(A)
(B)
(C)
Figure 4. Analysis of apoptosis in MCF-7 (A) and T-84 (B) cell viability with and without PTS or
PTSO for 48 h, revealed with annexin V-FITC/PI staining by cytometry. The graphics show the per-
centages of early apoptotic, late apoptotic, and necrotic cells after induction with PTS and PTSO at
a concentration of IC50 in MCF-7 cells. (C) Histograms of MCF-7 and T-84 cells. Control = untreated
control cells
.
The experiment was repeated independently three times yielding similar results. **** =
p < 0.0001; *** = p < 0.001.
Figure 4.
Analysis of apoptosis in MCF-7 (
A
) and T-84 (
B
) cell viability with and without PTS or
PTSO for 48 h, revealed with annexin V-FITC/PI staining by cytometry. The graphics show the
percentages of early apoptotic, late apoptotic, and necrotic cells after induction with PTS and PTSO
at a concentration of IC50 in MCF-7 cells. (
C
) Histograms of MCF-7 and T-84 cells. Control =
untreated control cells. The experiment was repeated independently three times yielding similar
results. **** = p< 0.0001; *** = p< 0.001.
3.4. Evaluation of Anti-Inflammatory Properties
To evaluate the anti-inflammatory properties of PTS and PTSO, the production of IL-8,
IL-6, and IL-17 was determined in HT-29 and T-84 cells after their incubation with LPS,
which has already been long demonstrated to be capable of eliciting responses associated
with inflammation
in vitro
, including the production of pro-inflammatory cytokines [
38
].
The concentrations of PTS and PTSO tested were selected considering the levels of cytotoxi-
city already determined in these lines considering their IC50. Both PTS and PTSO were able
to significantly inhibit the LPS-activated production of these cytokines (Figure 5). However,
no concentration–response relationship was observed since most of the concentrations
assayed showed a similar efficacy for both compounds with some exceptions: when the
production of IL-8 in HT-29 cells was considered, the most effective concentrations were 1
µ
M of PTS and 10
µ
M of PTSO. (Figure 5A); or when the production of IL-17 was consid-
ered in HT-29 and T-84 cells, both compounds showed more efficacy at the highest doses
assayed (10 µM and 25 µM) (Figure 5C).
Nutrients 2023, 15, x FOR PEER REVIEW 11 of 19
3.4. Evaluation of Anti-Inflammatory Properties
To evaluate the anti-inflammatory properties of PTS and PTSO, the production of IL-
8, IL-6, and IL-17 was determined in HT-29 and T-84 cells after their incubation with LPS,
which has already been long demonstrated to be capable of eliciting responses associated
with inflammation in vitro, including the production of pro-inflammatory cytokines [38].
The concentrations of PTS and PTSO tested were selected considering the levels of cyto-
toxicity already determined in these lines considering their IC50. Both PTS and PTSO were
able to significantly inhibit the LPS-activated production of these cytokines (Figure 5).
However, no concentration–response relationship was observed since most of the concen-
trations assayed showed a similar efficacy for both compounds with some exceptions:
when the production of IL-8 in HT-29 cells was considered, the most effective concentra-
tions were 1 μM of PTS and 10 μM of PTSO. (Figure 5A); or when the production of IL-17
was considered in HT-29 and T-84 cells, both compounds showed more efficacy at the
highest doses assayed (10 µM and 25 µM) (Figure 5C).
(A)
HT-29 cells
T-84 cells
(B)
HT-29 cells
T-84 cells
Figure 5. Cont.
Nutrients 2023,15, 1363 10 of 17
Nutrients 2023, 15, x FOR PEER REVIEW 11 of 19
3.4. Evaluation of Anti-Inflammatory Properties
To evaluate the anti-inflammatory properties of PTS and PTSO, the production of IL-
8, IL-6, and IL-17 was determined in HT-29 and T-84 cells after their incubation with LPS,
which has already been long demonstrated to be capable of eliciting responses associated
with inflammation in vitro, including the production of pro-inflammatory cytokines [38].
The concentrations of PTS and PTSO tested were selected considering the levels of cyto-
toxicity already determined in these lines considering their IC50. Both PTS and PTSO were
able to significantly inhibit the LPS-activated production of these cytokines (Figure 5).
However, no concentration–response relationship was observed since most of the concen-
trations assayed showed a similar efficacy for both compounds with some exceptions:
when the production of IL-8 in HT-29 cells was considered, the most effective concentra-
tions were 1 μM of PTS and 10 μM of PTSO. (Figure 5A); or when the production of IL-17
was considered in HT-29 and T-84 cells, both compounds showed more efficacy at the
highest doses assayed (10 µM and 25 µM) (Figure 5C).
(A)
HT-29 cells
T-84 cells
(B)
HT-29 cells
T-84 cells
Nutrients 2023, 15, x FOR PEER REVIEW 12 of 19
(C)
HT-29 cells
T-84 cells
Figure 5. Effect of PTS and PTSO on inflammatory status. (A) Impact of PTS and PTSO on IL-8
concentration in HT-29 cells (a) and T-84 cells (b) induced with LPS. (B) Effect of both treatments on
IL-6 concentration in HT-29 cells (a) and T-84 cells (b) induced with LPS. Cells were induced during
24 h with LPS (1 µg/mL) and increasing concentrations of PTS and PTSO (1–25 µM). (C) Evaluation
of PTS and PTSO on IL-17 concentration in HT-29 cells and T-84 cells induced with LPS. C = cells
not induced with LPS (control), C + LPS: cells induced with only LPS (1 µg/mL). Different letters
between columns indicate significant differences at p < 0.05.
4. Discussion
Treatment with the extracts or compounds derived from Allium has been the subject
of numerous studies and trials to establish a link with a reduced risk of cancer. In this
sense, our findings are in concordance with other assays previously published [39–41]. In
fact, several in vitro and in vivo studies have shown the potential antiproliferative activity
of the extracts or compounds derived from Allium in the same cell lines we have tested.
For instance, in one study conducted with quercetin from Allium cepa, this compound
showed cytotoxicity against MCF-7, HT-29, PC-3, and Jurkat cells [42]. The antitumor ca-
pacity of crude thiosulfinates from Allium tuberosum affected the viability of MCF-7 breast
tumor cells, with an IC50 of 155.1 μM in the case of S-methyl methanethiosulfonate and
51.1 μM for S-methyl 2-propene-1-thiosulfinate [43]. In another study conducted with 22
stabilized thiosulfinates derived from Allium vegetables, the IC50 of the compound with
the greatest anticancer activity in MCF-7 cells (S-4-methoxyphenyl 4-methoxybenzenesul-
finothioate) was 46.5 µM [20]. Despite the fact that in this article, PTS was synthesized to
carry out studies on the mechanism of action, the IC50 in the MCF-7 line was not reported
[20]. In our assays, both PTS and PTSO achieved lower IC50 values in MCF-7 (17.7 and 6.9
μM, respectively) than the mentioned compounds. Other Allium OSCs, whose IC50 values
at 72 h in MCF-7 have been reported, are allicin (10 μM) [44] and DADS (4.1 μM) [45],
showing an antiproliferative effect in this line similar or a little higher than the one ob-
tained in our assays with PTS and PTSO.
Some of the OSCs more commonly studied, as allicin, DADS, or diallyl trisulfide
(DATS), have also showed antitumor activity in colon cancer cell lines, including HT-29
[11,45–47]. A similar effect has been described for water-soluble garlic-derivatives, such
as S-allylmercaptocysteine. The effect of this compound on cell cycle progression and pro-
liferation was evaluated in colon cancer cell lines SW-480 and HT-29, achieving the growth
inhibition of both lines inducing apoptosis [48]. Our results showed that PTS and PTSO
exerted cytotoxicity in all of the colon tumor cells challenged, being especially remarkable
for PTS in T-84 and HT-29, with IC50 values below 20 μM (18.2 μM and 15.6 μM,
Figure 5.
Effect of PTS and PTSO on inflammatory status. (
A
) Impact of PTS and PTSO on IL-8
concentration in HT-29 cells (a) and T-84 cells (b) induced with LPS. (
B
) Effect of both treatments on
IL-6 concentration in HT-29 cells (a) and T-84 cells (b) induced with LPS. Cells were induced during
24 h with LPS (1
µ
g/mL) and increasing concentrations of PTS and PTSO (1–25
µ
M). (
C
) Evaluation
of PTS and PTSO on IL-17 concentration in HT-29 cells and T-84 cells induced with LPS. C = cells
not induced with LPS (control), C + LPS: cells induced with only LPS (1
µ
g/mL). Different letters
between columns indicate significant differences at p< 0.05.
4. Discussion
Treatment with the extracts or compounds derived from Allium has been the subject of
numerous studies and trials to establish a link with a reduced risk of cancer. In this sense,
our findings are in concordance with other assays previously published [
39
–
41
]. In fact,
several
in vitro
and
in vivo
studies have shown the potential antiproliferative activity of
the extracts or compounds derived from Allium in the same cell lines we have tested. For
instance, in one study conducted with quercetin from Allium cepa, this compound showed
cytotoxicity against MCF-7, HT-29, PC-3, and Jurkat cells [
42
]. The antitumor capacity of
crude thiosulfinates from Allium tuberosum affected the viability of MCF-7 breast tumor
cells, with an IC
50
of 155.1
µ
M in the case of S-methyl methanethiosulfonate and 51.1
µ
M
for S-methyl 2-propene-1-thiosulfinate [
43
]. In another study conducted with 22 stabilized
thiosulfinates derived from Allium vegetables, the IC
50
of the compound with the greatest
anticancer activity in MCF-7 cells (S-4-methoxyphenyl 4-methoxybenzenesulfinothioate)
was 46.5
µ
M [
20
]. Despite the fact that in this article, PTS was synthesized to carry out
Nutrients 2023,15, 1363 11 of 17
studies on the mechanism of action, the IC
50
in the MCF-7 line was not reported [
20
]. In
our assays, both PTS and PTSO achieved lower IC
50
values in MCF-7 (17.7 and 6.9
µ
M,
respectively) than the mentioned compounds. Other Allium OSCs, whose IC
50
values at
72 h in MCF-7 have been reported, are allicin (10
µ
M) [
44
] and DADS (4.1
µ
M) [
45
], showing
an antiproliferative effect in this line similar or a little higher than the one obtained in our
assays with PTS and PTSO.
Some of the OSCs more commonly studied, as allicin, DADS, or diallyl trisulfide
(DATS), have also showed antitumor activity in colon cancer cell lines, including HT-
29 [
11
,
45
–
47
]. A similar effect has been described for water-soluble garlic-derivatives,
such as S-allylmercaptocysteine. The effect of this compound on cell cycle progression
and proliferation was evaluated in colon cancer cell lines SW-480 and HT-29, achieving
the growth inhibition of both lines inducing apoptosis [
48
]. Our results showed that PTS
and PTSO exerted cytotoxicity in all of the colon tumor cells challenged, being especially
remarkable for PTS in T-84 and HT-29, with IC
50
values below 20
µ
M (18.2
µ
M and 15.6
µ
M,
respectively). Conversely, in SW-837 cells, the IC
50
of PTS was higher than for PTSO
(150.8 µM and 132.8 µM, respectively).
Regarding lung tumor cell lines, DADS and DATS have also shown antitumor activity
against A-549 by inducing apoptosis [
49
], though in this study, the IC
50
was not indicated.
In another
in vitro
assay, DADS (15–120
µ
M) was tested in AML HL-60 leukemic cells,
succeeding in suppressing cell growth [
50
]. These results are in accordance with those
obtained in our experiments since the IC
50
of PTS and PTSO against Jurkat cells were in
the same concentration range (10.6 and 15.7 µM, respectively).
In recent years, the use of blood cells from healthy volunteers has become a model
increasingly popular as a method to determine the toxicity of a compound, instead of
using established human cell lines [
51
]. In the assay to test the antiproliferative effect
of PTS and PTSO in PBMCs, high concentrations of both compounds were necessary to
affect the viability of these healthy cells (IC
50
> 200
µ
M). These concentrations lead to
high TIs. Another proof of the harmlessness of PTS and PTSO is the fact that certain
concentrations of these compounds could increase the population of hMSCs, as seen in
Figure 2. Consequently, our results are indicative of the large margin of safety of PTS and
PTSO and, therefore, of their potential to be tested in
in vivo
treatments against neoplastic
pathologies. These results in PBMCs are consistent with those obtained in previously
conducted
in vivo
assays, in which it was demonstrated that PTSO did not cause toxicity in
Sprague Dawley rats administered 55 mg PTSO/kg body weight/day for 90 days, without
showing liver damage, neither clinical signs nor mortality [27,52,53].
Oxidative stress can damage membrane lipids, proteins, and nuclear and mitochon-
drial DNA in cells. The assays related to oxidative stress revealed that populations of
MCF-7 and T-84 decreased significantly after treatment only with PTS or PTSO at IC
50
con-
centrations, compared to cells treated with the same concentrations but also pre-incubated
with NAC. This effect was more evident in MCF-7 cells, whose population increased around
40% compared to cells induced in the absence of NAC but only with both onion-derivative
compounds (Figure 3). These findings may be justified by the fact that NAC exerts an
antioxidant and protective effect against PTS and PTSO, which involved lower cell death.
In the assay performed with DCFH-DA, a ROS indicator, it was observed that MCF-7
cells incubated only with PTS or PTSO showed a lower fluorescence, which corresponds
to a lower cell density (Figure 4). As it is widely known, tumor cells have a high level
of oxidative stress compared to healthy cells, and this is related to an increase in ROS
production due to changes in their metabolism [
54
–
56
]. When MCF-7 cells were induced
with any of the compounds for 24 h but also preincubated with NAC, their viability was
hardly affected as their fluorescence increased. Therefore, this assay confirms the results
obtained in the proliferation assays conducted in the absence or presence of NAC (Figure 3).
It could be concluded, hence, that oxidative stress seems to be involved in the mechanism
of action of PTS and PTSO, as occurs with other known antitumor agents such as elesclomol
or paclitaxel, among others [57,58].
Nutrients 2023,15, 1363 12 of 17
Nevertheless, it must be considered that the cytotoxic activity of both compounds
could also be due to their pro-apoptotic action. Similar findings have been reported with
DADS in experiments conducted with A-549 and PC-3 cells [
59
], where the treatment
with NAC was able to block both the production of ROS (e.g., H
2
O
2
) and apoptosis.
Other authors have reported that DATS-induced apoptosis was associated with ROS
production in several of the lines tested in our trials, such as MCF-7 [
60
,
61
]. However,
there are other articles reporting that ROS generation appears to play only a secondary
role in the cytotoxicity of OSCs in tumor cells. For example, in an assay conducted with
esophageal cancer cells WHCO1 [
62
], it was observed that ROS was not the main cause of
cytotoxicity of garlic-related disulfides, although NAC was still able to interfere with the
assay. As oxidative stress and ROS levels seem to affect cancer development, personalized
treatments for patients should be addressed, considering the basal antioxidant status, type
of cancer, and mechanisms of action of drugs [
63
–
65
]. Moreover, various studies suggest
that the intake of supplements or foods with an antioxidant capacity may not be generally
recommended during chemotherapy treatments [66,67].
As previously stated, tumor cells used to be more sensitive to drugs that generate
large amounts of ROS, or that affect the ability of cells to eliminate them, which has
been associated with their death by apoptosis [
68
,
69
]. Apoptosis is the programmed cell
death characterized by a series of morphological events, including DNA fragmentation,
cell shrinkage, and the formation of membrane-bound apoptotic bodies that are rapidly
phagocytized by neighboring cells [
70
,
71
]. Our results revealed that there were significant
differences in the fraction of early and late apoptosis in MCF-7 and T-84 cells induced
with PTS and PTSO, indicating that both compounds would be able to induce apoptosis in
tumor cells.
According to the literature, there are studies that state that the regular intake of garlic
reduces neoplastic growth and tumor cells proliferation by inducing apoptosis [
72
,
73
]. Re-
garding, specifically, OSCs, it has been reported that DADS showed a significant induction
of apoptosis in a human gastric adenocarcinoma cell line [
74
], and allicin supplementation
induced death by apoptosis in several colon tumor cell lines, including HT-29 [
75
]. There-
fore, the way that PTS and PTSO exhibit their antitumor activity seems to be similar to
other OSCs. However, since the autophagy and apoptosis regulated by ROS are cellular
processes that can interact with each other [
76
], further studies would be necessary to
determine if autophagy is also involved in the mechanism of action of PTS and PTSO.
In summary, the reported IC
50
of PTS and PTSO in the tumor lines tested are in the
same range than those of the common OSCs whose antitumor effect has been proven.
Nevertheless, given their higher stability compared to substances such as allicin or DADS,
PTS and PTSO could be considered promising candidates to use in anticancer treatments,
alone or as adjuvants of chemotherapy drugs. This approach was described by Perez-
Ortiz et al., who co-administered a thiosulfinate-enriched garlic extract with 5-fluorouracil
(5-FU), achieving a greater effectiveness than standard chemotherapy with 5-FU and
oxaliplatin [
29
]. Similarly, other dietary compounds have also been used as adjuvants, such
as curcumin [77], epigallocatechin gallate (EGCG) [78], and lycopene [79].
In the anti-inflammatory assays, both PTS and PTSO were able to reduce the levels of
three pro-inflammatory cytokines usually involved in the development of cancer: IL-8, IL-6,
and IL-17. Some authors correlate IL-6 levels with tumor stage, the metastasis survival
rate, or apoptosis in various types of cancer, such as breast [
80
] or colon [
81
], while the
production of IL-8 has been linked to pro-tumorigenic roles which influence the tumor
microenvironment [
82
]. The IL-17 cytokine is widely recognized for its ability to modulate
the inflammatory response, contributing to the development of chronic inflammation [
83
],
and its level could increase in the serum and tissues of patients with CRC [
84
]. In fact,
in vivo
studies related to this type of cancer have shown that IL-17 plays an important role
in its prognosis and metastasis [
85
]. As previously stated, the production of this cytokine
and IL-8 was significantly reduced by PTS and PTSO in both cell lines, HT-29 and T-84.
However, compared to the control, the reduction of IL-6 was only achieved in HT-29 cells.
Nutrients 2023,15, 1363 13 of 17
Interestingly, most of the highest reductions in the production of pro-inflammatory
cytokines were obtained with the lowest concentrations of PTS and PTSO. Thus, these
OSCs would not act in a dose-dependent manner to exert their anti-inflammatory activity,
making their action dependent on the cancer cell line characteristics. In agreement with our
findings, PTS and PTSO have previously demonstrated their immunomodulatory effect
in several animal models. Concretely, PTSO was tested in two experimental models of
colitis, which were associated with the regulation of cytokines in inflamed colonic tissue,
leading to a reduction of pro-inflammatory cytokines IL-1
β
, TNF-
α
, and IL-6 [
86
]. In a
more recent work, PTSO showed its capacity of attenuating the obesity-associated systemic
inflammation, reducing the expression of the mentioned cytokines in adipose and hepatic
tissues in mice [
87
]. Moreover, in a murine model, PTS was able to normalize the levels of
IL-22 of animals fed an obesogenic diet [88].
5. Conclusions
PTS and PTSO were able to inhibit the growth of human tumor lines MCF-7, T-84, A-
549, HT-29, Panc-1, Jurkat, PC-3, SW-837, and T1-73. In addition, both compounds showed
high TIs and were able to induce hMSCs proliferation at low concentrations. Furthermore,
PTS and PTSO reduced the values of pro-inflammatory cytokines IL-6, IL-8, and IL-17 in
HT-29 and T-84 lines. The generation of ROS and apoptosis seems to be related to the
antiproliferative and anti-inflammatory activity of PTS and PTSO in tumor cells. This
work represents a promising new therapeutic application of these compounds, although
further investigation is needed to deepen the knowledge on the mechanisms of action and
demonstrate their efficacy in vivo.
Author Contributions:
All authors excepting E.G., J.G., A.M.-F. and A.C.-E. performed the exper-
iments and contributed to the acquisition and analysis of data. E.G., A.J.R.-M., N.M.-S., A.B. and
M.J.R.-S. performed the antitumor and anti-inflammatory studies and N.M.-S. and M.J.R.-S. con-
tributed to studies related with mechanism of action. E.G., A.R.-N., A.C.-E., A.B. and J.G. designed
the experiments. A.B., E.G., A.J.R.-M., N.M.-S., A.C.-E., A.R.-N. and J.G. wrote the manuscript and all
authors contributed to the revision. All authors have read and agreed to the published version of the
manuscript.
Funding:
This research was funded by the MISIONES-CDTI program (CDTI, Centre for the De-
velopment of Industrial Technology; CULTUREDMEAT Project MIG-20201012), by the Junta de
Andalucía (CTS 164) and by Instituto de Salud Carlos III (ISCIII)(PI19/01058) with funds from the
European Union. The CIBER-EHD is funded by the ISCIII. M.J.R.-S. is a predoctoral fellow from
ISCIII (IFI21/00030) (“Programa de Doctorado: Biomedicina”); A.J.R.-M. is a predoctoral fellow
from University of Granada (“Formación de Profesorado Universitario” Program) (“Programa de
Doctorado: Medicina Clínica y Salud Pública”).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
Data will be available to be shared upon publication by correspondence
with either Alba Rodriguez Nogales (albarn@ugr.es) or Julio Galvez (jgalvez@ugr.es), after approval
of a proposal, with a signed access agreement, and relevant ethics consent.
Conflicts of Interest: The authors declare that they do not have any competing interests.
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