Introduction
Physalis genus (Solanaceae family) comprises
~71 species distributed in America and Asian temperate regions
(1,2). Plants belonging to this genus are
recognized by the presence of calyces that cover the fruit
protecting it from external damage (birds or insects and adverse
climatic conditions) (2). The
edible fruit and beneficial properties of teas prepared with
different parts of the plants confer value within traditional
medicine worldwide to treat inflammation, hepatitis, dermatitis,
cancer and diabetes (2). Their
biological activity is associated with the presence of withanolides
(physalins, neophysalins, withaphysalins), labdane diterpenes,
sucrose esters, flavonoids and ceramides (2-4).
Among species of the genus Physalis, ground cherry (P.
angulata) is used in Colombian folk medicine, as well in Asian
and African countries, for its antimicrobial, antimalarial,
anti-inflammatory, antinociceptive, anti-proliferative and
immunomodulatory properties (4-7).
It is effective against cervix, skin, lung, liver, gastric and
colorectal cancer (CRC) (4,7). To the best of our knowledge, the
biological properties of P. angulata calyces have been
studied scarcely (2,4-6,8-10)
despite their high levels of secondary metabolites for defense. In
our previous research, fractionation of total ethanolic extract
obtained from calyces yielded P. angulata dichloromethane
fraction (PADF), which exhibited maintained or improved bioactivity
particularly when evaluating its anti-inflammatory effect (6,8,9). PADF
demonstrated a promising anti-inflammatory profile in experimental
models [lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages,
12-O-tetradecanoylphorbol-13-acetate-induced acute ear edema and
dextran sulfate sodium (DSS)-induced colitis] (6,9). The
present study aimed to evaluate the effect of PADF on CRC cell
lines and the azoxymethane (AOM)/DSS-induced colitis-associated
cancer (CAC) mouse model. CRC is a serious public health problem
that in 2020 caused worldwide nearly 1.9 million new cases and
915,880 deaths, being the third most common cancer diagnosed and
the second leading cause of cancer death (11). Chronic inflammation, such as that
found in inflammatory bowel disease, which includes ulcerative
colitis and Crohn's disease, is considered one of the most common
risk factors for the development of CRC, as it promotes tumor
growth and development (12). CRC
chemotherapy confronts serious challenges such as resistance, toxic
reactions and side effects (13,14).
Plants with biological activity may contain metabolites that
facilitate development of novel therapy (14).
Materials and methods
Reagents and chemicals
AOM, crystal violet, Eagle's Minimal Essential
Medium (EMEM), DMEM, hematoxylin-eosin (H&E), McCoy's 5A
medium, protease inhibitor cocktail, and phenylmethylsulfonyl
fluoride (PMSF) were purchased from Sigma-Aldrich (Merck KGaA). MTT
was obtained from Calbiochem. DSS (36-50 kDa) was obtained from MP
Biomedicals. Tissue Protein Extraction Reagent (TPER®)
was obtained from Thermo Fisher Scientific, Inc. and fetal bovine
serum (FBS) from Gibco (Thermo Fisher Scientific, Inc.). All
solvents (ethanol, methanol, dichloromethane, hexane and DMSO) were
reagent or HPLC grade.
Plant collection and preparation of
PADF
P. angulata calyces were collected at Loma de
Arena, Bolívar, Colombia. A sample of the whole plant was sent to
Herbarium of the Universidad de Antioquia, Medellín, Colombia and
authenticated according to a voucher previously deposited (no.
HUA213028) (6). Clean and dried
calyces were used for preparation of PADF according to the
procedure described in a previous article (6) (Data
S1).
Cell culture
Colorectal adenocarcinoma (HT-29 and Caco-2) and
healthy colonic epithelial cells (CCD 841 CoN) were obtained from
the American Type Culture Collection (ATCC) and maintained at 37˚C
and 5% CO2 in McCoy's 5A medium (HT-29) or EMEM (Caco-2
and CCD 841 CoN) supplemented with 10% FBS and 1.5 g/l sodium
bicarbonate (Sigma-Aldrich; Merck KGaA).
MTT assay
HT-29, Caco-2 or CCD 847 CoN cells were seeded
(5,000-15,000 cells/well) in 96-well plates (2D culture) or
Perfecta3D® Hanging Drop Plate (3D culture; 3D
Biomatrix) and incubated at 37˚C for 24 and 72 h, respectively, to
allow attachment or spheroid formation, respectively. After that,
cells were treated for 48 h at 37˚C with PADF (3.13, 6.25, 12.50
and 25.00 µg/ml), vehicle (MeOH), or Triton X-100 (1%) as a
positive control. Cell viability was measured using MTT (0.25-5.00
mg/ml). After 4 h, formazan crystals were dissolved in DMSO, and
the optical density (OD) at 550 nm was measured using a plate
reader (Multiskan GO; Thermo Fisher Scientific, Inc.). Selective
index (SI) was calculated as SI=IC50 of PADF in CCD 847
CoN cells/IC50 of the same compound in HT-29 or Caco-2
cells.
Clonogenic assay
The replicative capacity of HT-29 cells was
confirmed as previously described (15,16).
Cells (750 cells/well) were cultured into a 6-well plate at 37˚C
for 6 h and treated with PADF (3.13, 6.25, 12.50 and 25.00 µg/ml)
or vehicle for 48 h. Subsequently, the culture medium was refreshed
and colonies were allowed to form for 7 days at 37˚C. Finally,
colonies were fixed with 7:1 acetic acid-methanol at 24˚C for 30
min, stained with 0.5% crystal violet at 24˚C for 2 h, and counted
manually using light stereomicroscope (EZ4 HD, Leica Microsystems
GmbH; magnification, x10). A colony was considered to contain at
least 50 cells.
Gap closure assay
HT-29 cells (2x105 cells/well) were grown
with McCoy's 5A medium supplemented with 10% FBS (17,18),
on µDish35 mm,high plates (Ibidi) for 24 h (100%
confluence). The insert was removed to generate a scratch in the
monolayer, which was then treated with PADF (6.91 and 13.82 µg/ml)
or vehicle. Images were captured at 24, 48 and 72 h with an
inverted light microscope (Nikon Corporation; magnification x10).
The open area in each image was measured using ImageJ 1.47v
software (National Institutes of Health) and values were normalized
to the open area at 0 h.
Cell cycle analysis
The impact of PADF on cell cycle distribution was
determined by propidium iodide (PI) staining (cat. no. ab139418;
Abcam) and analyzed using a FACS Aria III flow cytometer (BD
Biosciences) and FlowJo X10.0.7 software (FlowJo LLC). HT-29 cells
(2x105 cells/ml) were exposed to PADF (13.8, 18.0 and
27.6 µg/ml) for 24-48 h at 37˚C. Cells were harvested by
trypsinization, fixed with 66% ethanol for 2 h at 4˚C, stained with
PI and incubated with RNase A for 30 min at 37˚C in the dark.
Doxorubicin (Doxo) was employed as a positive control.
Early and late apoptosis
detection
HT-29 cells (1-2x105 cells/ml) were
exposed to PADF (0, 13.8, 18.0 and 27.6 µg/ml) for 24-48 h at 37˚C.
In the first assay, cells were stained with Annexin V-FITC (cat.
no. BMS500FI-100; eBioscience) and analysis was performed using a
flow cytometer (FACS Aria III, BD Biosciences) and the images were
analyzed using FlowJo X10.0.7 software. For the second assay, DNA
electrophoresis was used to detect DNA fragmentation using the
apoptotic DNA ladder detection kit (cat. no. ab66093; Abcam)
following the manufacturer's instructions.
Mitochondrial membrane potential
(ΔΨm)
ΔΨm was monitored by JC-1 Mitochondrial Membrane
Potential kit (Item No. 10009172; Cayman Chemical Company). Cells
were incubated at 37˚C with JC-1 staining solution for 15 min and
rinsed according to the manufacturer's instructions. Measurement
was performed using a microplate fluorometer (Infinite 200 PRO,
Tecan Group Ltd.) to quantify the fluorescence of J-aggregates
(excitation 535 and emission 595 nm) and -monomers (excitation 485
and emission 585 nm).
Animals
Female Balb/c mice (mean weight, 19.41±0.13 g) were
obtained from the Instituto Nacional de Salud (Bogotá, Colombia).
Animals were housed in filtered-capped polycarbonate cages and kept
in a controlled environment (19.48±0.10˚C, 68.63±1.15% humidity
under a 12/12-h light/dark cycle) with access to food and water
ad libitum. All experiments were designed and conducted
following local and international regulations [European Union
regulations (CEC council 86/809), EU Directive 2010/63/EU,
protocols of the Organisation for Economic Cooperation and
Development] and approved by the Committee of Ethics in Research of
the University of Cartagena (Project Approval Minute No. 81 from
August 13, 2015).
AOM/DSS-induced CRC
The total experiment duration was 77 days (11
weeks). In brief, 5-6-week-old animals were randomized into control
(vehicle), AOM + DSS and treatment groups (PADF 10 and 20 mg/kg,
6/group). Carcinogenesis was induced by intraperitoneal (IP)
injection of AOM 10 mg/kg. One week later, animals received 2-4%
DSS in drinking water for 7 days. To promote chronic
inflammation-driven tumor progression, three DSS cycles at 2 weeks
intervals (normal drinking water) were performed (19,20).
Two doses of PADF (10 and 20 mg/kg, IP), selected according to
previous study (6), and the acute
toxicity test (Data S1), were
administered for 3 weeks after the final DSS cycle, while control
groups were treated with vehicle (saline). To check the safety of
PADF, toxicity control groups administered PADF daily (10 and 20
mg/kg/day) without AOM or DSS. Body weight was monitored daily,
while macroscopic parameters such as colon length and weight/length
ratio and tumor progression (tumor number, size and load) were
measured following sacrifice. Tumor load value was calculated as
the sum of the diameters of all the tumors in each animal. A
portion of the colon was rolled from the proximal to distal end.
Samples of colonic tissue containing tumors and adjacent normal
control tissue were also harvested for further analysis. The
experiments had two sets of replications using 197 experimental
animals in total. Sacrifice was performed by cervical dislocation
following anesthesia by 2% inhaled sevoflurane. Animal death was
verified by loss of heartbeat, reflexes and breathing. Humane
endpoints were excessive weight loss (>4 g), diarrhea or
hemorrhage. However, no animal was euthanized according to these
endpoints.
Histology analysis
Colon samples were fixed with 4% buffered formalin
for 30 days at 24˚C and embedded in paraffin; 5 µm sections were
cut and stained with H&E and examined by two blinded
researchers using light microscopy (cat. no. DM500, Leica
Microsystems GmbH; magnification x10). Severity of mucosal
inflammation, dysplasia and cancer were assessed. The epithelial
damage and cellular infiltration were scored from 0-4 as previously
described (21). Dysplastic
proliferation was graded as low- or high-grade and scored
considering the area of mucosal surface affected as absent (0),
low- (1), medium- (2) or high-grade (3). Cancer was scored as absent (0), early
invasive (1) or advanced (2).
Cell viability assay using conditioned
media
RAW 264.7 macrophages (ATCC; cat. no. TIB-71)
maintained in DMEM with 10% FBS at 37˚C and 5% CO2, were
cultured in 24-well plates (200,000 cells/well). After 48 h,
macrophages were treated with 12.5 µg/ml PADF for 1 h and then
stimulated for 6 h with LPS (1 µg/ml) or IL-4 (40 ng/ml) to induce
an inflammatory M(LPS) or alternative M(IL-4) profile,
respectively. Culture supernatant was collected and added to HT-29
cells seeded in 96-well plates (10,000 cells/well), which were
incubated for 48 h at 37˚C. HT-29 cell viability was measured using
the MTT method as aforementioned.
Protein expression analysis
Colonic tissue and HT-29 cells were homogenized
using TPER® extraction reagent supplemented with
protease inhibitor cocktail and PMFS for western blotting (Data S1) or cOmplete tablets Mini,
EDTA-free (Roche Diagnostics GmbH) for ELISA. The extracted
proteins were quantified with Bradford assay (Bio-Rad Laboratories,
Inc.) using BSA as standard. The levels of IL-1β (cat. no.
88-7261-88), IL-6 (ref 88-7066-88; both Invitrogen), and TNF-α (cat
no. 430204; BioLegend) in supernatant were measured using mouse
ELISA kits following the manufacturer's instructions.
Statistical analysis
All data are presented as the mean ± SEM of ≥2
independent experiments. The half-maximal inhibitory concentration
50 (IC50) was calculated using non-linear regression.
Statistical analysis was performed by one-way ANOVA followed by
Dunnett's multiple comparisons test. Statistical analyses were
performed using GraphPad Prism 6 demo version (GraphPad Software,
Inc.; Dotmatics). P<0.05 was considered to indicate a
statistically significant difference.
Results
PADF extraction and chemical
characterization
A total of 3.15 g PADF was obtained from 114 g dried
calyces (2.76% yield) of P. angulata. Based on the
information previously reported (6), four types of metabolites
(withanolides, sucrose esters, phenolic compounds and flavonoids)
were quantified for PADF chemical characterization. Only sucrose
derivatives, most likely sucrose esters, were detected in
significant amounts (Table SI;
Data S1). There were low levels of
withanolides and phenolic compounds and flavonoids were not
detected.
Cytotoxic effect of PADF
Fig. 1A shows the
inhibitory effect of PADF on the viability of colorectal
adenocarcinoma cell lines. HT-29 and Caco-2 cells were notably
sensitive to PADF, exhibiting IC50 values of 13.82±0.42
and 29.56±3.73 µg/ml, respectively. By contrast, normal colon
epithelial cells (CCD 841 CoN) were not significantly affected by
PADF at cytotoxic concentrations (12.5 µg/ml), displaying an
IC50 of 44.80±1.77 µg/ml and selectivity index of 3.24
(HT-29) and 1.52 (Caco-2).
Due to the higher susceptibility of HT-29 cells,
this cell line was selected to study the effect of PADF. Initially,
its cytotoxic effect was tested at different exposure times; PADF
exerted significant cytotoxicity at 6 h, which increased to a
maximum at 48 h (Fig. S1A;
Data S1). Due to the ability of
HT-29 cells to form spheroids, 3D culture was also employed to
mimic the cancer microenvironment better since it reflects
physiological relevant cell-cell interactions (22-24).
PADF showed a concentration-dependent effect, preserving the
cytotoxic effect (IC50=13.31±4.50 µg/ml; Fig. 1B) observed in the 2D model. Thus,
this fraction demonstrates its ability to affect HT-29 cell
viability in a tumor environment.
Effect of PADF on clonogenicity and
migration of HT-29 cells
HT-29 cells formed multiple colonies after 7 days
(Fig. 1C). This was inhibited by
PADF treatment in a concentration-dependent manner. Similarly, PADF
decreased gap closure of treated cells in comparison with control
cells. (Fig. 1D). Therefore, PADF
affected not only the clonogenicity but also the migration of CRC
cells.
Cell cycle and apoptosis analysis
Flow cytometry was employed to detect changes in
cell cycle distribution. PADF induced cell cycle arrest in G2/M
phase and significantly increased the proportion of sub-G1 cells,
suggesting apoptosis induction (Fig.
2), which was corroborated by ΔΨM, Annexin V-staining and
detection of DNA fragmentation. Annexin V-staining demonstrated
that PADF induced a significant increase in the proportion of
apoptotic cells (early and late), especially at the highest
concentration (Fig. 3A).
Consistently, this effect was accompanied by a significant
reduction of ΔΨM, expressed as JC-1 fluorescence ratio, compared
with control cells (Fig. 3B), as
well as the degradation of DNA in PADF-treated HT-29 cells
(Fig. 3C). To verify if apoptosis
was associated with induction of oxidative stress by PADF,
induction of reactive oxygen species (ROS) was measured; no change
in ROS production was detected (Fig.
S1B; Data S1).
In vivo assay
AOM-DSS produced a notable decrease in body weight
on day 56; this loss of weight was significantly improved by the
treatment with PADF at 10 mg/kg/day, while the higher dose (20
mg/kg/day) did not reverse this (Fig.
4A). Macroscopic assessment showed that 100% of animals in the
AOM + DSS developed tumors (mean, 10.82±0.96 tumors in the middle
and distal colon); by contrast, vehicle group showed no tumors
(Fig. 4B). PADF (10 and 20
mg/kg/day) markedly reduced the total tumor load, specifically
diminishing the number of tumors >1 mm in diameter. Furthermore,
PADF significantly reduced the tumor size and tumor load by 35-85%
(Fig. 4C-E). To assess the severity
of the disease, colon weight/length ratio was measured; this was
reduced in animals treated with PADF. Histological examination of
colon sections stained with H&E of the AOM + DSS group
confirmed large adenomas or adenocarcinomas inside the lumen and
prominent extension of dysplasia (both high- and low grade)
accompanied by slight inflammation of the epithelium and cellular
infiltration (Fig. 5A and B). Conversely, PADF (10 and 20 mg/kg)
promoted the recovery of tissue architecture by reducing tumor
growth and dysplasia, thus lowering the histology score from
3.48±0.35 for AOM + DSS to 2.21±0.19 for PADF 20 mg/kg group
(Fig. 5B).
During the study, the toxicity control group was
monitored to verify that the administration of PADF did not alter
the body weight of healthy mice; no signs of toxicity were detected
at any dose (Table SII; Fig. S2; Data
S1). Moreover, a detailed post-mortem analysis revealed no
notable changes during necropsy and hematological analysis
(Tables SII and SIII; Data
S1). Likewise, PADF did not induce genotoxic effects, as
established by the Ames test, micronucleus assay and comet assay
(Fig. S2; Data S1), indicating the safety of PADF at
effective dosage levels.
Protein expression analysis
PADF significantly reduced the levels of
pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in colon tissue
compared with the AOM + DSS group (Fig.
5C). Based on the immunomodulatory effect on macrophages of
PADF (6), it was assessed whether
PADF inhibited viability of CRC cells treated with
macrophage-conditioned media. Treatment with supernatants from
LPS-stimulated macrophages decreases viability (Fig. 5D). By contrast, treatment with
supernatants from IL-4-stimulated macrophages increased viability.
However, when PADF (12.5 µg/ml) was administered, the viability of
HT-29 cells was always significantly reduced, regardless of the
conditional media.
Western blot analysis of colonic samples showed that
PADF (10 and 20 mg/kg/day) significantly reduced the expression of
proliferating cell nuclear antigen (PCNA) while enhancing that of
phosphorylated MAPK in comparison with the AOM + DSS group
(Fig. 6A and B). The decreased levels of PCNA and
increased of p-p38 were confirmed in human CRC cells by western
blotting (Fig. 6C and D).
Discussion
The present study demonstrated therapeutic potential
of P. angulata calyces in CAC. Although the present study is
not the first to identify the promising anti-tumor effect of
derivatives from calyces of Physalis species (25), the present study combined in
vitro and in vivo experiments to assess the
effectiveness of PADF, as well as its mechanisms of action.
PADF chemical characterization revealed an enriched
fraction in sucrose esters, metabolites detected in other
Physalis species that exhibit anti-inflammatory properties
(3,26,27).
Although studies have reported sucrose esters with cytotoxic
activity against human cancer cells in Prunus tomentosa
(Rosaceae) and Echium angustifolium (Boraginaceae) (28-30),
to the best of our knowledge, the present study is the first to
report an anti-tumoral effect by a fraction enriched in sucrose
esters in the Physalis genus.
The present results support the anticancer potential
of PADF. PADF maintained cytotoxic activity on HT-29 cells in 2D
(cell culture in a monolayer) and 3D models (spheroids). In
addition, PADF demonstrated low cytotoxicity on CCD 841 CoN and
another non-cancerous cell line (PCS-201-012;
IC50=30.71±1.92 µg/ml), reported in a previous study
(8), further supporting its
selective inhibitory activity against CRC cell lines. Although
single-cell spheroids are a simple 3D approach to model CRC, the
present results suggested that PADF may be effective in more
complex experimental settings. The effectiveness as a cytotoxic
agent was confirmed by colony formation assay (31), which demonstrated an inhibitory
effect on the colony-forming ability at concentrations four times
lower than the IC50. Another technique used for
screening antitumor drugs is the gap closure assay, which is used
to evaluate the effects of selected drugs on cell migration, a
feature of tumor metastasis that allows neoplastic cells to invade
surrounding blood and lymphatic vessels and spread to other organs
(31,32). PADF showed a potent inhibitory
effect on HT-29 migration, making it a potentially useful
alternative to be used in advanced stages of CRC where cells tend
to invade other tissue.
To determine the mechanism of PADF underlying its
cytotoxic activity on HT-29 cells, flow cytometry was employed. The
results showed that PADF treatment induced apoptosis and G2/M
arrest. G2/M checkpoint blocks the entry into mitosis when DNA is
damaged to allow activation of repair mechanisms or the induction
of programmed cell death (33). To
confirm whether PADF triggered apoptosis, effects on early
apoptosis were assessed by ΔΨM quantify J-aggregates and monomers,
early/mid apoptosis assessing phosphatidylserine translocation by
staining with Annexin-V, and late apoptosis by DNA fragmentation
through electrophoresis (34). The
present results confirmed that apoptosis occurred after PADF
treatment of CRC cells.
Given the promising results in vitro as well
as the strong anti-inflammatory activity observed previously
(6), PADF was evaluated using a
model of CAC, a disease resulting from the
inflammation/dysplasia/carcinoma sequence where malignant cell
proliferation, pro-inflammatory cytokines [IL-6, IL-1β and tumor
necrosis factor (TNF)-α] and several signaling pathways (MAPK,
NF-κB and Wnt/β-catenin) are hyperactivated (22). Animal models are key to identify
carcinogens and pathogenesis and progression of cancer and screen
drug candidates during pre-clinical development. Despite its
limitations, the combination of AOM and DSS as a model of CAC has
popularity for its reproducibility, potency, low price and ease of
use (35-39).
The sustained release of pro-inflammatory cytokines and mediators
(IL-1β, IL-6, TNF-α, nitric oxide and prostaglandin E2) can
accelerate the process of colon carcinogenesis in both humans and
mice (40-43).
Therefore, these are important targets to assess when modeling CAC.
PADF significantly decreased the levels of pro-inflammatory
cytokines (TNF-α, IL-6 and IL-1β) in colon biopsies when compared
with the AOM + DSS group. This anti-inflammatory effect, which
promotes tissue recovery and mucosal regeneration, combined with
the harmful effect against CRC cell lines, makes PADF an attractive
alternative to treat CAC.
To assess the mechanism underlying with the
beneficial effect of PADF, expression of PCNA and MAPKs (ERK, JNK,
and p38) was determined as markers of abnormal cellular
proliferation, inflammation, and stress response (44,45).
PADF decreased PCNA phosphorylation and increased MAPK
phosphorylation. As PCNA is involved with DNA synthesis and
initiation of cell proliferation (40,43),
this supported the inhibitory effect of PADF against malignant
cells in CRC. However, increased MAPK phosphorylation by P.
angulata appears contradictory since these proteins are
traditionally associated with cancer pathogenesis (42,46,47).
Nonetheless, activation of this pathway is dynamic during the
inflammation/dysplasia/carcinoma sequence in the AOM-DSS model. For
example, p-p38 is highly expressed during inflammation, but when
carcinoma is established, expression of p-p38 decreases to normal
levels. The elevated levels of p-p38 observed after administration
of PADF in the high-grade dysplasia/carcinoma phase could be
related to the induction of apoptosis in malignant cells, similar
to that behavior observed with cisplatin, doxorubicin, and
camptothecin (48,49), while protecting normal epithelial
cells, a key role in the maintenance of epithelial homeostasis
(50). Numerous naturally derived
compounds (e.g., Phenethyl isothiocyanate, Evodiamine, Triptolide,
Quercetin, and Honokiol) have been described as apoptotic inducers
by increasing phosphorylation of JNK and ERK (49,51,52).
The inhibition of PCNA and activation of p-p38 was confirmed using
human CRC cells. β-catenin expression decreased, suggesting the
inhibition of the Wnt signaling pathway, activation of which
contributes to the initiation and progression of CRC (51).
In conclusion, PADF demonstrated potent cytotoxic,
anti-tumor and anti-inflammatory activity in experimental models of
CRC where viability of malignant cells was suppressed through
apoptosis, cell cycle arrest, inhibition of cytokine expression and
the modulation of key signaling pathways involving p38, PCNA, MAPKs
and β-catenin. This bioactivity may be related to the high content
of sucrose esters, metabolites with anti-inflammatory and
antibacterial properties. Further investigations are needed to
identify the compounds responsible for the activity.
Supplementary Material
Materials and methods
Biologic effects of PADF. (A) HT-29
cell viability. (B) Intracellular levels of reactive oxygen species
of HT-29 cells after PADF treatment. n=10/group.
*P<0.05, **P<0.01,
***P<0.001, and ****P<0.0001 vs. cells
without treatment. PADF, Physalis angulata dichloromethane
fraction.
Effect of PADF on genotoxic and
mutagenic parameters of BALB/c mice. (A) Proportion of MN-PCE,
MN-NCE and PCE/NCE ratio of micronucleated erythrocytes of
peripheral blood and bone marrow. (B) DNA damage measured by comet
assay in peripheral blood cells. (C) Mutagenesis in Salmonella
typhimurium TA100 measured by Ames test. n=10/group.
*P<0.05, ***P<0.001 and
****P<0.0001 vs. vehicle or control. PADF,
Physalis angulata dichloromethane fraction; MN,
Micronucleus; PCE, Polychromatic erythrocytes; NCE, Normochromatic
erythrocytes; MMC, Mitomycin c; 2-AA, 2-Aminoanthracene.
Content of representative secondary
metabolites of PADF.
Effect of PADF on weight of BALB/c
mice.
Effect of PADF on hematological
parameters of BALB/c mouse serum.
Acknowledgements
The authors would like to thank Professor Josefina
Zakzuk for support with flow cytometry and Ms. Jennifer Vazquez,
Ms. Catherine Meza, and Ms. Laura Ospina for their assistance with
the micronucleus assay (all Universidad de Cartagena, Cartagena,
Colombia).
Funding
Funding: The present study was supported by Minciencias and
Universidad de Cartagena (grant nos. 878-2015, 057-2018, and
025-2019).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
LF conceived, designed and supervised the study and
revised the manuscript. YO and RS designed the study. YO wrote the
manuscript, constructed figures and supervised the study. LF, YO,
IP, and RS confirm the authenticity of all the raw data. DC, DR, IP
and JC analyzed data. DC performed experiments and wrote the
manuscript. DR and JC performed experiments and revised the
manuscript. IP and RS revised the manuscript. DR and IP constructed
figures. All authors have read and approved the final
manuscript.
Ethics approval and consent to
participate
The animal study protocol was approved by
Institutional Ethics Committee of the Universidad de Cartagena
(protocol Minute No. 81 from August 13, 2015). All the experiments
were designed and conducted following local and international
regulations (European Union regulations (CEC council 86/809), EU
Directive 2010/63/EU, protocols of the Organisation for Economic
Cooperation and Development). In addition, tumor burden did not
exceed the recommended dimensions according to University of
Pennsylvania guidelines.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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