Myricetin treatment induces apoptosis in canine
osteosarcoma cells by inducing DNA fragmentation,
disrupting redox homeostass, and mediating loss of
mitochondrial membrane potential
Hahyun Park1 | Sunwoo Park1 | Fuller W. Bazer2 | Whasun Lim3 |
Gwonhwa Song1
1 Institute of Animal Molecular Biotechnology
and Department of Biotechnology, College of
Life Sciences and Biotechnology, Korea
University, Seoul, Republic of Korea
2Center for Animal Biotechnology and
Genomics and Department of Animal Science,
Texas A&M University, College Station, Texas
3 Department of Biomedical Sciences, Catholic
Kwandong University, Gangneung, Republic of
Korea
Correspondence
Whasun Lim, Department of Biomedical
Sciences, Catholic Kwandong University,
Gangneung, 25601, Republic of Korea.
E-mail: [email protected]
Gwonhwa Song, Department of
Biotechnology, College of Life Sciences and
Biotechnology, Korea University, Seoul,
02841, Republic of Korea.
Email: [email protected]
Funding information
Ministry of Health & Welfare, Republic of
Korea, Grant number: HI17C0929
Canine osteosarcoma is an aggressive primary bone tumor that shows metastasis to
distal regions and is associated with a high mortality rate. However, the pathophysiological mechanisms of canine osteosarcoma are not well characterized. In addition,
development of prognostic factors and novel therapeutic agents is necessary to
efficiently treat osteosarcoma. Therefore, we studied the effects of myricetin, an
antioxidant found in berries, nuts, teas, wine, and vegetables, on apoptosis and signal
transduction in the canine osteosarcoma cell lines, D-17 and DSN. Results of the
present study demonstrated thattreatment with myricetin decreased cell proliferation
and DNAreplication,while itincreased apoptotic DNAfragmentation in D-17 and DSN
cells. In addition, it increased generation of ROS, lipid peroxidation, and depolarization
of MMP in both D-17 and DSN cells. Myricetin treatment activated phosphorylation of
AKT, p70S6K, ERK1/2, JNK, and p90RSK in canine osteosarcoma cells. Moreover,
inhibition of PI3K and MAPK using LY294002, U0126, or SP600125, in addition to
myricetin treatment, effectively suppressed cell proliferation compared to treatment
with myricetin or each inhibitor alone. Therefore,we concluded that myricetin may be a
potentially effective and less toxic therapeutic agent to prevent and control
progression of canine osteosarcoma.
KEYWORDS
apoptosis, cell signaling, myricetin, osteosarcoma, treatment
1 | INTRODUCTION
Osteosarcoma is the most common primary malignant bone tumor,
accounting for approximately 85–98% of cases in dogs (Morello,
Martano, & Buracco, 2011). In humans and dogs, osteosarcoma
spontaneously occurs in diverse skeletal locations, such as the
appendicular skeleton, axial skeleton, and extraskeletal sites (Morello
et al., 2011; Szewczyk, Lechowski, & Zabielska, 2015). Tumor
localization at regions, such as proximal humerus, distal femur, or
proximal tibia increases incidence of metastasis, consequently, leading
to a high mortality rate (Schmidt et al., 2013). Conventionaltherapeutic
strategies against canine osteosarcoma include surgery, radiotherapy,
immunotherapy, and chemotherapy (Brodey & Abt, 1976; Morello
et al., 2011; Walter et al., 2005). Although adjuvant chemotherapy
Hahyun Park and Sunwoo Park contributed equally to this work. using doxorubicin, cisplatin, carboplatin, or a combination of these
J Cell Physiol. 2018;1–10. wileyonlinelibrary.com/journal/jcp © 2018 Wiley Periodicals, Inc. | 1
drugs improves survival rate, such therapy is limited by toxicity and
side effects (Fenger, London, & Kisseberth, 2014). Presently, the
etiology and pathology of osteosarcoma are not well characterized.
Therefore, development of prognostic factors and novel therapeutic
agents is necessary to efficiently treat osteosarcoma.
Natural remedies have been effectively used in the diverse
management of cancer and chronic diseases for centuries. Myricetin
is an antioxidant found in berries, nuts, vegetables, and teas that
exhibits anti-inflammatory, anti-neurodegenerative, anti-obesity,
anti-diabetic, and anti-cancer properties (Devi, Rajavel, Habtemariam, Nabavi, & Nabavi, 2015; Lee & Choi, 2008; Semwal, Semwal,
Combrinck, & Viljoen, 2016). In cancer cells, myricetin inhibits cell
proliferation by inducing cell cycle arrest via suppression of cell cycle
regulatory proteins (Yang, Lim, Bazer, & Song, 2017; Zhang, Zou, Xu,
Shen, & Li, 2011). Furthermore, myricetin activates release of
cytochrome c and cleavage of caspase-9 and caspase-3, leading to
mitochondrial-mediated apoptosis in leukemic and colon cancer cells
(Kim, Ha, Yoon, & Lee, 2014; Wang, Lin-Shiau, & Lin, 1999).
Moreover, it decreases activity of matrix metalloproteinase 2, a key
metastatic factor in colon cancer cells (Ko, Shen, Lee, & Chen, 2005).
However, there are no reports on the effect of myricetin on
osteosarcoma.
In our study, we analyzed whether myricetin regulated progression of canine osteosarcoma. More specifically, we investigated the
effects of myricetin on proliferation, apoptosis, mitochondrial
dysfunction, and signal transduction in the canine osteosarcoma cell
lines, D-17 and DSN. We demonstrated the therapeutic potential of
myricetin as it induced cell death in canine osteosarcoma.
2 | MATERIALS AND METHODS
2.1 | Chemicals
Myricetin was purchased from Sigma–Aldrich, Inc. (St. Louis, MO).
SP600125 and U0126 were purchased from Enzo Life Science
(Farmingdale, NY), and LY294002 was purchased from Cell Signaling
Technology (Beverly, MA). Antibodies against phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), 90 kDa
ribosomal protein S6 kinase (p90RSK) (Thr573), c-Jun N-terminal kinase
(JNK) (Thr183/Tyr185), AKT (Ser473), ribosomal protein S6 kinase
(p70S6K) (Thr421/Ser424), and ribosomal protein S6 (Ser235/236), and
total ERK1/2, RSK1/RSK2/RSK3, JNK, AKT, p70S6K, and S6 were
purchased from Cell Signaling Technology.
2.2 | Cell culture
D-17 and DSN cells originating from canine osteosarcoma epithelial
cells were purchased from the American Type Culture Collection
(ATCC) and maintained in Minimum Essential Medium (MEM; Cat No.
SH30024.01, HyClone, Logan, UT), supplemented with 10% fetal
bovine serum (FBS), at 37 °C in a 5% CO2 incubator. Cells were grown
to 70% confluency in 100 mm tissue culture dishes, serum starved for
24 hr, and then treated with myricetin and/or inhibitors against the
phosphoinositide 3-kinase (PI3K) and mitogen-activated protein
kinase (MAPK) signaling pathways.
2.3 | Proliferation assay
Proliferation assays were performed using the BrdU Cell Proliferation
ELISA kit(Cat No. 11647229001, Roche, Indianapolis, IN), according to
the manufacturer’s recommendations. D-17 and DSN cells were
seeded in a 96-well plate and incubated for 24 hr in serum-free MEM.
Cells were then treated with myricetin alone or with various inhibitors
(100 µl/well). After 48 hr, 10 µM 5-bromo-2ʹ-deoxyuridine (BrdU) was
added to the cell culture, and cells were incubated for an additional 2 hr
at 37 °C. After BrdU labeling, cells were fixed and incubated with antiBrdU-peroxidase (POD) for 90 min. Anti-BrdU-POD bound to BrdU
that was incorporated into newly synthesized cellular DNA, and these
immune complexes were detected by addition of the 3,3ʹ,5,5ʹ-
tetramethylbenzidine (TMB) substrate. Absorbance values of the
reaction product were determined by measuring absorbance at 370
and 492 nm using an ELISA reader.
2.4 | Immunofluorescence analysis
The effects of myricetin on the expression of proliferating cell nuclear
antigen (PCNA) were determined by immunofluorescence microscopy.
D-17 and DSN cells were probed with a mouse anti-human monoclonal
antibody against PCNA (Cat No. sc-56, Santa Cruz Biotechnology,
Santa Cruz, CA) at a final dilution of 1:100 (2 μg/ml). Cells were then
incubated with goat anti-mouse IgG Alexa 488 (Cat No. A-11001,
Invitrogen, Carlsbad, CA) at a final dilution of 1:200 for 1 hr at room
temperature. Purified nonimmune mouse immunoglobulin G (IgG) was
used as a negative control. Cells were washed using 0.1% bovine serum
albumin (BSA) in phosphate buffered saline (PBS) and overlaid with
4ʹ,6-diamidino-2-phenylindole (DAPI). Images were captured using a
LSM710 confocal microscope (Carl Zeiss, Thornwood, NY).
2.5 | Determination of apoptosis by annexin V and
propidium iodide (PI) staining
Apoptosis of D-17 and DSN cells induced by myricetin was analyzed
using fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ). Cells (4 × 105 cells) were
seeded in six-well plates and incubated for 24 hr in serum-free medium
to 70–80% confluency. Cells were treated with myricetin in a dosedependent manner for 48 hr at 37 °C in a CO2 incubator. Supernatants
were removed from culture dishes and adherent cells were detached
with trypsin-ethylenediaminetetraacetic acid (EDTA). Cells were
pelleted by centrifugation, washed with PBS, and resuspended in
binding buffer. Cell suspensions (100 µl) were transferred to 5 ml
culture tubes and incubated with FITC Annexin V (5 µl) and propidium
iodide (PI; 5 µl) for 15 min at room temperature in the dark. Binding
buffer (400 µl) in a 5-ml culture tube was used as a control.
Fluorescence intensity was analyzed using a flow cytometer (BD
Biosciences).
D-17 and DSN cells (3 × 104 cells per 300 µl) were seeded in confocal
dishes (Cat No. 100350, SPL Life Science, Republic of Korea) and
incubated for 24 hr in serum-free medium. Cells were treated with
100 μMmyricetin for48 hr at37 °Cin aCO2 incubator.Afterincubation,
cells were air dried and fixed in 4% paraformaldehyde in PBS for 1 hr at
room temperature. Fixed cells were briefly rinsed with PBS and
permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min
on ice. Cells were then subjected to terminal deoxynucleotidyl
transferase dNTP nick-end labeling (TUNEL) staining, using the In Situ
Cell Death Detection kit, TMR red (Roche), for 1 hr at 37°C in the dark.
Cells were washed with PBS and overlaid with DAPI. Fluorescence was
detected using a LSM710 confocal microscope (Carl Zeiss).
2.7 | Determination of cellular reactive oxygen
species (ROS) production
Intracellular reactive oxygen species (ROS) production was estimated
based on the conversion of 2ʹ, 7ʹ-dichlorofluorescin diacetate (DCFHDA, Sigma–Aldrich) to fluorescent 2ʹ, 7ʹ-dichlorofluorescin (DCF) in the
presence of peroxides. D-17 and DSN cells were detached on treatment
with trypsin-EDTA, pelleted by centrifugation, and washed with PBS.
Cellswere treatedwith 10 µMDCFH-DAfor 30 min at 37 °C.Cellswere
washed twice with PBS, and treated with myricetin in a dose-dependent
manner for 1 hr at 37 °C in a CO2 incubator. Myricetin-treated
osteosarcoma cells were washed with PBS again. Fluorescent DCF
intensity was analyzed by flow cytometry (BD Bioscience).
2.8 | Lipid peroxidation assay
Click-iT Lipid Peroxidation Imaging kit (Invitrogen) was used, according
to the manufacturer’s recommendation. D-17 and DSN cells (3 × 104
cells per 300 µl) were seeded in confocal dishes and treated with
100 µM myricetin and 50 µM linoleamide alkyne (LAA) for 2 hr at
37 °C in a CO2 incubator. After cell fixation (with 3.7% formaldehyde)
and permeabilization (with 0.5% Triton X-100), the nucleophilic side
chains of proteins modified by LAA oxidation were labeled by Alexa
Fluor 488 Azide for 30 min at room temperature. Fluorescence was
detected using a LSM710 confocal microscope (Carl Zeiss).
2.9 | JC-1 mitochondrial membrane potential assay
Changes in JC-1 mitochondrial membrane potential (MMP) were
determined using a Mitochondria Staining kit (Cat No. CS0390, Sigma–
Aldrich). D-17 and DSN cells (5 × 105 cells) were seeded in six-well
plates, incubated for 24 hr in serum-free medium, and cultured to 70%
confluency. Cells were treated with myricetin in a dose-dependent
manner for 48 hr at 37 °C in a CO2 incubator. Supernatants were
removed from culture dishes and adherent cells were detached by
treatment with trypsin-EDTA. Cells were pelleted by centrifugation
and resuspended in staining solution (200× JC-1 in 1× staining buffer)
and incubated at 37 °C in a CO2 incubator for 20 min. Stained cells
were collected by centrifugation, and washed once with 1× JC-1
staining buffer. After washing, cell suspensions were centrifuged and
resuspended in staining buffer (1 ml). Fluorescence intensity was
analyzed using FACSCalibur (BD Biosciences).
2.10 | Western blot analysis
Protein concentrations of whole-cell extracts from treated D-17 and
DSN cells were determined using the Bradford protein assay (Bio-Rad,
Hercules, CA) with BSA as the standard. Proteins were denatured,
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to nitrocellulose. Blots were
developed using enhanced chemiluminescence detection (SuperSignal
West Pico, Pierce, Rockford, IL), and quantified by measuring the
intensity of light emitted from correctly sized bands under ultraviolet
light using a ChemiDoc EQ system and Quantity One software (BioRad). Immunoreactive proteins were detected using goat anti-rabbit
polyclonal antibodies against phosphorylated and total proteins at
1:1,000 dilution. Total protein levels were used as loading controls to
normalize results from western blotting. All antibodies were purchased
from Cell Signaling Technology. Multiple exposures of western blots
were performed to ensure linearity of chemiluminescent signals. These
experiments were performed in triplicate.
2.11 | Statistical analysis
Data for proliferation assays and western blot analyses were subjected
to analysis of variance (ANOVA) according to the general linear model
(PROC-GLM) of the SAS program (SAS Institute, Cary, NC) to
determine significant effects of treatment on signal transduction
pathways in D-17 and DSN cells. Differences with p < 0.05 were
considered statistically significant. Data were presented as
mean ± standard error of the mean (SEM), unless otherwise stated.
3 | RESULTS
3.1 | Effects of myricetin on proliferation of canine
osteosarcoma cells
To investigate the effects of myricetin on cell proliferation in canine
osteosarcoma, we analyzed the proliferation of canine osteosarcoma cells
(D-17 and DSN) by using BrdU reagents (Figure 1a). Myricetin treatment
gradually decreased the cell proliferation of D-17 and DSN cells in a dosedependent manner, as proliferation of D-17 and DSN cells reduced to
40.3% (p < 0.001) and 38.2% (p < 0.001), respectively, after treatment
with 100 µM myricetin, as compared to vehicle-treated control cells
(100%). We then determined the expression of PCNA, a marker of
proliferation, in untreated D-17 and DSN cells and cells treated with
100 µM myricetin by immunofluorescence analysis (Figure 1b). Although
the expression of PCNA was strongly detected in nuclei of the canine
osteosarcoma cells, myricetin treatment significantly decreased expression of PCNA in cells. Thus,these results indicated that myricetin reduced
proliferation of canine osteosarcoma cells.
PARK ET AL.. | 3
3.2 | Cell death of canine osteosarcoma cells in
response to myricetin
Next, we determined the effects of myricetin on apoptosis of canine
osteosarcoma cells by using annexin V and PI staining assay (Figure 2a).
The population of apoptotic cells increased to approximately 157%
(p < 0.05) and 247% (p < 0.01) after treatment with 50 and 100 µM
myricetin, respectively, in D-17 cells, as compared to vehicle-treated
cells (100%). Similarly, it increased the apoptotic population to
approximately 169% (p < 0.05) and 273% (p < 0.001) after treatment
with 50 and 100 µM, respectively, in DSN cells, compared to vehicletreated control cells (100%). In addition, we performed TUNEL assay to
detect cell death-induced DNA fragmentation from myricetin treatment in both D-17 and DSN cells (Figure 2b). Apoptotic cells stained
with TMR red, indicated by red fluorescence, were predominantly
visualized in the nuclei of D-17 and DSN cells in response to myricetin
treatment, whereas red fluorescence was rarely detected in vehicletreated osteosarcoma cells. These results showed that myricetin
stimulated cell death of canine osteosarcoma cells.
3.3 | Effects of myricetin on generation of ROS and
depolarization of MMP in canine osteosarcoma cells
To analyze the functional effects of myricetin on mitochondrialmediated apoptosis, we measured the generation of ROS and
disruption of mitochondrial transmembrane by myricetin treatment
(Figures 3 and 4). Using cell-permeable, fluorogenic probe DCFH-DA,
we showed that addition of myricetin treatment significantly increased
the production of ROS to approximately 151% (50 µM, p < 0.05) and
230% (100 µM, p < 0.01) in D-17 cells, and approximately 399%
(50 µM, p < 0.01) and 1,164% (100 µM, p < 0.001) in DSN cells, as
compared to vehicle-treated cells (Figure 3a). In accordance with these
results, staining cells with LAA reagent showed that induction of lipid
peroxidation was higher in myricetin-treated osteosarcoma cells than
in vehicle-treated control cells (Figure 3b). Moreover, myricetin
stimulated MMP degradation to 261% (20 µM, p < 0.01), 399%
(50 µM, p < 0.01), and 596% (100 µM, p < 0.001) in D-17 cells, as
compared to vehicle-treated cells (Figure 4a). Similarly, disruption of
MMP was increased in myricetin-treated DSN cells to approximately
162% (20 µM, p < 0.05), 285% (50 µM, p < 0.01), and 4,554% (100 µM,
p < 0.001), as compared to vehicle-treated control cells (Figure 4b).
Taken together, these results indicated that myricetin induced
mitochondrial damage, leading to apoptosis in canine osteosarcoma.
3.4 | Myricetin-mediated signal transduction in
canine osteosarcoma cells
To identify signaling pathways related to cellular proliferation that
were regulated by myricetin, we performed western blot analysis
on signaling molecules belonging to the PI3K/AKT and MAPK
pathways (Figure 5). Myricetin treatment stimulated phosphorylation of ERK1/2 MAPK and its downstream molecule, p90RSK, in
FIGURE 1 Cellular proliferation of canine osteosarcoma in response to myricetin. (a) Proliferation of D-17 and DSN cells in response to
myricetin treatment was measured by staining cells with BrdU reagent and expressed as a percentage relative to vehicle-treated cells. (b)
Immunostained PCNA levels (green) were visualized in the nuclei of untreated D-17 and DSN cells and D-17 and DSN cells treated with
myricetin (100 µM). Nuclei were counterstained with DAPI (blue). The intensity of green fluorescence was estimated by Metamorph software.
Asterisks indicate a statistically significant effect (**p < 0.01 and ***p < 0.001). Scale bars represent 40 µm (first and third vertical panels) and
20 µm (second and fourth vertical panels)
4 | PARK ET AL.
D-17 and DSN cells in a dose-dependent manner (0, 20, 50, and
100 µM) (Figures 5a and 5b). In addition, it gradually activated
phosphorylation of JNK proteins in both canine osteosarcoma cell
lines (Figure 5c). In the PI3K/AKT pathway, myricetin treatment
increased phosphorylation of AKT, p70S6K, and S6 proteins in
dose-response experiments in D-17 and DSN cells (Figure 5d–f).
These results revealed that myricetin regulated MAPK and
PI3K/AKT signaling in canine osteosarcoma cells.
FIGURE 2 Cytotoxicity of myricetin in D-17 and DSN cells. (a) Apoptotic population in myricetin-treated D-17 and DSN cells was sorted
by flow cytometry after staining with annexin V and propidium iodide (PI) dye. The number of late apoptotic cells (upper right quadrant) was
expressed as a percentage relative to vehicle-treated control cells (100%). (b) TMR red-stained apoptotic cells (red) and nuclei counterstained
with DAPI (blue) were visualized in D-17 and DSN cells treated with myricetin. The intensity of red fluorescence was estimated by
Metamorph software. Asterisks indicate a statistically significant effect (*p < 0.01, **p < 0.01, and ***p < 0.001). Scale bars represent 40 µm
(first and third vertical panels) and 20 µm (second and fourth vertical panels)
FIGURE 3 Myricetin-induced oxidative stress in canine osteosarcoma cells. (a) Reactive oxygen species (ROS) production was analyzed in
myricetin-treated D-17 and DSN cells by flow cytometry based on DCFH-DA intensity, and data were indicated as percentages relative to
vehicle-treated control cells (100%). (b) Lipid peroxidation in response to myricetin in D-17 and DSN cells was visualized under a microscope
by using linoleamide alkyne (LAA) reagent conjugated with Alexa 488 (green). Nuclei were counterstained with DAPI (blue). The intensity of
green fluorescence was estimated by Metamorph software. Asterisks indicate a statistically significant effect (*p < 0.01, **p < 0.01, and
***p < 0.001). Scale bars represent 40 µm (first and third vertical panels) and 20 µm (second and fourth vertical panels)
PARK ET AL.. | 5
3.5 | Effects of combination treatment of myricetin
and small-molecule inhibitors on canine osteosarcoma
cells
We repeated the cell proliferation assay on D-17 and DSN cells treated
with myricetin with or without the addition of small-molecule
inhibitors, such as U0126 (ERK1/2 inhibitor), LY294002 (PI3K
inhibitor), and SP600125 (JNK inhibitor), to determine whether
combination treatment affected cell proliferation of canine osteosarcoma cells (Figure 6). Treatment with each inhibitor reduced cell
proliferation of both D-17 and DSN cells, as compared to non-treated
cells (p < 0.001). Moreover, D-17 cells treated with a combination of
myricetin and U0126, LY294002, or SP600125 exhibited a larger
reduction in cell proliferation than cells treated with myricetin alone.
Next, we analyzed phosphorylation levels of signaling molecules in D-
17 and DSN cells treated with each inhibitor in addition to myricetin
(Figure 7). Activated ERK1/2 phosphorylation was completely suppressed by combination treatment with myricetin and U0126, while
treatment with a combination of the other inhibitors had no significant
effect in canine osteosarcoma cells (Figure 7a). Although p90RSK
phosphorylation exhibited a similar trend to ERK1/2 phosphorylation
in treated D-17 cells, p90RSK activity did not significantly change in
treated DSN cells (Figure 7b). JNK phosphorylation activated by
myricetin treatment was inhibited by SP600125 in both canine
osteosarcoma cell lines, whereas combination treatment with the
other inhibitors additionally elevated phosphorylation levels
(Figure 7c). AKT phosphorylation upregulated by myricetin treatment
was weakly reversed by LY294002 in D-17 and DSN cells (Figure 7d).
In addition, p70S6K phosphorylation activated by myricetin treatment
was reduced by LY294002 in D-17 cells and by U0126 and LY294002
in DSN cells (Figure 7e). Finally, the significant increase in S6
phosphorylation on myricetin treatment was abrogated by all the
inhibitors in D-17 and DSN cells (Figure 7f). Taken together, we
demonstrated that treatment with myricetin synergistically added to
the effects of MAPK and PI3K inhibitors, ultimately reducing cell
proliferation in canine osteosarcoma.
4 | DISCUSSION
In our study, we identified the role and potential therapeutic effects of
myricetin in canine osteosarcoma. We demonstrated that myricetin
treatment decreased cell proliferation and increased apoptosis by
mediating abundant ROS production and depolarization of mitochondrial transmembrane in canine osteosarcoma cells. Myricetin regulated
cellular proliferation by activating ERK1/2, JNK, p90RSK, AKT,
p70S6K, and S6, which are proteins involved in the PI3K and MAPK
pathways. Moreover, combination treatment with myricetin and an
inhibitor for ERK1/2, JNK, or PI3K exhibited a synergistic cytotoxic
effect on canine osteosarcoma cells, as illustrated in Figure 8. These
FIGURE 4 Myricetin-induced disruption of mitochondrial membrane potential in canine osteosarcoma cells. Depolarization of
mitochondrial membrane potential (MMP) in myricetin-treated D-17 (a) and DSN (b) cells was analyzed by flow cytometry via JC-1 staining.
JC-1 aggregates were indicated in the upper right quadrant and JC-1 monomers were indicated in the lower right panel. The relative intensity
of JC-1 aggregates was normalized to that of JC-1 monomers, and data were indicated as a percentage relative to vehicle-treated control
cells (100%). Cells treated with valinomycin were used as a positive control. Asterisks indicate a statistically significant effect (*p < 0.01,
**p < 0.01, and ***p < 0.001)
6 | PARK ET AL.
results supported our hypothesis that myricetin may be used as an
alternative chemotherapeutic drug against canine osteosarcoma.
Canine osteosarcoma is a common and aggressive primary bone
tumor that frequently results in metastasis to distal organs (Fenger
et al., 2014). Due to similar histological features and spontaneous
pathogenesis of osteosarcoma between dogs and humans, canine
osteosarcoma serves as a valuable animal model for researching
pathophysiological mechanisms of the disease to improve diagnostic
biomarkers and therapeutic agents (Morello et al., 2011; Simpson et al.,
2017). However, due to the lack of pathogenetic history of canine
osteosarcoma, there are currently no effective therapeutic approaches
that do not require invasive surgery, such as limb amputation or limbsparing surgery. Therefore, we investigated the effects of myricetin on
canine osteosarcoma as a novel, alternative therapeutic drug.
Myricetin treatment significantly decreased cell viability by reducing
PCNA expression and inducing apoptotic DNA fragmentation in D-17
and DSN cells. Previous studies support our observations of the anticancer effects of myricetin treatment on different cancer cell lines. For
FIGURE 5 Signal transduction mediated by myricetin in canine osteosarcoma. Levels of phosphorylation of ERK1/2 (a), p90RSK (b), JNK
(c), AKT (d), p70S6K (e), and S6 (f) in D-17 and DSN cells were analyzed by immunoblotting. Protein levels were quantified relative to vehicletreated control cells. Asterisks indicate statistically significant differences compared to the vehicle-treated control (*p < 0.05, **p < 0.01, and
***p < 0.001)
FIGURE 6 Effects of pharmacological inhibitors in the presence or absence of myricetin treatment on proliferation of canine osteosarcoma
cells. Cell proliferation was analyzed in D-17 (a) and DNS (b) cells treated with pharmacological inhibitors, including U0126 (ERK1/2 inhibitor,
20 µM), LY294002 (PI3K inhibitor, 20 µM), and SP600125 (JNK inhibitor, 20 µM), in the presence and absence of myricetin. Data were
presented as percentage relative to non-treated control cells. Asterisks indicate statistically significant differences compared to the nontreated control (*p < 0.05 and ***p < 0.001)
PARK ET AL.. | 7
example, myricetin was shown to induce cell cycle arrest, leading to
apoptosis in gastric cancer cells (Feng et al., 2015). In addition,
myricetin was reported to reduce the invasive capacity of human
placental choriocarcinoma cells by decreasing cell migration, invasion,
and expression of genes related to invasive properties (Yang et al.,
2017). Moreover, polyphenols that originate from foods and plants
have been known to play a role in the prevention and treatment of
canine osteosarcoma. Turmeric and rosemary extracts were shown to
activate caspase-3, caspase-7, and phosphorylation of JNK, mediating
apoptosis in canine osteosarcoma cells (Levine, Bayle, Biourge, &
Wakshlag, 2017). Astaxanthin was also demonstrated to provide
beneficial effects in the treatment of canine osteosarcoma (Wakshlag,
FIGURE 7 Effects of pharmacological inhibitors in the presence and absence of myricetin treatment on PI3K and MAPK signaling pathways
in canine osteosarcoma. The level of phosphorylation of ERK1/2 (a), p90RSK (b), JNK (c), AKT (d), p70S6K (e), and S6 (f) was analyzed in D-17
and DSN cells treated with myricetin alone (100 µM), or a combination of myricetin and U0126 (20 µM), LY294002 (20 µM), or SP600125
(20 µM). Expression levels were quantified and presented relative to vehicle-treated control cells in a graph. Asterisks indicate statistically
significant differences compared to the vehicle-treated control (*p < 0.05, **p < 0.01, and ***p < 0.001)
FIGURE 8 Hypothetical schematic illustration of myricetin-induced apoptosis in canine osteosarcoma cells. Myricetin activated AKT/
p70S6K/S6 signaling, and the JNK MAPK and ERK1/2 MAPK pathways, mediating apoptotic events, including DNA fragmentation and
disruption of DNA replication in canine osteosarcoma cells. Furthermore, it increased ROS production and resulted in loss of MMP in a dosedependent manner in both D-17 and DSN cells. Finally, combination treatment of myricetin with pharmacological inhibitors conferred
synergistic effects on suppression of cellular proliferation in D-17 and DNS cells. Overall, myricetin proved to be an effective nutraceutical for
prevention and treatment of canine osteosarcoma
8 | PARK ET AL.
Balkman, Morgan, & McEntee, 2010). Thus, myricetin may be used in
adjuvant treatment as a “nutraceutical” for osteosarcoma in dogs.
Mitochondrial dysfunction is a distinct mechanistic feature of
apoptosis of cancers exposed to numerous extracellular stimuli,
including chemotherapy (Boland, Chourasia, & Macleod, 2013). During
apoptosis, opening of mitochondrial permeability transition pores
leads to depolarization of transmembrane potential, release of
cytochrome c and pro-apoptotic proteins, including Bax and Bak,
and disruption of redox and Ca2+ homeostasis (Boland et al., 2013;
Brooks et al., 2007; Weinberg et al., 2010). Therefore, regulation of
mitochondrialfunction is a majortargetfortreatment of cancers. In the
current study, myricetin treatment significantly increased ROS
production and mediated depolarization of transmembrane potential,
promoting apoptosis of canine osteosarcoma cells in dose-response
experiments. In accordance with our results, dihydroartemisinin and
tepoxalin were reported to activate cell death via oxidative damage by
inducing ROS production in canine osteosarcoma cells (Hosoya et al.,
2008; Loftus et al., 2016). In addition, a previous study showed that α-
mangostin exhibited anti-proliferative effects in D-17 cells by
increasing loss of MMP, leading to mitochondrial membrane collapse
(Krajarng, Nilwarankoon, Suksamrarn, & Watanapokasin, 2012).
Although there are few studies that investigate the therapeutic
potential of natural compounds via mitochondrial dysfunction in
canine osteosarcoma, a variety of in vitro and in vivo studies exist that
identify the role of flavonoids, such as myricetin, in mitochondrialmediated apoptosis in human osteosarcoma (Huang et al., 2010; Lin
et al., 2012; Zhang, Guo, Chen, & Chen, 2015). Therefore, it is
necessary to characterize these molecular mechanisms for development of new adjuvant therapy in canine osteosarcoma.
To develop therapeutic strategies to manage and treat cancers, it
is importantto understand the signaling pathways thatregulate cellular
proliferation, invasion, and apoptosis. Myricetin regulates diverse
intracellular signaling transduction pathways of apoptosis in different
types of cancers. Myricetin activates a mitochondria-dependent
apoptotic pathway mediated through cleavage of caspases, alteration
of Bax-to-Bcl-2 ratio, and cytochorome c release in thyroid, colon, and
liver cancer cells (Jo et al., 2017; Kim et al., 2014; Zhang et al., 2013).
Although constitutive activation of the PI3K/AKT signaling is
characteristic of many cancers, myricetin induces apoptosis of human
choriocarcinoma cells by increasing AKT and p70S6K phosphorylation
(Yang et al., 2017). Furthermore, upregulated ROS levels caused by
myricetin treatment result in the activation of various members of the
MAPK pathway in choriocarcinoma cells. Activation of MAPK pathways is closely associated with ROS production and depolarization of
transmembrane potential (Son et al., 2011; Yuan et al., 2013). Erianin
and celastrol induce apoptosis and autophagy, especially in osteosarcoma cells, through ROS/JNK signal transduction (Li et al., 2015; Wang
et al., 2016). Methyl protodioscin, a steroidal saponin, inhibits cell
growth via generation of ROS and loss of MMP by upregulating JNK
and p38 MAPK signaling (Tseng et al., 2017). In accordance with these
previous studies, our results showed that myricetin stimulated activity
of the ERK1/2 MAPK, JNK MAPK, and PI3K/AKT pathways in canine
osteosarcoma cells.
In conclusion, treatment with myricetin promoted apoptotic
events in canine osteosarcoma by inducing DNA fragmentation,
disrupting redox homeostasis, and mediating loss of MMP. Furthermore, it activated phosphorylation of AKT, p70S6K, S6, ERK1/2, and
JNK, and combination treatment with various small-molecule inhibitors conferred a synergistic effect, ultimately decreasing proliferation
of canine osteosarcoma MAPK Inhibitor Library cells. Our results showed that myricetin may
be an effective less toxic cytostatic drug for treatment of canine
osteosarcoma. However, further studies are required to determine the
prolonged survival rate of myricetin as an adjuvant chemotherapy
before administration in clinical trials.
ACKNOWLEDGMENT
The Korea Health Technology R&D Project through the Korea Health
Industry Development Institute funded by the Ministry of Health &
Welfare, Republic of Korea; Contract grant number: HI15C0810.
CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare.
ORCID
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How to cite this article: Park H, Park S, Bazer FW, Lim W,
Song G. Myricetin treatment induces apoptosis in canine
osteosarcoma cells by inducing DNA fragmentation,
disrupting redox homeostasis, and mediating loss of
mitochondrial membrane potential. J Cell Physiol. 2018;1–10