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. 2018 Apr 16;233(9):7457–7466. doi: 10.1002/jcp.26598

Myricetin treatment induces apoptosis in canine osteosarcoma cells by inducing DNA fragmentation, disrupting redox homeostasis, and mediating loss of mitochondrial membrane potential

Hahyun Park 1, Sunwoo Park 1, Fuller W Bazer 2, Whasun Lim 3,✉, Gwonhwa Song 1,✉
PMCID: PMC13482111  PMID: 29663365

Abstract

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 that treatment with myricetin decreased cell proliferation and DNA replication, while it increased apoptotic DNA fragmentation 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

Our results showed that Myricetin treatment decreased cell proliferation and increased apoptosis by mediating abundant ROS production and depolarization of mitochondrial transmembrane in canine osteosarcoma cells. Therefore myricetin may be used as an alternative chemotherapeutic drug against canine osteosarcoma.

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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). Conventional therapeutic strategies against canine osteosarcoma include surgery, radiotherapy, immunotherapy, and chemotherapy (Brodey & Abt, 1976; Morello et al., 2011; Walter et al., 2005). Although adjuvant chemotherapy using doxorubicin, cisplatin, carboplatin, or a combination of these 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‐2′‐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 anti‐BrdU‐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 3,3′,5,5′‐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 4′,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 dose‐dependent 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).

2.6. TUNEL assay

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 μM myricetin for 48 hr at 37 °C in a CO2 incubator. After incubation, 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 2′, 7′‐dichlorofluorescin diacetate (DCFH‐DA, Sigma–Aldrich) to fluorescent 2′, 7′‐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. Cells were treated with 10 µM DCFH‐DA for 30 min at 37 °C. Cells were 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 (Bio‐Rad). 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 dose‐dependent 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.

Figure 1.

Figure 1

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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)

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 vehicle‐treated 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 vehicle‐treated osteosarcoma cells. These results showed that myricetin stimulated cell death of canine osteosarcoma cells.

Figure 2.

Figure 2

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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)

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 mitochondrial‐mediated 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.

Figure 3.

Figure 3

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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)

Figure 4.

Figure 4

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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)

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 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 5.

Figure 5

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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 vehicle‐treated control cells. Asterisks indicate statistically significant differences compared to the vehicle‐treated control (*p < 0.05, **p < 0.01, and ***p < 0.001)

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.

Figure 6.

Figure 6

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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 non‐treated control (*p < 0.05 and ***p < 0.001)

Figure 7.

Figure 7

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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)

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 results supported our hypothesis that myricetin may be used as an alternative chemotherapeutic drug against canine osteosarcoma.

Figure 8.

Figure 8

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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 dose‐dependent 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

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 limb‐sparing 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 anti‐cancer effects of myricetin treatment on different cancer cell lines. For 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, 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 mitochondrial function is a major target for treatment 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 mitochondrial‐mediated 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 important to understand the signaling pathways that regulate 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 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.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare.

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.

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;233: 7457–7466. 10.1002/jcp.26598

Hahyun Park and Sunwoo Park contributed equally to this work.

Contributor Information

Whasun Lim, Email: wslim@cku.ac.kr.

Gwonhwa Song, Email: ghsong@korea.ac.kr.

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