Delphinidin induces apoptosis and inhibits epithelial‐to‐mesenchymal transition via the ERK/p38 MAPK‐signaling pathway in human osteosarcoma cell lines

Abstract Delphinidin is major anthocyanidin that is extracted from many pigmented fruits and vegetables. This substance has anti‐oxidant, anti‐inflammatory, anti‐angiogenic, and anti‐cancer properties. In addition, delphinidin strongly suppresses the migration and invasion of various cancer cells during tumorigenesis. Although delphinidin has anti‐cancer effects, little is known about its functional roles in osteosarcoma (OS). For these reasons, we have demonstrated the effects of delphinidin on OS cell lines. The effects of delphinidin on cell viability and growth of OS cells were assessed using the MTT assay and colony formation assays. Hoechst staining indicated that the delphinidin‐treated OS cells were undergoing apoptosis. Flow cytometry, confocal microscopy, and a western blot analysis also indicated evidence of apoptosis. Inhibition of cell migration and invasion was found to be associated with epithelial‐to‐mesenchymal transition (EMT), observed by using a wound healing assay, an invasion assay, and a western blot analysis. Furthermore, delphinidin treatment resulted in a profound reduction of phosphorylated forms of ERK and p38. These findings demonstrate that delphinidin treatment suppressed EMT through the mitogen‐activated protein kinase (MAPK) signaling pathway in OS cell lines. Taken together, our results suggest that delphinidin strongly inhibits cell proliferation and induces apoptosis. Delphinidin treatment also suppresses cell migration and prevents EMT via the MAPK‐signaling pathway in OS cell lines. For these reasons, delphinidin has anti‐cancer effects and can suppress metastasis in OS cell lines, and it might be worth using as an OS therapeutic agent.

Apoptosis plays a critical role in embryonic development and the elimination of damaged cells. [8][9][10] It is divided into two major pathways, known as intrinsic and extrinsic pathways. The intrinsic pathway is triggered by various molecular signals, which are mitochondrial stimuli. 11 When a problem occurs in this progression, it eventually leads to the development of cancer. 12 For this reason, it is considered very important to kill cancer cells through various molecular mechanisms, and many anti-cancer drugs, including natural products, are currently being studied. 13,14 Metastasis is defined as the ability of cancer cells to spread to distant organs in the body, and it accounts for more than 90% of cancerrelated deaths. 15 These progressions are modulated by the interactions of the cell signaling pathway among the transforming growth factor-b, the notch, the nuclear factor-jB, wnt, and epithelial mesenchymal transition (EMT). [15][16][17] EMT is a key step during embryonic morphogenesis and cancer metastasis. 18 In aggressive cancers, EMT is characterized by decreased E-cadherin and increased N-cadherin expression, contributing to a stroma-oriented adhesion with increased tumor cell motility and invasive properties in several cancers. 19,20 Additionally, several transcription factors, including the Snail/Slug family, respond to microenvironmental stimuli and function as molecular switches for the EMT process. 21 The mitogen-activated protein kinase (MAPK) pathway is a key signal transmission network in eukaryotes. 22 The MAPK family has been classified into three major subfamilies: extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK/SAPK), and p38 MAPK. 23,24 These all play important roles in cell proliferation, differentiation, survival or death, migration, and invasion. 22 In terms of metastasis, ERK, JNK, and p38 MAPK play an important role in various cancers. 25,26 ERK1/2 are serine/threonine protein kinases, and recent evidence demonstrates a specific role of ERK as a mediator of EMT in breast and colon tumors. 27 JNK is activated by environmental and genotoxic stresses, and it has key roles in inflammation, cell proliferation, differentiation, survival, and the migration of various cancer cells. 28,29 P38 MAPK is activated by the upstream MKK3 and MKK6 kinases, and activated p38 MAPK regulates cell migration and metastasis. 30 In addition, p38 MAPK has been implicated in the regulation of E-cadherin and Snail and Slug protein levels. 31 Delphinidin is a major anthocyanidin compound that is found in many pigmented fruits and vegetables, 32 and is known to have many beneficial health effects, including anti-oxidant, 33 anti-inflammatory, 34 anti-proliferation, 35 anti-cancer, and pro-apoptotic properties. 34,36 Delphinidin strongly suppresses transformation and migration of cells during tumorigenesis in various cancers. 32,37 However, it has not been reported that delphinidin prevents cancer metastasis and induces apoptosis via the MAPK-signaling pathway in OS.
Thus, in the present study, we demonstrate that delphinidin induces apoptosis through the mitochondria-dependent pathway, inhibits cell motility through the MAPK-signaling pathway, and regulates the expression of EMT-related factors in OS cell lines. Taken together, these findings suggest that the delphinidin is valuable as a therapy for OS patients, and further research is needed.  solution (500 lg/mL) was added to each well. The cells were incubated for 4 h at 378C. After 10 min of shaking in the SH30 orbital-shaker, the insoluble purple formazan product was dissolved in DMSO, and absorbance of each well was measured using an ELISA reader (Tecan, Mänedorf, Switzerland) at an excitatory emission wavelength of 620 nm.

| Colony-forming assay
The cells were seeded into six-well plates (3 3 10 2 cells/well) and incubated overnight. The cells were treated with various concentrations of delphinidin. After treatment, the cells were washed and allowed to grow for 10 days until colonies formed. Colonies were fixed with 100% methanol and stained with crystal violet for 10 min at room temperature. Next, after washing with sterile distilled water, they were dried at room temperature. Clones with >50 cells were counted with an ordinary optical microscope.

| Hoechst 33342 staining
HOS and U2OS cells were seeded into a 60 mm culture dish, treated with various concentrations of delphinidin, and incubated for 24 h.
Next, cells were harvested using trypsinization and centrifuged at 3000 rpm for 5 min onto a clean glass slide. After this, the cells were stained with 1 lg/mL Hoechst 33342 for 10 min at 378C. Following incubation, the cells were washed using phosphate buffered saline (PBS). Finally, the slides were mounted with glycerol. Each sample was observed under an epifluorescence microscope (Axioskop, Carl Zeiss, G€ oettingen, Germany).

| Flow cytometry analysis
70%-80% confluent cells were cultured in 60 mm culture dishes for 24 h, and then the cells were exposed to delphinidin for 24 h. Next, the cells were harvested using trypsinization, centrifuged at 3000 rpm for 5 min, fixed with ice-cold 70% ethanol, and stored overnight at 48C. The next day, the fixed cells were washed in 1% bovine serum albumin in PBS solution and re-suspended in a staining buffer of 1 mg/mL PI and 50 mg/mL RNase A, and then incubated at 48C for 30 min. The cells were then stained with 50 lg/mL propidium iodide. Finally, the stained cells were measured using a CYTOMICS FC500 flow cytometer system (Beckman Coulter, California), and the fractions in the G1 cell cycle phase were measured for statistical analysis using the flow cytometry system. The data were analyzed using Multi Cycle software.  When the cells grew to 95% confluence, each well was manually scratched with 200 lL sterile pipette tips. All the wells were removed the suspended cell and washed with PBS, and then the plates were washed and incubated with delphinidin at 378C. Images were obtained again after 24 h of incubation. The distance between two cell edges was analyzed using ImageJ software.

| Invasion assay
The Transwell assay was done using chambers with an 8.0 lm pore polycarbonate membrane (Corning Costar, Cambridge, Massachusetts).
Matrigel was coated with 40 lL on the transwell and incubated for 24 h. The next day, the cells were seeded (1.

| Western blot analysis
Cell lysates were prepared by extracting proteins with lysis buffer (pH 7.6, 50 mM Tris-Cl, 300 mM NaCl, 0.5% Triton X-100, 2 lL/mL aprotinin, 2 mM PMSF, and 2 lL/mL leupeptin). The lysate samples were centrifuged at a 13 200 rpm for 30 min at 48C. Protein amounts were measured using a Bradford protein assay (Bio-Rad, Richmond, California). Cell lysates were then exposed to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinyl difluoride membrane using a wet transfer apparatus. Next, the membranes were blocked with a 5% non-fat dry milk blocking buffer for 2 h at room temperature. After blocking, the membranes were kept at 48C for 24 h with the respective primary antibodies. The membranes were washed for 1 h and treated with secondary antibodies for 2 h at room temperature. Finally, the membranes were washed again for 1 h, and detection of each protein was performed using a Super Signal West Femto (Pierce, Rockford, Illinois) and analyzed using an Alpha Imager HP (Alpha Innotech, Santa Clara).  To determine the molecular mechanism of apoptosis with delphinidin treatment in HOS and U2OS cells, the apoptosis-related proteins were assessed using a western blot analysis. Delphinidin treatment in HOS and U2OS cells showed that the anti-apoptotic protein Bcl-2 was down-regulated, and the pro-apoptotic protein Bak was up-regulated in a time-dependent manner. Additionally, pro-caspase-3, cleavage caspase-3, and PARP were activated, and triggered the release of cytochrome c from the mitochondria to the cytosol in both cell lines ( Figure   2D-F). Overall, these results suggest that delphinidin-induced apoptosis occurs via a mitochondrial-dependent pathway.

| Delphinidin to inhibit the migration of OS cell lines via the MAPK-signaling pathway
To investigate the effect of delphinidin on HOS and U2OS cell migration, we performed the wound healing assay. In the delphinidin 75 lM treatment group, migration width was inhibited com-   Figure 5A,B). Therefore, the present study supports the hypothesis that inhibition of metastasis in OS cells with delphinidin is modulated by suppression via the MAPK-signaling pathway.

| D I SCUSSION
Metastasis is a very important process in human cancers. 38,39 This progression occurs when unstable cancer cells migrate to a tissue microenvironment that is distant from the primary tumor. 40 Cancer cells need to be able to migrate and invade surrounding local tissue, which usually involves a process referred to as EMT. 31 They then enter the microvasculature of the lymph and blood systems and translocate that through the bloodstream to the micro-vessels of distant tissues. Finally, they adapt to the foreign microenvironment of these tissues in ways that facilitate cell proliferation and the formation of a macroscopic secondary tumor. 41 OS is a malignant tumor that is observed mainly in children and is a highly aggressive tumor that metastasizes primarily to the lungs. 42 Thus, we have conducted various experiments to investigate the effect of delphinidin, which can inhibit OS metastasis. In other studies, delphinidin inhibits HER-2 and ERK1/2 signaling and induces apoptosis in breast cancer cell lines. 32 Furthermore, delphinidin suppresses proliferation and migration of human ovarian clear cell carcinoma cells. 43 In the present study, we performed a MTT assay to compare the cyto- The OS cells were pretreated with an ERK1/2 and p38 inhibitor, and were detected using a western blot analysis. The levels of b-actin were used as an internal standard for quantifying ERK, p38, and EMT factor expression proliferation effect of delphinidin, a very low concentration (0.1-10 lM) of delphinidin was used, and then a colony-forming assay was performed. As a result, delphinidin inhibited the proliferation of HOS and U2OS cells; in particular, a dose higher than 1 lM of delphinidin showed a sharp decrease. Based on these results, we found that delphinidin has a cytotoxicity effect in OS cell lines and that it inhibited cell proliferation at very low concentrations. When apoptosis progresses, the nucleus of the cell undergoes a morphological change. 44 These changes almost invariably involve chromatin condensation, DNA fragmentation, and cellular shrinkage and blebbing. 44,45 In another published study, delphinidin-induced DNA fragmentation and morphological changes in human ovarian clear cell carcinoma cells. 43  Delphinidin-treated OS cells had a time-dependent increase in the apoptosis ratio. In the apoptosis process, there are two main apoptotic pathways called the intrinsic and extrinsic pathways. 46 In the intrinsic pathway, mitochondrial dysfunction is an important key event of apoptosis that plays a vital role in the execution of both extrinsic and intrinsic apoptotic pathways. 47 Mitochondrial membrane depolarization is an early event of the apoptosis process, and a loss of the mitochondrial membrane potentially leads to the release of the pro-apoptotic protein Bax. 48 The cytochrome c in cytosol released by Bax interacts with the Apaf-1 and pro-caspase-9 complex and activates the downregulation of the caspase cascade. 48,49 Our results show a significant shift in the ratio of Bcl-2 and Bak expression, the anti-apoptotic protein Bcl-2 decreased, and the pro-apoptotic protein Bak increased timedependently with delphinidin treatment. Additionally, the expression levels of pro-caspase-3, cleavage caspase-3, and PARP caused significant time-dependent increases in OS cells. To observe the emission of cytochrome c from the mitochondria into the cytosol in OS cells, we used a confocal microscope. These findings clearly suggest delphinidininduced apoptosis occurs via the mitochondrial-signaling pathway.
The EMT process is regulated by E-cadherin (epithelial marker), Ncadherin (mesenchymal marker), and various transcription factors. 31 The master regulator, E-cadherin, is related to cell-cell adhesion, and the connection loss of E-cadherin in cancer cells which increases the tumor grade, metastasis, and mortality of various cancer cells. 50,51 Well-known EMT-related transcription factor, such as Snail, Slug, Twist, ZEB1, and ZEB2 respond to microenvironmental stimuli and function as molecular switches for the EMT process. 52 Snail is to encode a zincfinger transcriptional repressor controlling EMT during tumor progression. 53 Snail genes have subdivided into two subfamilies as Snail and Slug. 54 Recent evidence demonstrated that snail and slug were to affect extracellular matrix components regulation, a loss of intercellular cohesion, increased rate of cellular migration and invasion and increased resistance to apoptosis, and, it is important role in cancer metastasis. [55][56][57] Also, recently Snail and Slug showed to enhance the invasion in an in vitro invasion model. [58][59][60] In this study, we showed the effect of delphinidin on the metastatic ability of OS cells. The delphinidin-treated OS cells significantly decreased cell invasion and EMT-related transcription factors at a concentration of 75 lM ( Figure   3A). The expression of the epithelial marker E-cadherin was upregulated, and the expression of the mesenchymal marker N-cadherin was down-regulated. Moreover, the expression of the Slug and Snail transcription factors was decreased in a dose-dependent manner with delphinidin. In conclusion, our results demonstrate that delphinidin is able to inhibit cell invasion by regulating EMT-related protein and transcription factors.
Activation of the ERK-signaling pathways markedly reduced cellular migration, invasion, and metastasis of cancer cells. 61 The JNK is a master protein kinases that regulates many physiological processes, including cell proliferation, differentiation, survival, and death. 29,62 The p38 MAPK regulates cellular responses to the environment and controls gene expression, cell growth, cell migration, and invasion. 63 Several studies have shown that an increase in the p38 MAPKs plays an important role in the formation of metastatic tumors. 64 In this study, we performed the wound healing assay, which is the most commonly used assay for cell migration measurement. Compared to the control group, delphinidin treatment significantly inhibited cell migration in OS cell lines. As shown by the western blot analysis, ERK1/2 and p38 phosphorylation were significantly decreased with delphinidin treatment in a time-dependent manner, but there was no effect in the expression of JNK. These findings suggest that delphinidin can markedly suppress cell motility in OS cells via the MAPK-signaling pathway.
Based on these results, we confirmed the expression of EMT-related protein again after treatment with a MAPK inhibitor (p38 inhibitor, SB203580 and ERK inhibitor, PD38059). As expected, the expression of E-cadherin increased more when the inhibitor was treated with delphinidin than when treated alone whereas the expression of Ncadherin and Slug and Snail was decreased in HOS cells. The expression levels of E-cadherin and N-cadherin were lower in U2OS than HOS cells, however expression tendency of snail and slug were a similar with in HOS cells. Therefore, these results indicated that delphinidin suppressed the EMT via MAPK signaling pathway.
In conclusion, the present study demonstrates that delphinidin showed evidence of various types of apoptosis in OS cells, and it induced apoptosis through the mitochondrial pathway. Additionally, through the experiments on metastasis in OS cells, delphinidin was able to inhibit cell invasion and migration in OS cells. Furthermore, we found that delphinidin regulates metastasis-related protein expression levels via the MAPK-signaling pathway. As a result, we have demonstrated that delphinidin has the effect of inhibiting cancer cell metastasis in OS cells, and delphinidin is valuable as a therapeutic tool for OS patients.