Introduction

Osteosarcoma is the most common type of primary bone malignant tumour and causes serious health problems for adolescents (Daiy et al. 2012). In its early stage, osteosarcoma is very invasive and is mainly observed in actively growing long bone metaphysis (Daiy et al. 2012). Although the development of multimodality therapies such as the combination of neoadjuvant chemotherapy and surgery has significantly increased the survival rate of patients from 20 to 65% since the 1980s, the progress has slowed over the last 20 years (Fuchs and Pritchard 2002; Gibbs et al. 2002). Thus, the understanding of the molecular mechanism underlying osteosarcoma is one of the most important steps in the treatment process (Zao et al. 2011). New treatment strategies are strongly desirable to increase the survival of patients with osteosarcoma (Zao et al. 2011).

Apoptosis or programmed cell death plays an important role in eukaryotic tissue development and homeostasis (Kaufmann and Hengartner 2001). Apoptosis may progress through a death receptor-dependent extrinsic pathway or a mitochondria-dependent intrinsic pathway, and is induced by treatments with chemotherapeutic agents (Kaufmann and Earnshaw 2000; Reed 2001).

Reversine or 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (Fig. 1), a substituted purine, was originally identified to induce dedifferentiation of murine myoblasts into multipotent progenitor cells (Kim et al. 2007). Therefore, many studies have been focused on the role of this compound in cell reprogramming and regenerative medicine (Kim et al. 2007; Saraiya et al. 2010; Cheng et al. 2018). Several studies have shown that reversine acts as an anticancer agent and inhibits cell growth and induces cell death in various cancer cell types, including myeloid leukaemia, multiple myeloma, cervical carcinoma, thyroid cancer, breast cancer, oral squamous cell carcinoma, and prostate cancer (Hsieh et al. 2007; D’Alise et al. 2008; McMillin et al. 2010; Lee et al. 2012; Lu et al. 2012; Qin et al. 2013; Kuo et al. 2014). Furthermore, many studies have demonstrated the tumour suppressive activity of reversine against human cancer cells through the induction of cell apoptosis, cell cycle arrest, and autophagy (Hsieh et al. 2007; D’Alise et al. 2008; McMillin et al. 2010; Lee et al. 2012; Lu et al. 2012; Qin et al. 2013; Kuo et al. 2014). Hence, reversine may serve as a new anticancer agent for the treatment of various cancers (Cheng et al. 2018). Although reversine reduced the viability of cancer cells, the underlying mechanisms is yet questionable. In addition, the anticancer effects of reversine on osteosarcoma cells are unclear.

Fig. 1
figure 1

Chemical structure of reversine

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In the present study, we examined the anticancer effects of reversine on cell growth and investigated the mechanism underlying cell death using a human osteosarcoma cell line MG-63. This study reports for the first time that the induction of apoptosis by reversine is associated with the activation of caspases in osteosarcoma cells.

Materials and methods

Materials

Reversine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved poly (ADP-ribose) polymerase (PARP), B cell lymphoma (Bcl)-2, Bcl-xL, p53, and BAD were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-factor associated suicide ligand (FasL), anti-β-actin, and anti-Bcl-2-associated X protein (BAX) were supplied by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other analytical reagents procured were of analytical grade.

Cell culture

The human osteosarcoma cell line MG-63 was obtained from the American Type Culture Collection (ATCC). MG-63 cells were grown in Eagle’s minimum essential medium (EMEM; ATCC, Manassas, VA, USA) containing 10% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37 °C in an atmosphere containing 5% CO2.

Cell viability assay

The MG-63 osteosarcoma cells were seeded at a density of 5 × 105 cells/well in 96-well plates and allowed to attach to the well. After an overnight incubation, the cultured cells were treated with various concentrations of reversine in triplicates and incubated at 37 °C for 24 h in a 5% humidified CO2 incubator. MTT was added to each well and incubation was continued for further 4 h at 37 °C. The formazan crystals were dissolved in 200 µL of dimethyl sulfoxide (DMSO), and the optical density (OD) of the solution was determined using a spectrometer at 570 nm wavelength. The experiments were repeated thrice and the mean ± SEM of each group of replicates was calculated. Growth inhibition was calculated using the following equation: Percent growth inhibition = ([1 − OD extract treated]/[OD negative control]) × 100.

Cell survival assay

Cell survival was measured, as previously described (Bae et al. 2014), using calcein-AM (Molecular Probes, Eugene, OR, USA) to stain live cells and ethidium bromide homodimer-1 (Molecular Probes, Eugene, OR, USA) to stain dead cells. Briefly, MG-63 cells were plated in a chamber slide, incubated with reversine for 24 h, and stained with green calcein-AM and ethidium bromide homodimer-1 according to the manufacturer’s protocol. The cells were observed and photographed under an inverted phase-contrast microscope (Eclipse TE2000; Nikon Instruments, Melville, NY, USA).

Histology

MG-63 cells were collected after treatment with reversine (0, 20, and 50 µM), incubated for 24 h, and rinsed in phosphate-buffered saline (PBS). The cells were fixed with pre-chilled 4% paraformaldehyde for 30 min at 4 °C. Haematoxylin and eosin (H&E) staining was performed to observe morphological changes in MG-63 cells. The cells were observed and photographed under inverted phase-contrast microscope.

Nuclear staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)

MG-63 cells treated with 0, 20, and 50 µM reversine, incubated for 24 h, and fixed with 4% paraformaldehyde before washing with PBS. The washed cells were stained with 1 mg/mL of DAPI for 20 min. Nuclear condensation was observed by fluorescence microscopy.

Determination of caspase activation

The activity of caspase-3/7 was determined using the cell-permeable fluorogenic substrate, PhiPhiLux-G1D2 (OncoImmunin Inc., Gaithersburg, MD, USA), according to the manufacturer’s instructions. The cells were treated with 0, 20, and 50 µM reversine for 24 h and incubated with PhiPhiLux-G1D2. The activity of caspase-3/7 was examined by fluorescence microscopy.

Annexin V-fluorescein isothiocyanate (V-FITC), propidium iodide (PI), and flow cytometric analysis

MG-63 cells were cultured in culture dishes at a density of 1 × 106 cells/dish for 24 h and treated with 0, 20, and 50 µM reversine for 24 h. The cells were washed twice in PBS and resuspended in a binding buffer (BD Biosciences, San Diego, CA, USA). Annexin V-FITC and 7-amino-actinomycin D (BD Biosciences) were added to the culture, and the cells were incubated in the dark for 15 min. The cells were resuspended in 400 µL of binding buffer and analysed using a fluorescence-activated cell sorting Calibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Data analysis was performed using the standard CellQuest software (Becton-Dickinson).

Immunoblotting

MG-63 cells were treated with 0, 20, and 50 µM reversine for 24 h. Immunoblotting was performed using minor modifications in the method described elsewhere (Park et al. 2015). Anti-cleaved caspase-3/8/9, PARP, FasL, Bcl-2, Bcl-xL, p53, BAX, BAD, and β-actin antibodies were used as the primary antibodies.

Data analysis

All experiments were performed at least in triplicates. The results are presented as the mean ± SEM. Statistical significance was analysed using a Student’s t test for comparison between two groups or one-way analysis of variance for multi-group comparisons. A value of p < 0.05 was considered statistically significant.

Results

Reversine induces MG-63 cell death in a concentration-dependent manner

To verify whether reversine induces cytotoxic effects, the MTT assay was performed following treatment of MG-63 cells with various concentrations of reversine for 24 h. As shown in Fig. 2a, reversine inhibited the growth of cells in a dose-dependent manner, suggestive of reversine-induced MG-63 cell death. Based on these results, the IC50 value for reversine in MG-63 cancer cells was estimated to be approximately 17.9 µM. To confirm the reversine-induced death of MG-63 cells, the live-dead cell assay was performed using permeable green calcein-AM and red ethidium homodimer-1. As shown in Fig. 2b, the number of dead MG-63 cells stained with ethidium homodimer-1 (red fluorescence) increased following treatment with reversine (10, 20, and 50 µM) for 24 h. Taken together, these results indicate that reversine induces MG-63 cell death in a concentration-dependent manner. Also, reversine reduced the cell number and altered the typical morphology of MG-63 cancer cells in a dose-dependent manner (Fig. 2c).

Fig. 2
figure 2

Reversine treatment induces cytotoxicity in MG-63 human osteosarcoma cells. a Concentration-dependent cytotoxicity was observed in MG-63 cells treated with reversine for 24 h. MG-63 cells were cultured and treated with various concentrations of reversine for 24 h. MTT assay was performed to measure cytotoxicity. The percentage of cell viability was calculated as a ratio of absorbance at A570 nm wavelength for reversine-treated cells and untreated control cells. Each data point represents the mean ± SEM of four experiments. **p < 0.01 versus control (the control cells measured in the absence of reversine). b Reversine increased MG-63 cell death in a dose-dependent manner. MG-63 cells were cultured and treated with 10, 20, and 50 µM of reversine for 24 h. Live (green fluorescence) and dead cells (red fluorescence) were stained using green calcein AM and ethidium homodimer-1, respectively. c Reversine reduced the cell number and altered the typical morphology of MG-63 cells. MG-63 cells were treated with reversine (0, 20, and 50 µM) and fixed. Haematoxylin and eosin (H&E) staining was performed to observe the morphological changes in MG-63 cells

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Reversine-induced MG-63 cell death is mediated by apoptosis

To investigate the mechanism underlying reversine-induced MG-63 cell death, DAPI staining was performed to determine chromatin condensation in MG-63 cells following treatment with 20 and 50 µM reversine for 24 h. The number of MG-63 cancer cells with condensed nuclei was significantly increased following reversine treatment (Fig. 3a). To verify whether reversine-induced MG-63 cell death is mediated by apoptosis, caspase-3/7 activity was measured using the cell-permeable PhiPhiLux-G1D2. As shown in Fig. 3b, the activities of caspase-3/7 increased in MG-63 cells treated with reversine. To determine if the reversine-induced cell death is associated with the induction of apoptosis, MG-63 cells were treated with 20 and 50 µM reversine for 24 h and co-stained with Annexin V-FITC, an apoptotic marker, and PI, a necrotic marker. In comparison with the untreated control cells, those treated with 20 and 50 µM reversine showed 72.9% and 82.1% increase in the percentage of Annexin V-FITC-positive cells, respectively (Fig. 3c). Taken together, these results suggest that reversine induces cell death through apoptosis signaling pathways in MG-63 osteosarcoma cells.

Fig. 3
figure 3

Reversine-induced MG-63 cell death involves apoptosis through the activation of caspase-3/7. a DAPI staining was performed to observe chromatin condensation. MG-63 cells were cultured and treated with 20 and 50 µM reversine for 24 h. DAPI staining was performed to observe nuclear morphological alterations under an inverted phase-contrast microscope. Condensed nuclei, typical of apoptotic cells, were observed in MG-63 cells treated with reversine. b PhiPhiLux-caspase-3/7 substrate was cleaved in MG-63 cells treated with reversine. MG-63 cells were treated with reversine and the caspase-3/7 activity assay using PhiPhiLux-G1D2 was performed to observe the activation of caspase-3/7. The pro-apoptotic factor, cleaved caspase-3/7, was observed in MG-63 cells treated with reversine. c FACS analysis was used to assess reversine-induced apoptosis of MG-63 cells. To identify reversine-induced MG-63 cell apoptosis, FACS analysis was performed by Annexin V and PI staining. The MG-63 cells were cultured and incubated with 20 and 50 µM reversine for 24 h. After treatment, the cells were analysed by flow cytometry

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Reversine-induced apoptosis is mediated by the extrinsic death receptor-dependent and intrinsic mitochondria-dependent apoptotic signaling pathways

To determine the cellular apoptotic pathways associated with reversine-induced MG-63 cell death, immunoblotting was performed and the expression levels of apoptotic proteins were evaluated. As shown in Fig. 4a, the expression of FasL (48 kDa), an apoptotic ligand that triggers the extrinsic death receptor-mediated apoptotic pathway, was upregulated in a concentration-dependent manner in MG-63 cells treated with 20 and 50 µM reversine than in the non-treated control. The level of cleaved caspase-8 (18 kDa), the downstream target of pro-apoptotic factor FasL, increased following reversine treatment (Fig. 4a). These results suggest the involvement of the extrinsic death receptor-mediated apoptosis pathway in reversine-induced apoptosis.

Fig. 4
figure 4

Reversine-induced apoptosis is dependent on the extrinsic death receptor-mediated as well as the intrinsic mitochondria-dependent apoptotic signaling pathways in MG-63 cells. MG-63 cells were treated with 20 and 50 µM reversine for 24 h. Total proteins were extracted and western blotting was performed to verify the altered expression levels of pro-apoptotic and anti-apoptotic factors associated with apoptotic signaling pathways. a Reversine induces extrinsic death receptor-mediated apoptotic signaling pathway. Reversine upregulated the expression level of the death receptor ligand FasL and subsequently activated the extrinsic death receptor-mediated apoptotic signaling pathway through the cleavage of caspase-8 in MG-63 cells. b Reversine activates the intrinsic mitochondria-dependent apoptotic signaling pathway. Reversine downregulated anti-apoptotic factors Bcl-2 and Bcl-xL associated with the intrinsic mitochondria-dependent apoptotic pathway and upregulated the mitochondria-dependent pro-apoptotic factors BAX and BAD in MG-63 cells. c The activation of caspase-3 and PARP via reversine-induced extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis signaling pathways. Cleaved caspase-8 and cleaved caspase-9 induced the activation of caspase-3 and PARP in MG-63 cells treated with reversine

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The expression levels of Bcl-2 (26 kDa) and Bcl-xL (26 kDa), anti-apoptotic factors associated with the intrinsic mitochondria-dependent apoptosis pathway, were downregulated (Fig. 4b), while those of mitochondria-dependent pro-apoptotic factors such as BAX (21 kDa) and BAD (23 kDa) were upregulated (Fig. 4b) in MG-63 cells following reversine treatment in a concentration-dependent manner. Reversine treatment increased the expression levels of p53 (53 kDa) (Fig. 4b) and cleaved caspase-9 (35 kDa) in MG-63 cells (Fig. 4b). This result indicates that reversine-induced MG-63 cell death involves the intrinsic mitochondria-dependent apoptosis pathway. Both cleaved caspase-8 and caspase-9, acting in the extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptosis pathways, induced the expression of cleaved caspase-3 (17 and 19 kDa) and PARP (89 kDa) in MG-63 cells following reversine treatment (Fig. 4c). Therefore, reversine-induced MG-63 cell death is thought to be mediated by both the extrinsic death receptor-dependent and intrinsic mitochondria-dependent apoptotic signaling pathways.

Reversine-induced apoptosis depends on the activation of caspases in MG-63 cells

To verify whether reversine-induced apoptosis of MG-63 cells requires caspase activation, reversine-treated MG-63 cells were incubated with or without a pan caspase inhibitor Z-VAD-fmk for 24 h. And then, MTT assay and immunoblotting were performed. As shown in Fig. 5a, reversine treatment inhibited the cell growth by 46.0% ± 3.3% as compared with no treatment. The relative survival rate was 59.3% ± 4.6% for cells co-treated with reversine and Z-VAD-fmk (Fig. 5a). Furthermore, the expression levels of the cleaved forms of pro-apoptotic factors such as caspase-3 and PARP were significantly downregulated in the cells co-treated with reversine and Z-VAD-fmk than in those treated with reversine alone (Fig. 5b). These data indicate that reversine-induced apoptosis is dependent upon the activation of caspases in MG-63 cells.

Fig. 5
figure 5

Reversine-induced apoptosis is dependent on the activation of caspases in MG-63 cells. a A pan-caspase inhibitor, Z-VAD-fmk, partially reversed reversine-induced cell death in MG-63 cells. MG-63 cells were cultured and treated with 20 µM of reversine in the presence or absence of 20 µM Z-VAD-fmk for 24 h. MTT assay was performed to measure cytotoxicity. The values presented are mean ± SEM. **p < 0.01. b Z-VAD-fmk inhibited the activation of caspase-3 and PARP in MG-63 cells treated with reversine

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Discussion

Reversine is a small molecule first used as a dedifferentiation agent, and is known to induce dedifferentiation of a murine myoblast cell line into multipotent progenitor cells (Kim et al. 2007). The effect of reversine on cell differentiation is also applicable to cancer treatment (Cheng et al. 2018). Many studies have suggested that reversine has tumour-suppressive effects against several cancer cells (Hsieh et al. 2007; D’Alise et al. 2008; McMillin et al. 2010; Lee et al. 2012; Lu et al. 2012; Qin et al. 2013; Kuo et al. 2014). However, the studies evaluating the effects of reversine on osteosarcoma cells are rare. This study examined the cytotoxic effects of reversine and the underlying mechanism in MG-63 human osteosarcoma cells. Here, we demonstrated the potential application of reversine as an anti-osteosarcoma agent.

In the present study, reversine effectively inhibited the growth of MG-63 cells in a concentration-dependent manner (Fig. 2). This observation is in line with the results, wherein reversine exerted anticancer effects through the suppression of growth of several types of cancer cells (Hsieh et al. 2007; D’Alise et al. 2008; McMillin et al. 2010; Lee et al. 2012; Lu et al. 2012; Qin et al. 2013; Kuo et al. 2014). Furthermore, the IC50 value for reversine in MG-63 cancer cells was estimated to be approximately 17.9 µM. These results suggest that reversine exerts cytotoxicity against osteosarcoma cells and has potential applications for anticancer drug discovery.

As the induction of apoptosis in cancer cells is a useful strategy for the development of anticancer drugs, many studies have evaluated the effects of various compounds on apoptosis (Yang et al. 2014). To examine whether reversine-induced MG-63 cell death is mediated via apoptosis, DAPI staining was performed and nuclear condensation was evaluated in the present study. The treatment with reversine induced nuclear condensation and fragmentation, a hallmark of apoptosis, in MG-63 cells (Fig. 3a). To verify the reversine-induced apoptosis of MG-63 cells, we measured the activation of caspase-3/7, the central component of the apoptotic cascade. The activity of caspase-3/7 significantly increased in MG-63 cells treated with reversine (Fig. 3b). Flow cytometric analysis demonstrated the increase in the number of apoptotic MG-63 cells after treatment with reversine (Fig. 3c). Therefore, these results show that the cytotoxic activity of reversine against MG-63 cells is related to the induction of apoptosis.

Apoptosis occurs through the death receptor-mediated extrinsic pathway as well as the mitochondria-mediated intrinsic pathway (Kaufmann and Earnshaw 2000; Reed 2001). FasL, an important regulator of apoptosis, binds to the receptor FasR on the surface of the target cells and initiates the death receptor-mediated extrinsic apoptotic pathway through the activation of caspase-8 (Herrnring et al. 2000; Li et al. 2013). In the subsequent step, the active form of caspase-8 induces the cleavage of procaspase-3 to form active caspase-3 (Prokhorova et al. 2015; Zamaraev et al. 2015). In this study, the expression of FasL was significantly increased in MG-63 cells after reversine treatment in a concentration-dependent manner (Fig. 4a). Furthermore, the expression of cleaved caspase-8 was elevated in MG-63 cells treated with reversine (Fig. 4a). These results suggest that reversine-induced apoptosis is mediated through the death receptor-mediated extrinsic apoptosis pathway via the upregulation of FasL expression in MG-63 cells.

Next, we examined the effects of reversine on the expression of p53 in MG-63 cells. The molecular pathways underlying cancer growth inhibition typically involve tumour suppressor agents such as p53 (Kaufmann and Hengartner 2001; Lavin and Gueven 2006; Heath-Engel et al. 2008; Ryu et al. 2012). The level of p53 is constant in normal cells and the protein is phosphorylated in response to stress and interacts with the mouse double minute 2 (MDM2), leading to apoptosis (Fisher 2001; Kaufmann and Hengartner 2001; Lavin and Gueven 2006; Heath-Engel et al. 2008; Ryu et al. 2012). In this study, the level of p53 was higher in the MG-63 cells stimulated with reversine than in the control cells (Fig. 4b), indicating that p53 may mediate the reversine-induced apoptosis of MG-63 cells. In addition, the pro-apoptotic proteins BAX and BAD and the anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-xL are important regulators of cytochrome c release from the mitochondria (Kluck et al. 1997, 1999). The Bcl-2 family is localised onto the mitochondrial membrane and regulates apoptosis by permeabilising the mitochondrial membrane and releasing cytochrome c (Yu et al. 2012). In the present study, the expression of anti-apoptotic factors such as Bcl-2 and Bcl-xL was downregulated in MG-63 cells treated with reversine (Fig. 4b). However, the expression of pro-apoptotic factors such as BAX and BAD in MG-63 cells significantly increased following reversine treatment (Fig. 4b). Cytochrome c released from the mitochondrial outer membranes into the cytosol through the formation of apoptosomes comprised procaspase-9, which was cleaved to activate procaspase-3 (Kluck et al. 1999; Yu et al. 2012). In this study, the expression level of cleaved caspase-9 gradually increased in MG-63 cells treated with reversine in a concentration-dependent manner (Fig. 4b). On the other hand, the ratio of BAX/Bcl-2 is one of the indicators of the activation of the intrinsic mechanism of apoptosis in mitochondria (Oltvai et al. 1993). Reversine-induced apoptosis may involve the BAX/Bcl-2-mediated signal transduction pathway, as reversine treatment increased this ratio in MG-63 cells. Therefore, reversine is suggested to induce apoptosis in MG-63 cells through the mitochondria-mediated signal transduction pathways.

Reversine treatment in MG-63 cells resulted in an increase in the level of cleaved caspase-8 and cleaved caspase-9, which enhanced the level of cleaved caspase-3 through the cleavage of procaspase-3 (Fig. 4c). Cleaved caspase-3 subsequently triggered the cleavage of PARP and induced apoptosis through the access of endonucleases to chromatin (Fig. 4c). Taken together, these results (Fig. 4) suggest that reversine-induced MG-63 cell death is mediated by apoptosis through both the death receptor-mediated extrinsic pathway and mitochondria-mediated intrinsic pathway.

Apoptotic cell death is closely related to the cleavage of caspases such as caspase-3/8/9 (Yu et al. 2017). Therefore, whether reversine-induced MG-63 cell apoptosis is dependent on the cleavage of procaspases was confirmed with the pre-treatment of MG-63 cells with Z-VAD-fmk, a cell-permeable fluoromethyl ketone-derivatised peptide that acts as a caspase inhibitor, prior to reversine treatment (Yu et al. 2017). We performed MTT assay and immunoblotting analysis to measure cell cytotoxicity. Reversine-induced MG-63 cell death was significantly inhibited in the presence of Z-VAD-fmk (Fig. 5a). The expression level of cleaved caspase-3 and PARP was significantly suppressed in MG-63 cells co-treated with reversine and Z-VAD-fmk (Fig. 5b). These results demonstrate that reversine-induced MG-63 cell death depends on the cleavage of procaspase-3/8/9. The mechanisms underlying apoptosis induced by reversine in MG-63 cells are incompletely understood. Future studies are warranted to evaluate the precise cellular and molecular mechanisms of apoptosis induced by reversine in cancer cells.

In conclusion, the in vitro results presented in this study suggest that reversine inhibits proliferation and induces apoptotic death in MG-63 human osteosarcoma cells through the cleavage of procaspase-3/8/9 via mitochondria-mediated intrinsic pathway and death receptor-mediated extrinsic pathway. Further studies, including in vivo studies with xenograft animal models, are warranted for the complete understanding of the potential side effects of reversine and for the determination of the effective dose range.