Tanshinone I inhibits the growth and metastasis of osteosarcoma via suppressing JAK/STAT3 signalling pathway

Abstract Tanshinone I (Tan I) is a widely used diterpene compound derived from the traditional Chinese herb Danshen. Increasing evidence suggests that it exhibits anti‐cancer activity in various human cancers. However, the in vitro and in vivo effects of Tan I on osteosarcoma (OS) remain inadequately elucidated, especially those against tumour metastasis. Our results showed that Tan I significantly inhibited OS cancer cell proliferation, migration and invasion and induced cell apoptosis in vitro. Moreover, treatment with 10 and 20 mg/kg Tan I effectively suppressed tumour growth in subcutaneous xenografts and orthotopic xenograft mouse models. In addition, Tan I significantly inhibited tumour metastasis in intracardiac inoculation xenograft models. The results also showed that Tan I‐induced increased expression of the proapoptotic gene Bax and decreased expression of the anti‐apoptotic gene Bcl‐2 is the possible mechanism of its anti‐cancer effects. Tan I was also found to abolish the IL‐6‐mediated activation of the JAK/STAT3 signalling pathway. Conclusively, this study is the first to show that Tan I suppresses OS growth and metastasis in vitro and in vivo, suggesting it may be a potential novel and efficient drug candidate for the treatment of OS progression.


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Tanshinone I (Tan I), one of the most abundant diterpene compounds from the traditional Chinese herb Salvia miltiorrhiza Bunge (Danshen), has been approved for clinical treatment of coronary heart and cerebrovascular diseases. 14,15 Danshen, and especially its derivative tanshinone II A, reportedly performs comprehensive in vitro and in vivo activities against various human cancer cells, including OS. [16][17][18][19][20][21] Recently, the anti-cancer effects of Tan I in malignant cancers have also been elucidated. Substantial evidence indicates that Tan I induces apoptosis in myeloid leukaemia, colon, lung, prostate and gastric cancer cells in vitro by activating caspase-3. [22][23][24][25][26][27] Moreover, it reportedly suppresses cell growth by down-regulating adhesion molecules in breast cancer cells, 28 inhibiting vascular endothelial growth factor (VEGF) expression in lung cancer cells 29 and decreasing angiogenesis. 30 A small but growing number of researches have also suggested that Tan I could effectively inhibit lung and prostate cancer growth in vivo. [31][32][33] Nevertheless, its in vitro and in vitro effects on OS are inadequately elucidated, especially those against OS metastasis.
In this study, we therefore determined the effects of Tan I on the proliferation, apoptosis, migration and invasion of OS cell lines in vitro, and on tumour growth and metastasis in vivo, using xenografts and orthotopic implantation tumour models. We also analysed cellular and molecular biomarkers associated with its functions. For instance, Bcl-2/Bax is related to apoptosis, and matrix metalloproteinase (MMP)-2/MMP-9 is involved in migration and invasion. Moreover, we identified the JAK1/2-STAT3 signalling pathway as an anti-metastasis target of Tan I. These results imply the potential of Tan as a novel OS preventive and therapeutic agent.

| Cell lines, animals, and reagents
The human OS cell lines U2OS and MOS-J, and the mouse OS cell Tan I was purchased from Sigma, and a 50 mmol/L stock solution was prepared in dimethyl sulfoxide (DMSO; Sigma) and stored at −20°C. Different concentrations of Tan I were prepared by defined dilutions in culture medium. Most appropriate antibodies were purchased from Cell Signaling Technology, unless otherwise stated. Antibodies were used according to the manufacturer's instructions.

| Cell viability assay
Cell proliferation was determined using the sulforhodamine B assay. 34 Briefly, cells were seeded in 96-well plates at a density of 4 × 10 3 cells/well for 24 hours and then exposed to different Tan I concentrations for 72 hours. Then, the cells were fixed with 10% trichloroacetic acid for 1 hours at 4℃ and stained with 50 μL of 0.4% (w/v) SRB (Sigma) for 20 minutes at room temperature. Excess dye was removed by repeated washing with 1% (vol/vol) acetic acid, and 100 μL of 10 mmol/L Tris buffer was added for OD determination at 515 nm, using a microplate reader. All experiments were performed in triplicate.

| Colony formation assay
U2OS and MOS-J were seeded at a density of 2 × 10 3 and 4 × 10 3 cells/well, respectively, in 6-well plates for 24 hours recovery and then treated with different Tan I concentrations for a week. The cells were then fixed with 4% paraformaldehyde for 20 minutes at room temperature and stained with 0.2% crystal violet. The number of cell colonies was determined as the ratio of the number of treated to untreated samples. All experiments were performed in triplicate.

| Live/dead staining assay
The live/dead assay was conducted with the live/dead viability/ cytotoxicity kit (Molecular Probes), following the manufacturer's instructions. Briefly, cells were exposed to different Tan I concentrations for 24 hours. Then, Calcein-AM (living cell staining, green) and ethidium homodimer-1 (dead cell staining, red) were added. The green living and red dead cells were visualized using fluorescence microscopy and photographed, and the ratios of dead cells to total cells were calculated.

| Migration assay
The cell migration assay was conducted by Boyden inserts (8 μm; BD Biosciences) as previously described. 35 In brief, serum-starved U2OS and MOS-J cells in a medium containing 0.5% FBS were pre-treated with various Tan I concentrations (0, 0.08, 0.4 and 2.0 μmol/L) for 30 minutes. Then, the cells were seeded at a density of 1.5 × 10 4 cells/well in the upper chamber of a Transwell plate and allowed to migrate to the lower chamber. After incubation for 24 hours, non-migrated cells were removed while migrated cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The cells were then visualized and photographed with an inverted microscope (Olympus; magnification, ×100). The migrated cells in four random fields were quantified and manually counted, and the percentage of inhibition was expressed using 100% as that of the control well.

| Invasion assay
Cell invasion was determined using the wound-healing migration assay. Briefly, U2OS and MOS-J cells were seeded in 6-well plates.
After growing to confluent monolayers, "wounds" were carefully created on the cells using a sterile pipette tip. After sounding and washing, the cells were cultured in complete medium with various Tan I concentrations, incubated for 24 hours, fixed with 3.7% paraformaldehyde and photographed at ×100 magnification under a phasecontrast microscope. Migrated cells were manually quantified, and the percentage of inhibition of migrated cells was expressed using 100% as that of the untreated group. After staining, the cells were resuspended with binding buffer and immediately analysed with flow cytometry.

| Quantitative real-time PCR
Cells were cultured and treated with different concentrations of Tan I for 24 or 48 hours. After cell harvested, total RNA was extracted using TRIzol reagent (Takara), according to the manufacturer's instructions. cDNA was synthesized from RNA with a cDNA reverse transcription kit (Takara). Real-time PCR was performed in triplicate using gene-specific primers (Thermo Fisher Scientific), on the Stratagene Mx3005P PCR system (Agilent Technologies). mRNA levels were normalized to β-actin levels. The gene-specific primers used are listed in Table 1 and were checked for specificity before use.

| Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed to evaluate the interaction between STAT3 and Bcl2 or MMP2, as previously described. 36 Briefly, cells were treated with Tan I for 24 hours, fixed with 1% formaldehyde to crosslink chromatin and protein, immunoprecipitated with STAT3 or IgG antibodies and then incubated with protein A/G agarose beads.
After washing severally, the protein-DNA complex was reversed by proteinase K treatment, and DNA was recovered and isolated with

Gene
Primer set OMIM ID Abbreviations: MMP, matrix metalloproteinase; OMIM, Online Mendelian Inheritance in Man TA B L E 1 qRT-PCR primer sets phenol-chloroform. The DNA was analysed using qPCR with primers that are specific to regions spanning the Stat3-binding sites of the Bcl-2 and MMP2 promoters, listed in Table 1.

| Western blotting
Cells were exposed to various concentrations of Tan I for

| Xenograft model of human osteosarcoma cancer U2OS tumour
As previously described, 38

| OS-1-luc orthotopic transplantation xenograft model
Orthotopic implantation of tumour cells was performed as previously described. 39 Briefly, C57 mice were randomly assigned to sep-

| Statistical analysis
Data were presented as means ± SEM, and statistical analysis was performed using Microsoft Excel and GraphPad Prism 5 software with the Student's t test. All experiments were repeated at least three times. P < .05 was considered statistically significant.

| Tan I inhibits OS cell proliferation and colony formation
As shown in Figure 1A,B, the chemical structure of Tan I is similar to that of tanshinones, derived from Danshen. To investigate the effects of Tan I on tumour proliferation, we first performed sulforhodamine

| Tan I inhibits OS cell migration and invasion
Because of the association between OS cell migration, and invasion and metastatic potential, we conducted a chamber migra-

| Tan I inhibits U2OS subcutaneous tumour growth in vivo
Given that Tan I can inhibit OS cell proliferation and colony formation and induce apoptosis in vitro, we wondered whether Tan I could inhibit OS growth in vivo. U2OS cells were injected into nude mice to establish the OS subcutaneous tumour xenograft model. The mice were then treated with 10 or 20 mg/kg Tan I or vehicle control for 21 days, killed, and their tumour xenografts were dissected ( Figure 3A). As shown in Figure 3B and  were measured twice a week during the administration period. The mice were killed after 21 d, and their tumours were removed (A) and weighed (C). E, H&E stained liver, spleen, kidney, heart and lung. F, Organ/body mass ratios after the mice were killed. Ns, no significant difference and ***P < .001 at the given concentration had little effect on the bodyweight, compared with those of mice in the control group ( Figure 3D).
Pathological staining and organ/body ratio measurements showed no Tan 1-induced toxicity in the hearts, livers, spleens, lungs and kidneys of the treated mice, compared with those of the mice of in the control mice ( Figure 3E-F). These results therefore indicate that Tan I exerts potent anti-tumour efficacy in the U2OS tumour xenografts without obvious side effects.

| Tan I inhibits orthotopic tumour growth and metastasis in vivo
In an effort to closely mimic human disease, especially for confirming whether Tan I could  blocked tumour growth ( Figure 4A,B). Furthermore, daily treatment with 10 and 20 mg/kg Tan I significantly reduced tumour volume by 20% and 50%, respectively ( Figure 4C).
Using a tumour metastasis model, we investigated the effect of Tan I on OS metastasis in vivo. U2OS-luc cells were inoculated into the left ventricle of nude mice. Then, the mice were administered 10 or 20 mg/kg Tan I daily for 24 days, bioluminescence imaging was conducted, and the survival of mice was recorded. We found that both 10 and 20 mg/kg Tan I effectively inhibited tumour metastasis, as tumour metastasis was found in the control group mice ( Figure 4D-E). Moreover, Tan I treatment significantly increased mouse survival rates by 80% in the 20 mg/kg group and 60% in the F I G U R E 4 Tan I inhibits osteosarcoma cell metastasis in vivo. A, OS-1-luc cells were injected into the tibia of C57 mice, and the resulting tumour-bearing mice were treated with 10 or 20 mg/kg Tan I or vehicle daily. The OS-1-luc tumours were imaged using IVIS on treatment days 0, 10 and 20. B, Tumour volume was determined using the normalized photon flux. C, Tumour length and width were measured using a digital calliper after harvest, and tumour volume was calculated with the equation volume = length × width 2 × 0.52. D, U2OS-luc cells were injected through the left ventricle, and the resulting tumourbearing mice were treated with 10 or 20 mg/kg Tan I or vehicle daily. U2OS-luc tumours were imaged using IVIS on treatment days 0, 12 and 24. E, Tumour cell distribution was quantified using the normalized photon flux. F, Mouse survival curves during the treatment period. *P < .05, **P < .01 and ***P < .001 10 mg/kg group ( Figure 4F). These results confirm that Tan I efficaciously suppresses the growth and metastasis of OS in vivo.

| Tan I altered the expression of mRNAs and proteins involved in apoptosis and invasion
To clarify the anti-tumour mechanism of Tan I, we determined cell apoptosis and invasion-induced gene expression alterations, as Tan I-induced cell apoptosis and significantly inhibited cell migration and invasion. We first examined the mRNA and protein expression of apoptotic molecules belonging to the Bcl-2 family. We F and G, U2OS cells were incubated with Tan I and analysed using a quantitative ChIP assay with anti-STAT3 antibody. Ns, no significant difference, *P < .05, **P < .01 and ***P < .001 I significantly increased Bax expression, but drastically inhibited that of MMP9 ( Figure 5D).
Epidermal growth factor (EGF) signalling is well known to support the migration and invasion of OS cells, as well as those of many other cancers, 41 which we determined by examining the downstream JAK1/2-STAT3 signalling pathway. 42 As shown in Figure 5E,

| D ISCUSS I ON
Danshen, the traditional Chinese herb, and its derivatives are widely used to treat heart diseases and have no significant adverse effects. 43,44 Along with 30 diterpene compounds in Danshen, cryptotanshinone, tanshinone I and tanshinone IIA are the most abundant and well-studied, 14 and their anti-cancer effects have been widely demonstrated. 9,45 In the present study, Tan I (Figure 2A,B).
The induction of apoptosis is considered a crucial anti-tumour mechanism of drugs. 46 we investigated using a metastasis model, and found that Tan I decreased tumour metastasis and significantly increased survival time of mice ( Figure 4D,F). These results are exciting because the effects of tanshinones on tumour metastases have rarely been reported, although tanshinone IIA has been widely shown to inhibit OS cell growth in vitro and in vivo. 21,50 We therefore concluded that Tan I can effectively suppress OS growth and metastasis in mice.
Tumour-associated proteases are required for metastasis, invasion and migration. 51 Metalloproteinases (MMPs) are believed to be essential for extracellular matrix degradation, a significant characteristic of OS invasion and metastasis. 52 In the present study, Tan I suppressed the mRNA and protein expression of MMP-2 and MMP-9, possibly responsible for OS cell invasion and metastasis ( Figure 5B,C). Inflammatory cytokines and signalling pathways are well known to play pivotal roles in enhancing tumour metastasis and drug resistance. 53,54 IL-6 secreted in the tumour microenvironment activates the JAK/STAT3 signalling pathway, favouring tumour growth and metastasis. 55,56 In determining the potential mechanism, we found that Tan I blocked IL-6-induced JAK1/2 and STAT3 activation and down-regulated p-JAK1/2 and STAT3 protein levels ( Figure 5E-G). It is worth mentioning that a number of oncogenic signalling pathways are involved in OS progression, 47 and we could not exclude the association between Tan I treatment and the other signalling pathways. From these findings, it could be assumed that Tan I inhibits the JAK1/2-STAT3 signalling pathway and suppresses MMP-2 and MMP-9 expression, thereby blocking OS invasion and metastasis in mice.
This study had several limitations. Firstly, although Tan I inhibited OS cell proliferation, the detailed molecular mechanism such as regulation of cell cycle or microtubule dynamics, remained unclear. 57 In addition, the mechanism of Tan I-induced OS cell apoptosis and death remained unclear. 58 Besides, the drug metabolism, pharmacokinetics and pharmacodynamics of Tan I and other potential mechanisms involved in its anti-tumour in vivo effects needed further elucidation.
In summary, Tan I was efficacious in suppressing OS cell growth and metastasis both in vitro and in vivo. Tan I induced cell apoptosis by activating Bax and inhibited tumour proliferation and metastasis by inactivating the JAK-STAT3 signalling pathway. Therefore, Tan I could be considered and further investigated as a novel, efficient and safe drug candidate for the treatment of OS progression.

ACK N OWLED G EM ENTS
This work was supported by the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University (JY201510).