Unraveling the novel anti-osteosarcoma function of coptisine and its mechanisms

Toxicology Letters 226 (2014) 328–336

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Unraveling the novel anti-osteosarcoma function of coptisine and its mechanisms Di Yu 1 , Shilong Fu 1 , Zhifei Cao 1 , Meimei Bao, Gaochuan Zhang, Yanyan Pan, Wenming Liu, Quansheng Zhou ∗ Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, Soochow University, Suzhou, Jiangsu 215123, China

h i g h l i g h t s • Coptisine markedly inhibits osteosarcoma cell proliferation and tumor growth. • Coptisine impedes osteosarcoma cell migration, invasion, and tube formation. • Coptisine has very low toxicity with moderate increase in RBC and Hb levels.

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Article history: Received 11 October 2013 Received in revised form 15 February 2014 Accepted 22 February 2014 Available online 7 March 2014 Keywords: Coptisine Osteosarcoma Cancer Proliferation Angiogenesis Vasculogenic mimicry

a b s t r a c t Uncontrolled cell proliferation and robust angiogenesis play critical roles in osteosarcoma growth and metastasis. In this study we explored novel agents derived from traditional Chinese medicinal herbs that potently inhibit osteosarcoma growth and metastasis. Coptisine, an active component of the herb Coptidis rhizoma, markedly inhibited aggressive osteosarcoma cell proliferation. Coptisine induced cell cycle arrest at the G0/G1 phase through downregulation of CDK4 and cyclin D1 expression and effectively suppressed tumor growth in a xenografted mouse model. Coptisine significantly impeded osteosarcoma cell migration, invasion, and capillary-like network formation by decreasing the expression of VE-cadherin and integrin ␤3 , and diminishing STAT3 phosphorylation. Coptisine significantly elevated blood erythrocyte and hemoglobin levels while still remaining within the normal range. It also moderately increased white blood cell and platelet counts. These data suggest that coptisine exerts a strong anti-osteosarcoma effect with very low toxicity and is a potential anti-osteosarcoma drug candidate.

1. Introduction Osteosarcoma constitutes approximately 20% of all pediatric, solid, malignant tumors and is the most common malignant bone tumor in adolescents and young adults (Broadhead et al., 2011). Approximately 40% of osteosarcomas metastasize and the patients have a poor overall prognosis (Mirabello et al., 2009; AragonChing and Maki, 2012). The 5-year survival rate is reported to be 37% in osteosarcoma patients with tumor metastasis and 19% for those with more than five metastatic lung lesions. Uncontrolled cell proliferation and robust tumor angiogenesis are prominent fac-

∗ Corresponding author at: Cyrus Tang Hematology Center, Soochow University, 199 Ren Ai Road, Suzhou Industrial Park, Suzhou, 215123, China. Tel.: +86 512 65882116; fax: +86 512 65880929. E-mail address: [email protected] (Q. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.02.021 0378-4274/© 2014 Published by Elsevier Ireland Ltd.

© 2014 Published by Elsevier Ireland Ltd.

tors contributing to the poor prognosis of osteosarcoma patients. Aggressive osteosarcoma cells show increased angiogenesis and vasculogenic mimicry (Zhang et al., 2010). Several studies demonstrate that tumor cells can directly form tumor blood vessels through vasculogenic mimicry (Maniotis et al., 1999; Carmeliet and Jain, 2011; Seftor et al., 2012; Cao et al., 2013a,b; Liu et al., 2013a,b). This is closely associated with tumor metastasis and poor prognosis of various cancers, including osteosarcoma (Cao et al., 2013a,b). Accordingly, highly proliferative and vasculogenic osteosarcoma cells are a sensible target for novel anti-osteosarcoma drug discovery. For thousands of years the medicinal herb Coptidis rhizoma has been used in China as a treatment for various diseases. The herb contains many active alkaloids, including berberine, coptisine, palmatine, and jatrorrhizine (Yuan et al., 2012). Coptisine has been reported to display many therapeutic properties, including anti-oxidative stress (Gong et al., 2012), anti-diabetic (Chen et al.,

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2012), anti-bacterial (Yan et al., 2008), anti-fungal (Kong et al., 2009), anti-osteoporotic (Lee et al., 2012), and cardiovascular protection (Gong et al., 2012). Colombo et al. (2001) assessed the inhibitory effect of coptisine on the proliferation of several colon cancer and leukemia cell lines. Lin et al. (2004) evaluated the inhibitory effect of coptisine on liver cancer and leukemia cell lines. However, the anti-osteosarcoma effect of coptisine has not yet been reported yet, and the mechanism behind coptisine-mediated anticancer activity is currently unknown. In the current study, we used an aggressive osteosarcoma cell line, MG63, as a model to explore the anti-osteosarcoma effect of coptisine. We found that coptisine effectively inhibited osteosarcoma cell proliferation, migration, invasion, and angiogenesis in vitro. In vivo, coptisine inhibited tumor growth with very low toxicity, significantly elevated blood erythrocyte and hemoglobin levels, and also moderately increased white blood cell (WBC) and platelet counts. To our knowledge, this is the first report of the anti-osteosarcoma activity of coptisine. 2. Materials and methods 2.1. Materials Coptisine (purity ≥98%) was purchased from Chengdu Mansite Biotechnology Co., Ltd. (Chengdu, China). Other materials are listed in supplemental S1.

2.2. Cell culture Human osteosarcoma MG63 cells were cultured in DMEM (high glucose) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin G and 100 ␮g/mL streptomycin (complete medium) under a humidified atmosphere of 5% CO2 at 37 ◦ C as previously described (Bu et al., 2012). Fresh peripheral blood mononuclear cells (PBMNCs) from five healthy subjects were collected and separated by Ficoll-Hipaque density sedimentation and cultured as we previously reported (Bao et al., 2012; Liu et al., 2012). One human lung micro vessel endothelial cell line (HLMVEC) and three human osteosarcoma cell lines (SW1353, Saos-2, and U2OS) were cultured in the same DMEM (high glucose) complete medium mentioned above.

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cells were analyzed using a flow cytometry (Becton Dickinson FACSCalibur) as we previously reported (Feng et al., 2012; Cao et al., 2013a,b; Liu et al., 2013a,b). 2.6. Cell migration and invasion assays MG63 cells pre-treated with or without the coptisine for 24 h were wounded by a plastic tip. The wounded monolayer was then washed with PBS for three times to remove the detached cells and debris, and incubated with coptisine at concentrations 0–40 ␮M for 24 h and 48 h, respectively. The cells were imaged by OLYMPUS FSX-100 microscope. The relative migration of the cells in the wounded area was counted, and the inhibition ration was assessed by SPSS 16.0 software. Tumor cell invasion assay was carried out using trans-well chamber as we previous reported (Bao et al., 2012), and described in Supplemental Materials S5 in detail. 2.7. In vitro tube formation assay The tumor cell formation of capillary structure in vitro was tested as we previously described (Zhou et al., 2008, 2010; Liu et al., 2012, 2013a,b; Cao et al., 2013a,b). In brief, 0.15 mL Matrigel matrix (without supplement of growth factors) was transferred to a 48well plate, then the cells pre-treated with coptisine in DMEM with 5% FBS for 24 h were re-suspended at a density of 2 × 105 /mL. The cells were transferred to each well containing the Matrigel matrix. After the cells were incubated at 37 ◦ C, 5% CO2 for 8–10 h, the tubes were stained with Wright-Giesma solution and photographed by OLYMPUS FSX-100 microscope. 2.8. RT-PCR Total RNA was extracted from MG63 cells incubated with coptisine for 48 h and treated with RNase-free DNase I. The cDNA was generated by reverse transcription using RevertAidTM First Strand cDNA Synthesis Kit and oligo (dT) as we previously reported (Bao et al., 2012; Cao et al., 2013a,b), and described in Supplemental Materials S6. 2.9. Western blot analysis

2.3. Cell growth and apoptosis assays Cell growth was measured by the AlamarBlue assay in 96-well plates and cell apoptosis was detected as we described before (Feng et al., 2012) and in Supplemental Materials S2 and S3. The IC50 , defined as the drug concentration at which cell growth was inhibited by 50%, was assessed by SPSS 16.0 software. 2.4. Anti-osteosarcoma effect of coptisine in xenografted mice

MG63 cells were collected and the proteins were extracted by M-PER Mammalian Protein Extraction Kit, the protein concentration was determined by the BCA assay. Equal amounts of protein were loaded onto each lane and resolved by SDS-PAGE with Tris-glycine running buffer. The proteins were transferred to nitrocellulose membranes, followed by incubation with primary antibodies, HRP-coupled secondary antibody. The blots were visualized using enhanced chemiluminescence (ECL) detection reagents and exposed to X-ray film as we previously described (Bao et al., 2012; Cao et al., 2013a,b), and described in Supplemental Materials S7.

In brief, 6-week-old female BALB/C nude mice (18–22 g) were randomly divided into the control and coptisine groups (five mice per group). Each mouse was subcutaneously injected with 107 MG63 cells, and then intraperitoneally injected daily with either coptisine at the dose of 50 mg/kg/day or saline as control. Tumor growth was calculated according to the formula: tumor volume = 0.55 × length × width2 . After treatment with coptisine for 24 days, blood samples were collected and solid tumors were excised and analyzed as described in Supplemental Materials S4.

The data shown in this study represented the mean ± standard deviation (SD). Differences between the groups were assessed by one-way ANOVA using SPSS 16.0 software. The significance of differences was indicated as *P < 0.05 and **P < 0.01.

2.5. Cell cycle analysis

3. Results

MG63 cells were grown in T25 flasks and treated with coptisine at indicated concentration for 24 h. After the treatment, the cells were fixed with 70% ethanol overnight at 4 ◦ C, incubated with RNase and 10 mg/mL PI solutions. The DNA contents of PI-stained

To determine whether coptisine had anti-osteosarcoma activity, we first investigated its effect on the growth of four osteosarcoma cell lines. AlamarBlue assays showed that coptisine inhibited the growth of MG63, SW1353, Saos-2, and U-2OS cells with an IC50

2.10. Statistical analysis

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D. Yu et al. / Toxicology Letters 226 (2014) 328–336 Table 1 The effect of COP on the blood parameters of nude mice. Blood index parameters

Control

WBC (×109 /L) LYM (×109 /L) LYM (%) RBC (×1012 /L) HGB (g/dL) MCV (fL) MCH (pg) MCHC (g/dL) RDW-SD (fL) HCT (%) PLT (×109 /L) MPV (fL) PDW (fL) RDW-CV (%)

1.173 0.98 0.6 120.25 7.48 48.45 16.1 332.25 28.43 0.36 490 5.98 6.9 0.15

Coptisine ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.73 0.43 0.22 10.72 0.46 0.75 0.92 22.87 1.52 0.02 274.62 0.28 0.5 0.02

2.22 0.57 0.25 127 8.47 48.52 15.05 310.33 28.43 0.41 773.67 5.8 6.65 0.17

± ± ± ± ± ± ± ± ± ± ± ± ± ±

P-value 0.74 0.31 0.05 3.41 0.19 0.53 0.32 2.73 0.56 0.01 169.6 0.19 0.31 0.01

0.331 0.121 0.005** 0.179 0.001** 0.573 0.03* 0.043* 0.99 0.01* 0.075 0.264 0.349 0.104

The blood parameters were detected by Hematology Analyzer, and the data is shown as mean ± SD. WBC: white blood cell count; RBC: red blood cell; HGB: hemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW-SD: red blood cell distribution width-standard deviation; HCT: hematocrit; PLT: platelet count; MPV: mean platelet volume; PDW: platelet distribution width; RDW-CV: red blood cell volume distribution width. * P < 0.05 ** P < 0.01 versus control.

Fig. 1. Coptisine effectively inhibits human osteosarcoma cell growth in vitro and tumor growth in vivo. Coptisine (COP) is an isoquinoline alkaloid (A). Human osteosarcoma cell MG63 and human lung micro vascular endothelial cells (HLMVEC) were incubated with coptisine at concentrations indicated for 48 h, respectively; the cell growth was measured by AlamarBlue assay (mean ± SD, n = 5), representative of three repeats (B). Nude mice were subcutaneously injected with 107 MG63 cells per mouse, followed by daily treatment of coptisine (50 mg/kg) or saline as a control. The excised tumors were weighted and analyzed using Quantity One Software (mean ± SD, n = 5). *P < 0.05.

of 12.99 ± 0.77, 14.10 ± 2.17, 22.56 ± 2.94, and 28.54 ± 5.71 ␮M, respectively (Supplementary Table S1 and Supplementary Figure S1). Among the four cell lines tested, MG63 was the most sensitive to coptisine. Given the highly aggressive and metastatic characteristics of MG63, we selected these cells as a model to investigate the anti-tumor activity of coptisine and to study its mechanisms. 3.1. Coptisine effectively inhibits osteosarcoma cell proliferation and suppresses tumor growth with very low toxicity Coptisine is an isoquinoline alkaloid with a molecular weight of 355.77 g/mol (Fig. 1A). The AlamarBlue assay showed that coptisine inhibited MG63 cell growth in a dose-dependent manner with an IC50 of 12.99 ± 0.77 ␮M. It moderately affected the growth of human lung microvascular endothelial cells (HLMVEC), with an IC50 of 54.81 ± 8.17 ␮M. Next, we examined whether coptisine induced cell apoptosis. TUNEL assay indicated that coptisine did not induce significant apoptosis at 40 ␮M and promoted slight apoptosis at a higher dose (160 ␮M) (Supplementary Figure S2). These results indicate that coptisine selectively inhibits MG63 osteosarcoma cell growth, but does not have a significant cytotoxic effect. To evaluate the anti-tumor effects of coptisine in vivo, we used a MG63 osteosarcoma xenograft model. Nude mice were subcutaneously injected with MG63 cells followed by daily intraperitoneal injections of coptisine (50 mg/kg body weight) or saline (control). After 24 days of coptisine treatment the average tumor weight in the control and coptisine-treated groups were 42.0 ± 3.3 mg and 7.3 ± 2.1 mg, respectively. The tumor inhibitory rate of coptisine

reached 82.6% (Fig. 1C and D). It is noteworthy that coptisine treatment completely inhibited tumor growth in two of the five xenografted mice (Fig. 1C). The total body weight of the mice was similar between the coptisine-treated and control groups (Supplementary Figure S3). The organ index, which included heart, lung, liver, kidney, and spleen, showed no significant difference between the two groups (Supplemental Figure S4). This suggests that there is no obvious toxic effect of coptisine on mice treated at 50 mg/kg for 24 days. In light of that coptosine does not inhibit the growth of normal human PBMNC at the effective dosages tested, indicative of very low toxic to normal blood cells (Supplementary Figure S5); next, we examined the effect of coptosine on blood cell population in vivo, and found that the coptisine treatment regime significantly increased the red blood cell count (RBC), hemoglobin (HGB) levels, hematocrit (HCT) count, mean corpuscular volume (MCV), and red blood cell distribution width standard deviation (RDW-SD) when compared with the control (Table 1). Notably, all of the blood parameters were still within the normal range suggesting that coptisine improved both erythrocyte quantity and quality. In addition, coptisine mildly elevated the white blood cell count (WBC) and platelets, although it was statistically insignificant. Other hematological indicators were not significantly changed (Table 1). In addition to the anti-osteosarcoma activity, these data imply that coptisine could also be used to stimulate hematopoiesis. The in vitro and in vivo data demonstrate that coptisine exerts a novel anti-osteosarcoma activity with very low toxicity to normal cells and suggest that it could have a beneficial effect on hematopoiesis as a part of an anti-cancer therapy. 3.2. Mechanisms of coptisine-mediated, anti-osteosarcoma cell proliferation To elucidate the mechanism of coptisine-mediated, antiosteosarcoma cell proliferation, we analyzed the cell cycle changes in the absence or presence of coptisine. Coptisine treatment of MG63 cells increased the G0/G1 phase cell population and decreased the G2/M phase cell population in a dosage-dependent manner (Fig. 2A and B). Western-blot analysis showed that coptisine markedly decreased the CDK4 protein levels at doses of 2.5 ␮M or greater. It also reduced cyclin D1 levels at 10 ␮M or higher (Fig. 2C and D). Cyclin D3 levels did not increase until the dose reached

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Fig. 2. Coptisine induces osteosarcoma cell cycle arrest by down-regulation of CDK4 and cyclin D1. After treatment with coptisine at concentrations indicated for 24 h, DNA content of PI-stained MG63 cells was measured by flow cytometry (A), and the cell population in cell cycle phases was analyzed (B); the expression of cell cycle regulatory proteins was detected by Western blot (C). The data of CDK4 and cyclin D1 proteins were statistically analyzed using Quantity One Software (D), representative of three experiments, **P < 0.01.

40 ␮M and CDK6 protein levels were unaffected (Fig. 2C). These data indicate that coptisine induces MG63 osteosarcoma cell cycle arrest at the G0/G1 phase by selective diminution of CDK4 and cyclin D1 proteins. 3.3. Coptisine suppresses osteosarcoma cell invasion and angiogenesis In vitro MG63 cells directly form classic capillary-like networks (tubes) in the absence of coptisine. This effect was partially diminished by 5 ␮M coptisine and markedly suppressed at 10 to

20 ␮M doses (Fig. 3A and C). In addition, coptisine did not obviously inhibit HLMVEC tube formation at 5 ␮M concentration but significantly affected HLMVEC tube formation at 10 ␮M and above (Fig. 3B and C). These data imply that coptisine effectively inhibits tumor cell-dominant vasculogenic mimicry and endothelial cell-mediated angiogenesis, which play important roles in tumor growth and metastasis and lead to poor patient prognosis (Maniotis et al., 1999; Carmeliet and Jain, 2011; Seftor et al., 2012; Cao et al., 2013a,b). Wound healing assays of MG63 showed that the gap readily closed over 48 h in the absence of coptisine, but cell migration was impaired when the cells were treated with coptisine (Fig. 4A and

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Fig. 3. Coptisine suppresses osteosarcoma MG63 cell-mediated tube formation. Osteosarcoma MG63 cells and HLMVEC were treated by the indicated dosages of coptisine for 24 h, and subjected to a tube formation assay (A and B); the capillary-like structures were imaged and analyzed using Quantity One Software as described in Section 2 (C).

B). The invasion of MG63 cells significantly declined in the presence of coptisine. The invasion rate reduced 59.65 ± 6.72% with 10 ␮M coptisine and 73.68 ± 3.51% with 40 ␮M when compared to the control (Fig. 4C and D). In short, these data suggest that coptisine suppresses MG63 osteosarcoma cell angiogenesis, migration, and invasion. To isolate the mechanisms for the suppression of angiogenesis, migration, and invasion we performed semi-quantitative RT-PCR. This revealed that the expression of VE-cadherin and integrin ␤3 mRNAs was markedly inhibited by coptisine at doses of 2.5 ␮M and above (Fig. 5A and B). Other tumor angiogenic genes including VEGFA, MMP2, integrin ␣5 , and integrin ␤5 were slightly affected (Fig. 5A, Supplemental Figure S6). Consistent with the mRNA data, Western blot analysis showed that VE-cadherin protein levels were also reduced by coptisine in MG63 cells (Fig. 5C and D). Collectively, coptisine effectively inhibits MG63 cell invasion and capillary-like structure formation via the suppression of VE-cadherin and integrin ␤3 expression. We investigated the effect of coptisine on intracellular activation of four key signaling proteins including Akt, ERK1/2, NF-␬B, and STAT3. Treatment of MG63 cells with at least 5 ␮M coptisine significantly inhibited EGF-stimulated phosphorylation of STAT3 (Tyr 705) without altering total STAT3 protein levels; whereas, the phosphorylation of Akt (Thr308), ERK1/2 (Thr202/Tyr204), and NF-␬B (Ser536) was not obviously affected (Fig. 5E and F). 4. Discussion Normally cell cycle progression is precisely controlled by several cell cycle regulators (Fry et al., 2012). In most cancer cells CDKs and cyclins are overexpressed and/or activated, leading to uncontrolled

cell proliferation and aggressive tumorigenesis (Bao et al., 2012). In this study, we found that uncontrolled osteosarcoma growth can be effectively suppressed by coptisine through inhibition of CDK4 and cyclin D1 expression, inducing cell cycle arrest at the G0/G1 phase. This indicates that coptisine could potentially be used as an anti-proliferative agent for osteosarcoma. Metastatic osteosarcoma is a prevalent form of bone cancer and patients diagnosed with this disease have a poor prognosis. Currently there are no satisfactory clinical treatments for this disease (Geller and Gorlick, 2010). Tumor angiogenesis plays a pivotal role in osteosarcoma metastasis (Cao et al., 2013a,b). In this investigation, we found that coptisine inhibited osteosarcoma cell migration, invasion, and capillary-like network formation through the downregulation of VE-cadherin and integrin ␤3 expression. Therefore, coptisine could be used to suppress osteosarcoma tumor metastasis. VE-cadherin is a critical marker of tumor neovascularization. Specifically, it is a master gene in tumor cell-dominant vasculogenic mimicry (Wallez et al., 2006; Vestweber, 2008; Liu et al., 2012; Shang et al., 2012; Cao et al., 2013a,b,c). Under normal condition VE-cadherin is expressed exclusively in vascular endothelial cells. This gene is turned on in various tumor cells including the aggressive, metastatic MG63 osteosarcoma cells. Cell surface VE-cadherin acts as a bridge to connect cells and plays pivotal role in adherent junction among the vasculogenic tumor cells. Our data indicate that coptisine markedly suppresses VE-cadherin expression, which may be the main mechanism of coptisine-mediated, angiogenic and vasculogenic suppression effects. Coptisine is a small molecule and very stable. Its inhibitory mechanism is different from current antiangiogenic drugs such as Avastin and Sutent. This would potentially allow the combination of coptisine with traditional anti-angiogenic

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Fig. 4. Coptisine diminishes osteosarcoma MG63 cell migration and invasion. MG63 cell monolayer was wounded by a plastic tip, incubated with coptisine at concentrations indicated for 48 h, and imaged by OLYMPUS FSX-100 microscope (A). The relative migration cells in the wounded area were counted and the inhibitory rate was calculated (B). Cell invasion was performed in a trans-well system, stained with Wright-Giemsa solution, and imaged as described in method section (C); the cell numbers in the bottom well from six randomly chosen areas were counted under microscope, and the inhibitory rate was calculated from three independent experiments (D). *P < 0.05, **P < 0.01.

therapeutics to improve the efficacy of existing osteosarcoma treatments. Integrin ␤3 is an important cell adhesion molecule that promotes adhesion and migration in various cancers including osteosarcoma (Levinson et al., 2002; Karadag et al., 2004). Our study indicates that coptisine downregulates integrin ␤3 expression. This mechanism may explain the coptisine-mediated inhibition of cell migration and invasion in MG63 osteosarcoma cells. STAT3 is overexpressed and activated in many cancers. Robust activation of the STAT3 signaling pathway is associated with tumor progression and poor prognosis in osteosarcoma patients (Wang et al., 2011). Our data show that coptisine hinders the phosphorylation of STAT3. Inhibition of the STAT3 signaling pathway could explain the anti-tumor effect of coptisine. This suggests that coptisine could potentially be a new STAT3 activation inhibitor. Based on our current data, we propose a mechanistic model for the anti-osteosarcoma and anti-neovascularization activities of coptisine (Fig. 6). In brief, coptisine binds to an unknown receptor and inhibits osteosarcoma cell growth by reducing CDK4 and cyclin D1 protein levels leading to G0/G1 phase accumulation. It inhibits osteosarcoma cell-mediated tumor neovascularization by suppressing VE-cadherin and

integrin ␤3 expression, and diminution of the STAT3 signaling pathway. Notably, most anti-cancer chemotherapeutics are cytotoxic drugs. Doxorubicin has been the standard treatment for osteosarcoma for more than 40 years (Meschini et al., 2003). Many studies reported that doxorubicin induces cardiac toxicity in osteosarcoma patients (Longhi et al., 2007), and causes hematological system disorders, such as anemia, bleeding, and myeloid-suppression (Alberts et al., 1976; Kirshner et al., 2004; Longhi et al., 2007). Because of this there is a substantial demand for effective anti-osteosarcoma drugs with low toxicity and side effects. Specifically, an effective anti-cancer drug with cardiac and hematopoietic protection or improvement properties would have significant clinical relevance. In this investigation, we found that coptisine significantly improves both erythrocyte quantity and quality by elevation of RBC and hemoglobin levels (Table 1), while also moderately increasing the numbers of WBC and platelets in the blood. Of note, all of the blood indicators are still within the normal range and coptisine-mediated, mild RBC count elevation is unlikely to increase the risk of thrombosis. Hematopoiesis is a very complex process that involves multiple cell lineages, genes, and signaling pathways, hence, it is warrant to further investigate the beneficial or protective effects of coptisine on the toxicity of chemotherapeutic drugs.

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Fig. 5. Molecular mechanisms of the inhibitory effect of coptisine on MG63 cell-mediated tube formation and cell invasion. The expression of vasculogenic and angiogenic genes was determined by RT-PCR (A). The data of VE-cadherin and integrin ␤3 mRNAs from three independent experiments were statistically analyzed using Quantity One Software (B). VE-cadherin protein was detected by Western blot (C), and the data were also statistically analyzed (D). After incubation without or with coptisine for 48 h at indicated concentrations, MG63 cells were treated for 5 min either without or with 50 ng/mL EGF, the total and phosphorylated proteins of STAT3, Akt, Erk, and NF-␬B were detected by Western blot (E), and the bands were scanned and analyzed by Quantity One software (F); the data were from three independent experiments, *P < 0.05, **P < 0.01.

Collectively, our data indicate that coptisine possesses effective anti-osteosarcoma activity with very low toxicity. Therefore, the combination of coptisine with other chemotherapeutics such as doxorubicin, may act synergistically to increase potency,

decrease side effects such as hematological disorders, and improve the quality of life for cancer patients. Coptisine has the potential to be an excellent anti-osteosarcoma drug candidate.

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Fig. 6. Schematic model for molecular mechanisms of coptisine-mediated anti-osteosarcoma effect. Coptisine might bind to its unknown receptor either on cell member surface or in cell plasma, and it inhibits osteosarcoma cell growth through G0/G1 phase accumulation by reduction of CDK4 and cyclin D1 protein levels; additionally, coptisine impedes osteosarcoma cell-mediated tumor neovascularization by suppressing expression of VE-cadherin and integrin ␤3 , and diminishing STAT3 phosphorylation.

Conflict of interest

and Technology and Jiangsu Province, and Jiangsu Province’s Key Discipline of Medicine (XK201118).

The authors declare that there are no conflicts of interest. References Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgments This study was supported by grants from National Natural Science Foundation of China (Grants No. 81172087, No. 81372376), Suzhou City Scientific Research Funds (No. SS201004 and SS201138), a project funded by the priority academic program development of Jiangsu Higher Education Institutions (PAPD), Research and Innovation Project for College Graduates of Jiangsu Province (CXZZ13 0824), Cultivation base of State Key Laboratory of Stem Cell and Biomaterials built together by Ministry of Science

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