Galangin inhibits human osteosarcoma cells growth by inducing transforming growth factor-β1-dependent osteogenic differentiation

Biomedicine & Pharmacotherapy 89 (2017) 1415–1421

Available online at

ScienceDirect www.sciencedirect.com

Original article

Galangin inhibits human osteosarcoma cells growth by inducing transforming growth factor-b1-dependent osteogenic differentiation Chunhong Liua , Mingming Mab , Junde Zhanga , Shaoliu Guia , Xiaohai Zhanga , Shuangtao Xuea,* a b

Department of Orthopedic Surgery, The Second People’s Hospital of Wuhu, Anhui, China Department of Orthopedic Surgery, The People’s Hospital of Fuyang, Anhui, China

A R T I C L E I N F O

Article history: Received 15 December 2016 Received in revised form 28 February 2017 Accepted 9 March 2017 Keywords: Osteosarcoma Osteogenic differentiation Galangin TGF-b1/Smad2/3 Therapy

A B S T R A C T

Osteosarcoma is the most common primary malignancy of the musculoskeletal system, and is associated with excessive proliferation and poor differentiation of osteoblasts. Currently, despite the use of traditional chemotherapy and radiotherapy, no satisfactory and effective agent has been developed to treat the disease. Herein, we found that a flavonoid natural product, galangin, could significantly attenuate human osteosarcoma cells proliferation, without causing obvious cell apoptosis. Moreover, galangin enhanced the expression of osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin and osteopontin) remarkably and elevated the alkaline phosphatase activity in human osteosarcoma cells. And galangin could also attenuated osteosarcoma growth in vivo. These bioactivities of galangin resulted from its selective activation of the transforming growth factor (TGF)-b1/Smad2/3 signaling pathway, which was demonstrated by pathway blocking experiments. These findings suggested that galangin could be a promising agent to treat osteosarcoma. In addition, targeting TGF-b1 to induce osteogenic differentiation might represent a novel therapeutic strategy to treat osteosarcoma with minimal side effects. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Osteosarcoma, which arises from osteoblasts, is the most common malignancy of the musculoskeletal system in children and adolescents [1]. Despite modern surgical excision combined with chemotherapy and radiotherapy, the long-term survival rate of patients with osteosarcoma is still low: the 5-year survival rate is only 60–70% [2]. Cytotoxic side effects, including cardiac toxicity, nephrotoxicity, infertility, and drug-resistance also challenge current clinical therapy of osteosarcoma [3–5]. Thus, it is important to explore safer and more effective anti-osteosarcoma agents. Abnormal proliferation of osteosarcoma cells and disruption of differentiation are two crucial factors for osteosarcoma formation and development [6]. Histopathologically, more than 80% of osteosarcomas are either poorly differentiated or undifferentiated

* Corresponding author at: 259 Jiuhuazhong Road, Department of Orthopedic Surgery, The Second People’s Hospital of Wuhu, Anhui 241000, China. E-mail addresses: [email protected] (C. Liu), [email protected] (M. Ma), [email protected] (J. Zhang), [email protected] (S. Gui), [email protected] (X. Zhang), [email protected] (S. Xue). http://dx.doi.org/10.1016/j.biopha.2017.03.030 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

[7]. A previous study has shown that the loss of differentiation results in osteosarcoma progression and chemoresistance [8]. By contrast, osteogenic differentiation of osteosarcoma cells inhibits cell proliferation and tumor metastasis [9]. Therefore, novel therapies based on the induction of cell differentiation might represent a promising approach for osteosarcoma treatment. Galangin (3,5,7-trihydroxyflavone), a member of the flavonol subclass of flavonoids, is one of the main active components of Alpinia officinarum, a plant that has been used as a herbal medicine for a variety of ailments [10]. Modern pharmacological studies have shown that galangin has several pharmacological effects, such as anti-inflammatory [11] and anti-obesity effects [12], tumor growth inhibition [13], and attenuation of liver fibrosis induced by CCl4 in rats [14]. In orthopedic research, galangin can inhibit osteoclastic bone destruction and osteoclastogenesis by suppressing nuclear factor kappa B (NF-kB) in collagen-induced arthritis and bone marrow-derived macrophages [15]. Recently, several flavonoids (e.g., quercitrin and taxifolin), which have similar molecular structures to galangin, were shown to stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells [16]. Meanwhile, Wang et al. showed that galangin could activate the transforming growth

1416

C. Liu et al. / Biomedicine & Pharmacotherapy 89 (2017) 1415–1421

factor (TGF)-b1/Smads pathway [17], which plays crucial roles in bone tissue formation and remodeling [18]. Given this, we wondered what effect galangin would have on osteosarcoma cells and whether galangin could attenuates osteosarcoma cells growth in vivo. Thus, we examined its effects on proliferation and osteogenic differentiation in human osteosarcoma cells, and tested its function in the xenograft mouse model. We further investigated the potential biochemical mechanism that may be involved in the positive effects of galangin on osteosarcoma trans-differentiation.

50 -ACGTCAATGTCCCTGATGTTATG-30 (reverse); osteocalcin (OCN), 50 -CACTCCTCGCCCTATTGGC-30 (forward) and 50 -CCCTCCTGCTTGGACACAAAG-30 (reverse); osteopontin (OPN), 50 -GAAGTTTCGCAGACCTGACAT-30 (forward) and 50 -GTATGCACCATTCAACTCCTCG-30 (reverse); TGF-b1, 50 -CTAATGGTGGAAACCCACAACG-30 (forward) and 50 -TATCGCCAGGAATTGTTGCTG-30 (reverse); bone morphogenetic protein 2 (BMP-2), 50 -ACCCGCTGTCTTCTAGCGT-30 (forward) and 50 -TTTCAGGCCGAACATGCTGAG-30 (reverse). 2.6. Western blotting

2. Materials and Methods 2.1. Cell culture Human osteosarcoma cell lines MG-63 and U2-OS were purchased from the ATCC (American Type Culture Collection). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/ml penicillin, and 100 mg/L streptomycin. Both MG-63 and U2-OS cells were incubated at 37
C in a humidified atmosphere with 5% CO2. 2.2. MTT assay The thiazolyl blue tetrazolium bromide (MTT) assay measures mainly mitochondrial activity, which is representative of cell proliferation activity [19]. For MG-63 and U2-OS cells, 100 ml of cells (2  105 cells/ml) were seeded in a 96-well plate and treated with varying concentrations of galangin (cat. no. 282200; Sigma-Aldrich, St. Louis, MO, USA) or dimethyl sulfoxide (DMSO, Sigma-Aldrich). After incubation periods of 24, 48, or 72 h, the MTT assay was performed. The absorbance of the resulting solutions was read at 490 nm in a microplate reader (Tecan Group AG, Männedorf, Switzerland). 2.3. Colony Formation Anchorage-dependent growth of cells was investigated using a monolayer colony formation assay [20]. Cells were cultured in a 6-well plate (500 per well) and treated with 25–100 mM galangin or DMSO. After culture for 14 days, the surviving colonies were fixed with 4% paraformaldehyde and stained for 5 min with Gentian Violet (Sigma-Aldrich). 2.4. Cell apoptosis assay Cell apoptosis was assessed by flow cytometry using the Alexa Fluor1 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The stained cells were analyzed directly by flow cytometry using the Cell Quest Pro software (BD Biosciences, San Jose, CA). 2.5. RNA Isolation and Quantitative Real-Time PCR (qPCR) Total RNA from cells was isolated using the PureLink1 RNA Mini Kit (Invitrogen) and subjected to reverse transcription using an Oligo (dT) primer and M-MLV Reverse Transcriptase (Thermo Fisher Scientific). Quantitative PCR amplification was performed using the ViiATM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the SYBR quantitative PCR SuperMix W/ROX (Invitrogen). The primers used for PCR were as follows: collagen type I (Col I), 50 -GTGCGATGACGTGATCTGTGA-30 (forward) and 50 -CGGTGGTTTCTTGGTCGGT-30 (reverse); alkaline phosphatase (ALP), 50 -ACTGGTACTCAGACAACGAGAT-30 (forward) and

Osteosarcoma cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentrations were determined using the micro bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Equal amounts of protein samples (100 mg) were loaded and electrophoresed through 10% SDSpolyacrylamide gels and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was then blocked, incubated with primary antibodies overnight at 4
C, followed by washing and incubation with peroxidase-conjugated secondary antibodies for 2 hours at room temperature. The primary antibodies were as follows: anti-Col I (Millipore, Bedford, MA, USA, 1:1000), anti-OCN, anti-OPN, antiRunx2 (Abcam, Cambridge, UK, 1:1000), anti-Smads (Cell Signaling Technology, Beverly, MA, 1:1000). Immunoreactive bands were analyzed quantitatively using the Image J software. 2.7. Alkaline phosphatase (ALP) activity Osteosarcoma cells were seeded into a 12-well plate at 2  105 cells per well and treated with varying concentrations of galangin or DMSO for 72 h. Cells were harvested using 0.2% Nonidet P-40 and further disrupted by sonication for 10 seconds. Cell lysates were incubated with 10 mM p-nitrophenylphosphate (SigmaAldrich) and alkaline buffer containing 100 mM diethanolamine and 0.5 mM MgCl2 (Sigma-Aldrich) at 37
C for 30 min. The absorbance of each sample was read at 490 nm in a microplate reader (Tecan Group AG). 2.8. Enzyme linked immunosorbent assay (ELISA) The TGF-b1 and BMP-2 expression levels of osteosarcoma cells were determined using the Human TGF-b1 Quantikine1 ELISA Kit (R&D, Minneapolis, MN, USA) and the BMP-2 Quantikine1 ELISA Kit (R&D, Minneapolis, MN, USA), according to the manufacturer’s instructions. Based on the color reaction of the cytoplasm extract and antibodies, the absorbance values were detected at 450 nm in a microplate reader (Tecan Group A). 2.9. In vivo tumor inhibition assay Male BALB/c nude mice (4-6 weeks old; Slac, Shanghai, China) with an average body weight of 18 g were housed under a 12 h light/dark cycle with free access to food and water for 10 days prior to the experiment. All procedures were approved by the Institutional Animal Care and Use Committee of Anhui Medical University. The 1 107 MG-63 cells were subcutaneously injected into the flank and allowed to grow until tumors reached an average size of 80 mm3. Twenty-four tumor-bearing mice were equally randomized into three groups, with one group of mice were injected intraperitoneally with 0.5 ml vehicle (sterile saline plus 1% DMSO) while other two groups received 50 or 100 mg/kg galangin once a day for 4 weeks. Since galangin is practically insoluble in water, we obtained the galangin suspension through ultrasonic

C. Liu et al. / Biomedicine & Pharmacotherapy 89 (2017) 1415–1421

fragmentation, centrifugation and mixing with vortexer in order to dissolve as much galangin as possible. The vehicle was preheated at 42
C and the pH was adapted to 6-7 using sodium bicarbonate. The suspension was prepared fresh at the time of use. At the end of experiments, mice were sacrificed and tumor tissues were shown. Tumor volume was calculated as: (length  width2)/2. 2.10. Statistical Analysis Data were analyzed using Friedman’s analysis of variance (ANOVA) with a Bonferroni post hoc test, as appropriate. Quantitative data were processed by SPSS 20.0 (SPSS, Chicago, IL, USA) and expressed as the mean  SD. p values less than 0.05 were considered statistically significant.

1417

3. Results 3.1. Galangin inhibited cell proliferation, but did not induce apoptosis of osteosarcoma cells To study the anti-cancer effects of galangin (Fig. 1A) on osteosarcoma, galangin was administered to human osteosarcoma MG-63 and U-2 OS cells at different concentrations (25–100 mM) for 24, 48, or 72 h. MTT assays showed that galangin inhibited cell proliferation significantly in MG-63 and U-2 OS cells in a dosedependent manner (Fig. 1B), with IC50 values of 67.32 and 57.09 mM (72 h), respectively (Fig. 1C). The colony formation assay showed the same trend as the MTT assay. When MG-63 and U-2 OS cells were incubated with galangin for 14 days, their colony forming abilities were suppressed potently compared with the

Fig. 1. Galangin attenuated the proliferation of osteosarcoma cells. (A) The chemical structure of galangin. (B, C) MG-63 and U-2 OS cells were treated with serial concentrations of galangin for 24, 48 or 72 h and the IC50 (72 h) was determined using the MTT assay. (D) Both cell lines in were incubated with galangin for 14 days, and viable clones were stained with crystal violet. (E) Apoptosis was evaluated after treating MG-63 and U-2 OS cells with galangin or DMSO for 72 h. The flow cytometry profile represents Alexa Fluor1 488 Annexin V staining on the x-axis and PI on the y-axis. Data are presented as the mean  SD, NS = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

1418

C. Liu et al. / Biomedicine & Pharmacotherapy 89 (2017) 1415–1421

DMSO control (Fig. 1D). However, based on the flow cytometry analysis, no significant difference in the percentages of apoptotic cells was observed after exposure to galangin, indicating that galangin had limited cytotoxicity to osteosarcoma cells (Fig. 1E). 3.2. Galangin stimulated osteogenic differentiation of osteosarcoma cells We next investigated whether galangin could induce osteogenic differentiation in human osteosarcoma cells. Galangin was administered to MG-63 and U-2 OS cells at 25–100 mM for 3 days. QPCR analysis demonstrated that the mRNA levels of Col I, ALP, OPN, and OC, four confirmed osteoblastic differentiation markers [21], were upregulated remarkably in a dose-dependent fashion (Fig. 2A). Moreover, the protein levels of Col I, OPN, and OC increased dramatically in a similar manner to the changes in mRNA levels (Fig. 2B). Consistently, the ALP activity in both osteosarcoma cell lines was enhanced by galangin incubation compared with the DMSO control (Fig. 2C). Previous studies have proven that Runx2 is a crucial transcription factor of osteoblast differentiation [22]. Our western blotting analysis showed that galangin treatment could significantly elevate the protein level of Runx2 in a dosedependent manner (Fig. 2D). These results revealed that the defect in differentiation in osteosarcoma cells was restored by galangin treatment. 3.3. Galangin upregulated TGF-b1 production and the phosphorylation of Smad2 and Smad3 in osteosarcoma cells

the expression of BMP-2. We then studied the downstream signaling pathway of TGF-b1 and BMP-2. Western blotting analysis showed that phosphorylation of Smad2 and Smad3 was significantly upregulated in MG-63 and U-2 OS cells incubated with galangin (25–100 mM) for 72 h (Fig. 3C). However, the phosphorylation of Smad1/5/8, the downstream signaling protein of BMP-2, was not affected by galangin (Fig. 3D). These results indicated that galangin could activate the TGF-b1/Smad2/3 signaling pathway potently, which might lead to osteogenic differentiation of osteosarcoma cells. 3.4. TGF-b1/Smad2/3 is critical for galangin-mediated osteoblastic differentiation of osteosarcoma cells To further determine whether the galangin-induced osteogenic differentiation is dependent on the activation of the TGF-b1/ Smad2/3 signaling pathway, a specific inhibitor of this pathway, SB431542, was used. When osteosarcoma cells were incubated with galangin and SB431542 for 72 h, the inhibitory effect of galangin on cell growth was remarkably blocked (Fig. 4A). Moreover, the upregulation by galangin of the osteogenic biomarkers was almost complete abolished by SB431542 (Fig. 4B), and the enhancement of ALP activity was also blocked in a dose dependent manner (Fig. 4C). As shown in Fig. 4D, galangin-induced expression of Runx2 in both cell lines was diminished in the presence of SB431542. These data demonstrated that the cell growth inhibition and osteoblastic differentiation activities of galangin are mediated via the TGF-b1/Smad2/3 signaling pathway.

As two key cytokines regulating osteogenic differentiation, TGF-

b1 and BMP-2 have been reported to have a significant impact on

3.5. Galangin inhibited osteosarcoma growth in vivo

bone development [23]. Therefore, to further explore the underlying mechanism of how galangin induced osteosarcoma cell differentiation, the TGF-b1/Smad2/3 and BMP-2/Smad1/5/8 signaling pathways were analyzed. As shown in Fig. 3A and B, both the mRNA and protein levels of TGF-b1 increased gradually with increasing galangin concentrations, while galangin did not affect

To detect the anti-osteosarcoma effects of galangin in vivo, MG63 cells were subcutaneously injected into nude mice. After 28 days of galangin (50 and 100 mg/kg) administration, the tumor volume was obviously decreased at all examined time points compared with DMSO-treated mice (Fig. 5A, B). Galangin at

Fig. 2. Galangin induced osteogenic differentiation of osteosarcoma cells. (A) Dose-dependent effects of galangin on Col I, ALP, OPN, and OC mRNA expressions for 3 days and (B) the protein level of Col I, OPN, and OC at 5 days after galangin treatment. (C) ALP activity was examined after osteosarcoma cells were treated with galangin for 72 h. (D) The protein levels of Runx2 in MG-63 and U-2 OS cells after incubation with a series of concentrations of galangin for 5 days. Data are presented as the mean  SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

C. Liu et al. / Biomedicine & Pharmacotherapy 89 (2017) 1415–1421

1419

Fig. 3. Galangin increased TGF-b1 production and phosphorylation of Smad2 and Smad3 in osteosarcoma cells. (A) The mRNA levels of TGF-b1 and BMP-2 in both cell lines after incubation with galangin or DMSO for 72 h. (B) Osteosarcoma cells were treated with a series of concentrations of galangin for 72 h. The supernatant was collected to test TGF-b1 and BMP-2 production by ELISA. (C, D) Levels of phosphorylated and total Smad2, Smad3 and Smad1/5/8 were examined by western blotting after MG-63 and U-2 OS cells were incubated with 25 to 100 mM galangin or DMSO for 72 h. Data are presented as the mean  SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 4. TGF-b1/Smad2/3 is critical for galangin-mediated osteoblastic differentiation. (A) MTT assay in cultured MG-63 and U-2 OS cells after incubation with SB431542 and galangin at the indicated doses for 72 h. (B) Levels of Col I, OPN, and OC were examined by western blotting after both cell lines were incubated with SB431542 and galangin for 5 days. (C) ALP activity was examined after osteosarcoma cells were treated with SB431542 and galangin for 72 h. (D) The protein levels of Runx2 in both cell lines after incubation with SB431542 and galangin for 5 days. Data are presented as the mean  SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

100 mg/kg was more effective than the dose of 50 mg/kg (Fig. 5A, B). The results indicated that galangin significantly attenuated tumor growth in vivo. 4. Discussion Osteosarcoma is one of the most aggressive bone cancers and typically affects children and young adults. Although neo-adjuvant and adjuvant chemotherapies have improved the 5-year survival

rate, the prognosis for osteosarcoma remains poor [24]. Unfortunately, common chemotherapeutic agents frequently cause both acute and long-term toxicity [25]. Recently, differentiation therapy has been shown to hold great promise for cancer treatment and has yielded remarkable outcomes in certain malignancies [26]. For instance, all-trans retinoic acid (ATRA)-based differentiation therapy has become a standard first-line treatment in acute promyelocytic leukemia [27]. In solid tumors, retinoic acid could increase IkB kinase a (IKKa) expression by suppressing EZH2-

1420

C. Liu et al. / Biomedicine & Pharmacotherapy 89 (2017) 1415–1421

Fig. 5. Galangin inhibited osteosarcoma growth in vivo. (A) Images of tumors at day 28 after galangin (50 and 100 mg/kg) or DMSO administration. (B) Tumor volume was measured using a caliper at indicated time points. Data are presented as the mean  SD, * p < 0.05; ** p < 0.01; *** p < 0.001.

mediated H3K27 histone methylation, resulting in enhanced differentiation of nasopharyngeal carcinoma cells [28]. A lack of cellular differentiation is a key feature of osteosarcoma, which it is related markedly to osteosarcoma formation and progress [29]; therefore, induction of terminal differentiation might represent a potential strategy against osteosarcoma. In this study, we identified galangin as a novel anti-osteosarcoma compound. Our results indicated that galangin significantly inhibited the proliferation of osteosarcoma cells in a dose and time-dependent manner. However, in contrast to its anti-tumor mechanism in other malignancies [30], galangin did not induce apoptosis of osteosarcoma cells directly. Col I and ALP are two typical early osteoblastic differentiation markers, and their expression levels are much lower in osteosarcoma cells compared with those in normal osteoblasts [31]. As biomarkers of late stage of osteoblastic differentiation, the expression levels of OPN and OC increase with increasing mineralization [32]. Surprisingly, we revealed that after incubation with galangin, the expression levels of these four well-documented markers for osteogenic differentiation were upregulated remarkably. Furthermore, the expression of Runx2, an important transcription factor of osteogenic differentiation [22], increased dose dependently with increasing galangin concentrations. These results revealed that galangin induced significant osteogenic differentiation of osteosarcoma cells. Many nature-derived agents have been reported to have inhibitory effects of tumor growth and metastasis [33]. However, the anti-tumor activities of most natural compounds were attributed to their cytotoxicity [34]; only a few of them were able to stimulate tumor cell differentiation directly. Tachyplesin could reverse the malignant morphological and ultrastructural characteristics effectively, and regulated the expression of differentiation-associated oncogenes and tumor suppressor genes, consequently inducing hepatocarcinoma cell differentiation [35]. Shi et al. showed that Ginsenoside Rg1 could induce the differentiation of osteosarcoma MG-63 cells via suppressing prohibitin expression and enhancing its translocation from the nucleus to the cytoplasm [36]. We also revealed that the biochemical mechanism of how galangin induces osteosarcoma cell differentiation was dependent on its selective activation of the TGF-b1/Smad2/3 signaling pathway. There are several cytokines that control bone formation, among which TGF-b1 has been proven to be fundamental to osteoblastic differentiation and bone matrix synthesis, through Smads-dependent signaling [37]. Our data showed that TGF-b1 secretion was triggered in a dose-dependent manner after galangin treatment. The TGF-b1/Smads signaling cascade is initiated when TGF-b1 binds to its specific receptors Anaplastic lymphoma

kinases (ALKs) and triggers ALK-mediated phosphorylation of Smad2/3 [38]. Phosphorylated Smad2/3 then form a complex with Smad4 and enter the nucleus, where the complex acts as a transcription factor to control gene expression [38]. In the process of bone development, the Smads complexes interact with Runx2 to induce osteoblast-specific gene expression[37]. In the present study, we also analyzed the BMP-2/Smad1/5/8 signaling pathway, another crucial pathway in osteoblast differentiation [39]. However, galangin does not appear regulate the BMP-2 expression level or Smad1/5/8 phosphorylation. Although these findings are noteworthy, some limitations in our research must be recognized. In future work, these findings should be replicated in other cell lines and validated models of osteosarcoma. Moreover, future investigations are still essential to elucidate the precise mechanisms of how galangin activates the TGF-b1/Smad2/3 signaling pathway in osteosarcoma cells. Taken together, our results demonstrated that galangin could effectively attenuate cell proliferation, induce osteogenic differentiation of human osteosarcoma cells and inhibit osteosarcoma growth in vivo. Furthermore, our study revealed that these bioactivities of galangin were resulted from the activation of TGF-b1/Smad2/3 signaling pathway. These findings suggested that galangin could serve as a promising agent for osteosarcoma therapy, and that targeting TGF-b1 to induce osteogenic differentiation might represent a novel strategy for the treatment of osteosarcoma. Conflict of interest The authors declare that they have no conflict of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgment We are grateful to Dr. Bo Xie for constructive comments on the manuscript. References [1] L.J. Helman, P. Meltzer, Mechanisms of sarcoma development, Nat. Rev. Cancer 3 (9) (2003) 685–694. [2] A. Longhi, C. Errani, M. De Paolis, M. Mercuri, G. Bacci, Primary bone osteosarcoma in the pediatric age: state of the art, Cancer Treat. Rev. 32 (6) (2006) 423–436.

C. Liu et al. / Biomedicine & Pharmacotherapy 89 (2017) 1415–1421 [3] W.S. Ferguson, A.M. Goorin, Current treatment of osteosarcoma, Cancer Invest. 19 (3) (2001) 292–315. [4] A.J. Chou, R. Gorlick, Chemotherapy resistance in osteosarcoma: current challenges and future directions, Expert. Rev. Anticancer Ther. 6 (7) (2006) 1075–1085. [5] J.C. Clark, C.R. Dass, P.F. Choong, A review of clinical and molecular prognostic factors in osteosarcoma, J. Cancer Res. Clin. Oncol. 134 (3) (2008) 281–297. [6] N. Zhang, M.D. Ying, Y.P. Wu, Z.H. Zhou, Z.M. Ye, H. Li, D.S. Lin, Hyperoside, a flavonoid compound, inhibits proliferation and stimulates osteogenic differentiation of human osteosarcoma cells, PLoS one 9 (7) (2014) e98973. [7] D. Thomas, M. Kansara, Epigenetic modifications in osteogenic differentiation and transformation, J. Cell. Biochem. 98 (4) (2006) 757–769. [8] M. Ying, L. Zhang, Q. Zhou, X. Shao, J. Cao, N. Zhang, W. Li, H. Zhu, B. Yang, Q. He, The E3 ubiquitin protein ligase MDM2 dictates all-trans retinoic acid-induced osteoblastic differentiation of osteosarcoma cells by modulating the degradation of RARalpha, Oncogene (2016), doi:http://dx.doi.org/10.1038/ onc.2015.503. [9] T. Quist, H. Jin, J.F. Zhu, K. Smith-Fry, M.R. Capecchi, K.B. Jones, The impact of osteoblastic differentiation on osteosarcomagenesis in the mouse, Oncogene 34 (32) (2015) 4278–4284. [10] C.H. Jung, S.J. Jang, J. Ahn, S.Y. Gwon, T.I. Jeon, T.W. Kim, T.Y. Ha, Alpinia officinarum inhibits adipocyte differentiation and high-fat diet-induced obesity in mice through regulation of adipogenesis and lipogenesis, J. Med. Food 15 (11) (2012) 959–967. [11] Y.C. Jung, M.E. Kim, J.H. Yoon, P.R. Park, H.Y. Youn, H.W. Lee, J.S. Lee, Antiinflammatory effects of galangin on lipopolysaccharide-activated macrophages via ERK and NF-kappaB pathway regulation, Immunopharmacol. Immunotoxicol. 36 (6) (2014) 426–432. [12] S. Kumar, K.R. Alagawadi, Anti-obesity effects of galangin, a pancreatic lipase inhibitor in cafeteria diet fed female rats, Pharm Biol. 51 (5) (2013) 607–613. [13] M.A. Han, D.H. Lee, S.M. Woo, B.R. Seo, K.J. Min, S. Kim, J.W. Park, S.H. Kim, Y.H. Choi, T.K. Kwon, Galangin sensitizes TRAIL-induced apoptosis through downregulation of anti-apoptotic proteins in renal carcinoma Caki cells, Sci. Rep. 6 (2016) 18642. [14] X. Wang, G. Gong, W. Yang, Y. Li, M. Jiang, L. Li, Antifibrotic activity of galangin, a novel function evaluated in animal liver fibrosis model, Environ Toxicol. Pharmacol. 36 (2) (2013) 288–295. [15] J.E. Huh, I.T. Jung, J. Choi, Y.H. Baek, J.D. Lee, D.S. Park, D.Y. Choi, The natural flavonoid galangin inhibits osteoclastic bone destruction and osteoclastogenesis by suppressing NF-kappaB in collagen-induced arthritis and bone marrow-derived macrophages, Eur. J. Pharmacol. 698 (1-3) (2013) 57–66. [16] M. Satue, M. Arriero Mdel, M. Monjo, J.M. Ramis, Quercitrin and taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells, Biochem. Pharmacol. 86 (10) (2013) 1476–1486. [17] Y. Wang, J. Wu, B. Lin, X. Li, H. Zhang, H. Ding, X. Chen, L. Lan, H. Luo, Galangin suppresses HepG2 cell proliferation by activating the TGF-beta receptor/Smad pathway, Toxicology 326 (2014) 9–17. [18] G. Zhen, C. Wen, X. Jia, Y. Li, J.L. Crane, S.C. Mears, F.B. Askin, F.J. Frassica, W. Chang, J. Yao, J.A. Carrino, A. Cosgarea, D. Artemov, Q. Chen, Z. Zhao, X. Zhou, L. Riley, P. Sponseller, M. Wan, W.W. Lu, X. Cao, Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis, Nat. Med. 19 (6) (2013) 704–712. [19] D. Gerlier, N. Thomasset, Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods 94 (1-2) (1986) 57–63. [20] H. Jin, X. Wang, J. Ying, A.H. Wong, Y. Cui, G. Srivastava, Z.Y. Shen, E.M. Li, Q. Zhang, J. Jin, S. Kupzig, A.T. Chan, P.J. Cullen, Q. Tao, Epigenetic silencing of a Ca (2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers, Proc. Natl. Acad. Sci. U. S. A. 104 (30) (2007) 12353–12358. [21] A. Ongaro, A. Pellati, L. Bagheri, P. Rizzo, C. Caliceti, L. Massari, M. De Mattei, Characterization of Notch Signaling During Osteogenic Differentiation in

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37] [38] [39]

1421

Human Osteosarcoma Cell Line MG63, J. Cell. Physiol. (2016), doi:http://dx.doi. org/10.1002/jcp.25366. Y. Li, C. Ge, R.T. Franceschi, MAP Kinase-Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic Modification of Chromatin During Osteoblast Differentiation, J. Cell. Physiol. (2016), doi:http://dx.doi.org/10.1002/jcp.25517. A. Turri, I. Elgali, F. Vazirisani, A. Johansson, L. Emanuelsson, C. Dahlin, P. Thomsen, O. Omar, Guided bone regeneration is promoted by the molecular events in the membrane compartment, Biomaterials 84 (2016) 167–183. N. Tang, W.X. Song, J. Luo, R.C. Haydon, T.C. He, Osteosarcoma development and stem cell differentiation, Clin. Orthop. Relat. Res. 466 (9) (2008) 2114–2130. F. Ye, H. Wang, L. Zhang, Y. Zou, H. Han, J. Huang, Baicalein induces human osteosarcoma cell line MG-63 apoptosis via ROS-induced BNIP3 expression, Tumour Biol. 36 (6) (2015) 4731–4740. P. Luo, X. Yang, M. Ying, P. Chaudhry, A. Wang, H. Shimada, W.A. May, G.B. Adams, D. Mock, T.J. Triche, Q. He, L. Wu, Retinoid-suppressed phosphorylation of RARalpha mediates the differentiation pathway of osteosarcoma cells, Oncogene 29 (19) (2010) 2772–2783. M.S. Tallman, J.W. Andersen, C.A. Schiffer, F.R. Appelbaum, J.H. Feusner, A. Ogden, L. Shepherd, C. Willman, C.D. Bloomfield, J.M. Rowe, P.H. Wiernik, Alltrans-retinoic acid in acute promyelocytic leukemia, N. Engl. J. Med. 337 (15) (1997) 1021–1028. M. Yan, Y. Zhang, B. He, J. Xiang, Z.F. Wang, F.M. Zheng, J. Xu, M.Y. Chen, Y.L. Zhu, H.J. Wen, X.B. Wan, C.F. Yue, N. Yang, W. Zhang, J.L. Zhang, J. Wang, Y. Wang, L.H. Li, Y.X. Zeng, E.W. Lam, M.C. Hung, Q. Liu, IKKalpha restoration via EZH2 suppression induces nasopharyngeal carcinoma differentiation, Nat. Commun. 5 (2014) 3661. R.C. Haydon, H.H. Luu, T.C. He, Osteosarcoma and osteoblastic differentiation: a new perspective on oncogenesis, Clin. Orthop. Relat. Res. 454 (2007) 237–246. J. Cao, H. Wang, F. Chen, J. Fang, A. Xu, W. Xi, S. Zhang, G. Wu, Z. Wang, Galangin inhibits cell invasion by suppressing the epithelial-mesenchymal transition and inducing apoptosis in renal cell carcinoma, Mol. Med. Rep. 13 (5) (2016) 4238–4244. E.R. Wagner, G. Luther, G. Zhu, Q. Luo, Q. Shi, S.H. Kim, J.L. Gao, E. Huang, Y. Gao, K. Yang, L. Wang, C. Teven, X. Luo, X. Liu, M. Li, N. Hu, Y. Su, Y. Bi, B.C. He, N. Tang, J. Luo, L. Chen, G. Zuo, R. Rames, R.C. Haydon, H.H. Luu, T.C. He, Defective osteogenic differentiation in the development of osteosarcoma, Sarcoma 2011 (2011) 325238. C. Higuchi, A. Myoui, N. Hashimoto, K. Kuriyama, K. Yoshioka, H. Yoshikawa, K. Itoh, Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix, J. Bone Miner. Res. 17 (10) (2002) 1785–1794. V. Ravichandiran, K. Masilamani, B. Senthilnathan, A. Maheshwaran, W.T. Wui, P. Roy, Quercetin-decorated curcumin liposome design for cancer therapy: invitro and in-vivo studies, Curr. Drug Deliv. (2016), doi:http://dx.doi.org/ 10.2174/1567201813666160829100453. M.C. Cathcart, Z. Useckaite, C. Drakeford, V. Semik, J. Lysaght, K. Gately, K.J. O'Byrne, G.P. Pidgeon, Anti-cancer effects of baicalein in non-small cell lung cancer in-vitro and in-vivo, BMC Cancer 16 (2016) 707. G.L. Ouyang, Q.F. Li, X.X. Peng, Q.R. Liu, S.G. Hong, Effects of tachyplesin on proliferation and differentiation of human hepatocellular carcinoma SMMC7721 cells, World J. Gastroenterol. 8 (6) (2002) 1053–1058. S.L. Shi, Q.F. Li, Q.R. Liu, D.H. Xu, J. Tang, Y. Liang, Z.L. Zhao, L.M. Yang, Nuclear matrix protein, prohibitin, was down-regulated and translocated from nucleus to cytoplasm during the differentiation of osteosarcoma MG-63 cells induced by ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT), J. Cell. Biochem. 108 (4) (2009) 926–934. K. Janssens, P. ten Dijke, S. Janssens, W. Van Hul, Transforming growth factorbeta1 to the bone, Endocr. Rev. 26 (6) (2005) 743–774. C.H. Heldin, K. Miyazono, P. ten Dijke, TGF-beta signalling from cell membrane to nucleus through SMAD proteins, Nature 390 (6659) (1997) 465–471. G. Chen, C. Deng, Y.P. Li, TGF-beta and BMP signaling in osteoblast differentiation and bone formation, Int. J. Biol. Sci. 8 (2) (2012) 272–288.