Velvet Antler Compounds Targeting Major Cell Signaling

Open Chem., 2019; 17: 235–245

Review Article

Zhengyao Zhang, Pengfei Li, Tie Li, Changwei Zhao, Guoxiang Wang* Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma - a new insight into mediating the process of invasion and metastasis in OS https://doi.org/10.1515/chem-2019-0028 received May 17, 2018; accepted September 6, 2018. 1 Introduction

Abstract: Velvet antler is the only renewable bone tissue Osteosarcoma, derived from primitive bone-forming of mammalian animals, which consists of a variety mesenchymal cells, is the most common primary bone of growth factors, amino acids and polypeptides. But malignancy that occurs most frequently in adolescents the mechanism of high-speed proliferation without and elders over the age of 60 [1]. Osteosarcoma most carcinogenesis is still mystifying. The previous study frequently affects the growing ends of long bones and is of this work found that the velvet antler peptides (VAP) often located adjacent to joints. Approximately one-half could not only inhibit the proliferation and migration of all osteosarcomas affects the knee region, with the of osteosarcoma cell lines MG-63 and U2OS, but also distal femur being the most commonly affected site [2]. induced U2OS apoptosis and inhibited MG-63 epithelial- Osteosarcoma is characterized by destruction of bone mesenchymal transition (EMT) through TGF-β and Notch and soft tissue and is highly likely to be accompanied by pathways. These results lead us to conclude that VAP has metastasis of the cancer cells to the distal organ, where the potential ability to mediate osteosarcoma cells by metastasis to the lung accounts for approximately 80%, regulating related signaling pathways and growth factors. with a poor prognosis. If clinical metastasis occurs, the Therefore, finding a new appropriate inhibitor for OS is patient’s five-year survival rate is between 20%-30% by the a valuable research direction, which will give patients a combination of conventional surgery and chemotherapy better chance to receive proper therapy. From an applied [3]. Deer velvet antler is one of the most important perspective, this review summarized the effects of velvet conventional Chinese medicines, the application of antler, genes, growth factors and research progress of which was started two thousand years ago. It has been relative pathways and genes of osteosarcoma, which are extensively used in traditional Chinese medicine (TCM) to poised to help link regenerative molecular biology and treat a variety of diseases including degenerative disease regenerative medicine in osteosarcoma pathogenesis. (osteoarthritis), auto-immune or auto-inflammatory processes (rheumatoid arthritis and ankylosing Keywords: osteosarcoma; velvet antler; growth factors; spondylitis), infection (septic arthritis), idiopathic pathways; EMT. (juvenile idiopathic arthritis) and kidney diseases and so on. In a decade, researchers have extracted velvet antler peptides (VAP) by using ion exchange chromatography, gel filtration chromatography and high-performance

*Corresponding author: Guoxiang Wang, Cancer Center, The First liquid chromatography. Hence, the velvet antler contains Hospital of Jilin University, Changchun Jilin 130021, China, E-mail: a lot of growth factors such as insulin-like growth factor [email protected]; (IGF), nerve growth factor (NGF), epidermal growth Changwei Zhao, Department of Orthopedics, Changchun University factor (EGF) and transforming growth factor (TGF), of Chinese Medicine, Changchun Jilin 130021, China which have different influences on osteosarcoma cells Zhengyao Zhang, Pengfei Li: School of Life Science and Medicine, [4,5]. In addition, the progression of osteosarcoma is Dalian University of Technology, DaGong Road, Panjin Liaoning 124221, China regulated by signaling pathways such as TGF-β, which Tie Li: Acupuncture and Tuina Institute, Changchun University of can mediate the pro-apoptosis effects by doxorubicin [6] Chinese Medicine, Changchun Jilin 130021, China , and the inhibition of the Notch pathway can suppresses

Table 1: Summary of the validated growth factors in preclinical experiments of osteosarcoma.

Growth factors Target Functions in cancer

Sirtuin 6[17] MMP9 Migration and invasion ERK1/2

Fractalkine[18] CX3CR1 Osteosarcoma metastasis

EZH2[19] TUG1 tumorigenesis

NKD2[20] unclear Tumor growth and metastasis

HIF1α[21] COX2 Carcinogenesis

VEGF Metastasis

Endocannabinoid/Endovanilloid [22] CB1/2 Anti-proliferative, pro-apoptotic Anti-invasive CCL5/CCR5[23] VEGF Tumor angiogenesis BMP-9[24] Smad Cell apoptosis Bcl-2 Proliferation and metastasis MALAT1[25] PI3K/AKT Proliferation and metastasis

osteosarcoma growth [7]. Wnt-β-catenin and PI3K/Akt can related in OS and discusses some clinical options in order influence osteosarcoma cell proliferation and growth [8], to maintain or even improve progression-free survival. and NF-κB can be used as a target to induce osteosarcoma cells apoptosis [9], thus the molecule-targeted treatment of tumors should be widely considered. Pathway research provides the basis for study of osteosarcoma 2 Diagnosis and possible target diagnosis and drug targets. At the same time, during the therapies for osteosarcoma progress of osteosarcoma formation, many genes have mutated expression and modified abnormally, including In the recent years, the diagnosis and therapies of oncogene (Sema4d, Sema6d, ZNF217 and ZNF592) [10], osteosarcoma have been improved a lot by the efforts tumor suppressor gene (Period2, Bax and P53) [11,12] and of researchers. The conventional methods of diagnosis tumor migration gene (RANKL, CXCR4, RB1, MDM2) [13]. include performing biopsy of pathology tissue, magnetic Identified and screened mutated genes and abnormally resonance imaging (MRI), computed tomography (CT), expressed genes are very important to treat and diagnose positron emission tomography (PET) and so on. The osteosarcoma. Chen et.al have found the extracts of velvet new method which used to diagnose osteosarcoma antler had a dose-response relationship for osteosarcoma aims at detecting biomarkers including microRNAs, cell-line UMR-106, when the concentration was higher than long non-coding RNAs, circulating tumor cells, and 0.972mg/L and lower than 97.2mg/L, samples inhibited the circulating tumor DNA. There are a lot of advantages in proliferation of the cells, when the concentration reached the new method, called liquid biopsy, when compared 97.2ml/L the role will change to promote, and the effects to conventional methods, such as, low sample volume, are increased with an increase in protein concentration greater accuracy, less expensive, and easy detection [1,15]. [14]. However, its molecular mechanism is still unclear, Several pieces of evidence strongly support the which needs further study. Meanwhile, we have found potential capability of new therapies such as cellular that VAPs can inhibit the proliferation and migration of therapy and gene therapy to eradicate osteosarcoma. osteosarcoma cell lines MG-63 and U2OS. The VAP can Thus, clinical human trials using peptides, cytokines and also induce U2OS apoptosis and inhibit MG-63 epithelial- dendritic cells have been performed [16]. Investigators mesenchymal transition (EMT), while TGF-β and Notch have found a variety of growth factors and microRNAs pathways regulate these interactions. Thus, how far have which have effective impact on osteosarcoma. These we moved forward and what therapeutic strategy should findings can be innovative therapies for osteosarcoma to we prefer for anti-pathway therapy? This review provides improve survival and prognosis. an overview of the most updated pathways and genes Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma... 237

Table 2: Summary of the validated microRNA in preclinical experiments of osteosarcoma.

microRNA Target Functions in cancer

miR-137[26] FXYD6 Cell growth

miR-154[27] Wnt5a Tumor suppressor

miR-646[28] FGF2 Osteosarcoma cells metastasis miR-23a[29] PTEN Cell migration and invasion miR-153[30] TGF-β2 Cell proliferation and invasion miR-221[31] PI3K Osteosarcoma cells survival Cisplatin resistance

miR-29b[32] CDK6 Osteosarcoma cells proliferation Cells metastasis miR-150[33] ROCK1 Osteosarcoma cells proliferation Invasion and migration miR-543[34] PRMT9 Osteosarcoma cells proliferation and glycolysis HIF-1α miR-16[35] IGF1R Cell proliferation

3 Gene mutated in Cancer Bone osteosarcoma cells could increase event-free survival and overall survival significantly. Moreover, a decreased Disease level of ErbB2 was associated with poor prognosis of osteosarcoma patients. Thus, ErbB2 might serve as a There are numerous gene mutations and changes in potential therapeutic biomarker target for predicting expression in the process of osteosarcoma compared with the chemotherapy progress as an illustrative example normal people. It plays a pivotal role to identify and screen [41]. COX2 also known as PTGS encodes the inducible mutated genes for diagnosing and treating osteosarcoma. isozyme. It is required for tumoursphere formation, but It is widely believed that cell carcinogenesis and tumor tumourspheres increase invasiveness and tumourigenicity metastasis are caused by changes in genetic information. in osteosarcoma. Therefore, it could be a potential target to It has been found that some mutations and aberrant treat osteosarcoma. Furthermore, the expression of COX2 expressions can occur during osteosarcoma formation elevated 141-fold in a cancer stem cells (CSC) pool and plays including oncogenes (e.g.,HER2, c-myc, c-fos), tumor a vital role in various aspects of carcinogenesis including suppressor genes (e.g., TP53, Rb, and p16) and tumor the promotion of angiogenesis and the down-regulation migration genes (CD44, MMP-9, and nm23) [36,37]. In of apoptosis [42]. Therefore, COX-2 could be a biomarker recent years, some researchers tend to screen the changes in human osteosarcoma and the inhibition might be of massive genes in osteosarcoma to optimize its diagnosis a possible way to improve therapeutic outcome [43]. and treatment. Wang’s group screened for mutations in CUL4B gene is located on the X chromosome and largely 339 cancer-related genes from 10 osteosarcoma patients expressed in the nucleus. It can promote proliferation and through high-throughput sequencing and observed novel invasion and inhibit apoptosis of human osteosarcoma. In 85 mutinied genes in at least one patient, 39 mutinied addition, CUL4B can not only influence H2AK119 mono- genes in at least five patients (Table 2) [38]. In addition, 12 ubiquitination but also H3K9 tri-methylation and DNA osteosarcoma metastasis genes have been identified from methylation, thus suppressing the expression of relevant 31 patients by cDNA subtraction experiment (Table 3) [39]. genes including the tumor-suppressor IGFBP3 [44]. Since Also, more genes were found by other studies which are the gene mutation is a major cause in tumor occurrence related to osteosarcoma. MET one of the oncogenes, was and invasion, the possible applications of which can be causally involved in the pathogenesis of osteosarcoma. used as identification of new biomarkers for more accurate Overexpression of MET could promote the conversion and efficient diagnosis. of primary human osteoblasts into osteosarcoma cells, displaying phenotype of tumor in vitro and the distinguishing features of human osteosarcomas in vivo [40]. ErbB2 is another important gene in osteosarcoma. It has been demonstrated that high levels of ErbB2 in 238 Zhengyao Zhang et al.

Table 3: Changes in the expression of mutated genes in bone cancer [45].

Gene Function Gene Function

ALK oncogene GLTSCR1 tumor suppressor gene

ASPM cell division HSP90AA1 encodes heat shock protein90AA1

ATRX transcriptional regulation and chro- ITGB3 participate in cell adhesion and cell- matin remodeling surface mediated signaling

BCR activate GTPase KDR mediate endothelial proliferation, survival, migration

BL0C1S2 unknown LATS2 tumor suppressor gene

BRCA1 tumor suppressor gene MLH3 DNA mismatch repair genes

BRCA2 tumor suppressor gene MMP14 breakdown extracellular matrix

BRIP1 a target of germline cancer-inducing RCBTB1 induced cellular hypertrophy in mutations vascular smooth muscle cells

CCNA2 regulate cell cycle RECQL4 Maintain DNA stability of telomere and mitochondrial

CDKN2A tumor suppressor gene RNASEL mediate tumor cell apoptosis

CHAT catalyze the biosynthesis of the RNU6-28P Pseudogene and is affiliated with the acetylcholine snRNA class

CYP2D6 drug metabolism RPS6KB1 promote protein synthesis, cell growth, and cell proliferation

DLC1 tumor suppressor gene SRC proto-oncogene

DMBT1 tumor suppressor gene TMPRSS11A regulate cell growth and cell cycle arrest

DPYD pyrimidine catabolism TNC influence migration of neurons and axons, synaptic plasticity and neuro- nal regeneration

EGFR induce receptor dimerization, tyro- TP53 tumor suppressor gene sine autophosphorylation and cell proliferation.

EML4 participate in microtubule formation TRIM3 participate in myosin V-mediated cargo transport

FANCA participate in inter-strand DNA XPA DNA repair cross-link repair and maintain normal chromosome stability

FN1 cell adhesion and migration XPC DNA repair

GATA3 regulate cell proliferation

4 Pathways in a Physiological formation and bone resorption. After deregulating bone remolding, it is induced to release TGF-β in bone matrix and Pathological Context in the [47]. Antler polypeptides contain a variety of growth Osteosarcoma factors including TGF-β, they can become the source of exogenous TGF-β which can inhibit proliferation of 4.1 TGF-β pathways osteosarcoma and slow down the disease and make the prognosis of patients better. It has been confirmed that Latest research found that osteosarcoma can deregulate high concentration of exogenous TGF-β can inhibit the bone remolding and break the balance between bone proliferation of osteosarcoma MG-63 cells, however, the Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma... 239

Table 4: Gene name, functions of 12 reference metastasis genes involved in Osteosarcoma [46].

Gene Functions Gene Functions

SMAD signal transducers and transcriptional NSE2 mediate the attachment of a SUMO protein to proteins, modulators nuclear transport, transcription, chromosome segregation and DNA repair.

RANKL regulate osteoclast differentiation, T cell- RUNX2 regulate osteoblastic differentiation, skeletal gene expression dependent immune response and cell and skeletal morphogenesis and act as a scaffold for nucleic apoptosis acids

Ezrin participate in cell surface structure adhe- TGF-β regulate cell proliferation, differentiation and growth, and sion, migration and organization modulate expression and activation of other growth factors

IL-8 mediate inflammatory response and MAPK regulate cell proliferation, differentiation, signaling transcrip- induces chemotaxis and phagocytosis tion and survival and apoptosis

β4 integrin mediate cell-matrix or cell-cell adhesion SPP1 participate in biomineralization, bone remodeling and act as and regulate gene expression an anti-apoptotic factor

CLIC5 participate in hair cell stereocilia forma- TP53 tumor suppressor gene tion, myoblast proliferation

gene (TIEG), and finally inhibit osteocalcin synthesis. While numerous studies have demonstrated that high expression of bone calcium in bone cells and osteosarcoma cells can inhibit the synthesis of osteocalcin then inhibit the proliferation of osteosarcoma cells [49]. Although researchers have confirmed the inhibitive role of TGF-β in tumorigenesis, it has on the contrary been shown to enhance metastasis of tumor cells and promote advanced tumors invasiveness [50]. TGF-β is a double-edged sword in cancer, on the one hand it can suppress tumor growth potently, on the other hand, it can also enhance invasion and metastasis of cancer by suppressing miR-143, inducing epithelial-mesenchymal transition (EMT) [51,52], which plays a vital role in tumor invasion and metastasis [53]. Furthermore, it has been reported that TGF-β can induce the expression of Snail which could repress the expression of E-cadherin, an important tumor suppressor [54]. Though TGF-β is involved in the EMT process, VAP has been shown

Figure 1: Velvet antler polypeptides regulate TGF-β in osteosarcoma to block the binding of TGF-β1 with its receptors, TGF-β cells. Note: The VAP inhibit the expression of EMT transcriptional receptor1 and 2, and inhibit the downstream activated factors and E-cadherin by down-regulating the TGF-β pathway in pathway [55] (Figure1). It has also been confirmed that MG-63 cells, which interfere the invasion and migration afterwards. TGF-β1 not only stimulates the growth of osteosarcoma but also reduces the proliferative potential ability of osteosarcoma by inducing the expression of IGFBP-3 [56]. lower concentration of TGF-β had no significant effect on In addition to osteosarcoma, the antler polypeptide also it. Furthermore, exogenous TGF-β can inhibit the growth affects other tumors. Tang et al. demonstrated that the top of MG- 63 cells cultured in vitro, which can increase the VAP can inhibit the migration of prostate cancer cells by distribution of MG-63 cells in G1 phase and prevent the downregulating the expression of its relevant gene, such cells from entering S phase [48]. Exogenous TGF-β can as MMP-9 and VEGF [57]. make MG- 63 cells overexpressing a TGF-β inducible early 240 Zhengyao Zhang et al.

4.2 Notch signaling pathways induced recurrence in osteosarcoma by regulating notch and Wnt/β-catenin signaling pathways, so they can be The Notch signaling pathway includes Notch1, Notch2, targeted to inhibit tumor metastasis and recurrence in Jag1, DLL1, DLL4, Hey1/Hey2 and CSL. It is implicated as osteosarcoma [70]. miR-34a-5p, miR-26a and miR-199b-5p a key mediator in a number of various cancers [58]. It is also play an important role in osteosarcoma. The reduction more like a double-edged sword, which can be used as an of miR-26a can cause osteosarcoma metastasis and poor oncogene in one hand , while inhibiting tumor growth in survival of osteosarcoma patients, miR26-a can also some cases on the other hand [59]. In addition, regulating regulate cancer stem cells of osteosarcoma. But miR-26a hes1 and DTX1 in notch signaling pathways can affect only target Jagged1 which is a ligand in notch signaling migration and invasion of osteosarcoma cells [60]. It has pathways [71]. Moreover miR-34a-5p can promote multi- been confirmed that the expression of Notch and its target chemoresistance of osteosarcoma by down regulating gene are up-regulated in osteosarcoma. Inhibiting Notch DLL1 gene a ligand of notch signaling pathways and the signaling by chemical and genetic ways can reduce nude inhibition of miRNA-199b-5p can change expression of mices’ tumor burden in vivo and decrease the proliferation notch pathway components [72,73]. These findings reveal of osteosarcoma cells in vitro [61]. Furthermore, it has been that notch signaling pathways, and some microRNA can found that Notch pathways can regulate the osteosarcoma be novel targets for treating osteosarcoma and therapeutic cell cycle by influencing the expression of cyclin E1, cyclin options for osteosarcoma. E2, c-Myc and so on. Notch signaling pathways play a very important role in osteosarcoma cell progression [62]. Besides these roles we have referred to, it also participates 4.3 Wnt-β-catenin pathways in EMT. The EMT progression can be arrested by the inhibition of notch signaling pathways [63]. Furthermore, The Wnt signaling pathways are a group of signal notch pathways will become a mediator when some transduction pathways made of proteins that pass signals growth factors and drugs mitigate osteosarcoma. It into a cell through cell surface receptors. There are some has been found that BMP-9 can promote the growth of proteins in them such as frizzled, disheveled, β-catenin, osteosarcoma, which is mediated by the notch signaling GSK3β, and axin scaffolding protein. They play a key role pathway. The study also demonstrated BMP-9 has effects in cell cycle, apoptosis and tumorigenesis, and EMT [74]. on the receptors and ligands of notch signaling pathways It has been confirmed that it can decrease tumorigenicity, including hey1, notch1, DLL1, JAG1 and JAG2 at the metastasis and EMT of osteosarcoma by downregulating same time [64]. Moreover, some anticancer drugs exert LRP-5 which is a receptor of wnt in vivo and in nude anticancer effects by regulating notch signaling pathways. mice experiments [75]. Thus LRP-5 can be a new target It has been shown that the anticancer drug, doxorubicin, for inhibiting EMT. At the same time the activation of can inhibit the proliferation of osteosarcoma cells by Wnt pathways can promote the tumorigenic phenotypes, upregulating notch signaling pathways [65]. In addition, and some receptors, ligands also up-regulated in cinobufagin and curcumin can inhibit osteosarcoma osteosarcoma, therefore the treatment of osteosarcoma growth in vivo, induce osteosarcoma cell apoptosis, cell should aim at blocking Wnt pathways [76]. Rubin et al. growth inhibition, decrease cell survival, and improve provided Wnt Inhibitory Factor 1 can (WIF1) decreases mice survival by downregulating notch1 and its target gene tumorigenesis and metastasis in osteosarcoma cells [66,67]. Continuous medication can cause drug resistance line 143B cells and overexpression of WIF1 can inhibit in tumor cells, and the notch plays an important regulatory lung metastasis in vivo in an orthotopic mouse model of role in this process[68], which provides us a new way osteosarcoma. Therefore, WIF1 is a potential target for to cure osteosarcoma. In addition to growth factors and treating osteosarcoma [77]. IWR-1, a tankyrase inhibitor drugs, notch signaling pathways also participate in the which is a wnt/β-catenin signaling pathways inhibitor process where microRNAs affect osteosarcoma. MicroRNA and a specifically cytotoxic for osteosarcoma cancer is a small non-coding RNA molecule found in plants, stem calls. It has been confirmed that IWR-1 not only can animals and some viruses, that functions in RNA silencing inhibit the growth of cancer stem cells in osteosarcoma and post-transcriptional regulation of gene expression in vivo and in vitro by targeting wnt signaling pathways [69]. It is generally accepted that tumor recurrence often but also can eradicate the aggressive osteosarcoma appears during the treatment of the tumor and the cancer cancer stem cells and improve therapeutic outcomes stem cells play a key role in it. In recent years, researchers [78] . DKK-3 is a Secreted Wnt Antagonist and involved have found miR-135b can effect tumor metastasis and CSC- in embryonic development through its interactions with Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma... 241 the Wnt signaling pathway. Hoang et al. have confirmed tumor necrosis factor-α (TNF-α), known inducers of NF-κB that DDK-3 can decrease tumor growth and metastatic [86]. pulmonary nodules in nude mice. In addition, it can inhibit tumorigenic potential of osteosarcoma, decrease osteosarcoma cell motility in cells. So, we can make DDK3 4.5 PI3K/Akt Pathway as target to treat osteosarcoma [79]. Zhang et al. revealed that parathyroid hormone type 1 receptor (PTHR1) can PI3K-Akt pathway is a signal transduction pathway promote malignancy for osteosarcoma through activation that promotes survival and growth in response to of wnt and angiogenesis signaling pathways. They have extracellular signals. Key proteins involved are PI3K analyzed the microarray data extracted from the Gene (phosphatidylinositol 3-kinase) and Akt (Protein Kinase Expression Omnibus (GEO) database and compared with B). It is well known to be a major cell survival pathway PTHR1 knockdown samples [80]. However, a different and it can enhance resistance to apoptosis if activated opinion has been put forward that wnt signaling may act [87]. Apo2L/TRAIL is a member of the tumor necrosis as a tumor repressor in osteosarcoma which is in contrast factor (TNF) family, which can induce apoptosis of cancer with its oncogenic role in other tumors [81]. Some cells. It can induce osteosarcoma cell U2OS apoptosis by medicines also exert their role by targeting wnt/β-catenin inhibiting akt expression and then reduce expression signaling pathways. Triptolide(TPL) is a diterpenoid of Bcl-2 and activate caspase-9. Furthermore, it can also epoxide which is produced by the thunder god vine. It decrease osteosarcoma cell drug resistance by regulating can inhibit angiogenesis and induce cell apoptosis in Akt pathway [88,89]. Geraniin is an activated compound osteosarcoma cells in a dose dependent manner through isolated from Geranium sibiricum. It has been found that down-regulating Wnt/β-Catenin signaling [82]. Together, Geraniin suppresses matrix metalloproteinase-9 (MMP-9) these findings have revealed that wnt/βcatenin could be a expression in a dose dependent manner and then inhibits key pathway for treating osteosarcoma. the migration and invasion of osteosarcoma cells by suppressing the phosphorylation of the extracellular signal regulating kinase (ERK)1/2, phosphatidylinositide- 4.4 NF-κB pathway 3-kinase (PI3K), and Akt pathways [90]. miR-221 is an oncogenic microRNA and one of the most commonly NF-κB is a protein complex that controls transcription of upregulated miRNAs in cancer. It targets PTEN leading DNA, cytokine production and cell survival. NF-κB plays to activation of the Akt pathway, which is known as a a pivotal role in cell growth, apoptosis, migration and major cell survival pathway in many cancers. It has been invasion of osteosarcoma [83]. Aspirin is a medication confirmed that knockdown of miR-221 in osteosarcoma used to treat pain, fever, or inflammation. However, it has cells can downregulate p-Akt expression, promote been confirmed that aspirin can influence osteosarcoma osteosarcoma cells apoptosis and lower cisplatin procession too. Kang et al. have demonstrated that aspirin resistance through regulating PI3K/AKT pathway [91]. can reduce cell viability and the more doses and time the Some medicines can exert their roles through this pathway. better effects in osteosarcoma cells. Moreover, aspirin can Dryofragin is a phloroglucinol derivative extracted from also repress the migration and invasion of osteosarcoma Dryopteris fragrans. It has been found to be able to inhibit cells and decrease osteosarcoma metastases to the lungs tumor proliferation and induce apoptosis. Moreover, in nude mouse through regulating NF-κB pathway [84]. in recent years it has been confirmed that dryofragin Thymoquinone is a phytochemical compound found in can suppress the migration and invasive ability of U2OS the plant Nigella sativa. It can inhibit cell growth and cells, downregulate the expression of MMP-2 and MMP-9 effectively induce tumor cell apoptosis and exert anti- and upregulate the expression of TIMP-1 and TIMP-2 proliferative effects on several cancer cells in vitro. In through PI3K/AKT and p38 MAPK signaling pathways addition, thymoquinone can inhibit tumor angiogenesis [92]. Tricetin is a flavone, a type of flavonoid, that can and tumor growth by downregulating NF-κB and its also inhibit the metastasis of human osteosarcoma cells regulated molecules. Not only can thymoquinone inhibit by transcriptionally repressing MMP-9 via p38 and Akt osteosarcoma growth but also it can enhance sensitivity pathways [93]. All of them can be potentially used as anti- to chemotherapeutic agents [85]. Furthermore, NF-κB can cancer agents for osteosarcoma treatment and supplement contact with wnt pathway, wnt10b is a member of the wnt of osteosarcoma chemotherapy. family which could upregulate interleukin-1α (IL-1α) and 242 Zhengyao Zhang et al.

4.6 Other signaling pathways results suggested the involvement of VAP in regulation of proliferation and migration of osteosarcoma cells MG-63 Rheum palmatum L. is a common Chinese herb, also as well as U2OS. Information about molecular mechanism called Chinese rhubarb, ornamental rhubarb. Rheum and therapeutic targets of this reaction within the tumor palmatum L. has been used for anti-inflammatory and cells needs to be elucidated. Furthermore, various chronic liver diseases, and some cancer. In recent years, it growth factors such as sirtuin and NKD2 can regulate the has been indicated that Crude Extract of Rheum palmatum osteosarcoma cell-cycle, and biomarkers will be essential L. (CERP) can induce S phase arrest in osteosarcoma cells to advance into clinical development to obtain meaningful U2OS in a dose-dependent fashion, cause DNA damage and reliable answers on therapeutic ratios. Undoubtedly, and DNA condensation, up-regulate the expression of further advances in our understanding of the pathology pro-apoptosis proteins such as Bax, Bak, p21, and p27 of osteosarcoma, and in the techniques for extracting the and activate caspase-3, -8, and -9 through mitochondrial- velvet antler, will strengthen our understanding of the dependent pathways [94]. Aspidin PB is a phloroglucinol ways in which the Chinese medicine counteracts cancer, derivative isolated from Dryopteris fragrans (L.). Previous and will aid in the development of anti-cancer therapeutic studies have found it can inhibit fibrogenesis [95] and approaches. induce apoptosis in human hepatocarcinoma HepG2 cells [96]. Moreover, it also has been confirmed that Aspidin Conflicts of interest: The authors declared no potential PB can inhibit the proliferation of osteosarcoma cells in a conflicts of interest with respect to the research, dose-dependent and time-dependent manner and induce authorship, and/or publication of this article. osteosarcoma cells apoptosis and cell cycle arrest through the p53/p21 and mitochondria-dependent pathways [97]. Acknowledgements: This work was supported by the S-Adenosyl methionine (AdoMet) is a common National Natural Science Foundation of China (grant no. co-substrate involved in methyl group transfers, 81503177 and 81573999) and Jilin science and technology transsulfuration, and aminopropylation. Naviglio’s development plan project (20160101158JC). group has confirmed that it can inhibit osteosarcoma cell proliferation by slowing-down cell cycle progression and by inducing apoptosis. Furthermore, it can downregulate ERK1/2 and STAT3 pathways to arrest cell cycle and Reference induce osteosarcoma cell apoptosis [98]. Cucurbitacin B [1] Raimondi L., De A.L., Costa V., Amodio N., Carina V., Bellavia is class of tetracyclic triterpenoids and is extracted from D., et al. Circulating biomarkers in osteosarcoma: new Hemsleya endecaphylla (62 mg/72 g) and other plants translational tools for diagnosis and treatment. Oncotarget., [99]. It is the most abundant member of cucurbit family 2017, 8(59), 100831. and has extensive pharmacological activities. For human [2] Quan G.M., Slavin J.L., Schlicht S.M., Smith P.J., Powell G.J., Choong P.F. Osteosarcoma near joints: assessment and osteosarcoma cells, it can downregulate MMP-2 and MMP-9 implications. J Surg Oncol., 2005, 91(3), 159. which contribute to the invasion and metastasis of tumor [3] Anderson M.E. Update on Survival in Osteosarcoma. Orthop cells, upregulate the expression of pro-apoptotic proteins, Clin North Am., 2016, 47(1), 283-92. and modulate JAK2/STAT3 signaling pathway [100]. [4] Sadighi M., Haines S.R., Skottner A., Harris A.J., Suttie J.M. Effects of insulin-like growth factor-I (IGF-I) and IGF-II on the growth of antler cells in vitro. J Endocrinol., 1994, 143(3), 461-9. [5] Deng H.M., Ding Q.N., Wang C.M., Geng D., Dai J.D., Dong L. 5 Conclusion The Contents of the Growth Factors in Different Sections of Sika Deer (Cervus nippon) Velvet Antler Treated by Traditional Hot Processing. Special Wild Economic Animal & Plant Research., Osteosarcoma is a complex, heterogenous, and 2018. interpatient, between different individuals and their [6] Sun Y., Xia P., Zhang H., Liu B., Shi Y. P53 is required for living environment, disease. Therefore, successful Doxorubicin-induced apoptosis via the TGF-beta signaling treatment options are likely to arise from personalized pathway in osteosarcoma-derived cells. Am J Cancer Res., precise treatment. There is paramount preclinical and 2016, 6(1), 114-25. [7] Tanaka M., Setoguchi T., Hirotsu M., Gao H., Sasaki H., clinical evidence for potent efficient activity of velvet Matsunoshita Y., et al. Inhibition of Notch pathway prevents antler and its extracts for use as therapeutics in bone osteosarcoma growth by cell cycle regulation. Br J Cancer., fracture repair, osteoarthritis, osteoporosis and other bone 2009, 100(12), 1957-65. diseases. Relating to TGF-β and Notch pathways, previous Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma... 243

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