Velvet Antler compounds targeting major cell signaling pathways in osteosarcoma - a new insight into mediating the process of invasion and metastasis in OS

Abstract Velvet antler is the only renewable bone tissue of mammalian animals, which consists of a variety of growth factors, amino acids and polypeptides. But the mechanism of high-speed proliferation without carcinogenesis is still mystifying. The previous study of this work found that the velvet antler peptides (VAP) could not only inhibit the proliferation and migration of osteosarcoma cell lines MG-63 and U2OS, but also induced U2OS apoptosis and inhibited MG-63 epithelial-mesenchymal transition (EMT) through TGF-β and Notch pathways. These results lead us to conclude that VAP has the potential ability to mediate osteosarcoma cells by regulating related signaling pathways and growth factors. Therefore, finding a new appropriate inhibitor for OS is a valuable research direction, which will give patients a better chance to receive proper therapy. From an applied perspective, this review summarized the effects of velvet antler, genes, growth factors and research progress of relative pathways and genes of osteosarcoma, which are poised to help link regenerative molecular biology and regenerative medicine in osteosarcoma pathogenesis.


Introduction
Osteosarcoma, derived from primitive bone-forming mesenchymal cells, is the most common primary bone malignancy that occurs most frequently in adolescents and elders over the age of 60 [1]. Osteosarcoma most frequently affects the growing ends of long bones and is often located adjacent to joints. Approximately one-half of all osteosarcomas affects the knee region, with the distal femur being the most commonly affected site [2]. Osteosarcoma is characterized by destruction of bone and soft tissue and is highly likely to be accompanied by metastasis of the cancer cells to the distal organ, where metastasis to the lung accounts for approximately 80%, with a poor prognosis. If clinical metastasis occurs, the patient's five-year survival rate is between 20%-30% by the combination of conventional surgery and chemotherapy [3]. Deer velvet antler is one of the most important conventional Chinese medicines, the application of which was started two thousand years ago. It has been extensively used in traditional Chinese medicine (TCM) to treat a variety of diseases including degenerative disease (osteoarthritis), auto-immune or auto-inflammatory processes (rheumatoid arthritis and ankylosing spondylitis), infection (septic arthritis), idiopathic (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 liquid chromatography. Hence, the velvet antler contains a lot of growth factors such as insulin-like growth factor (IGF), nerve growth factor (NGF), epidermal growth factor (EGF) and transforming growth factor (TGF), which have different influences on osteosarcoma cells [4,5]. In addition, the progression of osteosarcoma is regulated by signaling pathways such as TGF-β, which can mediate the pro-apoptosis effects by doxorubicin [6] , and the inhibition of the Notch pathway can suppresses osteosarcoma growth [7]. Wnt-β-catenin and PI3K/Akt can influence osteosarcoma cell proliferation and growth [8], 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 diagnosis and drug targets. At the same time, during the progress of osteosarcoma formation, many genes have mutated expression and modified abnormally, including oncogene (Sema4d, Sema6d, ZNF217 and ZNF592) [10], tumor suppressor gene (Period2, Bax and P53) [11,12] and tumor migration gene (RANKL, CXCR4, RB1, MDM2) [13]. Identified and screened mutated genes and abnormally expressed genes are very important to treat and diagnose osteosarcoma. Chen et.al have found the extracts of velvet antler had a dose-response relationship for osteosarcoma cell-line UMR-106, when the concentration was higher than 0.972mg/L and lower than 97.2mg/L, samples inhibited the proliferation of the cells, when the concentration reached 97.2ml/L the role will change to promote, and the effects are increased with an increase in protein concentration [14]. However, its molecular mechanism is still unclear, which needs further study. Meanwhile, we have found that VAPs can inhibit the proliferation and migration of osteosarcoma cell lines MG-63 and U2OS. The VAP can also induce U2OS apoptosis and inhibit MG-63 epithelialmesenchymal transition (EMT), while TGF-β and Notch pathways regulate these interactions. Thus, how far have we moved forward and what therapeutic strategy should we prefer for anti-pathway therapy? This review provides an overview of the most updated pathways and genes related in OS and discusses some clinical options in order to maintain or even improve progression-free survival.

Diagnosis and possible target therapies for osteosarcoma
In the recent years, the diagnosis and therapies of osteosarcoma have been improved a lot by the efforts of researchers. The conventional methods of diagnosis include performing biopsy of pathology tissue, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET) and so on. The new method which used to diagnose osteosarcoma aims at detecting biomarkers including microRNAs, long non-coding RNAs, circulating tumor cells, and circulating tumor DNA. There are a lot of advantages in the new method, called liquid biopsy, when compared to conventional methods, such as, low sample volume, greater accuracy, less expensive, and easy detection [1,15].
Several pieces of evidence strongly support the potential capability of new therapies such as cellular therapy and gene therapy to eradicate osteosarcoma. Thus, clinical human trials using peptides, cytokines and dendritic cells have been performed [16]. Investigators have found a variety of growth factors and microRNAs which have effective impact on osteosarcoma. These findings can be innovative therapies for osteosarcoma to improve survival and prognosis.

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

TGF-β pathways
Latest research found that osteosarcoma can deregulate bone remolding and break the balance between bone formation and bone resorption. After deregulating bone remolding, it is induced to release TGF-β in bone matrix [47]. Antler polypeptides contain a variety of growth factors including TGF-β, they can become the source of exogenous TGF-β which can inhibit proliferation of osteosarcoma and slow down the disease and make the prognosis of patients better. It has been confirmed that high concentration of exogenous TGF-β can inhibit the proliferation of osteosarcoma MG-63 cells, however, the  [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 to block the binding of TGF-β1 with its receptors, TGF-β receptor1 and 2, and inhibit the downstream activated pathway [55] (Figure1). It has also been confirmed that 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].
In addition to osteosarcoma, the antler polypeptide also affects other tumors. Tang et al. demonstrated that the top VAP can inhibit the migration of prostate cancer cells by downregulating the expression of its relevant gene, such as MMP-9 and VEGF [57].

Notch signaling pathways
The Notch signaling pathway includes Notch1, Notch2, Jag1, DLL1, DLL4, Hey1/Hey2 and CSL. It is implicated as a key mediator in a number of various cancers [58]. It is more like a double-edged sword, which can be used as an oncogene in one hand , while inhibiting tumor growth in some cases on the other hand [59]. In addition, regulating hes1 and DTX1 in notch signaling pathways can affect migration and invasion of osteosarcoma cells [60]. It has been confirmed that the expression of Notch and its target gene are up-regulated in osteosarcoma. Inhibiting Notch signaling by chemical and genetic ways can reduce nude mices' tumor burden in vivo and decrease the proliferation of osteosarcoma cells in vitro [61]. Furthermore, it has been found that Notch pathways can regulate the osteosarcoma cell cycle by influencing the expression of cyclin E1, cyclin 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 in EMT. The EMT progression can be arrested by the inhibition of notch signaling pathways [63]. Furthermore, notch pathways will become a mediator when some growth factors and drugs mitigate osteosarcoma. It has been found that BMP-9 can promote the growth of osteosarcoma, which is mediated by the notch signaling pathway. The study also demonstrated BMP-9 has effects on the receptors and ligands of notch signaling pathways including hey1, notch1, DLL1, JAG1 and JAG2 at the same time [64]. Moreover, some anticancer drugs exert anticancer effects by regulating notch signaling pathways. It has been shown that the anticancer drug, doxorubicin, can inhibit the proliferation of osteosarcoma cells by upregulating notch signaling pathways [65]. In addition, cinobufagin and curcumin can inhibit osteosarcoma growth in vivo, induce osteosarcoma cell apoptosis, cell growth inhibition, decrease cell survival, and improve mice survival by downregulating notch1 and its target gene [66,67]. Continuous medication can cause drug resistance in tumor cells, and the notch plays an important regulatory role in this process [68], which provides us a new way to cure osteosarcoma. In addition to growth factors and drugs, notch signaling pathways also participate in the process where microRNAs affect osteosarcoma. MicroRNA is a small non-coding RNA molecule found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression [69]. It is generally accepted that tumor recurrence often appears during the treatment of the tumor and the cancer stem cells play a key role in it. In recent years, researchers have found miR-135b can effect tumor metastasis and CSC-induced recurrence in osteosarcoma by regulating notch and Wnt/β-catenin signaling pathways, so they can be targeted to inhibit tumor metastasis and recurrence in osteosarcoma [70]. miR-34a-5p, miR-26a and miR-199b-5p also play an important role in osteosarcoma. The reduction of miR-26a can cause osteosarcoma metastasis and poor survival of osteosarcoma patients, miR26-a can also regulate cancer stem cells of osteosarcoma. But miR-26a only target Jagged1 which is a ligand in notch signaling pathways [71]. Moreover miR-34a-5p can promote multichemoresistance of osteosarcoma by down regulating DLL1 gene a ligand of notch signaling pathways and the inhibition of miRNA-199b-5p can change expression of notch pathway components [72,73]. These findings reveal that notch signaling pathways, and some microRNA can be novel targets for treating osteosarcoma and therapeutic options for osteosarcoma.

Wnt-β-catenin pathways
The Wnt signaling pathways are a group of signal transduction pathways made of proteins that pass signals into a cell through cell surface receptors. There are some proteins in them such as frizzled, disheveled, β-catenin, GSK3β, and axin scaffolding protein. They play a key role in cell cycle, apoptosis and tumorigenesis, and EMT [74]. It has been confirmed that it can decrease tumorigenicity, metastasis and EMT of osteosarcoma by downregulating LRP-5 which is a receptor of wnt in vivo and in nude mice experiments [75]. Thus LRP-5 can be a new target for inhibiting EMT. At the same time the activation of Wnt pathways can promote the tumorigenic phenotypes, and some receptors, ligands also up-regulated in osteosarcoma, therefore the treatment of osteosarcoma should aim at blocking Wnt pathways [76]. Rubin et al. provided Wnt Inhibitory Factor 1 can (WIF1) decreases tumorigenesis and metastasis in osteosarcoma cells line 143B cells and overexpression of WIF1 can inhibit lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Therefore, WIF1 is a potential target for treating osteosarcoma [77]. IWR-1, a tankyrase inhibitor which is a wnt/β-catenin signaling pathways inhibitor and a specifically cytotoxic for osteosarcoma cancer stem calls. It has been confirmed that IWR-1 not only can inhibit the growth of cancer stem cells in osteosarcoma in vivo and in vitro by targeting wnt signaling pathways but also can eradicate the aggressive osteosarcoma cancer stem cells and improve therapeutic outcomes [78] . DKK-3 is a Secreted Wnt Antagonist and involved in embryonic development through its interactions with the Wnt signaling pathway. Hoang et al. have confirmed that DDK-3 can decrease tumor growth and metastatic 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 as target to treat osteosarcoma [79]. Zhang et al. revealed that parathyroid hormone type 1 receptor (PTHR1) can promote malignancy for osteosarcoma through activation of wnt and angiogenesis signaling pathways. They have analyzed the microarray data extracted from the Gene Expression Omnibus (GEO) database and compared with PTHR1 knockdown samples [80]. However, a different opinion has been put forward that wnt signaling may act as a tumor repressor in osteosarcoma which is in contrast with its oncogenic role in other tumors [81]. Some medicines also exert their role by targeting wnt/β-catenin signaling pathways. Triptolide(TPL) is a diterpenoid epoxide which is produced by the thunder god vine. It can inhibit angiogenesis and induce cell apoptosis in osteosarcoma cells in a dose dependent manner through down-regulating Wnt/β-Catenin signaling [82]. Together, these findings have revealed that wnt/βcatenin could be a key pathway for treating osteosarcoma.

NF-κB pathway
NF-κB is a protein complex that controls transcription of DNA, cytokine production and cell survival. NF-κB plays a pivotal role in cell growth, apoptosis, migration and invasion of osteosarcoma [83]. Aspirin is a medication used to treat pain, fever, or inflammation. However, it has been confirmed that aspirin can influence osteosarcoma procession too. Kang et al. have demonstrated that aspirin can reduce cell viability and the more doses and time the better effects in osteosarcoma cells. Moreover, aspirin can also repress the migration and invasion of osteosarcoma cells and decrease osteosarcoma metastases to the lungs in nude mouse through regulating NF-κB pathway [84]. Thymoquinone is a phytochemical compound found in the plant Nigella sativa. It can inhibit cell growth and effectively induce tumor cell apoptosis and exert antiproliferative effects on several cancer cells in vitro. In addition, thymoquinone can inhibit tumor angiogenesis and tumor growth by downregulating NF-κB and its regulated molecules. Not only can thymoquinone inhibit osteosarcoma growth but also it can enhance sensitivity to chemotherapeutic agents [85]. Furthermore, NF-κB can contact with wnt pathway, wnt10b is a member of the wnt family which could upregulate interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), known inducers of NF-κB [86].

PI3K/Akt Pathway
PI3K-Akt pathway is a signal transduction pathway that promotes survival and growth in response to extracellular signals. Key proteins involved are PI3K (phosphatidylinositol 3-kinase) and Akt (Protein Kinase B). It is well known to be a major cell survival pathway and it can enhance resistance to apoptosis if activated [87]. Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family, which can induce apoptosis of cancer cells. It can induce osteosarcoma cell U2OS apoptosis by inhibiting akt expression and then reduce expression of Bcl-2 and activate caspase-9. Furthermore, it can also decrease osteosarcoma cell drug resistance by regulating Akt pathway [88,89]. Geraniin is an activated compound isolated from Geranium sibiricum. It has been found that Geraniin suppresses matrix metalloproteinase-9 (MMP-9) expression in a dose dependent manner and then inhibits the migration and invasion of osteosarcoma cells by suppressing the phosphorylation of the extracellular signal regulating kinase (ERK)1/2, phosphatidylinositide-3-kinase (PI3K), and Akt pathways [90]. miR-221 is an oncogenic microRNA and one of the most commonly upregulated miRNAs in cancer. It targets PTEN leading to activation of the Akt pathway, which is known as a major cell survival pathway in many cancers. It has been confirmed that knockdown of miR-221 in osteosarcoma cells can downregulate p-Akt expression, promote osteosarcoma cells apoptosis and lower cisplatin resistance through regulating PI3K/AKT pathway [91]. Some medicines can exert their roles through this pathway. Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans. It has been found to be able to inhibit tumor proliferation and induce apoptosis. Moreover, in recent years it has been confirmed that dryofragin can suppress the migration and invasive ability of U2OS cells, downregulate the expression of MMP-2 and MMP-9 and upregulate the expression of TIMP-1 and TIMP-2 through PI3K/AKT and p38 MAPK signaling pathways [92]. Tricetin is a flavone, a type of flavonoid, that can also inhibit the metastasis of human osteosarcoma cells by transcriptionally repressing MMP-9 via p38 and Akt pathways [93]. All of them can be potentially used as anticancer agents for osteosarcoma treatment and supplement of osteosarcoma chemotherapy.

Other signaling pathways
Rheum palmatum L. is a common Chinese herb, also called Chinese rhubarb, ornamental rhubarb. Rheum palmatum L. has been used for anti-inflammatory and chronic liver diseases, and some cancer. In recent years, it has been indicated that Crude Extract of Rheum palmatum L. (CERP) can induce S phase arrest in osteosarcoma cells U2OS in a dose-dependent fashion, cause DNA damage and DNA condensation, up-regulate the expression of pro-apoptosis proteins such as Bax, Bak, p21, and p27 and activate caspase-3, -8, and -9 through mitochondrialdependent pathways [94]. Aspidin PB is a phloroglucinol derivative isolated from Dryopteris fragrans (L.). Previous studies have found it can inhibit fibrogenesis [95] and induce apoptosis in human hepatocarcinoma HepG2 cells [96]. Moreover, it also has been confirmed that Aspidin PB can inhibit the proliferation of osteosarcoma cells in a dose-dependent and time-dependent manner and induce osteosarcoma cells apoptosis and cell cycle arrest through the p53/p21 and mitochondria-dependent pathways [97]. S-Adenosyl methionine (AdoMet) is a common co-substrate involved in methyl group transfers, transsulfuration, and aminopropylation. Naviglio's 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 induce osteosarcoma cell apoptosis [98]. Cucurbitacin B is class of tetracyclic triterpenoids and is extracted from Hemsleya endecaphylla (62 mg/72 g) and other plants [99]. It is the most abundant member of cucurbit family and has extensive pharmacological activities. For human osteosarcoma cells, it can downregulate MMP-2 and MMP-9 which contribute to the invasion and metastasis of tumor cells, upregulate the expression of pro-apoptotic proteins, and modulate JAK2/STAT3 signaling pathway [100].

Conclusion
Osteosarcoma is a complex, heterogenous, and interpatient, between different individuals and their living environment, disease. Therefore, successful treatment options are likely to arise from personalized precise treatment. There is paramount preclinical and clinical evidence for potent efficient activity of velvet antler and its extracts for use as therapeutics in bone fracture repair, osteoarthritis, osteoporosis and other bone diseases. Relating to TGF-β and Notch pathways, previous results suggested the involvement of VAP in regulation of proliferation and migration of osteosarcoma cells MG-63 as well as U2OS. Information about molecular mechanism and therapeutic targets of this reaction within the tumor cells needs to be elucidated. Furthermore, various growth factors such as sirtuin and NKD2 can regulate the osteosarcoma cell-cycle, and biomarkers will be essential to advance into clinical development to obtain meaningful and reliable answers on therapeutic ratios. Undoubtedly, further advances in our understanding of the pathology of osteosarcoma, and in the techniques for extracting the velvet antler, will strengthen our understanding of the ways in which the Chinese medicine counteracts cancer, and will aid in the development of anti-cancer therapeutic approaches.

Conflicts of interest:
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.