Process Of Osteosarcoma Research Articles

Plumbagin induces G2/M arrest and apoptosis and ferroptosis via ROS/p38 MAPK pathway in human osteosarcoma cells

Plumbagin (PLB), a naphthoquinone compound, has been proven to have anti-cancer properties in different types of cancers. However, the anti-tumor actions as well as molecular processes in osteosarcoma (OS) remain unclear. This study investigates the underlying mechanisms of PLB in human OS cells in vitro. Our study showed that PLB significantly inhibited the proliferation of human OS cells in a time- and dosage-dependent manner. RNA sequencing data revealed that the efficacy of PLB is related to cell cycle, cell death, and p38 MAPK signaling pathway in KHOS-240S cells. Further studies indicated that PLB triggers G2/M phase arrest, mitochondrial-dependent apoptosis, as well as ferroptosis in human OS cells. Mechanistically, PLB treatment increased ROS generation that activates the p38 MAPK signaling pathway. BIRB 796, a p38 MAPK inhibitor, partly reversed PLB-induced phase arrest of G2/M and cell death. Furthermore, N-acetylcysteine (NAC), a ROS scavenger, significantly decreased G2/M phase arrest and cell death and reversed p38 MAPK activation caused by PLB. In conclusion, PLB induces arrest G2/M arrest, apoptosis as well as ferroptosis in human OS cells via ROS generation and p38 MAPK activation. There is potential for PLB to be an effective treatment for OS.

Antigen processing and presentation-related signature-derived BNIP3 is a novel oncogene and immunotherapy determinant in osteosarcoma based on machine learning and in vitro validation.

In recent decades, osteosarcoma has remained the most prevalent kind of malignant tumor. An important and crucial factor in immunotherapy is antigen processing and presentation (APP). The specific functions and pathogenic processes of APP in osteosarcoma have not, however, been studied. Patients with osteosarcoma were divided into groups using APP-related genes. Machine learning was used to further build the APP-related score. Investigated in-depth were the prognostic relevance of the score, mutation features, immunological aspects, and pharmacological prediction performance. Investigations of the prognostic utility, immunological traits, drug prediction effectiveness and immunotherapy prediction of BNIP3 were performed in-depth. Investigations by cell counting kit-8, Transwell and 5-ethynyl-2-deoxyuridine (EdU) demonstrated that BNIP3 is an osteosarcoma tumor accelerator. The osteosarcoma gene BNIP3 may promote macrophage migration. The APP-related score shows potential for clinical use. It was anticipated that more in vitro and in vivo studies would confirm BNIP3's tumorigenic and immunogenic processes in osteosarcoma.

Endosialin/CD248 may be a potential therapeutic target to prevent the invasion and metastasis in osteosarcoma.

Endosialin/CD248/tumor endothelial marker 1 is classified as a C-type lectin-like transmembrane receptor, found on the plasma membrane of activated mesenchymal cells, which binds to fibronectin. Although endosialin is expressed at high levels in stem-like cells of sarcomas, its role has not been fully uncovered. The present study aimed to determine whether endosialin expression is associated with tumor progression and metastasis, and whether endosialin has the potential to act as a novel therapeutic target in osteosarcoma (OS) using MORAb-004/ontuxizumab, a humanized monoclonal antibody, which targets the type C lectin domain of endosialin. The results demonstrated that endosialin was highly expressed in OSs with metastatic disease. Furthermore, MORAb-004 had no cytostatic effect on OS cells in vitro and did not change the expression of stem cells and differentiation markers; however, it inhibited migration of OS cells. Taken together, these results suggest that endosialin may play a role in migration, and may be involved in the metastatic process of OSs. Furthermore, MORAb-004 reduces the motility of OS cells, and suppresses invasion and the development of metastatic lesions.

MiR-663a, regulated by lncRNA GAS5, contributes to osteosarcoma development through targeting MYL9.

Osteosarcoma is characterized by high malignancy and high metastasis rate, resulting in high mortality and disability. MiR-663a has been reported in a variety of tumors to promote tumorigenesis. However, miR-663a has not been reported in the pathogenesis of osteosarcoma. Bioinformatics analysis and experiments including real-time quantitative polymerase chain reaction (RT-qPCR), luciferase reporter, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, RNA immunoprecipitation, and flow cytometry assay were applied to explore the function and mechanism of miR-663a in MG63, U2OS, Saos-2, SF-86, and hFOB1.19 cells. In this study, we found that miR-663a is highly expressed in osteosarcoma. At the same time, we discovered that miR-663a facilitates cell proliferation and migration, whereas suppresses cell apoptosis in osteosarcoma. Through a series of biological experiments, it was found that miR-663a regulates the cellular process in osteosarcoma by modulating the expression of MYL9. In addition, we also found that long noncoding RNA (lncRNA) GAS5 serves as a molecular sponge for miR-663a and regulates the progression of osteosarcoma via the ceRNA mechanism. We uncover that miR-663a promotes osteosarcoma development through targeting MYL9, which was regulated by lncRNA GAS5.

LncRNA SNHG16 sponges miR-98-5p to regulate cellular processes in osteosarcoma.

As has been illustrated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are potential regulators in the occurrence and progression of human cancers. LncRNA SNHG16 has been identified as an oncogene involved in the progression of human cancers. However, neither the function nor the underlying molecular mechanism of SNHG16 in osteosarcoma has been discovered. The aim of the study is to explore the role and molecular regulation mechanism of SNHG16 in osteosarcoma. The expression of SNHG16 in HNSCC tissues and cells was detected by RT-qPCR assay. The biological function of SNHG16 in osteosarcoma was measured by CCK-8, cell cycle, cell apoptosis and transwell assays. The interaction between SNHG16 and miR-98-5p was studied by luciferase reporter and RIP assays. The ectopic expression of SNHG16 was found in osteosarcoma tissues and cell lines, which indicated poor prognosis and lower overall survival rate of osteosarcoma patients. Knockdown of SNHG16 inhibited cell proliferation, migration, invasion, cell cycle and promoted apoptosis in osteosarcoma. It was demonstrated that SNHG16 directly interacts with miR-98-5p. What's more, we found a significantly negative correlation between SNHG16 and miR-98-5p expression. Finally, rescue experiments revealed that inhibition of miR-98-5p attenuated SNHG16 knockdown-mediated effects on cellular processes in osteosarcoma. LncRNA SNHG16 regulated cellular processes in osteosarcoma by sponging miR-98-5p, and SNHG16 may be a new and effective molecular therapeutic target for osteosarcoma.

LncRNA BLACAT1 accelerates the proliferation and migration of osteosarcoma cells through regulating STAT3

BackgroundLong non-coding RNAs (lncRNAs) have been reported in human cancers as regulators for biological processes. LncRNA bladder cancer associated transcript 1 (BLACAT1) has been found to exert oncogenic function in cervical cancer and lung cancer. However, whether it can regulate the biological processes in osteosarcoma (OS) is still unclear. This study aims to examine the potential effect of dysregulated BLACAT1 on the progression of OS. MethodsThe expression pattern of BLACAT1 in OS tissues and cell lines was detected by qRT-PCR assay. Gain or loss-of function assays were designed and conducted to determine the effect of BLACAT1 overexpression or knockdown on the OS cell proliferation, apoptosis, invasion and migration. RNA pull-down assay and western blot analysis were performed to identify the relationship between BLACAT1 and signal transducer and activator of transcription 3 (STAT3). ResultsBLACAT1 was upregulated in OS tissues and cells. Upregulation of BLACAT1 predicted unfavorable prognosis for patients with OS. Downregulation of BLACAT1 inhibited cell proliferation and invasion, whereas upregulation of BLACAT1 accelerated cell proliferation and invasion. More importantly, BLACAT1 could interact with STAT3 and regulat the phosphorylation of STAT3. ConclusionsLncRNA BLACAT1 contributes to the proliferation and migration of OS cells by regulating STAT3.

TCF3-activated LINC00152 exerts oncogenic role in osteosarcoma through regulating miR-1182/CDK14 axis

Long noncoding RNAs (lncRNAs) have been reported to participate in tumorigenesis and diverse cellular processes in osteosarcoma (OS). However, the role of lncRNA LINC00152 in OS remains elusive. In this study, LINC00152 was highly expressed in osteosarcoma tissues and cell lines. Moreover, MTT and colony formation assays revealed that knockdown of LINC00152 significantly suppressed cell proliferation. The inhibitory effect of LINC00152 knockdown on OS cell migration and invasion was analyzed and demonstrated by transwell assays. Additionally, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays suggested that LINC00152 was transcriptionally activated by the transcription factor TCF3. More importantly, mechanism investigation revealed that LINC00152 was predominantly located in the cytoplasm of OS cells and acted as a competing endogenous RNA (ceRNA) in OS by regulating miR-1182/CDK14 axis. Collectively, LINC00152 was activated by TCF3 and promotes cell proliferation and migration in osteosarcoma via miR-1182-CDK14 axis.

Toosendanin demonstrates promising antitumor efficacy in osteosarcoma by targeting STAT3

Signal transducer and activator of transcription 3(STAT3) is an emerging target for cancer therapy. In this study, we identify Toosendanin (TSN) is an effective inhibitor of STAT3, leading to the impediment of various oncogenic processes in osteosarcoma. TSN selectively inactivates phospho-STAT3 (Tyr-705); subsequent molecular docking and in vitro SPR analysis uncover TSN directly binds to the SH2 domain of STAT3. Consequently, TSN blocks STAT3 dimerization and impairs the complex formation of STAT3 and epidermal growth factor receptor (EGFR). In an animal tumor model study, TSN is well tolerated, inhibits osteosarcoma growth and metastasis. In another osteosarcoma patient-derived xenografts (PDX) model, we find TSN triggers strong inhibitory effects on patient-derived tumors. Further studies show that TSN also displays activity against other solid tumors. Our preclinical work therefore supports that TSN acts as a novel inhibitor of STAT3 that blocks tumorigenesis in ostoesarcoma.

Crude Fucoidan Extracts Impair Angiogenesis in Models Relevant for Bone Regeneration and Osteosarcoma via Reduction of VEGF and SDF-1.

The marine origin polysaccharide fucoidan combines multiple biological activities. As demonstrated by various studies in vitro and in vivo, fucoidans show anti-viral, anti-tumor, anti-oxidant, anti-inflammatory and anti-coagulant properties, although the detailed molecular action remains to be elucidated. The aim of the present study is to assess the impact of crude fucoidan extracts, on the formation of vascular structures in co-culture models relevant for bone vascularization during bone repair and for vascularization processes in osteosarcoma. The co-cultures consisted of bone marrow derived mesenchymal stem cells, respectively the osteosarcoma cell line MG63, and human blood derived outgrowth endothelial cells (OEC). The concentration dependent effects on the metabolic activity on endothelial cells and osteoblast cells were first assessed using monocultures of OEC, MSC and MG63 suggesting a concentration of 100 µg/mL as a suitable concentration for further experiments. In co-cultures fucoidan significantly reduced angiogenesis in MSC/OEC but also in MG63/OEC co-cultures suggesting a potential application of fucoidan to lower the vascularization in bone tumors such as osteosarcoma. This was associated with a decrease in VEGF (vascular endothelial growth factor) and SDF-1 (stromal derived factor-1) on the protein level, both related to the control of angiogenesis and furthermore discussed as crucial factors in osteosarcoma progression and metastasis. In terms of bone formation, fucoidan slightly lowered on the calcification process in MSC monocultures and MSC/OEC co-cultures. In summary, these data suggest the suitability of lower fucoidan doses to limit angiogenesis for instance in osteosarcoma.

Dysregulation of macrophage polarization is associated with the metastatic process in osteosarcoma.

Osteosarcoma (OS) is the most common bone sarcoma in adolescents, and has poor prognosis. A vicious cycle is established between OS cells and their microenvironment in order to facilitate the tumor growth and cell spreading. The present work aims to better characterize the tumor microenvironment in OS in order to identify new therapeutic targets relating to metastatic process. Tissue microarrays of pre-chemotherapy OS biopsies were used for characterizing the tumor niche by immunohistochemistry. Parameters studies included: immune cells (M1, M2-subtypes of tumor-associated macrophages (TAM); T, B lymphocytes; mast cells), vascularization (endothelial, perivascular cells), OPG, RANKL, and mitotic index. Two groups of patients were defined, 22 localized OS (OS Meta-) and 28 metastatic OS (OS Meta+). The OS Meta- group was characterized by a higher infiltration of INOS+ M1-polarizedmacrophages and upregulated OPG immunostaining. OS Meta+ tumors showed a significant increase in CD146+ cells. INOS+ M1-macrophages were correlated with OPG staining, and negatively with the presence of metastases. CD163+ M2-macrophages were positively correlated with CD146+ cells. In multivariate analysis, INOS and OPG were predictive factors for metastasis. An older age, non-metastatic tumor, good response to chemotherapy, and higher macrophage infiltration were significantly associated with better overall survival. TAMs are associated with better overall survival and a dysregulation of M1/M2 polarized-macrophages in favor of M1 subtype was observed in non-metastatic OS.

Abstract 3399: CTGF promotes tumor angiogenesis process in osteosarcoma through induction of angiopoietin-2

Abstract Osteosarcoma is the most common primary malignant bone tumor. Angiogenesis is essential for the tumor growth. Angiopoietin-2 (Angpt2) plays a critical role in angiogenesis and tumor progression. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the angiogenesis of certain human cancer cells. However, the effect of CTGF on Angpt2 expression in human osteosarcoma cells is mostly unknown. In this study, we found that overexpression of CTGF in osteosarcoma promoted angiogenesis in vitro and in vivo by tube formation assay, and Matrigel plug assay. Knockdown of CTGF obviously reduced expression of Angpt2 and angiogenesis. Recently, microRNAs (miRNAs) have been demonstrated that functions as oncogenes or angiogenesis regulators in human cancer. Here, we also found that overexpression of CTGF significantly decreased miR-543 in osteosarcoma cells. Transfection of miR-543 mimic reduced Angpt2 expression and angiogenesis. Taken together, CTGF promotes angiogenesis via up-regulation of Angpt2 by decreasing miR-543 in human osteosarcoma. These findings certify that CTGF functions as a tumor enhancer gene, suggesting that CTGF may be a potential therapeutic target for osteosarcoma patients. Citation Format: Yu-che Cheng, Hsiao-Chi Tsai, Tzu-Wei Tan, Chih-Hsin Tang. CTGF promotes tumor angiogenesis process in osteosarcoma through induction of angiopoietin-2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3399.

Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment.

Interleukin-34 (IL-34) was recently characterized as the M-CSF "twin" cytokine, regulating the proliferation/differentiation/survival of myeloid cells. The implication of M-CSF in oncology was initially suspected by the reduced metastatic dissemination in knock-out mice, due to angiogenesis impairment. Based on this observation, our work studied the involvement of IL-34 in the pathogenesis of osteosarcoma. The in vivo effects of IL-34 were assessed on tissue vasculature and macrophage infiltration in a murine preclinical model based on a paratibial inoculation of human osteosarcoma cells overexpressing or not IL-34 or M-CSF. In vitro investigations using endothelial cell precursors and mature HUVEC cells were performed to analyse the involvement of IL-34 in angiogenesis and myeloid cell adhesion. The data revealed that IL-34 overexpression was associated with the progression of osteosarcoma (tumor growth, lung metastases) and an increase of neo-angiogenesis. In vitro analyses demonstrated that IL-34 stimulated endothelial cell proliferation and vascular cord formation. Pre-treatment of endothelial cells by chondroitinases/heparinases reduced the formation of vascular tubes and abolished the associated cell signalling. In addition, IL-34 increased the in vivo recruitment of M2 tumor-associated macrophages into the tumor tissue. IL-34 increased in vitro monocyte/CD34(+) cell adhesion to activated HUVEC monolayers under physiological shear stress conditions. This work also demonstrates that IL-34 is expressed by osteosarcoma cells, is regulated by TNF-α, IL-1β, and contributes to osteosarcoma growth by increasing the neo-angiogenesis and the recruitment of M2 macrophages. By promoting new vessel formation and extravasation of immune cells, IL-34 may play a key role in tumor development and inflammatory diseases.

Resveratrol abrogates the effects of hypoxia on cell proliferation, invasion and EMT in osteosarcoma cells through downregulation of the HIF-1α protein.

Resveratrol has been shown to have antineoplastic effects in vivo and in vitro. However, the effect of resveratrol on the hypoxia-enhanced proliferation and invasion of osteosarcoma cells remains unclear. In this study, we investigated the role of resveratrol on regulating proliferation and invasion of osteosarcoma cells under hypoxic conditions. Saos-2 cells were cultured under controlled hypoxic conditions (3% O2) or normoxic conditions. Resveratrol (50 µM) was added in the medium, and hypoxia inducible factor-1α (HIF-1α) siRNA was used to inhibit HIF-1α transcription. Proliferation of Saos-2 cells was evaluated by the methabenzthiazuron (MTT) assay. The invasive ability of Saos-2 cells was determined by a Transwell assay. HIF-1α, E-cadherin and vimentin protein levels were detected by western blot analysis. HIF-1α, E-cadherin and vimentin mRNA levels were assessed by RT-PCR. Compared to the control group, hypoxia significantly increased the proliferation rate and invasive ability of Saos-2 cells. Moreover, hypoxia markedly increased the E-cadherin level and decreased vimentin expression. However, resveratrol or HIF-1α silencing reverted all the above effects of hypoxia in Saos-2 cells. Moreover, resveratrol inhibited HIF-1α protein accumulation without affecting the HIF-1α mRNA level. These data suggest that resveratrol can inhibit the hypoxia‑enhanced proliferation, invasion and epithelial to mesenchymal transition process in osteosarcoma via downregulation of the HIF-1α protein. Thus, HIF-1α may be a promising drug target of resveratrol in the context of development of anticancer therapy for human osteosarcoma.

Zinc Finger X-Chromosomal Protein (ZFX) Promotes Solid Agar Colony Growth of Osteosarcoma Cells

Zinc finger X-chromosomal protein (ZFX) is a member of the zinc finger family of proteins. The importance of ZFX in several cancer types, including prostate cancer, laryngeal squamous cell carcinoma, and glioma, has been addressed. However, the role of ZFX in human osteosarcoma remains unknown. Here we investigated the phenotype of ZFX knockdown on cell proliferation and in vitro tumorigenesis using lentivirus-mediated loss-of-function strategy. The results demonstrated that the proliferation and colony formation ability of human osteosarcoma Saos-2 and MG63 cells was impaired by ZFX small interfering RNA (siRNA)-expressing lentivirus. Moreover, loss of ZFX led to G0/G1 phase cell cycle arrest and a significant increase of cells in the sub-G1 fraction, indicating that ZFX functions as an oncogene in the malignant proliferation process in osteosarcoma. Furthermore, ZFX siRNA may have an antitumorigenic effect on osteosarcoma cells. Our findings hold important significance for RNA interference-mediated cancer gene therapy for human osteosarcoma.

Inhibition of Csn3 expression induces growth arrest and apoptosis of hepatocellular carcinoma cells

Csn3 (or CSN3) encodes the third subunit of an eight-subunit complex, the COP9 signalosome (CSN), which acts as a protein kinase and a deneddylase in mammalian cells. Previous studies have shown that Csn3 is essential for maintenance of cell proliferation in the mouse embryonic epiblast and associated with the tumorigenesis process in osteosarcoma. However, its correlation with hepatocellular carcinoma (HCC) has not been explored yet. The expression of Csn3 in HCC (n = 30), cirrhosis (n = 30), and normal tissues (n = 30) was detected using immunohistochemical analysis. The impacts of lentivirus-mediated inhibition of Csn3 on HCC cells were detected using MTT, BrdU incorporation assay, and flow cytometric analysis. In addition, the colony formation and tumor growth ability in nude mice were detected to define the role of Csn3 in tumorigenesis. Knockdown of Csn3 expression in HCC cell lines (SMMC-7721 and Hep3B) significantly inhibits the tumor growth both in vitro and in vivo. Further investigation indicates that this growth inhibition effect may be mediated through cell cycle arrest in G0/G1 phase and inductions of pro-apoptotic proteins BIK and Caspase-8. In addition, knockdown of Csn3 expression evidently suppresses tumor growth in a xenograft nude mice model. Collectively, this study demonstrates Csn3 as an oncogene that regulates the tumorigenesis process in HCC cells.

MicroRNA-21 is involved in osteosarcoma cell invasion and migration

MicroRNAs are involved in different cancer-related processes. MicroRNA-21 (miR-21), as an oncomiR, is overexpressed in all kinds of tumors and the role of miR-21 in carcinogenesis is elucidated in many cancers gradually. However, the function of miR-21 in osteosarcoma is still unclear. In our study, we found that miR-21 was significantly overexpressed in osteosarcoma tissues. More importantly, we confirmed that knockdown of miR-21 greatly decreased cell invasion and migration of MG-63. Furthermore, we identified that RECK (reversion-inducing-cysteine-rich protein with kazal motifs), a tumor suppressor gene, was a direct target of miR-21. Finally, the expression of RECK protein negatively correlated with the expression of miR-21 in human osteosarcoma tissues, indicating the potential regulation of RECK by miR-21. Our results suggest that miR-21 expression has a key role in regulating cellular processes in osteosarcoma, likely through regulating RECK and may serve as a therapeutic target.

Etiology of osteosarcoma.

Although the prognosis and quality of life of patients with osteosarcoma were improved significantly during the past decades, the pathogenesis and etiology of this disease remain obscure. Significant interest and effort in this cancer led to the identification of numerous etiologic agents. Several chemical agents such as beryllium, viruses such as FBJ, subsequently found to contain the src-oncogene, and radiation were shown to be potent inducers of osteosarcoma. Paget's disease, electrical burn, or trauma all are thought to be other factors that may contribute to the pathogenesis. More recently, patients with hereditary diseases such as Rothmund-Thomson syndrome, Bloom syndrome, and Li-Fraumeni syndrome were found to have an increased risk of having osteosarcoma develop. During the past few years, the molecular analysis brought a wealth of new information with numerous genes that were associated with osteosarcoma and its clinical disease progression. They can be categorized into self-sufficiency in growth signals, insensitivity to growth inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue evasion and metastasis. Although the understanding of these processes in osteosarcoma still is incomplete, it may have the potential to significantly affect the patient care in the future.

THE HEALING PROCESS OF OSTEOSARCOMA UNDER THE INFLUENCE OF THE ROENTGEN RAYS

The healing process of osteosarcoma is very similar to that observed in the healing of any other bone disease, and consists of a bone sclerosis. This is indicated by a progressive deposit of lime salts in the tumor area. This process continues over a period of from several months to a year or more, and progresses long after the roentgenotherapy has been discontinued. The deposit of lime salts does not depend, therefore, on stimulation by the Roentgen rays, but is simply Nature's method of replacing the tumor tissue which has been destroyed by their action. It is well known that the rays cause a granular degeneration of tumor-cells before affecting the surrounding healthy tissue-cells, probably because of their lower degree of vitality or resistance. This effect is due to the chemical action of the rays absorbed, just as these rays show their chemical action on the silver salts on photographic