Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS.

PubMed

Ben-Shoshan, Shirley Oren; Simon, Amos J; Jacob-Hirsch, Jasmine; Shaklai, Sigal; Paz-Yaacov, Nurit; Amariglio, Ninette; Rechavi, Gideon; Trakhtenbrot, Luba

2014-01-28

Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. By knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways

PubMed Central

Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

2015-01-01

Aim: Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Methods: Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Results: Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Conclusions: Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis. PMID:26722474

Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins

PubMed Central

LV, TING-ZHUO; WANG, GUANG-SHUN

2015-01-01

Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera. This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins. PMID:26170956

Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins.

PubMed

Lv, Ting-Zhuo; Wang, Guang-Shun

2015-07-01

Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera . This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins.

p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cappadone, C., E-mail: concettina.cappadone@unibo.it; Stefanelli, C.; Malucelli, E.

2015-11-13

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of themore » cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity. - Highlights: • The indole derivative NSC743420 induces antitumor effects on osteosarcoma cells. • p53 status could drive the activity of antitumor agents on osteosarcoma cells. • NSC743420 induces cytostatic and differentiating effects on U2OS cells. • NSC743420 causes apoptosis on p53-null SaOS2 cells.« less

Lung cells support osteosarcoma cell migration and survival.

PubMed

Yu, Shibing; Fourman, Mitchell Stephen; Mahjoub, Adel; Mandell, Jonathan Brendan; Crasto, Jared Anthony; Greco, Nicholas Giuseppe; Weiss, Kurt Richard

2017-01-25

Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased

Expression and prognostic relevance of PRAME in primary osteosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Pingxian; Zou, Changye; Yong, Bicheng

2012-03-23

Graphical abstract: High PRAME expression was associated with osteosarcoma patients' poor prognosis and lung metastasis. Highlights: Black-Right-Pointing-Pointer We analyzed and verified the role of PRAME in primary osteosarcoma. Black-Right-Pointing-Pointer High PRAME expression in osteosarcoma correlated to poor prognosis and lung metastasis. Black-Right-Pointing-Pointer PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. -- Abstract: The preferentially expressed antigen of melanoma (PRAME), a cancer-testis antigen with unknown function, is expressed in many human malignancies and is considered an attractive potential target for tumor immunotherapy. However, studies of its expression and function in osteosarcoma havemore » rarely been reported. In this study, we found that PRAME is expressed in five osteosarcoma cell lines and in more than 70% of osteosarcoma patient specimens. In addition, an immunohistochemical analysis showed that high PRAME expression was associated with poor prognosis and lung metastasis. Furthermore, PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. Our results suggest that PRAME plays an important role in cell proliferation and disease progression in osteosarcoma. However, the detail mechanisms of PRAME function in osteosarcoma require further investigation.« less

New Treatment Options for Osteosarcoma - Inactivation of Osteosarcoma Cells by Cold Atmospheric Plasma.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Weiss, Martin; Napp, Matthias; Daeschlein, Georg; Sckell, Axel; Ender, Stephan A; Kramer, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2016-11-01

Cold atmospheric plasma has been shown to inhibit tumor cell growth and induce tumor cell death. The aim of the study was to investigate the effects of cold atmospheric plasma treatment on proliferation of human osteosarcoma cells and to characterize the underlying cellular mechanisms. Human osteosarcoma cells (U2-OS and MNNG/HOS) were treated with cold atmospheric plasma and seeded in culture plates. Cell proliferation, p53 and phospho-p53 protein expression and nuclear morphology were assessed. The treated human osteosarcoma cell lines exhibited attenuated proliferation rates by up to 66%. The cells revealed an induction of p53, as well as phospho-p53 expression, by 2.3-fold and 4.5-fold, respectively, compared to controls. 4',6-diamidino-2-phenylindole staining demonstrated apoptotic nuclear condensation following cold atmospheric plasma treatment. Cold atmospheric plasma treatment significantly attenuated cell proliferation in a preclinical in vitro osteosarcoma model. The resulting increase in p53 expression and phospho-activation in combination with characteristic nuclear changes indicate this was through induction of apoptosis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Long non-coding RNA AK093407 promotes proliferation and inhibits apoptosis of human osteosarcoma cells via STAT3 activation

PubMed Central

Wang, Yongkun; Liang, Tingting; Wang, Yao; Huang, Yan; Li, Ye

2017-01-01

Osteosarcoma is a malignant tumor of the skeletal system. Long non-coding RNAs (lncRNAs) have been shown to play significant role in osteosarcoma. The present study evaluated the effects and mechanism of lncRNA AK093407 in osteosarcoma. The study included human osteosarcoma cell line, U-2OS. Cell proliferation, viability, and apoptosis were measured using Ki-67 proliferation assay, MTT assay, and Annexin V/PI staining assay, respectively. Relative mRNA and protein expressions were measured using qRT-PCR and western blot, respectively. Interaction between AK093407 and STAT3 was identified using mass spectrometry and RNA pull-down assay. Results revealed that AK093407 was highly expressed in osteosarcoma cells and tissues. Then we demonstrated that overexpression of AK093407 promoted cell proliferation and viability and inhibited apoptosis, whereas suppression of AK093407 showed opposite effects. In addition, AK093407 regulated the expression of genes and proteins (Bcl-2, TGF-β, NF-κB, and PCNA) involved in the cell proliferation, viability, and apoptosis. Furthermore, we showed that AK093407 interacted with STAT3, and promoted its phosphorylation. Lastly, we showed that STAT3 activation was essential for the effects of AK093407 on cell proliferation and apoptosis as the overexpression of AK093407 in the presence of STAT3 inhibitor did not promote cell proliferation and inhibit cell apoptosis. AK093407 is highly expressed in osteosarcoma cells and tissues, and promotes cell proliferation and viability and inhibits apoptosis of osteosarcoma cell line U-2OS via STAT3 activation. PMID:28469961

miR-34a increases cisplatin sensitivity of osteosarcoma cells in vitro through up-regulation of c-Myc and Bim signal.

PubMed

Li, Qi-Cai; Xu, Haiyan; Wang, Xiaohui; Wang, Ting; Wu, Jiang

2017-12-12

Osteosarcoma is the most common primary malignancy in bone. Patients who respond poorly to induction chemotherapy are at higher risk of adverse prognosis. The molecular basis for such poor prognosis remains unclear. Recently, increasing evidence has suggested decreased expression of miR-34a is observed in a number of cancer types, including human osteosarcoma, and decreased miR-34a is involved in drug resistance. However, the underlying molecular mechanisms of decreased miR-34a on cisplatin chemoresistance in osteosarcoma has not been reported. Osteosarcoma U2OS cells were transfected with miR-34a mimics for 48 h, then the cells were treated with 3.0 μm cisplatin for 24 h. Using siRNA targeting c-Myc and Bim to examine the relation between miR-34a, c-Myc and Bim expression exposure to cisplatin on cisplatin-induced apoptosis. Treatment of U2OS cells with cisplatin induced cell apoptosis by upregulation of c-Myc -dependent Bim expression; Osteosarcoma U2OS cells transfected with miR-34a mimics (miR-34a/U2OS) induced cell apoptosis and inhibited cell survival, and increased the sensitivity of U2OS cells to cisplatin. U2OS cells transfected with miR-34a mimics upregulated the protein expression of c-Myc and Bim. Targeting c-Myc downregulated the expression of Bim in the miR-34a/U2OS cells. In addition, Targeting Bim reversed the chemeresistance of miR-34a/U2OS cells to cisplatin. Our data indicated that miR-34a enhanced the sensitivity to cisplatin by upregulation of c-Myc and Bim pathway.

Glycogen Synthase Kinase-3β, NF-κB Signaling, and Tumorigenesis of Human Osteosarcoma

PubMed Central

Tang, Qing-Lian; Xie, Xian-Biao; Wang, Jin; Chen, Qiong; Han, An-Jia; Zou, Chang-Ye; Yin, Jun-Qiang; Liu, Da-Wei; Liang, Yi; Zhao, Zhi-Qiang; Yong, Bi-Cheng; Zhang, Ru-Hua; Feng, Qi-Sheng; Deng, Wu-Guo; Zhu, Xiao-Feng; Zhou, Binhua P.; Zeng, Yi-Xin

2012-01-01

Background Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, may function as a tumor suppressor or an oncogene, depending on the tumor type. We sought to determine the biological function of GSK-3β in osteosarcoma, a rare pediatric cancer for which the identification of new therapeutic targets is urgent. Methods We used cell viability assays, colony formation assays, and apoptosis assays to analyze the effects of altered GSK-3β expression in U2OS, MG63, SAOS2, U2OS/MTX300, and ZOS osteosarcoma cell lines. Nude mice (n = 5–8 mice per group) were injected with U2OS/MTX300, and ZOS cells to assess the role of GSK-3β in osteosarcoma growth in vivo and to evaluate the effects of inhibitors and/or anticancer drugs on tumor growth. We used an antibody array, polymerase chain reaction, western blotting, and a luciferase reporter assay to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Immunochemistry was performed on primary tumor specimens from osteosarcoma patients (n = 74) to determine the relationship of GSK-3β activity with overall survival. Results Osteosarcoma cells with low levels of inactive p-Ser9-GSK-3β formed colonies in vitro and tumors in vivo more readily than cells with higher levels and cells in which GSK-3β had been silenced formed fewer colonies and smaller tumors than parental cells. Silencing or pharmacological inhibition of GSK-3β resulted in apoptosis of osteosarcoma cells. Inhibition of GSK-3β resulted in inhibition of the NF-κB pathway and reduction of NF-κB-mediated transcription. Combination treatments with GSK-3β inhibitors, NF-κB inhibitors, and chemotherapy drugs increased the effectiveness of chemotherapy drugs in vitro and in vivo. Patients whose osteosarcoma specimens had hyperactive GSK-3β, and nuclear NF-κB had a shorter median overall survival time (49.2 months) compared with patients whose tumors had inactive GSK-3β and NF-κB (109.2 months). Conclusion GSK

Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Hyunjin; Bergeron, Eric; Senta, Helena

2010-08-27

Research highlights: {yields} We show for the first time the effect of sanguinarine (SA) on MG63 and SaOS-2 cells. {yields} SA altered osteosarcoma cell viability in a concentration and time dependent manner. {yields} SA induced osteosarcoma cell apoptosis and increased caspase-8 and -9 activities. {yields} SA decreased dose dependently the Bcl-2 protein level only in MG63 cells. {yields} SaOS-2 which are osteoblast-derived, seemed more resistant to SA than MG63. -- Abstract: The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma,more » a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 {mu}mol/L sanguinarine for 4 and 24 h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 {mu}mol/L sanguinarine killed almost 100% of both cell populations within 24 h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1 h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.« less

Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

PubMed

Hancock, Meaghan H; Cliffe, Anna R; Knipe, David M; Smiley, James R

2010-02-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

PubMed

Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

1989-07-01

A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

PubMed

Geiss, Carsten; Kis, Zoltán; Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane; Lacroix, Jeannine

2017-10-17

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

Silencing of VEGF inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via VEGF/PI3K/AKT signaling pathway

PubMed Central

Peng, Ningning; Gao, Shuming; Guo, Xu; Wang, Guangya; Cheng, Cai; Li, Min; Liu, Kehun

2016-01-01

Background: Osteosarcoma is a kind of highly malignant tumor and the growth and metastasis is closely related to angiogenesis. Vascular endothelial growth factor (VEGF) is an important angiogenesis-promoting factor. In the current study, we investigated the effects of suppressed VEGF on osteosarcoma and its molecular mechanism provided for a basis by targeting angiogenesis. Material/Methods: We established bearing human osteosarcoma Wistar rats model by subcutaneous inoculation of human SaOS-2 cells and the adenovirus vector Ad-VEGF-siRNA was constructed for further study. We assessed the efficiency of VEGF silencing and its influence on SaOS-2 cells. The expression of mRNA and protein were detected by RT-PCR and western blotting, respectively. Intratumoral microvessel density (MVD), VEGF and CD31 were evaluated by immunohistochemistry. We detected the cell apoptotic rates by flow cytometry. Results: Our results indicated that Ad-VEGF-siRNA could effectively suppressed the expression of VEGF expression, inhibited the proliferation capability and promoted apoptosis of SaOS-2 cells in vitro. Silencing of VEGF expression also suppress osteosarcoma tumor growth and reduce osteosarcoma angiogenesis in the Wistar rats model in vivo. Furthermore, We found that phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation were considerably reduced while inhibition VEGF expression in SaOS-2 cells. Conclusion: Our data demonstrated that VEGF silencing could suppress cells proliferation, promote cells apoptosis and reduce osteosarcoma angiogenesis through inactivation of VEGF/PI3K/AKT signaling pathway. PMID:27158386

Delocalized Claudin-1 promotes metastasis of human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jian, Yuekui; Chen, Changqiong; Li, Bo

2015-10-23

Tight junction proteins (TJPs) including Claudins, Occludin and tight junction associated protein Zonula occludens-1 (ZO-1), are the most apical component of junctional complex that mediates cell–cell adhesion in epithelial and endothelial cells. In human malignancies, TJPs are often deregulated and affect cellular behaviors of tumor cells. In this study, we investigated alternations of TJPs and related biological characteristics in human osteosarcoma (OS). Claudin1 was increased in the metastatic OS cells (KRIB and KHOS) compared with the normal osteoblast cells (hFOB1.19) or primary tumor cells (HOS and U2OS), whereas no significant difference was found in Occludin and ZO-1. Immunohistochemistry, immunofluorescence andmore » Western blotting revealed that Claudin1 was initially localized at cell junctions of normal osteoblasts, but substantially delocalized to the nucleus of metastatic OS cells. Phenotypically, inhibition of the nucleus Claudin1 expression compromised the metastatic potential of KRIB and KHOS cells. Moreover, we found that protein kinase C (PKC) but not PKA phosphorylation influenced Claudin1 expression and cellular functions, as PKC inhibitor (Go 6983 and Staurosporine) or genetic silencing of PKC reduced Claudin1 expression and decreased the motility of KRIB and KHOS cells. Taken together, our study implied that delocalization of claudin-1 induced by PKC phosphorylation contributes to metastatic capacity of OS cells. - Highlights: • Claudin1 is increased during the malignant transformation of human OS. • Delocalization of Claudin1 in metastatic OS cells. • Silencing nuclear Claudin1 expression inhibits cell invasion of OS. • Deregulated Claudin1 is regulated by PKC.« less

Small interfering RNA-mediated silencing of G-protein-coupled receptor 137 inhibits growth of osteosarcoma cells.

PubMed

Li, Hao; Fu, Xiaodong; Gao, Yingjian; Li, Xiaomiao; Shen, Yi; Wang, Weili

2018-06-01

Osteosarcoma is the most widespread primary carcinoma in bones. Osteosarcoma cells are highly metastatic and frequently develop resistance to chemotherapy making this disease harder to treat. This identifies an urgent need of novel therapeutic strategies for osteosarcoma. G-Protein-coupled receptor 137 (GPR137) is involved in several human cancers and may be a novel therapeutic target. The expression of GPR137 was assessed in one osteoblast and three human osteosarcoma cell lines via the quantitative real-time polymerase chain reaction and western blot assays. Stable GPR137 knockdown cell lines were established using an RNA interference lentivirus system. Viability, colony formation, and flow cytometry assays were performed to measure the effects of GPR137 depletion on cell growth. The underlying molecular mechanism was determined using signaling array analysis and western blot assays. GPR137 expression was higher in the three human osteosarcoma cell lines, Saos-2, U2OS, and SW1353, than in osteoblast hFOB 1.19 cells. Lentivirus-mediated small interfering RNA targeting GPR137 successfully knocked down GPR137 mRNA and protein expression in both Saos-2 and U2OS cells. In the absence of GPR137, cell viability and colony formation ability were seriously impaired. The extent of apoptosis was also increased in both cell lines. Moreover, AMP-activated protein kinase α, proline-rich AKT substrate of 40 kDa, AKT, and extracellular signal-regulated kinase phosphorylation levels were down-regulated in GPR137 knockdown cells. The results of this study highlight the crucial role of GPR137 in promoting osteosarcoma cell growth in vitro . GPR137 could serve as a potential therapeutic target against osteosarcoma.

PD-1 axis expression in musculoskeletal tumors and antitumor effect of nivolumab in osteosarcoma model of humanized mouse.

PubMed

Zheng, Bingxin; Ren, Tingting; Huang, Yi; Sun, Kunkun; Wang, Shidong; Bao, Xing; Liu, Kuisheng; Guo, Wei

2018-02-06

Immune checkpoint inhibitors have led to a breakthrough in solid tumor immunotherapy, but related studies on musculoskeletal tumors are few, especially for PD-L2. We examined expression of three molecular effectors of the PD-1 axis in 234 patients with musculoskeletal tumors, including osteosarcoma, chondrosarcoma, synovial sarcoma, and giant cell tumor. Survival analyses and potential mechanisms were investigated in osteosarcoma per the Gene Expression Omnibus (GEO) and immunohistochemistry analyses. In vivo, humanized mice were used to evaluate the effect of nivolumab on osteosarcoma. PD-L1, PD-L2, and PD-1 expression levels were significantly different between the histologic types of the musculoskeletal tumors. For osteosarcoma, PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with overall survival (OS). Meanwhile, PD-L2 had a positive correlation trend with OS. Nivolumab inhibited osteosarcoma metastasis in humanized mice by increasing CD4+ and CD8+ lymphocytes and the cytolytic activity of CD8 lymphocytes in the lung but did not affect primary osteosarcoma growth. We systematically detected the expression patterns of PD-L1, PD-L2, and PD-1 in musculoskeletal tumors for the first time and demonstrated the prognostic roles and underlying mechanisms of PD-1 axis in osteosarcoma. Furthermore, PD-1 blockade could effectively control osteosarcoma pulmonary metastasis in vivo. Therefore, the PD-1 axis may be a potential immunotherapeutic target for metastatic osteosarcoma.

Herpes Simplex Virus VP16, but Not ICP0, Is Required To Reduce Histone Occupancy and Enhance Histone Acetylation on Viral Genomes in U2OS Osteosarcoma Cells▿ †

PubMed Central

Hancock, Meaghan H.; Cliffe, Anna R.; Knipe, David M.; Smiley, James R.

2010-01-01

The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure. PMID:19939931

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

PubMed Central

Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R.; Rommelaere, Jean; Lacroix, Jeannine

2017-01-01

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo. PMID:29039746

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

Global Gene Expression Analysis of Canine Osteosarcoma Stem Cells Reveals a Novel Role for COX-2 in Tumour Initiation

PubMed Central

Pang, Lisa Y.; Gatenby, Emma L.; Kamida, Ayako; Whitelaw, Bruce A.; Hupp, Ted R.; Argyle, David J.

2014-01-01

Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation. PMID:24416158

Global gene expression analysis of canine osteosarcoma stem cells reveals a novel role for COX-2 in tumour initiation.

PubMed

Pang, Lisa Y; Gatenby, Emma L; Kamida, Ayako; Whitelaw, Bruce A; Hupp, Ted R; Argyle, David J

2014-01-01

Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

miR-144 suppresses proliferation and induces apoptosis of osteosarcoma cells via direct regulation of mTOR expression.

PubMed

Ren, Yuan-Fei; Zhang, Tie-Hui; Zhong, Sheng; Zhao, Yan-Tao; Lv, Ya-Nan

2018-01-01

Studied as a type of tumor suppressor, microRNA (miR) performs an important role in growth and apoptosis of various human carcinomas. However, the effects of miR-l44 on osteosarcoma growth and apoptosis, as well as possible underlying mechanisms, remain unclear. The present study investigated the expression of miR-144 in osteosarcoma MG-63 and U-2 OS cell lines compared with osteoblast cells. In order to elucidate the effects of miR-144 on osteosarcoma, miR-144 was upregulated in MG-63 and U-2 OS cells by transfecting chemically synthesized miR-144 mimics. Bioinformatics analysis of potential miR-144 target genes was performed using TargetScanHuman 7.0 and confirmed by luciferase assay. This analysis identified mammalian target of rapamycin (mTOR) as a target of miR-144. The present results indicated that the overexpression of miR-144 may significantly inhibit proliferation and promote apoptosis of MG-63 and U-2 cells compared with scramble control. Furthermore, the effects of miR-144 on osteosarcoma were associated with the mTOR signaling pathway via directly targeting the 3' untranslated region of mTOR mRNA, resulting in a decrease in the level of mTOR protein. In summary, miR-144 was demonstrated to act as a tumor suppressor, which inhibits proliferation and promotes apoptosis of osteosarcoma cell lines. In addition, this effect was mediated by direct targeting on mTOR following inhibition of the mTOR signaling pathway. The present study suggested that miR-144 may be a candidate for the gene therapy of osteosarcoma.

Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

PubMed

Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

2017-01-01

A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G 1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G 1 phase arrest. These results suggest that PANDA promotes G 1 -S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Experimental tumor growth of canine osteosarcoma cell line on chick embryo chorioallantoic membrane (in vivo studies).

PubMed

Walewska, Magdalena; Dolka, Izabella; Małek, Anna; Wojtalewicz, Anna; Wojtkowska, Agata; Żbikowski, Artur; Lechowski, Roman; Zabielska-Koczywąs, Katarzyna

2017-05-12

The chick embryo chorioallantoic membrane (CAM) model is extensively used in human medicine in preclinical oncological studies. The CAM model has several advantages: low cost, simple experimental approach, time saving and following "3R principles". Research has shown that the human osteosarcoma cell lines U2OS, MMNG-HOS, and SAOS can form tumors on the CAM. In veterinary medicine, this has been described only for feline fibrosarcomas, feline mammary carcinomas and canine osteosarcomas. However, in case of canine osteosarcomas, it has been shown that only non-adherent osteosarcoma stem cells isolated from KTOSA5 and CSKOS cell lines have the ability to form microtumors on the CAM after an incubation period of 5 days, in contrast to adherent KTOSA5 and CSKOS cells. In the presented study, we have proven that the commercial adherent canine osteosarcoma cell line (D-17) can form vascularized tumors on the CAM after the incubation period of 10 days.

Capillary electrophoresis coupled with mass spectrometry for the evaluation of substance P enzymatic degradation by SaOS-2 human osteosarcoma.

PubMed

Cavazza, Antonella; Corradini, Claudio; Marini, Mario; Roda, Luigi Giorgio; Valenti, Angela

2011-09-01

A new analytical method for the detection and the quantitative evaluation of the undecapeptide substance P by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) by a co-axial sheath liquid interface has been developed. Conditions of analysis employed an acidic buffer and a 60 cm fused silica capillary installed by overcoming the UV window position, thus allowing to perform the analysis in a brief time. The method has been applied to the evaluation of substance P enzymatic hydrolysis during incubation with the human osteosarcoma SaOS-2 cell line. The analysis of amino acids derived from the cleavage of substance P has been also carried out simultaneously under the same electrophoretic conditions allowing the description of a kinetic of amino acid formation, parallel with substance P disappearance. The amounts of intact substance P and of free amino acids were monitored along 600 s of incubation time. A steady decrease of substance P as function of reaction time was observed. Peptide's half-life was found to be about 4.3s, indicating an extremely fast hydrolysis in the presence of the SaOS-2 cells. Proline, phenilalanine and methionine were the predominant free amino acids recorded. Obtained results lead to hypothesize the occurrence of endopeptidases activity, followed by aminopeptidases responsible for the release of free amino acids originated after primary bond cleavage. Copyright © 2011 Elsevier B.V. All rights reserved.

Expression of Bcl-2 in canine osteosarcoma

PubMed Central

Piro, F.; Leonardi, L.

2015-01-01

Osteosarcoma (OS) is the most common primary malignancy of bone. It is responsible for 80-85% of the primary bone tumors affecting dogs and it is characterized by aggressive and invasive behavior, with a high metastatic potential. Several studies on cancer and related tumorigenesis, show an involvement of the mechanisms of programmed cell death and cell survival. Many signals seem to be involved in the related mechanism of autophagy and in particular, our interest is focused on the expression of a family of Bcl-2 that seems to be involved either in the control of biomolecular mechanisms like autophagy and apoptosis. In this study we investigated the expression of Bcl-2 in different cases of spontaneous canine osteosarcoma and the related preliminary results are described. We found Bcl-2 activity was increased in OS tissue compared to normal bone tissue. These results suggested that Bcl-2 activity may play an important role in the formation of OS and as a diagnostic for neoplastic activity. However, further research is needed to confirm the role of Bcl-2 activity in OS in canines. PMID:26623359

Comparative Aspects of Osteosarcoma Pathogenesis in Humans and Dogs

PubMed Central

Fan, Timothy M.; Khanna, Chand

2015-01-01

Osteosarcoma (OS) is a primary and aggressive bone sarcoma affecting the skeleton of two principal species, human beings and canines. The biologic behavior of OS is conserved between people and dogs, and evidence suggests that fundamental discoveries in OS biology can be facilitated through detailed and comparative studies. In particular, the relative genetic homogeneity associated with specific dog breeds can provide opportunities to facilitate the discovery of key genetic drivers involved in OS pathogenesis, which, to-date, remain elusive. In this review, known causative factors that predispose to the development OS in human beings and dogs are summarized in detail. Based upon the commonalities shared in OS pathogenesis, it is likely that foundational discoveries in one species will be translationally relevant to the other and emphasizes the unique opportunities that might be gained through comparative scientific approaches. PMID:29061942

CXCR6 promotes tumor cell proliferation and metastasis in osteosarcoma through the Akt pathway.

PubMed

Ma, Yunsheng; Xu, Xin; Luo, Mei

2017-01-01

Chemokine (C-X-C motif) receptor 6 (CXCR6) is up-regulated in many malignancies, indicating that CXCR6 plays an important role in tumor progression. However, the expression and function of CXCR6 in osteosarcoma (OS) remains unclear. This study aimed to explore the expression levels and function of CXCR6 in OS tissues and osteosarcoma cell lines MG-63, HOS and U2OS. The protein expression levels of CXCR6 in OS patient tissues and three osteosarcoma cell lines MG-63, HOS and U2OS were assessed. CXCR6-overexpression MG-63 cell lines were established and then the proliferation, invasion and the epithelial-mesenchymal transition (EMT) in those cells were assessed. CXCR6 mRNA levels in OS tissues were significantly higher than those in normal bone tissues. Consistently, both of the mRNA and protein levels of CXCR6 in OS cell lines MG-63, HOS and U2OS were higher than those in normal bone cells hFOB1.19. CXCR6 overexpression not only promoted cell proliferation, invasion and EMT, but also enhanced the phosphorylation of Akt in MG-63 cells. After inhibition of Akt-phosphorylation by Akt inhibitor, LY2940023, CXCR6-induced cell proliferation and invasion were dramatically attenuated. In conclusion, the present study demonstrated that CXCR6 enhances OS cell proliferation and invasion through the Akt pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Millimeter wave treatment induces apoptosis via activation of the mitochondrial-dependent pathway in human osteosarcoma cells.

PubMed

Wu, Guangwen; Chen, Xuzheng; Peng, Jun; Cai, Qiaoyan; Ye, Jinxia; Xu, Huifeng; Zheng, Chunsong; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2012-05-01

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment.

Paris polyphylla Suppresses Proliferation and Vasculogenic Mimicry of Human Osteosarcoma Cells and Inhibits Tumor Growth In Vivo.

PubMed

Yao, Nan; Ren, Ke; Wang, Yimin; Jin, Qiaomei; Lu, Xiao; Lu, Yan; Jiang, Cuihua; Zhang, Dongjian; Lu, Jun; Wang, Chen; Huo, Jiege; Chen, Yong; Zhang, Jian

2017-01-01

Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE. PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

Alpha-CaMKII plays a critical role in determining the aggressive behavior of human osteosarcoma.

PubMed

Daft, Paul G; Yuan, Kaiyu; Warram, Jason M; Klein, Michael J; Siegal, Gene P; Zayzafoon, Majd

2013-04-01

Osteosarcoma is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. Despite improvements in osteosarcoma treatment, more specific molecular targets are needed as potential therapeutic options. One target of interest is α-Ca(2+)/calmodulin-dependent protein kinase II (α-CaMKII), a ubiquitous mediator of Ca(2+)-linked signaling, which has been shown to regulate tumor cell proliferation and differentiation. Here, we investigate the role of α-CaMKII in the growth and tumorigenicity of human osteosarcoma. We show that α-CaMKII is highly expressed in primary osteosarcoma tissue derived from 114 patients, and is expressed in varying levels in different human osteosarcoma (OS) cell lines [MG-63, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)/HOS, and 143B). To examine whether α-CaMKII regulates osteosarcoma tumorigenic properties, we genetically inhibited α-CaMKII in two osteosarcoma cell lines using two different α-CaMKII shRNAs delivered by lentiviral vectors and overexpressed α-CaMKII by retrovirus. The genetic deletion of α-CaMKII by short hairpin RNA (shRNA) in MG-63 and 143B cells resulted in decreased proliferation (50% and 41%), migration (22% and 25%), and invasion (95% and 90%), respectively. The overexpression of α-CaMKII in HOS cells resulted in increased proliferation (240%), migration (640%), and invasion (10,000%). Furthermore, α-CaMKII deletion in MG-63 cells significantly reduced tumor burden in vivo (65%), whereas α-CaMKII overexpression resulted in tumor formation in a previously nontumor forming osteosarcoma cell line (HOS). Our results suggest that α-CaMKII plays a critical role in determining the aggressive phenotype of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat this devastating adolescent disease. ©2013 AACR.

Osthole inhibits proliferation and induces apoptosis in human osteosarcoma cells.

PubMed

Ding, Yong; Lu, Xiongwei; Hu, Xiaopeng; Ma, Jie; Ding, Huan

2014-02-01

The purpose of this study was to investigate the effect of osthole on osteosarcoma cell proliferation and apoptosis. Cell counting Kit-8 assay was performed to establish the effects of osthole on osteosarcoma MG-63 cell proliferation. Annexin V-FITC/PI was performed to analyze the apoptotic rate of the cells. The inhibitory effects of osthole on the expression of BCL-2, BAX, and caspase-3 were detected by Western blotting. Osthole inhibited the growth of human osteosarcoma MG-63 cells by inhibiting cell proliferation and induced cell apoptosis. Western blotting demonstrated that osthole downregulated the expressions of BCL-2 and caspase-3 and upregulated the expression of BAX in human osteosarcoma cells. Osthole can inhibit osteosarcoma cell proliferation and induced apoptosis effectively in a dose-dependent manner through downregulating the expression of BCL-2 and caspase-3 proteins levels and upregulating the expression of BAX proteins levels.

Selaginella tamariscina (Beauv.) possesses antimetastatic effects on human osteosarcoma cells by decreasing MMP-2 and MMP-9 secretions via p38 and Akt signaling pathways.

PubMed

Yang, Jia-Sin; Lin, Chiao-Wen; Hsieh, Yih-Shou; Cheng, Hsin-Lin; Lue, Ko-Huang; Yang, Shun-Fa; Lu, Ko-Hsiu

2013-09-01

Selaginella tamariscina is a traditional medicinal plant for treatment of some advanced cancers in the Orient. However, the effect of S. tamariscina on metastasis of osteosarcoma and the underlying mechanism remain unclear. We tested the hypothesis that S. tamariscina suppresses cellular motility, invasion and migration and also investigated its signaling pathways. This study demonstrates that S. tamariscina, at a range of concentrations (from 0 to 50 μg/mL), concentration-dependently inhibited the migration/invasion capacities of three osteosarcoma cell lines without cytotoxic effects. Zymographic and western blot analyses revealed that S. tamariscina inhibited the matrix metalloproteinase (MMP)-2 and MMP-9 enzyme activity, as well as protein expression. Western blot analysis also showed that S. tamariscina inhibits phosphorylation of p38 and Akt. Furthermore, SB203580 (p38 inhibitor) and LY294002 (PI3K inhibitor) showed the similar effects as S. tamariscina in U2OS cells. In conclusion, S. tamariscina possesses an antimetastatic activity in osteosarcoma cells by down-regulating MMP-2 and MMP-9 secretions and increasing TIMP-1 and TIMP-2 expressions through p38 and Akt-dependent pathways. S. tamariscina may be a powerful candidate to develop a preventive agent for osteosarcoma metastasis. Copyright © 2013 Elsevier Ltd. All rights reserved.

MicroRNA-127-3p inhibits proliferation and invasion by targeting SETD8 in human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jun; Hou, Wengen; Chai, Mingxiang

2016-01-22

MicroRNAs (miRNAs) play an essential role in cancer development. Several studies have indicated that miRNAs mediate tumorigenesis processes, such as, inflammation, proliferation, apoptosis and invasion. In the present study, we focused on the influence of the miR-127-3p on the proliferation, migration and invasion of osteosarcoma (OS). MiR-127-3p was found at reduced levels in OS tissues and cell lines. Overexpression of miR-127-3p in the OS cell lines significantly inhibited the cell proliferation, migration and invasion; however, inhibition of miR-127-3p increased the proliferation, migration and invasion of OS in vitro. SETD8 was identified as a direct target of miR-127-3p, and SETD8 expression decreasedmore » post miR-127-3p overexpression, while SETD8 overexpression could reverse the potential influence of miR-127-3p on the migration and invasion of OS cells. MiR-127-3p is suggested to act mainly via the suppression of SETD8 expression. Overall, the results revealed that miR-127-3p acts as a tumor suppressor and that its down-regulation in cancer may contribute to OS progression and metastasis, suggesting that miR-127-3p could be a potential therapeutic target in the treatment of OS. - Highlights: • MiR-127-3p is decreased in osteosarcoma tissues and cell lines. • MiR-127-3p overexpression suppresses cell migration and invasion in MG63 and U2OS. • SETD8 overexpression abolishes the roles of miR-127-3p in osteosarcoma.« less

Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

PubMed

Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

2014-01-01

Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.

Inhibition of human osteosarcoma cell migration and invasion by a gene silencer, pyrrole-imidazole polyamide, targeted at the human MMP9 NF-κB binding site.

PubMed

Kojima, Toshio; Wang, Xiaofei; Fujiwara, Kyoko; Osaka, Shunzo; Yoshida, Yukihiro; Osaka, Eiji; Taniguchi, Masashi; Ueno, Takahiro; Fukuda, Noboru; Soma, Masayoshi; Tokuhashi, Yasuaki; Nagase, Hiroki

2014-01-01

Osteosarcoma is one of the most prevalent bone tumors, occurring mostly in adolescence. However, no noticeable progress has been achieved in developing new therapeutic agents for this disease. Matrix metalloproteinase 9 (MMP9), a type IV collagenase, is a known anticancer target and is overexpressed in osteosarcomas. MMPs can degrade components of the extracellular matrix and are known to be involved in tumor invasion and metastasis. In the present study, we designed and synthesized a pyrrole-imidazole polyamide (HN.49), a gene-silencing agent that specifically targets the nuclear factor-kappa B (NF-κB) binding site of the human MMP9 promoter. We then examined the effect of HN.49 on the enzyme activity of MMP9 and the migration activity of osteosarcoma cells in vitro. It was clearly shown that HN.49 polyamide reduced the expression level of MMP9 mRNA and the enzymatic activity of MMP-9 in SaOS-2 cells. Moreover, HN.49 polyamide inhibited migration and invasion by SaOS-2 cells in in vitro wound-closure and matrigel-invasion assays. These results indicate that HN.49 may be a potential therapeutic agent for inhibiting the invasion and metastasis of osteosarcoma.

DHFR and MDR1 upregulation is associated with chemoresistance in osteosarcoma stem-like cells

PubMed Central

Lee, Yu-Hsien; Yang, Hui-Wen; Yang, Li-Chiu; Lu, Ming-Yi; Tsai, Lo-Lin; Yang, Shun-Fa; Huang, Yu-Feng; Chou, Ming-Yung; Yu, Cheng-Chia; Hu, Fang-Wei

2017-01-01

Tumor-initiating cells (TICs) are defined as a specialized subset of cells with tumor-initiating capacity that can initiate tumor growth, tumor relapse and metastasis. In the present study, osteosarcoma TICs (OS-TICs) were isolated and enriched from the osteosarcoma U2OS and MG-63 cell lines using sphere formation assays and serum-depleted media. These enriched OS-TICs showed the expression of several typical cancer stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, cluster of differentiation (CD)117, Nestin and CD133, and the expression of ATP binding cassette subfamily G member 2, multidrug resistance protein 1 (MDR1) and dihydrofolate reductase (DHFR). Notably, in vitro and in vivo tumorigenic properties were enhanced in these OS-TICs. Additionally, methotrexate and doxorubicin are the most widely used anticancer agents against osteosarcoma, and the observed enhanced chemoresistance of OS-TICs to these two agents could be associated with the upregulation of DHFR and MDR1. These findings suggest that the upregulation of DHFR and MDR1 is associated with the development of chemoresistance of OS-TICs. PMID:28693150

Raman spectroscopy for grading of live osteosarcoma cells.

PubMed

Chiang, Yi-Hung; Wu, Stewart H; Kuo, Yi-Chun; Chen, How-Foo; Chiou, Arthur; Lee, Oscar K

2015-04-18

Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells. We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma. We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p < 0.05). Matrix metalloproteinase MMP2, MMP9 were highly expressed in SaOS2, 143B and MSCs and decreased in human fetal osteoblast (FOB) and MG-63 cells as expected (p < 0.05). These results may highlight the inverse correlation between HA level and prognosis of osteosarcoma. The use of Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

Characterization of canine osteosarcoma by array comparative genomic hybridization and RT-qPCR: signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart.

PubMed

Angstadt, Andrea Y; Motsinger-Reif, Alison; Thomas, Rachael; Kisseberth, William C; Guillermo Couto, C; Duval, Dawn L; Nielsen, Dahlia M; Modiano, Jaime F; Breen, Matthew

2011-11-01

Osteosarcoma (OS) is the most commonly diagnosed malignant bone tumor in humans and dogs, characterized in both species by extremely complex karyotypes exhibiting high frequencies of genomic imbalance. Evaluation of genomic signatures in human OS using array comparative genomic hybridization (aCGH) has assisted in uncovering genetic mechanisms that result in disease phenotype. Previous low-resolution (10-20 Mb) aCGH analysis of canine OS identified a wide range of recurrent DNA copy number aberrations, indicating extensive genomic instability. In this study, we profiled 123 canine OS tumors by 1 Mb-resolution aCGH to generate a dataset for direct comparison with current data for human OS, concluding that several high frequency aberrations in canine and human OS are orthologous. To ensure complete coverage of gene annotation, we identified the human refseq genes that map to these orthologous aberrant dog regions and found several candidate genes warranting evaluation for OS involvement. Specifically, subsequenct FISH and qRT-PCR analysis of RUNX2, TUSC3, and PTEN indicated that expression levels correlated with genomic copy number status, showcasing RUNX2 as an OS associated gene and TUSC3 as a possible tumor suppressor candidate. Together these data demonstrate the ability of genomic comparative oncology to identify genetic abberations which may be important for OS progression. Large scale screening of genomic imbalance in canine OS further validates the use of the dog as a suitable model for human cancers, supporting the idea that dysregulation discovered in canine cancers will provide an avenue for complementary study in human counterparts. Copyright © 2011 Wiley-Liss, Inc.

PSMC2 is up-regulated in osteosarcoma and regulates osteosarcoma cell proliferation, apoptosis and migration

PubMed Central

Song, Mingzhi; Wang, Yong

2017-01-01

Proteasome 26S subunit ATPase 2 (PSMC2) is a recently identified gene potentially associated with certain human carcinogenesis. However, the expressional correlation and functional importance of PSMC2 in osteosarcoma is still unclear. Current study was focused on elucidating the significance of PSMC2 on malignant behaviors in osteosarcoma including proliferation, apoptosis, colony formation, migration as well as invasion. The high protein levels of PSMC2 in osteosarcoma samples were identified by tissue microarrays analysis. Besides, its expression in the levels of mRNA and protein was also detected in four different osteosarcoma cell lines by real-time PCR and western blotting separately. Silencing PSMC2 by RNA interference in osteosarcoma cell lines (SaoS-2 and MG-63) would significantly suppress cell proliferation, enhance apoptosis, accelerate G2/M phase and/or S phase arrest, and decrease single cell colony formation. Similarly, pharmaceutical inhibition of proteasome with MG132 would mimic the PSMC2 depletion induced defects in cell cycle arrest, apoptosis and colonies formation. Silencing of PSMC2 was able to inhibit osteosarcoma cell motility, invasion as well as tumorigenicity in nude mice. Moreover, the gene microarray indicated knockdown of PSMC2 notably changed a number of genes, especially some cancer related genes including ITGA6, FN1, CCND1, CCNE2 and TGFβR2, and whose expression changes were further confirmed by western blotting. Our data suggested that PSMC2 may work as an oncogene for osteosarcoma and that inhibition of PSMC2 may be a therapeutic strategy for osteosarcoma treatment. PMID:27888613

Raddeanin A, a natural triterpenoid saponin compound, exerts anticancer effect on human osteosarcoma via the ROS/JNK and NF-κB signal pathway.

PubMed

Ma, Bo; Zhu, Jianwei; Zhao, Ang; Zhang, Jie; Wang, Yu; Zhang, Hang; Zhang, Lifang; Zhang, Qi

2018-05-27

Osteosarcoma (OS) is the most frequent and high mortality primary bone tumor in the adolescent. And it is well-known for poor prognosis due to high incidence of metastasis. Raddeanin A (RA), an active component of Anemone raddeana Regel, showed potential anti-cancer activities. However, the anti-tumor effect and molecular mechanism(s) of RA on osteosarcoma are still unclear. The present research is the first in vitro and in vivo investigate systematically anticancer of RA on human osteosarcoma. Our study demonstrated that RA induced mitochondria-dependent apoptosis in osteosarcoma cell lines and markedly suppressed the metastasis of osteosarcoma cells in vitro. And, RA treatment markedly inhibits tumor growth in vivo. Further mechanism study demonstrated that RA caused a significant enhance reactive oxygen species (ROS) level to stimulate phosphorylation of JNK. Moreover, RA led to decrease of p-IκBα level in the cytosol and reduction of p65 level in the nucleus, which was associated with the inhibition of NF-κB transcriptional activity. When NF-κB signaling was inhibited by siRNA targeting p65, a significant increase in cell apoptosis activity was observed. In addition, non-toxic RA concentrations (0.25, 0.5 and 1 μM) inhibited the migration and invasion of OS by suppressing MMP-2/9 expression associated with NF-κB-dependent transcription in vitro. The silencing of p65 increased the sensitivity of the osteosarcoma cells to RA suppressed migration and invasion. These findings suggest RA induces apoptosis and inhibits metastasis in OS cells, involved in provoking ROS/JNK and inhibiting NF-κB signaling pathways. Therefore, it may be a potential anti-metastatic and anti-proliferative therapeutic agent for human osteosarcoma. Copyright © 2017. Published by Elsevier Inc.

Patient-derived osteosarcoma cells are resistant to methotrexate.

PubMed

Dos Santos Cavalcanti, Amanda; Meohas, Walter; Ribeiro, Gabriele de Oliveira; de Sá Lopes, Ana Cristina; Gholamin, Sharareh; Razavi, Mostafa; Hanae Kasai Brunswick, Taís; Avan, Amir; Matheus Guimarães, João Antonio; Leite Duarte, Maria Eugenia; Kahn, Suzana Assad

2017-01-01

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas.

Patient-derived osteosarcoma cells are resistant to methotrexate

PubMed Central

dos Santos Cavalcanti, Amanda; Meohas, Walter; Ribeiro, Gabriele de Oliveira; de Sá Lopes, Ana Cristina; Gholamin, Sharareh; Razavi, Mostafa; Hanae Kasai Brunswick, Taís; Avan, Amir; Matheus Guimarães, João Antonio; Leite Duarte, Maria Eugenia

2017-01-01

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas. PMID:28934267

Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

PubMed Central

Rastegar, Farbod; Gao, Jian-Li; Shenaq, Deana; Luo, Qing; Shi, Qiong; Kim, Stephanie H.; Jiang, Wei; Wagner, Eric R.; Huang, Enyi; Gao, Yanhong; Shen, Jikun; Yang, Ke; He, Bai-Cheng; Chen, Liang; Zuo, Guo-Wei; Luo, Jinyong; Luo, Xiaoji; Bi, Yang; Liu, Xing; Li, Mi; Hu, Ning; Wang, Linyuan; Luther, Gaurav; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan

2010-01-01

Background Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. Methodology/Principal Findings Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. Conclusions/Significance Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially

Synergistic effect of low-dose cucurbitacin B and low-dose methotrexate for treatment of human osteosarcoma

PubMed Central

Goff, Catherine; Iwanski, Gabriela B.; Forscher, Charles; Doan, Ngan B.; Said, Jonathan W.; Koeffler, H. Phillip

2016-01-01

We investigated the use of cucurbitacin B, a plant-derived tetracyclic triterpenoid, as a single agent or in combination with methotrexate (MTX) for human osteosarcoma (OS) treatment. Cucurbitacin B showed antiproliferative activity against seven human OS cell lines in vitro accompanying G2/M cell cycle arrest, apoptosis, and inhibition of ERK, Akt, and mTOR proteins. Cucurbitacin B in combination with MTX synergistically inhibited OS cell growth in vitro. Low-dose cucurbitacin B (LD-CuB, 0.5 mg/kg body weight) or low-dose MTX (LD-MTX, 150 mg/kg) failed to decrease the size of human OS xenografts in nude mice. However, combined therapy at identical concentrations inhibited tumor growth by 62% vs. LD-CuB and 81% vs. LD-MTX (p < 0.001). Strikingly, the effect persisted even when the dose of MTX was decreased by two thirds (VLD-MTX, 50 mg/kg). In conclusion, cucurbitacin B alone or in combination with MTX shows promising antiproliferative activity against human OS. PMID:21440986

Selective Cytotoxicity against Human Osteosarcoma Cells by a Novel Synthetic C-1 Analogue of 7-Deoxypancratistatin Is Potentiated by Curcumin

PubMed Central

Ma, Dennis; Tremblay, Phillip; Mahngar, Kevinjeet; Collins, Jonathan; Hudlicky, Tomas; Pandey, Siyaram

2011-01-01

The natural compound pancratistatin (PST) is a non-genotoxic inducer of apoptosis in a variety of cancers. It exhibits cancer selectivity as non-cancerous cells are markedly less sensitive to PST. Nonetheless, PST is not readily synthesized and is present in very low quantities in its natural source to be applied clinically. We have previously synthesized and evaluated several synthetic analogues of 7-deoxypancratistatin, and found that JC-TH-acetate-4 (JCTH-4), a C-1 acetoxymethyl analogue, possessed similar apoptosis inducing activity compared to PST. In this study, notoriously chemoresistant osteosarcoma (OS) cells (Saos-2, U-2 OS) were substantially susceptible to JCTH-4-induced apoptosis through mitochondrial targeting; JCTH-4 induced collapse of mitochondrial membrane potential (MMP), increased reactive oxygen species (ROS) production in isolated mitochondria, and caused release of apoptosis inducing factor (AIF) and endonuclease G (EndoG) from isolated mitochondria. Furthermore, JCTH-4 selectively induced autophagy in OS cells. Additionally, we investigated the combinatory effect of JCTH-4 with the natural compound curcumin (CC), a compound found in turmeric spice, previously shown to possess antiproliferative properties. CC alone had no observable effect on Saos-2 and U-2 OS cells. However, when present with JCTH-4, CC was able to enhance the cytotoxicity of JCTH-4 selectively in OS cells. Such cytotoxicity by JCTH-4 alone and in combination with CC was not observed in normal human osteoblasts (HOb) and normal human fetal fibroblasts (NFF). Therefore, this report illustrates a new window in combination therapy, utilizing a novel synthetic analogue of PST with the natural compound CC, for the treatment of OS. PMID:22205968

Liposomal short-chain C6 ceramide induces potent anti-osteosarcoma activity in vitro and in vivo.

PubMed

Zhai, Lei; Sun, Nan; Han, Zhe; Jin, Hai-chao; Zhang, Bo

Osteosarcoma (OS) remains one deadly disease for many affected patients. The search for novel and more efficient anti-OS agents is urgent. In the current study, we demonstrated that liposome-packed C6 ceramide exerted potent cytotoxic effect against established (U2OS and MG-63 lines) and primary human OS cells. Meanwhile, the liposomal C6 (ceramide) induced caspase-mediated apoptotic death in OS cells. Liposomal C6 was significantly more potent than conventional free C6 in inhibiting OS cells, yet it was safe to non-cancerous bone cells (primary murine osteoblasts or human MLO-Y4 osteocytic cells). At the signaling level, we showed that liposomal C6 potently inhibited Akt activation in OS cells. Further studies revealed that a low dose of liposomal C6 dramatically sensitized the in vitro anti-OS activity of two conventional chemodrugs: methotrexate (MTX) and doxorubicin. In vivo, intravenous injection of liposomal C6 inhibited Akt activation and suppressed U2OS xenograft growth in nude mice without causing apparent toxicities. Meanwhile, when given at a low-dose (5 mg/kg body weight), liposomal C6 dramatically sensitized MTX's anti-U2OS activity in vivo. Collectively, our data demonstrate that liposomal C6 exerts potent anti-tumor activity in preclinical OS models. Copyright © 2015 Elsevier Inc. All rights reserved.

MicroRNA-29b-1 impairs in vitro cell proliferation, self‑renewal and chemoresistance of human osteosarcoma 3AB-OS cancer stem cells.

PubMed

Di Fiore, Riccardo; Drago-Ferrante, Rosa; Pentimalli, Francesca; Di Marzo, Domenico; Forte, Iris Maria; D'Anneo, Antonella; Carlisi, Daniela; De Blasio, Anna; Giuliano, Michela; Tesoriere, Giovanni; Giordano, Antonio; Vento, Renza

2014-11-01

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR‑29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel

MicroRNA-182 downregulates Wnt/β-catenin signaling, inhibits proliferation, and promotes apoptosis in human osteosarcoma cells by targeting HOXA9

PubMed Central

Zhang, Zi-Feng; Wang, Yong-Jian; Fan, Shao-Hua; Du, Shi-Xin; Li, Xue-Dong; Wu, Dong-Mei; Lu, Jun; Zheng, Yuan-Lin

2017-01-01

We investigated the mechanisms by which microRNA (miR)-182 promotes apoptosis and inhibits proliferation in human osteosarcoma (OS) cells. Levels of miR-182 and Homeobox A9 (HOXA9) expression were compared between human OS and normal cells. Subjects were divided into OS and normal groups. We analyzed the target relationship of miR-182 and Homeobox A9 (HOXA9). Cells were then assigned into blank, negative control, miR-182 mimics, miR-182 inhibitors, siRNA-HOXA9, or and miR-182 inhibitors + siRNA-HOXA9 groups. Cell function was assayed by CCK-8, flow cytometry and wound healing assay. Additionally, we analyzed OS tumor growth in a xenograft mouse model. Dual-luciferase reporter assays indicated miR-182 directly targets HOXA9. Reverse transcription quantitative PCR and western blotting revealed elevated expression of miR-182, WIF-1, BIM, and Bax, and reduced expression of HOXA9, Wnt, β-catenin, Survivin, Cyclin D1, c-Myc, Mcl-1, Bcl-xL, and Snail in osteosarcoma cells treated with miR-182 mimic or siRNA-HOXA9 as compared to controls. Osteosarcoma cells also exhibited decreased cell proliferation, migration, and tumor growth, and increased apoptosis when treated with miR-182 mimic or siRNA-HOXA9. Correspondingly, in a xenograft mouse model, osteosarcoma tumor volume and growth were increased when cells were treated with miR-182 inhibitor and decreased by miR-182 mimic or siRNA-HOXA9. These results indicate that miR-182 downregulates Wnt/β-catenin signaling, inhibits cell proliferation, and promotes apoptosis in osteosarcoma cells by suppressing HOXA9 expression. PMID:29254169

MicroRNAs at the human 14q32 locus have prognostic significance in osteosarcoma

PubMed Central

2013-01-01

Background Deregulation of microRNA (miRNA) transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma. Methods Human and canine osteosarcoma patient’s samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. Results Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death) and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs) in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544. Conclusions We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease. PMID:23311495

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma.

PubMed

Rainusso, N; Brawley, V S; Ghazi, A; Hicks, M J; Gottschalk, S; Rosen, J M; Ahmed, N

2012-03-01

Despite radical surgery and multi-agent chemotherapy, less than one third of patients with recurrent or metastatic osteosarcoma (OS) survive. The limited efficacy of current therapeutic approaches to target tumor-initiating cells (TICs) may explain this dismal outcome. The purpose of this study was to assess the impact of modified T cells expressing a human epidermal growth factor receptor (HER2)-specific chimeric antigen receptor in the OS TIC compartment of human established cell lines. Using the sarcosphere formation assay, we found that OS TICs were resistant to increasing methotrexate concentrations. In contrast, HER2-specific T cells decreased markedly sarcosphere formation capacity and the ability to generate bone tumors in immunodeficient mice after orthotopic transplantation. In vivo, administration of HER2-specific T cells significantly reduced TICs in bulky tumors as judged by decreased sarcosphere forming efficiency in OS cells isolated from explanted tumors. We demonstrate that HER2-specific T cells target drug resistant TICs in established OS cell lines, suggesting that incorporating immunotherapy into current treatment strategies for OS has the potential to improve outcomes.

MicroRNA-198 inhibited tumorous behaviors of human osteosarcoma through directly targeting ROCK1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Shilian, E-mail: shilian_zhang@126.com; Zhao, Yuehua; Wang, Lijie

2016-04-08

Osteosarcoma is an aggressive primary sarcoma of bone and occurs mainly in adolescents and young adults. The prognosis of OS remains poor, and most of them will die due to local relapse or metastases. The discovery of microRNAs provides a new possibility for the early diagnosis and treatment of OS. Thus, the aim of this study was to explore the expression and functions of microRNA-198 (miR-198) in osteosarcoma. The expression levels of miR-198 were determined by qRT-PCR in osteosarcoma tissues and cell lines. Cell proliferation assays, migration and invasion assays were adopted to investigate the effects of miR-198 on tumorousmore » behaviors of osteosarcoma cells. The results showed that miR-198 expression levels were lower in osteosarcoma tissues and cell lines. In addition, low miR-198 expression levels were correlated with TNM stage and distant metastasis. After miR-198 mimics transfection, cell proliferation, migration and invasion were significantly suppressed in the osteosarcoma cells. Furthermore, ROCK1 was identified as a novel direct target of miR-198 in osteosarcoma. These findings suggested that miR-198 may act not only as a novel prognostic marker, but also as a potential target for molecular therapy of osteosarcoma.« less

2-methoxyestradiol-mediated anti-tumor effect increases osteoprotegerin expression in osteosarcoma cells.

PubMed

Benedikt, Michaela B; Mahlum, Eric W; Shogren, Kristen L; Subramaniam, Malayannan; Spelsberg, Thomas C; Yaszemski, Michael J; Maran, Avudaiappan

2010-04-01

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2-ME actions, we studied the effect of 2-ME treatment on OPG gene expression in human osteosarcoma cells. 2-ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2-ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3-, 1.9-, 2.8-, and 2.5-fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2-ME treatment. The effect of 2-ME on osteosarcoma cells was ligand-specific as parent estrogen, 17beta-estradiol and a tumorigenic estrogen metabolite, 16alpha-hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co-treating osteosarcoma cells with OPG protein did not further enhance 2-ME-mediated anti-tumor effects. OPG-released in 2-ME-treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2-ME-mediated anti-proliferative effects in osteosarcoma cells, but rather participates in anti-resorptive functions of 2-ME in bone tumor environment. Copyright 2010 Wiley-Liss, Inc.

Degraded iota-carrageenan can induce apoptosis in human osteosarcoma cells via the Wnt/β-catenin signaling pathway.

PubMed

Jin, Zhe; Han, Ya-Xin; Han, Xiao-Rui

2013-01-01

Osteosarcoma (OS) is a high-grade malignant bone tumor. Therefore, using both in vitro and in vivo assays, the effects of degraded iota-Carrageenan (ι-CGN) on a human osteosarcoma cell line, HOS, were examined. Degraded ι-CGN was observed to induce apoptosis and G(1) phase arrest in HOS cells. Moreover, degraded ι-CGN suppressed tumor growth in established xenograft tumor models. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumors treated with native ι-CGN or PBS. In addition, the formation of intratumoral microvessels was inhibited following treatment with degraded ι-CGN. In Western blot assays, degraded ι-CGN was found to inhibit the Wnt/β-catenin signaling pathway. Overall, these studies demonstrate the antitumor activity of degraded ι-CGN toward the OS cell line, HOS. Moreover, valuable insight into the mechanisms mediated by degraded ι-CGN was obtained, potentially leading to the identification of novel treatments for OS. However, additional studies are needed to confirm these results in other cell types, particularly in human umbilical vein endothelial cells.

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

PubMed Central

Giuliani, Anna Lisa; Colognesi, Davide; Ricco, Tiziana; Roncato, Carlotta; Capece, Marina; Amoroso, Francesca; Wang, Qi Guang; De Marchi, Elena; Gartland, Allison; Di Virgilio, Francesco; Adinolfi, Elena

2014-01-01

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca2+ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently

The long non-coding RNA PARTICLE is associated with WWOX and the absence of FRA16D breakage in osteosarcoma patients.

PubMed

O'Leary, Valerie Bríd; Maugg, Doris; Smida, Jan; Baumhoer, Daniel; Nathrath, Michaela; Ovsepian, Saak Victor; Atkinson, Michael John

2017-10-20

Breakage of the fragile site FRA16D disrupts the WWOX (WW Domain Containing Oxidoreductase) tumor suppressor gene in osteosarcoma. However, the frequency of breakage is not sufficient to explain the rate of WWOX loss in pathogenesis. The involvement of non-coding RNA transcripts is proposed due to their accumulation at fragile sites, where they are advocated to influence specific chromosomal regions associated with malignancy. The long ncRNA PARTICLE (promoter of MAT2A antisense radiation-induced circulating long non-coding RNA) is transiently elevated in response to irradiation and influences epigenetic silencing modification within WWOX . It now emerges that elevated PARTICLE levels are significantly associated with FRA16D non-breakage in OS patients. Although not associated with overall survival, high PARTICLE levels were found to be significantly linked to metastasis free outcome. The transcription of both PARTICLE and WWOX are transiently responsive to exposure to low doses of radiation in osteosarcoma cell lines. Herein, a relationship between WWOX and PARTICLE transcription is suggested in human osteosarcoma cell lines representing alternative genetic backgrounds. PARTICLE over-expression ameliorated WWOX promoter activity in U2OS harboring FRA16D non-breakage. It can be concluded that the lncRNA PARTICLE influences the WWOX tumor suppressor and in the absence of WWOX FRA16D breakage, it is associated with OS metastasis-free survival.

Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Ping; Mobasher, Maral E.; Alawi, Faizan, E-mail: falawi@upenn.edu

Highlights: • Dyskerin depletion triggers cellular senescence in U2OS osteosarcoma cells. • Dyskerin-depleted cells are resistant to apoptosis induced by genotoxic stress. • Chromatin relaxation sensitizes dyskerin-depleted cells to apoptosis. - Abstract: Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However,more » the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.« less

Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell motility in human osteosarcoma.

PubMed

Yang, Jilong; Annala, Matti; Ji, Ping; Wang, Guowen; Zheng, Hong; Codgell, David; Du, Xiaoling; Fang, Zhiwei; Sun, Baocun; Nykter, Matti; Chen, Kexin; Zhang, Wei

2014-10-10

The identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK and EWS-FLI1 in human sarcomas has provided important insight into the diagnosis and targeted therapy of sarcomas. No recurrent fusion has been reported in human osteosarcoma. Transcriptome sequencing was used to characterize the gene fusions and mutations in 11 human osteosarcomas. Nine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway. Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas. Expression of LRP1-SNRNP25 fusion gene promoted SAOS-2 osteosarcoma cell migration and invasion. Expression of KCNMB4-CCND3 fusion gene promoted SAOS-2 cell migration. Our study represents the first whole transcriptome analysis of untreated human osteosarcoma. Our discovery of two osteosarcoma specific fusion genes associated with osteosarcoma cellular motility highlights the heterogeneity of osteosarcoma and provides opportunities for new treatment modalities.

Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas

PubMed Central

Osaki, Shuhei; Tazawa, Hiroshi; Hasei, Joe; Yamakawa, Yasuaki; Omori, Toshinori; Sugiu, Kazuhisa; Komatsubara, Tadashi; Fujiwara, Tomohiro; Sasaki, Tsuyoshi; Kunisada, Toshiyuki; Yoshida, Aki; Urata, Yasuo; Kagawa, Shunsuke; Ozaki, Toshifumi; Fujiwara, Toshiyoshi

2016-01-01

Osteosarcoma is a rare disease diagnosed as malignant bone tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The reversal of chemoresistance is a major clinical challenge to improve the prognostic outcome of osteosarcoma patients. We developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301 (telomelysin) and assessed its synergistic effects with chemotherapeutic agents (cisplatin and doxorubicin) using human osteosarcoma cell lines and a xenograft tumor model. The molecular mechanism underlying the chemosensitizing effect of OBP-301 was evaluated in aspects of apoptosis induction. OBP-301 inhibits anti-apoptotic myeloid cell leukemia 1 (MCL1) expression, which in turn leads to chemosensitization in human osteosarcoma cells. The siRNA-mediated knockdown of MCL1 expression sensitized human osteosarcoma cells to common chemotherapeutic agents. We also found that upregulation of microRNA-29 targeting MCL1 via virally induced transcriptional factor E2F-1 activation was critical for the enhancement of chemotherapy-induced apoptosis in osteosarcoma cells. Telomerase-specific oncolytic adenovirus synergistically suppressed the viability of human osteosarcoma cells in combination with chemotherapeutic agents. The combination treatment also significantly inhibited tumor growth, as compared to monotherapy, in an osteosarcoma xenograft tumor model. Our data suggest that replicative virus-mediated tumor-specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients. PMID:27356624

Stimulators of Mineralization Limit the Invasive Phenotype of Human Osteosarcoma Cells by a Mechanism Involving Impaired Invadopodia Formation

PubMed Central

Cmoch, Anna; Podszywalow-Bartnicka, Paulina; Palczewska, Malgorzata; Piwocka, Katarzyna; Groves, Patrick; Pikula, Slawomir

2014-01-01

Background Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis). Results In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization. Conclusions Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma. PMID:25314307

Adenovirus small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells.

PubMed

Tao, Zhi-Wei; Zou, Ping-An

2018-06-13

Osteosarcoma is a disease prone to recurrence and metastasis, and adenovirus expression vector is frequently studied as a therapeutic target of osteosarcoma in recent year. This study attempts to explore the effect of adenovirus-mediated small interfering RNA (siRNA) targeting ezrin on the proliferation, migration, invasion and apoptosis of human osteosarcoma MG-63 cells. Human osteosarcoma MG-63 cell line was selected for construction of recombinant adenovirus vector. The mRNA and protein levels of ezrin, Bcl2-associated X protein (Bax), B cell lymphoma-2 (Bcl-2), p21, p53, Caspase-3, matrix metalloproteinase 2 (MMP-2) and MMP-9, Cyclin D1, and cyclin-dependent kinase 4a (CDK4a) were determined. Through ELISA, the levels of Caspase-3, MMP-2 and MMP-9 were examined. Finally, human osteosarcoma MG-63 cell viability, growth, invasion, migration, and apoptosis were detected. Initially, adenovirus expression vector of ezrin was constructed by ezrin 2 siRNA sequence. Adenovirus-mediated siRNA targeting ezrin reduced expression of ezrin in MG-63 cells. The results revealed that adenovirus-mediated siRNA targeting ezrin elevated expression levels of Bax, P21, P53, and Caspase-3, Cyclin D1, and CDK4a and reduced expression levels of Bcl-2, MMP-2, and MMP-9. Furthermore, adenovirus-mediated siRNA targeting ezrin inhibited human osteosarcoma MG-63 cell viability, growth, invasion, and migration, and promoted apoptosis. Our study demonstrates that adenovirus-mediated siRNA targeting ezrin can induce apoptosis and inhibit the proliferation, migration and invasion of human osteosarcoma MG-63 cells. ©2018 The Author(s).

Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function.

PubMed

Del Mare, Sara; Aqeilan, Rami I

2015-08-10

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases' seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease.

Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function

PubMed Central

Del Mare, Sara; Aqeilan, Rami I.

2015-01-01

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases’ seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease. PMID:26256646

Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma.

PubMed

Liu, Kuisheng; Ren, Tingting; Huang, Yi; Sun, Kunkun; Bao, Xing; Wang, Shidong; Zheng, Bingxin; Guo, Wei

2017-08-24

The cure rate of osteosarcoma has not improved in the past 30 years. The search for new treatments and drugs is urgently needed. Apatinib is a high selectivity inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, exerting promising antitumoral effect in various tumors. The antitumor effect of Apatinib in human osteosarcoma has never been reported. We investigated the effects of Apatinib in osteosarcoma in vitro and in vivo. Osteosarcoma patients with high levels of VEGFR2 have poor prognosis. Apatinib can inhibit cell growth of osteosarcoma cells. In addition to cycle arrest and apoptosis, Apatinib induces autophagy. Interestingly, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells. Immunoprecipitation confirmed direct binding between VEGFR2 and signal transducer and activator of transcription 3 (STAT3). Downregulation of VEGFR2 by siRNA resulted in STAT3 inhibition in KHOS cells. VEGFR2 and STAT3 are inhibited by Apatinib in KHOS cells, and STAT3 act downstream of VEGFR2. STAT3 and BCL-2 were downregulated by Apatinib. STAT3 knockdown by siRNA reinforced autophagy and apoptosis induced by Apatinib. BCL-2 inhibits autophagy and was apoptosis restrained by Apatinib too. Overexpression of BCL-2 decreased Apatinib-induced apoptosis and autophagy. Apatinib repressed the expression of STAT3 and BCL-2 and suppressed the growth of osteosarcoma in vivo. To sum up, deactivation of VEGFR2/STAT3/BCL-2 signal pathway leads to Apatinib-induced growth inhibition of osteosarcoma.

Ezrin and moesin expression in canine and feline osteosarcoma.

PubMed

Hlavaty, Juraj; Wolfesberger, Birgitt; Hauck, Marlene; Obermayer-Pietsch, Barbara; Fuchs-Baumgartinger, Andrea; Miller, Ingrid; Walter, Ingrid

2017-08-01

Biological features of canine osteosarcomas (OS) differ markedly from those found in feline and resemble more human osteosarcomas, in particular for their high rate of metastasis and poor prognosis. Ezrin, radixin and moesin are members of the ERM protein family and link the actin cytoskeleton with the cell membrane. Ezrin and moesin have been shown to be of prognostic significance in tumor progression due to their role in the metastatic process. The objective of this study was to analyze ezrin and moesin protein expression in a series of dog (n = 16) and cat (n = 8) osteosarcoma samples using immunohistochemistry and western blot techniques. We found that cat OS have a higher moesin expression compared to dog OS, however, the active phosphorylated forms of moesin and ezrin Tyr353 were more abundant in the dog samples. A statistically significant difference was found for the low and high immunohistochemical scores of ezrin and pan-phospho-ERM proteins between cat and dog. Although phospho-ezrin Thr567 was higher in feline OS, the membranous localization in dog OS samples indicates the presence of the biologically active form. Therefore, the observed differences in phosphorylated forms of ezrin and moesin status should be further studied to demonstrate if they are relevant for different biological behavior between dog and cat OS.

Positive effects of antitumor drugs in combination with propolis on canine osteosarcoma cells (spOS-2) and mesenchymal stem cells.

PubMed

Bernardino, Pedro Negri; Bersano, Paulo Ricardo Oliveira; Lima Neto, João Ferreira; Sforcin, José Maurício

2018-08-01

The combination of lower concentrations of antitumor drugs (carboplatin - CARB, doxorubicin - DOX, and methotrexate - MET) with propolis was investigated against canine osteosarcoma (spOS-2) and mesenchymal stem cells (MSC) in vitro. The mechanism of action in the combinations was analyzed. spOS-2 cells were incubated up to 72 h with propolis (50 μg/ml) alone or in combination with CARB (10-400 μmol/l), DOX (0.5-2 μmol/l) or MET (50-200 μmol/l). Cell viability was assessed by MTT assay, apoptosis/necrosis by flow cytometry, and MSC was incubated with the optimum combination. Propolis alone exerted no cytotoxic action against spOS-2 cells, whereas CARB (400, 200 and 100 μmol/l) exhibited the highest cytotoxic effects comparing to DOX and MET. The combination of propolis with the lowest concentrations of CARB led to better results comparing to CARB alone, which was not observed using DOX and MET. Apoptosis was involved in the action of propolis + CARB in spOS-2 cells. MSC were not affected by CARB/propolis, indicating that the cytotoxic action of the combination was specific to tumor cells but not to normal ones. Propolis improved the action of CARB against spOS-2 cells using lower concentrations of this drug, without affecting MSC. These findings are relevant and indicate a possible application of propolis in OSA treatment. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Osteosarcoma

Cancer.gov

The TARGET Osteosarcoma (OS) project elucidates comprehensive molecular characterization to determine the genetic changes that drive the initiation and progression of high-risk or hard-to-treat childhood cancers.The OS project has produced comprehensive genomic profiles of nearly 100 clinically annotated patient cases within the discovery dataset. Each fully-characterized TARGET OS case includes data from nucleic acid samples extracted from tumor and normal tissue.

HYPOXIA-ACTIVATED PRO-DRUG TH-302 EXHIBITS POTENT TUMOUR SUPPRESSIVE ACTIVITY AND COOPERATES WITH CHEMOTHERAPY AGAINST OSTEOSARCOMA

PubMed Central

Liapis, Vasilios; Labrinidis, Agatha; Zinonos, Irene; Hay, Shelley; Ponomarev, Vladimir; Panagopoulos, Vasilios; DeNichilo, Mark; Ingman, Wendy; Atkins, Gerald J.; Findlay, David M.; Zannettino, Andrew CW.; Evdokiou, Andreas

2015-01-01

Tumour hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumour hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumours. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS. PMID:25444931

Hypoxia-activated pro-drug TH-302 exhibits potent tumor suppressive activity and cooperates with chemotherapy against osteosarcoma.

PubMed

Liapis, Vasilios; Labrinidis, Agatha; Zinonos, Irene; Hay, Shelley; Ponomarev, Vladimir; Panagopoulos, Vasilios; DeNichilo, Mark; Ingman, Wendy; Atkins, Gerald J; Findlay, David M; Zannettino, Andrew C W; Evdokiou, Andreas

2015-02-01

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumor hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumors. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS. Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.

Near-Infrared Light Responsive Folate Targeted Gold Nanorods for Combined Photothermal-Chemotherapy of Osteosarcoma.

PubMed

Li Volsi, Anna; Scialabba, Cinzia; Vetri, Valeria; Cavallaro, Gennara; Licciardi, Mariano; Giammona, Gaetano

2017-04-26

Folate-targeted gold nanorods (GNRs) are proposed as selective theranostic agents for osteosarcoma treatment. An amphiphilic polysaccharide based graft-copolymer (INU-LA-PEG-FA) and an amino derivative of the α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide functionalized with folic acid (PHEA-EDA-FA), have been synthesized to act as coating agents for GNRs. The obtained polymer-coated GNRs were characterized in terms of size, shape, zeta potential, chemical composition, and aqueous stability. They protected the anticancer drug nutlin-3 and were able to deliver it efficiently in different physiological media. The ability of the proposed systems to selectively kill tumor cells was tested on U2OS cancer cells expressing high levels of FRs and compared with human bronchial epithelial cells (16HBE) and human dermal fibroblasts (HDFa). The property of the nanosystems of efficiently controlling drug release upon NIR laser irradiation and of acting as an excellent hyperthermia agent as well as Two Photon Luminescence imaging contrast agents was demonstrated. The proposed folate-targeted GNRs have also been tested in terms of chemoterapeutic and thermoablation efficacy on tridimensional (3-D) osteosarcoma models.

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma.

PubMed

Palmini, Gaia; Zonefrati, Roberto; Mavilia, Carmelo; Aldinucci, Alessandra; Luzi, Ettore; Marini, Francesca; Franchi, Alessandro; Capanna, Rodolfo; Tanini, Annalisa; Brandi, Maria Luisa

2016-10-14

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhi; Li, Youjun, E-mail: liyoujunn@126.com; Wang, Nan

miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and proteinmore » exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.« less

Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

PubMed

Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

2015-02-01

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma

PubMed Central

Liu, Kuisheng; Ren, Tingting; Huang, Yi; Sun, Kunkun; Bao, Xing; Wang, Shidong; Zheng, Bingxin; Guo, Wei

2017-01-01

The cure rate of osteosarcoma has not improved in the past 30 years. The search for new treatments and drugs is urgently needed. Apatinib is a high selectivity inhibitor of vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, exerting promising antitumoral effect in various tumors. The antitumor effect of Apatinib in human osteosarcoma has never been reported. We investigated the effects of Apatinib in osteosarcoma in vitro and in vivo. Osteosarcoma patients with high levels of VEGFR2 have poor prognosis. Apatinib can inhibit cell growth of osteosarcoma cells. In addition to cycle arrest and apoptosis, Apatinib induces autophagy. Interestingly, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells. Immunoprecipitation confirmed direct binding between VEGFR2 and signal transducer and activator of transcription 3 (STAT3). Downregulation of VEGFR2 by siRNA resulted in STAT3 inhibition in KHOS cells. VEGFR2 and STAT3 are inhibited by Apatinib in KHOS cells, and STAT3 act downstream of VEGFR2. STAT3 and BCL-2 were downregulated by Apatinib. STAT3 knockdown by siRNA reinforced autophagy and apoptosis induced by Apatinib. BCL-2 inhibits autophagy and was apoptosis restrained by Apatinib too. Overexpression of BCL-2 decreased Apatinib-induced apoptosis and autophagy. Apatinib repressed the expression of STAT3 and BCL-2 and suppressed the growth of osteosarcoma in vivo. To sum up, deactivation of VEGFR2/STAT3/BCL-2 signal pathway leads to Apatinib-induced growth inhibition of osteosarcoma. PMID:28837148

Doxorubicin induces ZAKα overexpression with a subsequent enhancement of apoptosis and attenuation of survivability in human osteosarcoma cells.

PubMed

Fu, Chien-Yao; Tseng, Yan-Shen; Chen, Ming-Cheng; Hsu, Hsi-Hsien; Yang, Jaw-Ji; Tu, Chuan-Chou; Lin, Yueh-Min; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-02-01

Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX-treated human OS cells. © 2017 Wiley Periodicals, Inc.

SOX2OT variant 7 contributes to the synergistic interaction between EGCG and Doxorubicin to kill osteosarcoma via autophagy and stemness inhibition.

PubMed

Wang, Wanchun; Chen, Ding; Zhu, Kewei

2018-02-23

Doxorubicin is the preferred chemotherapeuticdrug for osteosarcoma treatment of which clinical efficacy is limited because of its chemo-resistance and cardiac toxicity. It is necessary to develop the combination regimen with complementary molecular mechanisms to reduce the side effects and enhance sensitivity of Doxorubicin. EGCG is a polyphenol in green tea with antitumor bioactivity,which has been found that its combination with certain chemotherapeutic drugs could improve the antitumor efficiency. In this study, MTT assay was used to detect the cell growth inhibition The CD133+/CD44+ cells were isolated from U2OS and SaoS2 cell lines using magnetic-activated cell sorting and identified by flow cytometry analysis. qRT-PCR was used for determining the relative mRNA levels of key genes. Immunofluorescence was performed to evaluate the autophagy flux alterations. Self-renewal ability was accessed by sphere-forming assay. Tumorigenicity in nude mice was preformed to evaluate tumorigenicity in vivo. We found that EGCG targeting LncRNA SOX2OT variant 7 produced synergistic effects with Doxorubicin on osteosarcoma cell growth inhibition. On the one hand, EGCG could reduce the Doxorubicin-induced pro-survival autophagy through decreasing SOX2OT variant 7 to improve the growth inhibition of Doxorubicin. On the other hand, EGCG could partially inactivate Notch3/DLL3 signaling cascade targeting SOX2OT variant 7 to reduce the stemness then abated drug-resistance of osteosarcoma cells. This study will help to reveal the molecular mechanisms of synergistic effects of EGCG and Doxorubicin on OS chemotherapy and improve the clinical efficacy of chemotherapy as well as provide a basis for developing antitumor drugs targeting osteosarcoma stem cells.

Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Husmann, Knut, E-mail: khusmann@research.balgrist.ch; Ducommun, Pascal; Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich

2015-09-04

The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS andmore » 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.« less

MicroRNA-126 enhances the sensitivity of osteosarcoma cells to cisplatin and methotrexate

PubMed Central

JIANG, LIANGDONG; HE, AIYONG; HE, XIAOJIE; TAO, CHENG

2015-01-01

The establishment of novel chemotherapy drugs for osteosarcoma is urgently required, and the mechanisms and effects of cisplatin (DDP) and methotrexate (MTX) in the current treatment of osteosarcoma have not been fully elucidated. The present study aimed to observe the effect of DDP, MTX and rapamycin on osteosarcoma cell proliferation and apoptosis, and to investigate the association between miR-126 and the effects of DDP and MTX in osteosarcoma cells. miR-126-overexpressing and -silencing lentiviral vectors were constructed, and MG63 and U-2 OS osteosarcoma cells were infected. An MTT assay was conducted to detect transfected cell proliferation, and the effects of the chemotherapy drugs on transfected cell apoptosis were detected by flow cytometry. The cell cycle of the transfected cells was analyzed via flow cytometry. As the miR-126-overexpressing and -silencing osteosarcoma cell lines were successfully constructed, it was observed that DDP and MTX inhibited osteosarcoma cell proliferation. With the decreased expression of miR-126, the sensitivity of osteosarcoma cells to DDP and MTX was reduced at the same concentration. The flow cytometry suggested that DDP and MTX could promote the apoptosis of osteosarcoma cells with overexpressed miR-126, whereas they could not significantly impact the apoptosis of the miR-126-silenced osteosarcoma cells. Meanwhile, DDP inhibited the cell cycle of the miR-126-overexpressing osteosarcoma cells. In conclusion, DDP and MTX inhibited the proliferation and promoted the apoptosis of the osteosarcoma cells, and these processes were dependent upon the expression of miR-126. PMID:26788206

P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2.

PubMed

Zuffa, Elisa; Mancini, Manuela; Brusa, Gianluca; Pagnotta, Eleonora; Hattinger, Claudia Maria; Serra, Massimo; Remondini, Daniel; Castellani, Gastone; Corrado, Patrizia; Barbieri, Enza; Santucci, Maria Alessandra

2008-07-01

To investigate the impact of TP53 (tumor protein 53, p53) on genomic stability of osteosarcoma (OS). In first instance, we expressed in OS cell line SAOS-2 (lacking p53) a wild type (wt) p53 construct, whose protein undergoes nuclear import and activation in response to ionizing radiations (IR). Thereafter, we investigated genomic imbalances (amplifications and deletions at genes or DNA regions most frequently altered in human cancers) associated with radio-resistance relative to p53 expression by mean of an array-based comparative genomic hybridization (aCGH) strategy. Finally we investigated a putative marker of radio-induced oxidative stress, a 4,977 bp deletion at mitochondrial (mt) DNA usually referred to as 'common' deletion, by mean of a polimerase chain reaction (PCR) strategy. In radio-resistant subclones generated from wt p53-transfected SAOS-2 cells DNA deletions were remarkably reduced and the accumulation of 'common' deletion at mtDNA (that may let the persistence of oxidative damage by precluding detoxification from reactive oxygen species [ROS]) completely abrogated. The results of our study confirm that wt p53 has a role in protection of OS cell DNA integrity. Multiple mechanisms involved in p53 safeguard of genomic integrity and prevention of deletion outcome are discussed.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway.

PubMed

Tu, Bing; Peng, Zhao-Xiang; Fan, Qi-Ming; Du, Lin; Yan, Wei; Tang, Ting-Ting

2014-01-01

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells. © 2013 Published by Elsevier Inc.

Upregulation and biological function of transmembrane protein 119 in osteosarcoma

PubMed Central

Jiang, Zhen-Huan; Peng, Jun; Yang, Hui-Lin; Fu, Xing-Li; Wang, Jin-Zhi; Liu, Lei; Jiang, Jian-Nong; Tan, Yong-Fei; Ge, Zhi-Jun

2017-01-01

Osteosarcoma is suggested to be caused by genetic and molecular alterations that disrupt osteoblast differentiation. Recent studies have reported that transmembrane protein 119 (TMEM119) contributes to osteoblast differentiation and bone development. However, the level of TMEM119 expression and its roles in osteosarcoma have not yet been elucidated. In the present study, TMEM119 mRNA and protein expression was found to be up-regulated in osteosarcoma compared with normal bone cyst tissues. The level of TMEM119 protein expression was strongly associated with tumor size, clinical stage, distant metastasis and overall survival time. Moreover, gene set enrichment analysis (GSEA) of the Gene Expression Omnibus (GEO) GSE42352 dataset revealed TMEM119 expression in osteosarcoma tissues to be positively correlated with cell cycle, apoptosis, metastasis and TGF-β signaling. We then knocked down TMEM119 expression in U2OS and MG63 cells using small interfering RNA, which revealed that downregulation of TMEM119 could inhibit the proliferation of osteosarcoma cells by inducing cell cycle arrest in G0/G1 phase and apoptosis. We also found that TMEM119 knockdown significantly inhibited cell migration and invasion, and decreased the expression of TGF-β pathway-related factors (BMP2, BMP7 and TGF-β). TGF-β application rescued the inhibitory effects of TMEM119 knockdown on osteosarcoma cell migration and invasion. Further in vitro experiments with a TGF-β inhibitor (SB431542) or BMP inhibitor (dorsomorphin) suggested that TMEM119 significantly promotes cell migration and invasion, partly through TGF-β/BMP signaling. In conclusion, our data support the notion that TMEM119 contributes to the proliferation, migration and invasion of osteosarcoma cells, and functions as an oncogene in osteosarcoma. PMID:28496199

Nonmetastatic osteosarcoma of the extremity. Neoadjuvant chemotherapy with methotrexate, cisplatin, doxorubicin and ifosfamide. An Italian Sarcoma Group study (ISG/OS-Oss).

PubMed

Ferrari, Stefano; Meazza, Cristina; Palmerini, Emanuela; Tamburini, Angela; Fagioli, Franca; Cozza, Raffaele; Ferraresi, Virginia; Bisogno, Gianni; Mascarin, Maurizio; Cefalo, Graziella; Manfrini, Marco; Capanna, Rodolfo; Biagini, Roberto; Donati, Davide; Picci, Piero

2014-01-01

Based on the results of the ISG/OS-1 study, the MAP regimen (methotrexate [MTX], doxorubicin [ADM] and cisplatin [CDP] with the addition of ifosfamide [IFO] in poor-responder patients) was investigated in patients with nonmetastatic osteosarcoma of the extremity (ISG/OS-Oss study). Compared with the ISG/OS-1 study (cumulative doses: ADM 420 mg/m(2), MTX 120 g/m(2), CDP 600 mg/m(2), IFO 30 g/m(2)), the ISG/OS-Oss study reduced the number of MTX cycles from 10 to 5 (cumulative MTX dose: 60 g/m(2)) in order to diminish treatment duration and toxicity. From January 2007 to June 2011, 171 patients (median age 16 years, 60% males) were registered. The limb salvage rate was 94% and the good pathologic response rate 51% (these figures were 92% and 48%, respectively, in the ISG/OS-1 study). At a median follow-up of 39 months (range, 4-80), the 5-year overall survival rate was 80% (95% CI, 73%-87%) and the event-free survival was 50% (95% CI, 39%-59%). For comparison, the 5-year overall and event-free survival rates in ISG/OS-1 were 73% (95% CI, 65%-81%) and 64% (95% CI, 56%-73%), respectively. This study confirms that in nonmetastatic osteosarcoma of the extremity, conservative surgery in more than 90% and a good pathologic response rate of 50% can be expected with primary chemotherapy based on the MAP regimen. The response and resection rates in the ISG/OS-Oss study are in the same range as those of the previous study, whereas the event-free survival is lower than that previously achieved. Since the only difference between the two studies was the cumulative dose of postoperatively given MTX, our data support the importance of the cumulative dose of MTX in the MAP regimen.

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

PubMed

Liu, Chunhong; Ma, Mingming; Zhang, Junde; Gui, Shaoliu; Zhang, Xiaohai; Xue, Shuangtao

2017-05-01

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)-β1/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-β1 to induce osteogenic differentiation might represent a novel therapeutic strategy to treat osteosarcoma with minimal side effects. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Alpha-CaMKII plays a critical role in determining the aggressive behavior of human osteosarcoma

PubMed Central

Daft, Paul G.; Yuan, Kaiyu; Warram, Jason M.; Klein, Michael J.; Siegal, Gene P.; Zayzafoon, Majd

2013-01-01

Osteosarcoma is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. Despite improvements in osteosarcoma treatment, more specific molecular targets are needed as potential therapeutic options. One target of interest is alpha-Ca2+/calmodulin-dependent protein kinase II (α-CaMKII), a ubiquitous mediator of Ca2+-linked signaling, which has been shown to regulate tumor cell proliferation and differentiation. Here, we investigate the role of α-CaMKII in the growth and tumorigenicity of human osteosarcoma. We show that α-CaMKII is highly expressed in primary osteosarcoma tissue derived from 114 patients and is expressed in varying levels in different human osteosarcoma cell lines (HOS, MG-63, MNNG/HOS and 143B). To examine whether α-CaMKII regulates osteosarcoma tumorigenic properties, we genetically inhibited α-CaMKII in two osteosarcoma cell lines using two different α-CaMKII shRNAs delivered by lentiviral vectors and overexpressed α-CaMKII by retrovirus. The genetic deletion of α-CaMKII by shRNA in MG-63 and 143B cells resulted in decreased proliferation (50 and 41%), migration (22 and 25%) and invasion (95 and 90%), respectively. The overexpression of α-CaMKII in HOS cells resulted in increased proliferation (240%), migration (640%) and invasion (10,000%). Furthermore, α-CaMKII deletion in MG-63 cells significantly reduced tumor burden in vivo (65%), while α-CaMKII overexpression resulted in tumor formation in a previously non-tumor forming osteosarcoma cell line (HOS). Our results suggest that α-CaMKII plays a critical role in determining the aggressive phenotype of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat this devastating adolescent disease. PMID:23364534

Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

PubMed Central

Kurek, Kyle; Del Mare, Sara; Salah, Zaidoun; Abdeen, Suhaib; Sadiq, Hussain; Lee, Sukhee; Gaudio, Eugenio; Zanesi, Nicola; Jones, Kevin B.; DeYoung, Barry; Amir, Gail; Gebhardt, Mark; Warman, Matthew; Stein, Gary S.; Stein, Janet L.; Lian, Jane B.; Aqeilan, Rami I.

2011-01-01

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P< 0.0001). Compared to the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels, relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorgenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas Runx2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease. PMID:20530675

Multimodal transfer of MDR by exosomes in human osteosarcoma.

PubMed

Torreggiani, Elena; Roncuzzi, Laura; Perut, Francesca; Zini, Nicoletta; Baldini, Nicola

2016-07-01

Exosomes are extracellular vesicles released by both normal and tumour cells which are involved in a new intercellular communication pathway by delivering cargo (e.g., proteins, microRNAs, mRNAs) to recipient cells. Tumour-derived exosomes have been shown to play critical roles in different stages of tumour growth and progression. In this study, we investigated the potential role of exosomes to transfer the multidrug resistance (MDR) phenotype in human osteosarcoma cells. Exosomes were isolated by differential centrifugation of culture media from multidrug resistant human osteosarcoma MG-63DXR30 (Exo/DXR) and MG-63 parental cells (Exo/S). Exosome purity was examined by transmission electron microscopy and confirmed by immunoblot analysis for the expression of specific exosomal markers. Our data showed that exosomes derived from doxorubicin-resistant osteosarcoma cells could be taken up into secondary cells and induce a doxorubicin-resistant phenotype. The incubation of osteosarcoma cells with Exo/DXR decreased the sensitivity of parental cells to doxorubicin, while exposure with Exo/S was ineffective. In addition, we demonstrated that Exo/DXR expressed higher levels of MDR-1 mRNA and P-glycoprotein compared to Exo/S (p=0.03). Interestingly, both MDR-1 mRNA and P-gp increased in MG-63 cells after incubation with Exo/DXR, suggesting this as the main mechanism of exosome-mediated transfer of drug resistance. Our findings suggest that multidrug resistant osteosarcoma cells are able to spread their ability to resist the effects of doxorubicin treatment on sensitive cells by transferring exosomes carrying MDR-1 mRNA and its product P-glycoprotein.

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.

2005-07-01

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased inmore » association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.« less

The levels of bone alkaline phosphatase (BALP) and soluble epidermal growth factor receptor-2 (ECD/HER-2) in pediatric patients with osteosarcoma during clinical treatment.

PubMed

Rychłowska-Pruszyńska, Magdalena; Gajewska, Joanna; Ambroszkiewicz, Jadwiga; Karwacki, Marek; Szamotulska, Katarzyna

2018-01-01

Aim: The aim of this study was to assess the usefulness of bone-specific alkaline phosphatase (BALP) and the extracelluar domain of human epidermal growth factor receptor 2 (ECD/HER-2) measurements in pediatric patients with osteosarcoma as prospective prognostic and predictive markers for monitoring the treatment and early detection of disease recurrence. Material and methods: We studied 22 patients (5 girls, 17 boys) aged 7-20 years with osteosarcoma (OS) treated at the Institute of Mother and Child in Warsaw. All the patients were evaluated for the serum levels of BALP and ECD/HER-2 before treatment, during pre- and postoperative chemotherapy and after the completion of treatment. Healthy children (n=22) were the reference group. The levels of BALP and ECD/HER-2 were measured using immunoenzymatic methods. Results: The values of BALP and ECD/HER-2 proteins were higher (p<0.01; p<0.05, respectively) in patients with osteosarcoma at the time of diagnosis compared with the control group. The values of both markers significantly decreased during chemotherapy in most patients with remission. In contrast to ECD/HER-2, the value of BALP after therapy was higher in patients with progression than with remission (p<0.001). Conclusions: Our results demonstrate the different pattern of BALP and ECD/HER-2 proteins during clinical treatment in patients with osteosarcoma. Higher values of BALP may characterize the progression of the disease and unfavourable prognosis. Further longitudinal studies are necessary to confirm the prognostic values of BALP and ECD/HER-2 proteins in this group of patients.

ZD6474, a new treatment strategy for human osteosarcoma, and its potential synergistic effect with celecoxib

PubMed Central

Pan, Changchuan; Zhou, Yi; Du, Wuying; Chen, Jie-min; Zhu, Xiaofeng; Shen, Jingnan; Chen, Shuai; Liu, Ran-yi; Huang, Wenlin

2015-01-01

ZD6474, a small molecule VEGFR and EGFR tyrosine kinase inhibitor, has been considered as a promising tumor-targeted drug in various malignancies. EGFR and cyclooxygenase-2 (COX-2) were found overexpressed in osteosarcoma in previous reports, so here we tried to explore the anti-osteosarcoma effect of ZD6474 alone or combination with celecoxib, a COX-2 inhibitor. The data demonstrated that ZD6474 inhibited the growth of osteosarcoma cells, and promoted G1-phase cell cycle arrest and apoptosis by inhibiting the activity of EGFR tyrosine kinase, and consequently suppressing its downstream PI3k/Akt and MAPK/ERK pathway. Additionally, daily administration of ZD6474 produced a dose-dependent inhibition of tumor growth in nude mice. Celecoxib also significantly inhibited the growth of osteosarcoma cells in dose-dependent manner, while combination of ZD6474 and celecoxib displayed a synergistic or additive antitumor effect on osteosarcoma in vitro and in vivo. The possible molecular mechanisms to address the synergism are likely that ZD6474 induces the down-regulation of COX-2 expression through inhibiting ERK phosphorylation, while celecoxib promotes ZD6474-directed inhibition of ERK phosphorylation. In conclusion, ZD6474 exerts direct anti-proliferative effects on osteosarcoma cells, and the synergistic antitumor effect of the combination of ZD6474 with celecoxib may indicate a new strategy of the combinative treatment of human osteosarcoma. PMID:26050198

Meta-analysis of serum lactate dehydrogenase and prognosis for osteosarcoma.

PubMed

Fu, Yucheng; Lan, Tao; Cai, Hongliu; Lu, Anwei; Yu, Wei

2018-05-01

A large number of studies have reported the relationships between serum lactate dehydrogenase (LDH) and prognosis of osteosarcoma. However, the result is still controversial and no consensus has been reached. Therefore, we performed a meta-analysis to evaluate the prognostic role of serum LDH in osteosarcoma patients. We performed the systematic computerized search for eligible articles from PubMed, Embase, and Cochrane databases until December 21, 2017. The pooled hazard ratio (HR) and 95% confidence intervals (CIs) of overall survival (OS) and event-free survival (EFS) were obtained to assess the prognostic value of serum LDH. A total of 18 studies with 2543 osteosarcoma patients were included. Overall, 15 studies assessed the elevated serum LDH level on OS and the pooled HR was 1.87 (95% CI = 1.58-2.20). Meanwhile, the pooled HR to evaluate the relationship between serum LDH and EFS in 9 studies was 1.78 (95% CI = 1.51-2.10). The same results were acquired when these studies were stratified by Enneking stage, geographic region, and sample size. No heterogeneity existed between these subgroups (P > .05). Begg's funnel plot and Egger's test (OS: P = .04; EFS: P = .34) showed that possible publication bias might exist in OS studies. Sensitivity analysis suggested the pooled HR was robust. This meta-analysis demonstrates that elevated serum LDH level is apparently associated with lower EFS rate and serum LDH could be a prognostic biomarker for osteosarcoma patients.

CCL5/CCR5 axis induces vascular endothelial growth factor-mediated tumor angiogenesis in human osteosarcoma microenvironment.

PubMed

Wang, Shih-Wei; Liu, Shih-Chia; Sun, Hui-Lung; Huang, Te-Yang; Chan, Chia-Han; Yang, Chen-Yu; Yeh, Hung-I; Huang, Yuan-Li; Chou, Wen-Yi; Lin, Yu-Min; Tang, Chih-Hsin

2015-01-01

Chemokines modulate angiogenesis and metastasis that dictate cancer development in tumor microenvironment. Osteosarcoma is the most frequent bone tumor and is characterized by a high metastatic potential. Chemokine CCL5 (previously called RANTES) has been reported to facilitate tumor progression and metastasis. However, the crosstalk between chemokine CCL5 and vascular endothelial growth factor (VEGF) as well as tumor angiogenesis in human osteosarcoma microenvironment has not been well explored. In this study, we found that CCL5 increased VEGF expression and production in human osteosarcoma cells. The conditioned medium (CM) from CCL5-treated osteosarcoma cells significantly induced tube formation and migration of human endothelial progenitor cells. Pretreatment of cells with CCR5 antibody or transfection with CCR5 specific siRNA blocked CCL5-induced VEGF expression and angiogenesis. CCL5/CCR5 axis demonstrably activated protein kinase Cδ (PKCδ), c-Src and hypoxia-inducible factor-1 alpha (HIF-1α) signaling cascades to induce VEGF-dependent angiogenesis. Furthermore, knockdown of CCL5 suppressed VEGF expression and attenuated osteosarcoma CM-induced angiogenesis in vitro and in vivo. CCL5 knockdown dramatically abolished tumor growth and angiogenesis in the osteosarcoma xenograft animal model. Importantly, we demonstrated that the expression of CCL5 and VEGF were correlated with tumor stage according the immunohistochemistry analysis of human osteosarcoma tissues. Taken together, our findings provide evidence that CCL5/CCR5 axis promotes VEGF-dependent tumor angiogenesis in human osteosarcoma microenvironment through PKCδ/c-Src/HIF-1α signaling pathway. CCL5 may represent a potential therapeutic target against human osteosarcoma. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Spontaneous telangiectatic osteosarcoma in a rhesus macaque (Macaca mulatta).

PubMed

Goldschmidt, B; Calado, M I Z; Resende, F C; Caldas, R M; Pinto, L W; Lopes, C A A; França, F G O; Meireles, B S; Souza, I V

2017-04-01

Osteosarcoma (OS) is the most common type of bone cancer, especially in young. Telangiectatic osteosarcoma (TO) is a rare variant of OS, and hence, its occurrence, presentation, and prognosis are poorly understood. A 4-year-old female rhesus monkey presenting lameness and swelling was examined for a mass on the right humerus. Radiography revealed fracture and disorganized structure of bone tissue. Histopathological examination revealed malignant neoplasm composed of anaplastic osteoblasts, which invaded the bone marrow and surrounded blood-filled cysts in the epiphysis and diaphysis forming septa. Cytogenetic analysis showed aneuploid cells, supernumerary AgNORs, and a marker fragment. The neoplasm was diagnosed as TO. To our knowledge, the occurrence of TO and its cytogenetic analysis were reported for the first time in non-human primates. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biological characterization of preclinical Bioluminescent Osteosarcoma Orthotopic Mouse (BOOM) model: A multi-modality approach

PubMed Central

Garimella, Rama; Eskew, Jeff; Bhamidi, Priyanka; Vielhauer, George; Hong, Yan; Anderson, H. Clarke; Tawfik, Ossama; Rowe, Peter

2013-01-01

Osteosarcoma (OS) is a bone malignancy that affects children and adolescents. It is a highly aggressive tumor and typically metastasizes to lungs. Despite aggressive chemotherapy and surgical treatments, the current 5 year survival rate is 60–70%. Clinically relevant models are needed to understand OS pathobiology, metastatic progression from bones to lungs, and ultimately, to develop more efficacious treatment strategies and improve survival rates in OS patients with metastasis. The main goal of this study was to develop and characterize an in vivo OS model that will allow non-invasive tracking of tumor progression in real time, and aid in studying OS pathobiology, and screening of potential therapeutic agents against OS. In this study, we have used a multi-modality approach using bioluminescent imaging, electron microscopy, micro-computed tomography, and histopathology to develop and characterize a preclinical Bioluminescent Osteosarcoma Orthotopic Mouse (BOOM) model, using 143B human OS cell line. The results of this study clearly demonstrate that the BOOM model represents the clinical disease as evidenced by a spectrum of changes associated with tumor establishment, progression and metastasis, and detection of known OS biomarkers in the primary and metastatic tumor tissue. Key novel findings of this study include: (a) multimodality approach for extensive characterization of the BOOM model using 143B human OS cell line; (b) evidence of renal metastasis in OS orthotopic model using 143B cells; (c) evidence of Runx2 expression in the metastatic lung tissue; and (d) evidence of the presence of extracellular membrane vesicles and myofibroblasts in the BOOM model. PMID:25688332

Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Napp, Matthias; Daeschlein, Georg; Kramer, Axel; Sckell, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2017-03-01

To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

USP17 is upregulated in osteosarcoma and promotes cell proliferation, metastasis, and epithelial-mesenchymal transition through stabilizing SMAD4.

PubMed

Song, Chenyang; Liu, Wenge; Li, Jiandong

2017-07-01

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial-mesenchymal transition. SMAD4 has been found to regulate epithelial-mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

Combination of etoposide and fisetin results in anti-cancer efficiency against osteosarcoma cell models.

PubMed

Ferreira de Oliveira, José Miguel P; Pacheco, Ana Rita; Coutinho, Laura; Oliveira, Helena; Pinho, Sónia; Almeida, Luis; Fernandes, Eduarda; Santos, Conceição

2018-03-01

Osteosarcoma chemotherapy is often limited by chemoresistance, resulting in poor prognosis. Combined chemotherapy could, therefore, be used to prevent resistance to chemotherapeutics. Here, the effects of fisetin on osteosarcoma cells were investigated, as well as cytostatic potential in combination with the anti-cancer drug etoposide. For this, different osteosarcoma cell lines were treated with fisetin, with etoposide and with respective combinations. Fisetin was associated with decrease in colony formation in Saos-2 and in U2OS cells but not in MG-63 cells. Notwithstanding, upon evaluation of cellular growth by crystal violet assay, MG-63 and Saos-2 cells showed decreased cell proliferation at 40 and 20 µM fisetin, respectively. Depending on the relative concentrations, fisetin:etoposide combinations showed negative-to-positive interactions on the inhibition of cell proliferation. In addition, fisetin treatment up to 50 µM for 48 h resulted in G2-phase cell cycle arrest. Regardless of the combination, fisetin:etoposide increased % cells in G2-phase and decreased % cells in G1-phase. In addition, mixtures with more positive combined effects induced increased % cells in S-phase. Compared to etoposide treatment, these combinations resulted in decreased levels of cyclins B1 and E1, pointing to the role of these regulators in fisetin-induced cell cycle arrest. In conclusion, these results show that the combination of fisetin with etoposide has higher anti-proliferative effects in osteosarcoma associated with cell cycle arrest, allowing the use of lower doses of the chemotherapeutic agent, which has important implications for osteosarcoma treatment.

TIMP3 Overexpression Improves the Sensitivity of Osteosarcoma to Cisplatin by Reducing IL-6 Production

PubMed Central

Han, Xiu-guo; Mo, Hui-min; Liu, Xu-qiang; Li, Yan; Du, Lin; Qiao, Han; Fan, Qi-ming; Zhao, Jie; Zhang, Shu-hong; Tang, Ting-ting

2018-01-01

Osteosarcoma is the most common bone cancer in children and adolescents. Tissue inhibitors of metalloproteinases (TIMPs)-3 inhibit matrix metalloproteinases to limit extracellular matrix degradation. Cisplatin is a widely used chemotherapeutic drug used to cure osteosarcoma. Interleukin (IL)-6 and TIMP3 play important roles in the drug resistance of osteosarcoma; however, their relationship in this process remains unclear. This study aimed to explore the role of TIMP3 in the cisplatin sensitivity of osteosarcoma and its underlying molecular mechanisms in vitro and in vivo. We compared TIMP3 expression levels between patients with cisplatin-sensitive and -insensitive osteosarcoma. TIMP3 was overexpressed or knocked down in the Saos2-lung cell line, which is a Saos2 subtype isolated from pulmonary metastases that has higher cisplatin chemoresistance than Saos2 cells. IL-6 expression, cell proliferation, sensitivity to cisplatin, migration, and invasion after TIMP3 overexpression or knockdown were determined. The same experiments were performed using MG63 and U2OS cells. Subsequently, luciferase-labeled Saos2-lung cells overexpressing TIMP3 were injected into the tibiae of nude mice treated with cisplatin. The results showed that IL-6 inhibited TIMP3 expression in Saos2 and Saos2-lung cells via signal transducer and activator of transcription 3 (STAT3) activation. STAT3 knockdown reversed the effect of IL-6. The expression of TIMP3 was higher in patients with cisplatin-sensitive osteosarcoma than in those with insensitive osteosarcoma. IL-6 expression was downregulated upon TIMP3 overexpression, and upregulated by TIMP3 knockdown. TIMP3 overexpression suppressed cell proliferation and enhanced cisplatin sensitivity by activating apoptosis-related signal pathways and inhibiting IL-6 expression in vitro and in vivo. In conclusion, cisplatin sensitivity correlated positively with TIMP3 expression, which is regulated by the IL-6/TIMP3/caspase pathway. The TIMP3 pathway

Circular RNA GLI2 promotes osteosarcoma cell proliferation, migration, and invasion by targeting miR-125b-5p.

PubMed

Li, Ji-Feng; Song, Yu-Ze

2017-07-01

Circular RNAs are novel identified type of endogenous non-coding RNAs, which exert vital functions in human and animals. However, the in-depth role of circular RNAs in the progression of tumorigenesis, especially osteosarcoma, is still undefined. Our preliminary study had found that cir-GLI2 was significantly upregulated in osteosarcoma tissues compared to adjacent non-tumor tissue. Moreover, cir-GLI2 silencing could effectively suppress the proliferation, migration, and invasion capacity of osteosarcoma cells, indicating the tumor-promoting role. Besides, bioinformatics analysis and luciferase reporter assay predicted the direct binding to miR-125b-5p, which has been reported to function as a tumor suppressor in osteosarcoma. Furthermore, functional experiments validated that cir-GLI2 exerted the tumor-promoting effects on osteosarcoma cells via negatively targeting miR-125b-5p. In conclusion, our study demonstrated that cir-GLI2 acts as an oncogenic circular RNA in osteosarcoma genesis, providing a novel diagnostic and therapeutic target for osteosarcoma.

Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells.

PubMed

Yamanegi, Koji; Kawabe, Mutsuki; Futani, Hiroyuki; Nishiura, Hiroshi; Yamada, Naoko; Kato-Kogoe, Nahoko; Kishimoto, Hiromitsu; Yoshiya, Shinichi; Nakasho, Keiji

2015-05-01

The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.

[Effect of M007 mediated photodynamic therapy on proliferation of human osteosarcoma MG63 cells in vitro].

PubMed

Zhou, Yu-Kai; Wu, Wen-Zhi; Zhang, Lan; Yang, Chun-Hui; Wang, Yan-Ping

2012-01-01

To investigate the effect of a new photosensitizer, M007 mediated photodynamic therapy on proliferation of human osteosarcoma MG63 cells in vitro. Human osteosarcoma MG63 cells were prepared as 1 x 10(6) /mL single-cell suspension, and 1 mL cells were transferred into 60 mL culture dish, then treated with 5 different gradient dosages (0, 2, 4, 8, 16 micromol/L) of M007 followed by photodynamic therapy or dark reaction for 10 min. The survival rate of the cells and the mode of cell death were detected by flow cytometry with the stain of Annexin V-FITC/PI. The effect on proliferation of survival cells was observed by MTT assay and colony-forming assay. M007 mediated photodynamic therapy induced the inactivation of MG63 human osteosarcoma cells in the way of late apoptosis/necrosis or becoming naked nucleus predominately. More than 90% MG63 cells in M007-PDT group were dead under the treatment of 2-16 micromol/L M007. The survival rates of 4-16 micromol/L M007-PDT group were steadily less than 1%. The optical densities did not increase with extension of culture time in 2-8 micromol/L M007-PDT group (P > 0.05). There were 16 survival alive cells found occasionally in 2 micromol/L M007-PDT group, but no colonies found in other groups. M007 mediated photodynamic therapy totally inactivated human osteosarcoma MG63 cells in vitro with the dosage more than 4 micromol/L.

MicroRNA-143 Regulates Human Osteosarcoma Metastasis by Regulating Matrix Metalloprotease-13 Expression

PubMed Central

Osaki, Mitsuhiko; Takeshita, Fumitaka; Sugimoto, Yui; Kosaka, Nobuyoshi; Yamamoto, Yusuke; Yoshioka, Yusuke; Kobayashi, Eisuke; Yamada, Tesshi; Kawai, Akira; Inoue, Toshiaki; Ito, Hisao; Oshimura, Mitsuo; Ochiya, Takahiro

2011-01-01

Pulmonary metastases are the main cause of death in patients with osteosarcoma, however, the molecular mechanisms of metastasis are not well understood. To detect lung metastasis-related microRNA (miRNA) in human osteosarcoma, we compared parental (HOS) and its subclone (143B) human osteosarcoma cell lines showing lung metastasis in a mouse model. miR-143 was the most downregulated miRNA (P < 0.01), and transfection of miR-143 into 143B significantly decreased its invasiveness, but not cell proliferation. Noninvasive optical imaging technologies revealed that intravenous injection of miR-143, but not negative control miRNA, significantly suppressed lung metastasis of 143B (P < 0.01). To search for miR-143 target mRNA in 143B, microarray analyses were performed using an independent RNA pool extracted by two different comprehensive miR-143-target mRNA collecting systems. Western blot analyses revealed that MMP-13 was mostly protein downregulated by miR-143. Immunohistochemistry using clinical samples clearly revealed MMP-13-positive cells in lung metastasis-positive cases, but not in at least three cases showing higher miR-143 expression in the no metastasis group. Taken together, these data indicated that the downregulation of miR-143 correlates with the lung metastasis of human osteosarcoma cells by promoting cellular invasion, probably via MMP-13 upregulation, suggesting that miRNA could be used to develop new molecular targets for osteosarcoma metastasis. PMID:21427707

Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

PubMed

Pahl, Jens H W; Kwappenberg, Kitty M C; Varypataki, Eleni M; Santos, Susy J; Kuijjer, Marieke L; Mohamed, Susan; Wijnen, Juul T; van Tol, Maarten J D; Cleton-Jansen, Anne-Marie; Egeler, R Maarten; Jiskoot, Wim; Lankester, Arjan C; Schilham, Marco W

2014-03-10

In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our

Silver nanoparticles defeat p53-positive and p53-negative osteosarcoma cells by triggering mitochondrial stress and apoptosis

PubMed Central

Kovács, Dávid; Igaz, Nóra; Keskeny, Csilla; Bélteky, Péter; Tóth, Tímea; Gáspár, Renáta; Madarász, Dániel; Rázga, Zsolt; Kónya, Zoltán; Boros, Imre M.; Kiricsi, Mónika

2016-01-01

Loss of function of the tumour suppressor p53 observed frequently in human cancers challenges the drug-induced apoptotic elimination of cancer cells from the body. This phenomenon is a major concern and provides much of the impetus for current attempts to develop a new generation of anticancer drugs capable of provoking apoptosis in a p53-independent manner. Since silver nanoparticles (AgNPs) possess unique cytotoxic features, we examined, whether their activity could be exploited to kill tumour suppressor-deficient cancer cells. Therefore, we investigated the effects of AgNPs on osteosarcoma cells of different p53 genetic backgrounds. As particle diameters might influence the molecular mechanisms leading to AgNP-induced cell death we applied 5 nm and 35 nm sized citrate-coated AgNPs. We found that both sized AgNPs targeted mitochondria and induced apoptosis in wild-type p53-containing U2Os and p53-deficient Saos-2 cells. According to our findings AgNPs are able to kill osteosarcoma cells independently from their actual p53 status and induce p53-independent cancer cell apoptosis. This feature renders AgNPs attractive candidates for novel chemotherapeutic approaches. PMID:27291325

Bone formation in vitro and in nude mice by human osteosarcoma cells.

PubMed

Ogose, A; Motoyama, T; Hotta, T; Watanabe, H; Takahashi, H E

1995-01-01

Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.

Osteosarcoma subtypes: Magnetic resonance and quantitative diffusion weighted imaging criteria.

PubMed

Zeitoun, Rania; Shokry, Ahmed M; Ahmed Khaleel, Sahar; Mogahed, Shaimaa M

2018-03-01

Osteosarcoma (OS) is a primary bone malignancy, characterized by spindle cells producing osteoid. The objective of this study is to describe the magnetic resonance imaging (MRI) features of different OS subtypes, record their attenuation diffusion coefficient (ADC) values and to point to the relation of their pathologic base and their corresponding ADC value. We performed a retrospective observational lesion-based analysis for 31 pathologically proven osteosarcoma subtypes: osteoblastic (n = 9), fibroblastic (n = 8), chondroblastic (n = 6), para-osteal (n = 3), periosteal (n = 1), telangiectatic (n = 2), small cell (n = 1) and extra-skeletal (n = 1). On conventional images we recorded: bone of origin, epicenter, intra-articular extension, and invasion of articulating bones, skip lesions, distant metastases, pathological fractures, ossified matrix, hemorrhage and necrosis. We measured the mean ADC value for each lesion. Among the included OS lesions, 51.6% originated at the femur, 29% showed intra-articular extension, 16% invaded neighboring bone, 9% were associated with pathological fracture and 25.8% were associated with distant metastases. On MRI, all lesions showed ossified matrix, 35.5% showed hemorrhage and 58% showed necrosis. The mean ADC values for OS lesions ranged from 0.74 × 10 -3  mm 2 /s (recorded for conventional osteoblastic OS) to 1.50 × 10 -3  mm 2 /s (recorded for telangiectatic OS) with an average value of 1.16 ± 0.18 × 10 -3  mm 2 /s. Conventional chondroblastic OS recorded higher values compared to the other two conventional subtypes. Osteosarcoma has different pathologic subtypes which correspondingly vary in their imaging criteria and their ADC values. Copyright © 2018. Production and hosting by Elsevier B.V.

Amphiregulin enhances intercellular adhesion molecule-1 expression and promotes tumor metastasis in human osteosarcoma

PubMed Central

Liu, Ju-Fang; Tsao, Ya-Ting; Hou, Chun-Han

2015-01-01

Osteosarcoma is a common, high malignant, and metastatic bone cancer. Amphiregulin (AREG) has been associated with cancer cellular activities. However, the effect of AREG on metastasis activity in human osteosarcoma cells has yet to be determined. We determined that AREG increases the expression of intercellular adhesion molecule-1 (ICAM-1) through PI3K/Akt signaling pathway via its interaction with the epidermal growth factor receptor, thus resulting in the enhanced cell migration of osteosarcoma. Furthermore, AREG stimulation increased the association of NF-κB to ICAM-1 promoter which then up-regulated ICAM-1 expression. Finally, we observed that shRNA silencing of AREG decreased osteosarcoma metastasis in vivo. Our findings revealed a relationship between osteosarcoma metastatic potential and AREG expression and the modulating effect of AREG on ICAM-1 expression. PMID:26503469

Cardiomyocyte apoptosis vs autophagy with prolonged doxorubicin treatment: comparison with osteosarcoma cells.

PubMed

Tacar, Oktay; Indumathy, Sivanjah; Tan, Mei Lin; Baindur-Hudson, Swati; Friedhuber, Anna M; Dass, Crispin R

2015-02-01

Doxorubicin (Dox) is a frontline chemotherapeutic against osteosarcoma (OS) that is plagued by side effects, particularly in the heart. The specific objective of this article is to investigate whether low-dose Dox treatment had pro-autophagic effects in cardiomyocytes as well as osteosarcoma cells. This study characterises apoptotic (Bax) and autophagic (Beclin-1) biomarker levels in human OS and cardiomyocyte cell lines as well as in various tissues when mice are exposed to low (1 mg/kg, thrice weekly) and high (3 mg/kg thrice weekly) dose Dox for a month. There was a decrease in Bax and increase in Beclin-1 in cardiac tissue in the high-dose group. Dox decreased Beclin-1 in the skin and liver, with no clear indication in the stomach, small intestine and testis. At low Dox doses of 10 and 100 nm in cardiomyocytes and OS cells, there is a pro-apoptotic effect, with a quicker response in the 100-nm condition, and a slower but steady increase of a pro-apoptotic response at the lower 10-nm dose. However, electron microscopy images revealed changes to human OS cells that resembled autophagy. Human prostate, breast and colorectal cells treated with 10-nm Dox showed ∼ 40% reduction in cell viability after 24 h. In culture, cells of both cardiomyocytes and OS revealed a predominant pro-apoptotic response at the expense of autophagy, although both seemed to be occurring in vivo. © 2014 Royal Pharmaceutical Society.

Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ming; Wang, Dan, E-mail: danwangwdd@163.com; Li, Ning

2016-04-22

The transcriptional cofactor Che-1 is an RNA polymerase II (Pol II) which is involved in tumorigenesis, such as breast cancer and multiple myeloma. Che-1 can also regulate mutant p53 expression, which plays roles in many types of cancer. In this study, we aimed to investigate the effects and specific mechanism of Che-1 in the regulation of osteosarcoma (OS) cell growth. We found that Che-1 is highly expressed in several kinds of OS cells compared with osteoblast hFOB1.19 cells. MTT and flow cytometry assays showed that Che-1 depletion by siRNA markedly suppressed MG-63 and U2OS cell proliferation and promoted apoptosis. The chromatinmore » immunoprecipitation (ChIP) assay verified the presence of Che-1 on the p53 promoter in MG-63 and U2OS cells carrying mutant p53. Further studies showed that Che-1 depletion inhibited mutant p53 expression. Notably, our study showed that the loss of Che-1 inhibits proliferation and promotes apoptosis in MG-63 cells by decreasing the level of mutant p53. Therefore, these findings open the possibility that silencing of Che-1 will have therapeutic benefit in OS. - Highlights: • Che-1 is highly expressed in several kinds of OS cells. • Che-1 depletion suppressed MG-63 and U2OS cell growth. • Che-1 is existed in the p53 promoter in MG-63 and U2OS cells. • Che-1 depletion inhibited mutant p53 expression. • Che-1 depletion inhibits cell growth by decreasing the level of mutant p53.« less

HDAC inhibitor-loaded bone cement for advanced local treatment of osteosarcoma and chondrosarcoma.

PubMed

Tonak, Marcus; Becker, Marc; Graf, Claudine; Eckhard, Lukas; Theobald, Matthias; Rommens, Pol-Maria; Wehler, Thomas C; Proschek, Dirk

2014-11-01

The treatment of osteosarcoma, especially wide resection, is challenging. An additional local drug therapy after resection using anti-neoplastic bone cement (Polymethylmethacrylate (PMMA)) could help improve the outcome of therapy. In this study, we evaluated the effects of PMMA loaded with valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) on the cell activity of a SaOs-2 cell culture, as well as the elution rate of the drugs out of the bone cement. In our experiments, we used the SaOs-2 osteosarcoma and the SW1353 chondrosarcoma cell line. Bone cement clots (5 g) were prepared and loaded with different drug concentrations of VPA (25 mg and 50 mg) and SAHA (1 mg, 2.5 mg and 5 mg). Two control groups were established, one with a native cement clot, the other with human mesenchymal stem cells, in order to evaluate toxicity on non tumor-cells. Cell activity was measured using an Alamar Blue assay on days 1, 2, 3, 4 and 7. The cement clots were additionally examined in a material testing unit for biomechanical and structural changes. Tumor cells showed a significant and complete reduction of activity under therapy with VPA and SAHA. Drug release of VPA was extensive between days 0 and 3 and decreased progressively to day 7. Cumulative drug concentration in the medium continuously increased. Biomechanical testing of the cement clots showed no differences in stability and architecture compared to the control group. SaOs-2 and SW1353 cells with medium from native cement clots without drug therapy presented a cell activity of 100% in all groups and during all measurements. Human mesenchymal stem cells were not significantly affected during therapy with VPA and low concentrations of SAHA. In contrast, cell activity of human mesenchymal stem cells was significantly reduced under therapy with higher concentrations of SAHA, with an approximately linear decrease between days 0-3 and a rapidly decreasing activity between days 4-7. A local cytotoxic therapy in the

Anti-angiogenesis target therapy for advanced osteosarcoma

PubMed Central

Xie, Lu; Ji, Tao; Guo, Wei

2017-01-01

Osteosarcomas (OS), especially those with metastatic or unresectable disease, have limited treatment options. The greatest advancement in treatments occurred in the 1980s when multi-agent chemotherapy, including doxorubicin, cisplatin, high-dose methotrexate, and, in some regimens, ifosfamide, was demonstrated to improve overall survival compared with surgery alone. However, standard chemotherapeutic options have been limited by poor response rates in patients with relapsed or advanced cases. It has been reported that VEGFR expression correlates with the outcome of patients with osteosarcoma and circulating VEGF level has been associated with the development of lung metastasis. At present, it seems to us that progress has not been made since Grignani reported a phase II cohort trial of sorafenib and sorafenib combined with everolimus for advanced osteosarcoma, which, in a sense, have become a milestone as a second-line therapy for osteosarcoma. Although the recognization of muramyltripepetide phosphatidyl-ethanolamine has made some progress based on its combination with standard chemotherapy, its effect on refractory cases is controversial. Personalized comprehensive molecular profiling of high-risk osteosarcoma up to now has not changed the therapeutic prospect of advanced osteosarcoma significantly. Thus, how far have we moved forward and what therapeutic strategy should we prefer for anti-angiogenesis therapy? This review provides an overview of the most updated anti-angiogenesis therapy in OS and discusses some clinical options in order to maintain or even improve progression-free survival. PMID:28656259

SIRT1 promotes metastasis of human osteosarcoma cells

PubMed Central

Zhang, Ning; Xie, Tao; Xian, Miao; Wang, Yi-Jie; Li, Heng-Yuan

2016-01-01

Pulmonary metastasis is the leading cause of mortality in patients with osteosarcoma; however, the underlying mechanism remains unclear. The NAD+-dependent deacetylase, sirtuin 1 (SIRT1), has been reported to play a key role in carcinogenesis through deacetylation of important regulatory proteins. Here, we report that SIRT1 promotes osteosarcoma metastasis by regulating the expression of metastatic-associated genes. The SIRT1 protein was significantly upregulated in most primary osteosarcoma tumours, compared with normal tissues, and the SIRT1 expression level may be coupled with metastatic risk in patients with osteosarcoma. Moreover, the results of cell migration and wound-healing assays further suggested that higher expression of SIRT1 promoted invasive activity of osteosarcoma cells. Importantly, downregulating SIRT1 with shRNA inhibited the migration ability of osteosarcoma cells in vitro and suppressed tumour lung metastasis in mice. Finally, a gene expression analysis showed that knockdown of SIRT1 profoundly activated translation of its downstream pathway, particularly at migration and invasion. In summary, high levels of SIRT1 may be a biomarker for a high metastatic rate in osteosarcoma patients; inhibiting SIRT1 could be a potent therapeutic intervention for these patients. PMID:27793039

Three-dimensional alginate spheroid culture system of murine osteosarcoma.

PubMed

Akeda, Koji; Nishimura, Akinobu; Satonaka, Haruhiko; Shintani, Ken; Kusuzaki, Katsuyuki; Matsumine, Akihiko; Kasai, Yuichi; Masuda, Koichi; Uchida, Atsumasa

2009-11-01

Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis

PubMed Central

Roos, Alison; Satterfield, Laura; Zhao, Shuying; Fuja, Daniel; Shuck, Ryan; Hicks, M John; Donehower, Lawrence A; Yustein, Jason T

2015-01-01

Background: Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear. Methods: si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role. Results: Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways. Conclusions: Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS. PMID:26528706

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.

PubMed

Su, Y; Wagner, E R; Luo, Q; Huang, J; Chen, L; He, B-C; Zuo, G-W; Shi, Q; Zhang, B-Q; Zhu, G; Bi, Y; Luo, J; Luo, X; Kim, S H; Shen, J; Rastegar, F; Huang, E; Gao, Y; Gao, J-L; Yang, K; Wietholt, C; Li, M; Qin, J; Haydon, R C; He, T-C; Luu, H H

2011-09-15

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Strongly correlated electron behavior in single crystalline U2Os3Al9

NASA Astrophysics Data System (ADS)

Kumar, Neeraj; Das, Pranab Kumar; Kulkarni, Ruta; Thamizhavel, A.; Dhar, S. K.

2012-12-01

We report the magnetic properties of a single crystal of a new compound U2Os3Al9 which crystallizes in the well known Y2Co3Ga9 type orthorhombic structure with space group Cmcm. The susceptibility of U2Os3Al9 shows a peak at 7 K typical of antiferromagnetic ordering. The susceptibility in the paramagnetic state is anisotropic, the easy axis of magnetization lying in the ab-plane of the orthorhombic crystal lattice. The magnetization at 2 K, measured up to a maximum field of 160 kOe, shows a metamagnetic transition near 118 kOe when the field is aligned along [010] in addition to a small metamagnetic transition near 25 kOe. The bulk antiferromagnetic ordering of the uranium ions at TN = 7 K is confirmed by a peak in the heat capacity with ΔC nearly 7 J/U.mol K. An extrapolation of the heat capacity data from the paramagnetic regime to T = 0 gives an enhanced electronic specific heat coefficient of 120 mJ/U.mol K2. The electrical resistivity of U2Os3Al9 shows a negative temperature coefficient between 300 and TN which is a signature of spin fluctuations in a narrow band or a Kondo type of interaction. The data thus suggest the presence of strong electron correlations in this compound.

Understanding the Osteosarcoma Pathobiology: A Comparative Oncology Approach

PubMed Central

Varshney, Jyotika; Scott, Milcah C.; Largaespada, David A.; Subramanian, Subbaya

2016-01-01

Osteosarcoma is an aggressive primary bone tumor in humans and is among the most common cancer afflicting dogs. Despite surgical advancements and intensification of chemo- and targeted therapies, the survival outcome for osteosarcoma patients is, as of yet, suboptimal. The presence of metastatic disease at diagnosis or its recurrence after initial therapy is a major factor for the poor outcomes. It is thought that most human and canine patients have at least microscopic metastatic lesions at diagnosis. Osteosarcoma in dogs occurs naturally with greater frequency and shares many biological and clinical similarities with osteosarcoma in humans. From a genetic perspective, osteosarcoma in both humans and dogs is characterized by complex karyotypes with highly variable structural and numerical chromosomal aberrations. Similar molecular abnormalities have been observed in human and canine osteosarcoma. For instance, loss of TP53 and RB regulated pathways are common. While there are several oncogenes that are commonly amplified in both humans and dogs, such as MYC and RAS, no commonly activated proto-oncogene has been identified that could form the basis for targeted therapies. It remains possible that recurrent aberrant gene expression changes due to gene amplification or epigenetic alterations could be uncovered and these could be used for developing new, targeted therapies. However, the remarkably high genomic complexity of osteosarcoma has precluded their definitive identification. Several advantageous murine models of osteosarcoma have been generated. These include spontaneous and genetically engineered mouse models, including a model based on forward genetics and transposon mutagenesis allowing new genes and genetic pathways to be implicated in osteosarcoma development. The proposition of this review is that careful comparative genomic studies between human, canine and mouse models of osteosarcoma may help identify commonly affected and targetable pathways for

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

PubMed

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

Involvement of HIF-1α activation in the doxorubicin resistance of human osteosarcoma cells.

PubMed

Roncuzzi, Laura; Pancotti, Fabia; Baldini, Nicola

2014-07-01

Osteosarcoma is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, survival outcomes remain unsatisfactory, particularly in patients with metastatic and/or recurrent disease. Unfortunately, treatment failure is commonly due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. The aim of the present study was to investigate the role of hypoxia-inducible factor 1α (HIF‑1α) and its signalling pathways as mediators of drug-resistance in human osteosarcoma. Toward this aim, we established two osteosarcoma cell lines selected for resistance to doxorubicin, a drug of choice in the treatment of this tumour. Our results showed that the multidrug resistance (MDR) phenotype was also mediated by HIF-1α, the most important regulator of cell adaptation to hypoxia. Our data showed that this transcription factor promoted the outward transport of intracellular doxorubicin by activating the P-glycoprotein (P-gp) expression in osteosarcoma cells maintained in normoxic conditions. In addition, it hindered doxorubicin-induced apoptosis by regulating the expression of c-Myc and p21. Finally, we observed that the doxorubicin-resistant cells maintained for 2 months of continuous culture in a drug-free medium, lost their drug-resistance and this effect was associated with the absence of HIF-1α expression. The emerging role of HIF-1α in osteosarcoma biology indicates its use as a valuable therapeutic target.

Osteosarcoma Overview.

PubMed

Lindsey, Brock A; Markel, Justin E; Kleinerman, Eugenie S

2017-06-01

Osteosarcoma (OS) is the most common primary malignancy of bone and patients with metastatic disease or recurrences continue to have very poor outcomes. Unfortunately, little prognostic improvement has been generated from the last 20 years of research and a new perspective is warranted. OS is extremely heterogeneous in both its origins and manifestations. Although multiple associations have been made between the development of osteosarcoma and race, gender, age, various genomic alterations, and exposure situations among others, the etiology remains unclear and controversial. Noninvasive diagnostic methods include serum markers like alkaline phosphatase and a growing variety of imaging techniques including X-ray, computed tomography, magnetic resonance imaging, and positron emission as well as combinations thereof. Still, biopsy and microscopic examination are required to confirm the diagnosis and carry additional prognostic implications such as subtype classification and histological response to neoadjuvant chemotherapy. The current standard of care combines surgical and chemotherapeutic techniques, with a multitude of experimental biologics and small molecules currently in development and some in clinical trial phases. In this review, in addition to summarizing the current understanding of OS etiology, diagnostic methods, and the current standard of care, our group describes various experimental therapeutics and provides evidence to encourage a potential paradigm shift toward the introduction of immunomodulation, which may offer a more comprehensive approach to battling cancer pleomorphism.

Activation of Sonic Hedgehog Signaling Is Associated with Human Osteosarcoma Cells Radioresistance Characterized by Increased Proliferation, Migration, and Invasion.

PubMed

Qu, Wei; Li, Dichen; Wang, Yufei; Wu, Qining; Hao, Dingjun

2018-06-04

BACKGROUND Radioresistance restricts the application of radiotherapy in human osteosarcoma (OS). This study investigated the molecular mechanism of radioresistance in OS, which may provide clues to finding ideal targets for genetic therapy. MATERIAL AND METHODS The human OS cell line MG63 was employed as parent cells. After repeat low-dose X-ray irradiation of MG63, the radioresistant OS cell line MG63R was produced. Colony formation assay was used to assess the radioresistance. Cell viability was evaluated by CCK-8 assay. Flow cytometry was used to detect cell apoptosis, and wound healing assay was used to evaluate invasive capacity. The nuclear translocation was evaluated by fluorescent immunohistochemistry. Protein expression levels were assessed by Western blotting. Specific siRNA against Shh was used to silence Shh. RESULTS More survival colony formation, elevated cell viability, less cell apoptosis, and increased wound closure were found in MG63R than in MG63 cells exposed to irradiation. The nuclear translocation of Gli, expression levels of Shh, Smo, Ptch1, Bcl2, active MMP2, and active MMP9 were increased in MG63R cells compared with MG63 cells. Transfection of Shh-siRNA suppressed expression levels of Shh, Smo, Ptch1, Bcl2, active MMP2, and active MMP9, as well as the nuclear translocation of Gli in MG63R cells. The cell viability, survival colony formation, and wound closure were impaired, whereas cell apoptosis was increased, in siRNA-transfected MG63R cells than in control MG63R cells exposed to irradiation. CONCLUSIONS Activation of Shh signaling was involved in radioresistance of OS cells. Blocking this signaling can impair the radioresistance capacity of OS cells.

Generation of Osteosarcomas from a Combination of Rb Silencing and c‐Myc Overexpression in Human Mesenchymal Stem Cells

PubMed Central

Wang, Jir‐You; Wu, Po‐Kuei; Chen, Paul Chih‐Hsueh; Lee, Chia‐Wen

2016-01-01

Abstract Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c‐Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c‐Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum‐free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS‐like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient‐derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c‐Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS‐like cells by Rb knockdown and c‐Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512–526 PMID:28191765

A porcine model of osteosarcoma

PubMed Central

Saalfrank, A; Janssen, K-P; Ravon, M; Flisikowski, K; Eser, S; Steiger, K; Flisikowska, T; Müller-Fliedner, P; Schulze, É; Brönner, C; Gnann, A; Kappe, E; Böhm, B; Schade, B; Certa, U; Saur, D; Esposito, I; Kind, A; Schnieke, A

2016-01-01

We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53R167H and KRASG12D, and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7–8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease. PMID:26974205

Proteomic identification of 14-3-3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells.

PubMed

Kim, Kyung Ok; Hsu, Anny C; Lee, Heon Goo; Patel, Neel; Chandhanayingyong, Chandhanarat; Hickernell, Thomas; Lee, Francis Young-In

2014-06-01

Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Fluoroquinolone-mediated inhibition of cell growth, S-G2/M cell cycle arrest, and apoptosis in canine osteosarcoma cell lines.

PubMed

Seo, Kyoung won; Holt, Roseline; Jung, Yong-Sam; Rodriguez, Carlos O; Chen, Xinbin; Rebhun, Robert B

2012-01-01

Despite significant advancements in osteosarcoma research, the overall survival of canine and human osteosarcoma patients has remained essentially static over the past 2 decades. Post-operative limb-spare infection has been associated with improved survival in both species, yet a mechanism for improved survival has not been clearly established. Given that the majority of canine osteosarcoma patients experiencing post-operative infections were treated with fluoroquinolone antibiotics, we hypothesized that fluoroquinolone antibiotics might directly inhibit the survival and proliferation of canine osteosarcoma cells. Ciprofloxacin or enrofloxacin were found to inhibit p21(WAF1) expression resulting in decreased proliferation and increased S-G(2)/M accumulation. Furthermore, fluoroquinolone exposure induced apoptosis of canine osteosarcoma cells as demonstrated by cleavage of caspase-3 and PARP, and activation of caspase-3/7. These results support further studies examining the potential impact of quinolones on survival and proliferation of osteosarcoma.

miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1.

PubMed

Guo, Xiaodong; Yu, Ling; Zhang, Zhengpei; Dai, Guo; Gao, Tian; Guo, Weichun

2017-01-01

Evidence is accumulating to link cancer stem cells to the pathogenesis and progression of osteosarcoma. The aim of this study is to investigate the role of miR-335 in osteosarcoma stem cells. Tumor spheroid culture and flow cytometry were applied to screen out osteosarcoma stem cells. Real-time quantitative PCR was used to detect the expression level of miR-335 in MG63, U2OS and 143B osteosarcoma stem cells. The relationship of miR-335 expression with osteosarcoma stem cells was then analyzed. Transwell assay and transplantation assay were performed to elucidate biological effects of miR-335 on cell invasion and vivo tumor formation. Western Blot and luciferase assays were executed to investigate the regulation of POU5F1 by miR-335. The expression of miR-335 in osteosarcoma stem cells was lower than their differentiated counterparts. Cells expressing miR-335 possessed decreased stem cell-like properties. Gain or loss of function assays were applied to find that miR-335 antagonist promoted stem cell-like properties as well as invasion. Luciferase report and transfection assay showed that POU5F1 was downregulated by miR-335. Pre-miR-335 resulted in tumor enhanced sensitivity to traditional chemotherapy, whereas anti-miR-335 promoted chemoresistance. Finally, the inhibitory effect of miR-335 on in vivo tumor formation showed that combination of pre-miR-335 with cisplatin further reduced the tumor size, and miR-335 brought down the sphere formation capacity induced by cisplatin. The current study demonstrates that miR-335 negatively regulates osteosarcoma stem cell-like properties by targeting POU5F1, and miR-335 could target CSCs to synergize with traditional chemotherapeutic agents to overcome osteosarcoma.

Biology, diagnosis and treatment of canine appendicular osteosarcoma: similarities and differences with human osteosarcoma.

PubMed

Morello, Emanuela; Martano, Marina; Buracco, Paolo

2011-09-01

Osteosarcoma (OSA) is the most common primary bone tumour in dogs. The appendicular locations are most frequently involved and large to giant breed dogs are commonly affected, with a median age of 7-8 years. OSA is a locally invasive neoplasm with a high rate of metastasis, mostly to the lungs. Due to similarities in biology and treatment of OSA in dogs and humans, canine OSA represents a valid and important tumour model. Differences between canine and human OSAs include the age of occurrence (OSA is most commonly an adolescent disease in humans), localisation (the stifle is the most common site of localisation in humans) and limited use of neoadjuvant chemotherapy in canine OSA. Copyright © 2010 Elsevier Ltd. All rights reserved.

Inhibition of polo-like kinase 1 leads to the suppression of osteosarcoma cell growth in vitro and in vivo.

PubMed

Liu, Xianzhe; Choy, Edwin; Harmon, David; Yang, Shuhua; Yang, Cao; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

2011-06-01

Osteosarcoma is the most common type of primary bone cancer in children and adolescents. Treatment options for osteosarcoma may include surgery, chemotherapy, and radiotherapy. Unfortunately, many patients eventually relapse, resulting in an unsatisfactory outcome. The serine/threonine-specific polo-like kinase 1 (PLK1) is a kinase that plays an important role in mitosis and the maintenance of genomic stability. PLK1 has been found to be highly expressed in the malignant cells of osteosarcoma. Here, we describe the in-vitro and in-vivo effects of BI 2536, a small-molecule inhibitor of PLK1, which through inhibiting PLK1 enzymatic activity, causes mitotic arrest and eventually induces cancer cell apoptosis. In this study, we show that the PLK1 inhibitor, BI 2536, inhibits proliferation and induces apoptosis in two-dimensional and three-dimensional cultures of osteosarcoma cell lines, KHOS and U-2OS. A proliferation assay performed both in two-dimensional and three-dimensional culture showed that the growth of both cell lines was inhibited by BI 2536. Cell cycle analysis showed that the cells treated with BI 2536 were mainly arrested in the G2/M phase. Immunofluorescence and western blotting analysis confirmed that the administration of BI 2536 led to significant decrease of PLK1 and Mcl-1 protein expression levels in dose-dependent and time-dependent manners. Furthermore, BI 2536-induced apoptosis in the osteosarcoma cell lines was shown by poly (ADP-ribose) polymerase cleavage and caspase assay. Finally, in mouse osteosarcoma xenografts, BI 2536-treated mice had significantly smaller tumors compared with the control mice. These findings offer evidence of the potential role for targeting PLK1 in osteosarcoma therapy.

Anticancer Effects of Geopropolis Produced by Stingless Bees on Canine Osteosarcoma Cells In Vitro

PubMed Central

Cinegaglia, Naiara Costa; Bersano, Paulo Ricardo Oliveira; Araújo, Maria José Abigail Mendes; Búfalo, Michelle Cristiane; Sforcin, José Maurício

2013-01-01

Geopropolis is produced by indigenous stingless bees from the resinous material of plants, adding soil or clay. Its biological properties have not been investigated, such as propolis, and herein its cytotoxic action on canine osteosarcoma (OSA) cells was evaluated. OSA is a primary bone neoplasm diagnosed in dogs being an excellent model in vivo to study human OSA. spOS-2 primary cultures were isolated from the tumor of a dog with osteosarcoma and incubated with geopropolis, 70% ethanol (geopropolis solvent), and carboplatin after 6, 24, 48, and 72 hours. Cell viability was analyzed by the crystal violet method. Geopropolis was efficient against canine OSA cells in a dose- and time-dependent way, leading to a distinct morphology compared to control. Geopropolis cytotoxic action was exclusively due to its constituents since 70% ethanol (its solvent) had no effect on cell viability. Carboplatin had no effect on OSA cells. Geopropolis exerted a cytotoxic effect on canine osteosarcoma, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment. PMID:23690851

Anticancer effects of geopropolis produced by stingless bees on canine osteosarcoma cells in vitro.

PubMed

Cinegaglia, Naiara Costa; Bersano, Paulo Ricardo Oliveira; Araújo, Maria José Abigail Mendes; Búfalo, Michelle Cristiane; Sforcin, José Maurício

2013-01-01

Geopropolis is produced by indigenous stingless bees from the resinous material of plants, adding soil or clay. Its biological properties have not been investigated, such as propolis, and herein its cytotoxic action on canine osteosarcoma (OSA) cells was evaluated. OSA is a primary bone neoplasm diagnosed in dogs being an excellent model in vivo to study human OSA. spOS-2 primary cultures were isolated from the tumor of a dog with osteosarcoma and incubated with geopropolis, 70% ethanol (geopropolis solvent), and carboplatin after 6, 24, 48, and 72 hours. Cell viability was analyzed by the crystal violet method. Geopropolis was efficient against canine OSA cells in a dose- and time-dependent way, leading to a distinct morphology compared to control. Geopropolis cytotoxic action was exclusively due to its constituents since 70% ethanol (its solvent) had no effect on cell viability. Carboplatin had no effect on OSA cells. Geopropolis exerted a cytotoxic effect on canine osteosarcoma, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment.

Osteosarcoma of the mobile spine.

PubMed

Zils, K; Bielack, S; Wilhelm, M; Werner, M; Schwarz, R; Windhager, R; Hofmann-Wackersreuther, G; Andus, T; Kager, L; Kuehne, T; Reichardt, P; von Kalle, T

2013-08-01

The aims of this analysis were to investigate features and outcome of high-grade osteosarcomas of the mobile spine. Since 1977, 20 Cooperative Osteosarcoma Study Group patients had a diagnosis of high-grade osteosarcomas of the mobile spine and were included in this retrospective analysis of patient-, tumor- and treatment-related variables and outcome. The median age was 29 years (range 5-58). Most frequent tumor sites were thoracic and lumbar spine. All but three patients had nonmetastatic disease at diagnosis. Treatment included surgery and chemotherapy for all patients, 13 were also irradiated. Eight patients failed to achieve a macroscopically complete surgical remission (five local, one primary metastases, two both), six died, two are alive, both with radiotherapy. Of 12 patients with complete remission at all sites, three had a recurrence (two local, one metastases) and died. The median follow-up of the 11 survivors was 8.7 years (range 3.1-22.3), 5-year overall and event-free survival rates were 60% and 43%. Age <40 years, nonmetastatic disease at diagnosis and complete remission predicted for better overall survival (OS, P < 0.05). Osteosarcomas of the mobile spine are rare. With complete resection (and potentially radiotherapy) and chemotherapy, prognosis may be comparable with that of appendicular osteosarcomas.

Apatinib inhibits migration and invasion as well as PD-L1 expression in osteosarcoma by targeting STAT3.

PubMed

Zheng, Bingxin; Ren, Tingting; Huang, Yi; Guo, Wei

2018-01-08

The cure rate of osteosarcoma has not improved in the past 30 years. The new treatments and drugs is urgently needed, especially for metastatic osteosarcoma. Anti-angiogenesis therapy and immunotherapy has got promising anti-tumor effects in various tumors. It is hypothesised that combining checkpoint inhibitor immunotherapies with antiangiogenic treatment may have a synergistic effect and enhance the efficacy of both treatments. However, its underlying mechanism remain largely uninvestigated. To investigate the clinical significance of vascular endothelial growth factor receptor-2 (VEGFR2) and programmed death ligand-1 (PD-L1) in osteosarcoma, we analyzes their expression levels in 93 osteosarcoma specimens by immunohistochemistry. Meanwhile, we analyzes their correlation with the metastatic behavior and overall survival (OS). We also investigate the effects of Apatinib on migration and invasion of osteosarcoma cells and its underlying mechanism in vitro and in vivo. In our study, the positive rates of the VEGFR2 and PD-L1 expression are 64.5% (60/93) and 35.5% (33/93), respectively. A significant correlation is detected between VEGFR2 and PD-L1 expression (P = 0.009). Receiver-operating characteristic (ROC) curves analysis indicates the predictive value of the two markers in tumor metastasis, and both PD-L1 and VEGFR2 are negatively correlated with OS. Transwell assays reveals that VEGFR2 inhibition attenuates migration and invasion of osteosarcoma cells. Mechanistically, we demonstrate that Apatinib attenuates migration and invasion by suppressing epithelial-mesenchymal transition (EMT) and inactivating STAT3. Additionally, Apatinib reduces PD-L1 expression in osteosarcoma cells. Apatinib markedly weakens pulmonary metastatic potential of osteosarcoma in vivo. In conclusion, our study reveals a pro-metastatic functional mechanism for VEGFR2 in osteosarcoma. Furthermore, we demonstrate that Apatinib exerts anti-tumor effect not only through antiangiogenic

Gemcitabine and docetaxel in relapsed and unresectable high-grade osteosarcoma and spindle cell sarcoma of bone.

PubMed

Palmerini, E; Jones, R L; Marchesi, E; Paioli, A; Cesari, M; Longhi, A; Meazza, C; Coccoli, L; Fagioli, F; Asaftei, S; Grignani, G; Tamburini, A; Pollack, S M; Picci, P; Ferrari, S

2016-04-20

Few new compounds are available for relapsed osteosarcoma. We retrospectively evaluated the activity of gemcitabine (G) plus docetaxel (D) in patients with relapsed high-grade osteosarcoma and high-grade spindle cell sarcoma of bone (HGS). Patients receiving G 900 mg/m(2) d 1, 8; D 75 mg/m(2) d 8, every 21 days were eligible. Primary end-point: progression-free survival (PFS) at 4 months; secondary end-point: overall survival (OS) and response rate. Fifty-one patients were included, with a median age of 17 years (8-71), 26 (51%) were pediatric patients. GD line of treatment: 2nd in 14 patients, ≥3rd in 37. 25 (49%) patients had metastases limited to lungs, 26 (51%) multiple sites. 40 (78%) osteosarcoma, 11 (22%) HGS. Eight (16%) patients achieved surgical complete response (sCR2) after GD. Four-month PFS rate was 46%, and significantly better for patients with ECOG 0 (ECOG 0: 54% vs ECOG 1: 43% vs ECOG 2: 0%; p = 0.003), for patients undergoing metastasectomy after GD (sCR2 75% vs no-sCR2 40 %, p = 0.02) and for osteosarcoma (osteosarcoma 56% vs HGS 18%; p = 0.05), with no differences according to age, line of treatment, and pattern of metastases. Forty-six cases had RECIST measurable disease: 6 (13%) patients had a partial response (PR), 20 (43%) had stable disease (SD) and 20 (43%) had progressive disease (PD). The 1-year OS was 30%: 67% for PR, 54% for SD and 20% for PD (p = 0.005). GD is an active treatment for relapsed high-grade osteosarcoma, especially for ECOG 0 patients, and should be included in the therapeutic armamentarium of metastatic osteosarcoma.

Osteosarcoma: A Meta-Analysis and Review of the Literature.

PubMed

Friebele, Jill C; Peck, Jeffrey; Pan, Xueliang; Abdel-Rasoul, Mahmoud; Mayerson, Joel L

2015-12-01

Over the past 30 years, treatment advances and the addition of neoadjuvant chemotherapy have led to improved 5-year survival in patients with osteosarcoma. More recent literature suggests the overall prognosis remains highly variable, with little improvement since the introduction of neoadjuvant chemotherapy. Tumor necrosis is an important predictor of patient prognosis. Necrosis of more than 90% correlates with overall survival (OS) approaching 75%. We reviewed the history of osteosarcoma treatment and survival and performed a meta-analysis of the 2000-2011 literature. Forty articles were included in the study. Five-year OS was 63% (95% confidence interval, 60%-66%) in studies that included patients with metastatic and nonmetastatic disease and 71% (95% confidence interval, 67%-76%) in studies that included only patients with nonmetastatic disease. Fifty percent of the patients in the studies of those with nonmetastatic osteosarcoma achieved 90% necrosis on histology. Five-year OS and number of patients achieving 90% necrosis are consistent with previous reports. Research is needed to improve treatment regimens and patient outcomes.

Osteosarcoma Models: From Cell Lines to Zebrafish

PubMed Central

Mohseny, Alexander B.; Hogendoorn, Pancras C. W.; Cleton-Jansen, Anne-Marie

2012-01-01

High-grade osteosarcoma is an aggressive tumor most commonly affecting adolescents. The early age of onset might suggest genetic predisposition; however, the vast majority of the tumors are sporadic. Early onset, most often lack of a predisposing condition or lesion, only infrequent (<2%) prevalence of inheritance, extensive genomic instability, and a wide histological heterogeneity are just few factors to mention that make osteosarcoma difficult to study. Therefore, it is sensible to design and use models representative of the human disease. Here we summarize multiple osteosarcoma models established in vitro and in vivo, comment on their utilities, and highlight newest achievements, such as the use of zebrafish embryos. We conclude that to gain a better understanding of osteosarcoma, simplification of this extremely complex tumor is needed. Therefore, we parse the osteosarcoma problem into parts and propose adequate models to study them each separately. A better understanding of osteosarcoma provides opportunities for discovering and assaying novel effective treatment strategies. “Sometimes the model is more interesting than the original disease” PJ Hoedemaeker (1937–2007). PMID:22566751

Manipulation therapy prior to diagnosis induced primary osteosarcoma metastasis--from clinical to basic research.

PubMed

Wang, Jir-You; Wu, Po-Kuei; Chen, Paul Chih-Hsueh; Yen, Chuen-Chuan; Hung, Giun-Yi; Chen, Cheng-Fong; Hung, Shih-Chieh; Tsai, Shih-Fen; Liu, Chien-Lin; Chen, Tain-Hsiung; Chen, Wei-Ming

2014-01-01

Osteosarcoma (OS) patients who suffer manipulation therapy (MT) prior to diagnosis resulted in poor prognosis with increasing metastasis or recurrence rate. The aim of the study is to establish an in vivo model to identify the effects of MT on OS. The enrolled 235 OS patients were followed up in this study. In vivo nude mice model with tibia injection of GFP-labeled human OS cells were randomly allocated into MT(+) that with repeated massage on tumor site twice a week and no treatment as MT(-) group. The five-year survival, metastasis and recurrence rates were recorded in clinical subjects. X-ray plainfilm, micro-PET/CT scan, histopathology, serum metalloproteinase 2 (MMP2), metalloproteinase 9 (MMP9) level and human kinase domain insert receptor (KDR) pattern were assayed in mice model. The results showed that patient with MT decreased 5-year survival and higher recurrence or metastasis rate. Compatible with clinical findings, the decreased body weight (30.5 ± 0.65 g) and an increased tumor volume (8.3 ± 1.18 mm3) in MT(+) mice were observed. The increasing signal intensity over lymph node region of hind limb by micro-PET/CT and the tumor cells were detected in lung and bilateral lymph nodes only in MT(+) group. MMP2 (214 ± 9.8 ng/ml) and MMP9 (25.5 ± 1.81 ng/ml) were higher in MT(+) group than in MT(-) group (165 ± 7.8 ng/ml and 16.9 ± 1.40 ng/ml, individually) as well as KDR expression. Taking clinical observations and in vivo evidence together, MT treatment leads to poor prognosis of primary osteosarcoma; physicians should pay more attention on patients who seek MT before diagnosis.

Manipulation Therapy Prior to Diagnosis Induced Primary Osteosarcoma Metastasis—From Clinical to Basic Research

PubMed Central

Wang, Jir-You; Wu, Po-Kuei; Chen, Paul Chih-Hsueh; Yen, Chuen-Chuan; Hung, Giun-Yi; Chen, Cheng-Fong; Hung, Shih-Chieh; Tsai, Shih-Fen; Liu, Chien-Lin; Chen, Tain-Hsiung; Chen, Wei-Ming

2014-01-01

Osteosarcoma (OS) patients who suffer manipulation therapy (MT) prior to diagnosis resulted in poor prognosis with increasing metastasis or recurrence rate. The aim of the study is to establish an in vivo model to identify the effects of MT on OS. The enrolled 235 OS patients were followed up in this study. In vivo nude mice model with tibia injection of GFP-labeled human OS cells were randomly allocated into MT(+) that with repeated massage on tumor site twice a week and no treatment as MT(−) group. The five-year survival, metastasis and recurrence rates were recorded in clinical subjects. X-ray plainfilm, micro-PET/CT scan, histopathology, serum metalloproteinase 2 (MMP2), metalloproteinase 9 (MMP9) level and human kinase domain insert receptor (KDR) pattern were assayed in mice model. The results showed that patient with MT decreased 5-year survival and higher recurrence or metastasis rate. Compatible with clinical findings, the decreased body weight (30.5±0.65 g) and an increased tumor volume (8.3±1.18 mm3) in MT(+) mice were observed. The increasing signal intensity over lymph node region of hind limb by micro-PET/CT and the tumor cells were detected in lung and bilateral lymph nodes only in MT(+) group. MMP2 (214±9.8 ng/ml) and MMP9 (25.5±1.81 ng/ml) were higher in MT(+) group than in MT(−) group (165±7.8 ng/ml and 16.9±1.40 ng/ml, individually) as well as KDR expression. Taking clinical observations and in vivo evidence together, MT treatment leads to poor prognosis of primary osteosarcoma; physicians should pay more attention on patients who seek MT before diagnosis. PMID:24804772

Osteosarcoma: a comprehensive review

PubMed Central

Misaghi, Amirhossein; Goldin, Amanda; Awad, Moayd; Kulidjian, Anna A

2018-01-01

Osteosarcoma (OS) is a relatively rare tumor of bone with a worldwide incidence of 3.4 cases per million people per year. For most of the twentieth century, five-year survival rates for classic OS were very low. In the 1970s, the introduction of adjuvant chemotherapy in the treatment of OS increased survival rates dramatically. The current article reviews the various types of OS and analyzes the clinical and histological features. We also examine historical and current literature to present a succinct review of methods for diagnosis and staging, as well as treatment, and we also discuss some of the future directions of treatment. PMID:29629690

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, P.-S., E-mail: pslediting@mail.scu.edu.t; Chen, C.-Y.

2010-05-01

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in humanmore » osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.« less

HER-2 expression in biopsy and surgical specimen on prognosis of osteosarcoma: A systematic review and meta-analysis of 16 studies.

PubMed

Zhang, Qingyu; Liu, Fanxiao; Wang, Bomin; Li, Zhenfeng; Zhou, Dongsheng; Yang, Qiang; Dong, Jinlei; Li, Jianmin

2016-06-01

Numerous original clinical studies have attempted to investigate the prognostic value of HER-2 overexpression in osteosarcoma, but the results of these studies are not consistent. This meta-analysis and systematic review was performed to further assess the correlation between HER-2 expression and prognosis in patients with osteosarcoma. A detailed search of relevant publications was conducted using 7 electronic databases: PubMed, Embase, the Cochrane library, the Wanfang database, the China National Knowledge Internet (CNKI) database, the Chinese VIP database, and the Chinese Biological Medical (CBM) Database for publications through August 1, 2015, using the following keywords (HER-2 OR ErbB-2 OR C-erbB-2 OR neu) AND (osteosarcoma OR osteogenic tumor). The bibliographies of potentially relevant articles and identified articles were then searched by hand. Eligible studies were those that enrolled participants with osteosarcoma and provided survival outcome in HER-2 positive and negative groups. The hazard ratio (HR) and 95% confidence interval (CI) for each individual study was calculated and pooled to obtain integrated estimates, using random effects modeling. Sixteen studies involving 934 participants with osteosarcoma met our inclusion criteria. HER-2 overexpression was documented in 42.2% of patients with osteosarcoma. Compared with patients without HER-2 overexpression, those overexpressing HER-2 had decreased overall survival (HR = 2.03, 95% CI: 1.36-3.03, P < 0.001). Statistical associations between HER-2 overexpression and unfavorable overall survival (OS) were observed for both biopsy and surgical removal specimens (HR = 2.07, 95%CI: 1.16-3.72, P = 0.014; and HR = 2.02, 95%CI: 1.10-3.71, P = 0.024). Results for disease-free survival (DFS) were similar. Overexpression of HER-2 is significantly associated with poor outcome for patients with osteosarcoma and should be assessed at diagnosis and after surgery as a prognostic factor. However, larger

Pacific 187Os/188Os isotope chemistry and U-Pb geochronology: Synchroneity of global Os isotope change across OAE 2

NASA Astrophysics Data System (ADS)

Du Vivier, A. D. C.; Selby, D.; Condon, D. J.; Takashima, R.; Nishi, H.

2015-10-01

Studies of OAE 2 sections beyond the Atlantic Ocean, Western Interior Seaway (WIS) and European pelagic shelf are limited. Here, we present initial osmium isotope stratigraphy (187Os/188Os-Osi) from two proto-Pacific sites that span the Cenomanian-Turonian boundary interval (CTBI): the Yezo Group (YG) section, Hokkaido, Japan, and the Great Valley Sequence (GVS), California, USA; to evaluate the 187Os/188Os seawater chemistry of the proto-Pacific. Additionally we combine new 206Pb/238U zircon CA-ID-TIMS geochronology from five volcanic tuff horizons of the Yezo Group section to test and facilitate inter-basinal integration with the WIS using radio-isotopically constrained age-depth models for both sections, and quantitatively constrain the absolute timing and duration of events across the CTBI. The YG shows an almost identical Osi profile to that of the WIS, and very similar to that of other sites of the proto-Atlantic and European pelagic oceans (Turgeon and Creaser, 2008; Du Vivier et al., 2014). The characteristics of the Osi profile are radiogenic and heterogeneous (∼0.55-0.85) prior to the OAE 2, and synchronous with the inferred OAE 2 onset the Osi abruptly become unradiogenic and remain relatively homogeneous (∼0.20-0.30) before showing a gradual return to more radiogenic Osi (∼ 0.70) throughout the middle to late OAE 2. A206Pb/238U zircon age of an interbedded tuff (HK017) in the adjacent horizon to the first unradiogenic Osi value constrains the age of the Osi inflection at 94.44 ± 0.14 Ma. This age, including uncertainty, agrees with the interpolated age of the same point in the Osi profile (94.28 ± 0.25 Ma) in the only other dated OAE 2 section, the WIS; indicating a coeval shift in seawater chemistry associated with volcanism at the OAE 2 onset at the levels of temporal resolution (ca. 0.1 Myr). Further, prior to the onset of OAE 2 an enhanced radiogenic inflection in the Osi profile of the YG is correlative, within uncertainty, with a similar

A Potential Bone-Targeting Hypotoxic Platinum(II) Complex with an Unusual Cytostatic Mechanism toward Osteosarcoma Cells.

PubMed

Zhang, Zhenqin; Zhu, Zhenzhu; Luo, Cheng; Zhu, Chengcheng; Zhang, Changli; Guo, Zijian; Wang, Xiaoyong

2018-03-19

Osteosarcoma (OS) is the most common primary pediatric bone tumor lethal to children and adolescents. Chemotherapeutic agents such as cisplatin are not effective for OS because of their poor accessibility to this cancer and severe systemic toxicity. In this study, a lipophilic platinum(II) complex bearing a bisphosphonate bone-targeting moiety, cis-[PtL(NH 3 ) 2 Cl]NO 3 {BPP; L = tetraethyl [2-(pyridin-2-yl)ethane-1,1-diyl]bisphosphonate}, was prepared and characterized by NMR, electrospray ionization mass spectrometry, and single-crystal X-ray crystallography. The cytotoxicity of BPP toward OS cell lines U2OS and MG-63 was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BPP exhibits moderate inhibition against U2OS cells through a mechanism involving both DNA binding and a mevalonate pathway. The acute toxicity of BPP to mice is 7-fold lower than that of cisplatin. The relative low systemic toxicity may result from the steric hindrance of the ligand, which blocks BPP approaching the bases of DNA. The results suggest that incorporating bisphosphonates into a platinum complex not only enhances its bone-targeting property but also minimizes its reactivity toward DNA and thereby lowers the systematic toxicity of the complex. The diminished cytotoxicity of BPP could be compensated for by increasing the therapeutic dose with marginal harm. This strategy provides a new possibility for overcoming the ineffectiveness and systemic toxicity of platinum drugs in the treatment of OS.

In vitro cytotoxicity of galvanically coupled magnesium-titanium particles on human osteosarcoma SAOS2 cells: A potential cancer therapy.

PubMed

Kim, Jua; Gilbert, Jeremy L

2018-04-10

Osteosarcoma is a malignant bone cancer that occurs mostly in children and young adults. This study investigated the cytotoxicity of Mg and Mg-Ti microparticles to human osteosarcoma cells. Osteosarcoma cells were killed in a dosage-dependent manner when cells, with a cell seeding density of 30,000 cells/cm 2 , were cultured with 0 to 2500 µg/mL of Mg or Mg-Ti in cell culture media for 24-72 h. Mg-Ti killed cells more effectively, where 1250 µg/mL of Mg-Ti killed cells completely by 24 h, while 2500 µg/mL of Mg killed nearly all cells, but not all. Killing due to particle corrosion occurred mostly during the first 24 h, and so the percent cell viability between 24 and 72 h showed not much variability. However, the measurement of live and dead cell numbers, over the timeframe of 24-72 h, showed more insight, such as cell recovery. If particle concentrations were low, the number of live cells increased after 24 h, indicating cell proliferation. If particle concentrations were high, the number of live cells either remained steady or decreased, indicating cell quiescence or continued killing, respectively. Increase in the number of dead cells also indicated killing, while plateau meant discontinued killing. In addition, repeated killing of recovered cells exhibited the same dose-dependent killing profile as the initial experiment, implying little development of cell resistance to treatment. These results, together, show that osteosarcoma cells are susceptible to killing by way of exposure to corroding particles, showing highly effective killing using the galvanic couple of Mg-Ti. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc.

Proton pump inhibitor chemosensitization in human osteosarcoma: from the bench to the patients' bed.

PubMed

Ferrari, Stefano; Perut, Francesca; Fagioli, Franca; Brach Del Prever, Adalberto; Meazza, Cristina; Parafioriti, Antonina; Picci, Piero; Gambarotti, Marco; Avnet, Sofia; Baldini, Nicola; Fais, Stefano

2013-10-24

Major goals in translational oncology are to reduce systemic toxicity of current anticancer strategies and improve effectiveness. An extremely efficient cancer cell mechanism to avoid and/or reduce the effects of highly cytotoxic drugs is the establishment of an acidic microenvironment, an hallmark of all malignant tumors. The H +-rich milieu that anticancer drugs meet once they get inside the tumor leads to their protonation and neutralization, therefore hindering their access into tumor cells. We have previously shown that proton pump inhibitors (PPI) may efficiently counterattack this tumor advantage leading to a consistent chemosensitization of tumors. In this study, we investigated the effects of PPI in chemosensitizing osteosarcoma. MG-63 and Saos-2 cell lines were used as human osteosarcoma models. Cell proliferation after pretreatment with PPI and subsequent treatment with cisplatin was evaluated by using erythrosin B dye vital staining. Tumour growth was evaluated in xenograft treated with cisplatin after PPI pretreatment. Subsequently, a multi-centre historically controlled trial, was performed to evaluate the activity of a pre-treatment administration of PPIs as chemosensitizers during neoadjuvant chemotherapy based on methotrexate, cisplatin, and adriamycin. Preclinical experiments showed that PPI sensitize both human osteosarcoma cell lines and xenografts to cisplatin. A clinical study subsequently showed that pretreatment with PPI drug esomeprazole leads to an increase in the local effect of chemotherapy, as expressed by percentage of tumor necrosis. This was particularly evident in chondroblastic osteosarcoma, an histological subtype that normally shows a poor histological response. Notably, no significant increase in toxicity was recorded in PPI treated patients. This study provides the first evidence that PPI may be beneficially added to standard regimens in combination to conventional chemotherapy.

Liposomal nanoparticles as a drug delivery vehicle against osteosarcoma

NASA Astrophysics Data System (ADS)

Dhule, Santosh Subhashrao

The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-gamma-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded gamma-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. The second part of this study examines the anti-tumor potential of curcumin and C6 ceramide (C6) against osteosarcoma cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with systems with curcumin alone. Interestingly, C6-curcumin liposomes were found to be less toxic on untransformed human cells in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G 2/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G1 arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G2/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. Using pegylated liposomes to increase the plasma half-life and tagging

ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells

PubMed Central

Valabrega, G; Fagioli, F; Corso, S; Madon, E; Brach del Prever, A; Biasin, E; Linari, A; Aglietta, M; Giordano, S

2003-01-01

Osteosarcoma is the most common malignant bone neoplasia occurring in young patients in the first two decades of life, and represents 20% of all primitive malignant bone tumours. At present, treatment of metastatic osteosarcoma is unsatisfactory. High-dose chemotherapy followed by CD34+ leukapheresis rescue may improve these poor results. Neoplastic cells contaminating the apheresis may, however, contribute to relapse. To identify markers suitable for detecting osteosarcoma cells in aphereses we analysed the expression of bone-specific genes (Bone Sialoprotein (BSP) and Osteocalcin) and oncogenes (Met and ErbB2) in 22 patients with metastatic osteosarcoma and six healthy stem cell donors. The expression of these genes in aphereses of patients affected by metastatic osteosarcoma was assessed by RT–PCR and Southern blot analysis. Met and Osteocalcin proved to be not useful markers since they are positive in aphereses of both patients with metastatic osteosarcoma and healthy stem cell donors. On the contrary, BSP was expressed at significant levels in 85% of patients. Moreover, 18% of patients showed a strong and significantly positive (seven to 16 times higher than healthy stem cell donors) ErbB2 expression. In all positive cases, neoplastic tissue also expressed ErbB2. Our data show that ErbB2 can be a useful marker for tumour contamination in aphereses of patients affected by ErbB2-expressing osteosarcomas and that analysis of Bone Sialoprotein expression can be an alternative useful marker. PMID:12569382

Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells.

PubMed

Pelagalli, Alessandra; Nardelli, Anna; Fontanella, Raffaela; Zannetti, Antonella

2016-07-11

The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1.

Telangiectatic osteosarcoma of the rib: a rare entity and a potential diagnostic pitfall.

PubMed

Saguem, I; Ayadi, L; Kallel, R; Charfi, S; Bahri, I; Gouiaa, N; Sellami-Boudawara, T

2016-12-01

Osteosarcoma (OS) is a common primary malignant tumor of bones that produces osteoid matrix. Telangiectatic osteosarcoma (TOS) is a rare variant of OS. It affects the long bones especially the lower end of femur and the upper ends of tibia and humerus, a distribution similar to the conventional osteosarcoma. The rib involvement is very infrequent. We present a case of TOS of the rib that posed a diagnostic difficulty owing to its unusual location and to its resemblance to giant cell tumor and aneurysmal bone cyst. Correspondence. © Copyright Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology.

Energy stress-induced lncRNA HAND2-AS1 represses HIF1α-mediated energy metabolism and inhibits osteosarcoma progression

PubMed Central

Kang, Yao; Zhu, Xiaojun; Xu, Yanyang; Tang, Qinglian; Huang, Zongwen; Zhao, Zhiqiang; Lu, Jinchang; Song, Guohui; Xu, Huaiyuan; Deng, Chuangzhong; Wang, Jin

2018-01-01

During recent years, long noncoding RNAs (lncRNAs) have been recognized as key regulators in the development and progression of human cancers, however, their roles in osteosarcoma metabolism are still not well understood. The present study aims to investigate the expression profiles and potential modulation of specific lncRNA(s) in osteosarcoma metabolism. The high-throughput Hiseq sequencing was performed to screen for abnormally expressed lncRNAs in osteosarcoma cells cultured under glucose starvation condition, and lncRNA HAND2-AS1 was eventually identified as one that was significantly up-regulated when compared with normal cultured cells. Mechanistic investigations indicated that knockdown of HAND2-AS1 abrogated the energy stress-induced effect on cell apoptosis and proliferation, and promoted osteosarcoma progression. Moreover, knockdown of HAND2-AS1 promoted glucose uptake, lactate production, and the expression level of a serious of enzymes that involved in energy metabolism. Subsequently, RNA pull-down and RNA immuneprecipitation revealed that, upon energy stress, HAND2-AS1 regulated osteosarcoma metabolism through sequestering FBP1 from binding to HIF1α, thereby releasing HIF1α expression and promoting the protein level. Taken together, our integrated approach reveals a regulatory mechanism by lncRNA HAND2-AS1 to control energy metabolism and tumor development in osteosarcoma. Thus, HAND2-AS1 may be a potential biomarker and therapeutic target for the repression of osteosarcoma metabolism. PMID:29637006

Combinatorial Treatment of DNA and Chromatin-Modifying Drugs Cause Cell Death in Human and Canine Osteosarcoma Cell Lines

PubMed Central

Thayanithy, Venugopal; Park, ChangWon; Sarver, Aaron L.; Kartha, Reena V.; Korpela, Derek M.; Graef, Ashley J.; Steer, Clifford J.; Modiano, Jaime F.; Subramanian, Subbaya

2012-01-01

Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS. PMID:22957032

The effects of sulforaphane on canine osteosarcoma proliferation and invasion.

PubMed

Rizzo, V L; Levine, C B; Wakshlag, J J

2017-09-01

Recent evidence in in vitro and in vivo models suggests that sulforaphane (SFN), found in raw cruciferous vegetables, may have utility in chemoprevention, as an antineoplastic agent and as a free radical scavenger. The effects of SFN alone or with doxorubicin on cell viability were examined, as well as cell cycle kinetics, invasion capabilities and apoptosis in three canine osteosarcoma cell line (D17, OS 2.4 and HMPOS). Results showed that SFN could not induce cell death at potentially physiological concentrations (<50 μM), but significantly diminished cell invasion and downregulation of focal adhesion kinase (FAK) signaling. Modest cell cycle changes were observed in each cell line. When doxorubicin was used in conjunction with SFN, there was a protective effect to doxorubicin-induced cytotoxicity in D17 and OS 2.4 cells. Further studies examining SFN as a supplement are warranted, particularly in light of pro-proliferative and cytoprotective properties in canine osteosarcoma. © 2016 John Wiley & Sons Ltd.

Proton pump inhibitor chemosensitization in human osteosarcoma: from the bench to the patients’ bed

PubMed Central

2013-01-01

Background Major goals in translational oncology are to reduce systemic toxicity of current anticancer strategies and improve effectiveness. An extremely efficient cancer cell mechanism to avoid and/or reduce the effects of highly cytotoxic drugs is the establishment of an acidic microenvironment, an hallmark of all malignant tumors. The H + −rich milieu that anticancer drugs meet once they get inside the tumor leads to their protonation and neutralization, therefore hindering their access into tumor cells. We have previously shown that proton pump inhibitors (PPI) may efficiently counterattack this tumor advantage leading to a consistent chemosensitization of tumors. In this study, we investigated the effects of PPI in chemosensitizing osteosarcoma. Method MG-63 and Saos-2 cell lines were used as human osteosarcoma models. Cell proliferation after pretreatment with PPI and subsequent treatment with cisplatin was evaluated by using erythrosin B dye vital staining. Tumour growth was evaluated in xenograft treated with cisplatin after PPI pretreatment. Subsequently, a multi-centre historically controlled trial, was performed to evaluate the activity of a pre-treatment administration of PPIs as chemosensitizers during neoadjuvant chemotherapy based on methotrexate, cisplatin, and adriamycin. Results Preclinical experiments showed that PPI sensitize both human osteosarcoma cell lines and xenografts to cisplatin. A clinical study subsequently showed that pretreatment with PPI drug esomeprazole leads to an increase in the local effect of chemotherapy, as expressed by percentage of tumor necrosis. This was particularly evident in chondroblastic osteosarcoma, an histological subtype that normally shows a poor histological response. Notably, no significant increase in toxicity was recorded in PPI treated patients. Conclusion This study provides the first evidence that PPI may be beneficially added to standard regimens in combination to conventional chemotherapy. PMID

Vitamin D fails to prevent serum starvation- or staurosporine-induced apoptosis in human and rat osteosarcoma-derived cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witasp, Erika; Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm; Gustafsson, Ann-Catrin

2005-05-13

Previous studies have suggested that 1,25(OH){sub 2}D{sub 3}, the active form of vitamin D{sub 3}, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D{sub 3} has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH){sub 2}D{sub 3} induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in thismore » model. Moreover, 1,25(OH){sub 2}D{sub 3} failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D{sub 3}.« less

COX-2 Expression Correlates With Survival in Patients With Osteosarcoma Lung Metastases

PubMed Central

Rodriguez, Nidra I.; Hoots, William Keith; Koshkina, Nadezhda V.; Morales-Arias, Jaime A.; Arndt, Carola A.; Inwards, Carrie Y.; Hawkins, Douglas S.; Munsell, Mark F.; Kleinerman, Eugenie S.

2009-01-01

Summary The purpose of this study was to determine whether a correlation exists between tumor cyclooxygenase (COX)-2 expression and disease-specific survival in patients with osteosarcoma lung metastases. Thirty-six patients diagnosed with osteosarcoma lung metastases between the years 1990 and 2001 were included in this retrospective study. The majority of the patients (72%) presented newly -diagnosed osteosarcoma lung metastases whereas the remaining patients (28%) presented recurrent disease. Clinicopathologic parameters were obtained from patients’ clinical records. Tissue samples were obtained at the time of resection of the lung metastases and stained for COX-2 using immunohistochemistry. Samples were graded according to the intensity of COX-2 staining (grade 0: negative, grade 1: very weak, grade 2: weak, grade 3: moderate, and grade 4: strong). COX-2 staining was correlated with disease-specific survival and clinicopathologic parameters using the Jonckheere-Terpstra and the Kruskal-Wallis tests. All patients with grade 3 or 4 COX-2 expression died of osteosarcoma lung metastases. Ten percent of patients with grade 2 COX-2 expression and 29% of patients with grade 1 expression were alive and free of disease at the last follow-up. By contrast, 60% of the patients with grade 0 COX-2 expression were alive and free of disease at the last follow-up. No association between COX-2 expression and clinicopathologic parameters was found. However, COX-2 expression correlated inversely with disease-specific survival in patients with osteosarcoma lung metastases. Our data indicate that COX-2 expression in metastatic osteosarcoma may have prognostic significance. PMID:18797196

Osteosarcoma cells induce endothelial cell proliferation during neo-angiogenesis.

PubMed

de Nigris, Filomena; Mancini, Francesco Paolo; Schiano, Concetta; Infante, Teresa; Zullo, Alberto; Minucci, Pellegrino Biagio; Al-Omran, Mohammed; Giordano, Antonio; Napoli, Claudio

2013-04-01

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin-dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein-dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary-like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1-dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P < 0.01 vs. control). Finally, the finding that Roscovitine triggers apoptosis in SaOS cells as well as in HAECs by activating caspase-3/7 indicates multiple mechanisms for the potential antitumoral effect of Roscovitine. Present work suggests that Cdk2 and Cdk5 might be pharmacologically accessible targets for both antiangiogenic and antitumor therapy. Copyright © 2012 Wiley Periodicals, Inc.

Distribution and activity levels of matrix metalloproteinase 2 and 9 in canine and feline osteosarcoma.

PubMed

Gebhard, Christiane; Fuchs-Baumgartinger, Andrea; Razzazi-Fazeli, Ebrahim; Miller, Ingrid; Walter, Ingrid

2016-01-01

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma.

Tumoral immune-infiltrate (IF), PD-L1 expression and role of CD8/TIA-1 lymphocytes in localized osteosarcoma patients treated within protocol ISG-OS1.

PubMed

Palmerini, Emanuela; Agostinelli, Claudio; Picci, Piero; Pileri, Stefano; Marafioti, Teresa; Lollini, Pier-Luigi; Scotlandi, Katia; Longhi, Alessandra; Benassi, Maria Serena; Ferrari, Stefano

2017-12-19

We hypothesized that immune-infiltrates were associated with superior survival, and examined a primary osteosarcoma tissue microarrays (TMAs) to test this hypothesis. 129 patients (pts) with localized osteosarcoma treated within protocol ISG-OS1 were included in the study. Clinical characteristics, expression of CD8, CD3, FOXP3, CD20, CD68/CD163 (tumor associated macrophage, TAM), Tia-1 (cytotoxic T cell), CD303 (plasmacytoid dendritic cells: pDC), Arginase-1 (myeloid derived suppressor cells: MDSC), PD-1 on immune-cells (IC), and PD-L1 on tumoral cells (TC) and IC were analysed and correlated with outcome. Most of the cases presented tumor infiltrating lymphocytes (TILs) (CD3+ 90%; CD8+ 86%). Tia-1 was detected in 73% of the samples. PD-L1 expression was found in 14% patients in IC and 0% in TC; 22% showed PD-1 expression in IC.With a median follow-up of 8 years (range 1-13), the 5-year overall survival (5-year OS) was 74% (95% CI 64-85). Univariate analysis showed better 5-year OS for: a) pts with a good histologic response to neoadjuvant chemotherapy (p = 0.0001); b) pts with CD8/Tia1 tumoral infiltrates (p = 0.002); c) pts with normal alkaline phosphatas (sALP) (p = 0.04). After multivariate analysis, histologic response (p = 0.007) and CD8/Tia1 infiltration (p = 0.01) were independently correlated with survival. In the subset of pts with CD8+ infiltrate, worse (p 0.02) OS was observed for PD-L1(IC)+ cases. Our findings support the hypothesis that CD8/Tia1 infiltrate in tumor microenvironment at diagnosis confers superior survival for pts with localized osteosarcoma, while PD-L1 expression is associated with worse survival.

Role of RANK and Akt1 activation in human osteosarcoma progression: A clinicopathological study.

PubMed

Zhu, Jianxi; Liu, Yuwei; Zhu, Yong; Zeng, Min; Xie, Jie; Lei, Pengfei; Li, Kanghua; Hu, Yihe

2017-06-01

The receptor activator of nuclear factor κB (RANK) axis is the fundamental signaling pathway in bone formation as well as bone tumor pathophysiology. The aim of the present study was to evaluate the impact of the expression of RANK and its downstream signaling molecule Akt1 on tumor progression in patients with osteosarcoma. Expression of RANK and Akt1 was examined in 78 human osteosarcoma samples by immunohistochemistry using formalin-fixed samples. Following this, each graded immunohistochemistry result was correlated with clinicopathological parameters and patient survival. In total, 60 osteosarcomas (76.9%) expressed RANK and 58 cases (74.4%) showed expression of Akt1. In addition, expression of RANK was negatively correlated with disease-free survival by Kaplan-Meier analysis. A resistance was observed to chemotherapy in RANK-expressing cases, which was statistically significant (P<0.05). In addition, chemotherapy and staging of the tumor were found to independent factors that have an effect on patient survival (P<0.05). Thus, RANK was identified as a negative prognostic factor of osteosarcoma survival.

Survivin inhibition via EZN-3042 in canine lymphoma and osteosarcoma.

PubMed

Shoeneman, J K; Ehrhart, E J; Charles, J B; Thamm, D H

2016-06-01

Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Survivin, an inhibitor of apoptosis (IAP) family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs with LSA and OS, and canine LSA and OS cell lines express high levels of survivin. In this study, we demonstrate that survivin downregulation in canine LSA and OS cells using a clinically applicable locked nucleic acid antisense oligonucleotide (EZN-3042, Enzon Pharmaceuticals, Piscataway Township, NJ, USA) inhibits growth, induces apoptosis and enhances chemosensitivity in vitro, and inhibits survivin transcription and protein production in orthotopic canine OS xenografts. Our findings strongly suggest that survivin-directed therapies might be effective in treatment of canine LSA and OS and support evaluation of EZN-3042 in dogs with cancer. © 2014 John Wiley & Sons Ltd.

[gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

PubMed

Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

2010-12-01

To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

Distribution and activity levels of matrix metalloproteinase 2 and 9 in canine and feline osteosarcoma

PubMed Central

Gebhard, Christiane; Fuchs-Baumgartinger, Andrea; Razzazi-Fazeli, Ebrahim; Miller, Ingrid; Walter, Ingrid

2016-01-01

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma. PMID:26733734

Prognostic Significance of Serum Alkaline Phosphatase Level in Osteosarcoma: A Meta-Analysis of Published Data.

PubMed

Ren, Hai-Yong; Sun, Ling-Ling; Li, Heng-Yuan; Ye, Zhao-Ming

2015-01-01

Serum alkaline phosphatase (SALP) is commonly elevated in osteosarcoma patients. A number of studies have investigated the prognostic role of SALP level in patients with osteosarcoma but yielded inconsistent results. Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs) and relative risks (RRs) with corresponding confidence intervals (CIs) were calculated to assess the prognostic value of SALP level. Finally, 21 studies comprising 3228 patients were included. Overall, the pooled HRs of SALP suggested that elevated level had an unfavorable impact on osteosarcoma patients' overall survival (OS) (HR = 1.82; 95% CI: 1.61-2.06; p < 0.001) and event-free survival (EFS) (HR = 1.97; 95% CI: 1.61-2.42; p < 0.001). Combined RRs of SALP indicated that elevated level was associated with presence of metastasis at diagnosis (RR = 5.55; 95% CI: 1.61-9.49; p = 0.006). No significantly different results were obtained after stratified by variables of age range, cancer stage, sample size, and geographic region. This meta-analysis demonstrated that high SALP level is significantly associated with poor OS or EFS rate and presence of metastasis at diagnosis. SALP level is a convenient and effective biomarker of prognosis for osteosarcoma.

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B

PubMed Central

2013-01-01

Background Canine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible. Results Through genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors. Conclusions Mapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease. PMID:24330828

PI3K/Akt signaling mediated Hexokinase-2 expression inhibits cell apoptosis and promotes tumor growth in pediatric osteosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhuo, Baobiao; Li, Yuan; Li, Zhengwei

2015-08-21

Accumulating evidence has shown that PI3K/Akt pathway is frequently hyperactivated in osteosarcoma (OS) and contributes to tumor initiation and progression. Altered phenotype of glucose metabolism is a key hallmark of cancer cells including OS. However, the relationship between PI3K/Akt pathway and glucose metabolism in OS remains largely unexplored. In this study, we showed that elevated Hexokinase-2 (HK2) expression, which catalyzes the first essential step of glucose metabolism by conversion of glucose into glucose-6-phosphate, was induced by activated PI3K/Akt signaling. Immunohistochemical analysis showed that HK2 was overexpressed in 83.3% (25/30) specimens detected and was closely correlated with Ki67, a cell proliferationmore » index. Silencing of endogenous HK2 resulted in decreased aerobic glycolysis as demonstrated by reduced glucose consumption and lactate production. Inhibition of PI3K/Akt signaling also suppressed aerobic glycolysis and this effect can be reversed by reintroduction of HK2. Furthermore, knockdown of HK2 led to increased cell apoptosis and reduced ability of colony formation; meanwhile, these effects were blocked by 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor through its actions on hexokinase, indicating that HK2 functions in cell apoptosis and growth were mediated by altered aerobic glycolysis. Taken together, our study reveals a novel relationship between PI3K/Akt signaling and aerobic glycolysis and indicates that PI3K/Akt/HK2 might be potential therapeutic approaches for OS. - Highlights: • PI3K/Akt signaling contributes to elevated expression of HK2 in osteosarcoma. • HK2 inhibits cell apoptosis and promotes tumor growth through enhanced Warburg effect. • Inhibition of glycolysis blocks the oncogenic activity of HK2.« less

The immunotherapy of canine osteosarcoma: a historical and systematic review.

PubMed

Wycislo, K L; Fan, T M

2015-01-01

Osteosarcoma is a malignant mesenchymal neoplasm that accounts for the majority of primary bone tumors in dogs and shares biological and clinical similarities with osteosarcoma in humans. Despite dose intensification with conventional cytotoxic therapies, survival times for dogs and humans diagnosed with high-grade osteosarcoma have not changed in the past 20 years, with the principal cause of mortality being the development of pulmonary metastases. Given the therapeutic plateau reached for delaying metastatic progression with cytotoxic agents, exploration of alterative adjuvant therapies for improving management of osteosarcoma micrometastases is clinically justified. Evidence suggests that osteosarcoma is an immunogenic tumor, and development of immunotherapies for the treatment of microscopic lung metastases might improve long-term outcomes. In this review, the history and foundational knowledge of immune interactions to canine osteosarcoma are highlighted. In parallel, immunotherapeutic strategies that have been explored for the treatment of canine osteosarcoma are summarized. With a greater understanding and awareness for how the immune system might be redirected toward combating osteosarcoma metastases, the rational development of diverse immune strategies for managing osteosarcoma holds substantial promise for transforming the therapeutic landscape and improving disease management in both dogs and human beings. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Hydrogel-PLGA delivery system prolongs 2-methoxyestradiol-mediated anti-tumor effects in osteosarcoma cells.

PubMed

Maran, Avudaiappan; Dadsetan, Mahrokh; Buenz, Colleen M; Shogren, Kristen L; Lu, Lichun; Yaszemski, Michael J

2013-09-01

Osteosarcoma is a bone tumor that affects children and young adults. 2-Methoxyestradiol (2-ME), a naturally occurring estrogen metabolite, kills osteosarcoma cells, but does not affect normal osteoblasts. In order to effectively target osteosarcoma and improve the therapeutic index of the drug 2-ME, we have encapsulated 2-ME in a composite of oligo-(polyethylene glycol) fumarate (OPF) hydrogel and poly (lactic-co-glycolic acid) (PLGA) microspheres and investigated the effect of polymer composition on 2-ME release kinetics and osteosarcoma cell survival. The in vitro study shows that 2-ME can be released in a controlled manner over 21-days. The initial burst releases observed on day 1 were 50% and 32% for OPF and OPF/PLGA composites, respectively. The extended release kinetics show that 100% of the encapsulated 2-ME is released by day 12 from OPF, whereas the OPF/PLGA composites showed a release of 85% on day 21. 2-ME released from the polymers was biologically active and blocked osteosarcoma cell proliferation in vitro. Also, comparison of 2-ME delivery in osteosarcoma cells in culture, shows that direct treatment has no effect after 3 days, whereas polymer-mediated delivery produces anti-tumor effects that could be sustained for 21 days. These findings show that the OPF and PLGA polymeric system may prove to be useful in controlled and sustained delivery of 2-ME and could be further explored in the treatment of osteosarcoma. Copyright © 2012 Wiley Periodicals, Inc.

Prevalence of metastasis at diagnosis of osteosarcoma: an international comparison

PubMed Central

Marko, Tracy A.; Diessner, Brandon J.; Spector, Logan G.

2016-01-01

Background Osteosarcoma is the most common primary malignant bone tumor in many countries, with metastatic disease responsible for most patient deaths. This study compares the prevalence of metastatic osteosarcoma at diagnosis across countries to inform the critical question of whether diagnostic delay or tumor biology drives metastases development prior to diagnosis. Procedure A literature search of the PubMed database was conducted to compare the prevalence of metastatic disease at the time of OS diagnosis between countries. A pooled prevalence with 95% confidence intervals was calculated for each study meeting inclusion criteria. Studies were grouped for analysis based on human development index (HDI) scores. Results Our analysis found an 18% (95% CI: 15%, 20%) average global pooled proportion of metastasis at osteosarcoma diagnosis. The average prevalence of metastasis at diagnosis increased as HDI groupings decreased, with very high HDI, high HDI, and medium/ low HDI groups found to be 15% (95% CI: 13%, 17%), 20% (95% CI: 14%, 28%), and 31% (95% CI: 15%, 52%), respectively. Conclusions Our evidence suggests there is a biological baseline for metastatic OS at diagnosis, which is observed in countries with very high HDI. In countries with medium/ low HDI, where there are more barriers to accessing healthcare, the higher prevalence of metastasis may result from treatment delay or an artificial prevalence inflation due to patients with less severe symptoms not presenting to clinic. Additional research in countries with medium/ low HDI may reveal that earlier detection and treatment could improve patient outcomes in those countries. PMID:26929018

Comparisons of the skeletal locations of putative plutonium-induced osteosarcomas in humans with those in beagle dogs and with naturally occurring tumors in both species.

PubMed

Miller, Scott C; Lloyd, Ray D; Bruenger, Fred W; Krahenbuhl, Melinda P; Polig, Erich; Romanov, Sergey A

2003-11-01

Osteosarcomas occur from exposures to bone-seeking, alpha-particle-emitting isotopes, particularly plutonium. The skeletal distribution of putative 239Pu-induced osteosarcomas reported in Mayak Metallurgical and Radiochemical Plutonium Plant workers is compared with those observed in canine studies, and these are compared with distributions of naturally occurring osteosarcomas in both species. In the Mayak workers, 29% and 71% of the osteosarcomas were in the peripheral and central skeleton, respectively, with the spine having the most tumors (36%). An almost identical distribution of plutonium-induced osteosarcomas was reported for dogs injected with 239Pu as young adults. This distribution of osteosarcomas is quite different from the distributions of naturally occurring osteosarcomas for both species. In the Cooperative Osteosarcoma Study Group in humans (1,736 osteosarcomas from all ages), over 91% of the tumors occurred in the peripheral skeleton. In the Mayo Clinic group of older individuals (>40 years old), over 60% of the osteosarcomas appeared in the peripheral skeleton. The distribution of naturally occurring osteosarcomas in the canine is similar to that in the adult human. The similarities of the distributions of plutonium-associated osteosarcomas in the Mayak workers with those found in experimental studies suggest that many of the reported osteosarcomas may have been associated with plutonium exposures. These results also support the experimental paradigm that plutonium osteosarcomas have a preference for well vascularized cancellous bone sites. These sites have a greater initial deposition of plutonium, but also greater turnover due to elevated bone remodeling rates.

Correlation between E-cadherin-regulated cell adhesion and human osteosarcoma MG-63 cell anoikis.

PubMed

Lin, Ding-Sheng; Cai, Le-Yi; Ding, Jian; Gao, Wei-Yang

2014-01-01

The aim of this study was to investigate the relationship between cell adhesion and anoikis evasion among human osteosarcoma cells (MG-63), and to further study the molecular mechanisms. Human osteosarcoma cells (MG-63) were assessed for apoptosis, and caspase-3, E-cadherin and β-catenin expression in EDTA and control non-EDTA groups. MG-63 cells were predominantly aggregated when in suspension, and the suspended cells were more dispersed in the EDTA group. Following culture in suspension for 24 h, 48 h, or 72 h, the rates of apoptosis were 34.88%±3.64%, 59.3%±7.22% and 78.5%±5.21% in the experimental group and 7.34%±2.13%, 14.7%±3.69%, and 21.4%±3.60% in the control group, respectively. Caspase-3 expression progressively increased and E-cadherin and β-catenin were decreased in the experimental group, whereas there was no change in the control group. MG-63 cells could avoid anoikis through cell adhesion, and E-cadherin might play a role in this process.

Telangiectatic osteosarcoma.

PubMed

Sangle, Nikhil A; Layfield, Lester J

2012-05-01

Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Telangiectatic osteosarcoma is an unusual variant of osteosarcoma, forming 3% to 10% of all osteosarcomas. Radiographically, these tumors appear as purely lytic destructive lesions located in the metaphyses of long bones. The location and x-ray appearance of telangiectatic osteosarcomas are reminiscent of an aneurysmal bone cyst and can test the acumen of a diagnostic radiologist. Distinguishing between the two entities microscopically can also be quite challenging. Telangiectatic osteosarcoma shows dilated blood-filled spaces lined or traversed by septa containing atypical stromal cells, with or without production of a lacelike osteoid matrix. This review highlights the diagnostic features of telangiectatic osteosarcoma and discusses differential diagnostic considerations, treatment options, and prognostic implications.

Zinc finger X-chromosomal protein (ZFX) promotes solid agar colony growth of osteosarcoma cells.

PubMed

Jiang, Rui; Wang, Jin-cheng; Sun, Mei; Zhang, Xing-yi; Wu, Han

2012-01-01

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.

Development of a Model System to Evaluate Local Recurrence in Osteosarcoma and Assessment of the Effects of Bone Morphogenetic Protein-2.

PubMed

Geller, David S; Singh, Michael Y; Zhang, Wendong; Gill, Jonathan; Roth, Michael E; Kim, Mimi Y; Xie, Xianhong; Singh, Christopher K; Dorfman, Howard D; Villanueva-Siles, Esperanza; Park, Amy; Piperdi, Sajida; Gorlick, Richard

2015-07-01

It is increasingly relevant to better define what constitutes an adequate surgical margin in an effort to improve reconstructive longevity and functional outcomes following osteosarcoma surgery. In addition, nonunion remains a challenging problem in some patients following allograft reconstruction. Bone morphogenetic protein-2 (BMP-2) could enhance osseous union, but has been historically avoided due to concerns that it may promote tumor recurrence. An orthotopic xenograft murine model was utilized to describe the natural temporal course of osteosarcoma growth. Tumors were treated either with surgery alone, surgery and single-agent chemotherapy, or surgery and dual-agent chemotherapy to assess the relationship between surgical margin and local recurrence. The effect of BMP-2 on local recurrence was similarly assessed. Osteosarcoma tumor growth was categorized into reproducible phases. Margins greater than 997 μm resulted in local control following surgery alone. Margins greater than 36 μm resulted in local control following surgery and single-agent chemotherapy. Margins greater than 12 μm resulted in local control following surgery and dual-agent chemotherapy. The application of exogenous BMP-2 does not confer an increased risk of local recurrence. This model reliably reproduces the clinical, radiographic, and surgical conditions encountered in human osteosarcoma. It successfully incorporates relevant chemotherapy, further paralleling the human experience. Surgical margins required to achieve local control in osteosarcoma can be reduced using single-agent chemotherapy and further decreased using dual-agent chemotherapy. The application of BMP-2 does not increase local recurrence in this model. ©2014 American Association for Cancer Research.

PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

PubMed

Hirahata, Mio; Osaki, Mitsuhiko; Kanda, Yusuke; Sugimoto, Yui; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Kawai, Akira; Ito, Hisao; Ochiya, Takahiro; Okada, Futoshi

2016-05-01

Despite recent improvements in the therapy for osteosarcoma, 30-40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR-143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR-143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor-1 (PAI-1) as a direct target gene of miR-143. To determine the role of PAI-1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI-1 expression. An in vitro study showed that downregulation of PAI-1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI-1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA-injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI-1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase-13 (MMP-13), which is also a target gene of miR-143 and a proteolytic enzyme that regulates tumor-induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR-143 expressing cases showed poor expression of PAI-1 in the primary tumor cells. All such cases belonged to the lung metastasis-negative group. Moreover, the frequency of lung metastasis-positive cases was significantly higher in PAI-1 and MMP-13 double-positive cases than in PAI-1 or MMP-13 single-positive or double-negative cases (P < 0.05). These results indicated that PAI-1, a target gene of miR-143, regulates invasion and lung metastasis via enhancement of MMP-13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in

Structuring osteosarcoma knowledge: an osteosarcoma-gene association database based on literature mining and manual annotation.

PubMed

Poos, Kathrin; Smida, Jan; Nathrath, Michaela; Maugg, Doris; Baumhoer, Daniel; Neumann, Anna; Korsching, Eberhard

2014-01-01

Osteosarcoma (OS) is the most common primary bone cancer exhibiting high genomic instability. This genomic instability affects multiple genes and microRNAs to a varying extent depending on patient and tumor subtype. Massive research is ongoing to identify genes including their gene products and microRNAs that correlate with disease progression and might be used as biomarkers for OS. However, the genomic complexity hampers the identification of reliable biomarkers. Up to now, clinico-pathological factors are the key determinants to guide prognosis and therapeutic treatments. Each day, new studies about OS are published and complicate the acquisition of information to support biomarker discovery and therapeutic improvements. Thus, it is necessary to provide a structured and annotated view on the current OS knowledge that is quick and easily accessible to researchers of the field. Therefore, we developed a publicly available database and Web interface that serves as resource for OS-associated genes and microRNAs. Genes and microRNAs were collected using an automated dictionary-based gene recognition procedure followed by manual review and annotation by experts of the field. In total, 911 genes and 81 microRNAs related to 1331 PubMed abstracts were collected (last update: 29 October 2013). Users can evaluate genes and microRNAs according to their potential prognostic and therapeutic impact, the experimental procedures, the sample types, the biological contexts and microRNA target gene interactions. Additionally, a pathway enrichment analysis of the collected genes highlights different aspects of OS progression. OS requires pathways commonly deregulated in cancer but also features OS-specific alterations like deregulated osteoclast differentiation. To our knowledge, this is the first effort of an OS database containing manual reviewed and annotated up-to-date OS knowledge. It might be a useful resource especially for the bone tumor research community, as specific

Structuring osteosarcoma knowledge: an osteosarcoma-gene association database based on literature mining and manual annotation

PubMed Central

Poos, Kathrin; Smida, Jan; Nathrath, Michaela; Maugg, Doris; Baumhoer, Daniel; Neumann, Anna; Korsching, Eberhard

2014-01-01

Osteosarcoma (OS) is the most common primary bone cancer exhibiting high genomic instability. This genomic instability affects multiple genes and microRNAs to a varying extent depending on patient and tumor subtype. Massive research is ongoing to identify genes including their gene products and microRNAs that correlate with disease progression and might be used as biomarkers for OS. However, the genomic complexity hampers the identification of reliable biomarkers. Up to now, clinico-pathological factors are the key determinants to guide prognosis and therapeutic treatments. Each day, new studies about OS are published and complicate the acquisition of information to support biomarker discovery and therapeutic improvements. Thus, it is necessary to provide a structured and annotated view on the current OS knowledge that is quick and easily accessible to researchers of the field. Therefore, we developed a publicly available database and Web interface that serves as resource for OS-associated genes and microRNAs. Genes and microRNAs were collected using an automated dictionary-based gene recognition procedure followed by manual review and annotation by experts of the field. In total, 911 genes and 81 microRNAs related to 1331 PubMed abstracts were collected (last update: 29 October 2013). Users can evaluate genes and microRNAs according to their potential prognostic and therapeutic impact, the experimental procedures, the sample types, the biological contexts and microRNA target gene interactions. Additionally, a pathway enrichment analysis of the collected genes highlights different aspects of OS progression. OS requires pathways commonly deregulated in cancer but also features OS-specific alterations like deregulated osteoclast differentiation. To our knowledge, this is the first effort of an OS database containing manual reviewed and annotated up-to-date OS knowledge. It might be a useful resource especially for the bone tumor research community, as specific

Theranostic Profiling for Actionable Aberrations in Advanced High Risk Osteosarcoma with Aggressive Biology Reveals High Molecular Diversity: The Human Fingerprint Hypothesis.

PubMed

Egas-Bejar, Daniela; Anderson, Pete M; Agarwal, Rishi; Corrales-Medina, Fernando; Devarajan, Eswaran; Huh, Winston W; Brown, Robert E; Subbiah, Vivek

2014-03-12

The survival of patients with advanced osteosarcoma is poor with limited therapeutic options. There is an urgent need for new targeted therapies based on biomarkers. Recently, theranostic molecular profiling services for cancer patients by CLIA-certified commercial companies as well as in-house profiling in academic medical centers have expanded exponentially. We evaluated molecular profiles of patients with advanced osteosarcoma whose tumor tissue had been analyzed by one of the following methods: 1. 182-gene next-generation exome sequencing (Foundation Medicine, Boston, MA), 2. Immunohistochemistry (IHC)/PCR-based panel (CARIS Target Now, Irving, Tx), 3.Comparative genome hybridization (Oncopath, San Antonio, TX). 4. Single-gene PCR assays, PTEN IHC (MDACC CLIA), 5. UT Houston morphoproteomics (Houston, TX). The most common actionable aberrations occur in the PI3K/PTEN/mTOR pathway. No patterns in genomic alterations beyond the above are readily identifiable, and suggest both high molecular diversity in osteosarcoma and the need for more analyses to define distinct subgroups of osteosarcoma defined by genomic alterations. Based on our preliminary observations we hypothesize that the biology of aggressive and the metastatic phenotype osteosarcoma at the molecular level is similar to human fingerprints, in that no two tumors are identical. Further large scale analyses of osteosarcoma samples are warranted to test this hypothesis.

Expression and function of survivin in canine osteosarcoma.

PubMed

Shoeneman, Jenette K; Ehrhart, E J; Eickhoff, Jens C; Charles, J B; Powers, Barbara E; Thamm, Douglas H

2012-01-01

Osteosarcoma has a high mortality rate and remains in need of more effective therapeutic approaches. Survivin is an inhibitor of apoptosis family member protein that blocks apoptosis and drives proliferation in human cancer cells where it is commonly elevated. In this study, we illustrate the superiority of a canine osteosarcoma model as a translational tool for evaluating survivin-directed therapies, owing to the striking similarities in gross and microscopic appearance, biologic behavior, gene expression, and signaling pathway alterations. Elevated survivin expression in primary canine osteosarcoma tissue correlated with increased histologic grade and mitotic index and a decreased disease-free interval (DFI). Survivin attenuation in canine osteosarcoma cells inhibited cell-cycle progression, increased apoptosis, mitotic arrest, and chemosensitivity, and cooperated with chemotherapy to significantly improve in vivo tumor control. Our findings illustrate the utility of a canine system to more accurately model human osteosarcoma and strongly suggest that survivin-directed therapies might be highly effective in its treatment. ©2011 AACR.

TEAD1 mediates the oncogenic activities of Hippo-YAP1 signaling in osteosarcoma.

PubMed

Chai, Jiwei; Xu, Shijie; Guo, Fengbo

2017-06-24

Hippo signaling pathway is an evolutionarily conserved developmental network that governs the downstream transcriptional co-activators, YAP and TAZ, which bind to and activate the output of TEADs that responsible for cell proliferation, apoptosis, and stem cell self renewal. Emerging evidence has shown the tumor suppressor properties of Hippo signaling. However, limited knowledge is available concerning the downstream transcription factors of Hippo pathway in osteosarcoma (OS). In this study, we demonstrated that TEAD1 was the major transcription factor of Hippo signaling pathway in OS. Genetic silencing of TEAD1 suppressed multiple malignant phenotypes of OS cells including cell proliferation, apoptosis resistance, and invasive potential. Mechanistically, we showed that TEAD1 largely exerted its transcriptional control of its functional targets, PTGS2 and CYR61. Collectively, this work identifies the YAP1/TEAD1 complex as the representative dysregulated profile of Hippo signaling in OS and provides proof-of-principle that targeting TEAD1 may be a therapeutic strategy of osteosarcoma. Copyright © 2017. Published by Elsevier Inc.

Minichromosome maintenance protein 2 and 3 promote osteosarcoma progression via DHX9 and predict poor patient prognosis

PubMed Central

Cheng, Dong-dong; Zhang, Hui-zhen; Yuan, Jun-qing; Li, Shi-jie; Yang, Qing-cheng; Fan, Cun-yi

2017-01-01

A label free quantitative proteomic approach (SWATH™ experiment) was performed to identify tumor-associated nuclear proteins that are differentially expressed between osteosarcoma cells and osteoblast cells. By functional screening, minichromosome maintenance protein 2 (MCM2) and minichromosome maintenance protein 3 (MCM3) were found to be related to osteosarcoma cell growth. Here, we show that knockdown of MCM2 or MCM3 inhibits osteosarcoma growth in vitro and in vivo. In co-immunoprecipitation and co-localization experiments, MCM2 and MCM3 were found to interact with DExH-box helicase 9 (DHX9) in osteosarcoma cells. A rescue study showed that the decreased growth of osteosarcoma cells by MCM2 or MCM3 knockdown was reversed by DHX9 overexpression, indicating that MCM2 and MCM3 activity was DHX9-dependent. In addition, the depletion of DHX9 hindered osteosarcoma cell proliferation. Notably, MCM2 and MCM3 expression levels were positively correlated with the DHX9 expression level in tumor samples and were associated with a poor prognosis in patients with osteosarcoma. Taken together, these results suggest that the MCM2/MCM3–DHX9 axis has an important role in osteosarcoma progression. PMID:28460433

Prognostic Significance of Serum Alkaline Phosphatase Level in Osteosarcoma: A Meta-Analysis of Published Data

PubMed Central

Ren, Hai-Yong; Sun, Ling-Ling; Li, Heng-Yuan; Ye, Zhao-Ming

2015-01-01

Background. Serum alkaline phosphatase (SALP) is commonly elevated in osteosarcoma patients. A number of studies have investigated the prognostic role of SALP level in patients with osteosarcoma but yielded inconsistent results. Method. Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs) and relative risks (RRs) with corresponding confidence intervals (CIs) were calculated to assess the prognostic value of SALP level. Results. Finally, 21 studies comprising 3228 patients were included. Overall, the pooled HRs of SALP suggested that elevated level had an unfavorable impact on osteosarcoma patients' overall survival (OS) (HR = 1.82; 95% CI: 1.61–2.06; p < 0.001) and event-free survival (EFS) (HR = 1.97; 95% CI: 1.61–2.42; p < 0.001). Combined RRs of SALP indicated that elevated level was associated with presence of metastasis at diagnosis (RR = 5.55; 95% CI: 1.61–9.49; p = 0.006). No significantly different results were obtained after stratified by variables of age range, cancer stage, sample size, and geographic region. Conclusion. This meta-analysis demonstrated that high SALP level is significantly associated with poor OS or EFS rate and presence of metastasis at diagnosis. SALP level is a convenient and effective biomarker of prognosis for osteosarcoma. PMID:26618165

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

PubMed

Blattmann, Claudia; Thiemann, Markus; Stenzinger, Albrecht; Roth, Eva K; Dittmar, Anne; Witt, Hendrik; Lehner, Burkhard; Renker, Eva; Jugold, Manfred; Eichwald, Viktoria; Weichert, Wilko; Huber, Peter E; Kulozik, Andreas E

2015-04-30

Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Histologic prognosticators in feline osteosarcoma: a comparison with phenotypically similar canine osteosarcoma.

PubMed

Dimopoulou, Maria; Kirpensteijn, Jolle; Moens, Hester; Kik, Marja

2008-07-01

To investigate the histologic characteristics of feline osteosarcoma (OS) and compare the histologic data with phenotypically comparable canine OS. The effects of histologic and clinical variables on survival statistics were evaluated. Retrospective study. Cats (n=62) and dogs (22). Medical records of 62 cats with OS were reviewed for clinically relevant data. Clinical outcome was obtained by telephone interview. Histologic characteristics of OS were classified using a standardized grading system. Histologic characteristics in 22 feline skeletal OS were compared with 22 canine skeletal OS of identical location and subtype. Prognostic variables for clinical outcome were determined using multivariate analysis. Feline OS was characterized by moderate to abundant cellular pleomorphism, low mitotic index, small to moderate amounts of matrix, high cellularity, and a moderate amount of necrosis. There was no significant difference between histologic variables in feline and canine OS. Histologic grade, surgery, and mitotic index significantly influenced clinical outcome as determined by multivariate analysis. Tumor invasion into vessels was not identified as a significant prognosticator. Feline and canine skeletal OS have similar histologic but different prognostic characteristics. Prognosis for cats with OS is related to histologic grade and mitotic index of the tumor.

Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression.

PubMed

Ahmed, Nabil; Salsman, Vita S; Yvon, Eric; Louis, Chrystal U; Perlaky, Laszlo; Wels, Winfried S; Dishop, Meghan K; Kleinerman, Eugenie E; Pule, Martin; Rooney, Cliona M; Heslop, Helen E; Gottschalk, Stephen

2009-10-01

Human epidermal growth factor receptor 2 (HER2) is expressed by the majority of human osteosarcomas and is a risk factor for poor outcome. Unlike breast cancer, osteosarcoma cells express HER2 at too low, a level for patients to benefit from HER2 monoclonal antibodies. We reasoned that this limitation might be overcome by genetically modifying T cells with HER2-specific chimeric antigen receptors (CARs), because even a low frequency of receptor engagement could be sufficient to induce effector cell killing of the tumor. HER2-specific T cells were generated by retroviral transduction with a HER2-specific CAR containing a CD28.zeta signaling domain. HER2-specific T cells recognized HER2-positive osteosarcoma cells as judged by their ability to proliferate, produce immunostimulatory T helper 1 cytokines, and kill HER2-positive osteosarcoma cell lines in vitro. The adoptive transfer of HER2-specific T cells caused regression of established osteosarcoma xenografts in locoregional as well as metastatic mouse models. In contrast, delivery of nontransduced (NT) T cells did not change the tumor growth pattern. Genetic modification of T cells with CARs specific for target antigens, expressed at too low a level to be effectively recognized by monoclonal antibodies, may allow immunotherapy to be more broadly applicable for human cancer therapy.

Effect of bevacizumab on angiogenesis and growth of canine osteosarcoma cells xenografted in athymic mice.

PubMed

Scharf, Valery F; Farese, James P; Coomer, Alastair R; Milner, Rowan J; Taylor, David P; Salute, Marc E; Chang, Myron N; Neal, Dan; Siemann, Dietmar W

2013-05-01

Objective-To investigate the effects of bevacizumab, a human monoclonal antibody against vascular endothelial growth factor, on the angiogenesis and growth of canine osteosarcoma cells xenografted in mice. Animals-27 athymic nude mice. Procedures-To each mouse, highly metastasizing parent osteosarcoma cells of canine origin were injected into the left gastrocnemius muscle. Each mouse was then randomly allocated to 1 of 3 treatment groups: high-dose bevacizumab (4 mg/kg, IP), low-dose bevacizumab (2 mg/kg, IP), or control (no treatment). Tumor growth (the number of days required for the tumor to grow from 8 to 13 mm), vasculature, histomorphology, necrosis, and pulmonary metastasis were evaluated. Results-Mice in the high-dose bevacizumab group had significantly delayed tumor growth (mean ± SD, 13.4 ± 3.8 days; range, 9 to 21 days), compared with that for mice in the low-dose bevacizumab group (mean ± SD, 9.4 ± 1.5 days; range, 7 to 11 days) or control group (mean ± SD, 7. 2 ± 1.5 days; range, 4 to 9 days). Mice in the low-dose bevacizumab group also had significantly delayed tumor growth, compared with that for mice in the control group. Conclusions and Clinical Relevance-Results indicated that bevacizumab inhibited growth of canine osteosarcoma cells xenografted in mice, which suggested that vascular endothelial growth factor inhibitors may be clinically useful for the treatment of osteosarcoma in dogs. Impact for Human Medicine-Canine osteosarcoma is used as a research model for human osteosarcoma; therefore, bevacizumab may be clinically beneficial for the treatment of osteosarcoma in humans.

Overexpression of EZH2 is associated with the poor prognosis in osteosarcoma and function analysis indicates a therapeutic potential

PubMed Central

Sun, Ranran; Shen, Jacson; Gao, Yan; Zhou, Yubing; Yu, Zujiang; Hornicek, Francis; Kan, Quancheng; Duan, Zhenfeng

2016-01-01

Osteosarcoma is a primary malignant bone tumor that has a poor prognosis due to local recurrence, metastasis, and chemotherapy resistance. Therefore, there is an urgent need to develop novel potential therapeutic targets for osteosarcoma. Enhancer of zeste homologue 2 (EZH2) is a member of the polycomb group of proteins, which has important functions in epigenetic silencing and cell cycle regulation. Overexpression of EZH2 has been found in several malignancies, however, its expression and the role of EZH2 in osteosarcoma is largely unknown. In this study, we examined EZH2 expression by immunohistochemistry in a large series of osteosarcoma tissues in association with tumor characteristics and patient outcomes. EZH2 expression was also analyzed in a microarray dataset of osteosarcoma. Results showed that higher expression of EZH2 was significantly associated with more aggressive tumor behavior and poor patient outcomes of osteosarcoma. We subsequently investigated the functional and therapeutic relevance of EZH2 as a target in osteosarcoma. Immunohistochemical analysis indicated that EZH2 expression was significantly associated with more aggressive tumor behavior and poorer patient outcomes of osteosarcoma. EZH2 silencing by siRNA inhibited osteosarcoma cell growth, proliferation, migration, and invasion. Moreover, suppression of EZH2 attenuated cancer stem cell functions. Similar results were observed in osteosarcoma cells treated with EZH2 specific inhibitor 3-deazaneplanocin A (DZNep), which exhausted cellular levels of EZH2. These results suggest that EZH2 is critical for the growth and metastasis of osteosarcoma, and an epigenetic therapy that pharmacologically targets EZH2 via specific inhibitors may constitute a novel approach to the treatment of osteosarcoma. PMID:27223261

Molecular subtypes of osteosarcoma identified by reducing tumor heterogeneity through an interspecies comparative approach

PubMed Central

Scott, Milcah C.; Sarver, Aaron L.; Gavin, Katherine J.; Thayanithy, Venugopal; Getzy, David M.; Newman, Robert A.; Cutter, Gary R.; Lindblad-Toh, Kerstin; Kisseberth, William C.; Hunter, Lawrence E.; Subramanian, Subbaya; Breen, Matthew; Modiano, Jaime F.

2011-01-01

The heterogeneous and chaotic nature of osteosarcoma has confounded accurate molecular classification, prognosis, and prediction for this tumor. The occurrence of spontaneous osteosarcoma is largely confined to humans and dogs. While the clinical features are remarkably similar in both species, the organization of dogs into defined breeds provides a more homogeneous genetic background that may increase the likelihood to uncover molecular subtypes for this complex disease. We thus hypothesized that molecular profiles derived from canine osteosarcoma would aid in molecular subclassification of this disease when applied to humans. To test the hypothesis, we performed genome wide gene expression profiling in a cohort of dogs with osteosarcoma, primarily from high-risk breeds. To further reduce inter-sample heterogeneity, we assessed tumor-intrinsic properties through use of an extensive panel of osteosarcoma-derived cell lines. We observed strong differential gene expression that segregated samples into two groups with differential survival probabilities. Groupings were characterized by the inversely correlated expression of genes associated with G2/M transition and DNA damage checkpoint and microenvironment-interaction categories. This signature was preserved in data from whole tumor samples of three independent dog osteosarcoma cohorts, with stratification into the two expected groups. Significantly, this restricted signature partially overlapped a previously defined, predictive signature for soft tissue sarcomas, and it unmasked orthologous molecular subtypes and their corresponding natural histories in five independent data sets from human patients with osteosarcoma. Our results indicate that the narrower genetic diversity of dogs can be utilized to group complex human osteosarcoma into biologically and clinically relevant molecular subtypes. This in turn may enhance prognosis and prediction, and identify relevant therapeutic targets. PMID:21621658

[Experimental research in vitro of TK/GCV system for osteosarcoma MG-63 cell damage].

PubMed

Zhang, Hua-Dong; Lu, Zhi; Feng, Yi; Liu, Xiao-Li; Hou, Hui-Ming

2014-03-01

To study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects. Liposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group. The TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P < 0.05), and in the experimental group 2 that of the 1/10 and 1/7 of concentration ratio of mixed culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased. The experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

Polyoxometalates as antitumor agents: Bioactivity of a new polyoxometalate with copper on a human osteosarcoma model.

PubMed

León, I E; Porro, V; Astrada, S; Egusquiza, M G; Cabello, C I; Bollati-Fogolin, M; Etcheverry, S B

2014-10-05

Polyoxometalates (POMs) are early transition metal oxygen anion clusters. They display interesting biological effects mainly related to their antiviral and antitumor properties. On the other hand, copper compounds also show different biological and pharmacological effects in cell culture and in animal models. We report herein for the first time, a detailed study of the mechanisms of action of a copper(II) compound of the group of HPOMs with the formula K7Na3[Cu4(H2O)2(PW9034)2]20H2O (PW9Cu), in a model of human osteosarcoma derived cell line, MG-63. The compound inhibited selectively the viability of the osteosarcoma cells in the range of 25-100μM (p<0.01). Besides, we have clearly shown a more deleterious action of PW9Cu on tumor osteoblasts than in normal cells. Cytotoxicity studies also showed deleterious effects for PW9Cu. The increment of reactive oxygen species (ROS) and the decrease of the GSH/GSSG ratio were involved in the antiproliferative effects of PW9Cu. Moreover, the compound caused cell cycle arrest in G2 phase, triggering apoptosis as determined by flow cytometry. As a whole, these results showed the main mechanisms of the deleterious effects of PW9Cu in the osteosarcoma cell line MG-63, demonstrating that this compound is a promissory agent for cancer treatments. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Canine tumor cross-species genomics uncovers targets linked to osteosarcoma progression

PubMed Central

2009-01-01

Background Pulmonary metastasis continues to be the most common cause of death in osteosarcoma. Indeed, the 5-year survival for newly diagnosed osteosarcoma patients has not significantly changed in over 20 years. Further understanding of the mechanisms of metastasis and resistance for this aggressive pediatric cancer is necessary. Pet dogs naturally develop osteosarcoma providing a novel opportunity to model metastasis development and progression. Given the accelerated biology of canine osteosarcoma, we hypothesized that a direct comparison of canine and pediatric osteosarcoma expression profiles may help identify novel metastasis-associated tumor targets that have been missed through the study of the human cancer alone. Results Using parallel oligonucleotide array platforms, shared orthologues between species were identified and normalized. The osteosarcoma expression signatures could not distinguish the canine and human diseases by hierarchical clustering. Cross-species target mining identified two genes, interleukin-8 (IL-8) and solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3), which were uniformly expressed in dog but not in all pediatric osteosarcoma patient samples. Expression of these genes in an independent population of pediatric osteosarcoma patients was associated with poor outcome (p = 0.020 and p = 0.026, respectively). Validation of IL-8 and SLC1A3 protein expression in pediatric osteosarcoma tissues further supported the potential value of these novel targets. Ongoing evaluation will validate the biological significance of these targets and their associated pathways. Conclusions Collectively, these data support the strong similarities between human and canine osteosarcoma and underline the opportunities provided by a comparative oncology approach as a means to improve our understanding of cancer biology and therapies. PMID:20028558

Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bin Hafeez, Bilal; Ahmed, Salahuddin; Wang, Naizhen

2006-10-01

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-{kappa}B (NF-{kappa}B) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20-60 {mu}g/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-{kappa}B/p65 and lowering of NF-{kappa}B/p65 and p50 levels in the cytoplasm and nucleus. GTPmore » treatment of cells reduced I{kappa}B-{alpha} phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-{alpha} and IKK-{beta}, the upstream kinases that phosphorylate I{kappa}B-{alpha}. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-{kappa}B.« less

Expression of the Nrf2 and Keap1 proteins and their clinical significance in osteosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jihong, E-mail: zhangjihong63@163.com; Wang, Xiaojuan, E-mail: yangjian142@163.com; Wu, Wuzhou, E-mail: jiangchunli68@163.com

Objective: To investigate the expression and clinical significance of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in osteosarcoma tissue. Methods: The data of 102 osteosarcoma patients who underwent surgical treatment at our hospital from June 2000 to March 2009 were collected. The expression levels of the Nrf2 and Keap1 proteins in osteosarcoma tissue and normal peritumour tissues were detected by immunohistochemistry, and the relationship between the expression level and the clinical and pathological features as well as the prognosis was explored. Results: The nuclear expression rate of Nrf2 was 77.5% in osteosarcoma tissue, which wasmore » significantly higher than the rate in normal peritumour bone tissue (9.8%) (P < 0.05). The expression rate of the Keap1 protein in osteosarcoma tissue was 13.7%, which was significantly lower than the rate in normal peritumour tissue (80.4%). In addition, Nrf2/Keap1 expression was unrelated to patient gender and age, tumour site, and histological type and was related to metastasis and patient response to chemotherapy (P < 0.05). The five-year survival rate was significantly lower in patients with positive Nrf2 expression than in those with negative Nrf2 expression (p = 0.023), and it was significantly higher in patients with positive Keap1 expression than in those with negative Keap1 expression (P = 0.018). Conclusion: The expression of Nrf2-Keap1 is abnormal in osteosarcoma tissue and shows significant clinical relevance for determining the prognosis of osteosarcoma.« less

EFEMP1 promotes the migration and invasion of osteosarcoma via MMP-2 with induction by AEG-1 via NF-κB signaling pathway

PubMed Central

Ke, Zun-Fu; Luo, Can-Jiao; Lin, Zhong-Wei; Wang, Fen; Zhang, Yuan-Qi; Wang, Lian-Tang

2015-01-01

The role of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in osteosarcoma remains unknown. Then applying EFEMP1 siRNA, plasmids transfection and adding purified EFEMP1 protein in human osteosarcoma cell lines, and using immunohistochemistry on 113 osteosarcoma tissues, demonstrated that EFEMP1 was a poor prognostic indicator of osteosarcoma; EFEMP1 was specifically upregulated in osteosarcoma and associated with invasion and metastasis in vitro and in vivo. At the same time, we found a direct regulatory effect of EFEMP1 on MMP-2. Moreover, we firstly found the marked induction of EFEMP1 by oncogenic AEG-1. And EFEMP1 expression was inhibited by the selective inhibitor of NF-κB (PDTC) in osteosarcoma cells. Then we thought that NF-κB pathways might be one of the effective ways which EFEMP1 was induced by AEG-1. Thus, we suggested that EFEMP1 played a part as the mediator between AEG-1 and MMP-2. And NF-κB signaling pathway played an important role in this process. In summary, EFEMP1 was associated with invasion, metastasis and poor prognosis of osteosarcoma patients. EFEMP1 might indirectly enhance the expression of MMP-2, providing a potential explanation for the role of AEG-1 in metastasis. NF-κB pathways might be one of the effective ways which EFEMP1 was induced by AEG-1. PMID:25987128

IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling

PubMed Central

Lu, Jinchang; Song, Guohui; Tang, Qinglian; Zou, Changye; Han, Feng; Zhao, Zhiqiang; Yong, Bicheng; Yin, Junqiang; Xu, Huaiyuan; Xie, Xianbiao; Kang, Tiebang; Lam, YingLee; Yang, Huiling; Shen, Jingnan; Wang, Jin

2015-01-01

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis–free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis. PMID:25822025

Effectiveness of nanoencapsulated methotrexate against osteosarcoma cells: in vitro cytotoxicity under dynamic conditions.

PubMed

Mitxelena-Iribarren, O; Hisey, C L; Errazquin-Irigoyen, M; González-Fernández, Y; Imbuluzqueta, E; Mujika, M; Blanco-Prieto, M J; Arana, S

2017-06-01

Cancer is a leading cause of mortality in the world, with osteosarcoma being one of the most common types among children between 1 and 14 years old. Current treatments including preoperative chemotherapy, surgery and postoperative chemotherapy produce several side effects with limited effectiveness. The use of lipid nanoparticles as biodegradable shells for controlled drug delivery shows promise as a more effective and targeted tumor treatment. However, in vitro validation of these vehicles is limited due to fluid stagnation in current techniques, in which nanoparticles sediment onto the bottom of the wells killing the cells by asphyxiation. In the current series of experiments, results obtained with methotrexate-lipid nanoparticles under dynamic assay conditions are presented as a promising alternative to current free drug based therapies. Effects on the viability of the U-2 OS osteosarcoma cell line of recirculation of cell media, free methotrexate and blank and methotrexate containing lipid nanoparticles in a 11 μM concentration were successfully assessed. In addition, several designs for the microfluidic platform used were simulated using COMSOL-Multiphysics, optimized devices were fabricated using soft-lithography and simulated parameters were experimentally validated. Nanoparticles did not sediment to the bottom of the platform, demonstrating the effectiveness of the proposed system. Moreover, encapsulated methotrexate was the most effective treatment, as after 72 h the cell population was reduced nearly 40% while under free methotrexate circulation the cell population doubled. Overall, these results indicate that methotrexate-lipid nanoparticles are a promising targeted therapy for osteosarcoma treatment.

Imprinting defects at human 14q32 locus alters gene expression and is associated with the pathobiology of osteosarcoma

PubMed Central

Shu, Jingmin; Li, Lihua; Sarver, Anne E.; Pope, Emily A.; Varshney, Jyotika; Thayanithy, Venugopal; Spector, Logan; Largaespada, David A.; Steer, Clifford J.; Subramanian, Subbaya

2016-01-01

Osteosarcoma is the most common primary bone malignancy affecting children and adolescents. Although several genetic predisposing conditions have been associated with osteosarcoma, our understanding of its pathobiology is rather limited. Here we show that, first, an imprinting defect at human 14q32-locus is highly prevalent (87%) and specifically associated with osteosarcoma patients < 30 years of age. Second, the average demethylation at differentially methylated regions (DMRs) in the 14q32-locus varied significantly compared to genome-wide demethylation. Third, the 14q32-locus was enriched in both H3K4-me3 and H3K27-me3 histone modifications that affected expression of all imprinted genes and miRNAs in this region. Fourth, imprinting defects at 14q32 - DMRs are present in triad DNA samples from affected children and their biological parents. Finally, imprinting defects at 14q32-DMRs were also observed at higher frequencies in an Rb1/Trp53 mutation-induced osteosarcoma mouse model. Further analysis of normal and tumor tissues from a Sleeping Beauty mouse model of spontaneous osteosarcoma supported the notion that these imprinting defects may be a key factor in osteosarcoma pathobiology. In conclusion, we demonstrate that imprinting defects at the 14q32 locus significantly alter gene expression, may contribute to the pathogenesis of osteosarcoma, and could be predictive of survival outcomes. PMID:26802029

Immunohistochemical expression of vegf and her-2 proteins in osteosarcoma biopsies

PubMed Central

Becker, Ricardo Gehrke; Galia, Carlos Roberto; Morini, Sandra; Viana, Cristiano Ribeiro

2013-01-01

OBJECTIVES: To identify the prevalence of erbB-2 and vascular endothelial growth factor (VEGF) in osteosarcoma biopsies and to correlate them with possible prognosis factors. METHODS: Retrospective study conducted at the Hospital do Câncer de Barretos-SP including 27 osteosarcoma biopsies immunohistochemically stained for VEGF and erbB-2. The pathological characteristics were collected from medical records of patients to correlate with markers. RESULTS: In 27 biopsies, four overexpressed VEGF and three overexpressed erbB-2. Two thirds of patients had no metastases. Almost all patients with overexpression of VEGF showed metastases. Overexpression of erbB-2 was inversely related to the presence of metastases. There was no significant association between markers and prognosis. CONCLUSION: We identified a low prevalence of erbB-2 and VEGF in the sample. There was no significant association between overexpression of markers and pathological features. A larger sample and a longer follow-up, in addition to using new laboratory techniques can determine the real expression of VEGF and erbB-2 and its role in osteosarcoma. Level of Evidence III, Case-Control Study. PMID:24453675

The Effect of Krill Oil and n-3 Polyunsaturated Fatty Acids on Human Osteosarcoma Cell Proliferation and Migration.

PubMed

Su, Xiao; Tanalgo, Philline; Bustos, Marcel; Dass, Crispin R

2018-01-01

Osteosarcoma (OS) is a neoplastic condition afflicting mostly the young, as the lesion usually occurs in areas of bone growth with tumour cells metastasising to the lungs in advanced disease. There is no real cure for the disease, with conventional drugs causing side-effects that decrease the quality of life of sufferers. Newer and safer drugs are needed, and one avenue is to use natural compounds that can stunt the growth of the tumour. In this study, two such biological entities were evaluated: krill oil and fish oil. Human OS cells were exposed to krill oil, fish oil, EPA and DHA in time-course assays lasting up to 72h. Krill oil inhibited 23, 50 and 64% of cell proliferation at 24, 48 and 72h respectively, while fish oil resulted in no significant changes although an increase was observed at 24h. Interestingly EPA and DHA promoted OS cell proliferation and migration in this neoplasia. The inhibitory effect of krill oil was comparable to 0.5 and 1µM doxorubicin, a commonly used clinical drug for OS treatment. These results indicate that krill oil may be used in combination with standard clinical practices to control primary tumour growth, and more importantly, metastasis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Aplasia Ras homologue member Ⅰ overexpression inhibits tumor growth and induces apoptosis through inhibition of PI3K/Akt survival pathways in human osteosarcoma MG-63 cells in culture.

PubMed

Ye, Kaishan; Wang, Shuanke; Yang, Yong; Kang, Xuewen; Wang, Jing; Han, Hua

2015-09-01

Aplasia Ras homologue member Ⅰ (ARHI), an imprinted tumor-suppressor gene, is downregulated in various types of cancer. However, the expression, function and specific mechanisms of ARHI in human osteosarcoma (OS) cells remain unclear. The aim of the present study was to assess the effect of ARHI on OS cell proliferation and apoptosis and its associated mechanism. In the study, ARHI mRNA and protein levels were markedly downregulated in OS cells compared with the human osteoblast precursor cell line hFOB1.19. By generating stable transfectants, ARHI was overexpressed in OS cells that had low levels of ARHI. Overexpression of ARHI inhibited cell viability and proliferation and induced apoptosis. However, caspase‑3 activity was not changed by ARHI overexpression. In addition, phosphorylated Akt protein expression decreased in the ARHI overexpression group compared to that in the control vector group. The knockdown of ARHI also resulted in the promotion of cell proliferation and the attenuation of apoptosis in MG‑63 cells. Additionally, ARHI silencing increased the level of p‑Akt. The present results indicate that ARHI inhibits OS cell proliferation and may have a key role in the development of OS.

Tetrahydrocurcumin induces mesenchymal-epithelial transition and suppresses angiogenesis by targeting HIF-1α and autophagy in human osteosarcoma

PubMed Central

Zhang, Yan; Liu, Ying; Zou, Jilong; Yan, Lixin; Du, Wei; Zhang, Yafeng; Sun, Hanliang; Lu, Peng; Geng, Shuo; Gu, Rui; Zhang, Hongyue; Bi, Zhenggang

2017-01-01

Human osteosarcoma is considered a malignant tumor with poor prognosis that readily metastasizes. Tetrahydrocurcumin (THC) has been reported to have anti-tumor activity in numerous tumors. In addition, hypoxia-inducible factor-1α (HIF-1α) has been demonstrated to be associated with tumor metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of THC in osteosarcoma remains uncertain. Therefore, this study aimed to elucidate the potential mechanisms. We found that THC significantly reduced the growth of osteosarcoma cells and suppressed migration and invasion, as tested in a nude mouse lung metastasis model. Additionally, the mesenchymal-epithelial transition (MET) process was facilitated by THC. Mechanistically, our study showed that HIF-1α had a pivotal role in the anti-metastatic effect of THC. Importantly, HIF-1α expression was downregulated by THC by inhibiting Akt/mTOR and p38 MAPK pathways. Moreover, THC exhibited a remarkable inhibitory effect on HIF-1α expression and angiogenesis under hypoxic conditions. Furthermore, THC activated autophagy and induced MET and suppressed angiogenesis in a HIF-1α-related manner. Taken together, our findings suggest that THC suppresses metastasis and invasion and this may be associated with HIF-1α and autophagy, which would potentially provide therapeutic strategies for human osteosarcoma. PMID:29207631

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells.

PubMed

Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza

2016-08-23

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells

PubMed Central

Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza

2016-01-01

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88–92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88–92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream. PMID:27323409

Factors predicting survival following complete surgical remission of pulmonary metastasis in osteosarcoma

PubMed Central

SALAH, SAMER; TOUBASI, SAMAR

2015-01-01

Pulmonary metastasectomy (PM) is associated with improved survival of patients with metastatic osteosarcoma; however, the factors affecting survival following achievement of complete surgical remission remain controversial. The main objective of this study was to report the outcomes and prognostic factors of osteosarcoma patients who achieved complete remission (CR) following PM. We analyzed the effect of demographic and disease-related characteristics on the overall survival (OS) of consecutive patients with metastatic osteosarcoma who were treated at a single institution and achieved CR following PM, through univariate and multivariate analyses. Between January, 2000 and August, 2013, 62 patients with metastatic osteosarcoma were treated and followed up at our institution. A total of 25 patients achieved CR following PM and were included in this analysis. The 5-year OS and disease-free survival following PM were 30 and 21%, respectively. The factors correlated with inferior OS in the univariate analysis included chondroblastic subtype, post-chemotherapy necrosis <90% in the primary tumor, metastasis detected during neoadjuvant or adjuvant chemotherapy and pathological identification of tumor cells reaching the visceral pleural surface of any of the resected nodules. In the multivariate analysis, the chondroblastic subtype was the sole independent adverse prognostic factor (HR=4.6, 95% CI: 1.0–21.3, P=0.044). Therefore, factors associated with tumor biology, including poor tumor necrosis in the primary tumor and detection of metastasis during primary chemotherapy, are associated with poor post-metastasectomy survival. In addition, chondroblastic subtype and visceral pleural involvement predicted poor prognosis in our series. PMID:25469287

Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

PubMed Central

Salyards, Gregory W; Blas-Machado, Uriel; Mishra, Sasmita; Harvey, Stephen B; Butler, Abigail M

2013-01-01

Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil. PMID:23561939

Abnormal DNA methylation may contribute to the progression of osteosarcoma.

PubMed

Chen, Xiao-Gang; Ma, Liang; Xu, Jia-Xin

2018-01-01

The identification of optimal methylation biomarkers to achieve maximum diagnostic ability remains a challenge. The present study aimed to elucidate the potential molecular mechanisms underlying osteosarcoma (OS) using DNA methylation analysis. Based on the GSE36002 dataset obtained from the Gene Expression Omnibus database, differentially methylated genes were extracted between patients with OS and controls using t‑tests. Subsequently, hierarchical clustering was performed to segregate the samples into two distinct clusters, OS and normal. Gene Ontology (GO) and pathway enrichment analyses for differentially methylated genes were performed using the Database for Annotation, Visualization and Integrated Discovery tool. A protein‑protein interaction (PPI) network was established, followed by hub gene identification. Using the cut‑off threshold of ≥0.2 average β‑value difference, 3,725 unique CpGs (2,862 genes) were identified to be differentially methylated between the OS and normal groups. Among these 2,862 genes, 510 genes were differentially hypermethylated and 2,352 were differentially hypomethylated. The differentially hypermethylated genes were primarily involved in 20 GO terms, and the top 3 terms were associated with potassium ion transport. For differentially hypomethylated genes, GO functions principally included passive transmembrane transporter activity, channel activity and metal ion transmembrane transporter activity. In addition, a total of 10 significant pathways were enriched by differentially hypomethylated genes; notably, neuroactive ligand‑receptor interaction was the most significant pathway. Based on a connectivity degree >90, 7 hub genes were selected from the PPI network, including neuromedin U (NMU; degree=103) and NMU receptor 1 (NMUR1; degree=103). Functional terms (potassium ion transport, transmembrane transporter activity, and neuroactive ligand‑receptor interaction) and hub genes (NMU and NMUR1) may serve as potential

Effects of aurothiomalate treatment on canine osteosarcoma in a murine xenograft model.

PubMed

Scharf, Valery F; Farese, James P; Siemann, Dietmar W; Abbott, Jeffrey R; Kiupel, Matti; Salute, Marc E; Milner, Rowan J

2014-03-01

Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan-Meier method with the log-rank test and one-way analysis of variance with the Tukey's test or Dunn's method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate's established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate.

Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells

PubMed Central

Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B.

2014-01-01

The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors. PMID:24810746

Blocking signaling at the level of GLI regulates downstream gene expression and inhibits proliferation of canine osteosarcoma cells.

PubMed

Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B

2014-01-01

The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.

Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma*

PubMed Central

Scott, Milcah C.; Sarver, Aaron L.; Tomiyasu, Hirotaka; Cornax, Ingrid; Van Etten, Jamie; Varshney, Jyotika; O'Sullivan, M. Gerard; Subramanian, Subbaya; Modiano, Jaime F.

2015-01-01

We previously identified two distinct molecular subtypes of osteosarcoma through gene expression profiling. These subtypes are associated with distinct tumor behavior and clinical outcomes. Here, we describe mechanisms that give rise to these molecular subtypes. Using bioinformatic analyses, we identified a significant association between deregulation of the retinoblastoma (RB)-E2F pathway and the molecular subtype with worse clinical outcomes. Xenotransplantation models recapitulated the corresponding behavior for each osteosarcoma subtype; thus, we used cell lines to validate the role of the RB-E2F pathway in regulating the prognostic gene signature. Ectopic RB resets the patterns of E2F regulated gene expression in cells derived from tumors with worse clinical outcomes (molecular phenotype 2) to those comparable with those observed in cells derived from tumors with less aggressive outcomes (molecular phenotype 1), providing a functional association between RB-E2F dysfunction and altered gene expression in osteosarcoma. DNA methyltransferase and histone deacetylase inhibitors similarly reset the transcriptional state of the molecular phenotype 2 cells from a state associated with RB deficiency to one seen with RB sufficiency. Our data indicate that deregulation of RB-E2F pathway alters the epigenetic landscape and biological behavior of osteosarcoma. PMID:26378234

Pleiotropic effects of bisphosphonates on osteosarcoma.

PubMed

Ohba, Tetsuro; Cates, Justin M M; Cole, Heather A; Slosky, David A; Haro, Hirotaka; Ichikawa, Jiro; Ando, Takashi; Schwartz, Herbert S; Schoenecker, Jonathan G

2014-06-01

Osteosarcoma is the most common primary malignant tumor of bone and accounts for half of all primary skeletal malignancies in children and teenagers. The prognosis for patients who fail or progress on first-line chemotherapy protocols is poor, therefore, additional adjuvant therapeutic strategies are needed. A recent feasibility study has demonstrated that the nitrogen-containing bisphosphonate zoledronic acid (ZOL) can be combined safely with conventional chemotherapy. However, the pharmacodynamics of bisphosphonate therapy is not well characterized. Osteosarcoma is a highly angiogenic tumor. Recent reports of the anti-angiogenic effects of bisphosphonates prompted us to determine whether nitrogen-containing bisphosphonate (ZOL and alendronate) treatment attenuates osteosarcoma growth by inhibition of osteoclast activity, tumor-mediated angiogenesis, or direct inhibitory effects on osteosarcoma. Here, we demonstrate that bisphosphonates directly inhibit VEGFR2 expression in endothelial cells, as well as endothelial cell proliferation and migration. Additionally, bisphosphonates also decrease VEGF-A expression in osteosarcoma (K7M3) cells, resulting in reduced stimulation of endothelial cell migration in co-culture assays. ZOL also decreases VEGFR1 expression in aggressive osteosarcoma cell lines (K7M3, 143B) and induces apoptosis of these cells, but has negligible effects on less aggressive osteosarcoma cell lines (K12 and TE85). In vivo ZOL treatment results in significant reduction in osteosarcoma-initiated angiogenesis and tumor growth in a murine model of osteosarcoma. In conclusion, bisphosphonates have diverse growth inhibitory effects on osteosarcoma through: (1) activation of apoptosis and inhibition of cell proliferation, (2) inhibition of VEGF-A and VEGFR1 expression by tumor cells, (3) inhibition of tumor-induced angiogenesis, and (4) direct inhibitory actions on endothelial cells. Published by Elsevier Inc.

Apurinic/apyrimidinic endonuclease 1 regulates angiogenesis in a transforming growth factor β-dependent manner in human osteosarcoma.

PubMed

Jiang, Xuan; Shan, Jinlu; Dai, Nan; Zhong, Zhaoyang; Qing, Yi; Yang, Yuxing; Zhang, Shiheng; Li, Chongyi; Sui, Jiangdong; Ren, Tao; Li, Mengxia; Wang, Dong

2015-10-01

Angiogenesis plays an important role in tumor growth and metastasis and has been reported to be inversely correlated with overall survival of osteosarcoma patients. It has been shown that apurinic/apyrimidinic endonuclease 1 (APE1), a dually functional protein possessing both base excision repair and redox activities, is involved in tumor angiogenesis, although these mechanisms are not fully understood. Our previous study showed that the expression of transforming growth factor β (TGFβ) was significantly reduced in APE1-deficient osteosarcoma cells. Transforming growth factor β promotes cancer metastasis through various mechanisms including immunosuppression, angiogenesis, and invasion. In the current study, we initially revealed that APE1, TGFβ, and microvessel density (MVD) have pairwise correlation in osteosarcoma tissue samples, whereas TGFβ, tumor size, and MVD were inversely related to the prognosis of the cohort. We found that knocking down APE1 in osteosarcoma cells resulted in TGFβ downregulation. In addition, APE1-siRNA led to suppression of angiogenesis in vitro based on HUVECs in Transwell and Matrigel tube formation assays. Reduced secretory protein level of TGFβ of culture medium also resulted in decreased phosphorylation of Smad3 of HUVECs. In a mouse xenograft model, siRNA-mediated silencing of APE1 downregulated TGFβ expression, tumor size, and MVD. Collectively, the current evidence indicates that APE1 regulates angiogenesis in osteosarcoma by controlling the TGFβ pathway, suggesting a novel target for anti-angiogenesis therapy in human osteosarcoma. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

A novel interplay between HOTAIR and DNA methylation in osteosarcoma cells indicates a new therapeutic strategy.

PubMed

Li, Xingang; Lu, Hongming; Fan, Guilian; He, Miao; Sun, Yu; Xu, Kai; Shi, Fengjun

2017-11-01

Osteosarcoma (OS) is one of the most prevalent primary malignant bone tumors in adolescent. HOTAIR is highly expressed and associated with the epigenetic modifications, especially DNA methylation, in cancer. However, the regulation mechanism between HOTAIR and DNA methylation and the biological effects of them in the pathogenesis of osteosarcoma remains elusive. Through RNA-sequencing and computational analysis, followed by a variety of experimental validations, we report a novel interplay between HOTAIR, miR-126, and DNA methylation in OS. We found that HOTAIR is highly expressed in OS cells and the knockdown of HOTAIR leads to the down-regulation of DNMT1, as well as the decrease of global DNA methylation level. RNA-sequencing analysis of HOTAIR-regulated gene shows that CDKN2A is significantly repressed by HOTAIR. A series of experiments show that HOTAIR represses the expression of CDKN2A through inhibiting the promoter activity of CDKN2A by DNA hypermethylation. Further evidence shows that HOTAIR activates the expression of DNMT1 through repressing miR-126, which is the negative regulator of DNMT1. Functionally, HOTAIR depletion increases the sensibility of OS cells to DNMT1 inhibitor through regulating the viability and apoptosis of OS cells via HOTAIR-miR126-DNMT1-CDKN2A axis. These results not only enrich our understanding of the regulation relationship between non-coding RNA, DNA methylation, and gene expression, however, also provide a novel direction in developing more sophisticated therapeutic strategies for OS patients.

Decreased RECQL5 correlated with disease progression of osteosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Junlong; Zhi, Liqiang; Dai, Xin

Human RecQ helicase family, consisting of RECQL, RECQL4, RECQL5, BLM and WRN, has critical roles in genetic stability and tumorigenesis. Although RECQL5 has been reported to correlate with the susceptibility to malignances including osteosarcoma, the specific effect on tumor genesis and progression is not yet clarified. Here we focused on the relationship between RECQL5 expression and osteosarcoma disease progression, and further investigated the function of RECQL5 on MG-63 cell proliferation and apoptosis. By immunohistochemical analysis, qRT-PCR and western blot, we found that RECQL5 expression was downregulated in osteosarcoma tissues and cells. Patients with advanced tumor stage and low grade expressedmore » lower RECQL5. To construct a stable RECQL5 overexpression osteosarcoma cell line (MG-63-RECQL5), RECQL5 gene was inserted into the human AAVS1 safe harbor by CRISPR/Cas9 system. The overexpression of RECQL5 was verified by qRT-PCR and western blot. Cell proliferation, cell cycle and apoptosis assay revealed that RECQL5 overexpression inhibited proliferation, induced G1-phase arrest and promoted apoptosis in MG-63 cells. Collectively, our results suggested RECQL5 as a tumor suppressor in osteosarcoma and may be a potential therapeutic target for osteosarcoma treatment. - Highlights: • The expression of RECQL5 was downregulated in osteosarcoma tissues and cells. • Decreased RECQL5 correlated with osteosarcoma Enneking surgical classification. • We constructed a stable RECQL5 overexpression cell line by CRISPR/Cas9 system. • RECQL5 overexpression inhibited proliferation of MG-63 cells. • RECQL5 overexpression promoted apoptosis of MG-63 cells.« less

Genomic Heterogeneity of Osteosarcoma - Shift from Single Candidates to Functional Modules

PubMed Central

Maugg, Doris; Eckstein, Gertrud; Baumhoer, Daniel; Nathrath, Michaela; Korsching, Eberhard

2015-01-01

Osteosarcoma (OS), a bone tumor, exhibit a complex karyotype. On the genomic level a highly variable degree of alterations in nearly all chromosomal regions and between individual tumors is observable. This hampers the identification of common drivers in OS biology. To identify the common molecular mechanisms involved in the maintenance of OS, we follow the hypothesis that all the copy number-associated differences between the patients are intercepted on the level of the functional modules. The implementation is based on a network approach utilizing copy number associated genes in OS, paired expression data and protein interaction data. The resulting functional modules of tightly connected genes were interpreted regarding their biological functions in OS and their potential prognostic significance. We identified an osteosarcoma network assembling well-known and lesser-known candidates. The derived network shows a significant connectivity and modularity suggesting that the genes affected by the heterogeneous genetic alterations share the same biological context. The network modules participate in several critical aspects of cancer biology like DNA damage response, cell growth, and cell motility which is in line with the hypothesis of specifically deregulated but functional modules in cancer. Further, we could deduce genes with possible prognostic significance in OS for further investigation (e.g. EZR, CDKN2A, MAP3K5). Several of those module genes were located on chromosome 6q. The given systems biological approach provides evidence that heterogeneity on the genomic and expression level is ordered by the biological system on the level of the functional modules. Different genomic aberrations are pointing to the same cellular network vicinity to form vital, but already neoplastically altered, functional modules maintaining OS. This observation, exemplarily now shown for OS, has been under discussion already for a longer time, but often in a hypothetical manner, and

Aven-mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma.

PubMed

Baranski, Zuzanna; Booij, Tijmen H; Cleton-Jansen, Anne-Marie; Price, Leo S; van de Water, Bob; Bovée, Judith V M G; Hogendoorn, Pancras C W; Danen, Erik H J

2015-07-01

Conventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti-apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G2 cell-cycle arrest; Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

PubMed

Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

2013-08-01

The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Identification of osteosarcoma-related specific proteins in serum samples using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry.

PubMed

Gu, Jianli; Li, Jitian; Huang, Manyu; Zhang, Zhiyong; Li, Dongsheng; Song, Guoying; Ding, Xingpo; Li, Wuyin

2014-01-01

Osteosarcoma (OS) is the most common malignant bone tumor. To identify OS-related specific proteins for early diagnosis of OS, a novel approach, surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF-MS) to serum samples from 25 OS patients, 16 osteochondroma, and 26 age-matched normal human volunteers as controls, was performed. Two proteins showed a significantly different expression in OS serum samples from control groups. Proteomic profiles and external leave-one-out cross-validation analysis showed that the correct rate of allocation, the sensitivity, and the specificity of diagnosis were 100%. These two proteins were further identified by searching the EPO-KB database, and one of the proteins identified as Serine rich region profile is involved in various cellular signaling cascades and tumor genesis. The presence of these two proteins in OS patients but absence from premalignant and normal human controls implied that they can be potential biomarkers for early diagnosis of OS.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

PubMed

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-05-10

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats

PubMed Central

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-01-01

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function. PMID:28489060

A critical role of Notch signaling in osteosarcoma invasion and metastasis

PubMed Central

Zhang, Pingyu; Yang, Yanwen; Zweidler-McKay, Patrick A.; Hughes, Dennis P.M.

2010-01-01

Purpose Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown. Experimental Design We assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. We then employed pharmacologic and retroviral manipulation of the Notch pathway and studied the impact on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion and metastasis in vitro and in vivo. Results Notch pathway genes, including Notch ligand DLL1, Notch 1 and 2, and the Notch target gene HES1 were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of gamma secretase eliminated invasion in matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling demonstrated a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo. Conclusion These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Since the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma. PMID:18483362

Growth Inhibition of Osteosarcoma Cell Lines in 3D Cultures: Role of Nitrosative and Oxidative Stress.

PubMed

Gorska, Magdalena; Krzywiec, Pawel Bieniasz; Kuban-Jankowska, Alicja; Zmijewski, Michal; Wozniak, Michal; Wierzbicka, Justyna; Piotrowska, Anna; Siwicka, Karolina

2016-01-01

3D cell cultures have revolutionized the understanding of cell behavior, allowing culture of cells with the possibility of resembling in vivo intercellular signaling and cell-extracellular matrix interaction. The effect of limited oxygen penetration into 3D culture of highly metastatic osteosarcoma 143B cells in terms of expression of nitro-oxidative stress markers was investigated and compared to standard 2D cell culture. Human osteosarcoma (143B cell line) cells were cultured as monolayers, in collagen and Matrigel. Cell viability, gene expression of nitro-oxidative stress markers, and vascular endothelial growth factor were determined using Trypan blue assay, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Three-dimensional environments modify nitro-oxidative stress and influence gene expression and cell proliferation of OS 143B cells. Commercial cell lines might not constitute a good model of 3D cultures for bone tissue engineering, as they are highly sensitive to hypoxia, and hypoxic conditions can induce oxidation of the cellular environment. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

3D-printed guiding templates for improved osteosarcoma resection

NASA Astrophysics Data System (ADS)

Ma, Limin; Zhou, Ye; Zhu, Ye; Lin, Zefeng; Wang, Yingjun; Zhang, Yu; Xia, Hong; Mao, Chuanbin

2016-03-01

Osteosarcoma resection is challenging due to the variable location of tumors and their proximity with surrounding tissues. It also carries a high risk of postoperative complications. To overcome the challenge in precise osteosarcoma resection, computer-aided design (CAD) was used to design patient-specific guiding templates for osteosarcoma resection on the basis of the computer tomography (CT) scan and magnetic resonance imaging (MRI) of the osteosarcoma of human patients. Then 3D printing technique was used to fabricate the guiding templates. The guiding templates were used to guide the osteosarcoma surgery, leading to more precise resection of the tumorous bone and the implantation of the bone implants, less blood loss, shorter operation time and reduced radiation exposure during the operation. Follow-up studies show that the patients recovered well to reach a mean Musculoskeletal Tumor Society score of 27.125.

Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

PubMed Central

2012-01-01

Background Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. Methods CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. Results The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. Conclusions MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma. PMID:22475227

Multiple supervised residual network for osteosarcoma segmentation in CT images.

PubMed

Zhang, Rui; Huang, Lin; Xia, Wei; Zhang, Bo; Qiu, Bensheng; Gao, Xin

2018-01-01

Automatic and accurate segmentation of osteosarcoma region in CT images can help doctor make a reasonable treatment plan, thus improving cure rate. In this paper, a multiple supervised residual network (MSRN) was proposed for osteosarcoma image segmentation. Three supervised side output modules were added to the residual network. The shallow side output module could extract image shape features, such as edge features and texture features. The deep side output module could extract semantic features. The side output module could compute the loss value between output probability map and ground truth and back-propagate the loss information. Then, the parameters of residual network could be modified by gradient descent method. This could guide the multi-scale feature learning of the network. The final segmentation results were obtained by fusing the results output by the three side output modules. A total of 1900 CT images from 15 osteosarcoma patients were used to train the network and a total of 405 CT images from another 8 osteosarcoma patients were used to test the network. Results indicated that MSRN enabled a dice similarity coefficient (DSC) of 89.22%, a sensitivity of 88.74% and a F1-measure of 0.9305, which were larger than those obtained by fully convolutional network (FCN) and U-net. Thus, MSRN for osteosarcoma segmentation could give more accurate results than FCN and U-Net. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mediator of RNA polymerase II transcription subunit 19 promotes osteosarcoma growth and metastasis and associates with prognosis.

PubMed

Yu, Wenxi; Zhang, Zhichang; Min, Daliu; Yang, Qingcheng; Du, Xuefei; Tang, Lina; Lin, Feng; Sun, Yuanjue; Zhao, Hui; Zheng, Shuier; He, Aina; Li, Hongtao; Yao, Yang; Shen, Zan

2014-04-01

Osteosarcoma (OS) is the most common primary malignant tumour of bone. Nearly 30-40% of OS patients have a poor prognosis despite multimodal treatments. Because the carcinogenesis of OS remains unclear, the identification of new oncogenes that control the tumourigenesis and progression of OS is crucial for developing new therapies. Here, we found that the expression of Mediator of RNA polymerase II transcription subunit 19 (Med19) was increased in OS samples from patients compared to normal bone tissues. Cyclin D1 and cyclin B1 are upregulated in Med19 positive OS tissues. Importantly, among 97 OS patients of Enneking stage IIB or IIIB, Med19 expression was correlated with metastasis (P<0.05) and poor prognosis (P<0.01). Med19 knockdown significantly induced growth inhibition, reduced colony-forming ability and suppressed migration in the OS cell lines Saos-2 and U2OS, along with the downregulated expression of cyclin D1 and cyclin B1. Med19 knockdown also induced apoptosis in Saos-2 cells via induction of caspase-3 and poly ADP-ribose polymerase (PARP). In addition, Med19 knockdown significantly suppressed tumour growth in an OS xenograft nude mouse model via suppression of cyclin D1 and cyclin B1. Simultaneously, Med19 downregulation decreased the expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumour samples from OS xenograft nude mice. Med19 depletion remarkably reduced tumour metastasis in a model of OS metastatic spreading. Taken together, our data suggest that Med19 acts as an oncogene in OS via a possible cyclin D1/cyclin B1 modulation pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pharmacological or genetic inhibition of LDHA reverses tumor progression of pediatric osteosarcoma.

PubMed

Gao, Shan; Tu, Dan-Na; Li, Heng; Jiang, Jian-Xin; Cao, Xin; You, Jin-Bin; Zhou, Xiao-Qin

2016-07-01

Reprogrammed energy metabolism is an emerging hallmark of cancer. Lactate dehydrogenase A (LDHA), a key enzyme involved in anaerobic glycolysis, is frequently deregulated in human malignancies. However, limited knowledge is known about its roles in the progression of osteosarcoma (OS). In this study, we found that LDHA is commonly upregulated in four OS cell lines compared with the normal osteoblast cells (hFOB1.19). Treatment with FX11, a specific inhibitor of LDHA, significantly reduced LDHA activity, and inhibited cell proliferation and invasive potential in a dose dependent manner. Genetic silencing of LDHA resulted in a decreased lactate level in the culture medium, reduced cell viability and decreased cell invasion ability. Meanwhile, silencing of LDHA also compromised tumorigenesis in vivo. Furthermore, knockdown of LDHA remarkably reduced extracellular acidification rate (ECAR) as well as glucose consumption. In the presence of 2-DG, a glycolysis inhibitor, LDHA-mediated cell proliferation and invasion were completely blocked, indicating the oncogenic activities of LDHA may dependent on Warburg effect. Finally, pharmacological inhibition of c-Myc or HIF1α significantly attenuated LDHA expression. Taken together, upregulated LDHA facilitates tumor progression of OS and might be a potential target for OS treatment. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Spinal Osteosarcoma

PubMed Central

Katonis, P.; Datsis, G.; Karantanas, A.; Kampouroglou, A.; Lianoudakis, S.; Licoudis, S.; Papoutsopoulou, E.; Alpantaki, K.

2013-01-01

Although osteosarcoma represents the second most common primary bone tumor, spinal involvement is rare, accounting for 3%–5% of all osteosarcomas. The most frequent symptom of osteosarcoma is pain, which appears in almost all patients, whereas more than 70% exhibit neurologic deficit. At a molecular level, it is a tumor of great genetic complexity and several genetic disorders have been associated with its appearance. Early diagnosis and careful surgical staging are the most important factors in accomplishing sufficient management. Even though overall prognosis remains poor, en-block tumor removal combined with adjuvant radiotherapy and chemotherapy is currently the treatment of choice. This paper outlines histopathological classification, epidemiology, diagnostic procedures, and current concepts of management of spinal osteosarcoma. PMID:24179411

MALAT1 predicts poor survival in osteosarcoma patients and promotes cell metastasis through associating with EZH2

PubMed Central

Huo, Yanqing; Li, Qingbo; Wang, Xiqian; Jiao, Xiejia; Zheng, Jiachun; Li, Zhiqiang; Pan, Xiaohan

2017-01-01

Osteosarcoma is the most common type of bone cancer, especially in children and young adults. Recently, long noncoding RNAs (lncRNAs) have emerged as new prognostic markers and gene regulators in several cancers, including osteosarcoma. In this study, we investigated the contributions of the lncRNA MALAT1 in osteosarcoma with a specific focus on its transcriptional regulation and its interaction with EZH2. Our results showed that MALAT1 was significantly increased in osteosarcoma specimens and cell lines. ROC curve analysis showed that MALAT1 had a higher area under the curve than alkaline phosphatase, and Kaplan-Meier survival analysis indicated that patients with high serum levels of MALAT1 showed reduced survival rate. Knockdown of MALAT1 decreased osteosarcoma cell invasion and promoted E-cadherin expression. Mechanistic investigations showed that MALAT1 was transcriptionally activated by TGF-β. Additionally, EZH2 is highly expressed and associated with the 3’ end region of lncRNA MALAT1 in osteosarcoma, and this association finally suppressed the expression of E-cadherin. Subsequently, our gain and loss function assay showed that MALAT1 overexpression promoted cell metastasis and decreased E-cadherin level, however, this effect was partially reversed by EZH2 knockdown. In conclusion, our work illuminates that lncRNA MALAT1 is a potential diagnostic and prognostic factor in osteosarcoma and further demonstrates how MALAT1 confers an oncogenic function. Thus, lncRNA MALAT1 may serve as a promising prognostic and therapeutic target for osteosarcoma patients. PMID:28388584

Biodegradable bisphosphonate nanoparticles for imaging and therapeutic applications in osteosarcoma

NASA Astrophysics Data System (ADS)

Rudnick-Glick, S.; Corem-Salkmon, E.; Grinberg, I.; Gluz, E.; Margel, S.

2015-08-01

Osteosarcoma (OS) is amongst the most commonly diagnosed bone tumors occurring in adolescence, young adults and adults over the age of 65. Current treatment is based on a combination of surgery and chemotherapy. Chemotherapy has improved the survival rate, however it is associated with severe side effects due to the use of high dosages, nonspecific uptake and poor bone blood supply. At present bisphosphonates (BP) are widely used in the treatment of bone disorders including OS. We have engineered a unique biodegradable BP nanoparticle that possesses a dual functionality: 1) covalent attachment of a dye (e.g., NIR dye) or drug to the nanoparticles through the primary amine groups on the surface of the nanoparticle; 2) chelation to the bone mineral hydroxyapatite through the BP on the surface of the nanoparticle. Due to a high concentration of PEG in the BP nanoparticles they possess a relatively long plasma half-life time. Therefore, the nanoparticle has potential for use both in diagnosis and therapy of OS. Doxorubicin was conjugated to the free amine on the surface of the BP nanoparticles. In vitro experiments on osteosarcoma cells demonstrated that the doxorubicin-conjugated BP nanoparticles possess a higher efficacy than the free doxorubicin. Further investigation in vivo in a chicken embryo model confirmed that the doxorubicin-conjugated nanoparticle was significantly more effective in inhibiting tumor growth compared to free doxorubicin at a similar concentration. Additionally, we have shown that these BP nanoparticles preferentially target OS tumor tissue, thus increasing anti-cancer drug bioavailability at targeted site.

Characterization of osteosarcoma cells from two sibling large-breed dogs.

PubMed

Norrdin, R W; Powers, B E; Torgersen, J L; Smith, R E; Withrow, S J

1989-11-01

Neoplastic cells were isolated from 2 sibling Great Dane/Labrador Retriever mixed-breed dogs in which telangiectatic type osteosarcomas arose concurrently. Cells from various sites in the same osteosarcoma appeared similar in culture, but there were differences between the 2 osteosarcomas in growth characteristics and appearance of cells. Cells from 1 osteosarcoma had a small, but significant (P less than 0.05), cyclic adenosine monophosphate response to parathyroid hormone stimulation, indicating a low order of osteoblastic differentiation. Cells from the other osteosarcoma had no response to parathyroid hormone stimulation. Cells from both osteosarcomas and a concentrated cell-free filtrate from the osteosarcoma with osteoblastic differentiation were injected into nude mice, but osteosarcomas were not induced. Results of ultrastructural examination of osteosarcoma samples for viral particles were negative and supernatant fluids from cultured cells were considered negative for viral reverse transcriptase activity.

Long noncoding RNA LINC00858 promotes osteosarcoma through regulating miR-139-CDK14 axis.

PubMed

Gu, Zenghui; Hou, Zhenhai; Zheng, Longbao; Wang, Xinqiang; Wu, Liangbang; Zhang, Cheng

2018-06-23

Long noncoding RNAs (lncRNAs) have been identified to modulate the tumorigenesis of human cancers. The in-depth of lncRNAs on human osteosarcoma oncogenesis is still ambiguous. In present study, functional and mechanism experiments were conducted to investigate the role of long intergenic non-protein coding RNA 00858 (LINC00858) on human osteosarcoma tumorigenesis. Results demonstrated that LINC00858 expression was significantly upregulated in both osteosarcoma tissues and cell lines. Mechanism assays presented that LINC00858 silencing significantly repressed osteosarcoma cells' proliferation and invasion in vitro, and inhibited the tumor growth in vivo. In further experiments, LINC00858 was identified to sponge miR-139 to form RNA-induced silencing complex (RISC) using luciferase reporter assay and RNA immunoprecipitation (RIP). Besides, CDK14 was validated to be the target protein the miR-139. Rescue experiments confirmed the role of LINC00858/miR-139/CDK14 pathway on osteosarcoma cells' phenotype. In summary, these data prove that LINC00858/miR-139/CDK14 axis promotes the tumorigenesis of osteosarcoma, providing a new mechanism or target for osteosarcoma. Copyright © 2018. Published by Elsevier Inc.

Intra- and interspecies gene expression models for predicting drug response in canine osteosarcoma.

PubMed

Fowles, Jared S; Brown, Kristen C; Hess, Ann M; Duval, Dawn L; Gustafson, Daniel L

2016-02-19

Genomics-based predictors of drug response have the potential to improve outcomes associated with cancer therapy. Osteosarcoma (OS), the most common primary bone cancer in dogs, is commonly treated with adjuvant doxorubicin or carboplatin following amputation of the affected limb. We evaluated the use of gene-expression based models built in an intra- or interspecies manner to predict chemosensitivity and treatment outcome in canine OS. Models were built and evaluated using microarray gene expression and drug sensitivity data from human and canine cancer cell lines, and canine OS tumor datasets. The "COXEN" method was utilized to filter gene signatures between human and dog datasets based on strong co-expression patterns. Models were built using linear discriminant analysis via the misclassification penalized posterior algorithm. The best doxorubicin model involved genes identified in human lines that were co-expressed and trained on canine OS tumor data, which accurately predicted clinical outcome in 73 % of dogs (p = 0.0262, binomial). The best carboplatin model utilized canine lines for gene identification and model training, with canine OS tumor data for co-expression. Dogs whose treatment matched our predictions had significantly better clinical outcomes than those that didn't (p = 0.0006, Log Rank), and this predictor significantly associated with longer disease free intervals in a Cox multivariate analysis (hazard ratio = 0.3102, p = 0.0124). Our data show that intra- and interspecies gene expression models can successfully predict response in canine OS, which may improve outcome in dogs and serve as pre-clinical validation for similar methods in human cancer research.

Selective sensitiveness of mesenchymal stem cells to shock waves leads to anticancer effect in human cancer cell co-cultures.

PubMed

Foglietta, Federica; Duchi, Serena; Canaparo, Roberto; Varchi, Greta; Lucarelli, Enrico; Dozza, Barbara; Serpe, Loredana

2017-03-15

Mesenchymal stem cells (MSC) possess the distinctive feature of homing in on and engrafting into the tumor stroma making their therapeutic applications in cancer treatment very promising. Research into new effectors and external stimuli, which can selectively trigger the release of cytotoxic species from MSC toward the cancer cells, significantly raises their potential. Shock waves (SW) have recently gained recognition for their ability to induce specific biological effects, such as the local generation of cytotoxic reactive oxygen species (ROS) in a non-invasive and tunable manner. We thus investigate whether MSC are able to generate ROS and, in turn, affect cancer cell growth when in co-culture with human glioblastoma (U87) or osteosarcoma (U2OS) cells and exposed to SW. MSC were found to be the cell line that was most sensitive to SW treatment as shown by SW-induced ROS production and cytotoxicity. Notably, U87 and U2OS cancer cell growth was unaffected by SW exposure. However, significant decreases in cancer cell growth, 1.8 fold for U87 and 2.3 fold for U2OS, were observed 24h after the SW treatment of MSC co-cultures with cancer cells. The ROS production induced in MSC by SW exposure was then responsible for lipid peroxidation and cell death in U87 and U2OS cells co-cultured with MSC. This experiment highlights the unique ability of MSC to generate ROS upon SW treatment and induce the cell death of co-cultured cancer cells. SW might therefore be proposed as an innovative tool for MSC-mediated cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Relationship of peak serum methotrexate concentration to prognosis and drug tolerance in non-metastatic extremity osteosarcomas.

PubMed

Wang, Bo; Yao, Hao; Xie, Xianbiao; Yin, Junqiang; Zou, Changye; Huang, Gang; Shen, Jingnan

2018-05-28

This study aimed to explore whether peak serum methotrexate concentration (C max ) correlated with adverse events, overall survival (OS) and event-free survival (EFS) in patients with primary extremity osteosarcoma. Patients with extremity osteosarcoma who were treated at our center between 2005 and 2015 were retrospectively studied. All the patients were Enneking stage II and had received standard perioperative chemotherapy composed of high-dose methotrexate, doxorubicin, cisplatin and ifosfamide. C max and treatment-associated toxicities of each cycle were recorded. OS and EFS were estimated and compared by Kaplan-Meier survival analysis, and Cox regression models were performed for univariate comparisons. In total, 567 patients were followed for an average of 53 months (24-104 months). The estimated 3- and 5-year EFS were 71.7 and 63.1%, and the 3- and 5-year OS were 78.2 and 72.9%, respectively. C max ranged from 527 to 2495 µmol/L with a mean value of 931 ± 106 µmol/L. No significant differences in EFS and OS (p = 0.18 and p = 0.28) were observed among patients with a mean C max  > 1500, > 1000, > 700 and < 700 µmol/L. However, patients with a mean C max  > 1500 µmol/L had significantly increased rates of grade 3-5 toxicity. In the univariate analysis, C max was not a prognostic factor for EFS (p = 0.08) or OS (p = 0.16). C max did not correlate significantly with the oncologic prognosis of non-metastatic extremity osteosarcoma patients treated by multi-agent chemotherapy; however, C max correlated closely with toxicities and complications. The persistent inclusion of methotrexate in classical multidisciplinary chemotherapy was questioned and should be examined in future trials.

On the importance of considering disease subtypes: Earliest detection of a parosteal osteosarcoma? Differential diagnosis of an osteosarcoma in an Anglo-Saxon female.

PubMed

Ferrante di Ruffano, Lavinia; Waldron, Tony

2016-12-30

A case of potentially dedifferentiated parosteal osteosarcoma was found in the proximal humerus of an adult female buried in the late Anglo-Saxon cemetery of Cherry Hinton, Cambridgeshire, UK. Key features include a large, dense, lobulated mass attached to the medial metaphysis of the proximal humerus by a broad-based attachment, accompanied by cortical destruction and widespread spiculated periosteal reaction. Radiographic images confirm medullary involvement, lack of continuity between the cortex and external mass, a radiolucent cleavage plane and possible radiolucent zones within the bony masses. Differential diagnoses considered include osteochondroma, myositis ossificans, fracture callus, as well as the primary malignancies of osteosarcoma and chondrosarcoma, and their various subtypes. The macroscopic and radiographic analysis of the tumor is described and discussed within clinical and paleopathological contexts. One of only 19 uncontested examples of osteosarcoma from past human populations, most of which remain unconfirmed, this case represents what we believe to be the earliest, and probably singular, bioarcheological example of parosteal osteosarcoma in human history. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

Effectiveness of multi-drug regimen chemotherapy treatment in osteosarcoma patients: a network meta-analysis of randomized controlled trials.

PubMed

Wang, Xiaojie; Zheng, Hong; Shou, Tao; Tang, Chunming; Miao, Kun; Wang, Ping

2017-03-29

Osteosarcoma is the most common malignant bone tumour. Due to the high metastasis rate and drug resistance of this disease, multi-drug regimens are necessary to control tumour cells at various stages of the cell cycle, eliminate local or distant micrometastases, and reduce the emergence of drug-resistant cells. Many adjuvant chemotherapy protocols have shown different efficacies and controversial results. Therefore, we classified the types of drugs used for adjuvant chemotherapy and evaluated the differences between single- and multi-drug chemotherapy regimens using network meta-analysis. We searched electronic databases, including PubMed (MEDLINE), EmBase, and the Cochrane Library, through November 2016 using the keywords "osteosarcoma", "osteogenic sarcoma", "chemotherapy", and "random*" without language restrictions. The major outcome in the present analysis was progression-free survival (PFS), and the secondary outcome was overall survival (OS). We used a random effect network meta-analysis for mixed multiple treatment comparisons. We included 23 articles assessing a total of 5742 patients in the present systematic review. The analysis of PFS indicated that the T12 protocol (including adriamycin, bleomycin, cyclophosphamide, dactinomycin, methotrexate, cisplatin) plays a more critical role in osteosarcoma treatment (surface under the cumulative ranking (SUCRA) probability 76.9%), with a better effect on prolonging the PFS of patients when combined with ifosfamide (94.1%) or vincristine (81.9%). For the analysis of OS, we separated the regimens to two groups, reflecting the disconnection. The T12 protocol plus vincristine (94.7%) or the removal of cisplatinum (89.4%) is most likely the best regimen. We concluded that multi-drug regimens have a better effect on prolonging the PFS and OS of osteosarcoma patients, and the T12 protocol has a better effect on prolonging the PFS of osteosarcoma patients, particularly in combination with ifosfamide or vincristine

Sensitization of interferon-γ induced apoptosis in human osteosarcoma cells by extracellular S100A4

PubMed Central

Pedersen, Kjetil Boye; Andersen, Kristin; Fodstad, Øystein; Mælandsmo, Gunhild Mari

2004-01-01

Background S100A4 is a small Ca2+-binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension. Methods Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B. Results In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB). Conclusion In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown. PMID:15318945

Telangiectatic osteosarcoma of the patella.

PubMed

Shehadeh, Ahmad M; Haiba, Moutaz A; Henshaw, Robert M; Lack, Ernest

2008-08-01

Although osteosarcoma is the most common primary malignancy of bone, it has only been reported to arise from the patella in a handful of cases. Telangiectatic osteosarcoma accounts for <5% of all osteosarcomas, and it is distinguished histologically by spaces, often blood filled, separated by septa containing highly malignant cells and radiographically by a predominately lytic and/or expansile component. Telangiectatic osteosarcoma can be radiologically confused with aneurysmal bone cyst or giant cell tumor. A 22-year-old otherwise healthy man presented with increasing pain, swelling, and limited flexion of the right knee after failing physical therapy for anterior knee pain. Standard anteroposterior and lateral radiographs demonstrated a diffuse destructive process involving the majority of the patella (including loss of the inferior patellar cortex) and a lytic lesion of the proximal tibia. Apparent osteoid matrix was visible in the soft tissue extension along the inferior pole of the patella. A computed tomography scan of the chest showed 2 pulmonary nodules consistent with metastatic disease. Evaluation of core needle biopsy showed osteosarcoma with telangiectatic features. Given that the majority of the tumor involved the patella/extensor mechanism, it was clear that the tumor originated in the patella. This case presents the first published report of a telangiectatic osteosarcoma arising from the patella.

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

PubMed

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-03-16

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 ( FGF1 and FGF12 ), bone morphogenetic factor-1 ( BMP1 ), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 ( SMARCA4 ), Matrix extracellular phosphoglycoprotein ( MEPE ), Integrin, β4 ( ITGBP4 ), Matrix Metalloproteinase -1, -28 ( MMP1 and MMP28 ), and signal transducer and activator of transcription-4 ( STAT4 ) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1 , MMP28 and kallikrein related peptidase-7 ( KLK7 ), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

PubMed Central

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-01-01

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

Cytogenetic and molecular characterization of plutonium-induced rat osteosarcomas.

PubMed

Roch-Lefevre, Sandrine; Daino, Kazuhiro; Altmeyer-Morel, Sandrine; Guilly, Marie-Noëlle; Chevillard, Sylvie

2010-01-01

The association between ionizing radiation and the subsequent development of osteosarcoma has been well described, but little is known about the cytogenetic and molecular events, which could be involved in the formation of radiation-induced osteosarcomas. Here, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in a series of 16 rat osteosarcomas induced by injection of plutonium-238. Recurrent gains/amplifications were observed at chromosomal regions 3p12-q12, 3q41-qter, 4q41-qter, 6q12-q16, 7q22-q34, 8q11-q23, 9q11-q22, 10q32.1-qter, and 12q, whereas recurrent losses were observed at 1p, 1q, 3q23-q35, 5q21-q33, 8q24-q31, 10q22-q25, 15p, 15q, and 18q. The gained region at 7q22-q34 was homologous to human chromosome bands 12q13-q15/8q24/22q11-q13, including the loci of Mdm2, Cdk4, c-Myc and Pdgf-b genes. The lost regions at 5q21-q33, 10q22-q25 and 15q contained tumor suppressor genes such as p16INK4a/p19ARF, Tp53 and Rb1. To identify potential target gene(s) for the chromosomal aberrations, we compared the expression levels of several candidate genes, located within the regions of frequent chromosomal aberrations, between the tumors and normal osteoblasts by using quantitative RT-PCR analysis. The Cdk4, c-Myc, Pdgf-b and p57KIP2 genes were thought to be possible target genes for the frequent chromosomal gain at 7q22-34 and loss at 1q in the tumors, respectively. In addition, mutations of the Tp53 gene were found in 27% (4 of 15) osteosarcomas. Our data may contribute to further understanding of the molecular mechanisms underlying osteosarcomas induced by ionizing radiation in human.

Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy.

PubMed

Tao, Yong; Ou, Yunsheng; Yin, Hang; Chen, Yanyang; Zhong, Shenxi; Gao, Yongjian; Zhao, Zenghui; He, Bin; Huang, Qiu; Deng, Qianxing

2017-11-01

The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester‑mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved‑PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human

Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy

PubMed Central

Tao, Yong; Ou, Yunsheng; Yin, Hang; Chen, Yanyang; Zhong, Shenxi; Gao, Yongjian; Zhao, Zenghui; He, Bin; Huang, Qiu; Deng, Qianxing

2017-01-01

The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved-PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human osteosarcoma

Antimetastatic Efficacy of the Combination of Caffeine and Valproic Acid on an Orthotopic Human Osteosarcoma Cell Line Model in Nude Mice.

PubMed

Igarashi, Kentaro; Kawaguchi, Kei; Kiyuna, Tasuku; Murakami, Takashi; Yamamoto, Norio; Hayashi, Katsuhiro; Kimura, Hiroaki; Miwa, Shinji; Tsuchiya, Hiroyuki; Hoffman, Robert M

2017-03-01

We have previously reported that caffeine can enhance chemotherapy efficacy of bone and soft tissue sarcoma via cell-cycle perturbation. Valproic acid has histone deacetylase (HDAC) inhibitory activity. We have also reported the anti-tumor efficacy of combination treatment with caffeine and valproic acid against osteosarcoma primary tumors in a cell-line orthotopic mouse model. In this study, we performed combination treatment of caffeine and valproic acid on osteosarcoma cell lines in vitro and in spontaneous and experimental lung metastasis mouse models of osteosarcoma. Survival of 143B-RFP human osteosarcoma cells after exposure to caffeine and valproic acid for 72 hours was determined using the WST-8 assay. IC 50 values and combination indices were calculated. Mouse models of primary osteosarcoma and spontaneous lung metastasis were obtained by orthotopic intra-tibial injection of 143B-RFP cells. Valproic acid, caffeine, and combination of both drugs were administered from day 7, five times a week, for four weeks. Six weeks after orthotopic injection, lung samples were excised and observed with a fluorescence imaging system. A mouse model of experimental lung metastasis was obtained by tail vein injection of 143B-RFP cells. The mice were treated with these agents from day 0, five times a week for four weeks. Both caffeine and valproic acid caused concentration-dependent cell kill in vitro. Synergistic efficacy of the combination treatment was observed. In the spontaneous lung-metastasis model, the number of lung metastasis was 9.0±2.6 in the untreated group (G1); 10.8±2.9 in the caffeine group (G2); 10.0±3.1 in the valproic-acid group (G3); and 3.0±1.1 in the combination group (G4); (p=6.78E-5 control vs. combination; p=0.006 valproic acid vs. combination; p=0.003 caffeine vs. combination). In the experimental lung-metastasis model, the combination group significantly reduced lung metastases and improved overall survival (p=0.0005). Efficacy of the

In vitro and in vivo characterization of stem-like cells from canine osteosarcoma and assessment of drug sensitivity.

PubMed

Gatti, Monica; Solari, Agnese; Pattarozzi, Alessandra; Campanella, Chiara; Thellung, Stefano; Maniscalco, Lorella; De Maria, Raffaella; Würth, Roberto; Corsaro, Alessandro; Bajetto, Adriana; Ratto, Alessandra; Ferrari, Angelo; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2018-02-01

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs. Copyright © 2018 Elsevier Inc. All rights reserved.

Central Telangiectatic Osteosarcoma of the Mandible in a Paediatric Patient: A Rarity

PubMed Central

Tomar, Divya; Thayath, Muhamad Nishad; Zaidi, Iram; Singh, Shikha

2016-01-01

Osteosarcoma (OS) involving the head and neck region is rare and is a diagnostic challenge due to its aggressive nature and varied presentations. Although OS constitute 40% to 60% of all bone tumours, only 10% of these occur in the head and neck region, most commonly in the maxilla and mandible. OS of the jaw bone has a different pattern and behaviour when compared to OS at other anatomic location. It occurs in a more elderly population, less aggressive and usually spreads locally rather than distant metastases. Telangiectatic Osteosarcoma (TOS) of jaws in a paediatric patient is still a very rare variant and till date only two cases have been reported in literature. Radiographic evaluation plays an important role as the clinical symptoms are not specific to the condition. Conventional radiography should always be supplemented by advanced imaging modalities for the diagnosis, which gives a three dimensional assessment and also prevents superimposition of surrounding structures. Early diagnosis and radical surgery are the keys to high survival rates. Herein, we present a third ever reported case of TOS occurring in mandible of a nine-year-old male patient. PMID:28208989

Central Telangiectatic Osteosarcoma of the Mandible in a Paediatric Patient: A Rarity.

PubMed

Tomar, Divya; Dhillon, Manu; Thayath, Muhamad Nishad; Zaidi, Iram; Singh, Shikha

2016-12-01

Osteosarcoma (OS) involving the head and neck region is rare and is a diagnostic challenge due to its aggressive nature and varied presentations. Although OS constitute 40% to 60% of all bone tumours, only 10% of these occur in the head and neck region, most commonly in the maxilla and mandible. OS of the jaw bone has a different pattern and behaviour when compared to OS at other anatomic location. It occurs in a more elderly population, less aggressive and usually spreads locally rather than distant metastases. Telangiectatic Osteosarcoma (TOS) of jaws in a paediatric patient is still a very rare variant and till date only two cases have been reported in literature. Radiographic evaluation plays an important role as the clinical symptoms are not specific to the condition. Conventional radiography should always be supplemented by advanced imaging modalities for the diagnosis, which gives a three dimensional assessment and also prevents superimposition of surrounding structures. Early diagnosis and radical surgery are the keys to high survival rates. Herein, we present a third ever reported case of TOS occurring in mandible of a nine-year-old male patient.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

PubMed

Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest

PubMed Central

Gascoyne, Duncan M.; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E.; Croucher, Peter I.; Banham, Alison H.

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology. PMID:26034982

Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

PubMed

Costa-Rodrigues, J; Teixeira, C A; Fernandes, M H

2011-08-01

Although in the past little attention has been paid to the influence of osteosarcoma cells in osteoclast function, recent studies suggest a close relationship between osteosarcoma aggressiveness and osteoclastic activity. The present study addresses the paracrine effects of MG63 cells, a human osteosarcoma-derived cell line, on the differentiation of peripheral blood osteoclast precursor cells (PBMC). PBMC were cultured for 21 days in the presence of conditioned media from MG63 cell cultures (CM) collected at 48 h (CM_MG1), 7 days (CM_MG2) and 14 days (CM_MG3). MG63 cell cultures displayed the expression of ALP and BMP-2 and, also, the osteoclastogenic genes M-CSF and RANKL, although with a low expression of RANKL. PBMC cultures supplemented with CM presented an evident osteoclastogenic behavior, which was dependent on the culture period of the MG63 cells. The inductive effect appeared to be more relevant for the differentiation and activation genes, c-myc and c-src, and lower for genes associated with osteoclast function. In addition, PBMC cultures displayed increased functional parameters, including calcium phosphate resorbing activity. Assessment of the PBMC cultures in the presence of U0126, PDTC, and indomethacin suggested that in addition to MEK and NFkB pathways, other signaling mechanisms, probably not involving RANKL/RANK interaction, might be activated in the presence of conditioned medium from MG63. In conclusion, MG63 cell line appears to induce a significant paracrine-mediated osteoclastogenic response. Understanding the mechanisms underlying the interaction of osteosarcoma cells and osteoclasts may contribute to the development of new potential approaches in the treatment of such bone metabolic diseases.

Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

PubMed Central

Hsieh, I-Shan; Yang, Rong-Sen; Fu, Wen-Mei

2014-01-01

Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients. PMID:25310823

In vitro and in vivo antitumor effects of the VO-chrysin complex on a new three-dimensional osteosarcoma spheroids model and a xenograft tumor in mice.

PubMed

León, Ignacio E; Cadavid-Vargas, Juan F; Resasco, Agustina; Maschi, Fabricio; Ayala, Miguel A; Carbone, Cecilia; Etcheverry, Susana B

2016-12-01

Osteosarcoma (OS) is the most common primary tumor of bone, occurring predominantly in the second decade of life. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized disease. Vanadium is an ultra-trace element that after being absorbed accumulates in bone. Besides, vanadium compounds have been studied during recent years to be considered as representative of a new class of non-platinum antitumor agents. Moreover, flavonoids are a wide family of polyphenolic compounds that display many interesting biological effects. Since coordination of ligands to metals can improve the pharmacological properties, we report herein, for the first time, the in vitro and in vivo effects of an oxidovanadium(IV) complex with the flavonoid chrysin on the new 3D human osteosarcoma and xenograft osteosarcoma mice models. The pharmacological results show that VOchrys inhibited the cell viability affecting the shape and volume of the spheroids and VOchrys suppressed MG-63 tumor growth in the nude mice without inducing toxicity and side effects. As a whole, the results presented herein demonstrate that the antitumor action of the complex was very promissory on human osteosarcoma models, whereby suggesting that VOchrys is a potentially good candidate for future use in alternative antitumor treatments.

[Magnetic resonance for the study of osteosarcoma].

PubMed

Spina, V; Romagnoli, R; Manfrini, M; Cerofolini, E; Capanna, R; Gaiani, L; Calandra Buonaura, P; Picci, P; Campanacci, M

1991-01-01

The authors report their experience with MR imaging in the study of osteosarcoma. Two main elements were evaluated: signal characteristics and loco-regional staging. Seventy-one patients were studied: 65 of them had central long-bone osteosarcoma, and 6 had telangiectatic long-bone osteosarcoma. T1- and T2-weighted spin-echo sequences were employed and all cases were scanned on 3 planes (sagittal, coronal, and axial). In 28 patients MR imaging was performed both before and after preoperative chemotherapy. The obtained data were compared to surgical and pathological findings. With the exception of the typical signal patterns of quite-osteoblastic osteosarcoma (which presents with low signal on both T1- and T2-weighted sequences), no particular signal features were observed which could help distinguish the different types of osteosarcoma. MR imaging is the method of choice in loco-regional staging for, in our series, it allowed a rational and adequate surgical planning. For this purpose, at least a longitudinal T1- and an axial T2-weighted images are required.

EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance.

PubMed

Sevelda, Florian; Mayr, Lisa; Kubista, Bernd; Lötsch, Daniela; van Schoonhoven, Sushilla; Windhager, Reinhard; Pirker, Christine; Micksche, Michael; Berger, Walter

2015-11-02

Enhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines. We have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed. Osteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3β). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential. Our data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.

Murine models of osteosarcoma: A piece of the translational puzzle.

PubMed

Walia, Mannu K; Castillo-Tandazo, Wilson; Mutsaers, Anthony J; Martin, Thomas John; Walkley, Carl R

2018-06-01

Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome. © 2017 Wiley Periodicals, Inc.

Elevated expression of CXC chemokines in pediatric osteosarcoma patients.

PubMed

Li, Yiting; Flores, Ricardo; Yu, Alexander; Okcu, M Fatih; Murray, Jeffrey; Chintagumpala, Murali; Hicks, John; Lau, Ching C; Man, Tsz-Kwong

2011-01-01

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis. The authors used an antibody microarray to identify chemokines that were elevated in the plasma samples of osteosarcoma patients. The results were validated using enzyme-linked immunosorbent assays on an independent set of samples. The tumor expressions of 3 chemokines were examined in 2 sets of osteosarcoma tissue arrays. The authors also evaluated the proliferative effect of the chemokines in 4 osteosarcoma cell lines. The authors found that the plasma levels of CXCL4, CXCL6, and CXCL12 in the osteosarcoma patients were significantly higher than those in the controls, and the results were validated by an independent osteosarcoma cohort (P < .05). However, CXCL4 (100%) and CXCL6 (91%) were frequently expressed in osteosarcoma, whereas CXCL12 was only expressed in 4%. Survival analysis further showed that higher circulating levels of CXCL4 and CXCL6, but not CXCL12, were associated with a poorer outcome of osteosarcoma patients. Addition of exogenous chemokines significantly promoted the growth of different osteosarcoma cells (P < .05). The results demonstrate that CXCL4 and CXCL6 are frequently expressed in osteosarcoma, and that the plasma levels of these 2 chemokines are associated with patient outcomes. Further study of these circulating chemokines may provide a promising approach for prognostication of osteosarcoma. Targeting these chemokines or their receptors may also lead to a novel therapeutic invention. © 2010 American Cancer Society.

The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin α5β1, p38 MAPK, and matrix metalloproteinases.

PubMed

Odagiri, Haruki; Kadomatsu, Tsuyoshi; Endo, Motoyoshi; Masuda, Tetsuro; Morioka, Masaki Suimye; Fukuhara, Shigetomo; Miyamoto, Takeshi; Kobayashi, Eisuke; Miyata, Keishi; Aoi, Jun; Horiguchi, Haruki; Nishimura, Naotaka; Terada, Kazutoyo; Yakushiji, Toshitake; Manabe, Ichiro; Mochizuki, Naoki; Mizuta, Hiroshi; Oike, Yuichi

2014-01-21

The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation-related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin α5β1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. Expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

Immunotherapy with a HER2-Targeting Listeria Induces HER2-Specific Immunity and Demonstrates Potential Therapeutic Effects in a Phase I Trial in Canine Osteosarcoma.

PubMed

Mason, Nicola J; Gnanandarajah, Josephine S; Engiles, Julie B; Gray, Falon; Laughlin, Danielle; Gaurnier-Hausser, Anita; Wallecha, Anu; Huebner, Margie; Paterson, Yvonne

2016-09-01

Recombinant Listeria vaccines induce tumor-specific T-cell responses that eliminate established tumors and prevent metastatic disease in murine cancer models. We used dogs with HER2/neu(+) appendicular osteosarcoma, a well-recognized spontaneous model for pediatric osteosarcoma, to determine whether a highly attenuated, recombinant Listeria monocytogenes expressing a chimeric human HER2/neu fusion protein (ADXS31-164) could safely induce HER2/neu-specific immunity and prevent metastatic disease. Eighteen dogs that underwent limb amputation or salvage surgery and adjuvant chemotherapy were enrolled in a phase I dose escalation clinical trial and received either 2 × 10(8), 5 × 10(8), 1 × 10(9), or 3.3 × 10(9) CFU of ADXS31-164 intravenously every 3 weeks for 3 administrations. Only low-grade, transient toxicities were observed. ADXS31-164 broke peripheral tolerance and induced antigen-specific IFNγ responses against the intracellular domain of HER2/neu in 15 of 18 dogs within 6 months of treatment. Furthermore, ADXS31-164 reduced the incidence of metastatic disease and significantly increased duration of survival time and 1-, 2-, and 3-year survival rates when compared with a historical control group with HER2/neu(+) appendicular osteosarcoma treated with amputation and chemotherapy alone. These findings demonstrate that ADXS31-164 administered in the setting of minimal residual disease can induce HER2/neu-specific immunity and may reduce the incidence of metastatic disease and prolong overall survival in a clinically relevant, spontaneous, large animal model of cancer. These findings, therefore, have important translational relevance for children with osteosarcoma and adults with other HER2/neu(+) cancers. Clin Cancer Res; 22(17); 4380-90. ©2016 AACR. ©2016 American Association for Cancer Research.

[Jaw osteosarcomas].

PubMed

Steve, M; Ernenwein, D; Chaine, A; Bertolus, C; Goudot, P; Ruhin-Poncet, B

2011-11-01

Osteosarcoma (OS) is the most frequent bone malignant tumor. It is usually found on long bones, 5 to 10% are located on jaws, accounting for 0.5 to 1% of all facial tumors. There is little published data which concerns only few patients. Our aim was to study retrospectively cases of facial bone OS in adults, and to compare our results with published data to suggest an optimal management scheme. Thirty-three patients were managed for an OS, from January 1997 to January 2007. Fourteen patients with a maxillary and mandibular OS, treated in first-intention in our unit, were included. The following data were analyzed: age; personal history; circumstance of discovery; clinical, functional, and physical signs; loco-regional extension and metastasis radiological investigation. The histological slides were systematically reviewed. The protocol, therapeutic outcome, and follow-up were studied. The mean age at diagnosis was 43. Swelling was the most frequent functional sign. The mean delay before management was 3.4 months. The most frequent radiological presentation was a lytic and hyperdense image. The diagnosis was suggested after CT scan in 57.1% of cases. The biopsy was correlated to the anatomopathological analysis in 78.6% of cases. The most common treatment was surgical exeresis completed by chemotherapy. The 5-year survival rate was 50%. Jaw OS are specific because of their localization and specific bone ultrastructure. Their management remains controversial: should they be managed like limb OS or treated more specifically? Neoadjuvant chemotherapy, even if it delays exeresis for 3 months, seems to stop the growth or reduce the tumor. An early anatomopathological analysis of the surgical piece determines adjuvant therapy. The negative prognostic factors are: maxillary localization because of limited exeresis margins, tumoral size, and osteoblastic sub-type. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Efficient drug delivery using SiO2-layered double hydroxide nanocomposites.

PubMed

Li, Li; Gu, Zi; Gu, Wenyi; Liu, Jian; Xu, Zhi Ping

2016-05-15

MgAl-layered double hydroxide (MgAl-LDH) nanoparticles have great potentials in drug and siRNA delivery. In this work, we used a nanodot-coating strategy to prepare SiO2 dot-coated layered double hydroxide (SiO2@MgAl-LDH) nanocomposites with good dispersibility and controllable size for drug delivery. The optimal SiO2@MgAl-LDH nanocomposite was obtained by adjusting synthetic parameters including the mass ratio of MgAl-LDH to SiO2, the mixing temperature and time. The optimal SiO2@MgAl-LDH nanocomposite was shown to have SiO2 nanodots (10-15nm in diameter) evenly deposited on the surface of MgAl-LDHs (110nm in diameter) with the plate-like morphology and the average hydrodynamic diameter of 170nm. We further employed SiO2@MgAl-LDH nanocomposite as a nanocarrier to deliver methotrexate (MTX), a chemotherapy drug, to the human osteosarcoma cell (U2OS) and found that MTX delivered by SiO2@MgAl-LDH nanocomposite apparently inhibited the U2OS cell growth. Copyright © 2016 Elsevier Inc. All rights reserved.

Pigment epithelium-derived factor upregulates collagen I and downregulates matrix metalloproteinase 2 in osteosarcoma cells, and colocalises to collagen I and heat shock protein 47 in fetal and adult bone.

PubMed

Alcantara, Marice B; Nemazannikova, Natalie; Elahy, Mina; Dass, Crispin R

2014-11-01

Pigment epithelium-derived factor (PEDF) has proven anti-osteosarcoma activity. However, the mechanism(s) underpinning its ability to reduce primary bone tumour (osteosarcoma) metastasis is unknown. Adult and fetal murine bone were immunostained for PEDF, collagen I (major protein in bone) and its processing proteins, heat shock protein 47 (HSP47, a chaperone protein for collagen I), membrane type I matrix metalloproteinase (MT1-MMP, a collagenase), and matrix metalloproteinase 2 (MMP-2, which is activated by MT1-MMP). Immunoblotting and immunocytochemistry were used to observe levels of the above biomarkers when human osteosarcoma cells were treated with PEDF. Immunohistochemical staining in adult and fetal bone mirrors collagen I. PEDF localised to ridges of trabecular bone in tibial cortex and to megakaryocytes within bone marrow. Second, we observed that PEDF upregulates collagen I, HSP47 and MT1-MMP, while downregulating MMP-2 in osteosarcoma cells in vitro. PEDF is a promising antagonist to osteosarcoma cell metastasis via downregulation of MMP-2, and can induce tumour cells to further adopt differentiative properties, thereby possibly reducing their aggressive growth in vitro and in vivo. © 2014 Royal Pharmaceutical Society.

Histone Deacetylase Inhibitor Trichostatin a Promotes the Apoptosis of Osteosarcoma Cells through p53 Signaling Pathway Activation

PubMed Central

Deng, Zhantao; Liu, Xiaozhou; Jin, Jiewen; Xu, Haidong; Gao, Qian; Wang, Yong; Zhao, Jianning

2016-01-01

Purpose: The purpose of this study was to investigate the profile of histone deacetylase (HDAC) activity and expression in osteosarcoma cells and tissues from osteosarcoma patients and to examine the mechanism by which a histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), promotes the apoptosis of osteosarcoma cells. Methods: HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of MG63 cells, hFOB 1.19 cells and tissues from 6 patients with primary osteosarcoma. The protein expression of Class I HDACs (1, 2, 3 and 8) and the activation of the p53 signaling pathway were examined by Western blot. Cell growth and apoptosis were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. Results: Nuclear HDAC activity and class I HDAC expression were significantly higher in MG63 cells than in hFOB 1.19 cells, and a similar trend was observed in the human osteosarcoma tissues compared with the paired adjacent non-cancerous tissues. TSA significantly inhibited the growth of MG63 cells and promoted apoptosis in a dose-dependent manner through p53 signaling pathway activation. Conclusion: Class I HDACs play a central role in the pathogenesis of osteosarcoma, and HDAC inhibitors may thus have promise as new therapeutic agents against osteosarcoma. PMID:27877082

Verification of TREX1 as a promising indicator of judging the prognosis of osteosarcoma.

PubMed

Feng, Jinyi; Lan, Ruilong; Cai, Guanxiong; Lin, Jinluan; Wang, Xinwen; Lin, Jianhua; Han, Deping

2016-11-24

The study aimed to explore the correlation between the expression of TREX1 and the metastasis and the survival time of patients with osteosarcoma as well as biological characteristics of osteosarcoma cells for the prognosis judgment of osteosarcoma. The correlation between the expression of TREX1 protein and the occurrence of pulmonary metastasis in 45 cases of osteosarcoma was analyzed. The CD133 + and CD133 - cell subsets of osteosarcoma stem cells were sorted by the flow cytometry. The tumorsphere culture, clone formation, growth curve, osteogenic and adipogenic differentiation, tumor-formation ability in nude mice, sensitivity of chemotherapeutic drugs, and other cytobiology behaviors were compared between the cell subsets in two groups; the expressions of stem cell-related genes Nanog and Oct4 were compared; The expressions of TREX1 protein and mRNA were compared between the cell subsets in two groups. The data was statistically analyzed. The measurement data between the two groups were compared using t test. The count data between the two groups were compared using χ 2 test and Kaplan-Meier survival analysis. A P value <0.05 indicated that the difference was statistically significant. The expression of TREX1 protein in patients with osteosarcoma in the metastasis group was significantly lower than that in the non-metastasis group. The difference was statistically significant (P < 0.05). Up to the last follow-up visit, the former average survival time was significantly lower than that of the latter, and the difference was statistically significant (P < 0.05). The expression of TREX1 in human osteosarcoma CD133 + cell subsets was significantly lower than that in CD133 - cell subsets. Stemness-related genes Nanog and Oct4 were highly expressed in human osteosarcoma CD133 + cell subsets with lower expression of TREX1; the biological characteristics identification experiment showed that human CD133 + cell subsets with low TREX1 expression could form

Sensitivity of osteosarcoma cells to HDAC inhibitor AR-42 mediated apoptosis.

PubMed

Murahari, Sridhar; Jalkanen, Aimee L; Kulp, Samuel K; Chen, Ching-Shih; Modiano, Jaime F; London, Cheryl A; Kisseberth, William C

2017-01-21

Osteosarcoma (OS) is the most common primary bone tumor in both humans and dogs and is the second leading cause of cancer related deaths in children and young adults. Limb sparing surgery along with chemotherapy has been the mainstay of treatment for OS. Many patients are not cured with current therapies, presenting a real need for developing new treatments. Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents. In this study, we investigated the activity of the novel HDAC inhibitor AR-42 in a panel of human and canine OS cell lines. The effect of AR-42 and suberoylanilide hydroxamic acid (SAHA) alone or in combination with doxorubicin on OS cell viability was assessed. Induction of histone acetylation after HDAC inhibitor treatment was confirmed by Western blotting. Drug-induced apoptosis was analyzed by FACS. Apoptosis was assessed further by measuring caspase 3/7 enzymatic activity, nucleosome fragmentation, and caspase cleavage. Effects on Akt signaling were demonstrated by assessing phosphorylation of Akt and downstream signaling molecules. AR-42 was a potent inhibitor of cell viability and induced a greater apoptotic response compared to SAHA when used at the same concentrations. Normal osteoblasts were much less sensitive. The combination of AR-42 with doxorubicin resulted in a potent inhibition of cell viability and apparent synergistic effect. Furthermore, we showed that AR-42 and SAHA induced cell death via the activation of the intrinsic mitochondrial pathway through activation of caspase 3/7. This potent apoptotic activity was associated with the greater ability of AR-42 to downregulate survival signaling through Akt. These results confirm that AR-42 is a potent inhibitor of HDAC activity and demonstrates its ability to significantly inhibit cell survival through its pleiotropic effects in both canine and human OS cells and suggests that spontaneous OS in pet dogs may be a useful large animal model for preclinical

A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies.

PubMed

Lee, N; Smolarz, A J; Olson, S; David, O; Reiser, J; Kutner, R; Daw, N C; Prockop, D J; Horwitz, E M; Gregory, C A

2007-12-03

Canonical Wnt signalling is an osteoinductive signal that promotes bone repair through acceleration of osteogenic differentiation by progenitors. Dkk-1 is a secreted inhibitor of canonical Wnt signalling and thus inhibits osteogenesis. To examine a potential osteoinhibitory role of Dkk-1 in osteosarcoma (OS), we measured serum Dkk-1 in paediatric patients with OS (median age, 13.4 years) and found it to be significantly elevated. We also found that Dkk-1 was maximally expressed by the OS cells at the tumour periphery and in vitro, Dkk-1 and RANKL are coexpressed by rapidly proliferating OS cells. Both Dkk-1 and conditioned media from OS cells reduce osteogenesis by human mesenchymal cells and by immunodepletion of Dkk-1, or by adding a GSK3beta inhibitor, the effects of Dkk-1 were attenuated. In mice, we found that the expression of Dkk-1 from implanted tumours was similar to the human tumour biopsies in that human Dkk-1 was present in the serum of recipient animals. These data demonstrate that systemic levels of Dkk-1 are elevated in OS. Furthermore, the expression of Dkk-1 by the OS cells at the periphery of the tumour probably contributes to its expansion by inhibiting repair of the surrounding bone. These data demonstrate that Dkk-1 may serve as a prognostic or diagnostic marker for evaluation of OS and furthermore, immunodepletion of Dkk-1 or administration of GSK3beta inhibitors could represent an adjunct therapy for this disease.

PERSPECTIVES ON CANCER STEM CELLS IN OSTEOSARCOMA

PubMed Central

Basu-Roy, Upal; Basilico, Claudio; Mansukhani, Alka

2012-01-01

Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. These mesenchymal tumors are derived from progenitor cells in the osteoblast lineage that have accumulated mutations to escape cell cycle checkpoints leading to excessive proliferation and defects in their ability to differentiate appropriately into mature bone-forming osteoblasts. Like other malignant tumors, osteosarcoma is often heterogeneous, consisting of phenotypically distinct cells with features of different stages of differentiation. The cancer stem cell hypothesis posits that tumors are maintained by stem cells and it is the incomplete eradication of a refractory population of tumor-initiating stem cells that accounts for drug resistance and tumor relapse. In this review we present our current knowledge about the biology of osteosarcoma stem cells from mouse and human tumors, highlighting new insights and unresolved issues in the identification of this elusive population. We focus on factors and pathways that are implicated in maintaining such cells, and differences from paradigms of epithelial cancers. Targeting of the cancer stem cells in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating childhood cancer. PMID:22659734

MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.

PubMed

Fenger, Joelle M; Roberts, Ryan D; Iwenofu, O Hans; Bear, Misty D; Zhang, Xiaoli; Couto, Jason I; Modiano, Jaime F; Kisseberth, William C; London, Cheryl A

2016-10-10

MicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS. We evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion. We identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified

Expression of nociceptive ligands in canine osteosarcoma.

PubMed

Shor, S; Fadl-Alla, B A; Pondenis, H C; Zhang, X; Wycislo, K L; Lezmi, S; Fan, T M

2015-01-01

Canine osteosarcoma (OS) is associated with localized pain as a result of tissue injury from tumor infiltration and peritumoral inflammation. Malignant bone pain is caused by stimulation of peripheral pain receptors, termed nociceptors, which reside in the localized tumor microenvironment, including the periosteal and intramedullary bone cavities. Several nociceptive ligands have been determined to participate directly or indirectly in generating bone pain associated with diverse skeletal abnormalities. Canine OS cells actively produce nociceptive ligands with the capacity to directly or indirectly activate peripheral pain receptors residing in the bone tumor microenvironment. Ten dogs with appendicular OS. Expression of nerve growth factor, endothelin-1, and microsomal prostaglandin E synthase-1 was characterized in OS cell lines and naturally occurring OS samples. In 10 dogs with OS, circulating concentrations of nociceptive ligands were quantified and correlated with subjective pain scores and tumor volume in patients treated with standardized palliative therapies. Canine OS cells express and secrete nerve growth factor, endothelin-1, and prostaglandin E2. Naturally occurring OS samples uniformly express nociceptive ligands. In a subset of OS-bearing dogs, circulating nociceptive ligand concentrations were detectable but failed to correlate with pain status. Localized foci of nerve terminal proliferation were identified in a minority of primary bone tumor samples. Canine OS cells express nociceptive ligands, potentially permitting active participation of OS cells in the generation of malignant bone pain. Specific inhibitors of nociceptive ligand signaling pathways might improve pain control in dogs with OS. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of American College of Veterinary Internal Medicine.

A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis

PubMed Central

Moriarity, Branden S; Otto, George M; Rahrmann, Eric P; Rathe, Susan K; Wolf, Natalie K; Weg, Madison T; Manlove, Luke A; LaRue, Rebecca S; Temiz, Nuri A; Molyneux, Sam D; Choi, Kwangmin; Holly, Kevin J; Sarver, Aaron L; Scott, Milcah C; Forster, Colleen L; Modiano, Jaime F; Khanna, Chand; Hewitt, Stephen M; Khokha, Rama; Yang, Yi; Gorlick, Richard; Dyer, Michael A; Largaespada, David A

2016-01-01

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. PMID:25961939

Minnelide reduces tumor burden in preclinical models of osteosarcoma.

PubMed

Banerjee, Sulagna; Thayanithy, Venugopal; Sangwan, Veena; Mackenzie, Tiffany N; Saluja, Ashok K; Subramanian, Subbaya

2013-07-28

Osteosarcoma is the most common bone cancer in children and adolescents with a 5-year survival rate of about 70%. In this study, we have evaluated the preclinical therapeutic efficacy of the novel synthetic drug, Minnelide, a prodrug of triptolide on osteosarcoma. Triptolide was effective in significantly inducing apoptosis in all osteosarcoma cell lines tested but had no significant effect on the human osteoblast cells. Notably, Minnelide treatment significantly reduced tumor burden and lung metastasis in the orthotopic and lung colonization models. Triptolide/Minnelide effectively downregulated the levels of pro-survival proteins such as heat shock proteins, cMYC, survivin and targets the NF-κB pathway. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

MicroRNAs for osteosarcoma in the mouse: a meta-analysis

PubMed Central

Chang, Junli; Yao, Min; Li, Yimian; Zhao, Dongfeng; Hu, Shaopu; Cui, Xuejun; Liu, Gang; Shi, Qi; Wang, Yongjun; Yang, Yanping

2016-01-01

Osteosarcoma (OS) is the most common primary malignant bone carcinoma with high morbidity that happens mainly in children and young adults. As the key components of gene-regulatory networks, microRNAs (miRNAs) control many critical pathophysiological processes, including initiation and progression of cancers. The objective of this study is to summarize and evaluate the potential of miRNAs as targets for prevention and treatment of OS in mouse models, and to explore the methodological quality of current studies. We searched PubMed, Web of Science, Embase, Wan Fang Database, VIP Database, China Knowledge Resource Integrated Database, and Chinese BioMedical since their beginning date to 10 May 2016. Two reviewers separately screened the controlled studies, which estimate the effects of miRNAs on osteosarcoma in mice. A pair-wise analysis was performed. Thirty six studies with enough randomization were selected and included in the meta-analysis. We found that blocking oncogenic or restoring decreased miRNAs in cancer cells could significantly suppress the progression of OS in vivo, as assessed by tumor volume and tumor weight. This meta-analysis suggests that miRNAs are potential therapeutic targets for OS and correction of the altered expression of miRNAs significantly suppresses the progression of OS in mouse models, however, the overall methodological quality of studies included here was low, and more animal studies with the rigourous design must be carried out before a miRNA-based treatment could be translated from animal studies to clinical trials. PMID:27852052

The effects of baicalein on canine osteosarcoma cell proliferation and death.

PubMed

Helmerick, E C; Loftus, J P; Wakshlag, J J

2014-12-01

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti-inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage-independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response. © 2012 Blackwell Publishing Ltd.

Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression.

PubMed

Zhou, Yan; Zhao, Rui-hua; Tseng, Kuo-fu; Li, Kun-peng; Lu, Zhi-gang; Liu, Yuan; Han, Kun; Gan, Zhi-hua; Lin, Shu-chen; Hu, Hai-yan; Min, Da-liu

2016-04-01

Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro. Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays. Sirolimus (1-100 nmol/L) dose-dependently suppressed the cell proliferation (IC50=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC50 values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group. Sirolimus increases the sensitivity of human OS cells to anticancer drugs in vitro by

Cathepsin K expression and activity in canine osteosarcoma.

PubMed

Schmit, J M; Pondenis, H C; Barger, A M; Borst, L B; Garrett, L D; Wypij, J M; Neumann, Z L; Fan, T M

2012-01-01

Cathepsin K (CatK) is a lysosomal protease with collagenolytic activity, and its secretion by osteoclasts is responsible for degrading organic bone matrix. People with pathologic bone resorption have higher circulating CatK concentrations. Canine osteosarcoma (OS) cells will possess CatK, and its secretion will be cytokine inducible. Circulating CatK concentrations will be increased in dogs with OS, and will be a surrogate marker of bone resorption. Fifty-one dogs with appendicular OS and 18 age- and weight-matched healthy control dogs. In a prospective study, expressions of CatK mRNA and protein were investigated in OS cells. The inducible secretion and proteolytic activity of CatK from OS cells was assessed in vitro. Serum CatK concentrations were quantified in normal dogs and dogs with OS and its utility as a bone resorption marker was evaluated in dogs with OS treated with palliative radiation and antiresorptive agents. Canine OS cells contain preformed CatK within cytoplasmic vesicles. In OS cells, TGFβ1 induced the secretion of CatK, which degraded bone-derived type I collagen in vitro. CatK concentrations were higher in dogs with OS than healthy dogs (11.3 ± 5.2 pmol/L versus 8.1 ± 5.0 pmol/L, P = .03). In a subset of dogs with OS, pretreatment CatK concentrations gradually decreased after palliative radiation and antiresorptive treatment, from 9.3 ± 3.2 pmol/L to 5.0 ± 3.1 pmol/L, P = .03. Canine OS is associated with pathologic bone resorption, and CatK inhibitors might aid in the management of canine OS-related malignant osteolysis. Copyright © 2011 by the American College of Veterinary Internal Medicine.

Phase-II trials in osteosarcoma recurrences: A systematic review of past experience.

PubMed

Omer, Natacha; Le Deley, Marie-Cécile; Piperno-Neumann, Sophie; Marec-Berard, Perrine; Italiano, Antoine; Corradini, Nadège; Bellera, Carine; Brugières, Laurence; Gaspar, Nathalie

2017-04-01

The most appropriate design of Phase-II trials evaluating new therapies in osteosarcoma remains poorly defined. To study consistency in phase-II clinical trials evaluating new therapies for osteosarcoma recurrences with respect to eligibility criteria, response assessment, end-points, statistical design and reported results. Systematic review of clinical trials registered on clinicaltrials.gov, clinicaltrialsregister.eu and French National Cancer Institute website or referenced in PubMed and American Society of Clinical Oncology websites, between 2003 and 2016, using the following criteria: (osteosarcoma OR bone sarcoma) AND (Phase-II). Among the 99 trials identified, 80 were Phase-II, 17 I/II and 2 II/III, evaluating mostly targeted therapy (n = 40), and chemotherapy alone (n = 26). Results were fully (n = 28) or partially (abstract, n = 6) published. Twenty-four trials were dedicated to osteosarcoma, 22 had an osteosarcoma stratum. Twenty-eight out of 99 trials refer to the age range observed at recurrence (28%). Overall, 65 trials were run in multicentre settings, including 17 international trials. Only 9 trials were randomised. The primary end-point was tumour response in 71 trials (response rate, n = 40 or best response, n = 31), with various definitions (complete + partial ± minor response and stable disease), mainly evaluated with RECIST criteria (n = 69); it was progression-free survival in 24 trials and OS in 3. In single-arm trials evaluating response rate, the null hypothesis tested (when available, n = 12) varied from 5% to 25%. No robust historical data can currently be derived from past efficacy Phase-II trials. There is an urgent need to develop international randomised Phase-II trials across all age ranges with standardised primary end-point. Copyright © 2017 Elsevier Ltd. All rights reserved.

Oxymatrine extracted from Sophora flavescens inhibited cell growth and induced apoptosis in human osteosarcoma MG-63 cells in vitro.

PubMed

Wei, Jianghua; Zhu, Yin; Xu, Gang; Yang, Fan; Guan, Zhe; Wang, Mao; Fang, Yonghong

2014-11-01

Oxymatrine, one of the most active components of the ethanol extracts from Sophora flavescens, is known for its potent antitumor activity both in vitro and in vivo. However, the mechanism of its action in mediating the cell apoptosis remains elusive. In this study, we investigated the proliferation inhibitory and apoptotic activities of oxymatrine against human osteosarcoma MG-63 cells. The compound was found to markedly and dose-dependently inhibit the cell proliferation determined by 5-bromo-2-deoxyuridine incorporation. Oxymatrine also induced the cell apoptosis in a dose- and time-dependent manner as showed by the annexin V-FITC/PI double staining and TUNEL assay. Furthermore, a disruption of mitochondrial membrane potential and an up-regulation of cleaved caspases-3, and-9 and downregulation of Bax/Bcl-2 was evidenced in the oxymatrine-treated cells. These proteins have been known to play a pivotal role in the regulation of apoptosis. In conclusion, these observations indicate of the oxymatrine potential as an effective antitumor agent against osteosarcoma. Moreover, the compound appears to exert its anti-tumor action by stimulating the caspase-triggered signaling pathway.

Proteomic Analysis of Exosomes and Exosome-Free Conditioned Media From Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression.

PubMed

Jerez, Sofía; Araya, Héctor; Thaler, Roman; Charlesworth, M Cristine; López-Solís, Remigio; Kalergis, Alexis M; Céspedes, Pablo F; Dudakovic, Amel; Stein, Gary S; van Wijnen, Andre J; Galindo, Mario

2017-02-01

Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Impact of chemotherapy on the outcome of osteosarcoma of the head and neck in adults

PubMed Central

Boon, Eline; van der Graaf, Winette T. A.; Gelderblom, Hans; Tesselaar, Margot E. T.; van Es, Robert J. J.; Oosting, Sjoukje F.; de Bree, Remco; van Meerten, Esther; Hoeben, Ann; Smeele, Ludi E.; Willems, Stefan M.; Witjes, Max J. H.; Buter, Jan; Baatenburg de Jong, Robert J.; Flucke, Uta E.; Peer, Petronella G. M.; Bovée, Judith V. M. G.

2016-01-01

Abstract Background There is an ongoing debate about the value of (neo‐)adjuvant chemotherapy in high‐ and intermediate‐grade osteosarcoma of the head and neck. Methods All records of patients older than 16 years diagnosed with osteosarcoma of the head and neck in the Netherlands between 1993 and 2013 were reviewed. Results We identified a total of 77 patients with an osteosarcoma of the head and neck; the 5‐year overall survival (OS) was 55%. In 50 patients with surgically resected high‐ or intermediate‐grade osteosarcoma of the head and neck younger than 75 years, univariate and multivariable analysis, adjusting for age and resection margins, showed that patients who had not received chemotherapy had a significantly higher risk of local recurrence (hazard ratio [HR] = 3.78 and 3.66, respectively). Conclusion In patients younger than 75 years of age with surgically resected high‐ and intermediate‐grade osteosarcoma of the head and neck, treatment with (neo‐)adjuvant chemotherapy resulted in a significantly smaller risk of local recurrence. Therefore, we suggest (neo‐)adjuvant chemotherapy in patients amenable to chemotherapy. © 2016 The Authors Head & Neck Published by Wiley Periodicals, Inc. Head Neck 39: 140–146, 2017 PMID:27507299

Overexpressing circular RNA hsa_circ_0002052 impairs osteosarcoma progression via inhibiting Wnt/β-catenin pathway by regulating miR-1205/APC2 axis.

PubMed

Wu, Zhen; Shi, Wangping; Jiang, Chendi

2018-08-25

Circular RNAs (circRNAs) are a novel class of noncoding RNAs, whose importance in cancer has been gradually acknowledged. However, the functions of circRNAs in tumorigenesis have not been fully understood. In the present study, we identified a novel circRNA hsa_circ_0002052 significantly downregulated in osteosarcoma (OS) tissues and cell lines. Moreover, we found that hsa_circ_0002052 could act as a biomarker to indicate the prognosis of OS patients. Functionally, we showed that hsa_circ_0002052 overexpression significantly suppressed OS cell proliferation, migration and invasion while promoting apoptosis in vitro. Similarly, in vivo assay indicated that ectopic expression of hsa_circ_0002052 impaired OS cell growth. In terms of mechanism, we found that hsa_circ_0002052 inhibited miR-1205 while miR1205 targeted APC2, a negative regulator of Wnt/β-catenin signaling pathway. By releasing the inhibition of miR-1205 on APC2 expression, hsa_circ_0002052 suppressed the activation of Wnt/β-catenin signaling pathway, leading to attenuated OS progression. Taken together, our study for the first time revealed a suppressive circRNA hsa_circ_0002052 involved in OS progression. Our study suggested hsa_circ_0002052/miR-1205/APC2/Wnt/β-catenin axis might be a potential target for OS therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

Systematic screening identifies dual PI3K and mTOR inhibition as a conserved therapeutic vulnerability in osteosarcoma

PubMed Central

Gupte, Ankita; Baker, Emma K.; Wan, Soo-San; Stewart, Elizabeth; Loh, Amos; Shelat, Anang A.; Gould, Cathryn M.; Chalk, Alistair M.; Taylor, Scott; Lackovic, Kurt; Karlström, Åsa; Mutsaers, Anthony J.; Desai, Jayesh; Madhamshettiwar, Piyush B.; Zannettino, Andrew CW.; Burns, Chris; Huang, David CS.; Dyer, Michael A.; Simpson, Kaylene J.; Walkley, Carl R.

2015-01-01

Purpose Osteosarcoma (OS) is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of OS, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for OS patients. Experimental Design We have used genetically engineered murine models of human OS in a systematic, genome wide screen to identify new candidate therapeutic targets. We performed a genome wide siRNA screen, with or without doxorubicin. In parallel a screen of therapeutically relevant small molecules was conducted on primary murine and primary human OS derived cell cultures. All results were validated across independent cell cultures and across human and mouse OS. Results The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary OS cell cultures in vitro in both human and mouse OS, while specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on OS cell cultures. Conclusions The approaches described here have identified dual inhibition of the PI3K/mTOR pathway as a sensitive, druggable target in OS and provide rationale for translational studies with these agents. PMID:25862761

CXCR7 maintains osteosarcoma invasion after CXCR4 suppression in bone marrow microenvironment.

PubMed

Han, Yan; Wu, Chunlei; Wang, Jing; Liu, Na

2017-05-01

The major cause of death in osteosarcoma is the invasion and metastasis. Better understanding of the molecular mechanism of osteosarcoma invasion is essential in developing effective tumor-suppressive therapies. Interaction between chemokine receptors plays a crucial role in regulating osteosarcoma invasion. Here, we investigated the relationship between CXCR7 and CXCR4 in osteosarcoma invasion induced by bone marrow microenvironment. Human bone marrow mesenchymal stem cells were co-cultured with osteosarcoma cells to mimic actual bone marrow microenvironment. Osteosarcoma cell invasion and CXCL12/CXCR4 activation were observed within this co-culture model. Interestingly, in this co-culture model, osteosarcoma cell invasion was not inhibited by suppressing CXCR4 expression with neutralizing antibody or specific inhibitor AMD3100. Downstream signaling extracellular signal-regulated kinase and signal transducer and activator of transcription 3 were not significantly affected by CXCR4 inhibition. However, suppressing CXCR4 led to CXCR7 upregulation. Constitutive expression of CXCR7 could maintain osteosarcoma cell invasion when CXCR4 was suppressed. Simultaneously, inhibiting CXCR4 and CXCR7 compromised osteosarcoma invasion in co-culture system and suppressed extracellular signal-regulated kinase and signal transducer and activator of transcription 3 signals. Moreover, bone marrow microenvironment, not CXCL12 alone, is required for CXCR7 activation after CXCR4 suppression. Taken together, suppressing CXCR4 is not enough to impede osteosarcoma invasion in bone marrow microenvironment since CXCR7 is activated to sustain invasion. Therefore, inhibiting both CXCR4 and CXCR7 could be a promising strategy in controlling osteosarcoma invasion.

Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line.

PubMed

Qiang, Weiguang; Wu, Qinqin; Zhou, Fuxiang; Xie, Conghua; Wu, Changping; Zhou, Yunfeng

2014-03-07

Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined. Copyright © 2014 Elsevier Inc. All rights reserved.

Induction of osteosarcoma and acute myeloid leukaemia in CBA/H mice by the alpha-emitting nuclides, uranium-233, plutonium-239 and amercium-241.

PubMed

Ellender, M; Harrison, J D; Pottinger, H; Thomas, J M

2001-01-01

To compare tumour induction in CBA/H mice, principally osteosarcoma and acute myeloid leukaemia, resulting from exposure to the alpha-emitting nuclides, uranium-233, plutonium-239 and americium-241, and to relate differences between the three nuclides to the pattern of dose delivery within tissues. Each nuclide was administered intraperitoneally in citrate solution to three groups of adult male CBA/H mice at levels of activity which gave estimated life-time average skeletal doses of about 0.25-0.3 Gy, 0.5-1 Gy and 1-2 Gy. Animals were carefully monitored and sacrificed as soon as they showed signs of ill health; tumours were identified by standard histopathological techniques. Statistical modelling by Cox regression showed that, considering all three nuclides together, there was a highly significant increase in risk of death from osteosarcoma or myeloid leukaemia with increasing dose rate. For osteosarcoma, the effect was significantly greater for 239Pu than 241Am, while separate analysis for 233U showed no significant increase with increasing dose rate. For example, the increase in relative risk of death from osteosarcoma for an increase in life-time average dose rate to bone of 1 mGyd(-1) was 4.2 (2.7-6.5) for 239Pu, 2.3 (1.4-3.4) for 241Am and 1.1 (0.4-3.1) for 233U. For myeloid leukaemia, there was no significant difference between 239Pu and 241Am in the effect of dose rate. The increase in relative risk from myeloid leukaemia for an increase in average dose rate of 1 mGyd(-1) was 1.8 (1.1-2.8) for 239Pu, 2.0 (1.4-2.9) for 241Am and 1.5 (0.8-2.7) for 233U. Significant increases in renal and hepatic carcinomas were also recorded in animals exposed to 233U and 241Am, respectively. Studies of the distribution of the nuclides within the skeleton, published separately, have shown differences in their retention in individual bones and within bone. The proportions of decays occurring near to endosteal bone surfaces and throughout bone marrow were in the order: 239Pu

Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times

PubMed Central

Selvarajah, Gayathri T; Kirpensteijn, Jolle; van Wolferen, Monique E; Rao, Nagesha AS; Fieten, Hille; Mol, Jan A

2009-01-01

Background Gene expression profiling of spontaneous tumors in the dog offers a unique translational opportunity to identify prognostic biomarkers and signaling pathways that are common to both canine and human. Osteosarcoma (OS) accounts for approximately 80% of all malignant bone tumors in the dog. Canine OS are highly comparable with their human counterpart with respect to histology, high metastatic rate and poor long-term survival. This study investigates the prognostic gene profile among thirty-two primary canine OS using canine specific cDNA microarrays representing 20,313 genes to identify genes and cellular signaling pathways associated with survival. This, the first report of its kind in dogs with OS, also demonstrates the advantages of cross-species comparison with human OS. Results The 32 tumors were classified into two prognostic groups based on survival time (ST). They were defined as short survivors (dogs with poor prognosis: surviving fewer than 6 months) and long survivors (dogs with better prognosis: surviving 6 months or longer). Fifty-one transcripts were found to be differentially expressed, with common upregulation of these genes in the short survivors. The overexpressed genes in short survivors are associated with possible roles in proliferation, drug resistance or metastasis. Several deregulated pathways identified in the present study, including Wnt signaling, Integrin signaling and Chemokine/cytokine signaling are comparable to the pathway analysis conducted on human OS gene profiles, emphasizing the value of the dog as an excellent model for humans. Conclusion A molecular-based method for discrimination of outcome for short and long survivors is useful for future prognostic stratification at initial diagnosis, where genes and pathways associated with cell cycle/proliferation, drug resistance and metastasis could be potential targets for diagnosis and therapy. The similarities between human and canine OS makes the dog a suitable pre

P-glycoprotein binds to ezrin at amino acid residues 149-242 in the FERM domain and plays a key role in the multidrug resistance of human osteosarcoma.

PubMed

Brambilla, Daria; Zamboni, Silvia; Federici, Cristina; Lugini, Luana; Lozupone, Francesco; De Milito, Angelo; Cecchetti, Serena; Cianfriglia, Maurizio; Fais, Stefano

2012-06-15

Overexpression of the mdr1 gene encoding P-glycoprotein (Pgp) exerts a major role in reducing the effectiveness of cytotoxic therapy in osteosarcoma. The interaction between actin and Pgp has been shown to be instrumental in the establishment of multidrug resistance (MDR) in human tumor cells. The cytoskeleton linker ezrin exerts a pivotal role in maintaining the functional connection between actin and Pgp. We investigated the role of ezrin in a human multidrug-resistant osteosarcoma cell line overexpressing Pgp and compared it to its counterpart that overexpresses an ezrin deletion mutant. The results showed that Pgp binds at amino acid residues 149-242 of the N-terminal domain of ezrin. The interaction between ezrin and Pgp occurs in the plasma membrane of MDR cells, where they also co-localize with the ganglioside G(M1) located in lipid rafts. The overexpression of the ezrin deletion mutant entirely restored drug susceptibility of osteosarcoma cells, consistent with Pgp dislocation to cytoplasmic compartments and abrogation of G(M1) /Pgp co-localization at the plasma membrane. Our study provides evidence that ezrin exerts a key role in MDR of human osteosarcoma cells through a Pgp-ezrin-actin connection that is instrumental for the permanence of Pgp into plasma membrane lipid rafts. We also show for the first time that Pgp-binding site is localized to amino acid residues 149-242 of the ezrin Band 4.1, Ezrin/Radixin/Moesin (FERM) domain, thus proposing a specific target for future molecular therapy aimed at counteracting MDR in osteosarcoma patients. Copyright © 2011 UICC.

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

PubMed Central

Kim, Mihwa; Jung, Ji-Yeon; Choi, Seungho; Lee, Hyunseung; Morales, Liza D.; Koh, Jeong-Tae; Kim, Sun Hun; Choi, Yoo-Duk; Choi, Chan; Slaga, Thomas J.; Kim, Won Jae; Kim, Dae Joon

2017-01-01

ABSTRACT Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma. PMID:27754745

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy.

PubMed

Kim, Mihwa; Jung, Ji-Yeon; Choi, Seungho; Lee, Hyunseung; Morales, Liza D; Koh, Jeong-Tae; Kim, Sun Hun; Choi, Yoo-Duk; Choi, Chan; Slaga, Thomas J; Kim, Won Jae; Kim, Dae Joon

2017-01-02

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

[Amplification of γδ T cells in PBMCs of healthy donors and osteosarcoma patients stimulated by zoledronate].

PubMed

Li, Zhao-xu; Sun, Ling-ling; Cheng, Rui-lin; Sun, Zheng-wang; Ye, Zhao-ming

2012-08-01

To investigate the amplification and cytotoxicity of γδ T cells in peripheral blood mononuclear cells (PBMCs) of healthy donors and osteosarcoma patients stimulated by zoledronate (Zol) and IL-2. PBMCs from healthy donors and osteosarcoma patients were stimulated with IL-2 and Zol+IL-2, respectively. After 14-day culture, the purity of γδ T cells was assessed by flow cytometry. The cytotoxicity of γδ T cells against target cells was analyzed using a standard lactate dehydrogenase release assay with γδ T lymphocyte-sensitive Daudi cells, γδ T lymphocyte-resistant Raji cells and human osteoblast cell line, hFOB, as the target cells. After 2-week culture ex vivo of PBMCs from healthy donors and osteosarcoma patients, compared with stimulation of IL-2, Zol+IL-2 significantly promoted the amplification of γδ T cells. In addition, γδ T cells showed the higher cytotoxicity against Daudi cells, but no cytotoxic effect on normal cells like hFOB. γδ T cells of high purity and high cytotoxicity can be obtained by the stimulation of Zol combined with IL-2 on PBMCs from healthy donors and osteosarcoma patients.

Serum 25-hydroxyvitamin D concentrations in dogs with osteosarcoma do not differ from those of age- and weight-matched control dogs.

PubMed

Willcox, Jennifer L; Hammett-Stabler, Catherine; Hauck, Marlene L

2016-11-01

Vitamin D concentrations show an inverse correlation with incidence of certain tumors in people and dogs. Additionally, human osteosarcoma has been associated with dysregulation of vitamin D-dependent pathways. The study objective was to compare serum 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 in 20 dogs with osteosarcoma to age- and weight-matched control dogs. We hypothesized that dogs with osteosarcoma would have lower serum 25-hydroxyvitamin D than control dogs. The mean 25-hydroxyvitamin D 3 concentrations for dogs with osteosarcoma and matched-controls were 34.95 ng/mL and 33.85 ng/mL, respectively (P = 0.784). Based on these data, 25-hydroxyvitamin D insufficiency might not be important in the pathogenesis of canine osteosarcoma. Published by Elsevier Ltd.

Osteosarcoma: Diagnostic dilemmas in histopathology and prognostic factors

PubMed Central

Wadhwa, Neelam

2014-01-01

Osteosarcoma (OS), the commonest malignancy of osteoarticular origin, is a very aggressive neoplasm. Divergent histologic differentiation is common in OS; hence triple diagnostic approach is essential in all cases. 20% cases are atypical owing to lack of concurrence among clinicoradiologic and pathologic features necessitating resampling. Recognition of specific anatomic and histologic variants is essential in view of better outcome. Traditional prognostic factors of OS do stratify patients for short term outcome, but often fail to predict their long term outcome. Considering the negligible improvement in the patient outcome during the last 20 years, search for novel prognostic factors is in progress like ezrin vascular endothelial growth factor, chemokine receptors, dysregulation of various micro ribonucleic acid are potentially promising. Their utility needs to be validated by long term followup studies before they are incorporated in routine clinical practice. PMID:24932029

The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

PubMed

Daft, Paul G; Yang, Yang; Napierala, Dobrawa; Zayzafoon, Majd

2015-01-01

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

The Growth and Aggressive Behavior of Human Osteosarcoma Is Regulated by a CaMKII-Controlled Autocrine VEGF Signaling Mechanism

PubMed Central

Daft, Paul G.; Yang, Yang; Napierala, Dobrawa; Zayzafoon, Majd

2015-01-01

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease. PMID:25860662

Characterizing biomarkers in osteosarcoma metastasis based on an ego-network.

PubMed

Liu, Zhen; Song, Yan

2017-06-01

To characterize biomarkers that underlie osteosarcoma (OS) metastasis based on an ego-network. From the microarray data, we obtained 13,326 genes. By combining PPI data and microarray data, 10,520 shared genes were found and constructed into ego-networks. 17 significant ego-networks were identified with p < 0.05. In the pathway enrichment analysis, seven ego-networks were identified with the most significant pathway. These significant ego-modules were potential biomarkers that reveal the potential mechanisms in OS metastasis, which may contribute to understanding cancer prognoses and providing new perspectives in the treatment of cancer.

A Rb1 promoter variant with reduced activity contributes to osteosarcoma susceptibility in irradiated mice

PubMed Central

2014-01-01

Background Syndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility. Methods Tumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay. Results A common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation. Conclusion This is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant

Triple primary malignancies of surface osteosarcoma of jaw, myelodysplastic syndrome and colorectal cancer as a second primary cancer detected by PET2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography: A case report.

PubMed

Maruyama, Nobuyuki; Nishihara, Kazuhide; Nakasone, Toshiyuki; Saio, Masanao; Maruyama, Tessho; Tedokon, Iori; Ohira, Tetsuya; Nimura, Fumikazu; Matayoshi, Akira; Karube, Ken-Nosuke; Yoshimi, Naoki; Arasaki, Akira

2018-06-01

Second primary malignancy (SPM) is a severe issue for cancer survivors, particularly for osteosarcoma (OS) survivors. To date, the associations between subsequent SPM and OS have been well reported. Hematogenic and solid malignancies tend to occur following OS treatment. Reportedly, 2-[ 18 F]-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) is mainly used in OS patients for initial cancer staging, to evaluate the response of neoadjuvant chemotherapy, and when recurrence or metastasis is clinically suspected. The present case report describes a 70-year-old man diagnosed with three primary malignancies: jaw OS, myelodysplastic syndrome and colorectal adenocarcinoma. To the best of our knowledge, this combination of malignancies has not been reported previously. Until now, there is no specific protocol of postoperative FDG-PET for OS patients. Few studies have described OS follow-up methods; therefore, there is no consensus on proper follow-up methods. In the present case report, the colorectal early-stage SPM was observed, without any symptoms, by FDG-PET/computed tomography. To avoid overlooking solid SPMs, it is suggested that FDG-PET should be performed in the long-term follow-up of OS patients.

Does ATRX germline variation predispose to osteosarcoma? Three additional cases of osteosarcoma in two ATR-X syndrome patients.

PubMed

Masliah-Planchon, Julien; Lévy, Dominique; Héron, Delphine; Giuliano, Fabienne; Badens, Catherine; Fréneaux, Paul; Galmiche, Louise; Guinebretierre, Jean-Marc; Cellier, Cécile; Waterfall, Joshua J; Aït-Raïs, Khadija; Pierron, Gaëlle; Glorion, Christophe; Desguerre, Isabelle; Soler, Christine; Deville, Anne; Delattre, Olivier; Michon, Jean; Bourdeaut, Franck

2018-04-30

Osteosarcoma is the most common malignant bone tumor in adolescents and young adults. Most osteosarcomas are sporadic but the risk of osteosarcoma is also increased by germline variants in TP53, RB1 and RECQL4 genes. ATRX germline variations are responsible for the rare genetic disorder X-linked alpha-thalassemia mental retardation (ATR-X) syndrome characterized by severe developmental delay and alpha-thalassemia but no obvious increased risk of cancer. Here we report two children with ATR-X syndrome who developed osteosarcoma. Notably, one of the children developed two osteosarcomas separated by 10 years. Those two cases raise the possibility that ATRX germline variant could be associated with an increased risk of osteosarcoma.

Retrospective evaluation of toceranib (Palladia) treatment for canine metastatic appendicular osteosarcoma.

PubMed

Kim, Changseok; Matsuyama, Arata; Mutsaers, Anthony J; Woods, J Paul

2017-10-01

This retrospective study evaluated the outcomes of dogs with macroscopic pulmonary metastasis of appendicular osteosarcoma (OSA) treated with toceranib. Medical records of 20 dogs with macroscopic pulmonary metastasis of OSA that received toceranib were reviewed. The median dose and duration of toceranib administration were 2.52 mg/kg (range: 2.12 to 2.72 mg/kg) and 60 days (range: 17 to 231 days). The median progression free survival (PFS) and overall survival (OS) were 36 days (range: 17 to 231 days) and 90 days (range: 17 to 433 days), respectively. The clinical benefit rate was 10% (2/20; 1 partial response and 1 stable disease). The longest length of initial pulmonary nodules had significant impact on both PFS ( P = 0.01) and OS ( P = 0.02). The prognosis for dogs with metastatic OSA was poor with only 10% of dogs showing clinical benefit from toceranib. These results suggest that toceranib may not improve outcome in dogs with macroscopic pulmonary metastasis of OSA.

Effect of p27 gene combined with Pientzehuang ([characters: see text]) on tumor growth in osteosarcoma-bearing nude mice.

PubMed

Ren, Shou-song; Yuan, Fang; Liu, Ying-hong; Zhou, Le-tian; Li, Jun

2015-11-01

To observe the effect of p27 gene recombinant adenovirus combined with Chinese medicine Pientzehuang ([characters: see text]) on the growth of xenografted human osteosarcoma in nude mice. Tissue transplantation was used to construct the orthotopic model of human osteosarcoma Saos-2 cell in nude mice. Thirty tumor-bearing nude mice were randomly divided into 5 groups with 6 mice in each group: blank control group (model of osteosarcoma), empty vector group (recombinant adeno-associated virus-multiple cloning site), Pientzehuang group, p27 gene group and combined treatment group (p27 gene combined with Pientzehuang). The effect of combined treatment on human osteosarcoma was analyzed through the tumor formation, tumor volume and inhibition rate of tumor growth. The expression of p27 was measured by immunohistochemical staining and Western blot. The orthotopic model of osteosarcoma in nude mice was successfully constructed. The general appearance of tumor-bearing nude mice in Pientzehuang and p27 gene groups was markedly improved compared with the blank control group; and in the combined treatment group it was significantly improved compared with the Pientzehuang and p27 gene groups. The tumor growth in the Pientzehuang and p27 gene groups was significantly inhibited compared with the blank control group P<0.05); while in the combined treatment group it was markedly inhibited compared with the Pientzehuang and p27 gene groups (P<0.05). The rates of tumor growth inhibition were 34.1%, 56.5% and 63.8% in the Pientzehuang, p27 gene and combined treatment groups, respectively. Meanwhile, the protein expression of p27 gene in the p27 gene group was significantly increased compared with the blank control group (P<0.05); and it was significantly increased in the combined treatment group compared with the p27 gene and Pientzehuang groups (P<0.05). p27 gene introduced by adenovirus combined with Pientzehuang can inhibit the growth of human osteosarcoma cell Saos-2 in nude mice.

The effects of taurolidine alone and in combination with doxorubicin or carboplatin in canine osteosarcoma in vitro.

PubMed

Marley, Kevin; Helfand, Stuart C; Edris, Wade A; Mata, John E; Gitelman, Alix I; Medlock, Jan; Séguin, Bernard

2013-01-18

Osteosarcoma (OS) affects over 8000 dogs/year in the United States. The disease usually arises in the appendicular skeleton and metastasizes to the lung. Dogs with localized appendicular disease benefit from limb amputation and chemotherapy but most die within 6-12 months despite these treatments. Taurolidine, a derivative of taurine, has anti-tumor and anti-angiogenic effects against a variety of cancers. The following in vitro studies tested taurolidine as a candidate for adjuvant therapy for canine OS. Tests for p53 protein status and caspase activity were used to elucidate mechanisms of taurolidine-induced cell death. Taurolidine was cytotoxic to osteosarcoma cells and increased the toxicity of doxorubicin and carboplatin in vitro. Apoptosis was greatly induced in cells exposed to 125 μM taurolidine and less so in cells exposed to 250 μM taurolidine. Taurolidine cytotoxicity appeared caspase-dependent in one cell line; with apparent mutant p53 protein. This cell line was the most sensitive to single agent taurolidine treatment and had a taurolidine-dependent reduction in accumulated p53 protein suggesting taurolidine's effects may depend on the functional status of p53 in canine OS. Taurolidine's cytotoxic effect appears dependent on cell specific factors which may be explained, in part, by the functional status of p53. Taurolidine initiates apoptosis in canine OS cells and this occurs to a greater extent at lower concentrations. Mechanisms of cell death induced by higher concentrations were not elucidated here. Taurolidine combined with doxorubicin or carboplatin can increase the toxicity of these chemotherapy drugs and warrants further investigation in dogs with osteosarcoma.

Establishment and characterization of in vivo orthotopic bioluminescent xenograft models from human osteosarcoma cell lines in Swiss nude and NSG mice.

PubMed

Marques da Costa, Maria Eugenia; Daudigeos-Dubus, Estelle; Gomez-Brouchet, Anne; Bawa, Olivia; Rouffiac, Valerie; Serra, Massimo; Scotlandi, Katia; Santos, Conceição; Geoerger, Birgit; Gaspar, Nathalie

2018-03-01

Osteosarcoma is one of the most common primary bone tumors in childhood and adolescence. Metastases occurrence at diagnosis or during disease evolution is the main therapeutic challenge. New drug evaluation to improve patient survival requires the development of various preclinical models mimicking at best the complexity of the disease and its metastatic potential. We describe here the development and characteristics of two orthotopic bioluminescent (Luc/mKate2) cell-derived xenograft (CDX) models, Saos-2-B-Luc/mKate2-CDX and HOS-Luc/mKate2-CDX, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system allowed both longitudinal computed tomography (CT) and bioluminescence real-time follow-up of primary tumor growth and metastatic spread, which was confirmed by histology. The murine immune context influenced tumor engraftment, primary tumor growth, and metastatic spread to lungs, bone, and spleen (an unusual localization in humans). Engraftment in NSG mice was found superior to that found in nude mice and intratibial bone environment more favorable to engraftment compared to paratibial injection. The genetic background of the two CDX models also led to distinct primary tumor behavior observed on CT scan. Saos-2-B-Luc/mKate2-CDX showed osteocondensed, HOS-Luc/mKate2-CDX osteolytic morphology. Bioluminescence defined a faster growth of the primary tumor and metastases in Saos-2-B-Luc/mKate2-CDX than in HOS-Luc/mKate2-CDX. The early detection of primary tumor growth and metastatic spread by bioluminescence allows an improved exploration of osteosarcoma disease at tumor progression, and metastatic spread, as well as the evaluations of anticancer treatments. Our orthotopic models with metastatic spread bring complementary information to other types of existing osteosarcoma models. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture.

PubMed

Jhaveri, Ankur; Walsh, Stephen J; Wang, Yatzen; McCarthy, MaryBeth; Gronowicz, Gloria

2008-11-01

Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line. (c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Nb2OsB2, with a new twofold superstructure of the U3Si2 type: Synthesis, crystal chemistry and chemical bonding

NASA Astrophysics Data System (ADS)

Mbarki, Mohammed; Touzani, Rachid St.; Fokwa, Boniface P. T.

2013-07-01

The new ternary metal-rich boride, Nb2OsB2, was synthesized by arc-melting the elements in a water-cooled copper crucible under an argon atmosphere. The compound was characterized from single-crystal X-ray data and EDX measurements. It crystallizes as a new superstructure (space group P4/mnc, no. 128) of the tetragonal U3Si2-structure type with lattice parameters a=5.922(1) Å and c=6.879(2) Å. All of the B atoms are involved in B2 dumbbells with B-B distances of 1.89(4) Å. Structure relaxation using VASP (Vienna ab intio Simulation Package) has confirmed the space group and the lattice parameters. According to electronic structure calculations (TB-LMTO-ASA), the homoatomic B-B interactions are optimized and very strong, but relatively strong heteroatomic Os-B, Nb-B and Nb-Os bonds are also found: These interactions, which together build a three-dimensional network, are mainly responsible for the structural stability of this new phase. The density of state at the Fermi level predicts metallic behavior, as expected, from this metal-rich boride.

Pharmacokinetic study and evaluation of the safety of taurolidine for dogs with osteosarcoma

PubMed Central

2013-01-01

Background Osteosarcoma in dogs and humans share many similarities and the dog has been described as an excellent model to study this disease. The median survival in dogs has not improved in the last 25 years. Taurolidine has been shown to be cytotoxic to canine and human osteosarcoma in vitro. The goals of this study were to determine the pharmacokinetics and safety of taurolidine in healthy dogs and the safety of taurolidine in combination with doxorubicin or carboplatin in dogs with osteosarcoma. Methods Two percent taurolidine was infused into six healthy dogs (150 mg/kg) over a period of two hours and blood samples were taken periodically. One dog received taurolidine with polyvinylpyrrolidone (PVP) as its carrier and later received PVP-free taurolidine as did all other dogs in this study. Serum taurolidine concentrations were determined using high-performance liquid chromatography (HPLC) online coupled to ESI-MS/MS in the multiple reaction monitoring mode. Subsequently, the same dose of taurolidine was infused to seven dogs with osteosarcoma also treated with doxorubicin or carboplatin. Results Taurolidine infusion was safe in 6 healthy dogs and there were no significant side effects. Maximum taurolidine serum concentrations ranged between 229 to 646 μM. The dog that received taurolidine with PVP had an immediate allergic reaction but recovered fully after the infusion was stopped. Three additional dogs with osteosarcoma received doxorubicin and taurolidine without PVP. Toxicities included dilated cardiomyopathy, protein-losing nephropathy, renal insufficiency and vasculopathy at the injection site. One dog was switched to carboplatin instead of doxorubicin and an additional 4 dogs with osteosarcoma received taurolidine-carboplatin combination. One incidence of ototoxicity occurred with the taurolidine- carboplatin combination. Bone marrow and gastro-intestinal toxicity did not appear increased with taurolidine over doxorubicin or carboplatin alone

Pharmacokinetic study and evaluation of the safety of taurolidine for dogs with osteosarcoma.

PubMed

Marley, Kevin; Helfand, Stuart C; Simpson, Jennifer; Mata, John E; Tracewell, William G; Brownlee, Lisa; Bracha, Shay; Séguin, Bernard

2013-10-11

Osteosarcoma in dogs and humans share many similarities and the dog has been described as an excellent model to study this disease. The median survival in dogs has not improved in the last 25 years. Taurolidine has been shown to be cytotoxic to canine and human osteosarcoma in vitro. The goals of this study were to determine the pharmacokinetics and safety of taurolidine in healthy dogs and the safety of taurolidine in combination with doxorubicin or carboplatin in dogs with osteosarcoma. Two percent taurolidine was infused into six healthy dogs (150 mg/kg) over a period of two hours and blood samples were taken periodically. One dog received taurolidine with polyvinylpyrrolidone (PVP) as its carrier and later received PVP-free taurolidine as did all other dogs in this study. Serum taurolidine concentrations were determined using high-performance liquid chromatography (HPLC) online coupled to ESI-MS/MS in the multiple reaction monitoring mode. Subsequently, the same dose of taurolidine was infused to seven dogs with osteosarcoma also treated with doxorubicin or carboplatin. Taurolidine infusion was safe in 6 healthy dogs and there were no significant side effects. Maximum taurolidine serum concentrations ranged between 229 to 646 μM. The dog that received taurolidine with PVP had an immediate allergic reaction but recovered fully after the infusion was stopped. Three additional dogs with osteosarcoma received doxorubicin and taurolidine without PVP. Toxicities included dilated cardiomyopathy, protein-losing nephropathy, renal insufficiency and vasculopathy at the injection site. One dog was switched to carboplatin instead of doxorubicin and an additional 4 dogs with osteosarcoma received taurolidine-carboplatin combination. One incidence of ototoxicity occurred with the taurolidine- carboplatin combination. Bone marrow and gastro-intestinal toxicity did not appear increased with taurolidine over doxorubicin or carboplatin alone. Taurolidine did not

High lung-metastatic variant of human osteosarcoma cells, selected by passage of lung metastasis in nude mice, is associated with increased expression of α(v)β(3) integrin.

PubMed

Tome, Yasunori; Kimura, Hiroaki; Maehara, Hiroki; Sugimoto, Naotoshi; Bouvet, Michael; Tsuchiya, Hiroyuki; Kanaya, Fuminori; Hoffman, Robert M

2013-09-01

Altered expression of αvβ3 integrin is associated with tumor progression and metastasis in several types of cancer, including metastatic osteosarcoma. In this study, we demonstrate that in vivo passaging of lung metastasis in nude mice can generate an aggressive variant of human osteosarcoma cells. Experimental metastases were established by injecting 143B human osteosarcoma cells, expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, in the tail vein of nude mice. Lung metastases were harvested under fluorescence microscopy from nude mice to establish cell lines which were then injected via the tail vein of additional nude mice. This procedure was repeated for four passages in order to isolate highly metastatic variant sublines. When the parental and metastatic variants were transplanted orthotopically into the tibia of nude mice, the 143B-LM4 variant had the highest metastatic rate, approximately 18-fold higher than the parent (p<0.01). αvβ3 integrin expression was increased approximately 5.6-fold in 143B-LM4 compared to parental cells (p<0.05). Thus, serial passage of lung metastases created a highly metastatic variant of human osteosarcoma cells which had increased expression of αvβ3 integrin, suggesting that αvβ3 integrin plays an essential role in osteosarcoma metastasis. With this highly metastatic variant overexpressing αvβ3 integrin, it will now be possible to further investigate the mechanism by which αvβ3 integrin facilitates metastasis.