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Sample records for osteosarcoma cell lines

  1. Establishment and characterization of a novel osteosarcoma cell line: CHOS.

    PubMed

    Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

    2016-12-01

    Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  2. Ferulic acid promoting apoptosis in human osteosarcoma cell lines

    PubMed Central

    Zhang, Xu-dong; Wu, Qiang; Yang, Shu-hua

    2017-01-01

    Objective: To explore the promoting apoptosis and antitumor activities of ferulic acid (FA) in human osteosarcoma and its potential mechanism. Methods: The SaOS-2 and MG63 osteosarcoma cell lines were opted to experiment and these cells were, respectively, cultured with various concentrations of FA (0 μM, 10 μM, 20 μM, 40 μM) for 72 hours at 37°C. The viabilities of the FA treated cells were monitored by MTT. Apoptosis cells were evaluated using annexin V/PI by flow cytometry. Apoptosis proteins caspase-3, procaspase-3, Bcl-2 and Bax were detected by western blot. Expressions of apoptotic genes Bcl-2 and Bax were quantified by qPCR. Results: The cell viabilities were critically declined in the concentration-dependent manner in FA groups (P < 0.01). The apoptosis cells were increased proportionately with the concentration of FA (P < 0.05). The procaspase-3 protein contents, and Bcl-2 mRNA and protein contents were significantly decreased while caspase-3 protein contents, and Bax mRNA and protein contents were concomitantly increased in the concentration-dependent manner in FA groups (P < 0.05). The response to FA by the SaOS-2 osteosarcoma cell was similar with the MG63 osteosarcoma cell (P > 0.05). Conclusion: Ferulic acid could significantly descend osteosarcoma cell viability through the promoting apoptosis pathway in which FA activates both caspase-3 and Bax and inactivates Bcl-2. PMID:28367185

  3. Role of glutathione in cisplatin resistance in osteosarcoma cell lines.

    PubMed

    Komiya, S; Gebhardt, M C; Mangham, D C; Inoue, A

    1998-01-01

    This study was designed to examine whether and how glutathione and catalase increase the resistance of osteosarcoma cells to the toxicity of cisplatin. Eight osteosarcoma cell lines were exposed to varying concentrations of cisplatin, and a [3H]thymidine incorporation study then estimated their drug sensitivity. Cells were pretreated with aminotriazole and buthionine sulfoximine to depress catalase and glutathione activities and then entered into the same protocol to assess their sensitivity to cisplatin. Intracytoplasmic levels of catalase and glutathione were measured before and after the treatments. Cisplatin-glutathione conjugates were created to examine how glutathione might depress the toxicity of cisplatin. Although the cell lines differed in the magnitude of their response to cisplatin, there was a statistical correlation between intrinsic glutathione content and cisplatin resistance. Pretreatment with aminotriazole reduced catalase activity by 84% but did not change the sensitivity to cisplatin. Depletion of glutathione activity by 70% increased the sensitivity of the cells to the cytotoxicity of cisplatin. In addition, cisplatin was detoxified following conjugation with glutathione. The increased sensitization to cisplatin toxicity caused by the depletion of glutathione and cisplatin detoxification after the in vitro reaction of glutathione to cisplatin indicated that the formation of the glutathione-cisplatin conjugate was an important mechanism in the cellular resistance to cisplatin. These data also demonstrated that catalase activity did not contribute to resistance to cisplatin and suggested that H2O2-induced oxidative stress did not significantly contribute to the cytotoxicity of cisplatin in osteosarcoma cells.

  4. MicroRNA-132 inhibits cell growth and metastasis in osteosarcoma cell lines possibly by targeting Sox4

    PubMed Central

    LIU, YULONG; LI, YE; LIU, JINGCHEN; WU, YUNTAO; ZHU, QINGSAN

    2015-01-01

    Increasing evidence has confirmed that dysregulation of microRNAs (miRNAs) can contribute to the progression and metastasis of human tumors. Previous studies have shown that dysregulation of microRNAs (miRNAs) can contribute to the progression and metastasis of human tumors. However, the precise mechanisms of miR-132 in osteosarcoma have not been well clarified. Real-time PCR was performed to detect the expression of miR-132 in osteosarcoma cell lines. miR-132 mimic, miR-132 inhibitor and negative control were transfected into osteosarcoma cells and the effects of miR-132 on the cell growth and metastasis were investigated. Furthermore, protein level of Sox4 was measured by western blotting. Luciferase assays were performed to validate Sox4 as miR-132 target in osteosarcoma cells. We found that miR-132 was downregulated in osteosarcoma cell lines. Introduction of miR-132 significantly inhibited proliferation, arrested cell cycle and induced apoptosis in osteosarcoma cells. Besides, invasion and epithelial-mesenchymal transition (EMT) of osteosarcoma cells was suppressed by overexpressing miR-132. However, downregulation of miR-132 promoted cell growth and metastasis in osteosarcoma cells. Bioinformatics analysis predicted that Sox4 was a potential target gene of miR-132. Luciferase reporter assay demonstrated that miR-132 could directly target Sox4. Moreover, the low level of miR-132 was associated with increased expression of Sox4 in osteosarcoma cells. Sox4 inhibition suppressed cell malignant behaviors. Overexpression of Sox4 in osteosarcoma cells transfected with miR-132 mimic partially reversed the inhibitory effect of miR-132. In conclusion, miR-132 inhibited cell growth and metastasis in osteosarcoma cells by downregulation of Sox4, and knockdown of Sox4 was essential for the miR-132-inhibited cell growth and metastasis in osteosarcoma cells. PMID:26352673

  5. The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

    PubMed Central

    Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

    2013-01-01

    Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

  6. The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

    PubMed

    Lucero, Claudia M J; Vega, Oscar A; Osorio, Mariana M; Tapia, Julio C; Antonelli, Marcelo; Stein, Gary S; van Wijnen, Andre J; Galindo, Mario A

    2013-04-01

    Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. Copyright © 2012 Wiley Periodicals, Inc.

  7. Establishment and characterization of a new highly metastatic human osteosarcoma cell line derived from Saos2

    PubMed Central

    Du, Lin; Fan, Qiming; Tu, Bing; Yan, Wei; Tang, Tingting

    2014-01-01

    Osteosarcoma is the most common primary malignancy of bone in adolescents and young adults. There is a shortage of tumorigenic and highly metastatic human osteosarcoma cell lines that can be used for metastasis study. Here we establish and characterize a highly metastatic human osteosarcoma cell line that is derived from Saos2 cell line based on bioluminescence. The occasional pulmonary metastatic cells developed from Saos2 were isolated, harvested, characterized and named Saos2-l. The parental Saos2 and Saos2-l cells were further characterized both in vitro and in vivo. Results showed that Saos2-l cells demonstrated increased cell adhesion, migration and invasion compared to the parental Saos2 cells. Conversely, Saos2-l cells grew at a slightly slower rate than that of the parental cells. When injected into nude mice, Saos2-l cells had a greater increase in developing pulmonary metastases compared to the parental Saos2 cells. Further transcriptional profiling analysis revealed that some gene expression were up-regulated or down-regulated in the highly metastatic Saos2-l cells, indicating possible influencing factors of metastasis. Thus, we have established and characterized a highly metastatic human osteosarcoma cell line that should serve as a valuable tool for future investigations on the pathogenesis, metastasis and potential treatments of human osteosarcoma. PMID:25031706

  8. Genomic Instability of Osteosarcoma Cell Lines in Culture: Impact on the Prediction of Metastasis Relevant Genes

    PubMed Central

    Muff, Roman; Rath, Prisni; Ram Kumar, Ram Mohan; Husmann, Knut; Born, Walter; Baudis, Michael; Fuchs, Bruno

    2015-01-01

    Background Osteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages. Principal Findings The osteosarcoma cell lines showed altered gene expression during in vitro culture, and it was more pronounced in two metastatic cell lines compared to the respective parental cells. Chromosomal instability contributed in part to the altered gene expression in SAOS and LM5 cells with low and high metastatic potential. To identify metastasis-relevant genes in a background of passage-dependent altered gene expression, genes involved in "Pathways in cancer" that were consistently regulated under all passage comparisons were evaluated. Genes belonging to "Hedgehog signaling pathway" and "Wnt signaling pathway" were significantly up-regulated, and IHH, WNT10B and TCF7 were found up-regulated in all three metastatic compared to the parental cell lines. Conclusions Considerable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture. PMID:25992885

  9. Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology

    PubMed Central

    Zonefrati, Roberto; Mavilia, Carmelo; Franchi, Alessandro; Capanna, Rodolfo

    2016-01-01

    Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA. PMID:27651797

  10. Experimental models for the study of drug resistance in osteosarcoma: P-glycoprotein-positive, murine osteosarcoma cell lines.

    PubMed

    Takeshita, H; Gebhardt, M C; Springfield, D S; Kusuzaki, K; Mankin, H J

    1996-03-01

    P-glycoprotein is an adenosine triphosphate-dependent drug-efflux pump that extrudes drugs from cells and causes drug-resistance. P-glycoprotein is believed to mediate drug-resistance in a wide variety of tumors. In this study, we developed two P-glycoprotein-positive, murine osteosarcoma cell lines that were resistant to Adriamycin (doxorubicin) (MOS/ADR1 and MOS/ADR2). We created the cell lines by short-term pulse exposures of the parent cell line to Adriamycin followed by single-cell cloning. The MOS/ADR1 and MOS/ADR2 cells were sevenfold and eighteenfold more resistant to Adriamycin than the cells from the parent line. Expression of P-glycoprotein, as examined with an immunofluorescence method, was detected in most of the MOS/ADR1 and MOS/ADR2 cells but not in the parent cells. After the cells had been incubated with Adriamycin for one hour, there was less accumulation of the drug in the resistant cell lines than in the parent cell line. The reduced accumulation was due to the increased efflux of Adriamycin. The Adriamycin-resistant cell lines demonstrated greater alkaline phosphatase activity than the parent cell line and produced more differentiated osteoblastic sarcomas in mice. Dose survival studies with use of a tetrazolium colorimetric assay showed that the MOS/ADR1 cells were cross-resistant to vincristine, vinblastine, etoposide, bleomycin, mitomycin C, and actinomycin D but not to dacarbazine, cisplatin, carboplatin, cytosine arabinoside, carmustine, cyclophosphamide, ifosfamide, methotrexate, and 5-fluorouracil. Although the MOS/ADR2 cells exhibited a similar spectrum of cross-resistance, they were more resistant than the MOS/ADR1 cells. We also tested the effect of three different resistance-modifying agents on the reversal of resistance to Adriamycin. We found that verapamil and trifluoperazine substantially reversed resistance to Adriamycin in the P-glycoprotein positive cell lines, whereas cyclosporin A was relatively ineffective. Because these

  11. mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts

    PubMed Central

    2011-01-01

    Background Conventional high-grade osteosarcoma is a primary malignant bone tumor, which is most prevalent in adolescence. Survival rates of osteosarcoma patients have not improved significantly in the last 25 years. Aiming to increase this survival rate, a variety of model systems are used to study osteosarcomagenesis and to test new therapeutic agents. Such model systems are typically generated from an osteosarcoma primary tumor, but undergo many changes due to culturing or interactions with a different host species, which may result in differences in gene expression between primary tumor cells, and tumor cells from the model system. We aimed to investigate whether gene expression profiles of osteosarcoma cell lines and xenografts are still comparable to those of the primary tumor. Methods We performed genome-wide mRNA expression profiling on osteosarcoma biopsies (n = 76), cell lines (n = 13), and xenografts (n = 18). Osteosarcoma can be subdivided into several histological subtypes, of which osteoblastic, chondroblastic, and fibroblastic osteosarcoma are the most frequent ones. Using nearest shrunken centroids classification, we generated an expression signature that can predict the histological subtype of osteosarcoma biopsies. Results The expression signature, which consisted of 24 probes encoding for 22 genes, predicted the histological subtype of osteosarcoma biopsies with a misclassification error of 15%. Histological subtypes of the two osteosarcoma model systems, i.e. osteosarcoma cell lines and xenografts, were predicted with similar misclassification error rates (15% and 11%, respectively). Conclusions Based on the preservation of mRNA expression profiles that are characteristic for the histological subtype we propose that these model systems are representative for the primary tumor from which they are derived. PMID:21933437

  12. Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

    PubMed

    de Sá Rodrigues, L C; Holmes, K E; Thompson, V; Newton, M A; Stein, T J

    2017-03-01

    An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells.

  13. Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency

    PubMed Central

    de Sá Rodrigues, L. C.; Holmes, K. E.; Thompson, V.; Newton, M. A.; Stein, T. J.

    2016-01-01

    An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells. PMID:25689105

  14. Effects of naproxen on cell proliferation and genotoxicity in MG-63 osteosarcoma cell line.

    PubMed

    Correia, Isabel; Arantes-Rodrigues, Regina; Pinto-Leite, Rosário; Gaivão, Isabel

    2014-01-01

    The purpose of this study was to determine the efficacy of naproxen, a nonsteroidal anti-inflammatory drug, on the MG-63 human osteosarcoma cell line. MG-63 cells were exposed to naproxen in a wide range of concentrations of 0.03, 0.05, 0.1, 0.42, 0.83, and 1.67 mg/ml for 72 h. The activity of naproxen was assessed by the following assays: cell morphology; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay; comet assay; and acridine orange and monodansylcadaverine (MDC) staining. Naproxen exerted a significant inhibitory effect on MG-63 cell proliferation, in a concentration-dependent manner, in all treatment groups compared with untreated cells. An increase in frequency of DNA damage, apoptotic bodies, apoptotic cells, and autophagic vacuoles was observed in MG-63-treated cells. Although future studies are needed, these findings suggest that naproxen may lead to improvements in treatment of patients with osteosarcoma.

  15. Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

    Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced an inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.

  16. A proteomic study on a human osteosarcoma cell line Saos-2 treated with diallyl trisulfide.

    PubMed

    Zhang, Yong Kui; Zhang, Xu Hua; Li, Jian Min; Sun, De Sheng; Yang, Qiang; Diao, Dong Mei

    2009-09-01

    Garlic is generally used as a therapeutic reagent against various diseases, and numerous studies have indicated that garlic and its derivatives can reduce the risk of various types of human cancer. Diallyl trisulfide (DATS), a major member of garlic derivatives, could inhibit the cell proliferation by triggering either cell cycle arrest or apoptosis in a variety of cancer cell lines as shown in many studies. However, whether DATS has the same effect on human osteosarcoma cells remains unknown. In this study, we have attempted to analyze the effects of DATS on cell proliferation, cell cycle, induction of apoptosis, global protein expression pattern in a human osteosarcoma cell line Saos-2 cells, and the potential molecular mechanisms of the action of DATS. Saos-2 cells, a human osteosarcoma cell line, were treated with or without 25, 50, and 100 micromol/l DATS for various time intervals. The cell proliferation, cell cycle progression, and apoptosis were examined in this study. Then, after treatment with or without 50 micromol/l DATS for 48 h, protein add pattern in Saos-2 cells were systematically studied using two-dimensional electrophoresis and mass spectrometry. DATS could inhibit the proliferation of Saos-2 cells in a dose-dependent and time-dependent manner. Moreover, the percentage of apoptotic cell and cell arrest in G0/G1 phase was also dose-dependent and time-dependent upon DATS treatment. A total of 27 unique proteins in Saos-2 cells, including 18 downregulated proteins and nine upregulated proteins, were detected with significant changes in their expression levels corresponding to DATS administration. Interestingly, almost half of these proteins (13 of 27) are related to either the cell cycle or apoptosis. DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in a dose and time-dependent manner. The proteomic results presented, therefore, provide additional support to the hypothesis

  17. Suramin inhibits growth and transforming growth factor-beta 1 (TGF-beta 1) binding in osteosarcoma cell lines.

    PubMed

    Kloen, P; Jennings, C L; Gebhardt, M C; Springfield, D S; Mankin, H J

    1994-01-01

    Autocrine production of growth factors has been shown to be involved in the multistep process of tumorigenesis. The ability of suramin, a polyanionic anti-parasitic drug, to block growth factor-induced cell proliferation makes it a potential antineoplastic drug. We studied the effects of suramin on seven osteosarcoma cell lines. Using clinically achievable concentrations of suramin (50-400 micrograms/ml), we found a time- and dose-dependent inhibition of [3H]thymidine incorporation. We also showed that suramin is able, dose-dependently, to prevent binding of transforming growth factor (TGF)-beta 1 to its receptors. DNA synthesis inhibition by suramin was attenuated by TGF-beta 1 in some cell lines. Two cell lines that were inhibited by TGF-beta 1 were affected similarly by suramin as cell lines that were stimulated by TGF-beta 1. In conclusion, in five out of seven osteosarcoma cell lines, we showed a correlation between inhibition of growth factor-stimulated mitogenesis and binding of TGF-beta 1 to its receptor. Similar effects in TGF-beta 1-inhibited osteosarcoma cell lines suggest involvement of other mechanisms and/or growth factors. However, suramin proves to be a potent inhibitor of osteosarcoma cell proliferation in vitro.

  18. Paclitaxel-induced apoptosis in osteosarcoma cell line U-2 OS.

    PubMed

    Guo, Wei; Zeng, Chun; Dong, Fengquan; Lei, Wei

    2002-12-01

    To observe the in vitro growth inhibitory and apoptosis-inducing effects of paclitaxel on the human osteosarcoma cell line U-2 OS. U-2 OS cells were treated with various concentrations of paclitaxel. Proliferation was determined by cell count in a neubauer cytometer chamber. Viability was assessed by trypan blue dye exclusion. Paclitaxel-induced morphologic alterations were visualized using light and transmission electron microscopy. The extent of paclitaxel-induced apoptosis was determined by flow cytometry and immunohistochemical detection (TdT-mediated dUTP nick end labeling technique, TUNEL). The cells treated with paclitaxel initially showed G(2)/M arrest, which was followed by apoptosis. The characteristic apoptotic changes, including nuclear disintegration and chromatin agglomeration, were displayed. Large amounts of micronuclei cells appeared, something not observed in those cells treated with cisplatin and adriamycin for contrast. Also, extensive DNA-cleavage was detected using TUNEL. This study demonstrates that paclitaxel has an apoptotic-inducing effect on osteosarcoma cells through the initiation of G(2)/M arrest and inhibiting mitosis in both a time-dependent and dose-dependent manner.

  19. Dealcoholated red wine induces autophagic and apoptotic cell death in an osteosarcoma cell line.

    PubMed

    Tedesco, I; Russo, M; Bilotto, S; Spagnuolo, C; Scognamiglio, A; Palumbo, R; Nappo, A; Iacomino, G; Moio, L; Russo, G L

    2013-10-01

    Until recently, the supposed preventive effects of red wine against cardiovascular diseases, the so-called "French Paradox", has been associated to its antioxidant properties. The interest in the anticancer capacity of polyphenols present in red wine strongly increased consequently to the enormous number of studies on resveratrol. In this study, using lyophilized red wine, we present evidence that its anticancer effect in a cellular model is mediated by apoptotic and autophagic cell death. Using a human osteosarcoma cell line, U2Os, we found that the lyophilized red wine was cytotoxic in a dose-dependent manner with a maximum effect in the range of 100-200 μg/ml equivalents of gallic acid. A mixed phenotype of types I/II cell death was evidenced by means of specific assays following treatment of U2Os with lyophilized red wine, e.g., autophagy and apoptosis. We found that cell death induced by lyophilized red wine proceeded through a mechanism independent from its anti-oxidant activity and involving the inhibition of PI3K/Akt kinase signaling. Considering the relative low concentration of each single bioactive compound in lyophilized red wine, our study suggests the activation of synergistic mechanism able to inhibit growth in malignant cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Expression Levels and Activation of a PXR Variant Are Directly Related to Drug Resistance in Osteosarcoma Cell Lines

    PubMed Central

    Mensah-Osman, Edith J.; Thomas, Dafydd G.; Tabb, Michelle M.; Larios, Jose M.; Hughes, Dennis P.; Giordano, Thomas J.; Lizyness, Michelle L.; Rae, James M.; Blumberg, Bruce; Hollenberg, Paul F.; Baker, Laurence H.

    2011-01-01

    BACKGROUND Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors. METHODS Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining. RESULTS Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole. CONCLUSION The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance. PMID:17279585

  1. MicroRNA-21 Inhibits the Apoptosis of Osteosarcoma Cell Line SAOS-2 via Targeting Caspase 8.

    PubMed

    Xu, Bin; Xia, Hehuan; Cao, Junming; Wang, Zhihong; Yang, Yipeng; Lin, Yongsheng

    2017-08-07

    Currently, multiple microRNAs (miRNAs) have been found to play vital roles in the pathogenesis of osteosarcoma. This study aimed to investigate the role of miR-21 in osteosarcoma. The level of miR-21 in 20 pairs of osteosarcoma and corresponding adjacent tissues was monitored by qPCR. Human osteosarcoma cell line SAOS-2 was transfected with either miR-21 mimic or miR-21 inhibitor, and then cell viability, survival, and apoptosis were measured by MTT, colony formation assay, and flow cytometry. A target of miR-21 was predicted by the microRNA.org database and verified in vitro by using luciferase reporter, qPCR, and Western blot analyses. Finally, cells were cotransfected with siRNA against caspase 8 and miR-21 inhibitor, and the apoptotic cell rate was determined again. Results showed that the mRNA level of miR-21 was highly expressed in osteosarcoma tissues compared with adjacent tissues. Overexpression of miR-21 improved cell viability and survival but suppressed apoptosis. Caspase 8 was a direct target of miR-21, and it was negatively regulated by miR-21. Moreover, miR-21 suppression attenuated caspase 8 silencing and induced the decrease in apoptosis. In conclusion, overexpression of miR-21 suppressed SAOS-2 cell apoptosis via directly targeting caspase 8.

  2. Nucleosome-binding protein HMGN2 exhibits antitumor activity in human SaO2 and U2-OS osteosarcoma cell lines.

    PubMed

    Liang, Guojun; Xu, Enjie; Yang, Chaoqun; Zhang, Chenglin; Sheng, Xiaolong; Zhou, Xuhui

    2015-03-01

    High mobility group N (HMGNs) are members of the high mobility group protein family, and are involved in the development and progression of several tumors. HMGN1 and HMGN5 were previously shown to be associated with the bioactivities of osteosarcoma. However, the effects and molecular mechanisms of HMGN2 on osteosarcoma progression remain to be determined. In order to characterize the endogenous expression of HMGN2 in osteosarcoma cell lines, RT-PCR and western blot analysis were performed. Recombinant HMGN2 lentivirus was used to infect the osteosarcoma cell lines with relatively low HMGN2 expression to determine the functional relevance of HMGN2 overexpression in osteosarcoma cell growth and migration in vitro and in vivo, and to investigate the expression levels of Ki-67, PCNA, cyclin D1 and cyclin E. The results showed that osteosarcoma cell proliferation and migration were significantly reduced by HMGN2, as indicated by cell count and wound-healing assays. Cell apoptosis was markedly induced and HMGN2 increased the sensitivity to chemotherapy. When HMGN2 expression was enhanced, the expression of cyclin D1 and PCNA was downregulated in osteosarcoma cells. In addition, the tumor volumes in SaO2 and U2-OS subcutaneous nude mouse models treated with HMGN2 lentivirus were significantly decreased as compared to those of the GFP group. These results suggested that the enhanced expression of HMGN2 in osteosarcoma cells by HMGN2 lentivirus, exerts inhibitory effects on growth and migration of osteosarcoma cells.

  3. The proteome profile of the human osteosarcoma U2OS cell line.

    PubMed

    Niforou, Katerina M; Anagnostopoulos, Athanasios K; Vougas, Konstantinos; Kittas, Christos; Gorgoulis, Vassilis G; Tsangaris, George T

    2008-01-01

    The human osteosarcoma U2OS cell line is one of the first generated cell lines and is used in various areas of biomedical research. Knowledge of its protein expression is limited and no comprehensive study on the proteome of this cell line has been reported to date. Proteomics technology was used in order to analyse the proteins of the U2OS cell line. Total protein extracts were separated by two-dimensional gel electrophoresis (2-DE) and analysed by matrix-assisted laser desorption ionisation-mass spectrometry (MALDI-MS) and MALDI--MS-MS following in-gel digestion with trypsin and, finally, protein identification was carried out by peptide mass fingerprint (PMF) and post source decay (PSD), respectively. Approximately 3,000 spots were excised from two 2-DE gels and were analysed, resulting in the identification of 237 different gene products. The majority of the identified proteins were enzymes, regulatory proteins and RNA-associated proteins, while leukocyte markers and oncogenes were also present. Our findings include 11 protooncogenes (FKBP4, SRC8, PSD10, FUBP1, PARK7, NPM, PDIA1, OXRP, SET, TCTP and GRP75) related to the cancerous state of the U2OS cell line. The U2OS 2-DE database provides the basis for future protein studies.

  4. Anti-tumor necrosis factor therapy inhibits lung metastasis in an osteosarcoma cell line.

    PubMed

    Kato, Hiroaki; Wakabayashi, Hiroki; Naito, Yohei; Kato, Sho; Nakagawa, Taro; Matsumine, Akihiko; Sudo, Akihiro

    2015-01-01

    Osteosarcoma is the most common primary malignancy of bone, and patients often develop pulmonary metastases. In a previous study, tumor necrosis factor (TNF)-α treatment of human osteosarcoma cells increases their metastatic ability in an animal model. TNF-α can act as a tumor necrosis factor and also as a tumor-promoting factor. In the present study, the effect of a TNF-α inhibitor on osteosarcoma aggressiveness and pulmonary metastases was investigated in vitro and in vivo. The effect of infliximab, a TNF-α inhibitor, on a metastatic osteosarcoma 143B cell growth and motility was investigated in vitro. An orthotopic xenograft model of 143B cell growth and spontaneous metastasis in SCID mice was used to assess the in vivo effect of infliximab. Infliximab greatly reduced cell motility and pulmonary metastases in 143B cells. The mechanism of pulmonary metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4), Rho (small GTPase protein), and its effector. These results suggest a novel role for TNF-α inhibition in the reduction or prevention of pulmonary metastases of osteosarcoma in this animal model. TNF-α inhibition may become a preventive therapeutic option for the pulmonary metastases of osteosarcoma. © 2014 S. Karger AG, Basel.

  5. Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

    PubMed

    Viall, A K; Goodall, C P; Stang, B; Marley, K; Chappell, P E; Bracha, S

    2016-06-01

    Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target.

  6. Mechanisms of methotrexate resistance in osteosarcoma cell lines and strategies for overcoming this resistance.

    PubMed

    Wang, Jianjun; Li, Guojun

    2015-02-01

    The aim of the present study was to investigate the underlying mechanisms of methotrexate (MTX) resistance in the human osteosarcoma cell line, Saos-2/MTX4.4, and to evaluate various methods of overcoming the resistance to this chemotherapeutic agent. MMT assays were performed to determine the resistance of the primary (Saos-2) and resistant (Saos-2/MTX4.4) cell lines to MTX, cisplatin [cis-diamminedichloroplatinum II (DDP)], ifosfamide (IFO), Adriamycin (ADM), epirubicin (EPI) and theprubicin (THP). The Saos-2/MTX4.4 cells exhibited a low resistance to IFO, ADM, EPI and THP; however, no resistance to DDP was identified. Overall, the Saos-2/MTX4.4 cells exhibited a greater resistance to all the chemotherapeutic agents investigated compared with the Saos-2 cells. Rhodamine 123 (R123) fluorescence was measured in the Saos-2/MTX4.4 and Saos-2 cells 30 and 60 min after the addition of R123, and R123 plus verapamil (VER). VER administration increased the intracellular accumulation of R123. In addition, reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of multidrug resistance gene 1 (MDR1) in the two cell lines. Although the Saos-2/MTX4.4 cells were more resistant to the chemotherapeutic agents than the Saos-2 cells, no significant difference was identified between the relative mRNA expression levels of MDR1 in the Saos-2/MTX4.4 and Saos-2 cells (0.4350±0.0354 vs. 0.3886±0.0456; P>0.05).

  7. Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2.

    PubMed

    Tanaka, Hideki; Tanabe, Natsuko; Suzuki, Naoto; Shoji, Maiko; Torigoe, Hirotaka; Sugaya, Atsuto; Motohashi, Masafumi; Maeno, Masao

    2005-09-16

    Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.

  8. Morphological characterization of a newly established human osteosarcoma cell line, HS-Os-1, revealing its distinct osteoblastic nature.

    PubMed

    Sonobe, H; Mizobuchi, H; Manabe, Y; Furihata, M; Iwata, J; Hikita, T; Oka, T; Ohtsuki, Y; Goto, T

    1991-01-01

    A newly established human osteosarcoma cell line, HS-Os-1, from an osteoblastic tumor arising in the left humerus of an 11-year-old girl was morphologically characterized in vitro and in vivo. HS-Os-1 cells in a monolayer have been maintained for more than 2 years since the initial cultivation, and were round or polygonal in shape with marked pleomorphism. Their cytoplasm was strongly positive for specific markers of osteoblasts, such as alkaline phosphatase and osteocalcin. Tumors induced in nude mice by HS-Os-1 cell inoculation at passage 12 or 23 revealed typical histological features of osteoblastic osteosarcoma, similar to those observed in the original tumor, producing prominent osteoid matrix with calcification. Ultrastructurally, HS-Os-1 cells in vitro and tumor cells in vivo showed similar well-developed, markedly dilated rough endoplasmic reticulum, polysomes and microfilaments in their cytoplasm. Additionally, many collagen fibers associated with deposition of electron-dense material were detected in the stroma featuring osteoid matrix. Thus, the HS-Os-1 cell line was shown to exhibit its osteoblastic nature in vitro and in vivo, and therefore might become an extremely useful tool for various pathomorphological investigations on human osteosarcomas.

  9. Homeodomain-containing gene 10 inhibits cell apoptosis and promotes cell invasion and migration in osteosarcoma cell lines.

    PubMed

    Xiong, Wen; Zhou, Quan; Liu, Gang; Liu, Xiang-Sheng; Li, Xin-Yu

    2017-05-01

    Homeodomain-containing gene 10 (HOXC10) belongs to the homeobox family, which encodes a highly conserved family of transcription factors that plays an important role in morphogenesis in all multicellular organisms. Altered expressions of HOXC10 have been reported in several malignancies. This study was aimed to reveal the expression profile of HOXC10 in osteosarcoma and evaluated whether HOXC10 is a molecular target for cancer therapy. We found that HOXC10 was up-regulated in osteosarcoma tissues compared with bone cyst specimens from The Cancer Genome Atlas database. Osteosarcoma MG63 cells were infected with HOXC10 shRNA expressing vector, and 143B cells were infected with HOXC10 expressing vector. We found that reduced expression of HOXC10 markedly impaired the ability of proliferation, invasion, and migration, and promoted cell apoptosis in vitro and in vivo. Up-regulated expression of HOXC10 promoted the proliferation, invasion, and migration, and inhibited apoptosis of 143B cells. Additionally, HOXC10 regulated apoptosis and migration via modulating expression of Bax/Bcl-2, caspase-3, MMP-2/MMP-9, and E-cadherin in both MG63 and 143B cells and in vivo. These results indicated that HOXC10 might be a diagnostic marker for osteosarcoma and could be a potential molecular target for the therapy of osteosarcoma.

  10. Canine osteosarcoma cell lines contain stem-like cancer cells: biological and pharmacological characterization.

    PubMed

    Gatti, Monica; Wurth, Roberto; Vito, Guendalina; Pattarozzi, Alessandra; Campanella, Chiara; Thellung, Stefano; Maniscalco, Lorella; De Maria, Raffaella; Villa, Valentina; Corsaro, Alessandro; Nizzari, Mario; Bajetto, Adriana; Ratto, Alessandra; Ferrari, Angelo; Barbieri, Federica; Florio, Tullio

    2016-05-01

    Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

  11. Comparative study of cytotoxicity of detonation nanodiamond particles with an osteosarcoma cell line and primary mesenchymal stem cells

    PubMed Central

    Keremidarska, Milena; Ganeva, Aneliya; Mitev, Dimitar; Hikov, Todor; Presker, Radina; Pramatarova, Lilyana; Krasteva, Natalia

    2014-01-01

    Recently, nanodiamonds (NDs) have attracted great interest due to their unique physical and chemical properties that could be used in various biological applications. However, depending on the origin, NDs often contain different impurities which may affect cellular functions and viability. Therefore, before their biomedical application, the cytotoxicity of newly produced NDs should be assessed. In the present study, we have evaluated cytotoxicity of four types of ND particles with two cell models: a human osteosarcoma cell line, MG-63, and primary rat mesenchymal stem cells (rMSCs). Detonation-generated nanodiamond (DND) particles were purified with different acid oxidizers and impurities’ content was determined by elemental analysis. The particles size distribution was measured revealing that the DND particles have an average size in the range of 51–233 nm. Cytotoxicity was assessed by optical microscopy and proliferation assay after 72 hours exposure of the cells to nanoparticles. We observed cell-specific and material-specific toxicity for all tested particles. Primary stem cells demonstrated higher sensitivity to DND particles than osteosarcoma cells. The most toxic were the DND particles with the smallest grain size and slight content of non-diamond carbon, while DNDs with higher grain size and free from impurities had no significant influence on cell proliferation and morphology. In addition, the smaller DND particles were found to form large aggregates mainly during incubation with rMSCs. These results demonstrate the role of the purification method on the properties of DND particles and their cytotoxicity as well as the importance of cell types used for evaluation of the nanomaterials. PMID:26019557

  12. Inhibition of lung metastasis of osteosarcoma cell line POS-1 transplanted into mice by thigh ligation.

    PubMed

    Kamijo, Akira; Koshino, Tomihisa; Uesugi, Masaaki; Nitto, Hironori; Saito, Tomoyuki

    2002-12-15

    Using a model with external ligation of the thigh, the effect of ischemia-reperfusion injury on tumor growth and the activity of lung metastasis was investigated in mice inoculated a spontaneous murine osteosarcoma cell line (POS-1) in vivo. POS-1 cell suspension was inoculated into the right hind footpad of 70 mice. Four weeks after inoculation, the ipsilateral thigh was ligated for 3 h in 15 mice and the contralateral thigh in 15 mice. Another ten mice were inoculated with POS-1 without ligating the thigh. The number of metastatic foci on the lung surface 6 weeks after inoculation was 2.29+/-0.98 (mean+/-SE) foci/lungs in mice with ipsilateral ligation and 6.25+/-2.41 in mice with contralateral ligation, which were significantly lower than control (13.40+/-1.42 in mice no ligation) (P<0.01). The number of metastatic foci on the lung surface in mice with intraperitoneal injection of superoxide dismutase (SOD) and catalase was 3.25+/-0.65 (mean+/-SE) foci/lungs in mice with ligation which was significantly greater than that in mice without SOD and catalase injection 1.29+/-0.97 (P=0.04). Cell viability was 9.12+/-4.07% with 100 microM H(2)O(2) in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. It revealed that at concentrations of 100 microM H(2)O(2) or higher was cytotoxic to POS-1. In cell invasion assay, the number of invading cells with 10 microM H(2)O(2) was 2.80+/-0.53 cells/field, which was significantly lower than control (5.93+/-0.18) (mean+/-SE), indicating that low-dose H(2)O(2) suppressed invasion of POS-1. These results suggested that reperfusion injury had selective cytotoxicity to POS-1 through producing reactive oxygen species. Activated oxygen was considered to inhibit the regional growth and the ability of lung metastasis of POS-1 cells.

  13. Expression of different phenotypes in cell lines from canine mammary spindle-cell tumours and osteosarcomas indicating a pluripotent mammary stem cell origin.

    PubMed

    Hellmén, E; Moller, M; Blankenstein, M A; Andersson, L; Westermark, B

    2000-06-01

    Mammary spindle-cell tumours and sarcomas seem to be restricted to dogs and humans. Two cell lines from spontaneous primary canine mammary spindle-cell tumours (CMT-U304 and CMT-U309) and two cell lines from spontaneous primary canine mammary osteosarcomas (CMT-U334 and CMT-U335) were established to study the mesenchymal phenotypes of mammary tumours in the female dog. The cells from the spindle-cell tumours expressed cytokeratin, vimentin and smooth muscle actin filaments. When these cells were inoculated subcutaneously into female and male nude mice they formed different types of mesenchymal tumours such as spindle-cell tumours, fibroma and rhabdomyoid tumours (n = 6/8). The cells from the osteosarcomas expressed vimentin filaments and also formed different types of mesenchymal tumours such as chondroid, rhabdomyoid, smooth muscle-like and spindle-cell tumours (n = 6/10). The cell lines CMT-U304, CMT-U309 and CMT-U335 had receptors for progesterone but none of the four cell lines had receptors for estrogen. All four cell lines and their corresponding primary tumours showed identical allelic patterns in microsatellite analysis. By in situ hybridization with genomic DNA we could verify that all formed tumours but one were of canine origin. Our results support the hypothesis that canine mammary tumours are derived from pluripotent stem cells.

  14. 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.

  15. The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines.

    PubMed

    Stratford, Eva Wessel; Daffinrud, Jeanette; Munthe, Else; Castro, Russell; Waaler, Jo; Krauss, Stefan; Myklebost, Ola

    2014-02-01

    Wnt/β-catenin is a major regulator of stem cell self-renewal and differentiation and this signaling pathway is aberrantly activated in a several cancers, including osteosarcoma (OS). Attenuation of Wnt/β-catenin activity by tankyrase inhibitors is an appealing strategy in treatment of OS. The efficacy of the tankyrase inhibitor JW74 was evaluated in three OS cell lines (KPD, U2OS, and SaOS-2) both at the molecular and functional level. At the molecular level, JW74 induces stabilization of AXIN2, a key component of the β-catenin destruction complex, resulting in reduced levels of nuclear β-catenin. At the functional level, JW74 induces reduced cell growth in all three tested cell lines, in part due to a delay in cell cycle progression and in part due to an induction of caspase-3-mediated apoptosis. Furthermore, JW74 induces differentiation in U2OS cells, which under standard conditions are resistant to osteogenic differentiation. JW74 also enhances differentiation of OS cell lines, which do not harbor a differentiation block. Interestingly, microRNAs (miRNAs) of the let-7 family, which are known tumor suppressors and inducers of differentiation, are significantly upregulated following treatment with JW74. We demonstrate for the first time that tankyrase inhibition triggers reduced cell growth and differentiation of OS cells. This may in part be due to an induction of let-7 miRNA. The presented data open for novel therapeutic strategies in the treatment of malignant OS. © 2013 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  16. Cancer stem cells in osteosarcoma.

    PubMed

    Brown, Hannah K; Tellez-Gabriel, Marta; Heymann, Dominique

    2017-02-01

    Osteosarcoma is the most common primary bone tumour in children and adolescents and advanced osteosarcoma patients with evidence of metastasis share a poor prognosis. Osteosarcoma frequently gains resistance to standard therapies highlighting the need for improved treatment regimens and identification of novel therapeutic targets. Cancer stem cells (CSC) represent a sub-type of tumour cells attributed to critical steps in cancer including tumour propagation, therapy resistance, recurrence and in some cases metastasis. Recent published work demonstrates evidence of cancer stem cell phenotypes in osteosarcoma with links to drug resistance and tumorigenesis. In this review we will discuss the commonly used isolation techniques for cancer stem cells in osteosarcoma as well as the identified biochemical and molecular markers. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Genes Regulated in Metastatic Osteosarcoma: Evaluation by Microarray Analysis in Four Human and Two Mouse Cell Line Systems

    PubMed Central

    Muff, Roman; Ram Kumar, Ram Mohan; Botter, Sander M.; Born, Walter; Fuchs, Bruno

    2012-01-01

    Osteosarcoma (OS) is a rare bone neoplasm that affects mainly adolescents. It is associated with poor prognosis in case of metastases formation. The search for metastasis predicting markers is therefore imperative to optimize treatment strategies for patients at risk and important for the search of new drugs for the treatment of this devastating disease. Here, we have analyzed by microarray the differential gene expression in four human and two mouse OS cell line systems consisting of parental cell lines with low metastatic potential and derivatives thereof with increased metastatic potential. Using two osteoblastic cell line systems, the most common OS phenotype, we have identified forty-eight common genes that are differentially expressed in metastatic cell lines compared to parental cells. The identified subset of metastasis relevant genes in osteoblastic OS overlapped only minimally with differentially expressed genes in the other four preosteoblast or nonosteoblastic cell line systems. The results imply an OS phenotype specific expression pattern of metastasis regulating proteins and form a basis for further investigation of gene expression profiles in patients' samples combined with survival analysis with the aim to optimize treatment strategies to develop new drugs and to consequently improve the survival of patients with the most common form of osteoblastic OS. PMID:23213280

  18. Autocrine Transforming Growth Factor-β Growth Pathway in Murine Osteosarcoma Cell Lines Associated with Inability to Affect Phosphorylation of Retinoblastoma Protein

    PubMed Central

    Letterio, John J.; Yeung, Choh L.; Pegtel, Michiel; Helman, Lee J.

    2000-01-01

    Purpose. Production of active transforming growth factor-β (TGF-β ) by human osteosarcoma may contribute to malignant progression through mechanisms that include induction of angiogenesis, immune suppression and autocrine growth stimulation of tumor cell growth.To study events associated with induction of cell proliferation by TGF-β , we have evaluated the TGF-β pathway in two murine osteosarcoma cell lines, K7 and K12. Results. Northern and immunohistochemical analyses show that each cell line expressesTGF-β1 and TGF-β3 mRNA and protein. Both cell lines secrete activeTGF-β 1 and display a 30–50% reduction in growth when cultured in the presence of a TGF-β blocking antibody. Expression of TGF-β receptors TβRI, TβRII and TβRIII is demonstrated by affinity labeling with 125 -TGF-β 1, and the intermediates, Smads 2, 3 and 4, are uniformly expressed. Smads 2 and 3 are phosphorylated in response toTGF-β , while pRb phosphorylation in each osteosarcoma cell line is not affected by either exogenousTGF-β or TGF-β antibody. Conclusions. The data implicate events downstream of Smad activation, including impaired regulation of pRb, in the lack of a growth inhibitory response toTGF-β , and indicate that this murine model of osteosarcoma is valid for investigating the roles of autocrineTGF-β in vivo. PMID:18521287

  19. CYR61 downregulation reduces osteosarcoma cell invasion, migration, and metastasis.

    PubMed

    Fromigue, Olivia; Hamidouche, Zahia; Vaudin, Pascal; Lecanda, Fernando; Patino, Ana; Barbry, Pascal; Mari, Bernard; Marie, Pierre J

    2011-07-01

    Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and the poor survival rate for patients. We showed previously that the HMGCoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitor statin exhibits antitumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify Cyr61 as a new target of statins. Transcriptome and molecular analyses revealed that statins downregulate Cyr61 expression in human and murine osteosarcoma cells. Cyr61 silencing in osteosarcoma cell lines enhanced cell death and reduced cell migration and cell invasion compared with parental cells, whereas Cyr61 overexpression had opposite effects. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients. Tissue microarray analysis revealed that Cyr61 protein expression was higher in human osteosarcoma than in normal bone tissue and was further increased in metastatic tissues. Finally, tumor behavior and metastasis occurrence were analyzed by intramuscular injection of modified osteosarcoma cells into BALB/c mice. Cyr61 overexpression enhanced lung metastasis development, whereas cyr61 silencing strongly reduced lung metastases in mice. The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma. Copyright © 2011 American Society for Bone and Mineral Research.

  20. Kinome and mRNA expression profiling of high-grade osteosarcoma cell lines implies Akt signaling as possible target for therapy

    PubMed Central

    2014-01-01

    Background High-grade osteosarcoma is a primary malignant bone tumor mostly occurring in adolescents and young adults, with a second peak at middle age. Overall survival is approximately 60%, and has not significantly increased since the introduction of neoadjuvant chemotherapy in the 1970s. The genomic profile of high-grade osteosarcoma is complex and heterogeneous. Integration of different types of genome-wide data may be advantageous in extracting relevant information from the large number of aberrations detected in this tumor. Methods We analyzed genome-wide gene expression data of osteosarcoma cell lines and integrated these data with a kinome screen. Data were analyzed in statistical language R, using LIMMA for detection of differential expression/phosphorylation. We subsequently used Ingenuity Pathways Analysis to determine deregulated pathways in both data types. Results Gene set enrichment indicated that pathways important in genomic stability are highly deregulated in these tumors, with many genes showing upregulation, which could be used as a prognostic marker, and with kinases phosphorylating peptides in these pathways. Akt and AMPK signaling were identified as active and inactive, respectively. As these pathways have an opposite role on mTORC1 signaling, we set out to inhibit Akt kinases with the allosteric Akt inhibitor MK-2206. This resulted in inhibition of proliferation of osteosarcoma cell lines U-2 OS and HOS, but not of 143B, which harbors a KRAS oncogenic transformation. Conclusions We identified both overexpression and hyperphosphorylation in pathways playing a role in genomic stability. Kinome profiling identified active Akt signaling, which could inhibit proliferation in 2/3 osteosarcoma cell lines. Inhibition of PI3K/Akt/mTORC1 signaling may be effective in osteosarcoma, but further studies are required to determine whether this pathway is active in a substantial subgroup of this heterogeneous tumor. PMID:24447333

  1. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells.

    PubMed

    Mamo, Tewodros; Mladek, Ann C; Shogren, Kris L; Gustafson, Carl; Gupta, Shiv K; Riester, Scott M; Maran, Avudaiappan; Galindo, Mario; van Wijnen, Andre J; Sarkaria, Jann N; Yaszemski, Michael J

    2017-04-29

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Effects of transplantation sites on tumour growth, pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice.

    PubMed

    Jaroensong, T; Endo, Y; Lee, S-J; Kamida, A; Mochizuki, M; Nishimura, R; Sasaki, N; Nakagawa, T

    2012-12-01

    To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT-xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC-xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin-radixin-moesin (p-ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p-ERM expression at later time points. The expression of ezrin and p-ERM in the primary tumours of IT-xenografted mice was higher than those in SC-xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells. © 2011 Blackwell Publishing Ltd.

  3. Human cytomegalovirus immediate early gene expression in the osteosarcoma line U2OS is repressed by the cell protein ATRX.

    PubMed

    McFarlane, Steven; Preston, Chris M

    2011-04-01

    The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. 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

  5. Salinomycin inhibits osteosarcoma by targeting its tumor stem cells.

    PubMed

    Tang, Qing-Lian; Zhao, Zhi-Qiang; Li, Jin-Chun; Liang, Yi; Yin, Jun-Qiang; Zou, Chang-Ye; Xie, Xian-Biao; Zeng, Yi-Xin; Shen, Jing-Nan; Kang, Tiebang; Wang, Jin

    2011-12-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents and is typically associated with a poor prognosis. Tumor stem cells (TSCs) are presumed to drive tumor initiation and tumor relapse or metastasis. Hence, the poor prognosis of osteosarcoma likely results from a failure to target the osteosarcoma stem cells. Here, we have utilized three different methods to enrich TSCs in osteosarcoma and further evaluated whether salinomycin could selectively target TSCs in osteosarcoma. Our results indicated that sarcosphere selection, chemotherapy selection and stem cell marker OCT4 or SOX2 over-expression are all effective in the enrichment of TSCs from osteosarcoma cell lines. Further investigation found that salinomycin inhibited osteosarcoma by selectively targeting its stem cells both in vitro and in vivo without severe side effects, and the Wnt/β-catenin signaling pathway may be involved in this inhibition of salinomycin. Taken together, we have identified that salinomycin is an effective inhibitor of osteosarcoma stem cells, supporting the use of salinomycin for elimination of osteosarcoma stem cells and implying a need for further clinical evaluation.

  6. Analysis of Selenium Levels in Osteosarcoma Patients and the Effects of Se-Methylselenocysteine on Osteosarcoma Cells In Vitro.

    PubMed

    Huang, Gang; Yong, Bi-cheng; Xu, Ming-hong; Li, Jing-chun; Guo, Hai-hua; Shen, Jing-nan

    2015-01-01

    The form of selenium appears to be important for preventing cancer in humans. Here, we evaluated selenium levels in the serum and bone tissue samples from osteosarcoma patients using atomic absorption spectrometry. The in vitro effects of Se-methylselenocysteine (Se-MSC) on growth, cell cycle status, and apoptosis of osteosarcoma cells were assessed using the WST-1 assay, Hoechst 33342/propidium iodide staining, and flow cytometry, respectively. In osteosarcoma cases, the mean serum selenium levels in osteosarcoma tissue and normal bone were 0.08 mg/kg and 0.03 mg/kg, respectively (P < 0.05). Serum selenium levels in osteosarcoma and non-osteosarcoma cases were 0.09 mg/L and 0.08 mg/L, respectively (P > 0.05). Se-MSC-treated MG63 cells showed altered cellular morphology, decreased viability in a time- and dose-dependent manner, and an increase in the sub-G1 cell population. Se-MSC also downregulated Bcl-2 expression and upregulated Bax. Se-MSC inhibited the proliferation of the drug-resistant osteosarcoma cell line Saos-2/MTX300 and enhanced the inhibitory effect of pirarubicin on MG63 cells. Our data demonstrate that selenium levels are significantly higher in osteosarcoma tissue than normal bone tissue in osteosarcoma patients. The results also support the anticancer effects of Se-MSC in osteosarcoma. Further development of Se-MSC as an ancillary chemotherapy agent in osteosarcoma is warranted.

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

    SciTech Connect

    Witasp, Erika; Gustafsson, Ann-Catrin; Cotgreave, Ian; Lind, Monica . E-mail: monica.lind@imm.ki.se; Fadeel, Bengt . E-mail: bengt.fadeel@imm.ki.se

    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 this 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}.

  8. The influence of elementary silver versus titanium on osteoblasts behaviour in vitro using human osteosarcoma cell lines.

    PubMed

    Hardes, Jendrik; Streitburger, Arne; Ahrens, Helmut; Nusselt, Thomas; Gebert, Carsten; Winkelmann, Winfried; Battmann, Achim; Gosheger, Georg

    2007-01-01

    Purpose. The antimicrobial effect of a silver-coated tumor endoprosthesis has been proven in clinical and experimental trials. However, in the literature there are no reports concerning the effect of elementary silver on osteoblast behaviour. Therefore, the prosthetic stem was not silver-coated because of concerns regarding a possible inhibition of the osseointegration. The aim of the present study was to investigate the effect of 5-25 mg of elementary silver in comparison to Ti-6Al-4V on human osteosarcoma cell lines (HOS-58, SAOS). Methods. Cell viability was determined by measuring the MTT proliferation rate. Cell function was studied by measuring alkaline phosphatase (AP) activity and osteocalcine production. Results. In the HOS-58 cells, the AP activity was statistically significant (P < 0.05) higher at a supplement of 5-10 mg of silver than of Ti-6 Al-4V at the same doses. For both cell lines, a supplement above 10 mg of silver resulted in a reduced AP activity in comparision to the Ti-6 Al-4V group, but a statistically significant difference (P < 0.05) was observed at a dose of 25 mg for the SAOS cells only. At doses of 20-25 mg in the HOS-58 cells and 10-25 mg in the SAOS cells, the reduction of the proliferation rate by silver was statistically significant (P < 0.05) compared to the Ti-6 Al-4V supplement. Discussion. In conclusion, elementary silver exhibits no cytotoxicity at low concentrations. In contrast, it seems to be superior to Ti-6 Al-4V concerning the stimulation of osteogenic maturation at these concentrations, whereas at higher doses it causes the known cytotoxic properties.

  9. The Influence of Elementary Silver Versus Titanium on Osteoblasts Behaviour In Vitro Using Human Osteosarcoma Cell Lines

    PubMed Central

    Hardes, Jendrik; Streitburger, Arne; Ahrens, Helmut; Nusselt, Thomas; Gebert, Carsten; Winkelmann, Winfried; Battmann, Achim; Gosheger, Georg

    2007-01-01

    Purpose. The antimicrobial effect of a silver-coated tumor endoprosthesis has been proven in clinical and experimental trials. However, in the literature there are no reports concerning the effect of elementary silver on osteoblast behaviour. Therefore, the prosthetic stem was not silver-coated because of concerns regarding a possible inhibition of the osseointegration. The aim of the present study was to investigate the effect of 5–25 mg of elementary silver in comparison to Ti-6Al-4V on human osteosarcoma cell lines (HOS-58, SAOS). Methods. Cell viability was determined by measuring the MTT proliferation rate. Cell function was studied by measuring alkaline phosphatase (AP) activity and osteocalcine production. Results. In the HOS-58 cells, the AP activity was statistically significant (P < 0.05) higher at a supplement of 5–10 mg of silver than of Ti-6 Al-4V at the same doses. For both cell lines, a supplement above 10 mg of silver resulted in a reduced AP activity in comparision to the Ti-6 Al-4V group, but a statistically significant difference (P < 0.05) was observed at a dose of 25 mg for the SAOS cells only. At doses of 20–25 mg in the HOS-58 cells and 10–25 mg in the SAOS cells, the reduction of the proliferation rate by silver was statistically significant (P < 0.05) compared to the Ti-6 Al-4V supplement. Discussion. In conclusion, elementary silver exhibits no cytotoxicity at low concentrations. In contrast, it seems to be superior to Ti-6 Al-4V concerning the stimulation of osteogenic maturation at these concentrations, whereas at higher doses it causes the known cytotoxic properties. PMID:17680031

  10. Vanadium and cancer treatment: antitumoral mechanisms of three oxidovanadium(IV) complexes on a human osteosarcoma cell line.

    PubMed

    León, I E; Butenko, N; Di Virgilio, A L; Muglia, C I; Baran, E J; Cavaco, I; Etcheverry, S B

    2014-05-01

    We report herein the antitumor actions of three oxidovanadium(IV) complexes on MG-63 human osteosarcoma cell line. The three complexes: VO(oda), VO(oda)bipy and VO(oda)phen (oda=oxodiacetate), caused a concentration dependent inhibition of cell viability. The antiproliferative action of VO(oda)phen could be observed in the whole range of concentrations (at 2.5 μM), while VO(oda)bipy and VO(oda) showed a decrease of cell viability only at higher concentrations (at 50 and 75 μM, respectively) (p<0.01). Moreover, VO(oda)phen caused a decrease of lysosomal and mitochondrial activities at 2.5 μM, while VO(oda) and VO(oda)bipy affected neutral red uptake and mitochondrial metabolism at 50 μM (p<0.01). On the other hand, no DNA damage studied by the Comet assay could be observed in MG-63 cells treated with VO(oda) at 2.5-10 μM. Nevertheless, VO(oda)phen and VO(oda)bipy induced DNA damage at 2.5 and 10 μM, respectively (p<0.01). The generation of reactive oxygen species increased at 10 μM of VO(oda)phen and only at 100 μM of VO(oda) and VO(oda)bipy (p<0.01). Besides, VO(oda)phen and VO(oda)bipy triggered apoptosis as determined by externalization of the phosphatidylserine. The determination of DNA cleavage by agarose gel electrophoresis showed that the ability of VO(oda)(bipy) is similar to that of VO(oda), while VO(oda)(phen) showed the highest nuclease activity in this series. Overall, our results showed a good relationship between the bioactivity of the complexes and their structures since VO(oda)phen presented the most potent antitumor action in human osteosarcoma cells followed by VO(oda)bipy and then by VO(oda) according to the number of intercalating heterocyclic moieties.

  11. Expression and function of the ACE2/angiotensin(1-7)/Mas axis in osteosarcoma cell lines U-2 OS and MNNG-HOS.

    PubMed

    Ender, Stephan Albrecht; Dallmer, Andrea; Lässig, Florian; Lendeckel, Uwe; Wolke, Carmen

    2014-08-01

    The renin-angiotensin-system (RAS), via its classical angiotensin-converting enzyme (ACE)/angiotensin II/angiotensin II type 1 receptor (AT1R)-axis, is associated with proliferation and metastasis of numerous types of solid tumor. AT1R blockers reduce tumor volume and decrease liver and lung metastasis in murine models of osteosarcoma. Expression and function of the alternative ACE2/Ang(1-7)/Mas axis in osteosarcoma is yet to be studied. In the present study, the basic and interleukin (IL)-1β-stimulated expression of components of this alternative RAS axis were analyzed and the impact of Mas on proliferation and/or migration of U-2 OS and MNNG-HOS osteosarcoma cells was studied. Quantitative polymerase chain reaction revealed that the two cell lines expressed the Ang(1‑7)-generating peptidases ACE2, neutral endopeptidase 24.11 and prolyl-endopeptidase together with the putative receptor for Ang(1-7), Mas. IL-1β provoked an induction of Mas mRNA and protein expression which was associated with a reduction of proliferation and migration. By contrast, small interfering RNA-mediated knockdown of Mas expression led to increased cell proliferation. In conclusion, osteosarcoma cells express a functional active alternative ACE2/Ang(1-7)/Mas axis. The induction and reinforcement of this axis may be beneficial for the treatment of osteosarcoma by reducing growth and preventing cancer metastasis. These effects may be achieved directly by the administration of Mas agonists or, indirectly, via blocking the classical AngII RAS axis via ACE inhibitors or AT1R antagonists.

  12. 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. IC50 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

  13. Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63.

    PubMed

    Marella, Narasimharao V; Zeitz, Michael J; Malyavantham, Kishore S; Pliss, Artem; Matsui, Sei-ichi; Goetze, Sandra; Bode, Juergen; Raska, Ivan; Berezney, Ronald

    2008-01-01

    The organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of approximately 6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5-7 'bands' spanning a single chromosome termed the 'IFN chromosome'. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage-fusion-bridge events.

  14. 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

  15. Alopecurone B reverses doxorubicin-resistant human osteosarcoma cell line by inhibiting P-glycoprotein and NF-kappa B signaling.

    PubMed

    Xia, Yuan-Zheng; Ni, Kai; Guo, Chao; Zhang, Chao; Geng, Ya-Di; Wang, Zhen-Dong; Yang, Lei; Kong, Ling-Yi

    2015-03-15

    Doxorubicin (DOX) was first used in osteosarcoma in the early 1970s as a first-line antineoplastic drug. However, the occurrence of drug resistance in chemotherapeutic treatment has greatly restricted its use. When resistance to DOX treatment occurs, osteosarcoma may become not only resistant to the drug originally administered but also to a wide variety of structurally and mechanistically unrelated drugs. Thus, there is an urgent need to find ways of reversing DOX chemotherapy resistance in osteosarcoma. Plant-derived agents have great potential in preventing the onset of the carcinogenic process and enhancing the efficacy of conventional antitumor drugs. Alopecurone B (ALOB), a flavonoid, is isolated from Traditional Chinese Medicine Sophora alopecuroides L., and is reported to have potent inhibitory effect on multidrug resistance associated protein 1. In this study, a DOX-resistant osteosarcoma cell line (MG-63/DOX) was established by increasing the concentration gradient of DOX in a stepwise manner. MTT assay, flow cytometry analysis, dual-luciferase reporter gene assay, quantitative real-time polymerase chain reaction and Western blot analysis were applied to investigate the reversing effect of ALOB and its underlying mechanisms. The results indicated that ALOB mediated the resistance of MG-63/DOX cells to DOX by inhibiting P-glycoprotein function, transcription and expression. Besides, ALOB also enhanced the sensitivity of MG-63/DOX cells to other conventional chemotherapeutic drugs. Cell viability assay confirmed the reversing activity of ALOB. Furthermore, ALOB increased DOX-induced apoptosis at nontoxic concentration. In addition, ALOB showed inhibitory effect on NF-κB transcription in a DOX-independent manner. Furthermore, NF-κB signaling was suppressed by ALOB in an IKK-dependent manner. These studies not only demonstrate that ALOB is a potential agent for reversal of drug resistant cancers, but also testify that ALOB reverses multidrug resistance by

  16. Quantitative comparison of PTH1R in breast cancer MCF7 and osteosarcoma SaOS-2 cell lines.

    PubMed

    Alokail, Majed S; Peddie, Margaret J

    2008-06-01

    The aim of the present study was to compare the classical parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptors in MCF7 breast cancer cells with SaOS-2 osteosarcoma cell line. Quantitative binding showed that (125)I-PTHrP-1-34(Tyr) binds with a single binding site in both cells. However (125)I-PTHrP-1-34(Tyr) has higher affinity binding in MCF7 (K(D) = 1.88 +/- 0.08 nM) than in SaOS-2 cells (K(D) = 4.4 +/- 0.185 nM). The competitive binding using 3.3 nM (125)I-PTHrP-1-34(Tyr) with increasing amounts (0.33-33 nM) of unlabelled human PTHrP-1-34, PTHrP-7-34, PTHrP-1-86 His(5)-PTHrP-1-36, His(5)-Phe(23)-PTHrP-1-36 or PTH-1-34 revealed different displacements. In SaOS-2 the PTHrP-7-34 and PTHrP-1-86 caused similar displacement compared with 73% by PTH-1-34 and 70% by PTHrP-1-34. However, in MCF7, PTHrP-7-34, PTHrP-1-86 and PTH-1-34 displaced by 54%, 72% and 67%, respectively, compared to 87% by PTHrP-1-34. The His(5)-Phe(23)-PTHrP-1-36 caused an increase in the K(D) from 2.0 +/- 0.03 nM to 2.75 +/- 0.045 nM in MCF7 cells, but had no significant effect in SaOS-2 cells. The PTH/PTHrP receptor in both cell lines revealed a single 85 KDa band with different intensity. Our results suggest that the PTH/PTHrP receptor in MCF7 cells has higher binding affinity for PTHrP than PTH compared to the receptor in SaOS-2 cells. (c) 2008 John Wiley & Sons, Ltd.

  17. Platelets Inhibit Migration of Canine Osteosarcoma Cells.

    PubMed

    Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

    2017-01-01

    The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

  18. Aloe-emodin-mediated photodynamic therapy induces autophagy and apoptosis in human osteosarcoma cell line MG‑63 through the ROS/JNK signaling pathway.

    PubMed

    Tu, Pinghua; Huang, Qiu; Ou, Yunsheng; Du, Xing; Li, Kaiting; Tao, Yong; Yin, Hang

    2016-06-01

    The present study was carried out to investigate the effect and mechanisms of aloe‑emodin (AE)-mediated photodynamic therapy (AE-PDT) on the human osteosarcoma cell line MG-63. After treatment with AE-PDT, the human osteosarcoma cell line MG-63 was tested for levels of viability, autophagy, reactive oxygen species (ROS) and apoptosis and changes in cell morphology with the Cell Counting Kit-8 (CCK‑8), monodansylcadaverine (MDC) and Hoechst staining and transmission electron microscopy. The expression of proteins including LC-3, cleaved caspase-3, Beclin-1, Bcl-2, p-JNK, t-JNK and β-actin was examined with western blotting. AE-PDT significantly inhibited the viability of the MG-63 cells in an AE-concentration- and PDT energy density-dependent manner. Autophagy and apoptosis of MG-63 cells was substantially promoted in the AE-PDT group compared to the control group, the AE alone group and the light emitting diode (LED) alone group. Inhibition of autophagy by 3-methyladenine (3-MA) (5 mM) and chloroquine (CQ) (15 µM) significantly promoted the apoptosis rate and improved the sensitivity of the MG-63 cells to AE-PDT. AE-PDT was found to induce the expression of ROS and p-JNK. ROS scavenger, N-acetyl-L-cysteine (NAC, 5 mM), was able to hinder the autophagy, apoptosis and phosphorylation of JNK, and JNK inhibitor (SP600125, 10 µM) significantly inhibited the autophagy and apoptosis, and attenuated the sensitivity of MG63 cells to AE-PDT. In conclusion, AE-PDT induced the autophagy and apoptosis of human osteosarcoma cell line MG-63 through the activation of the ROS-JNK signaling pathway. Autophagy may play a protective role during the early stage following treatment of AE-PDT.

  19. Effect of troglitazone on tumor growth and pulmonary metastasis development of the mouse osteosarcoma cell line LM8.

    PubMed

    Aizawa, Junichi; Sakayama, Kenshi; Kamei, Setsuya; Kidani, Teruki; Yamamoto, Haruyasu; Norimatsu, Yoshiaki; Masuno, Hiroshi

    2010-02-22

    Osteosarcoma often develops micrometastases in the lung prior to diagnosis, causing a fatal outcome. Therefore, the prevention of pulmonary metastases is critical for the improvement of the prognosis of patients with osteosarcoma. The purpose of this study was to investigate whether troglitazone (TGZ) is considered as possible therapeutics in the treatment of growth and metastasis of osteosarcoma. LM8 cells were treated for 3 days with various concentrations of TGZ. The effect of TGZ on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine incorporation study. The assay of cell invasion and motility was performed using either the Matrigel-coated cell culture inserts or the uncoated cell culture inserts in the invasion chambers. The effect of TGZ on Akt signaling was assessed by Western blot analysis of Akt and p-Akt. The effects of oral administration of either TGZ (TGZ group) or ethanol (control group) on the growth of primary tumor and the development of pulmonary metastasis were examined in nude mice implanted with LM8 cells on their backs. The expression and activity of matrix metalloproteinase 2 (MMP-2) within the tumor were determined by immunohistochemistry and zymography. The microvessel density (MVD) within the tumor was determined by immunohistochemistry for CD34. TGZ dose-dependently inhibits cell proliferation. TGZ-treated cells were less invasive and less motile than untreated cells. The activity of MMP-2 secreted by TGZ-treated cells was lower than that secreted by untreated cells. TGZ decreased the level of p-Akt. The primary tumor mass was smaller in the TGZ group than in the control group. The TGZ group had less metastatic tumors in the lung compared with the control group. The expression and activity of MMP-2 within the tumor of the TGZ group were lower than those of the control group. The MVD within the tumor of the TGZ group was lower than that of the control group. Inhibition of Akt signaling by TGZ

  20. 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

  1. Possible contribution of aminopeptidase N (APN/CD13) to migration and invasion of human osteosarcoma cell lines.

    PubMed

    Liang, Wenqing; Gao, Bo; Xu, Guojian; Weng, Dong; Xie, Minghua; Qian, Yu

    2014-12-01

    Osteosarcoma is the most common primary malignancy of the bone. Aminopeptidase N (APN/CD13), a Zn+2-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion mechanism. This study explores the potential involvement of APN in migration and invasion of human osteosarcoma cells in vitro using inhi-bitors and activators of APN. Cells treated with APN inhibitor bestatin displayed decreased migration and invasion in a Boyden chamber Transwell assay. Western blotting revealed reduced levels of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway proteins, reduced phosphorylation of p38, ERK1/2 and JNK and decreased levels of NF-κB. Bestatin treatment also lowered APN, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and their mRNA expression. Reduced MMP-2 and -9 protein levels were also observed. By comparison, cells treated with cytokine interleukin-6 (IL-6), a stimulator of APN, displayed increased migration and invasion. Western blotting revealed increased levels of MAPK and PI3K pathway proteins, phosphorylated p38, ERK1/2 and JNK, and NF-κB. IL-6 treatment also increased APN and MMP-2 and -9 enzymatic activity. An increase of APN, MMP-2 and -9 mRNA levels, and MMP-2 and -9 protein levels was also observed. Together these experiments reveal potential enzymatic and signalling roles for APN in osteosarcoma and establish a starting point for an in-depth analysis of the role of APN in regulating invasiveness. A deeper knowledge about the regulatory mechanisms of APN may contribute to the development of anti-metastatic therapies.

  2. A possible role of oxidative stress in the vanadium-induced cytotoxicity in the MC3T3E1 osteoblast and UMR106 osteosarcoma cell lines.

    PubMed

    Cortizo, A M; Bruzzone, L; Molinuevo, S; Etcheverry, S B

    2000-06-08

    The cytotoxicity and free radical production induced by vanadium compounds were investigated in an osteoblast (MC3T3E1) and an osteosarcoma (UMR106) cell lines in culture. Vanadate induced cell toxicity, reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) increased in a concentration-dependent manner (0.1-10 mM) after 4 h. The concentration-response curve of vanadate-induced cytotoxicity and oxidative stress in MC3T3E1 cells was shifted to the left of the UMR106 curve, suggesting a greater sensitivity of the non-transformed cells in comparison to the osteosarcoma UMR106 cells. Supplementing with vitamin E acetate (80 microM) significantly inhibited ROS and TBARS formation but did not improve the vanadate-dependent decrease in cell number. Other vanadium compounds (vanadyl, pervanadate, and VO/Aspi, a complex of vanadyl(IV) with aspirin) showed different degrees of cell toxicity and induced oxidative stress. Altogether these results suggest that oxidative stress is involved in vanadium induced osteoblastic cytotoxicity, although the mechanism is unknown.

  3. Identification and gene expression profiling of tumor-initiating cells isolated from human osteosarcoma cell lines in an orthotopic mouse model

    PubMed Central

    Rainusso, Nino; Man, Tsz-Kwong; Lau, Ching C; Hicks, John; Shen, Jianhe J; Yu, Alexander; Wang, Lisa L

    2011-01-01

    In the cancer stem cell model a cell hierarchy has been suggested as an explanation for intratumoral heterogeneity and tumor formation is thought to be driven by this tumor cell subpopulation. The identification of cancer stem cells in osteosarcoma (OS) and the biological processes dysregulated in this cell subpopulation, also known as tumor-initiating cells (TICs), may provide new therapeutic targets. The goal of this study, therefore, was to identify and characterize the gene expression profiles of TICs isolated from human OS cell lines. We analyzed the self-renewal capacity of OS cell lines and primary OS tumors based upon their ability to form sphere-like structures (sarcospheres) under serum-starving conditions. TICs were identify from OS cell lines using the long-term label retention dye PKH26. OS TICs and the bulk of tumor cells were isolated and used to assess their ability to initiate tumors in NOD/SCID mice. Gene expression profiles of OS TICs were obtained from fresh orthotopic tumor samples. We observed that increased sarcosphere efficiency correlated with an enhanced tumorigenic potential in OS. PKH26Hi cells were shown to constitute OS TICs based upon their capacity to form more sarcospheres, as well as to generate both primary bone tumors and lung metastases efficiently in NOD/SCID mice. Genomic profiling of OS TICs revealed that both bone development and cell migration processes were dysregulated in this tumor cell subpopulation. PKH26 labeling represents a valuable tool to identify OS TICs and gene expression analysis of this tumor cell compartment may identify potential therapeutic targets. PMID:21617384

  4. 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.

  5. MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1.

    PubMed

    Geng, Shuo; Gu, Lina; Ju, Fang; Zhang, Hepeng; Wang, Yiwen; Tang, Han; Bi, ZhengGang; Yang, Chenglin

    2016-09-01

    Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR-224 in the development and progression of osteosarcoma. We demonstrated that miR-224 was down-regulated in osteosarcoma cell lines and tissues. Lower miR-224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR-224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG-63 cells to cisplatin. We identified Rac1 as a direct target gene of miR-224 in osteosarcoma. Rac1 expression was up-regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR-224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR-224-overexpressing MG-63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR-224-overexpressing MG-63 cells. These data suggest that miR-224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  6. CD271+ Osteosarcoma Cells Display Stem-Like Properties

    PubMed Central

    Tian, Jiguang; Li, Xin; Si, Meng; Liu, Ting; Li, Jianmin

    2014-01-01

    Cancer stem cell (CSC) theory has been proposed and verified in many cancers. The existence of osteosarcoma CSCs has been confirmed for many years and multiple surface markers have been employed to identify them. In this study, we identified CD271+ subpopulation of osteosarcoma displaying stem-like properties. CD271, known as the neural crest nerve growth factor receptor, is the marker of bone marrow mesenchymal stem cells (MSCs) and human melanoma-initiating cells. We discovered that CD271 was expressed differentially in diverse types of human osteosarcoma and stabilized cell lines. CD271+ osteosarcoma cells displayed most of the properties of CSC, such as self-renewal, differentiation, drug resistance and tumorigenicity in vivo. Nanog, Oct3/4, STAT3, DNA-PKcs, Bcl-2 and ABCG2 were more expressed in CD271+ cells compared with CD271− cells. Our study supported the osteosarcoma CSC hypothesis and, to a certain extent, revealed one of the possible mechanisms involved in maintaining CSCs properties. PMID:24893164

  7. CD271+ osteosarcoma cells display stem-like properties.

    PubMed

    Tian, Jiguang; Li, Xin; Si, Meng; Liu, Ting; Li, Jianmin

    2014-01-01

    Cancer stem cell (CSC) theory has been proposed and verified in many cancers. The existence of osteosarcoma CSCs has been confirmed for many years and multiple surface markers have been employed to identify them. In this study, we identified CD271(+) subpopulation of osteosarcoma displaying stem-like properties. CD271, known as the neural crest nerve growth factor receptor, is the marker of bone marrow mesenchymal stem cells (MSCs) and human melanoma-initiating cells. We discovered that CD271 was expressed differentially in diverse types of human osteosarcoma and stabilized cell lines. CD271(+) osteosarcoma cells displayed most of the properties of CSC, such as self-renewal, differentiation, drug resistance and tumorigenicity in vivo. Nanog, Oct3/4, STAT3, DNA-PKcs, Bcl-2 and ABCG2 were more expressed in CD271(+) cells compared with CD271- cells. Our study supported the osteosarcoma CSC hypothesis and, to a certain extent, revealed one of the possible mechanisms involved in maintaining CSCs properties.

  8. Hydrogen peroxide overload increases adriamycin-induced apoptosis of SaOS(2)FM, a manganese superoxide dismutase-overexpressing human osteosarcoma cell line.

    PubMed

    Wang, Yadi; Kuroda, Masahiro; Gao, Xian-Shu; Asaumi, Jun-Ichi; Shibuya, Kohichi; Kawasaki, Shoji; Akaki, Shiro; St Clair, Daret; Hiraki, Yoshio; Kanazawa, Susumu

    2005-05-01

    We previously developed a new microscopic observation system that enables time-lapse quantitative analysis of apoptosis and necrosis. With this system we quantitatively analyzed adriamycin (ADR)-induced cell death using manganese superoxide dismutase (MnSOD)- and wild-type p53-gene transfectants on SaOS(2), a p53-deficient human osteosarcoma cell line. A highly MnSOD-overexpressing cell line, SaOS(2)FM(H), acquired ADR-tolerance compared to the parent cell line SaOS(2). The ADR-tolerance of SaOS(2)FM(H) diminished by L-buthionine-[S,R]-sulfoximine (BSO), which did not change ADR-sensitivity of SaOS(2), to the similar ADR-sensitivity of SaOS(2). A wild-type p53-expressing cell line, SaOS(2)wtp53, significantly increased in ADR-sensitivity compared to SaOS(2). This ADR-sensitivity of SaOS(2)wtp53 was enhanced by BSO. When isosorbide 5-mononitrate was combined with BSO, isosorbide 5-mononitrate increased ADR sensitivity of a moderately MnSOD-overexpressing cell line, SaOS(2)FM(L), decreased that of SaOS(2) FM(H), and did not change those of SaOS(2) and SaOS(2)wtp53 compared to BSO alone. Time-lapse microscopic observations during ADR treatment for 24 h indicated that the most cells of each cell line underwent apoptosis, and a few cells (less than 11%) died by necrosis. When cells were treated with iso-concentration of ADR, apoptosis of SaOS(2)FM(H) was less than that of SaOS(2). BSO, which did not change ADR-sensitivity of SaOS(2), increased appearance rate of ADR-induced apoptosis, but not necrosis of MnSOD-overexpressing cell lines. When iso-survival dose of ADR, which reduced surviving fraction to 0.01, was given for each cell line, no difference was observed in appearance of either apoptosis or necrosis between SaOS(2) and MnSOD-overexpressing cell lines. On the other hands, appearance of both apoptosis and the following secondary necrosis of SaOS(2) wtp53 was significantly accelerated compared to those of SaOS(2). These findings indicate that hydrogen peroxide

  9. Programmed cell death ligand 1 expression in osteosarcoma.

    PubMed

    Shen, Jacson K; Cote, Gregory M; Choy, Edwin; Yang, Pei; Harmon, David; Schwab, Joseph; Nielsen, G Petur; Chebib, Ivan; Ferrone, Soldano; Wang, Xinhui; Wang, Yangyang; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

    2014-07-01

    Programmed cell death ligand 1 (PDL1, also known as B7H1) is a cell-surface protein that suppresses the cytotoxic CD8(+) T-cell-mediated immune response. PDL1 expression and its clinical relevance in sarcomas are not well understood. Therefore, we sought to measure RNA expression levels for PDL1 in 38 clinically annotated osteosarcoma tumor samples and aimed to determine if PDL1 expression correlates with clinical features and tumor-infiltrating lymphocytes (TIL). Quantitative real-time RT-PCR for PDL1 was optimized in 18 cell lines, of which 5 were osteosarcoma derived. qRT-PCR results were validated via flow cytometry and immunohistochemistry (IHC) in select cell lines. Total RNA was isolated from 38 human osteosarcoma samples for qRT-PCR analysis. Clinical data were sorted, and significance was determined by the Student t test. TILs were examined in patient samples by tissue microarray hematoxylin-eosin staining. We confirmed the constitutive PDL1 mRNA expression in cell lines by qRT-PCR, flow cytometry, and IHC. Across human osteosarcoma samples, PDL1 mRNA gene expression ranged over 4 log (>5,000-fold difference). Relative expression levels were evaluated against clinical factors such as age/gender, metastasis, recurrence, chemotherapy, percentage of necrosis, and survival; no significant associations were identified. The presence of TILs was associated with high PDL1 expression (R(2) = 0.37; P = 0.01). In summary, we developed an RNA-based assay to determine PDL1 expression levels, and we show, for the first time, that high levels of PDL1 are expressed in a subset of osteosarcoma, and PDL1 expression is positively correlated with TILs. Multiple agents targeting PD1/PDL1 are in clinical development, and this may be a novel immunotherapeutic strategy for osteosarcoma clinical trials.

  10. Deciphering the effect of an oxovanadium(iv) complex with the flavonoid chrysin (VOChrys) on intracellular cell signalling pathways in an osteosarcoma cell line.

    PubMed

    León, Ignacio E; Díez, Paula; Etcheverry, Susana B; Fuentes, Manuel

    2016-08-01

    Vanadium complexes were studied during recent years and considered as a representative of a new class of non-platinum metal antitumor agents in combination with their low toxicity. However, a few challenges still remain in the discovery of new molecular targets for these novel metal-based drugs. The study of cell signaling pathways related to vanadium drugs, which is highly critical for identifying specific targets that play an important role in the antitumor activity of vanadium compounds, is scarce. This research deals with the alterations in intracellular signaling pathways promoted by an oxovanadium(iv) complex with the flavonoid chrysin [VO(chrysin)2EtOH]2 (VOChrys) in a human osteosarcoma cell line (MG-63). Herein we report for the first time the effect of [VO(chrysin)2EtOH]2 on the relative abundance of 224 proteins, which are involved in the most common intracellular pathways. Besides, full-length human recombinant (FAK and AKT1) kinases are produced using an in situ IVTT system and then we have evaluated the variation of relative tyrosine-phosphorylation levels caused by the [VO(chrysin)2EtOH]2 compound. The results of the differential protein expression levels reveal that several proteins such as PKB/AKT, PAK, DAPK, Cdk 4, 6 and 7, FADD, AP2, NAK, and JNK, among others, were altered. Moreover, cell signaling pathways related to the PTK2B, FAK, PKC families suggests an important role associated with the antitumor activity of [VO(chrysin)2EtOH]2 was demonstrated. Finally, the effect of this compound on in situ expressed FAK and AKT1 is validated by determining the phosphorylation level, which decreased in the former and increased in the latter.

  11. Downregulation of microRNA-586 Inhibits Proliferation, Invasion and Metastasis and Promotes Apoptosis in Human Osteosarcoma U2-OS Cell Line.

    PubMed

    Yang, Lei; Liu, Zong-Ming; Rao, Yan-Wei; Cui, Shao-Qian; Wang, Huan; Jia, Xiao-Jing

    2015-01-01

    In this study, we aim to examine the association of microRNA-586 (miR-586) with osteosarcoma (OS) cell proliferation, apoptosis, invasion, and metastasis. U2-OS cell lines were divided into 4 groups: an miR-586 group, anti-miR-586 group, control group (empty plasmid) and blank group (no plasmid). qRT-PCR was used to detect miR-586 expression, cell counting kit-8 and EdU assays to detect cell proliferation, flow cytometry to detect cell cycle distribution, Annexin V/PI double staining to detect cell apoptosis, and the Transwell assay to detect cell invasion and metastasis. miR-586 expression was significantly higher in the miR-586 group but significantly lower in the anti-miR-586 group compared with the control and blank groups. Cell proliferation at 2-5 days after cell transfection and the EdU-positive cell number increased obviously in the miR-586 group but decreased clearly in the anti-miR-586 group. In the miR-586 group, cells at G0/G1 stage and apoptosis cells significantly decreased, while cells at G2/M and S stages and invasive and metastatic cells significantly increased compared to the control and blank groups; however, opposite trends were found in the anti-miR-586 group. Downregulation of miR-586 expression in OS may inhibit cell proliferation, invasion and metastasis, and promote cell apoptosis. © 2015 S. Karger AG, Basel.

  12. Mir-150 Up-Regulates Glut1 and Increases Glycolysis in Osteosarcoma Cells

    PubMed

    Yuan, Guangke; Zhao, Yanqing; Wu, Dongjin; Gao, Chunzheng

    2017-04-01

    Objective: Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Many studies have shown that microRNAs play a critical role in proliferation and metastasis with this tumour type. However, whether aberrant expression might contribute to a metabolism switch in osteosarcoma cases is not clearly understood. In this study, we explored expression and function of miR-150 in osteosarcoma cells. Materials and methods: Expression of miR-150 was assessed by real-time PCR in cell lines and human patient tissues. Scramble siRNA, miR-150 inhibitor, and miR-150 mimics were transfected into osteosarcoma cells to determine their effects on proliferation rate, glucose uptake and lactate secretion. Finally, the relationship between Glut1 and the miR-150 level was explored by luciferase reporter assay and western blotting. Result: miR-150 was consistently decreased in cell lines and osteosarcoma tissues as compared to osteoblast cells and normal bone. Ectopic overexpression of miR-150 inhibited osteosarcoma cell proliferation and suppressed glucose uptake and lactate secretion. Loss of function of miR-150, on the other hand, enhanced osteosarcoma cell proliferation and increased glucose uptake and lactate secretion. Western blot and luciferase reporter assays showed that miR-150 may function by regulating Glut1 expression. Conclusion: These data suggest that miR-150 is involved in regulation of glycolysis in osteosarcoma cells by influencing Glut1 expression. Creative Commons Attribution License

  13. Troglitazione affects survival of human osteosarcoma cells.

    PubMed

    Lucarelli, Enrico; Sangiorgi, Luca; Maini, Veronica; Lattanzi, Giovanna; Marmiroli, Sandra; Reggiani, Matteo; Mordenti, Marina; Alessandra Gobbi, Giuliana; Scrimieri, Francesca; Zambon Bertoja, Annarosa; Picci, Piero

    2002-03-20

    Activation of PPAR gamma, a transcription factor member of the family of peroxisome proliferator-activated receptors, induces apoptosis in several normal and tumor cell lines. In our study, we investigated whether treatment with troglitazone (TRO), a known PPAR gamma agonist, induced apoptosis in the human osteosarcoma (OS) cell lines G292, MG63, SAOS and U2OS that express PPAR gamma. In our experiments, TRO never induced apoptosis of OS cells; on the contrary, TRO increased cell number, based on MTT proliferation assay. Remarkably, the TRO-induced cell number increase depended on a decrease of apoptosis that naturally occurred in the culture and was not due to an increased cell proliferation rate. TRO also prevented staurosporin-induced apoptosis. The TRO-mediated survival effect correlated with the activation of Akt, a well-known mediator of survival stimuli. Our work describes a new function for TRO and indicates that the Akt survival pathway may be a mediator of TRO-induced increase of survival. Copyright 2002 Wiley-Liss, Inc.

  14. Genetically modified T cells targeting interleukin-11 receptor α-chain kill human osteosarcoma cells and induce the regression of established osteosarcoma lung metastases.

    PubMed

    Huang, Gangxiong; Yu, Ling; Cooper, Laurence Jn; Hollomon, Mario; Huls, Helen; Kleinerman, Eugenie S

    2012-01-01

    The treatment of osteosarcoma pulmonary metastases remains a challenge. T cells genetically modified to express a chimeric antigen receptor (CAR), which recognizes a tumor-associated antigen, have shown activity against hematopoietic malignancies in clinical trials, but this requires the identification of a specific receptor on the tumor cell. In the current study, we found that interleukin (IL)-11Rα was selectively expressed on 14 of 16 osteosarcoma patients' lung metastases and four different human osteosarcoma cell lines, indicating that IL-11Rα may be a novel target for CAR-specific T-cell therapy. IL-11Rα expression was absent or low in normal organ tissues, with the exception of the gastrointestinal tract. IL-11Rα-CAR-specific T cells were obtained by non-viral gene transfer of Sleeping Beauty DNA plasmids and selectively expanded ex vivo using artificial antigen-presenting cells derived from IL-11Rα + K562 cells genetically modified to coexpress T-cell costimulatory molecules. IL-11Rα-CAR(+) T cells killed all four osteosarcoma cell lines in vitro; cytotoxicity correlated with the level of IL-11Rα expression on the tumor cells. Intravenous injection of IL-11Rα-CAR(+) T cells into mice resulted in the regression of osteosarcoma pulmonary metastases with no organ toxicity. Together, the data suggest that IL-11Rα-CAR T cells may represent a new therapy for patients with osteosarcoma pulmonary metastases. ©2011 AACR.

  15. High expression of long non-coding RNA XIST in osteosarcoma is associated with cell proliferation and poor prognosis.

    PubMed

    Li, G-L; Wu, Y-X; Li, Y-M; Li, J

    2017-06-01

    Osteosarcoma is one of the most common primary bone malignancies. Long non-coding RNAs (lncRNAs) have recently emerged as key regulators of osteosarcoma. The aim of present study was to explore the prognostic value of long non-coding RNA XIST (XIST) in osteosarcoma and XIST's relation to the cell proliferation in osteosarcoma in vitro. The XIST expressions were detected in osteosarcoma tissues and their paired adjacent normal tissues from 145 osteosarcoma patients by using qRT-PCR. The association between XIST expression and clinicopathological factors, as well as survival rates, was analyzed. The possibility of XIST as a prognostic biomarker for osteosarcoma was examined by Cox proportional hazard regression model. MTT assays were conducted to explore the impact of XIST overexpression on the proliferation of osteosarcoma cells. The results showed that XIST was significantly up-regulated in osteosarcoma tissues and cell lines, and high XIST expression was significantly associated with advanced tumor size (p=0.009), advanced clinical stage (p=0.001) and present distant metastasis (p=0.009). Kaplan-Meier analysis showed that increased XIST expression was associated with poor overall survival of patients. Univariate and multivariate analysis suggested that XIST expression was an independent prognostic factor for the survival of patients with osteosarcoma. Furthermore, we found that knockdown of XIST significantly suppressed the proliferation of osteosarcoma cells in vitro. XIST was suggested to have a tumor promoter effect, and thus, to be a predictor of outcome in patients with osteosarcoma.

  16. MicroRNA-448 suppresses osteosarcoma cell proliferation and invasion through targeting EPHA7.

    PubMed

    Wu, Xiangkun; Yan, Lihua; Liu, Yongxi; Xian, Wenfeng; Wang, Liuyu; Ding, Xunmeng

    2017-01-01

    Osteosarcoma is the most common type of malignant bone tumor, often affecting adolescents and children. MicroRNAs (miRNAs) are a group of small, non-protein coding, endogenous RNAs that play critical roles in osteosarcoma tumorigenesis. In our study, we demonstrated that miR-448 expression was downregulated in osteosarcoma tissues and cell lines. Overexpression of miR-448 suppressed osteosarcoma cell proliferation, colony formation and migration. Moreover, we found that EPHA7 was a direct target gene of miR-448 in osteosarcoma cells. We further demonstrated that the EPHA7 expression level was upregulated in osteosarcoma tissues. Interestingly, the expression level of EPHA7 was inversely correlated with the expression level of miR-448 in osteosarcoma tissues. In addition, elevated expression of miR-448 suppressed osteosarcoma cell proliferation and invasion through targeting EPHA7. Taken together, these findings suggest that miR-448 functioned as a tumor suppressor gene in the development of osteosarcoma through targeting EPHA7.

  17. Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

    PubMed

    Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

    2015-03-01

    We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted.

  18. Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

    PubMed

    Mantovani, Fernanda B; Morrison, Jodi A; Mutsaers, Anthony J

    2016-05-31

    Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of

  19. Runx2, p53 and pRB status as diagnostic parameters for deregulation of osteoblast growth and differentiation in a new pre-chemotherapeutic osteosarcoma cell line (OS1)

    PubMed Central

    Pereira, Barry P.; Zhou, Yefang; Gupta, Anurag; Leong, David T.; Aung, Khin Zarchi; Ling, Ling; Pho, Robert W. H.; Galindo, Mario; Salto-Tellez, Manuel; Stein, Gary S.; Cool, Simon M.; van Wijnen, Andre J.; Nathan, Saminathan S.

    2009-01-01

    Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, western blotting and flow cytometry. OS1 have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio (p<0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63 and CD71 to SAOS-2. (p<0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin-E and increased cyclin-A (p<0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS-2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre-chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development. PMID:19746444

  20. AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway

    PubMed Central

    Morishita, Masayuki; Kawamoto, Teruya; Hara, Hitomi; Onishi, Yasuo; Ueha, Takeshi; Minoda, Masaya; Katayama, Etsuko; Takemori, Toshiyuki; Fukase, Naomasa; Kurosaka, Masahiro; Kuroda, Ryosuke; Akisue, Toshihiro

    2017-01-01

    The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma. PMID:27878239

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

    PubMed Central

    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

    2014-01-01

    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. PMID:25348612

  2. 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.

  3. AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway.

    PubMed

    Morishita, Masayuki; Kawamoto, Teruya; Hara, Hitomi; Onishi, Yasuo; Ueha, Takeshi; Minoda, Masaya; Katayama, Etsuko; Takemori, Toshiyuki; Fukase, Naomasa; Kurosaka, Masahiro; Kuroda, Ryosuke; Akisue, Toshihiro

    2017-01-01

    The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) modulates cellular energy metabolism, and promotes mitochondrial proliferation and apoptosis. Previous studies have shown that AICAR has anticancer effects in various cancers, however the roles of AMPK and/or the effects of AICAR on osteosarcoma have not been reported. In the present study, we evaluated the effects of AICAR on tumor growth and mitochondrial apoptosis in human osteosarcoma both in vitro and in vivo. For in vitro experiments, two human osteosarcoma cell lines, MG63 and KHOS, were treated with AICAR, and the effects of AICAR on cell growth and mitochondrial apoptosis were assessed by WST assays, TUNEL staining, and immunoblot analyses. In vivo, human osteosarcoma-bearing mice were treated with AICAR, and the mitochondrial proliferation and apoptotic activity in treated tumors were assessed. In vitro experiments revealed that AICAR activated AMPK, inhibited cell growth, and induced mitochondrial apoptosis in both osteosarcoma cell lines. In vivo, AICAR significantly reduced osteosarcoma growth without apparent body weight loss and AICAR increased both mitochondrial proliferation and apoptotic activity in treated tumor tissues. AICAR showed anticancer effects in osteosarcoma cells through an AMPK-dependent peroxisome proliferator‑activated receptor-γ coactivator-1α (PGC-1α)/mitochondrial transcription factor A (TFAM)/mitochondrial pathway. The findings in this study strongly suggest that AICAR could be considered as a potent therapeutic agent for the treatment of human osteosarcoma.

  4. 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

  5. Knockdown of DDX46 Inhibits the Invasion and Tumorigenesis in Osteosarcoma Cells.

    PubMed

    Jiang, Feng; Zhang, Dengfeng; Li, Guojun; Wang, Xiao

    2017-03-13

    DDX46, a member of the DEAD-box (DDX) helicase family, is involved in the development of several tumors. However, the exact role of DDX46 in osteosarcoma and the underlying mechanisms in tumorigenesis remain poorly understood. Thus, in the present study, we explored the role of DDX46 in osteosarcoma and the underlying mechanisms. Our results demonstrated that the expression levels of DDX46 in both mRNA and protein were greatly elevated in human osteosarcoma tissues and cell lines. Knockdown of DDX46 obviously inhibited osteosarcoma cell proliferation and tumor growth in vivo. In addition, knockdown of DDX46 also significantly suppressed migration and invasion in osteosarcoma cells. Furthermore, knockdown of DDX46 substantially downregulated the phosphorylation levels of PI3K and Akt in SaOS2 cells. In summary, the present results have revealed that DDX46 plays an important role in osteosarcoma growth and metastasis. Knockdown of DDX46 inhibited osteosarcoma cell proliferation, migration, and invasion in vitro and tumor growth in vivo. Therefore, DDX46 may be a potential therapeutic target for the treatment of osteosarcoma.

  6. Long noncoding RNA ATB promotes osteosarcoma cell proliferation, migration and invasion by suppressing miR-200s

    PubMed Central

    Han, Feng; Wang, Changhe; Wang, Yi; Zhang, Liang

    2017-01-01

    Long noncoding RNA activated by transforming growth factor-β (lncRNA-ATB) is a novel lncRNA, which is recently reported to have critical roles in carcinogenesis and progression of several cancers. However, the expression, clinical values, biological roles, and underlying molecular mechanisms of lncRNA-ATB in osteosarcoma are still known. In this study, we measured lncRNA-ATB expression in serum and osteosarcoma tissues of osteosarcoma patients, analyzed its diagnostic and prognostic values. Serum lncRNA-ATB is increased in osteosarcoma patients and could accurately discriminate osteosarcoma patients from healthy controls. LncRNA-ATB is also upregulated in osteosarcoma tissues and cell lines, and positively associated with Enneking stage, metastasis and recurrence. Increased lncRNA-ATB level indicates poor recurrence-free survival and overall survival. Functional experiments demonstrated that overexpression of lncRNA-ATB enhances osteosarcoma cells proliferation, migration, and invasion, and while depletion of lncRNA-ATB inhibits osteosarcoma cells proliferation, migration, and invasion. Mechanistically, we found that lncRNA-ATB inhibits miR-200s, and upregulates miR-200s target genes ZEB1 and ZEB2. Additionally, the roles of lncRNA-ATB on osteosarcoma cells proliferation, migration, and invasion in vitro, and osteosarcoma tumor growth in vivo are dependent on the regulation of miR-200s. Taken together, this study suggests that lncRNA-ATB may be a potential diagnostic and prognostic biomarker and a therapeutic target for osteosarcoma. PMID:28469952

  7. NSD2 promotes osteosarcoma cell proliferation and metastasis by inhibiting E-cadherin expression.

    PubMed

    Lu, M-H; Fan, M-F; Yu, X-D

    2017-03-01

    Osteosarcoma is one of the most common malignant bone tumors. The mechanisms of osteosarcoma development and invasion have been studied for periods of time, yet targeted therapy for improving survival has not been well established. Histone lysine methyltransferase NSD2 was frequently overexpressed in multiple types of cancer such as multiple myeloma, stomach and colon cancer, and the overexpression of it usually associated with aggressiveness tumor type. However, the expression status and function of NSD2 are still ambiguous in osteosarcoma. Here, we evaluate the abnormal expression levels of NSD2 in osteosarcoma samples and cell lines. The higher expression of NSD2 in tumors resulted in a poorer outcome and a worse 5-year overall survival. To investigate the role of NSD2 in osteosarcoma cell proliferation and invasion in vitro, MTT assay, cell cycle distribution, wound healing, transwell assay was performed in relative cell lines, using a recombinant lentivirus expressing NSD2 short hairpin RNA or NSD2 construction. Our results imply that NSD2 promotes osteosarcoma cell proliferation and invasion, and the mechanism was possibly through the suppression of E-cadherin and induction of the epithelial mesenchymal transition, further to proceed invasion of osteosarcoma cells.  NSD2 may work as a novel repression of E-cadherin; therefore, NSD2 has potential as a target of OS therapy. In the future, the monitoring of NSD2 in the serum/plasma from the RNA level may be used as a non-invasive method for selecting patients for target therapy.

  8. An assay to measure adriamycin binding in osteosarcoma cells.

    PubMed

    Gebhardt, M C; Kusuzaki, K; Mankin, H J; Springfield, D S

    1994-09-01

    Adjuvant chemotherapy is currently employed in the treatment of patients with osteosarcoma, but the drug regimens, although effective in improving disease-free survival, are unsuccessful in 20-40% of patients and very toxic. It would be useful to know whether tumor cells are sensitive to a given drug prior to its use. To this end, we developed a method of assessing Adriamycin (doxorubicin) binding to tumor nuclei as a possible means of detecting sensitivity to the drug. Adriamycin-sensitive murine osteosarcoma cells were used to develop the assay. The in vitro conditions (drug concentration, duration of incubation, and temperature) were optimized with use of the murine osteosarcoma cells in culture. After the cells had been incubated with Adriamycin, cell viability was determined and Adriamycin fluorescence intensity was measured with a cytofluorometer. The optimal parameters for Adriamycin binding were found to be a 30-minute incubation in a 10 micrograms/ml concentration of Adriamycin at 37 degrees C; the frequency of cells that emitted Adriamycin fluorescence from the nucleus compared with the total number of living cells reached 100% under these conditions. In a murine leukemia cell line with known sensitivity to Adriamycin, the cells emitted red fluorescence from the nucleus and cytoplasm, whereas in a resistant line the cells emitted Adriamycin fluorescence from only the cytoplasm. We demonstrated that it is possible to differentiate nuclear from cytoplasmic concentration of Adriamycin in a tumor cell with use of a fluorescent microscope and that resistant cell lines can be distinguished from sensitive cell lines by this method.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53.

    PubMed

    York, D; Withers, S S; Watson, K D; Seo, K W; Rebhun, R B

    2017-09-01

    Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity. © 2016 John Wiley & Sons Ltd.

  10. Memory T Cells Expressing an NKG2D-CAR Efficiently Target Osteosarcoma Cells.

    PubMed

    Fernández, Lucía; Metais, Jean-Yves; Escudero, Adela; Vela, María; Valentín, Jaime; Vallcorba, Isabel; Leivas, Alejandra; Torres, Juan; Valeri, Antonio; Patiño-García, Ana; Martínez, Joaquín; Leung, Wing; Pérez-Martínez, Antonio

    2017-10-01

    Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8(+) T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA(-) memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR).Experimental Design: CD45RA(-) cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR(+) CD45RA(-) T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rg(null) mice.Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA(-) cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR(+) memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR(+) memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective.Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824-35. ©2017 AACR. ©2017 American Association for Cancer Research.

  11. MicroRNA-497 inhibits cell proliferation, migration, and invasion by targeting AMOT in human osteosarcoma cells

    PubMed Central

    Ruan, Wen-Dong; Wang, Pei; Feng, Shiqing; Xue, Yuan; Zhang, Bin

    2016-01-01

    MicroRNAs (miRNAs) have a role in the development and progression of human malignancy. The expression of miR-497 is decreased in malignant tumors, which suggests a role for miR-497 as a tumor suppressor. Angiomotin is encoded by the AMOT gene, which is a target for miR-497. Angiomotin has a role in angiogenesis, cell proliferation, and invasion in human malignancies, including osteosarcoma. However, the role of miR-497 in human osteosarcoma is unknown. This preliminary study included human osteosarcoma tissues and normal tissues from 20 patients, the osteosarcoma cell lines, MG-63, SAOS-2, U-2 OS, and the human osteoblast cell line hFOB (OB3). Western blots for angiomotin and quantitative real-time polymerase chain reaction for the expression of miR-497 and AMOT were performed. Knockdown studies were performed using RNA interference and transfection studies used miR-497 mimics. Quantitative cell migration assays were performed, and cell apoptosis was studied by flow cytometry. Osteosarcoma cells and cell lines showed reduced expression of miR-497 and increased expression of angiomotin. Transfection of osteosarcoma cells with miR-497 mimics suppressed the expression of angiomotin. Results from a dual-luciferase reporter system supported AMOT as a direct target gene of miR-497. Knockdown of AMOT using RNA interference resulted in inhibition of osteosarcoma cell proliferation, migration, and invasion. These preliminary studies support a role for miR-497 as a suppressor of AMOT gene expression in human osteosarcoma cells, resulting in suppression of tumor cell proliferation and invasion. Further studies are recommended to investigate the role of miR-497 in osteosarcoma and other malignant mesenchymal tumors. PMID:26855583

  12. Effects of melatonin combined with Cis-platinum or methotrexate on the proliferation of osteosarcoma cell line SaOS-2.

    PubMed

    Wang, Ya-peng; Yang, Zhi-ping

    2015-04-01

    To evaluate the effects of melatonin (Mel) combined with cis-platinum (DDP) or methotrexate (MTX) on the proliferation of osteosarcoma cell line SaOS-2, and to explore whether Mel combined with DDP or MTX could play a synergistic antitumor effect. SaOS-2 was treated with Mel alone or Mel combined with DDP or MTX. Cell counting kit-8 assay was used to measure the cell activities. Combination index(CI) value was used to evaluate the combined effects: CI<1 indicating synergetic effect, CI=1 additive, and CI>1 antagonistic.Flow cytometry was used to analyze cell cycle distribution and cell apoptosis. After treated with Mel (0.5,1,2,4,5 mmol/L), DDP (6.67, 16.67, 33.33, 66.66 μmol/L) or MTX(0.1, 0.5, 1, 2, 4 mmol/L)alone,SaOS-2 cell activities decreased in a dose-dependent manner (all P<0.05). The activities of SaOS-2 cell treated with both Mel (1 mmol/L) and DDP or MTX were significantly lower than that of DDP or MTX alone (all P<0.05).CI values of cells exposed to 1 mmol/L Mel plus 6.67, 16.67, 33.33, and 66.66 μmol/L DDP were 1.18, 1.21, 1.09, and 0.84, respectively,and CI values of cells exposed to 1 mmol/L Mel plus 0.1, 0.5, 1, 2, and 4 mmol/L MTX were 0.88, 0.88 ,0.83, 0.78, and 0.81, respectively. The G1-stage cells were increased and the S-stage cells were reduced when the cells were treated with Mel (1 mmol/L) alone or combined with MTX (0.5 mmol/L) (P<0.05). The S-stage cells were increased when the cells treated with MTX (0.5 mmol/L) (P<0.05). The apoptotic cells were increased when they treated with Mel (1 mmol/L) alone or combined with DDP (16.67 μmol/L) or MTX (0.5 mmol/L) (P<0.05). When the cells were treated with Mel combined with DDP or MTX, the apoptotic cells were more than that of DDP or Mel alone(P<0.05). Mel can inhibit SaOS-2 cells activity,block the cell cycle at G1-stage,and induce apoptosis. Mel has an antagonistic effect with lower concentration of DDP but a synergistic effect with MTX or higher concentration of DDP.

  13. Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions.

    PubMed

    Lussier, Danielle M; O'Neill, Lauren; Nieves, Lizbeth M; McAfee, Megan S; Holechek, Susan A; Collins, Andrea W; Dickman, Paul; Jacobsen, Jeffrey; Hingorani, Pooja; Blattman, Joseph N

    2015-04-01

    Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

  14. Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2

    PubMed Central

    Vaidya, Himani; Rumph, Candie; Katula, Karen S.

    2016-01-01

    WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a

  15. Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2.

    PubMed

    Vaidya, Himani; Rumph, Candie; Katula, Karen S

    2016-01-01

    WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a

  16. Effects of PEMF on a murine osteosarcoma cell line: drug-resistant (P-glycoprotein-positive) and non-resistant cells.

    PubMed

    Miyagi, N; Sato, K; Rong, Y; Yamamura, S; Katagiri, H; Kobayashi, K; Iwata, H

    2000-02-01

    After pulsed exposure of Dunn osteosarcoma cells (nonresistant cells) to Adriamycin (ADR) at increasing concentrations and single-cell cloning of surviving cells, ADR-resistant cells were obtained. These resistant cells expressed P-glycoprotein and had resistance more than 10 times that of their nonresistant parent cells. Compared to the nonresistant cells not exposed to pulsing electromagnetic fields (PEMF) in ADR-free medium, their growth rates at ADR concentrations of 0.01 and 0.02 micrograms/ml, which were below IC50, were 83.0% and 61.8%, respectively. On the other hand, in the nonresistant cells exposed to PEMF (repetition frequency, 10 Hz; rise time, 25 microsec, peak magnetic field intensity, 0.4-0.8 mT), the growth rate was 111.9% in ADR-free medium, 95.5% at an ADR concentration of 0.01 micrograms/ml, and 92.2% at an ADR concentration of 0.02 micrograms/ml. This promotion of growth by PEMF is considered to be a result of mobilization of cells in the non-proliferative period of the cell cycle due to exposure to PEMF. However, at ADR concentrations above the IC50, the growth rate tended to decrease in the cells not exposed to PEMF. This may be caused by an increase in cells sensitive to ADR resulting from mobilization of cells in the non-proliferative period to the cell cycle. The growth rate in the resistant cells exposed to PEMF was significantly lower than that in the non-exposed resistant cells at all ADR concentrations, including ADR-free culture (Pcells but progressively suppresses the growth of more differentiated cells, i.e., PEMF controls cell growth depending on the degree of cell differentiation. This study also shows the potentiality of PEMF as an adjunctive treatment method for malignant tumors. Copyright 2000 Wiley-Liss, Inc.

  17. MicroRNA-93 promotes cell proliferation by directly targeting P21 in osteosarcoma cells

    PubMed Central

    He, Yu; Yu, Bo

    2017-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that are key regulators of gene expression by directly binding to the 3′-untranslated region of their target mRNAs, resulting in translational repression or degradation of mRNA. It has been demonstrated that miRNAs have key roles in a variety of human malignancies, including osteosarcoma. The present study aimed to assess the molecular mechanism of miR-93 in the regulation of osteosarcoma cell proliferation. Reverse-transcription quantitative PCR and western blot assays were used to examine mRNA and protein expression. An MTT assay and flow cytometry were performed to determine the cell proliferation and cell cycle distribution. A luciferase reporter assay was performed to confirm the direct targeting of cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as P21, by miR-93, which was suggested by a bioinformatics analysis. The results showed that the expression of miR-93 was frequently and significantly increased in a total of 19 osteosarcoma tissues compared to their matched adjacent non-tumor tissues, and the upregulation of miR-93 was associated with the malignant progression of osteosarcoma. Furthermore, miR-93 was also upregulated in the human osteosarcoma cell lines Saos-2, U2OS, SW1353 and MG63 when compared with that in the human osteoblast cell line hFOB1.19. Transfection with miR-93 inhibitor significantly reduced the miR-93 levels and inhibited the proliferation of U2OS and MG63 osteosarcoma cells. The protein levels of P21 were negatively regulated by miR-93 in U2OS and MG63 cells. Knockdown of miR-93 caused cell cycle arrest at G1 stage in U2OS and MG63 cells, identical to the effect of P21 overexpression. Finally, P21 was found to be significantly downregulated in osteosarcoma tissues compared to their matched adjacent non-tumor tissues, suggesting that the inhibition of P21 may be due to increased miR-93 expression in osteosarcoma tissues. In conclusion, the present study demonstrated that miR-93

  18. Quercetin induces apoptosis in the methotrexate-resistant osteosarcoma cell line U2-OS/MTX300 via mitochondrial dysfunction and dephosphorylation of Akt.

    PubMed

    Xie, Xianbiao; Yin, Junqiang; Jia, Qiang; Wang, Jin; Zou, Changye; Brewer, Kari J; Colombo, Chiara; Wang, Yaofei; Huang, Gang; Shen, Jingnan

    2011-09-01

    Quercetin is the most abundant polyphenolic flavonoid found in plants. Several studies suggest that it has potent anticancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of quercetin in a methotrexate (MTX)-resistant osteosarcoma model. Our results showed that quercetin inhibited cell viability in a dose-dependent manner and there was no cross-resistance between MTX and quercetin in U2-OS/MTX300 cells. The induction of apoptosis was observed by flow cyto-metry and fluorescence staining experiments. Quercetin-induced apoptosis was accompanied by a significant reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to the cytosol, activation of caspase-3, down-regulation of Bcl-2, p-Bad and up-regulation of Bax. A remarkable dephospho-rylation of Akt was also detected after quercetin treatment. Furthermore, transduction with constitutively active Akt protected against the quercetin-induced dephosphorylation of Akt and Bad as well as poly(ADP-ribose)polymerase (PARP) degradation, while combined treatment with quercetin and LY294002 enhanced the dephosphorylation of Akt, Bad and PARP cleavage in U2-OS/MTX300 cells. Taken together, our results demonstrate that quercetin-induced apoptosis in the MTX-resistant osteosarcoma cells U2-OS/MTX300 was mediated via mitochondrial dysfunction and dephosphorylation of Akt.

  19. Quercetin Inhibits Cell Migration and Invasion in Human Osteosarcoma Cells.

    PubMed

    Lan, Haifeng; Hong, Wei; Fan, Pan; Qian, Dongyang; Zhu, Jianwei; Bai, Bo

    2017-09-21

    Osteosarcoma is a malignant tumor associated with high mortality; however, no effective therapies for the disease have been developed. Several studies have focused on elucidating the pathogenesis of osteosarcoma and have aimed to develop novel therapies for the disease. Quercetin is a vital dietary flavonoid that has been shown to have a variety of anticancer effects, as it induces cell cycle arrest, apoptosis, and differentiation and is involved in cell adhesion, metastasis and angiogenesis. Herein, we aimed to investigate the effects of quercetin on osteosarcoma migration and invasion in vitro and in vivo and to explore the molecular mechanisms underlying its effects on osteosarcoma migration and invasion. Cell viability, cell cycle activity and cell apoptosis were measured using CCK-8 assay and flow cytometry, and cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The mRNA and protein expression levels of several proteins of interest were assessed by real-time quantitative PCR and western blotting, respectively. Moreover, a nude mouse model of human osteosarcoma lung metastasis was established to assess the anti-metastatic effects of quercetin in vivo. We noted no significant differences in cell cycle activity and apoptosis between HOS and MG63 cells and control cells. Treatment with quercetin significantly attenuated cell migration and invasion in HOS and MG63 cells compared with treatment with control medium. Moreover HIF-1α, VEGF, MMP2, and MMP9 mRNA and protein expression levels were significantly downregulated in HOS cells treated with quercetin compared with HOS cells treated with controls. Additionally, treatment with quercetin attenuated metastatic lung tumor formation and growth in the nude mouse model of osteosarcoma compared with treatment with controls. Our findings regarding the inhibitory effects of quercetin on cell migration and invasion suggest that quercetin may have potential as a therapy for human

  20. Small cell osteosarcoma: cytopathologic characteristics and differential diagnosis.

    PubMed

    Bishop, Justin A; Shum, Chung H; Sheth, Sheila; Wakely, Paul E; Ali, Syed Z

    2010-05-01

    Small cell osteosarcoma may present a challenging primary diagnosis on cytologic assessment owing to its rarity and its morphologic similarity to other small round blue cell tumors. Five cases of small cell osteosarcoma from our cytopathology archives were identified and reviewed and cytologic features elaborated. Three cases were fine-needle aspirations from bony lesions in the classic location for osteosarcoma (2 distal femur and 1 proximal tibia), and 2 aspirations were from metastases. Common cytomorphologic features included relatively small to intermediate cell size, high nuclear/cytoplasmic ratios, round nuclei, minimal anisonucleosis, finely granular nuclear chromatin, fine cytoplasmic vacuoles, and only rare osteoid. Small cell osteosarcoma shares many of the well-described cytomorphologic features of classic osteosarcoma, but the relatively small cells, round hyperchromatic nuclei, and scant osteoid constitute the common denominator. Correlation with radiographic findings and ancillary tests can aid in definitive diagnosis.

  1. TRAF4 enhances osteosarcoma cell proliferation and invasion by Akt signaling pathway.

    PubMed

    Yao, Weitao; Wang, Xin; Cai, Qiqing; Gao, Songtao; Wang, Jiaqiang; Zhang, Peng

    2014-01-01

    TRAF4, or tumor necrosis factor receptor-associated factor 4, is overexpressed in several cancers, suggesting a specific role in cancer progression. However, its functions in osteosarcoma are unclear. This study aimed to explore the expression of TRAF4 in osteosarcoma tissues and cells, the correlation of TRAF4 to clinical pathology of osteosarcoma, as well as the role and mechanism of TRAF4 in osteosarcoma metastasis. The protein expression levels of TRAF4 in osteosarcoma tissues and three osteosarcoma cell lines, MG-63, HOS, and U2OS, were assessed. Constructed TRAF4 overexpression vectors and established TRAF4 overexpression of the U2OS cell line. Cell proliferation, cell invasion, protein levels, and TRAF4 phosphorylations were assessed following TRAF4 transfection, as well as the effects of TRAF4 siRNA on cell proliferation and invasion. The results show that TRAF4 protein levels in osteosarcoma tissues were significantly higher than that in normal bone tissues. Importantly, an obvious upregulation of TRAF4 was found in carcinoma tissues from patients with lung metastasis compared with patients without lung metastasis. Consistently, a similar increase in TRAF4 mRNA and protein was also demonstrated in the osteosarcoma cell lines MG-63, HOS, and U2OS compared to normal bone cells, hFOB1.19. When TRAF4 was overexpressed in U2OS cells, cell proliferation was significantly enhanced, accompanied by an increase in Ki67 expression and colony formation. Compared with the control and vector-treated groups, TRAF4 transfection increased the invasion potential of U2OS cells (p < 0.05). Interestingly, TRAF4 transfection significantly enhanced the phosphorylation of Akt. After blocking Akt with its specific siRNA, TRAF4-induced cell proliferation and invasion were dramatically attenuated. In summary, our findings demonstrated that TRAF4 enhances osteosarcoma cell proliferation and invasion partially by the Akt pathway. This work suggests that TRAF4 might be an important

  2. Polyploidization induced by acridine orange in mouse osteosarcoma cells.

    PubMed

    Kusuzaki, K; Takeshita, H; Murata, H; Gebhardt, M C; Springfield, D S; Mankin, H J; Ashihara, T; Hirasawa, Y

    2000-01-01

    This study was undertaken to clarify the in vitro effect of acridine orange (AO) on the cell kinetics of mouse osteosarcoma cells, as well as the mechanism of cell growth inhibition induced by AO. A mouse osteosarcoma cell line (MOS), established from a radiation-induced mouse osteosarcoma, was cultured under exposure to 0.05, 0.5, 5, and 50 micrograms/ml of AO, either continuously or for 10 minutes. The cell kinetic analysis was performed using the following parameters: tumor cell growth by trypan blue exclusion test, mitotic activity, DNA synthetic activity by BrdU labeling and DNA ploidy by cytofluorometry. The results showed that continuous exposure to 5 and 50 micrograms/ml of AO or 10 minute exposure to 50 micrograms/ml of AO quickly killed the tumor cells within 12 hours, whereas continuous exposure to 0.5 microgram/ml of AO or 10 minute exposure to 5 micrograms/ml of AO gradually inhibited tumor cell growth. Under the latter conditions, mitotic activity was rapidly and completely inhibited within 48 hours but DNA synthetic activity was not completely inhibited even after 96 hours. DNA ploidy analysis demonstrated that most of the tumor cells arrested at the S-G2 phase after 12 hours, followed by G2 phase arrest after 24 hours and progressive DNA synthesis to a higher DNA ploidy class after 48 to 96 hours. We therefore concluded that a high concentration of AO has a strong cytocidal effect due to cytotoxicity whilst a moderate concentration of AO induces progressive and synchronous polyploidization by mitotic inhibition without DNA damage in MOS cells. We presume that this in vitro effect on MOS cells may be caused by protein synthetic inhibition after transfer RNA inactivation caused by AO binding.

  3. Plumbagin induces apoptosis via the p53 pathway and generation of reactive oxygen species in human osteosarcoma cells.

    PubMed

    Tian, Linqiang; Yin, Delong; Ren, Ye; Gong, Chen; Chen, Anmin; Guo, Feng-Jin

    2012-01-01

    Osteosarcoma, which is the most common primary bone tumor, occurs most frequently in adolescents. A number of studies have indicated that plumbagin (PL) (5-hydroxy-2-methyl-1, 4-naphthoquinone), a compound found in the plants of the Plumbaginaceae and Droseraceae families, possesses anticancer activity. However, its anticancer effects and mechanisms against osteosarcoma have not been explored. To determine the anticancer effect of PL on osteosarcoma cell lines MG-63 and U2OS, cell viability, apoptosis, cell cycle distribution, caspase-3 and caspase-9 activity and intracellular reactive oxygen species (ROS) generation were measured, and Western blot analyses were performed. PL significantly inhibited the growth of osteosarcoma cells, particularly U2OS cells. PL up-regulated the expression of p53 in U2OS cells and p21 in the two osteosarcoma cell lines causing cell cycle arrest by decreasing the expression of murine double minute 2 (MDM2)/cyclin B1 and cyclin D1. Furthermore, PL altered the ratio of Bax/Bcl-2, and may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and caspase-9 activation. We also found that PL induced the generation of ROS in osteosarcoma cell lines. To conclude, PL exerted anticancer activity on osteosarcoma cells by inducing pro-apoptotic signaling and modulating the intracellular ROS that causes induction of apoptosis. These effects may relate to the p53 status.

  4. Metabolic markers of MG-63 osteosarcoma cell line response to doxorubicin and methotrexate treatment: comparison to cisplatin.

    PubMed

    Lamego, Inês; Duarte, Iola F; Marques, M Paula M; Gil, Ana M

    2014-12-05

    A high resolution magic angle spinning NMR metabolomics study of the effects of doxorubicin (DOX), methotrexate (MTX) and cisplatin (cDDP) on MG-63 cells is presented and unveils the cellular metabolic adaptations to these drugs, often used together in clinical protocols. Although cDDP-treated cells were confirmed to undergo extensive membrane degradation accompanied by increased neutral lipids, DOX- and MTX-treated cells showed no lipids increase and different phospholipid signatures, which suggests that (i) DOX induces significant membrane degradation, decreased membrane synthesis, and apparent inhibition of de novo lipid synthesis, and (ii) MTX induces decreased membrane synthesis, while no membrane disruption or de novo lipid synthesis seem to occur. Nucleotide signatures were in apparent agreement with the different drug action mechanisms, a link having been found between UDP-GlcNAc and the active pathways of membrane degradation and energy metabolism, for cDDP and DOX, with a relation to oxidative state and DNA degradation, for cDDP. Correlation studies unveiled drug-specific antioxidative signatures, which pinpointed m- and s-inositols, taurine, glutamate/glutamine, and possibly creatine as important in glutathione metabolism. These results illustrate the ability of NMR metabolomics to measure cellular responses to different drugs, a first step toward understanding drug synergism and the definition of new biomarkers of drug efficacy.

  5. Natural killer cell therapy and aerosol interleukin-2 for the treatment of osteosarcoma lung metastasis.

    PubMed

    Guma, Sergei R; Lee, Dean A; Yu, Ling; Gordon, Nancy; Hughes, Dennis; Stewart, John; Wang, Wei Lien; Kleinerman, Eugenie S

    2014-04-01

    Survival of patients with osteosarcoma lung metastases has not improved in 20 years. We evaluated the efficacy of combining natural killer (NK) cells with aerosol interleukin-2 (IL-2) to achieve organ-specific NK cell migration and expansion in the metastatic organ, and to decrease toxicity associated with systemic IL-2. Five human osteosarcoma cell lines and 103 patient samples (47 primary and 56 metastatic) were analyzed for NKG2D ligand (NKG2DL) expression. Therapeutic efficacy of aerosol IL-2 + NK cells was evaluated in vivo compared with aerosol IL-2 alone and NK cells without aerosol IL-2. Osteosarcoma cell lines and patient samples expressed various levels of NKG2DL. NK-mediated killing was NKG2DL-dependent and correlated with expression levels. Aerosol IL-2 increased NK cell numbers in the lung and within metastatic nodules but not in other organs. Therapeutic efficacy, as judged by tumor number, size, and quantification of apoptosis, was also increased compared with NK cells or aerosol IL-2 alone. There were no IL-2-associated systemic toxicities. Aerosol IL-2 augmented the efficacy of NK cell therapy against osteosarcoma lung metastasis, without inducing systemic toxicity. Our data suggest that lung-targeted IL-2 delivery circumvents toxicities induced by systemic administration. Combining aerosol IL-2 with NK cell infusions, may be a potential new therapeutic approach for patients with osteosarcoma lung metastasis. © 2013 Wiley Periodicals, Inc.

  6. MiR-133b Is Down-Regulated in Human Osteosarcoma and Inhibits Osteosarcoma Cells Proliferation, Migration and Invasion, and Promotes Apoptosis

    PubMed Central

    Zhao, Huafu; Li, Mei; Li, Lihua; Yang, Xiaoming; Lan, Guobo; Zhang, Yu

    2013-01-01

    MicroRNAs (miRNAs) decrease the expression of specific target oncogenes or tumor suppressor genes and thereby play crucial roles in tumorigenesis and tumor growth. To date, the potential miRNAs regulating osteosarcoma growth and progression are not fully identified yet. In this study, the miRNA microarray assay and hierarchical clustering analysis were performed in human osteosarcoma samples. In comparison with normal human skeletal muscle, 43 miRNAs were significantly differentially expressed in human osteosarcomas (fold change ≥2 and p≤0.05). Among these miRNAs, miR-133a and miR-133b expression was decreased by 135 folds and 47 folds respectively and the decreased expression was confirmed in both frozen and paraffin-embedded osteosarcoma samples. The miR-133b precursor expression vector was then transfected into osteosarcoma cell lines U2-OS and MG-63, and the stable transfectants were selected by puromycin. We found that stable over-expression of miR-133b in osteosarcoma cell lines U2-OS and MG-63 inhibited cell proliferation, invasion and migration, and induced apoptosis. Further, over-expression of miR-133b decreased the expression of predicted target genes BCL2L2, MCL-1, IGF1R and MET, as well as the expression of phospho-Akt and FAK. This study provides a new insight into miRNAs dysregulation in osteosarcoma, and indicates that miR-133b may play as a tumor suppressor gene in osteosarcoma. PMID:24391788

  7. Knockdown of Long Noncoding RNA TUG1 Inhibits the Proliferation and Cellular Invasion of Osteosarcoma Cells By Sponging MiR-153.

    PubMed

    Wang, Heping; Yu, Yanzhang; Fan, Shuxin; Luo, Leifeng

    2017-04-12

    Long noncoding RNA (lncRNA) Taurine-upregulated gene 1 (TUG1) has been confirmed to be involved in the progression of various cancers, however, its mechanism of action in osteosarcoma has not been well addressed. In our study, TUG1 was overexpressed and miR-153 was downregulated in osteosarcoma tissues and cell lines. Loss-of-function assay showed that TUG1 knockdown suppressed the viability, colony formation, and invasion of osteosarcoma cells in vitro. Moreover, TUG1 was confirmed to be a miR-153 sponge. Ectopic expression of TUG1 reversed the inhibitory effect of miR-153 on the proliferation and invasion of osteosarcoma cells. Further transplantation experiment proved the carcinogenesis of TUG1 in osteosarcoma in vivo. Collectively, our study elucidated that TUG1 contributed to the osteosarcoma development by sponging miR-153. These findings may provide a novel lncRNA-targeted therapy for patients with osteosarcoma.

  8. Reoxygenation using a novel CO2 therapy decreases the metastatic potential of osteosarcoma cells.

    PubMed

    Harada, Risa; Kawamoto, Teruya; Ueha, Takeshi; Minoda, Masaya; Toda, Mitsunori; Onishi, Yasuo; Fukase, Naomasa; Hara, Hitomi; Sakai, Yoshitada; Miwa, Masahiko; Kuroda, Ryosuke; Kurosaka, Masahiro; Akisue, Toshihiro

    2013-08-01

    Osteosarcoma is the most common primary solid malignant bone tumor. Despite substantial improvements in surgery and chemotherapy, metastasis remains a major cause of fatal outcomes, and the molecular mechanisms of metastasis are still poorly understood. Hypoxia, which is common in malignant tumors including osteosarcoma, increases expressions of hypoxia inducible factor (HIF)-1α, matrix metalloproteinase (MMP)-2 and MMP-9, and can induce invasiveness. As we previously showed a novel transcutaneous CO2 application to decrease HIF-1α expression and induce apoptosis in malignant fibrous histiocytoma, we hypothesize that transcutaneous CO2 application could suppress metastatic potential of osteosarcoma by improving hypoxic conditions. Here, we examined the effects of transcutaneous CO2 application on apoptosis, and development of pulmonary metastasis using a highly metastatic osteosarcoma cell line, LM8. Transcutaneous CO2 application significantly decreased tumor growth and pulmonary metastasis in LM8 cells. Apoptotic activity increased, and intratumoral hypoxia was improved with decreased expressions of HIF-1α, MMP-2 and MMP-9, significantly, in the CO2-treated tumors. In conclusion, we found that transcutaneous CO2 application can induce tumor cell apoptosis and might suppress pulmonary metastasis by improvement of hypoxic conditions with decreased expressions of HIF-1α and MMPs in highly metastatic osteosarcoma cell. These findings strongly indicate that this novel transcutaneous CO2 therapy could be a therapeutic breakthrough for osteosarcoma patients. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    SciTech Connect

    Nishida, Yoshihiro . E-mail: ynishida@med.nagoya-u.ac.jp; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    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 in 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.

  10. Inhibition of miR-664a interferes with the migration of osteosarcoma cells via modulation of MEG3.

    PubMed

    Sahin, Yunus; Altan, Zekiye; Arman, Kaifee; Bozgeyik, Esra; Koruk Ozer, Meltem; Arslan, Ahmet

    2017-08-26

    Osteosarcoma is the most common primary bone tumor in children and adolescents. Understanding the basic molecular mechanisms in developing cancer can be helpful in developing alternative treatment strategies. The relationship between dysregulated non-coding RNAs' (ncRNA) expression level and osteosarcoma was detected. Among those ncRNAs, the expression levels of miR-664a were detected to be upregulated and MEG3 long non-coding RNA levels were detected to be downregulated in osteosarcoma tissue and cell lines. In this study, miR-664a inhibitor was used in order to investigate the changes in the expression levels of MEG3 gene and miR-664a in osteosarcoma cancer cell line (U2-OS) and human osteoblast cell line (hFOB 1.19). According to our results, the expression level of MEG3 gene was increased while the expression level of miR-664a was decreased, as expected. In addition, changes in expression level of MEG3 and miR-644a interferes with the migration of osteosarcoma cells migration speed of osteosarcoma cells. These results are found to be statistically significant (p < 0.05). As a result of this study, it was shown that the upregulated expression of miR-664a could have an inhibitory effect on MEG3 gene expression and migration of osteosarcoma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    SciTech Connect

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

    2014-03-07

    Highlights: • Down-regulation of TPP1 shortened telomere length in telomerase-negative cells. • Down-regulation of TPP1 induced cell apoptosis in telomerase-negative cells. • Down-regulation of TPP1 increased radiosensitivity in telomerase-negative cells. - Abstract: 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.

  12. MiR-138 Acts as a Tumor Suppressor by Targeting EZH2 and Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells

    PubMed Central

    Wang, Jianqiang; Duan, Gang; Zhou, Lei; Zhou, Xiaoqing

    2016-01-01

    Chemotherapeutic insensitivity remains a major obstacle to treating osteosarcoma effectively. Recently, increasing evidence has suggested that microRNAs (miRNAs) are involved in drug resistance. However, the effect of miR-138 on cisplatin chemoresistance in osteosarcoma has not been reported. We used real-time PCR to detect the expression of mature miR-138 in osteosarcoma tissues and cell lines. Cell proliferation, invasion, and migration assays were used to observe changes to the osteosarcoma malignant phenotype. MiR-138 was downregulated in osteosarcoma tissues and cell lines, and miR-138 overexpression negatively regulated osteosarcoma cell proliferation, migration, and invasion. We also verified that EZH2 is a direct target of miR-138. Furthermore, enhancing EZH2 expression reduced the inhibitory effects of miR-138 on osteosarcoma. Proliferation, apoptosis assays and caspase-3 activity assay confirmed that elevated miR-138 expression enhanced osteosarcoma cell chemosensitivity to cisplatin by targeting EZH2. In conclusion, the present study demonstrates that miR-138 acts as a tumor suppressor by enhancing osteosarcoma cell chemosensitivity and supports its potential application for treating osteosarcoma in the future. PMID:27019355

  13. MiR-19a regulates the cell growth and apoptosis of osteosarcoma stem cells by targeting PTEN.

    PubMed

    Zhao, Di; Chen, Youbin; Chen, Shunliang; Zheng, Chuangyi; Hu, Jun; Luo, Shaowei

    2017-05-01

    MicroRNAs are small, endogenous, and non-coding RNAs that play important regulatory roles in multiple biological processes in cancers. Recent evidence has indicated that miR-19a participates in the cancer tumorigenic progression. However, the functional roles of miR-19a in cancer stem cells are still unclear. As the cancer stem cells are considered to be responsible for the tumor recurrence and treatment failure in osteosarcoma, the aim of this study is to investigate the molecular mechanism of miR-19a underlying osteosarcoma tumorigenesis. In this study, we observed significant upregulation of miR-19a in osteosarcoma patients' tumor tissues as well as the osteosarcoma cell lines in vitro. We showed that knockdown of miR-19a by its antisense oligonucleotide (anti-miR-19a) significantly decreased the population of cancer stem cells in osteosarcoma cell lines. Furthermore, we found the miR-19a regulated the cell proliferation, migration, and viability in the human osteosarcoma-cancer stem cells. The gene of phosphatase and tensin homolog deleted on chromosome 10, which is an important tumor suppressor, was found to be directly regulated by miR-19a in human osteosarcoma-cancer stem cells. We demonstrated that knockdown of miR-19a increased the expression of phosphatase and tensin homolog deleted on chromosome 10. As the anti-miR-19a inhibited the phosphatidylinositol 3-kinase/AKT pathway and induced apoptosis of human osteosarcoma-cancer stem cells, the phosphatase and tensin homolog deleted on chromosome 10 small interfering RNA inhibited the effect of it. Meanwhile, the phosphatase and tensin homolog deleted on chromosome 10 small interfering RNA also abolished the effect of anti-miR-19a on inhibiting the cell proliferation, migration, and viability in the human osteosarcoma-cancer stem cells. In conclusion, our findings demonstrated that dysregulation of miR-19a plays critical roles in the osteosarcoma stem cells, at least in part via targeting the phosphatase and

  14. Opposite Effects of Soluble Factors Secreted by Adipose Tissue on Proliferating and Quiescent Osteosarcoma Cells.

    PubMed

    Avril, Pierre; Duteille, Franck; Ridel, Perrine; Heymann, Marie-Françoise; De Pinieux, Gonzague; Rédini, Françoise; Blanchard, Frédéric; Heymann, Dominique; Trichet, Valérie; Perrot, Pierre

    2016-03-01

    Autologous adipose tissue transfer may be performed for aesthetic needs following resection of osteosarcoma, the most frequent primary malignant tumor of bone, excluding myeloma. The safety of autologous adipose tissue transfer regarding the potential risk of cancer recurrence must be addressed. Adipose tissue injection was tested in a human osteosarcoma preclinical model induced by MNNG-HOS cells. Culture media without growth factors from fetal bovine serum were conditioned with adipose tissue samples and added to two osteosarcoma cell lines (MNNG-HOS and MG-63) that were cultured in monolayer or maintained in nonadherent spheres, favoring a proliferation or quiescent stage, respectively. Proliferation and cell cycle were analyzed. Adipose tissue injection increased local growth of osteosarcoma in mice but was not associated with aggravation of lung metastasis or osteolysis. Adipose tissue-derived soluble factors increased the in vitro proliferation of osteosarcoma cells up to 180 percent. Interleukin-6 and leptin were measured in higher concentrations in adipose tissue-conditioned medium than in osteosarcoma cell-conditioned medium, but the authors' results indicated that they were not implicated alone. Furthermore, adipose tissue-derived soluble factors did not favor a G0-to-G1 phase transition of MNNG-HOS cells in nonadherent oncospheres. This study indicates that adipose tissue-soluble factors activate osteosarcoma cell cycle from G1 to mitosis phases, but do not promote the transition from quiescent G0 to G1 phases. Autologous adipose tissue transfer may not be involved in the activation of dormant tumor cells or cancer stem cells.

  15. Intrinsic resistance to chemotherapeutic agents in murine osteosarcoma cells.

    PubMed

    Takeshita, H; Kusuzaki, K; Ashihara, T; Gebhardt, M C; Mankin, H J; Hirasawa, Y

    2000-07-01

    There are two general categories of drug resistance: acquired and intrinsic. The mechanisms involved in acquired drug resistance have been extensively studied, and several mechanisms have been described. However, the mechanisms responsible for intrinsic drug resistance have not been elucidated, to our knowledge. The purpose of the present study was to investigate the cytological and biochemical differences between acquired and intrinsic drug resistance in osteosarcoma cells. We previously isolated a clonal cell line (MOS/ADR1) to study acquired resistance in osteosarcoma by exposure of parental murine osteosarcoma cells (MOS) to doxorubicin. In the present study, we cloned a new, intrinsically resistant cell line (MOS/IR1) by single-cell culture of MOS cells and we investigated the differences in cell phenotype and the mechanisms of resistance in both of these resistant clones. The MOS/ADR1 and MOS/IR1 cells were sevenfold and fivefold more resistant to doxorubicin than the parental murine osteosarcoma cells. Morphologically, the MOS/ADR1 cell line was composed of polygonal cells, whereas the MOS/IR1 cell line consisted of plump spindle cells with long cytoplasmic processes. The MOS/IR1 cells showed a much lower level of alkaline phosphatase activity than did the MOS/ ADR1 and MOS cells. There were no substantial differences in the cellular DNA content or the doubling time among these three lines. Overexpression of the P-glycoprotein involved in the function of an energy-dependent drug-efflux pump was detected in the MOS/ADR1 cells but not in the MOS/ IR1 cells. After the cells were incubated with doxorubicin for one hour, the two resistant lines had less accumulation of the drug than did the parent line (p < 0.05). The addition of a P-glycoprotein antagonist, verapamil, or the depletion of cellular adenosine triphosphate resulted in a marked increase in the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0.05) but not in the MOS/ IR1 cells. The MOS/ADR1

  16. Stem cell growth factor receptor in canine vs. feline osteosarcomas

    PubMed Central

    Wolfesberger, Birgitt; Fuchs-Baumgartinger, Andrea; Hlavaty, Juraj; Meyer, Florian R.; Hofer, Martin; Steinborn, Ralf; Gebhard, Christiane; Walter, Ingrid

    2016-01-01

    Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of KIT was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10–50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma. PMID:27698817

  17. 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.

  18. Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells.

    PubMed

    Cifuentes, Manuel; García, Maria A; Arrabal, Pilar M; Martínez, Fernando; Yañez, María J; Jara, Nery; Weil, Bernardo; Domínguez, Dolores; Medina, Rodolfo A; Nualart, Francisco

    2011-06-01

    Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.

  19. Icarisid II inhibits the proliferation of human osteosarcoma cells by inducing apoptosis and cell cycle arrest.

    PubMed

    Tang, Yuanyuan; Xie, Mao; Jiang, Neng; Huang, Feifei; Zhang, Xiao; Li, Ruishan; Lu, Jingjing; Liao, Shijie; Liu, Yun

    2017-06-01

    Icarisid II, one of the main active components of Herba Epimedii extracts, shows potent antitumor activity in various cancer cell lines, including osteosarcoma cells. However, the anticancer mechanism of icarisid II against osteosarcoma U2OS needs further exploration. This study aims to investigate further antitumor effects of icarisid II on human osteosarcoma cells and elucidate the underlying mechanism. We cultivated human osteosarcoma USO2 cells in vitro using different concentrations of icarisid II (0-30 µM). Cell viability was detected at 24, 48, and 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis. Cell cycle was tested by flow cytometry after treatment with icarisid II for 48 h. Annexin V-allophycocyanin and 7-aminoactinomycin D staining were conducted to detect cell apoptosis. Quantitative real-time polymerase chain reaction and Western blot assay were performed to measure the levels of genes and proteins related to cell cycle and apoptosis. Results showed that icarisid II significantly inhibited the proliferation and induced apoptosis of human osteosarcoma U2OS cells. The half maximal inhibitory concentration values were 14.44, 11.02, and 7.37 µM at 24, 48, and 72 h, respectively. Cell cycle was arrested in the G2/M phase in vitro. In addition, icarisid II upregulated the expression levels of P21 and CyclinB1 whereas downregulated the expression levels of CyclinD1, CDC2, and P-Cdc25C, which were related to cell cycle arrest in U2OS cells. The cell apoptotic rate increased in a dose-dependent manner after treatment with icarisid II for 48 h. Icarisid II induced apoptosis by upregulating Bax, downregulating Bcl-2, and activating apoptosis-related proteins, including cleaved caspase-3, caspase-7, caspase-9, and poly (ADP-ribose) polymerase. These data indicate that icarisid II exhibits an antiproliferation effect on human osteosarcoma cells and induces apoptosis by activating the caspase family in a time- and dose

  20. SIRT1 promotes metastasis of human osteosarcoma cells.

    PubMed

    Zhang, Ning; Xie, Tao; Xian, Miao; Wang, Yi-Jie; Li, Heng-Yuan; Ying, Mei-Dan; Ye, Zhao-Ming

    2016-11-29

    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.

  1. 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

  2. Impaired cell cycle regulation of osteoblast-related transcription factor Runx2/Cbfa1 in osteosarcoma cells

    PubMed Central

    San Martin, Inga; Varela, Nelson; Gaete, Marcia; Villegas, Karina; Osorio, Mariana; Tapia, Julio C.; Antonelli, Marcelo; Mancilla, Edna; Lian, Jane B.; Stein, Janet L.; Stein, Gary S; van Wijnen, Andre J.; Galindo, Mario

    2011-01-01

    In mammals, bone differentiation requires the functional expression of the Runx2/Cbfβ heterodimeric complex. Our previous results indicate that Runx2 is also a suppressor of pre-osteoblast proliferation by affecting cell cycle progression at G1. Runx2 levels are cell cycle regulated, oscillating from a maximum during early G1 to a minimum during late G1, S and mitosis phases in proliferating pre-osteoblasts Nevertheless, there is no information concerning Cbfβ gene expression during the cell cycle nor on Runx2 cell cycle expression in bone cancer cells. We analyzed Runx2 and Cbfβ gene expression during cell cycle progression in the pre-osteoblast MC3T3 and osteosarcoma ROS and SaOS cell lines. The expected reduction of Runx2 protein level was observed in MC3T3 cells arrested in late G1 or M phase using mimosine or nocodazole, respectively. However, this reduction was not observed in the cell cycle arrested osteosarcoma cells. Cbfβ protein levels were not regulated during the cell cycle in pre-osteoblasts and osteosarcoma cells. Using cells synchronized in late G1 and mitosis we found that Runx2 levels, but not Cbfβ levels, were cell cycle regulated in MC3T3 osteoblasts. Interestingly, both factors showed a constitutively elevated expression throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 prevented cell cycle-dependent downregulation of Runx2 protein levels in osteoblasts, but not in osteosarcoma. We propose that Runx2 is involved in tumoral osteosarcoma progression. Altogether, deregulated Runx2 expression throughout the cell cycle seems to constitute a central mechanism in the pathogenesis of osteosarcoma. PMID:19739101

  3. Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-D gel electrophoresis and MALDI-TOF MS.

    PubMed

    Kang, Jeong Han; Park, Kwan-Kyu; Lee, In-Seon; Magae, Junji; Ando, Kunio; Kim, Cheorl-Ho; Chang, Young-Chae

    2006-10-01

    Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.

  4. LDOC1 regulates Wnt5a expression and osteosarcoma cell metastasis and is correlated with the survival of osteosarcoma patients.

    PubMed

    Yong, Bi-Cheng; Lu, Jin-Chang; Xie, Xian-Biao; Su, Qiao; Tan, Ping-Xian; Tang, Qing-Lian; Wang, Jing; Huang, Gang; Han, Ju; Xu, Hong-Wen; Shen, Jing-Nan

    2017-02-01

    Osteosarcomas are common bone malignancies in children and adolescents. LDOC1 (leucine zipper, down-regulated in cancer 1), a tumor suppressor, is down-regulated in many cancers. In this study, we investigated the role of LDOC1 in tumor metastasis and its prognostic significance in osteosarcomas. We established osteosarcoma cells stably expressing LDOC1, driven by an HIV-based lentiviral system. We investigated the impact of LDOC1 on migration and invasion abilities in these cells using a transwell assay. LDOC1-associated changes in expression of metastasis-promoting genes were analyzed with a quantitative real-time polymerase chain reaction primer array. A xenograft tumor model (n = 7 mice/group) was used to assess the effect of LDOC1 on osteosarcoma metastasis in vivo. The overall survival and disease-free survival of osteosarcoma patients (n = 74) were analyzed retrospectively based on immunohistochemical analysis of LDOC1 levels in tumors and Kaplan-Meier analysis. LDOC1-expressing osteosarcoma cells displayed decreased migration and invasion in vitro. The quantitative real-time polymerase chain reaction primer array data showed that increased LDOC1 expression up-regulated many metastasis-suppressor genes. In the xenograft model, micro-computed tomography imaging data indicated that increased LDOC1 expression is associated with weaker lung metastasis ability. The Wnt5a signaling pathway promotes osteosarcoma metastasis; LDOC1 expression decreased Wnt5a levels in osteosarcoma cells. Kaplan-Meier analysis showed that higher LDOC1 expression was associated with improved osteosarcoma patient overall survival and disease free survival (p = 0.022). Our data show that LDOC1 is a tumor suppressor in osteosarcoma, and that it regulates metastasis of osteosarcoma cells. Furthermore, LDOC1 might be a valuable prognostic marker in osteosarcomas.

  5. [MicroRNA-150 inhibits osteosarcoma cell proliferation by targeting RUNX2 gene].

    PubMed

    Wang, Longfei; Wang, Weiguo; Li, Jinsong; Chen, Shijie; Zhan, Ruisen

    2016-12-28

    To investigate the microRNA (miR)-150 expression level in human osteosarcoma cell lines (Saos-2, MG-63) and its function in cell proliferation, and to explore the potential molecular mechanisms. 
 Methods: MiR-150 expression levels in human osteosarcoma cell lines (Saos-2, MG-63) and normal osteoblast cell line (NHOst) were detected by relative quantitative real-time PCR (qRT-PCR). MiR-150 was overexpressed in Saos-2 and MG-63 cells by lentivirus infection. Cell proliferation rates were monitored by MTS assay. RUNX2 and β-actin protein levels were examined by Western blot. Inhibitory effect of miR-150 on binding RUNX2 3'-UTR was detected by Dual-Luciferase assay.
 Results: MiR-150 expression level is lower in human osteosarcoma cell lines (Saos-2, MG-63) compared to the normal osteoblast cell line (NHOst) (0.23±0.02 and 0.32±0.03 vs 1.00±0.02), which showed statistical significance (P<0.01). After lentivirus infection, miR-150 level increased in Saos-2 (P<0.01) and MG-63 cells (P<0.01). Overexpression of miR-150 decreased cell proliferation and RUNX2 protein level in Saos-2 and MG-63 cells. The binding of miR-150 to RUNX2 3'-UTR decreased luciferase activity by 69% in Saos-2 cells (P<0.05) and 59% in MG-63 cells (P<0.05). Administration of exogenous RUNX2 recovered the cell proliferation in miR-150 overexpressed Saos-2 and MG-63 cell lines (P<0.01).
 Conclusion: MiR-150 inhibites proliferation in human osteosarcoma cell lines through binding to RUNX2 3'-UTR, resulting in the reducion of RUNX2 protein level.

  6. Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment

    PubMed Central

    Li, Dongqi; Li, Huiling; Ren, Mingyan; Liao, Yedan; Yu, Shunling; Chen, Yanjin; Yang, Yihao; Zhang, Ya

    2016-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. Although combined therapy including surgery and multi-agent chemotherapy have resulted in great improvements in the overall survival of patients, chemoresistance remains an obstacle for the treatment of osteosarcoma. Molecular targets or effective agents that are actively involved in cell death including apoptosis, autophagy and necroptosis have been studied. We summarized how these agents (novel compounds, miRNAs, or proteins) regulate apoptotic, autophagic and necroptotic pathways; and discussed the current knowledge on the role of these new agents in chemotherapy resistance in osteosarcoma. PMID:27007056

  7. TRIM14 regulates cell proliferation and invasion in osteosarcoma via promotion of the AKT signaling pathway

    PubMed Central

    Xu, Guoxing; Guo, Yongfei; Xu, Dabo; Wang, Yi; Shen, Yafeng; Wang, Feifei; Lv, Yuanyuan; Song, Fanglong; Jiang, Dawei; Zhang, Yinquan; Lou, Yi; Meng, Yake; Yang, Yongji; Kang, Yifan

    2017-01-01

    Recent studies have shown that some members of the tripartite motif-containing protein (TRIM) family serve as important regulators of tumorigenesis. However, the biological role of TRIM14 in osteosarcoma remains to be established. In this study, we showed that TRIM14 is upregulated in human osteosarcoma specimens and cell lines, and correlated with osteosarcoma progression and shorter patient survival times. Functional studies demonstrated that overexpression of TRIM14 enhances osteosarcoma cell proliferation, clone formation, cell cycle procession, migration and invasion in vitro and promotes tumor growth in vivo, and conversely, its silencing has the opposite effects. Furthermore, TRIM14 overexpression induced activation of the AKT pathway. Inhibition of AKT expression reversed the TRIM14-mediated promotory effects on cell growth and mobility, in addition to TRIM14-induced epithelial-to-mesenchymal transition (EMT) and cyclin D1 upregulation. Our findings collectively suggest that TRIM14 functions as an oncogene by upregulating the AKT signaling pathway in osteosarcoma cells, supporting its potential utility as a therapeutic target for this disease. PMID:28205534

  8. T-Cell-Based Immunotherapy for Osteosarcoma: Challenges and Opportunities

    PubMed Central

    Wang, Zhan; Li, Binghao; Ren, Yingqing; Ye, Zhaoming

    2016-01-01

    Even though combining surgery with chemotherapy has significantly improved the prognosis of osteosarcoma patients, advanced, metastatic, or recurrent osteosarcomas are often non-responsive to chemotherapy, making development of novel efficient therapeutic methods an urgent need. Adoptive immunotherapy has the potential to be a useful non-surgical modality for treatment of osteosarcoma. Recently, alternative strategies, including immunotherapies using naturally occurring or genetically modified T cells, have been found to hold promise in the treatment of hematologic malignancies and solid tumors. In this review, we will discuss possible T-cell-based therapies against osteosarcoma with a special emphasis on combination strategies to improve the effectiveness of adoptive T cell transfer and, thus, to provide a rationale for the clinical development of immunotherapies. PMID:27683579

  9. LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma

    PubMed Central

    Xiao-Po, Liu; Xiao-Li, Li; Guo-Long, Cao; Pei, Zhang; Fa-Ming, Tian

    2016-01-01

    Abstract Objective: To examine the expression and function of long non-coding RNA taurine up-regulated 1 (TUG1) in human osteosarcoma cells. Methods: Real-time quantitive PCR was used to detect the transcription level of TUG1 in a series of osteosarcoma cell lines. Knockdown of TUG1 in U2OS cells was carried out by transient transfection of siRNAs. MTT assay was performed to access the cell growth rates. Afterwards, RNA and protein of these cells were extracted to analyze the transfection efficient as well as the expression of other molecules. Results: Compared to the normal cell line, TUG1 exhibited a significant upregulation in osteosarcoma cells. Phenotyping analysis showed the growth-promotion activity of TUG1, since knockdown of TUG1 resulted in declined proliferation. We also found that AKT phosphorylation was impaired after TUG1 was inhibited, suggesting that the AKT pathway was involved in the regulation of TUG1 in U2OS cells. Conclusion: Our data provided evidence that TUG1 was upregulated and acted as a possible oncogene via positively regulating cell proliferation in osteosarcoma cells.

  10. CXCL8 promotes the invasion of human osteosarcoma cells by regulation of PI3K/Akt signaling pathway.

    PubMed

    Jiang, Hai; Wang, Xiaowei; Miao, Wusheng; Wang, Bing; Qiu, Yusheng

    2017-09-01

    Chemokine cysteine-X-cysteine motif ligand 8 (CXCL8) is up-regulated in many malignancies, indicating that CXCL8 takes part in tumor progression. However, the expression and function of CXCL8 in osteosarcoma remained not fully elucidated. In this study, expressions of 12 cytokines and chemokines were measured in the serum from 12 of normal controls (NCs) and 25 of osteosarcoma patients. The human osteosarcoma cell line MG-63 was stimulated by recombinant CXCL8 to further analyze invasion, proliferation, apoptosis, cell cycles, cytokine secretions, and signaling pathways. We found that serum concentrations of CXCL8 and vascular endothelial growth factor were elevated in osteosarcoma patients in comparison with those in NCs. CXCL8 stimulation led to enhancement of invasion and suppression of late stage apoptosis in MG-63 cells. Moreover, secretions of MMPs by MG-63 cells were also increased upon stimulation. However, early stage apoptosis, proliferation, and cell cycles were not affected by CXCL8 treatment. Furthermore, CXCL8 stimulation induced elevations of phosphorylated PI3K and Akt, but not PKC or FAK. In conclusion, our findings suggested that CXCL8 enhanced the invasion and suppressed late stage apoptosis of osteosarcoma cells probably via influencing PI3K/Akt signaling pathway and elevating the expression of MMPs. CXCL8 may promote disease progression of osteosarcoma as a protumorigenic molecule, and may be served as a new therapeutic target for osteosarcoma. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  11. 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

  12. Imatinib Mesylate Exerts Anti-Proliferative Effects on Osteosarcoma Cells and Inhibits the Tumour Growth in Immunocompetent Murine Models

    PubMed Central

    Ory, Benjamin; Charrier, Céline; Brion, Régis; Blanchard, Frederic; Redini, Françoise; Heymann, Dominique

    2014-01-01

    Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma), a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1). Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma) and POS-1 (undifferentiated osteosarcoma). Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRβ, Axl, RYK, EGFR, EphA2 and 10, IGF1R), appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor status of patients

  13. The Downregulation of MiR-182 Is Associated with the Growth and Invasion of Osteosarcoma Cells through the Regulation of TIAM1 Expression

    PubMed Central

    Hu, Jun; Lv, Guohua; Zhou, Shuguang; Zhou, Yucheng; Nie, Bangxu; Duan, Hong; Zhang, Yunfeng; Yuan, Xiaofeng

    2015-01-01

    Background Osteosarcoma is the most common primary bone malignancy in children and young adults. Increasing results suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for osteosarcoma. Methods MiR-182 expression level in osteosarcoma cell lines and tissues were assayed by qRT-PCR. MiRNA mimics or inhibitor were transfected for up-regulation or down-regulation of miR-182 expression. Cell function was assayed by CCK8, migration assay and invasion assay. The target genes of miR-182 were predicated by bioinformatics algorithm (TargetScan Human). Results MiR-182 was down-regulated in osteosarcoma tissues and cell lines. Overexpression of miR-182 inhibited tumor growth, migration and invasion. Subsequent investigation revealed that TIAM1 was a direct and functional target of miR-182 in osteosarcoma cells. Overexpression of miR-182 impaired TIAM1-induced inhibition of proliferation and invasion in osteosarcoma cells. Conclusions Down-expression of miR-182 in osteosarcoma promoted tumor growth, migration and invasion by targeting TIAM1. MiR-182 might act as a tumor suppressor gene whose down-regulation contributes to the progression and metastasis of osteosarcoma, providing a potential therapy target for osteosarcoma patients. PMID:25973950

  14. The Downregulation of MiR-182 Is Associated with the Growth and Invasion of Osteosarcoma Cells through the Regulation of TIAM1 Expression.

    PubMed

    Hu, Jun; Lv, Guohua; Zhou, Shuguang; Zhou, Yucheng; Nie, Bangxu; Duan, Hong; Zhang, Yunfeng; Yuan, Xiaofeng

    2015-01-01

    Osteosarcoma is the most common primary bone malignancy in children and young adults. Increasing results suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for osteosarcoma. MiR-182 expression level in osteosarcoma cell lines and tissues were assayed by qRT-PCR. MiRNA mimics or inhibitor were transfected for up-regulation or down-regulation of miR-182 expression. Cell function was assayed by CCK8, migration assay and invasion assay. The target genes of miR-182 were predicated by bioinformatics algorithm (TargetScan Human). MiR-182 was down-regulated in osteosarcoma tissues and cell lines. Overexpression of miR-182 inhibited tumor growth, migration and invasion. Subsequent investigation revealed that TIAM1 was a direct and functional target of miR-182 in osteosarcoma cells. Overexpression of miR-182 impaired TIAM1-induced inhibition of proliferation and invasion in osteosarcoma cells. Down-expression of miR-182 in osteosarcoma promoted tumor growth, migration and invasion by targeting TIAM1. MiR-182 might act as a tumor suppressor gene whose down-regulation contributes to the progression and metastasis of osteosarcoma, providing a potential therapy target for osteosarcoma patients.

  15. Oridonin synergizes with Nutlin-3 in osteosarcoma cells by modulating the levels of multiple Bcl-2 family proteins.

    PubMed

    Wang, Xiao-Hui; Zhang, Shu-Feng; Bao, Jun-Tao; Liu, Fu-Yun

    2017-06-01

    The small-molecule inhibitors of p53-murine double minute 2 interaction, such as Nutlin-3, are effective against cancers bearing wild-type p53. However, murine double minute 2 inhibitors often are unable to completely eliminate solid tumor cells. To address this issue, we investigated the anticancer effects of Nutlin-3 in combination with Oridonin in osteosarcoma cells. We found that Oridonin at sub-toxic concentrations synergistically enhanced Nutlin-3-mediated cell viability inhibition in wild-type p53 U2OS and SJSA-1, but not in p53-mutant MNNG/HOS and in null-p53 Saos-2 osteosarcoma cell lines. Importantly, in the presence of Oridonin, Nutlin-3 could completely abolish cell viability in the wild-type p53 osteosarcoma cell lines. Western blotting analysis showed that Oridonin treatment rapidly and distinctly increased the levels of all three forms of Bim and also markedly reduced the levels of Bcl-2 and Bcl-xl in osteosarcoma cells. Western blotting analysis further showed that Oridonin considerably enhanced Nutlin-3-triggered activation of caspases-9 and -3 and poly(ADP-ribose) polymerase cleavage. Flow cytometry assay showed that Oridonin significantly enhanced Nutlin-3-mediated apoptosis in wild-type p53 osteosarcoma cells. Overall, our results suggest that the combined treatment of Nutlin-3 plus Oridonin may offer a novel therapeutic strategy for osteosarcoma.

  16. Formation of calcifying matrix by osteosarcoma cells in diffusion chambers in vivo.

    PubMed

    Shteyer, A; Gazit, D; Passi-Even, L; Bab, I; Majeska, R; Gronowicz, G; Lurie, A; Rodan, G

    1986-07-01

    The potential of clonal rat osteosarcoma (ROS) cell lines to form mineralized matrices was assessed in diffusion chambers in vivo. Diffusion chambers were inoculated with osteoblastic (ROS 17/2 and its subclone ROS 17/2.8) and non-osteoblastic (ROS 24/1) clonal lines and implanted either intraperitoneally into athymic mice or subcutaneously into syngeneic ACI rats. Control chamber cultures of rabbit marrow or spleen cells were also incubated in athymic mice. Light and electron microscopy of chambers with ROS 17/2 and ROS 17/2.8 cells revealed production of mineralized matrices typical of osteosarcoma and characterized by abundance of collagen fibrils and associated mineralizing nodules. ROS 24/1 cells produced similar collagenous matrices, but these were devoid of mineral. The present experiments, carried out independently in two different laboratories, demonstrate the potential of ROS cells to produce a mineralized matrix. This corroborates previous studies on other osteoblastic features of these cell lines.

  17. Antitumor activity of dobutamine on human osteosarcoma cells

    PubMed Central

    YIN, JUN; DONG, QIRONG; ZHENG, MINQIAN; XU, XIAOZU; ZOU, GUOYOU; MA, GUOLIN; LI, KEFENG

    2016-01-01

    Dobutamine has been widely used for the treatment of heart failure and cardiogenic shock since the 1970s. Osteosarcoma is the most commonly observed malignant bone tumor in children. Currently, there are no effective drugs for the treatment of osteosarcoma. In the present study, the potential anticancer activity of dobutamine on human osteosarcoma cells was examined. Human osteosarcoma MG-63 cells were treated with dobutamine at various concentrations and for various incubation times. The inhibition of cell growth by dobutamine was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was utilized to evaluate the effect of dobutamine on cell apoptosis and the cell cycle. Furthermore, the expression levels of caspase-3 and caspase-9 were assessed by western blot analysis. The influence of dobutamine on cancer cell migration and invasion was additionally evaluated using wound-healing assay and the Boyden Chamber migration method. Dobutamine significantly inhibited the growth of MG-63 cells at a concentration of 10 µM or higher when incubated for 12 h or longer (P=0.023). Dobutamine augmented cell apoptosis and arrested the cell cycle in the G2/M phase. Western blot analysis revealed that dobutamine induces expression of caspase-3 and caspase-9. In addition, the invasiveness and migration of MG-63 cells was inhibited by dobutamine in a concentration-dependent manner. The results of the present study may lead to novel applications for dobutamine in the treatment of osteosarcoma. PMID:27284371

  18. 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.

  19. MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the PI3K/Akt pathway.

    PubMed

    Dong, Yongqiang; Liang, Guojun; Yuan, Bo; Yang, Chaoqun; Gao, Rui; Zhou, Xuhui

    2015-03-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), one of the first found cancer-associated long noncoding RNAs (lncRNAs), involves in the development and progression of many types of tumors. An aberrant expression of MALAT1 was observed in hepatocellular carcinoma, cervical cancer, breast cancer, ovarian cancer, and colorectal cancer. However, the exact effects and molecular mechanisms of MALAT1 in osteosarcoma progression are still unknown up to now. Here, we investigated the role of MALAT1 in human osteosarcoma cell lines and clinical tumor samples in order to determine the function of this molecule. In our research, the MALAT1 messenger RNA (mRNA) was highly expressed in human osteosarcoma tissues, and its expression level was closely correlated with pulmonary metastasis. Then, we employed lentivirus-mediated knockdown of MALAT1 in U-2 OS and SaO2 to determine the role of MALAT1 in osteosarcoma cell lines. Lentivirus-mediated MALAT1 small interfering RNA (siRNA) could efficiently downregulated the expression level of MALAT1 in osteosarcoma cell lines. Knockdown of MALAT1 inhibited the proliferation and invasion of human osteosarcoma cell and suppressed its metastasis in vitro and vivo. At the same time, the proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 (MMP-9), phosphorylated PI3Kp85α, and Akt expressions were significantly inhibited in MALAT1-deleted cells. These findings indicated that MALAT1 might suppress the tumor growth and metastasis via PI3K/AKT signaling pathway. Taken together, our data indicated that MALAT1 might be an oncogenic lncRNA that promoted proliferation and metastasis of osteosarcoma and could be regarded as a therapeutic target in human osteosarcoma.

  20. Heat shock transcription factor 1 promotes the proliferation, migration and invasion of osteosarcoma cells.

    PubMed

    Zhou, Zhenhua; Li, Yan; Jia, Qi; Wang, Zhiwei; Wang, Xudong; Hu, Jingjing; Xiao, Jianru

    2017-08-01

    Osteosarcoma is the most commonly diagnosed primary malignancy of bone and its overall survival rate is still very low. The molecular mechanisms underlying the progression of osteosarcoma have not been clearly illuminated. Heat shock transcription factor 1 (HSF1) is a key regulator of the heat shock response and also plays important roles in many cancers, but its function in osteosarcoma remains unexplored. In this study, the proliferation of osteosarcoma cells was determined by Cell Counting Kit-8 assays and colony formation assays. Transwell assays were used to demonstrate the migration and invasion abilities of osteosarcoma cells. A tumour formation assay in a nude mouse model was performed to assess the effect of HSF1 on osteosarcoma cell growth in vivo. The protein levels of HSF1 were analysed with immunohistochemical staining in samples from osteosarcoma patients. We demonstrated that knockdown of HSF1 reduced the proliferation, migration and invasion of osteosarcoma cells, while overexpression of HSF1 promoted the proliferation, migration and invasion of osteosarcoma cells. Furthermore, HSF1 promoted the proliferation of osteosarcoma cells in vivo. In addition, high levels of HSF1 were associated with a poor prognosis in osteosarcoma. These data highlight an important role of HSF1 in proliferation, migration and invasion of osteosarcoma cells. Moreover, the expression of HSF1 was associated with prognosis in osteosarcoma. © 2017 John Wiley & Sons Ltd.

  1. p53-Dependent Activation of microRNA-34a in Response to Etoposide-Induced DNA Damage in Osteosarcoma Cell Lines Not Impaired by Dominant Negative p53 Expression

    PubMed Central

    Novello, Chiara; Pazzaglia, Laura; Conti, Amalia; Quattrini, Irene; Pollino, Serena; Perego, Paola; Picci, Piero; Benassi, Maria Serena

    2014-01-01

    Osteosarcoma (OS) is the most common primary malignant bone tumor and prevalently occurs in the second decade of life. Etoposide, a chemotherapeutic agent used in combined treatments of recurrent human OS, belongs to the topoisomerase inhibitor family and causes DNA breakage. In this study we evaluated the cascade of events determined by etoposide-induced DNA damage in OS cell lines with different p53 status focusing on methylation status and expression of miR-34a that modulate tumor cell growth and cell cycle progression. Wild-type p53 U2-OS cells and U2-OS cells expressing dominant-negative form of p53 (U2- OS175) were more sensitive to etoposide than p53-deficient MG63 and Saos-2 cells, showing increased levels of unmethylated miR-34a, reduced expression of CDK4 and cell cycle arrest in G1 phase. In contrast, MG63 and Saos-2 cell lines presented aberrant methylation of miR-34a promoter gene with no miR-34a induction after etoposide treatment, underlining the close connection between p53 expression and miR-34a methylation status. Consistently, in p53siRNA transfected U2-OS cells we observed loss of miR-34a induction after etoposide exposure associated with a partial gain of gene methylation and cell cycle progress towards G2/M phase. Our results suggest that the open and unmethylated conformation of the miR-34a gene may be regulated by p53 able to bind the gene promoter. In conclusion, cell response to etoposide-induced DNA damage was not compromised in cells with dominant-negative p53 expression. PMID:25490093

  2. Cell Cycle Arrest and Apoptosis Induced by Kinamycin F in Human Osteosarcoma Cells.

    PubMed

    Bavelloni, Alberto; Focaccia, Enrico; Piazzi, Manuela; Errani, Costantino; Blalock, William; Faenza, Irene

    2017-08-01

    Kinamycin F is a bacterial metabolite which contains an unusual and potentially reactive diazo group that is known for its ability to inhibit cell growth. In this study, the potential anti-tumor activity of kinamycin F was investigated in three human osteosarcoma cell lines, MG-63, U-2 OS and HOS as an antitumor agent with a potentially novel target. Proliferation and cell viability were measured in three human osteosarcoma cell lines by commercially available kits. We also evaluated the effects of the drug on cell cycle progression using the Muse™ Cell Analyzer. Caspase-3 activity was determined by a fluorometric EnzChek assay kit. Finally, following treatment with kinamycin F the protein levels of cyclin D3, cyclin A and cdK-2 were examined. Kinamycin F induced a concentration-dependent cell death in all the three cell lines. Flow cytometry revealed that kinamycin F treatment at 1 μM concentration significantly increased the cell population in the G2/M-phase (60-65%). Kinamycin F activated caspase 3 in all the three cell lines, clearly demonstrating that the growth inhibitory effect of kinamycin F can be attributed to apoptosis induction. Finally, kinamycin F suppressed osteosarcoma cell proliferation affecting cyclin A and D3 expression. Understanding the mechanism by which kinamycin F exerts its ability to inhibit cell growth may be a step forward in the development of new therapeutic strategies for the treatment of OS. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  3. In vitro antitumor activity of the ethyl acetate extract of Potentilla chinensis in osteosarcoma cancer cells

    PubMed Central

    Wan, Guang; Tao, Jin-Gang; Wang, Guo-Dong; Liu, Shen-Peng; Zhao, Hong-Xing; Liang, Qiu-Dong

    2016-01-01

    The aim of the current study was to evaluate the anticancer effect of the ethanol extract of Potentilla chinensis, a Chinese medicinal plant. An MTT assay was used to evaluate the cell viability of MG-63 human osteosarcoma cancer cells and fR-2 cells. Furthermore, the effect of the extract on apoptosis induction, cell cycle phase distribution and inhibition of cell migration in the MG63 human osteosarcoma cancer cell line was evaluated. The effect of the extract on cell cycle phase distribution was assessed by flow cytometry using propidium iodide (PI). Phase contrast microscopy detected the morphological changes in MG63 cancer cells following extract treatment. The results of the study demonstrated that the extract was cytotoxic to MG63 cancer cells, while the normal cell line (epithelial cell line) showed lower susceptibility. Phase contrast microscopy showed distinguishing morphological features, such as cell shrinkage and blebbing induced by the extract treatment in osteosarcoma cancer cells. The average proportion of Annexin V-positive cells (total apoptotic cells) significantly increased from 5.6% in the control to 24.2, 38.8 and 55.7% in the 40, 80 and 150 µg/ml groups, respectively. The extract induced early and late apoptosis in the cancer cells. Flow cytometric analysis revealed that the extract induced G0/G1-cell cycle arrest, which also showed significant dose-dependence. The extract induced a significant and concentration-dependent reduction in cell migration. Moreover, DNA fragmentation was also examined by observation of the formation of DNA ladders. It was demonstrated that DNA fragmentation was increased with extract concentration compared with that in the control. Taken together, EEPC may serve as potential therapeutic agent against osteosarcoma, provided that the toxicity profile and in vivo investigations demonstrate that it is safe. PMID:27573158

  4. Curcumin inhibits hypoxia-induced proliferation and invasion of MG-63 osteosarcoma cells via downregulating Notch1.

    PubMed

    Wang, Zhan; Zhang, Kun; Zhu, Yangjun; Wang, Dengfeng; Shao, Yuxiong; Zhang, Jun

    2017-04-01

    Curcumin is a biologically active ingredient abundantly present in the ground rhizomes of Curcuma longa with a wide range of bioactive properties, including antitumor effects. Hypoxia is a common characteristic of solid tumors, including osteosarcoma. However, whether curcumin has antitumor effects on osteosarcoma under hypoxic conditions, and its underlying molecular mechanisms, remain unclear. The present study demonstrated that the MG‑63 osteosarcoma cell line exhibited increased proliferation and enhanced invasiveness upon exposure to hypoxic conditions. However, these effects were prevented by curcumin treatment. Further investigation revealed that curcumin may inhibit Notch1 upregulation induced by hypoxia. Overexpression of Notch1 via Notch1 cDNA transfection ameliorated curcumin‑inhibited MG‑63 cell growth under hypoxic conditions. Taken together, these data revealed that curcumin may suppress the growth of osteosarcoma cells in hypoxia via inhibiting Notch1 signaling.

  5. Silencing of carboxypeptidase E inhibits cell proliferation, tumorigenicity, and metastasis of osteosarcoma cells

    PubMed Central

    Fan, Shuli; Li, Xu; Li, Leiming; Wang, Liguo; Du, Zhangzhen; Yang, Yan; Zhao, Jiansong; Li, Yan

    2016-01-01

    Carboxypeptidase E (CPE), a prohormone processing enzyme, has been implicated in the progression of multiple malignancies. However, the biological role and molecular mechanisms of CPE in osteosarcoma remain elusive. In this study, we assessed the effects of CPE on cell proliferation, tumorigenicity, migration, and invasion in osteosarcoma. Our results showed that silencing of CPE significantly inhibited cell proliferation, caused cell cycle arrest at G0/G1 phase, decreased the expression levels of cell cycle protein, cyclin D1, and inhibited tumorigenicity in vivo. Additionally, CPE downregulation repressed the migratory and invasive capacities of osteosarcoma cells in vitro. Furthermore, overexpression of CPE-ΔN (a splice variant of CPE) enhanced the cell growth, migration, and invasion of osteosarcoma cells. It is possible that both CPE forms are involved in the tumorigenesis and development of osteosarcoma, and therefore CPE may provide a promising biological target for osteosarcoma therapy. PMID:27274275

  6. miR-34a inhibits the metastasis of osteosarcoma cells by repressing the expression of CD44.

    PubMed

    Zhao, Haien; Ma, Baoan; Wang, Yucai; Han, Tao; Zheng, Lianhe; Sun, Cong; Liu, Tao; Zhang, Yinglong; Qiu, Xiuchun; Fan, Qingyu

    2013-03-01

    Osteosarcoma is the most common type of malignant bone tumor in children and adolescents and approximately 30% of patients develop lung metastasis, which is the leading cause of mortality. In this study, we investigated the role of miR-34a in the invasion and metastasis of osteosarcoma cells by examining its expression level and functional pattern in these cells. miR-34a mimics were transfected into the highly metastatic subline, F5M2, and into the F4 subline with low metastatic potential of the paired human osteosarcoma cell line, SOSP‑9607. Cell viability patterns, cell migration and alterations in gene expression levels were assessed by real-time PCR, and changes in protein levels were assessed by immunocytochemistry and western blot analysis. The ectopic overexpression of miR-34a significantly inhibited the migration and invasive ability of osteosarcoma cells by repressing the expression of CD44. These data suggest that miR-34a plays a tumor suppressor role in the metastasis of osteosarcoma cells by repressing the expression of CD44. Of note, studies have also suggested that the CD44 protein correlates with the metastatic potential of several malignant tumors. Therefore, it can be concluded that through the inhibition of CD44 expression levels, miR-34a plays a significant role in the migration and invasion of osteosarcoma cells.

  7. MicroRNA-101 inhibits proliferation, migration and invasion in osteosarcoma cells by targeting ROCK1

    PubMed Central

    Jiang, Rui; Zhang, Chao; Liu, Guangyao; Gu, Rui; Wu, Han

    2017-01-01

    Osteosarcoma is a rare malignant bone tumor in adolescents, with high degree of malignancy, and highly incidence of recurrence and metastasis. Our study aimed to explore the role of miR-101 in osteosarcoma cells by targeting ROCK1. In the present study, reverse transcription-quantitative polymerase chain reaction data revealed that miR-101 was down-regulated in the tissue samples of 20 patients with osteosarcoma compared with their matched adjacent non-tumor tissues (P < 0.01). Furthermore, miR-101 was significantly down-regulated in three common OS cell lines, MG63, U2OS, and OS732 compared with the human osteoblast cell line, hFOB1.19 (P < 0.01). MiR-101 was shown to target the ROCK1 3’-UTR in dual-luciferase reporter assays in MG63 cells. Overexpression of miR-101 significantly suppressed the protein expression levels of ROCK1, while knockdown of miR-101 significantly enhanced the formers’ expression levels in MG63 cells (P < 0.05). Overexpression of miR-101 inhibited cell viability, migration, and invasion while promoted apoptosis. Independent inhibition of ROCK1 and knockdown of miR-101 expression levels significantly promoted MG63 cell proliferation, migration and invasion while inhibited apoptosis (P < 0.01). Moreover, knockdown of ROCK1 reversed the promotion effect of miR-101 knockdown on proliferation, migration, and invasion while promoted apoptosis of MG63 cells, suggesting that miR-101 acts as a tumor suppressor in osteosarcoma cells via targeting ROCK1. Furthermore, overexpression of miR-101 inhibited tumor growth and motion by inactivating PI3K/AKT and JAK/STAT signaling pathways via downregulation of ROCK1. To conclude, miR-101/ROCK1 may be a potential therapeutic target for osteosarcoma therapy. PMID:28123850

  8. Aminolevulinic Acid-Mediated Photodynamic Therapy Causes Cell Death in MG-63 Human Osteosarcoma Cells.

    PubMed

    White, Bradley; Rossi, Vince; Baugher, Paige J

    2016-09-01

    The aim of this study was to test the efficacy of aminolevulinic acid-mediated photodynamic therapy (PDT) against the human osteosarcoma cell line MG-63. Osteosarcoma is the most common type of primary malignant bone tumor diagnosed in the United States among adolescents and children. Treatments for osteosarcoma often result in diminished limb use or amputation. Because ALA-mediated PDT exhibits dual specificity in the context of tumor killing, this therapy could represent a less invasive, but effective, treatment for this disease. To assess ALA dark toxicity in MG-63 cells, cells were incubated with varying concentrations of ALA, and cell viability was determined by crystal violet assay. Protoporphyrin IX (PpIX) accumulation was assessed subsequent to ALA incubation at various concentrations using spectrofluorometry. Cell death subsequent to ALA-PDT was determined by illuminating cells at a wavelength of 635 nm at various light intensities subsequent to ALA incubation. Cell viability was assessed using the MTT assay. ALA dark toxicity was observed only at the highest concentrations of 2, 5, and 10 mM. Maximal PpIX concentration was observed at 0.5 and 1 mM ALA, subsequent to a 24-h incubation. Maximal cell death with minimal light toxicity was observed at 0.5 and 1 mM ALA after illumination with 0.6 and 3 J/cm(2) light. Collectively, our data indicate that ALA-PDT can result in the death of MG-64 human osteosarcoma cells in vitro.

  9. miR-24 represses metastasis of human osteosarcoma cells by targeting Ack1 via AKT/MMPs pathway.

    PubMed

    Liu, Zhendong; Liu, Zhitao; Zhang, Yuanjun; Li, Yan; Liu, Bo; Zhang, Kexiang

    2017-04-29

    The expression levels of the protein tyrosine kinase Ack1 has been reported to be dysregulated in various cancers and involve in oncogenesis and progression. However, the expression and role of Ack1 in osteosarcoma remains unknown. In this study, we found that Ack1 were evidently upregulated in human osteosarcoma tissues and cell lines. In addition, the clinical data showed that high expression level of Ack1 is closely associated with clinical stage and positive distant metastasis, and negatively correlated with overall survival. Then, bioinformatics prediction and luciferase reporter assay indicated Ack1 as a direct target of miR-24, and Ack1 could be downregulated by miR-24 at both the mRNA and protein expression levels. Moreover, Ack1 expression levels were inversely correlated with that of miR-24 in osteosarcoma tissues. Furthermore, functional assay showed that miR-24 significantly suppressed osteosarcoma progression partially mediated by inhibiting Ack1 expression. Finally, western bolt assay revealed that miR-24 regulate AKT/MMPs pathway via Ack1 in osteosarcoma cells. In conclusion, our study demonstrated the suppression of miR-24 on osteosarcoma metastasis by targeting Ack1 via AKT/MMPs pathways, providing a novel strategy for the diagnosis and treatment of osteosarcoma patients. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis

    PubMed Central

    Ji, Guang-rong; Yu, Nai-chun; Xue, Xiang; Li, Zong-guang

    2015-01-01

    Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy. PMID:26078722

  11. PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis.

    PubMed

    Ji, Guang-rong; Yu, Nai-chun; Xue, Xiang; Li, Zong-guang

    2015-01-01

    Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy.

  12. microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R.

    PubMed

    Han, Kang; Zhao, Tingbao; Chen, Xiang; Bian, Na; Yang, Tongtao; Ma, Qiong; Cai, Chengkui; Fan, Qingyu; Zhou, Yong; Ma, Baoan

    2014-10-01

    Studies have shown that miR-194 functions as a tumor suppressor and is associated with tumor growth and metastasis. We studied the effects of miR-194 in osteosarcoma and the possible mechanism by which miR-194 affected the survival, apoptosis and metastasis of osteosarcoma. Both human osteosarcoma cell lines SOSP-9607 and U2-OS were transfected with recombinant lentiviruses to regulate miR-194 expression. Overexpression of miR-194 partially inhibited the proliferation, migration, and invasion of osteosarcoma cells in vitro, as well as tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. Potential miR-194 target genes were predicted using bioinformatics. Luciferase reporter assay, real-time quantitative PCR and western blotting confirmed that CDH2 (N-cadherin) and IGF1R were targets of miR-194. Using real-time quantitative PCR, we evaluated the expression of miR-194 and two miR-194 target genes, CDH2 and IGF1R in osteosarcoma samples from 107 patients and 99 formalin- or paraformalin-fixed paraffin-embedded tissues. The expressions of the target genes were also examined in osteosarcoma samples using immunohistochemistry. Overexpression of miR-194 inhibited tumor growth and metastasis of osteosarcoma probably by downregulating CDH2 and IGF1R. miR-194 may prove to be a promising therapeutic agent for osteosarcoma.

  13. microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R

    PubMed Central

    HAN, KANG; ZHAO, TINGBAO; CHEN, XIANG; BIAN, NA; YANG, TONGTAO; MA, QIONG; CAI, CHENGKUI; FAN, QINGYU; ZHOU, YONG; MA, BAOAN

    2014-01-01

    Studies have shown that miR-194 functions as a tumor suppressor and is associated with tumor growth and metastasis. We studied the effects of miR-194 in osteosarcoma and the possible mechanism by which miR-194 affected the survival, apoptosis and metastasis of osteosarcoma. Both human osteosarcoma cell lines SOSP-9607 and U2-OS were transfected with recombinant lentiviruses to regulate miR-194 expression. Overexpression of miR-194 partially inhibited the proliferation, migration, and invasion of osteosarcoma cells in vitro, as well as tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. Potential miR-194 target genes were predicted using bioinformatics. Luciferase reporter assay, real-time quantitative PCR and western blotting confirmed that CDH2 (N-cadherin) and IGF1R were targets of miR-194. Using real-time quantitative PCR, we evaluated the expression of miR-194 and two miR-194 target genes, CDH2 and IGF1R in osteosarcoma samples from 107 patients and 99 formalin- or paraformalin-fixed paraffin-embedded tissues. The expressions of the target genes were also examined in osteosarcoma samples using immunohistochemistry. Overexpression of miR-194 inhibited tumor growth and metastasis of osteosarcoma probably by downregulating CDH2 and IGF1R. miR-194 may prove to be a promising therapeutic agent for osteosarcoma. PMID:25096247

  14. miR-125b suppresses the proliferation and migration of osteosarcoma cells through down-regulation of STAT3

    SciTech Connect

    Liu, Li-hong; Li, Hui; Li, Jin-ping; Zhong, Hui; Zhang, Han-chon; Chen, Jia; Xiao, Tao

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer miR-125b is frequently down-regulated in osteosarcoma samples and human osteosarcoma cell lines. Black-Right-Pointing-Pointer Ectopic restoration of miR-125b suppresses cell proliferation and migration in vitro. Black-Right-Pointing-Pointer STAT3 is the direct and functional downstream target of miR-125b. Black-Right-Pointing-Pointer STAT3 can bind to the promoter region of miR-125b and serves as a transactivator. -- Abstract: There is accumulating evidence that microRNAs are involved in multiple processes in development and tumor progression. Abnormally expressed miR-125b was found to play a fundamental role in several types of cancer; however, whether miR-125b participates in regulating the initiation and progress of osteosarcoma still remains unclear. Here we demonstrate that miR-125b is frequently down-regulated in osteosarcoma samples and human osteosarcoma cell lines. The ectopic restoration of miR-125b expression in human osteosarcoma cells suppresses proliferation and migration in vitro and inhibits tumor formation in vivo. We further identified signal transducer and activator of transcription 3 (STAT3) as the direct and functional downstream target of miR-125b. Interestingly, we discovered that the expression of miR-125b is regulated by STAT3 at the level of transcription. STAT3 binds to the promoter region of miR-125b in vitro and serves as a transactivator. Taken together, our findings point to an important role in the molecular etiology of osteosarcoma and suggest that miR-125b is a potential target in the treatment of osteosarcoma.

  15. Pirarubicin inhibits multidrug-resistant osteosarcoma cell proliferation through induction of G2/M phase cell cycle arrest

    PubMed Central

    Zheng, Shui-er; Xiong, Sang; Lin, Feng; Qiao, Guang-lei; Feng, Tao; Shen, Zan; Min, Da-liu; Zhang, Chun-ling; Yao, Yang

    2012-01-01

    Aim: Pirarubicin (THP) is recently found to be effective in treating patients with advanced, relapsed or recurrent high-grade osteosarcoma. In this study, the effects of THP on the multidrug-resistant (MDR) osteosarcoma cells were assessed, and the underlying mechanisms for the disruption of cell cycle kinetics by THP were explored. Methods: Human osteosarcoma cell line MG63 and human MDR osteosarcoma cell line MG63/DOX were tested. The cytotoxicity of drugs was examined using a cell proliferation assay with the Cell Counting Kit-8 (CCK-8). The distribution of cells across the cell cycle was determined with flow cytometry. The expression of cell cycle-regulated genes cyclin B1 and Cdc2 (CDK1), and the phosphorylated Cdc2 and Cdc25C was examined using Western blot analyses. Results: MG63/DOX cells were highly resistant to doxorubicin (ADM) and gemcitabine (GEM), but were sensitive or lowly resistant to THP, methotrexate (MTX) and cisplatin (DDP). Treatment of MG63/DOX cells with THP (200–1000 ng/mL) inhibited the cell proliferation in time- and concentration-dependent manners. THP (50–500 ng/mL) induced MG63/DOX cell cycle arrest at the G2/M phase in time- and concentration-dependent manners. Furthermore, the treatment of MG63/DOX cells with THP (200–1000 ng/mL) downregulated cyclin B1 expression, and decreased the phosphorylated Cdc2 at Thr161. Conversely, the treatment increased the phosphorylated Cdc2 at Thr14/Tyr15 and Cdc25C at Ser216, which led to a decrease in Cdc2-cyclin B1 activity. Conclusion: The cytotoxicity of THP to MG63/DOX cells may be in part due to its ability to arrest cell cycle progression at the G2/M phase, which supports the use of THP for managing patients with MDR osteosarcoma. PMID:22580740

  16. 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 G1 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 G1 phase arrest. These results suggest that PANDA promotes G1-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.

  17. Phyllostachys edulis extract induces apoptosis signaling in osteosarcoma cells, associated with AMPK activation.

    PubMed

    Chou, Chi-Wen; Cheng, Ya-Wen; Tsai, Chung-Hung

    2014-01-01

    Bamboo is distributed worldwide, and its different parts are used as foods or as a traditional herb. Recently, antitumoral effects of bamboo extracts on several tumors have been increasingly reported; however, antitumoral activity of bamboo extracts on osteosarcoma remains unclear. In the present study, we investigated effects of an aqueous Phyllostachys edulis leaf extract (PEE) on osteosarcoma cells and the underlying mechanism of inhibition. The growth of human osteosarcoma cell lines 143 B and MG-63 and lung fibroblast MRC-5 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Apoptosis was demonstrated using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay and flow cytometric analysis. Phosphorylation and protein levels were determined by immunoblotting. After treatment with PEE, viability of 143 B and MG-63 cells was dose-dependently reduced to 36.3% ± 1.6% of control values, which were similar to AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) treatments. In parallel, ratios of apoptotic cells and cells in the sub-G1 phase were significantly increased. Further investigation showed that PEE treatments led to activation of caspase cascades and changes of apoptotic mediators Bcl2, Bax, and p53. Consistently, our results revealed that PEE activated adenosine monophosphate-activated protein kinase (AMPK) signaling, and the AMPK activation was associated with the induction of apoptotic signaling. Our results indicated that PEE suppressed the growth of 143 B and MG-63 cells but moderately affected MRC-5 cells. PEE-induced apoptosis may attribute to AMPK activation and the following activation of apoptotic signaling cascades. These findings revealed that PEE possesses antitumoral activity on human osteosarcoma cells by manipulating AMPK signaling, suggesting that PEE alone or combined with regular antitumor drugs may be beneficial as osteosarcoma treatments.

  18. Quercetin Enhances Cisplatin Sensitivity of Human Osteosarcoma Cells by Modulating microRNA-217-KRAS Axis

    PubMed Central

    Zhang, Xian; Guo, Qinggong; Chen, Jingtao; Chen, Zhaohui

    2015-01-01

    Quercetin can suppress osteosarcoma cell growth and metastasis. However, other effects of quercetin on osteosarcoma remain largely unknown. This research aims to evaluate the effects of quercetin in combination with cisplatin as treatment for osteosarcoma and investigate its regulatory mechanism. Cell viability and apoptosis in 143B cell line were determined after treatment with quercetin and/or cisplatin. RT-PCR and Western blot analysis were performed to determine the RNA or protein expression levels. Moreover, transwell assay was used to evaluate metastasis. Furthermore, rescue experiments were performed to investigate the potential regulatory mechanism of the treatment. Results showed that quercetin with concentration that was equal to or greater than 10 μM inhibited 143B proliferation, while 5 μM quercetin enhanced the cisplatin sensitivity of 143B cells. Expression of miR-217 was upregulated after quercetin and/or cisplatin treatment, while its target KRAS was downregulated both at mRNA and protein levels. MiR-217 knockdown led to the loss of enhanced cisplatin sensitivity while miR-217 overexpression showed the opposite effects, indicating that quercetin regulated cisplatin sensitivity by modulating the miR-217-KRAS axis. In conclusion, 5 μM quercetin enhanced the cisplatin sensitivity by modulating the miR-217-KRAS axis. This finding suggests that quercetin may be administered with cisplatin to improve the treatment for osteosarcoma. PMID:26062553

  19. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    SciTech Connect

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. ); Mattei, M.G. ); Seldin, M.F. ); Riviere, M.; Szpirer, J. )

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 8; by in situ hybridization, the gene was mapped to human chromosome 3p21.1-p22 and to mouse chromosome 9 band F; and by interspecific backcross analysis, the Pthr gene segregated with the transferrin (Trf) gene in chromosome 9 band F. Mouse chromosome 9 and rat chromosome 8 are known to be highly homologous and to also show synteny conservation with human chromosome 3. These three chromosomes share the transferrin gene (TF), the myosin light polypeptide 3 gene (MYL3), and the acelpeptide hydrolase gene (APEH). These results add a fourth gene, the PTHR gene, to the synteny group conserved in these chromosomes. 34 refs., 7 figs. 1 tab.

  20. miR-10b promotes invasion by targeting KLF4 in osteosarcoma cells.

    PubMed

    Wang, Jing; Wang, Bing; Chen, Ling-Qiang; Yang, Jin; Gong, Zhi-Qiang; Zhao, Xue-Ling; Zhang, Chun-Qiang; Du, Kai-Li

    2016-12-01

    Osteosarcoma is a common malignancy with high rate of metastasis. miR-10b has been reported to be expressed in many types of tumors abnormally and be associated with cancer carcinogenesis and progression. But the function of miR-10b in osteosarcoma is still unknown. So this study was aimed to investigate the role of miR-10b in osteosarcoma development. miR-10b expression in osteosarcoma tissues and osteosarcoma cells were detected using real time PCR. The effects of miR-10b on osteosarcoma cells proliferation, apoptosis, migration and invasion were detected using CCK-8 assay, flow cytometry, wound-healing assay and transwell assay, respectively. The relationship between miR-10b and KLF4 was evaluated using dual-luciferase assay, correlation analysis. miR-10b was highly expressed in osteosarcoma tissues and osteosarcoma cells. Furthermore, inhibition of miR-10b in osteosarcoma cells depressed the cells proliferation, migration and invasion but promoted cells apoptosis. In addition, KLF4 was down-regulated by miR-10b and miR-10b expression was negatively related to KLF4 expression in osteosarcoma tissue, miR-10b participated in the process of osteosarcoma cells invasion by regulating KLF4 expression. miR-10b is overexpressed in osteosarcoma and KLF4 is the direct target gene of miR-10b. Furthermore, miR-10b promotes osteosarcoma cells progression by downregulating KLF4 expression. These results suggest that miR-10b functions as an oncomiR and play an important role in osteosarcoma cellular processes at least partially through regulating KLF4; miR-10b may be a therapeutic target for osteosarcoma treatment. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  1. A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells

    PubMed Central

    1992-01-01

    In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation). PMID:1469057

  2. RASSF4 Overexpression Inhibits the Proliferation, Invasion, EMT, and Wnt Signaling Pathway in Osteosarcoma Cells.

    PubMed

    Zhang, Minglei; Wang, Dapeng; Zhu, Tongtong; Yin, Ruofeng

    2017-01-02

    RASSF4, a member of the RASSF family, is broadly expressed in normal tissues but often inactivated in human cancers. Despite various studies on RASSF4, its role in osteosarcoma remains unclear. Therefore, in this study, we investigated the effects of RASSF4 expression on osteosarcoma cells and explored the underlying mechanism. The results of our study showed that RASSF4 was lowly expressed in osteosarcoma tissues and cells. RASSF4 overexpression significantly inhibited proliferation, migration, and invasion as well as the EMT process in osteosarcoma cells. Meanwhile, we found that RASSF4 overexpression markedly decreased the protein expression of β-catenin, cyclin D1, and c-Myc in osteosarcoma cells. In conclusion, our findings showed that RASSF4 overexpression inhibits proliferation, invasion, EMT, and Wnt signaling pathway in osteosarcoma cells. Thus, RASSF4 may be considered a novel target for osteosarcoma treatment.

  3. Long non-coding RNA small nucleolar RNA host gene 12 (SNHG12) promotes cell proliferation and migration by upregulating angiomotin gene expression in human osteosarcoma cells.

    PubMed

    Ruan, Wendong; Wang, Pei; Feng, Shiqing; Xue, Yuan; Li, Yulin

    2016-03-01

    The long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) has a role in cell proliferation and migration. Angiomotin, encoded by the AMOT gene, is a protein that regulates the migration and organization of endothelial cells. SNHG12 and AMOT have been shown to play a role in a variety of human cancers but have yet to be studied in detail in human osteosarcoma. Tissue samples from primary osteosarcoma (n = 20) and adjacent normal tissues (n = 20), the osteosarcoma cell lines, SAOS-2, MG-63, U-2 OS, and the human osteoblast cell line hFOB (OB3) were studied using Western blot for angiomotin, and quantitative real-time polymerase chain reaction for the expression of SNHG12 and AMOT. The expression of SNHG12 was knocked down using RNA interference. Cell migration assays were performed. Cell apoptosis was studied using flow cytometry. SNHG12 and AMOT messenger RNA (mRNA) expression was upregulated in osteosarcoma tissues and cell lines when compared with normal tissues and cells. Upregulation of AMOT mRNA was associated with upregulation of SNHG12. Knockdown of SNHG12 reduced the expression of angiomotin in osteosarcoma cells and suppressed cell proliferation and migration but did not affect cell apoptosis. This preliminary study has shown that the lncRNA SNHG12 promotes cell proliferation and migration by upregulating AMOT gene expression in osteosarcoma cells in vivo and in vitro. Further studies are recommended to investigate the role of SNHG12 and AMOT expression in tumor cell proliferation and migration and angiogenesis in osteosarcoma and a range of malignant mesenchymal tumors.

  4. MicroRNA-195-5p suppresses osteosarcoma cell proliferation and invasion by suppressing naked cuticle homolog 1.

    PubMed

    Qu, Qiang; Chu, Xiangdong; Wang, Peng

    2017-03-01

    MiR-195-5p has been shown to play an essential role in human cancer progression. Nevertheless, the biological role of miR-195-5p in osteosarcoma development remains unclear. In this study, real-time PCR was performed to examine the miR-195-5p expression in human osteosarcoma cell lines. CCK-8 assay and Transwell assay were carried out to measure the effect of miR-195-5p on cell proliferation and invasion. Luciferase reporter assay was used to identify the targets of miR-195-5p. The results showed that miR-195-5p was significantly downregulated in osteosarcoma cells. Forced expression of miR-195-5p significantly inhibited cell proliferation, suppressed cell migration and invasion, compared with wild-type and control-transfected osteosarcoma cells. Luciferase reporter assay revealed that miR-195-5p binds to the 3'-untranslated region (UTR) of Naked cuticle homolog 1 (NKD1), indicating that NKD1 was a novel target of miR-195-5p. NKD1 mRNA and protein levels were reduced after overexpression of miR-195-5p. Moreover, silencing of NKD1 significantly inhibited the proliferation and invasion of osteosarcoma cells. Accordingly, our results support a tumor suppressor role of miR-195-5p in osteosarcoma through inhibiting NKD1, and it may be a promising therapeutic target for osteosarcoma. © 2017 International Federation for Cell Biology.

  5. MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1.

    PubMed

    Han, Kang; Chen, Xiang; Bian, Na; Ma, Baoan; Yang, Tongtao; Cai, Chengkui; Fan, QingYu; Zhou, Yong; Zhao, Ting Bao

    2015-04-20

    Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma.

  6. MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1

    PubMed Central

    Ma, Baoan; Yang, Tongtao; Cai, Chengkui; Fan, QingYu; Zhou, Yong; Zhao, TingBao

    2015-01-01

    Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, Using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inverse relate to each other. In summary, our study suggests miR-195 function as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma. PMID:25823925

  7. Enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides GD2/GD3.

    PubMed

    Shibuya, Hidenobu; Hamamura, Kazunori; Hotta, Hiroshi; Matsumoto, Yasuyuki; Nishida, Yoshihiro; Hattori, Hisashi; Furukawa, Keiko; Ueda, Minoru; Furukawa, Koichi

    2012-09-01

    The expression and implications of gangliosides in human osteosarcomas have not been systematically analyzed. In this study, we showed that gangliosides GD3 and GD2 are highly expressed in the majority of human osteosarcoma cell lines derived from oral cavity regions. Introduction of GD3 synthase cDNA into a GD3/GD2-negative (GD3/GD2-) human osteosarcoma subline resulted in the establishment of GD3/GD2+ transfectant cells. They showed increased cell migration and invasion activities in wound healing and Boyden chamber invasion assays, respectively, compared to the control cells. When treated with serum, GD3/GD2+ cells showed stronger tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin than GD3/GD2- cells. In particular, paxillin underwent much stronger phosphorylation, suggesting its role in cell motility. Furthermore, we tried to dissect the roles of GD3 and GD2 in the malignant properties of the transfectant cells by establishing single ganglioside-expressing cells, that is, either GD3 or GD2. Although GD3/GD2+ cells showed the most malignant properties, GD2+ cells showed almost equivalent levels to GD3/GD2+ cells in invasion and migration activities, and in the intensities of tyrosine phosphorylation of paxillin. Among Src family kinases, Lyn was expressed predominantly, and was involved in the invasion and motility of GD3- and/or GD2-expressing transfectants. Furthermore, it was elucidated by gene silencing that Lyn was located in a different pathway from that of FAK to eventually lead paxillin activation. These results suggested that GD2/GD3 are responsible for the enhancement of the malignant features of osteosarcomas, and might be candidate targets in molecular-targeted therapy. © 2012 Japanese Cancer Association.

  8. Inhibition of hyaluronan retention by 4-methylumbelliferone suppresses osteosarcoma cells in vitro and lung metastasis in vivo.

    PubMed

    Arai, E; Nishida, Y; Wasa, J; Urakawa, H; Zhuo, L; Kimata, K; Kozawa, E; Futamura, N; Ishiguro, N

    2011-12-06

    Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma. We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS). In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 mM). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU. These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.

  9. Inhibition of hyaluronan retention by 4-methylumbelliferone suppresses osteosarcoma cells in vitro and lung metastasis in vivo

    PubMed Central

    Arai, E; Nishida, Y; Wasa, J; Urakawa, H; Zhuo, L; Kimata, K; Kozawa, E; Futamura, N; Ishiguro, N

    2011-01-01

    Background: Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma. Methods: We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS). Results: In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 m). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU. Conclusion: These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention. PMID:22045192

  10. In vitro photodynamic therapy of MG-63 osteosarcoma cells mediated by aminolevulinic acid

    NASA Astrophysics Data System (ADS)

    Rossi, Vincent M.; White, Bradley M.; Newton, Mariko J.; Jacques, Steven L.; Baugher, Paige J.

    2011-02-01

    This is an in vitro study of photodynamic therapy (PDT) in the MG-63 line of human osteosarcoma cells, as mediated by aminolevulinic acid (ALA). The primary goal of this work is to determine the feasibility and effectiveness of treating osteosarcoma through PDT. In addition, this work is aimed at determining whether the resulting cell death occurs through apoptosis or cellular necrosis. The MG-63 cells are treated with increasing concentrations of ALA from 0.1-10 mM ALA, leading to the accumulation of the photosensitizer protoporphyrin IX (PpIX) within the cells. After incubation periods of 4 and 24 hours in ALA, the cells are illuminated by 0-10 J/cm2 of 636 nm light in order to activate the PpIX and induce oxidative damage to the cells. Light is administered by an 8x12 array of LED's, which are controlled by an Arduino Duemilanove microcontroller board in order to assure ease of use along with accurate levels of exposure. Controls for this experiment include 0 J/cm2 of light exposure for all experimental concentrations of ALA, as well as illuminating cells that have not been incubated in ALA at all experimental levels of illumination. MG-63 cells are analyzed through fluorimetry and MTT assays in order to determine the effectiveness of ALA mediated PDT of osteosarcoma.

  11. Inhibitory effects of low-intensity pulsed ultrasound sonication on the proliferation of osteosarcoma cells.

    PubMed

    Matsuo, Toshihiro; Sato, Keiji; Matsui, Takuya; Sawada, Shigeyuki; Muramatsu, Yoshitaka; Kawanami, Katsuhisa; Deie, Masataka

    2017-09-01

    To date, there is limited data on the biological effects of low-intensity pulsed ultrasound (LIPUS) on primary malignant bone tumors. The purpose of the present study was to investigate the antitumor effects of LIPUS on osteosarcoma cells. The effects of LIPUS on cell viability, induction of apoptosis, mitochondrial membrane potential and intracellular signaling molecules in the LM8 osteosarcoma cell line were investigated. LIPUS inhibited cell viability (P=0.0022) and mitochondrial membrane potential (P=0.0019) in LM8 cells. Flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed significantly higher numbers of apoptotic (P<0.0001) and necrotic cells (P=0.0091) compared with cells without treatment. LIPUS treatment significantly increased phosphorylated Akt (P<0.0001) and IκBα (P=0.0001) levels, and reduced phosphorylated mitogen-activated protein kinase 7 (P<0.0001) and phosphorylated checkpoint kinase 1 (P=0.0008) levels. These results suggest that LIPUS is a non-invasive adjuvant therapy that is able to inhibit cellular proliferation in osteosarcoma cells.

  12. Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells.

    PubMed

    Troyer, Ryan M; Ruby, Carl E; Goodall, Cheri P; Yang, Liping; Maier, Claudia S; Albarqi, Hassan A; Brady, Jacqueline V; Bathke, Kallan; Taratula, Oleh; Mourich, Dan; Bracha, Shay

    2017-09-15

    Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells. Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-β, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree. Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    SciTech Connect

    Park, Hyunjin; Bergeron, Eric; Senta, Helena; Guillemette, Kim; Beauvais, Sabrina; Blouin, Richard; Sirois, Joel; Faucheux, Nathalie

    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, 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.

  14. Cobalt-Doped Brushite Cement: Preparation, Characterization, and In Vitro Interaction with Osteosarcoma Cells

    NASA Astrophysics Data System (ADS)

    Cummings, Haley; Han, Weiguo; Vahabzadeh, Sahar; Elsawa, Sherine F.

    2017-08-01

    Brushite cement (BrC) is being widely used in bone and dental tissue engineering application because of its significant biocompatibility, bioresorbability, and moldability. Here, we have reported the effects of cobalt (Co) and its concentration on physical and biological properties of BrC. Our results show that Co addition stabilizes the tricalcium phosphate structure and decreases the amount of BrC phase in the final product. The in vitro interaction of samples with osteosarcoma MG-63 cells proved the cytocompatibility of all compositions in both normoxic and hypoxic conditions. Although the cell viability increased under hypoxia, the change was insignificant compared with normoxic conditions. Our data show that Co addition reduced hypoxia inducible factor-1α and glioma-associated oncogene family zinc finger 2 expression in MG-63, suggesting Co may provide the benefit of reducing the effects of hypoxia on gene expression in the osteosarcoma cell line.

  15. CXCR4-mediated osteosarcoma growth and pulmonary metastasis is promoted by mesenchymal stem cells through VEGF.

    PubMed

    Zhang, Peng; Dong, Ling; Yan, Kang; Long, Hua; Yang, Tong-Tao; Dong, Ming-Qing; Zhou, Yong; Fan, Qing-Yu; Ma, Bao-An

    2013-10-01

    Chemokines and chemokine receptor 4 (CXCR4) play an important role in metastasis. CXCR4 is also expressed in the human osteosarcoma cell line 9607-F5M2 (F5M2), which has a high tumorigenic ability and potential for spontaneous pulmonary metastasis. Mesenchymal stem cells (MSCs) contribute to the formation of the tumor stroma and promote metastasis. However, mechanisms underlying the promotion of osteosarcoma growth and pulmonary metastasis by MSCs are still elusive. Our study co-injected the human MSCs and F5M2 cells into the caudal vein of nude mice. The total number of tumor nodules per lung was significantly increased in the F5M2+MSC group compared to the other groups (control, F5M2 cells alone and MSCs alone) at week six. Moreover, a high number of Dil-labeled MSCs was present also at the osteosarcoma metastasis sites in the lung. Using Transwell assays, we found that F5M2 cells migrate towards MSCs, while the CXCR4 inhibitor AMD3100 decreased the migration potential of F5M2 cells towards MSCs. Furthermore, upon treatment with F5M2-conditioned medium, MSCs expressed and secreted higher levels of VEGF as determined by immunohistochemistry, western blotting and ELISA, respectively. Importantly, co-cultured with F5M2 cells, MSCs expressed and secreted higher VEGF levels, while AMD3100 dramatically decreased the VEGF secretion by MSCs. However, CXCR4 expression on F5M2 cells was not significantly increased in the co-culture system. Additionally, VEGF increased the proliferation of both MSCs and F5M2 cells. These findings suggest that CXCR4-mediated osteosarcoma growth and pulmonary metastasis are promoted by MSCs through VEGF.

  16. Down-regulation of RBP-J mediated by microRNA-133a suppresses dendritic cells and functions as a potential tumor suppressor in osteosarcoma.

    PubMed

    Gao, Xuren; Han, Dong; Fan, Weimin

    2016-12-10

    In recent years, immunotherapy for the treatment of tumors have been established. Dendritic cells (DCs) are extremely efficient and professional antigen presenting cells (APCs), which are an important target for immune therapeutic interventions in cancer. In present study, we investigated whether RBP-J signaling regulated by miR-133a was involved in the DCs mediated tumor suppressor in osteosarcoma. DCs were isolated from 30 osteosarcoma patients and 30 healthy subjects. Mouse macrophage-like cell line RAW264.7 were cultured and osteosarcoma mouse model with injection of murine osteosarcoma cell line S180 were established. In osteosarcoma patients, miR-133a expression level of DCs was increased, and RBP-J expression in mRNA and protein levels were decreased. MiR-133a inhibitor promoted maturation and activation of DCs in osteosarcoma patients. In osteosarcoma mouse model, miR-133a mimic suppressed the maturation and activation of spleen DCs, while miR-133a inhibitor promoted them. Overexpression of miR-133a decreased therapeutic effect of DCs on osteosarcoma mice. In RAW264.7 cells, miR-133a was observed to target RBP-J and regulate its expression. MiR-133a mimic inhibited the maturation of DCs in cells exposed to LPS, the effect of which was reversed by overexpression of RBP-J. RBP-J mediated by miR-133a probably contributed to the regulation of DCs maturation and activation in osteosarcoma, which functioned as a therapeutic target for the immunotherapy in cancers. Copyright © 2016. Published by Elsevier Inc.

  17. Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

    PubMed

    Cavazza, Antonella; Marini, Mario; Roda, L Giorgio; Tarantino, Umberto; Valenti, Angela

    2011-12-01

    The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

  18. MicroRNA-21 promotes proliferation, invasion and suppresses apoptosis in human osteosarcoma line MG63 through PTEN/Akt pathway.

    PubMed

    Lv, Chen; Hao, Yuehan; Tu, Guanjun

    2016-07-01

    Osteosarcoma, which accounts for 5 % of pediatric tumor, remains the major cause of death among orthopedic malignancies. However, the factors associated with its malignant biological behavior are still poorly understood. MicroRNAs are a class of small noncoding RNAs, which have been considered to associate with malignant progression including cell differentiation, proliferation, apoptosis, invasion, and distant metastasis. In our research, we found that microRNA-21 (miR-21) was significantly overexpressed in human osteosarcoma cell line MG63 compared to human fetal osteoblastic cell line hFOB1.19 by using quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, miR-21 overexpression in MG63 caused a significant raise in cell proliferation and invasion and a significant reduction in cell apoptosis. However, miR-21 underexpression in MG63 caused an opposite result. Western blotting displayed that proteins related with proliferation, apoptosis, and invasion were significantly changed in different groups, respectively. Furthermore, we demonstrated that PTEN may be a potential target of miR-21 in MG63 cells and miR-21 could activate PI3K/Akt pathway by suppressing PTEN expression. In summary, our findings suggested that miR-21 played an active role in osteosarcoma and it could predict the occurrence and development of osteosarcoma.

  19. 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.

  20. Invariant NKT cells increase drug-induced osteosarcoma cell death

    PubMed Central

    Fallarini, S; Paoletti, T; Orsi Battaglini, N; Lombardi, G

    2012-01-01

    BACKGROUND AND PURPOSE In osteosarcoma (OS) patients, only a limited number of drugs are active and the regimens currently in use include a combination of at least two of these drugs: doxorubicin, cisplatin, methotrexate and ifosfamide. Today, 30–40% of patients still die of OS highlighting the urgent need for new treatments. Invariant NKT (iNKT) cells are a lymphocyte lineage with features of both T and NK cells, playing important roles in tumour suppression. Our aim was to test whether the cytoxicity induced by cisplatin, doxorubicin and methotrexate against OS cells can be enhanced by iNKT cell treatment. EXPERIMENTAL APPROACH iNKT cells were purified from human peripheral blood mononuclear cells by cell sorting (Vα24Vβ11+ cells) and used as effector cells against OS cells (U2-OS, HOS, MG-63). Cell death (calcein-AM method), perforin/granzyme B and Fas/FasL expressions were determined by flow cytometry. CD1d expression was analysed at both the gene and protein level. KEY RESULTS iNKT cells were cytotoxic against OS cells through a CD1d-dependent mechanism. This activity was specific for tumour cells, because human CD1d+ mesenchymal stem cells and CD1d- osteoblasts were not affected. iNKT cell treatment enhanced drug-induced OS cell death in a concentration-dependent manner and this effect was reduced in CD1d-silenced OS cells. CONCLUSION AND IMPLICATIONS iNKT cells kill malignant, but not non-malignant, cells. iNKT cell treatment enhances the cytotoxicity of anti-neoplastic drugs against OS cells in a CD1d-dependent manner. The present data encourage further studies on the use of iNKT cells in OS therapy. PMID:22817659

  1. miR-22 promotes apoptosis of osteosarcoma cells via inducing cell cycle arrest.

    PubMed

    Gai, Pengzhou; Sun, Hongliang; Wang, Guangda; Xu, Qiang; Qi, Xiaojun; Zhang, Zuofu; Jiang, Lei

    2017-04-01

    To study the effects of miR-22 on the proliferation and the apoptosis of osteosarcoma MG-63 cell line and to explore the potential molecular mechanism that miR-22 regulates this biological process. Quantitive real-time polymerase chain reaction (RT-qPCR) was performed to explore the miRNA level of miR-22. The MG-63 cell line was infected with miR-22 mimics for establishment of miR-22 overexpression. Non-infected cells were in blank group and cells infected with empty vector were served as negative control (NC group). MTT assay was conducted to measure cell viability. The cell cycle and apoptosis were explored using flow cytometry and the apoptosis-related markers were detected by western blotting. RT-qPCR results revealed that the miR-22 miRNA level in the MG-63 cells was significantly lower than that in osteoblasts (P<0.05). MTT assay showed that the MG-63 cells infected with miR-22 mimics exhibited markedly decreased proliferation ability compared with blank and empty vector (NC) groups. Next, we found that overexpression of miR-22 remarkably increased the apoptosis of the MG-63 cells, evidenced from the flow cytometry results and elevated Bax and reduced Bcl-2. Furthermore, results revealed that percentage of the cells at G0/G1 phase in miR-22 mimic group (66.75±3.67%) was significantly higher than blank (52.9±2.58%) and NC (50.5±2.45%) groups. miR-22 attenuated the proliferation and induced the apoptosis of the MG-63 cells via promoting G0/G1 cell cycle arrest. Thus, miR-22 may have the potential to be a novel therapeutic in treatment of osteosarcoma.

  2. Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

    PubMed Central

    2012-01-01

    Background The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion We

  3. Expression of transforming growth factor-beta (TGF-beta) receptors, TGF-beta 1 and TGF-beta 2 production and autocrine growth control in osteosarcoma cells.

    PubMed

    Kloen, P; Jennings, C L; Gebhardt, M C; Springfield, D S; Mankin, H J

    1994-08-01

    Transforming growth factor-beta (TGF-beta) is a polypeptide with multiple physiological functions. Isoforms of this growth factor have important roles in control of the cell cycle, in regulation of cell-cell interactions and in growth and development. Malignant transformation has been shown to be associated with increased expression of TGF-beta. Since bone is the largest storage site and producer of TGF-beta, we speculated on the existence of an autocrine mechanism in osteosarcoma, a malignant bone tumor. Expression of TGF-beta cell surface receptors, effects on growth of TGF-beta and TGF-beta antibodies and production of 2 TGF-beta isoforms were studied in a panel of 7 osteosarcoma cell lines. In contrast to most previous reports on the effects of TGF-beta on osteosarcoma cell growth, we found a mitogenic effect of TGF-beta 1 in 4 of 7 osteosarcoma cell lines. Receptor profiles for TGF-beta were aberrant in 5 of the 7 cell lines tested, and production of TGF-beta 1 and TGF-beta 2 varied among cell lines. Addition of anti-TGF-beta antagonized the effects of endogenous TGF-beta. Our results suggest a potential role of TGF-beta in autocrine growth control of osteosarcoma cells.

  4. [Effects of total flavones of Chrysanthemum indicum on proliferation and apoptosis of human osteosarcoma Saos-2 cells].

    PubMed

    Wei, Qiang-qiang; Yin, Chang-chang; Zhou, Hu-yan; He, Ding-wen; Liang, Guang-sheng; Liu, Yu-liang; Yin, Ming

    2013-11-01

    To study the effects of total flavones of Chrysanthemum indicum on proliferation and apoptosis of human osteosarcoma Saos-2 cells and its mechanism. The effect of the total flavones of Chrysanthemum indicum on the proliferation of human osteosarcoma Saos-2 cells was detected by CCK assay, and the morphological changes of cells treated with total flavones of Chrysanthemum indicum were observed using contrast microscope. Flow cytomerty was performed to analyze the apoptotic rate of the cells, and the gene expression levels of Caspase-3, BCL-2, BAX were detected by RT-PCR. The total flavones of Chrysanthemum indicum suppressed the proliferation of osteosarcoma cells in a dose-and time-dependent manner. Under a microscope observation of cell morphology, the volume became smaller ,the number of internal particles was increased. Cell apoptosis rate was positively related to the drug concentration. After treated for 48 hours, Caspase-3 and BAX expression were up-regulated, BCL-2 and BCL-2/BAX were decreased. The total flavones of Chrysanthemum indicum can inhibit the proliferation of osteosarcoma cell line Saos-2 by inducing cell apoptosis,the mechanism of which might be related with reducing BCL-2/BAX and activating Caspase-3.

  5. Voacamine Modulates the Sensitivity to Doxorubicin of Resistant Osteosarcoma and Melanoma Cells and Does Not Induce Toxicity in Normal Fibroblasts

    PubMed Central

    2015-01-01

    In previous studies it has been demonstrated that the plant alkaloid voacamine (1), used at noncytotoxic concentrations, enhanced the cytotoxicity of doxorubicin and exerted a chemosensitizing effect on cultured multidrug-resistant (MDR) U-2 OS-DX osteosarcoma cells. The in vitro investigations reported herein gave the following results: (i) the chemosensitizing effect of 1, in terms of drug accumulation and cell survival, was confirmed using SAOS-2-DX cells, another MDR osteosarcoma cell line; (ii) compound 1 enhanced the cytotoxic effect of doxorubicin also on the melanoma cell line Me30966, intrinsically drug resistant and P-glycoprotein-negative; (iii) at the concentrations used to sensitize tumor cells, 1 was not cytotoxic to normal cells (human fibroblasts). These findings suggest possible applications of voacamine (1) in integrative oncologic therapies against resistant tumors. PMID:24720452

  6. Targeting programmed cell death ligand 1 in osteosarcoma: an auto-commentary on therapeutic potential.

    PubMed

    Shen, Jacson K; Cote, Gregory M; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    Programmed cell death ligand 1 (PDL1) expression was recently shown to correlate with tumor-infiltrating lymphocytes (TILs) in a subset of osteosarcoma patients. Among clinical factors evaluated across human osteosarcoma samples, a pulmonary origin of metastases correlated with high PDL1 expression and prominent TILs. Considering that multiple agents targeting PD-1/PDL1 are under development, targeting this immune checkpoint may be a novel immunotherapeutic route for osteosarcoma in future clinical trials.

  7. MicroRNA-33b Inhibits the Proliferation and Migration of Osteosarcoma Cells via Targeting Hypoxia-Inducible Factor-1α.

    PubMed

    Zhou, Yong; Yang, Chuandong; Wang, Kunpeng; Liu, Xuefeng; Liu, Quan

    2017-03-13

    Recently, microRNA (miR)-33b has been demonstrated to act as a tumor suppressor in osteosarcoma. However, the regulatory mechanism of miR-33b in osteosarcoma cell proliferation and migration remains largely unknown. In this study, real-time PCR showed that miR-33b was significantly downregulated in osteosarcoma tissues compared to their matched adjacent nontumor tissues. Its expression was also decreased in several common osteosarcoma cell lines, including Saos-2, MG63, U2OS, and SW1353, when compared to normal osteoblast cell line hFOB. Overexpression of miR-33b suppressed U2OS cell proliferation and migration. HIF-1α was further identified as a target of miR-33b, and its protein levels were reduced after overexpression of miR-33b in U2OS cells. Moreover, overexpression of HIF-1α significantly reversed the suppressive effect of miR-33b on U2OS cell proliferation and migration. In addition, HIF-1α was found to be significantly upregulated in osteosarcoma tissues compared to adjacent nontumor tissues, and their expression levels were inversely correlated to the miR-33b levels in osteosarcoma tissues. According to these findings, miR-33b plays a suppressive role in the regulation of osteosarcoma cell proliferation and migration via directly targeting HIF-1α. Therefore, we suggest that the miR-33b/HIF-1α axis may become a promising therapeutic target for osteosarcoma.

  8. Targeting Glycoprotein NMB With Antibody-Drug Conjugate, Glembatumumab Vedotin, for the Treatment of Osteosarcoma.

    PubMed

    Roth, Michael; Barris, David M; Piperdi, Sajida; Kuo, Vicky; Everts, Stephanie; Geller, David; Houghton, Peter; Kolb, E Anders; Hawthorne, Thomas; Gill, Jonathan; Gorlick, Richard

    2016-01-01

    Cure rates for children and young adults with osteosarcoma have remained stagnant over the past three decades. Targeting glycoprotein non-metastatic b (GPNMB) with the antibody-drug conjugate glembatumumab vedotin has improved outcomes for patients with melanoma and breast cancer. The potential utility of targeting GPNMB in osteosarcoma was explored. GPNMB protein expression was evaluated by immunohistochemistry in human osteosarcoma tumor samples and by enzyme-linked immunosorbent assay (ELISA) in osteosarcoma cell lines. mRNA expression was measured by quantitative PCR in primary osteosarcoma samples and cell lines. Surface GPNMB expression was evaluated by flow cytometry and correlated with in vitro and in vivo cytotoxicity of glembatumumab vedotin. Sixty seven human osteosarcoma samples were evaluated by immunohistochemistry, including 12 samples from initial biopsy, 38 samples from definitive surgery, and 17 from the time of disease recurrence. GPNMB was expressed in 92.5% (62/67) of osteosarcoma samples. All primary osteosarcoma samples expressed high levels of GPNMB mRNA. Glembatumumab induced cytotoxic effects in 74% (14/19) of osteosarcoma cell lines, and GPNMB protein levels correlated with glembatumumab in vitro cytotoxicity (r = -0.46, P = 0.04). All osteosarcoma cell lines demonstrated surface GPNMB expression. GPNMB is expressed in osteosarcoma and targeting GPNMB with the antibody-drug conjugate glembatumumab vedotin demonstrates osteosarcoma cytotoxic activity. Clinical trials are indicated to assess the efficacy of targeting GPNMB in patients with osteosarcoma. © 2015 Wiley Periodicals, Inc.

  9. Genetically Modified T-Cell Therapy for Osteosarcoma

    PubMed Central

    DeRenzo, Christopher

    2015-01-01

    T-cell immunotherapy may offer an approach to improve outcomes for patients with osteosarcoma, who fail current therapies. In addition, it has the potential to reduce treatment-related complications for all patients. Generating tumor-specific T cells with conventional antigen presenting cells ex vivo is time consuming and often results in T-cell products with a low frequency of tumor-specific T cells. In addition, the generated T cells remain sensitive to the immunosuppressive tumor microenvironment. Genetic modification of T cells is one strategy to overcome these limitations. For example, T cells can be genetically modified to render them antigen specific, resistant to inhibitory factors, or increase their ability to home to tumor sites. Most genetic modification strategies have only been evaluated in preclinical models, however early phase clinical trials are in progress. In this chapter we review the current status of gene-modified T-cell therapy with special focus on osteosarcoma, highlighting potential antigenic targets, preclinical and clinical studies, and strategies to improve current T-cell therapy approaches. PMID:24924183

  10. SPAG9 is overexpressed in osteosarcoma, and regulates cell proliferation and invasion through regulation of JunD

    PubMed Central

    Xiao, Chi; Fu, Lin; Yan, Chongnan; Shou, Fenyong; Liu, Qi; Li, Lei; Cui, Shaoqian; Duan, Jingzhu; Jin, Guoxin; Chen, Jianhua; Bian, Yuanming; Wang, Xu; Wang, Huan

    2016-01-01

    Sperm-associated antigen 9 (SPAG9) is a recently characterized oncoprotein that is considered to be involved in several forms of malignant tumor. However, its biological function and expression pattern in human osteosarcoma have not yet been elucidated. In the present study, SPAG9 expression was analyzed in 58 cases of human osteosarcoma by immunohistochemistry. The results demonstrated that SPAG9 was overexpressed in 63.8% (37/58) of osteosarcoma tissues, while normal bone tissues exhibited negative SPAG9 expression. SPAG9 small interfering RNA was employed in the U2OS cell line, which has high endogenous expression, and SPAG9 transfection was performed in the MG63 cell line, which has low endogenous expression. MTT and Matrigel invasion assays demonstrated that SPAG-9-knockdown significantly reduced U2OS cell invasion and proliferation, while SPAG9 transfection enhanced MG63 cell proliferation and invasion. Furthermore, it was observed that SPAG9 positively regulated cyclin D1, phosphorylated-c-Jun NH2-terminal kinase (JNK) and JunD expression. Treatment with the JNK inhibitor, SP600125, abolished the upregulatory effect of SPAG9 on JunD. Taken together, the present study identified SPAG9 as a critical oncoprotein involved in osteosarcoma proliferation and invasion, possibly functioning through JNK-JunD signaling. PMID:27698841

  11. miR-542-3p overexpression is associated with enhanced osteosarcoma cell proliferation and migration ability by targeting Van Gogh-like 2

    PubMed Central

    LI, HUAZHUANG; LIU, HONGTAO; PEI, JINGFANG; WANG, HAIYAN; LV, HONGLIN

    2015-01-01

    Osteosarcoma is the most common histological form of primary bone cancer, which arises from osteoid tissue. It occurs predominantly in infants and adolescents, with an incidence of 4–5 cases/100,000,000. The 5-year survival rate of patients with osteosarcoma has significantly improved over time; however, there remains a significant proportion of patients that respond poorly to chemotherapy. An improved understanding of the pathology of osteosarcoma is required to provide more effective treatment strategies, identify biomarkers and develop novel chemotherapeutic agents. Disturbance in microRNA (miRNA) expression has been identified in osteosarcoma tissues and cell lines; however, the roles of miRNA during osteosarcoma pathogenesis remain to be elucidated. In the present study, the expression levels of eight selected miRNAs were investigated in osteosarcoma tissues and the results revealed that the expression levels of miR-542-3p and miR-542-5p were significantly upregulated and the expression of miR-199-3p was significantly downregulated. Using a dual luciferase assay and western blot analysis, the present study confirmed that Van Gogh-like 2, which is a non-canonical Wnt pathway suppressor, was a target gene of miR-542-3p. Subsequently, the biological function of miR-542-3p in U2OS cells was examined, which revealed that overexpression of miR-542-3p can enhance the cell proliferation and migration ability of U2OS cells. This indicated that miR-542-3p may act as an oncogene in osteosarcoma pathogenesis. The findings of the present study may provide assistance in understanding the development of osteosarcoma and aid in the development of strategies for the diagnosis and treatment of osteosarcoma. PMID:25352048

  12. Bortezomib sensitizes human osteosarcoma cells to adriamycin-induced apoptosis through ROS-dependent activation of p-eIF2α/ATF4/CHOP axis.

    PubMed

    Xian, Miao; Cao, Handi; Cao, Ji; Shao, Xuejing; Zhu, Difeng; Zhang, Ning; Huang, Ping; Li, Weixu; Yang, Bo; Ying, Meidan; He, Qiaojun

    2017-09-01

    Osteosarcoma is the most common bone cancer, and chemotherapy is currently indispensable for its treatment. Adriamycin has been claimed to be the most effective agent for osteosarcoma, however, the outcome of adriamycin chemotherapy remains unsatisfactory. Here, we reported a potent combination therapy that bortezomib, a proteasome inhibitor, enhances adriamycin-induced apoptosis to eliminate osteosarcoma cells and we revealed that the activation of p-eIF2α/ATF4/CHOP axis is the underlying associated mechanisms. First, we observed that bortezomib enhances adriamycin-mediated inhibition of cell proliferation and enhances the apoptosis in osteosarcoma cell lines. Moreover, this drug combination produced more potent tumor-growth inhibitory effects in human osteosarcoma cell line KHOS/NP xenografts. Our study showed that reactive oxygen species (ROS) plays an important role in apoptosis induced by adriamycin plus bortezomib, whereas ROS scavenger NAC could almost completely block the apoptosis induced by the combination treatment. Meanwhile, p-eIF2α is remarkably elevated in the combination group. As a result, ATF4 exhibits strong activation which consequently induces the activation of CHOP and leads to the cell death. Finally, 13 primary osteosarcoma cells demonstrated potent response to the combination treatment. In a human osteosarcoma patient-derived xenograft (PDX) model, our finding suggests that when combined with bortezomib, a relatively low dose of adriamycin produced more potent tumor-growth inhibitory effects without increased toxicity. Thus, our findings not only provide a promising combination strategy to overcome osteosarcoma but also shed new light on the strategy of combining increased ROS and inhibited proteasome to open up new opportunities for the clinical development of chemotherapy regimens. © 2017 UICC.

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

    SciTech Connect

    Cappadone, C.; Stefanelli, C.; Malucelli, E.; Zini, M.; Onofrillo, C.; Locatelli, A.; Rambaldi, M.; Sargenti, A.; Merolle, L.; Farruggia, G.; Graziadio, A.; Montanaro, L.; Iotti, S.

    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 the 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.

  14. HMGB1 Osteo-Modulatory Action on Osteosarcoma SaOS-2 Cell Line: An Integrated Study From Biochemical and -Omics Approaches.

    PubMed

    Martinotti, Simona; Patrone, Mauro; Manfredi, Marcello; Gosetti, Fabio; Pedrazzi, Marco; Marengo, Emilio; Ranzato, Elia

    2016-11-01

    High mobility group box protein-1 (HMGB1) is released from cells under various pathological conditions and it plays a pivotal role as an alarmin signaling tissue damage. Little is known about the impact of HMGB1 in bone repair and remodeling. To this aim, we focused on HMGB1-induced effects on the in vitro osteoblast model SaOS-2. Cell proliferation was stimulated with a maximum at concentration of 2.5 nM, and such a dose also stimulated cell migration and scratch wound healing. We then characterized the modulatory effect of HMGB1 on bone biology, by using osteogenesis/mineralization assays, a PCR array, and the analysis of a series of osteogenic markers. We performed also a proteomic screening using SWATH-MS on SaOS-2 cell exposed to HMGB1 and we provide evidence for proteins modulated in HMGB1 exposed cells. Taken together, our data demonstrate that SaOS-2 cell proliferation, migration, and osteogenic differentiation were increased by HMGB1. We, therefore, propose that HMGB1 could be a potent bone-remodeling signal but the physiological meaning of this property remains to be more ascertained. J. Cell. Biochem. 117: 2559-2569, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Polymeric nanoparticle-based delivery of microRNA-199a-3p inhibits proliferation and growth of osteosarcoma cells

    PubMed Central

    Zhang, Linlin; lyer, Arun K; Yang, Xiaoqian; Kobayashi, Eisuke; Guo, Yuqi; Mankin, Henry; Hornicek, Francis J; Amiji, Mansoor M; Duan, Zhenfeng

    2015-01-01

    Our prior screening of microRNAs (miRs) identified that miR-199a-3p expression is reduced in osteosarcoma cells, one of the most common types of bone tumor. miR-199a-3p exhibited functions of tumor cell growth inhibition, suggesting the potential application of miR-199a-3p as an anticancer agent. In the study reported here, we designed and developed a lipid-modified dextran-based polymeric nanoparticle platform for encapsulation of miRs, and determined the efficiency and efficacy of delivering miR-199a-3p into osteosarcoma cells. In addition, another potent miR, let-7a, which also displayed tumor suppressive ability, was selected as a candidate miR for evaluation. Fluorescence microscopy studies and real-time polymerase chain reaction results showed that dextran nanoparticles could deliver both miR-199a-3p and let-7a into osteosarcoma cell lines (KHOS and U-2OS) successfully. Western blotting analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that dextran nanoparticles loaded with miRs could efficiently downregulate the expression of target proteins and effectively inhibit the growth and proliferation of osteosarcoma cells. These results demonstrate that a lipid-modified dextran-based polymeric nanoparticle platform may be an effective nonviral carrier for potential miR-based anticancer therapeutics. PMID:25931818

  16. Overexpression of miR-664 is associated with enhanced osteosarcoma cell migration and invasion ability via targeting SOX7.

    PubMed

    Bao, Yongzheng; Chen, Bin; Wu, Qiang; Hu, Konghe; Xi, Xinhua; Zhu, Wengang; Zhong, Xueren; Chen, Jianting

    2017-02-01

    Osteosarcoma (OS) is one of the most common types of primary sarcoma of bone in children and young adults, and the long-term prognosis for OS patients still remains dismal due to the lack of effective early diagnostic biomarkers. Identifying sensitive and specific biomarkers in carcinogenesis may improve diagnostic and therapeutic strategies for this malignancy. The expression of miR-664 in osteosarcoma cell lines and osteosarcoma tissues was examined using real-time PCR. The effects of miR-664 on osteosarcoma cell migration and invasion were evaluated by cell invasion assays, migration assays, and three-dimension spheroid invasion assay. The effect of miR-664 on SOX7 was determined by luciferase assays and Western blot assay. The clinical association between miR-664 and SOX7 was analyzed by real-time PCR and Western blot assay. Expression of miR-664 was found to be upregulated in OS cell lines and tissues. Overexpression of miR-664 was associated with increased migration and invasive abilities of OS cells in vitro, whereas downregulation of miR-664 appeared to inhibit their migration and invasive potential. Furthermore, using biological approaches, we showed that miR-664 directly targeted and suppressed expression of the tumor suppressor SOX7. Additionally, the expression of miR-664 was negatively correlated with SOX7 expression in OS clinical tissues. Our findings suggest that miR-664 functions as an oncogene miRNA and has an important role in promoting human OS cell invasion and migration by suppressing SOX7 expression. Consequently, miR-664 may have potential as a novel diagnostic and therapeutic target of osteosarcoma.

  17. Berberine induces apoptosis and DNA damage in MG‑63 human osteosarcoma cells.

    PubMed

    Zhu, Yu; Ma, Nan; Li, Hui-Xiang; Tian, Lin; Ba, Yu-Feng; Hao, Bin

    2014-10-01

    Berberine, an isoquinoline alkaloid extracted from the dry root of Coptidis Rhizoma, has been found to exhibit marked anticancer effects on a panel of established cancer cells. Among the human osteosarcoma lines treated, MG‑63 cells were found to be the most sensitive. The present study investigated the potential genotoxic effect of berberine on MG‑63 human osteosarcoma cells. The effect of berberine on cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5‑diphenyltetrazolium bromide assay and cell apoptosis was analyzed by flow cytometry and a DNA ladder assay. γH2AX focus formation was used to detect DNA damage in MG-63 cells. Berberine induced a significant increase in apoptosis in MG-63 cells in a concentration- and time-dependent manner, as determined by DNA fragmentation analysis and flow cytometry. Furthermore, berberine induced significant concentration- and time-dependent increases in DNA damage compared with that in the negative control. In conclusion, these observations indicated that berberine induced apoptosis and DNA damage in MG‑63 cells.

  18. Berberine induces apoptosis and DNA damage in MG-63 human osteosarcoma cells

    PubMed Central

    ZHU, YU; MA, NAN; LI, HUI-XIANG; TIAN, LIN; BA, YU-FENG; HAO, BIN

    2014-01-01

    Berberine, an isoquinoline alkaloid extracted from the dry root of Coptidis Rhizoma, has been found to exhibit marked anticancer effects on a panel of established cancer cells. Among the human osteosarcoma lines treated, MG-63 cells were found to be the most sensitive. The present study investigated the potential genotoxic effect of berberine on MG-63 human osteosarcoma cells. The effect of berberine on cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell apoptosis was analyzed by flow cytometry and a DNA ladder assay. γH2AX focus formation was used to detect DNA damage in MG-63 cells. Berberine induced a significant increase in apoptosis in MG-63 cells in a concentration- and time-dependent manner, as determined by DNA fragmentation analysis and flow cytometry. Furthermore, berberine induced significant concentration- and time-dependent increases in DNA damage compared with that in the negative control. In conclusion, these observations indicated that berberine induced apoptosis and DNA damage in MG-63 cells. PMID:25050485

  19. Inhibition of cell growth and telomerase activity in osteosarcoma cells by DN-hTERT.

    PubMed

    Xu, Tao; Rao, Yaojian; Zhu, Wentao; Guo, Fengjin

    2006-01-01

    In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45-0.11 in MG63/DN cells, while 3.40+/-0.12 in the cells transfected with blank vector (MG63/I), (P<0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P<0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

  20. 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.

  1. Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

    SciTech Connect

    Hodge, B.O.; Kream, B.E.

    1988-05-01

    We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of (/sup 3/H)proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis.

  2. Enrichment of human osteosarcoma stem cells based on hTERT transcriptional activity

    PubMed Central

    Yu, Ling; Liu, Shiqing; Zhang, Chun; Zhang, Bo; Simões, Bruno M.; Eyre, Rachel; Liang, Yi; Yan, Huichao; Wu, Zheng; Guo, Weichun; Clarke, Robert B.

    2013-01-01

    Telomerase is crucial for the maintenance of stem/progenitor cells in adult tissues and is detected in most malignant cancers, including osteosarcoma. However, the relationship between telomerase expression and cancer stem cells remains unknown. We observed that sphere-derived osteosarcoma cells had higher telomerase activity, indicating that telomerase activity might be enriched in osteosarcoma stem cells. We sorted subpopulations with high or low telomerase activity (TEL) using hTERT transcriptional promoter-induced green fluorescent protein (GFP). The TELpos cells showed an increased sphere and tumor propagating capacity compared to TELneg cells, and enhanced stem cell-like properties such as invasiveness, metastatic activity and resistance to chemotherapeutic agents both in vitro and in vivo. Furthermore, the telomerase inhibitor MST312 prevented tumorigenic potential both in vitro and in vivo, preferentially targeting the TELpos cells. These data support telomerase inhibition as a potential targeted therapy for osteosarcoma stem-like cells. PMID:24334332

  3. 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.

  4. Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells

    PubMed Central

    2012-01-01

    Background Osteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells. Methods 2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α). Results 2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls. Conclusions 2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment

  5. A phase II trial of second-line pemetrexed in adults with advanced/metastatic osteosarcoma.

    PubMed

    Duffaud, Florence; Egerer, Gerlinde; Ferrari, Stefano; Rassam, Hisham; Boecker, Ulrike; Bui-Nguyen, B

    2012-03-01

    Osteosarcoma is the most common primary malignant tumour in young adults. An effective treatment strategy for relapsed patients is still not defined. Pemetrexed is a multitargeted antifolate with a mode of action similar to, and a range of action broader than that of methotrexate. The primary objective of this phase II study was to determine tumour response rate in patients with high-grade, advanced/metastatic osteosarcoma. Secondary end-points included progression-free survival (PFS), overall survival (OS) and safety. Pemetrexed 500mg/m(2) was administered on day 1 of 21-day cycles with folic acid and vitamin B(12) supplementation. At least 5 tumour responses in a targeted population of 32 were required to consider further investigation. Thirty-two patients (median age, 43.3 years; range, 18.6-76.0) with 1 prior chemotherapy regimen for high-grade advanced/metastatic osteosarcoma were enrolled. Thirty (93.8%) patients had an ECOG performance status ≤ 1 and 29 (90.6%) had metastases in the lung. One patient had partial response (3.1%) and 5 (15.6%) had stable disease. Median PFS and OS were 1.4 months (95% CI: 1.4-1.7) and 5.5 months (95% CI: 2.3-10.5), respectively. The most common drug-related grade 3/4 toxicities were leukopaenia, asthaenia and elevated alanine aminotransferase in 3 (9.4%) patients each. One patient died due to multi-organ failure considered possibly related to the study drug. Pemetrexed 500mg/m(2) administered on day 1 of 21-day cycles as second-line treatment to patients with advanced/metastatic high-grade osteosarcoma was generally well tolerated but did not meet minimal response expectations for further investigation in this patient population. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells

    PubMed Central

    Bonuccelli, Gloria; Avnet, Sofia; Grisendi, Giulia; Salerno, Manuela; Granchi, Donatella; Dominici, Massimo; Kusuzaki, Katsuyuki; Baldini, Nicola

    2014-01-01

    The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma. PMID:25277190

  7. Induction of G2/M phase cell cycle arrest and apoptosis by ginsenoside Rf in human osteosarcoma MG‑63 cells through the mitochondrial pathway.

    PubMed

    Shangguan, Wen-Ji; Li, He; Zhang, Yue-Hui

    2014-01-01

    Ginsenosides, extracted from the traditional Chinese herb ginseng, are a series of novel natural anticancer products known for their favorable safety and efficacy profiles. The present study aimed to investigate the cytotoxicity of ginsenoside Rf to human osteosarcoma cells and to explore the anticancer molecular mechanisms of ginsenoside Rf. Five human osteosarcoma cell lines (MG-63, OS732, U-2OS, HOS and SAOS-2) were employed to investigate the cytotoxicity of ginsenoside Rf by MTT and colony forming assays. After treatment with ginsenoside Rf, MG-63 cells which were the most sensitive to ginsenoside Rf, were subjected to flow cytometry to detect cell cycle distribution and apoptosis, and nuclear morphological changes were visualized by Hoechst 33258 staining. Caspase-3, -8 and -9 activities were also evaluated. The expression of cell cycle markers including cyclin B1 and Cdk1 was detected by RT-PCR and western blotting. The expression of apoptotic genes Bcl-2 and Bax and the release of cytochrome c were also examined by western blotting. Change in the mitochondrial membrane potential was observed by JC-1 staining in situ. Our results demonstrated that the cytotoxicity of ginsenoside Rf to these human osteosarcoma cell lines was dose-dependent, and the MG-63 cells were the most sensitive to exposure to ginsenoside Rf. Additionally, ginsenoside Rf induced G2/M phase cell cycle arrest and apoptosis in MG-63 cells. Furthermore, we observed upregulation of Bax and downregulation of Bcl-2, Cdk1 and cyclin B1, the activation of caspase-3 and -9 and the release of cytochrome c in MG-63 cells following treatment with ginsenoside Rf. Our findings demonstrated that ginsenoside Rf induces G2/M phase cell cycle arrest and apoptosis in human osteosarcoma MG-63 cells through the mitochondrial pathway, suggesting that ginsenoside Rf, as an effective natural product, may have a therapeutic effect on human osteosarcoma.

  8. 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

  9. Fibular giant cell-rich osteosarcoma virtually indistinguishable radiographically and histopathologically from giant cell tumor-analysis of subtle differentiating features.

    PubMed

    Chow, Louis T C

    2015-06-01

    Giant cell-rich osteosarcoma by its abundance of osteoclastic giant cells and paucity of tumor osteoid, leads to its easy confusion with giant cell tumor during biopsy interpretation. In this report, we describe a unique case of upper fibular metaphyseal giant cell-rich osteosarcoma in a 12-year-old boy; the radiographic and histopathologic features of the biopsy and initial resected tumor are virtually indistinguishable from conventional giant cell tumor. The tumor rapidly recurred 7 months after resection with metastasis to the groin lymph nodes, was resistant to first-line chemotherapy and pursued an aggressive course, developing disseminated metastasis to the lung, liver, pelvis, scapula and clavicle, and resulted in the death of the patient 21 months after initial presentation. The subtle features alerting one to the possibility of giant cell-rich osteosarcoma are retrospectively evaluated in comparison with cases of metaphyseal conventional giant cell tumors, four from our records and those from literature review. We conclude that the occurrence of a giant cell-rich lesion in the metaphysis of a skeletally immature individual merits careful assessment for the presence of periosteal reaction, permeative infiltrative margins, lacelike osteoid formation, high mitotic activity or Ki67 proliferative index, and extra-tumoral lymphovascular permeation, since the possibility of an aggressive lesion notably giant cell-rich osteosarcoma probably increases with the number of such features. © 2015 APMIS. Published by John Wiley & Sons Ltd.

  10. MicroRNA-184 Modulates Doxorubicin Resistance in Osteosarcoma Cells by Targeting BCL2L1

    PubMed Central

    Lin, Bo-chuan; Huang, Dong; Yu, Chao-qun; Mou, Yong; Liu, Yuan-hang; Zhang, Da-wei; Shi, Feng-jun

    2016-01-01

    Background Early metastasis of osteosarcoma (OS) is highly lethal and responds poorly to drug and radiation therapies. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, the detailed functions of specific miRNAs are not entirely understood. The aim of the present study was to investigate the role of miR-184 as a mediator of drug resistance in human osteosarcoma. Material/Methods qRT-PCR was used to analyze the expression level of miR-184 in OS cell line U-2 OS and MG-63 treated with doxorubicin. MiR-184 agomir or miR-184 antagomir was transferred into cells to regulated miR-184. The target of miR-184 was predicted by TargetScan and confirmed by luciferase reporter assay. Bcl-2-like protein 1 (BCL2L1) expression was detected by Western blot. Cell apoptosis was determined by Annexin V staining and analysis by flow cytometry. Results Doxorubicin induced time-dependent expression of miR-184 in OS cell line U-2 OS and MG-63. Luciferase reporter assay identified BCL2L1 as the direct target gene of miR-184. Furthermore, doxorubicin reduced BCL2L1 expression, which was reversed by miR-184 overexpression and further decreased by miR-184 inhibition in OS cells. In addition, miR-184 agomir reduced doxorubicin-induced cell apoptosis, whereas miR-184 antagomir enhanced apoptosis in OS cells, suggesting that up-regulation of miR-184 contributes to chemoresistance of the OS cell line. Conclusions Our data show that miR-184 was up-regulated in OS patients treated with doxorubicin therapy and leads to poor response to drug therapy by targeting BCL2L1. PMID:27222034

  11. Focal adhesion kinase is involved in the migration of human osteosarcoma cells

    PubMed Central

    FENG, SITAN; SHI, XIN; REN, KE; WU, SUJIA; SUN, XIAOLIANG

    2015-01-01

    The aim of the present study was to analyze the expression of focal adhesion kinase (FAK) in osteosarcoma (OS) cell lines with different migration abilities in order to determine the role of FAK in migration. A number of different 143B subclone cell lines (A1, A2, A3, A4 and A5) were obtained by a limiting dilution method, and the expression of FAK was detected using western blot analysis. The role of FAK in the migration of OS cells was investigated using small interfering RNA (siRNA), and the ratio of the number of lamellipodia was compared by immunofluorescence staining. The A2 and A3 OS 143B subclone cell lines demonstrated a stronger migration ability and exhibited higher FAK expression compared with the A1 cell line (P<0.05). Following transfection with FAK-siRNA, the migration ability of the A3 cells was significantly decreased (P<0.05), and the ratio of the number of lamellipodia formed was reduced from 35 to 11% (P<0.05). In conclusion, the level of FAK expression was higher in the cell lines with a stronger migration ability. FAK affects the migration ability of OS cells by suppressing the formation of lamellipodia. PMID:26137126

  12. Galangin suppresses human osteosarcoma cells: An exploration of its underlying mechanism.

    PubMed

    Yang, Zhifan; Li, Xiucheng; Han, Weiqi; Lu, Xuanyuan; Jin, Songtao; Yang, Wanlei; Li, Jianlei; He, Wei; Qian, Yu

    2017-01-01

    Osteosarcoma is the most common malignant bone tumor that frequently affects adolescents. Osteosarcoma cells tend to proliferate and invade other tissues such as those of the lungs. Currently, neoadjuvant chemotherapy is the primary strategy to prevent tumor progression. However, its adverse effects result in poor long-term outcomes. Previous research has shown that galangin exhibits antitumor properties on several types of cancer cells; however its effect on osteosarcoma cells is yet unknown. The aims of this study were to evaluate the effects of galangin on the proliferation, apoptosis, migration, and invasion of osteosarcoma cells and to explore the underlying mechanisms. We found that the proliferation of MG63 and U20S osteosarcoma cells decreased significantly, while the apoptosis of MG63 cells accelerated significantly after exposure to galangin. In addition, the migration and invasion of MG63 cells were significantly inhibited by galangin. Moreover, phosphoinositide 3-kinase (PI3K) and Aktp-Thr308 expression levels were found to be significantly lower in galangin-treated MG63 cells than in the control cells, and the protein expression levels of their downstream regulators cyclin D1 and matrix metalloproteinase 2/9 were also downregulated in galangin-treated groups, while those of p27Kip1, caspase-3, and caspase-8 were upregulated. These findings suggest that galangin suppresses osteosarcoma cells by inhibiting their proliferation and invasion and accelerating their apoptosis, and the mechanism may be associated with the inhibition of PI3K and its downstream signaling pathway.

  13. Evaluation of quercetin as a potential drug in osteosarcoma treatment.

    PubMed

    Berndt, Kersten; Campanile, Carmen; Muff, Roman; Strehler, Emanuel; Born, Walter; Fuchs, Bruno

    2013-04-01

    Osteosarcoma is the most common malignant bone tumor in children and young adults. Since the introduction of chemotherapy, the 5-year survival rate of patients with non-metastatic osteosarcoma is ~70%. The main problems in osteosarcoma therapy are the occurrence of metastases, severe side-effects and chemoresistance. Antiproliferative and apoptotic effects of quercetin were shown in several types of cancers, including breast cancer and lung carcinoma. The present study investigates the cytotoxic potential of quercetin, a dietary flavonoid, in a highly metastasizing human osteosarcoma cell line, 143B. We found that quercetin induces growth inhibition, G2/M phase arrest, and apoptosis in the 143B osteosarcoma cell line. We also observed impaired adhesion and migratory potential after the addition of quercetin. Since quercetin has already been shown to have low side effects in a clinical phase I trial in advanced cancer patients, this compound may have considerable potential for osteosarcoma treatment.

  14. SPAG9 controls the cell motility, invasion and angiogenesis of human osteosarcoma cells

    PubMed Central

    YANG, XIAORONG; ZHOU, WENLAI; LIU, SHIQING

    2016-01-01

    Sperm-associated antigen 9 (SPAG9) is an oncoprotein involved in the progression of various human malignancies; however, its role in osteosarcoma (OS) remains poorly evaluated. The present study used Matrigel™ cell migration and invasion assays, tube formation assay, Cell Counting kit-8, quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay to investigate the role of SPAG9 in OS cell motility, invasion and angiogenesis. The results of the present study demonstrated that SPAG9 expression was upregulated in OS tissues, as compared with adjacent normal tissues, and knockdown of SPAG9 in an OS cell line inhibited cell motility and invasion via inactivation of metalloproteinase (MMP)-2 and MMP-9. Furthermore, the present study demonstrated that silencing of SPAG9 in OS cells inhibited tube formation, the proliferation of human umbilical vascular endothelial cells, and suppressed vascular endothelial growth factor (VEGF) expression and secretion, contributing to a reduction in angiogenesis. The results of the present study indicated that SPAG9 may be an important regulator in OS and may be involved in metastasis. Therefore SPAG9 may be a promising target for the treatment of metastatic OS. PMID:26893659

  15. CCL5 and CCR5 Interaction Promotes Cell Motility in Human Osteosarcoma

    PubMed Central

    Liu, Shih-Chia; Wang, Po-Chuan; Ou, Wen-Chieh; Chou, Wen-Yi; Shen, Yung-Shuen; Tang, Chih-Hsin

    2012-01-01

    Background Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown. Methodology/Principal Findings Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells. Conclusions/Significance CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells. PMID:22506069

  16. Effects of ZEB1 on regulating osteosarcoma cells via NF-κB/iNOS.

    PubMed

    Xu, X-M; Liu, W; Cao, Z-H; Liu, M-X

    2017-03-01

    Osteosarcoma is one common malignant bone tumors, as it frequently has invasion, metastasis and recurrence, causing unfavorable prognosis of patients. Osteosarcoma has complicated pathogenesis, which has not been elucidated fully. Therefore, the identification of effective molecular target of osteosarcoma onset can help to improve treatment efficacy and prognosis of osteosarcoma. Zinc finger E-box binding homeobox 1 (ZEB1) protein is one member of zinc finger E-box binding protein family, and participates in embryonic genesis and development. A recent study found the participation of ZEB1 in mediating multiple tumor onset and its up-regulation of osteosarcoma. The regulatory mechanism of ZEB1 in osteosarcoma has not been illustrated yet. In vitro cultured osteosarcoma MG-63 cells were transfected with ZEB1 siRNA. Real-time PCR and Western blot were tested for ZEB1 mRNA/protein expression. MTT was used to test MG-63 cell proliferation, whilst cell invasion was used to describe the effect of ZEB1 on MG-63 cells. Caspase-3 activity assay was employed to test MG-63 cell apoptosis. Western blot was employed to detect nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) protein expression. After transfecting with ZEB1 siRNA, MG-63 cell proliferation or invasion was inhibited accompanied with lower ZEB1 mRNA/protein expression. Caspase3 activity was also increased after transfection (p < 0.05), along with down-regulation of NF-kB and iNOS proteins in MG-63 cells (p < 0.05). Inhibition of ZEB1 can facilitate osteosarcoma cell apoptosis and inhibit cell proliferation or invasion via down-regulating NF-kB/iNOS signal pathway.

  17. Imaging the inhibition by anti-β1 integrin antibody of lung seeding of single osteosarcoma cells in live mice.

    PubMed

    Kimura, Hiroaki; Tome, Yasunori; Momiyama, Masashi; Hayashi, Katsuhiro; Tsuchiya, Hiroyuki; Bouvet, Michael; Hoffman, Robert M

    2012-11-01

    Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line, expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, was established. Such double-labeled cells enable imaging of apoptosis and mitosis and other nuclear-cytoplasmic dynamics. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti-β1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung (experimental metastasis) while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically growing 143B-RFP. Copyright © 2012 UICC.

  18. Identification of CBX3 and ABCA5 as putative biomarkers for tumor stem cells in osteosarcoma.

    PubMed

    Saini, Vaibhav; Hose, Curtis D; Monks, Anne; Nagashima, Kunio; Han, Bingnan; Newton, Dianne L; Millione, Angelena; Shah, Jalpa; Hollingshead, Melinda G; Hite, Karen M; Burkett, Mark W; Delosh, Rene M; Silvers, Thomas E; Scudiero, Dominic A; Shoemaker, Robert H

    2012-01-01

    Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.

  19. Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma

    PubMed Central

    Saini, Vaibhav; Hose, Curtis D.; Monks, Anne; Nagashima, Kunio; Han, Bingnan; Newton, Dianne L.; Millione, Angelena; Shah, Jalpa; Hollingshead, Melinda G.; Hite, Karen M.; Burkett, Mark W.; Delosh, Rene M.; Silvers, Thomas E.; Scudiero, Dominic A.; Shoemaker, Robert H.

    2012-01-01

    Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma. PMID:22870217

  20. Mesenchymal stem cells promote osteosarcoma cell survival and drug resistance through activation of STAT3

    PubMed Central

    Liu, Shen; Wang, Lei; Fan, Qiming; Hao, Yongqiang; Fan, Cunyi; Tang, Ting-Ting

    2016-01-01

    Increasing evidence suggests that the tumor microenvironment plays a key role in the development of drug resistant tumor cells. In this study, we tried to determine whether the mesenchymal stem cells (MSCs) in the tumor microenvironment contribute to the increased chemoresistance of osteosarcoma. We found that exposure of Saos-2 and U2-OS cells to MSCs conditioned medium (CM) increased the viable cells in the presence of therapeutic concentrations of doxorubicin or cisplatin. Meanwhile, the MSC CM-associated pro-proliferative effects were accompanied by reduced caspase 3/7 activity and Annexin V binding. We confirmed that STAT3 activation by IL-6 regulates MSCs-induced chemoresistance. Blockade of this signal re-sensitized drug-resistant Saos-2 cells to drug treatment. Using a osteosarcoma mouse model with co-injection of MSCs with Saos-2cells, we found that inhibition of STAT3 prolonged the survival time of tumor bearing mice by suppressing tumor growth and increasing the sensitivity of tumor cells to doxorubicin. Finally, we demonstrated that increased expression of p-STAT3, multidrug resistance protein (MRP) and P-glycoprotein (MDR-1) was associated with high chemotherapy resistance in clinical osteosarcoma samples. Collectively, our findings suggest that MSCs within the tumor microenvironment may represent a new target to enhance chemotherapeutic efficacy in osteosarcoma patients. PMID:27340780

  1. Overexpression of BMI-1 Promotes Cell Growth and Resistance to Cisplatin Treatment in Osteosarcoma

    PubMed Central

    Chen, Dafu; Hao, Dongsheng; Duan, Yuanhui; Qiu, Guixing; Wang, Yipeng

    2011-01-01

    Background BMI-1 is a member of the polycomb group of genes (PcGs), and it has been implicated in the development and progression of several malignancies, but its role in osteosarcoma remains to be elucidated. Methodology/Principal Findings In the present study, we found that BMI-1 was overexpressed in different types of osteosarcomas. Downregulation of BMI-1 by lentivirus mediated RNA interference (RNAi) significantly impaired cell viability and colony formation in vitro and tumorigenesis in vivo of osteosarcoma cells. BMI-1 knockdown sensitized cells to cisplatin-induced apoptosis through inhibition of PI3K/AKT pathway. Moreover, BMI-1-depletion-induced phenotype could be rescued by forced expression of BMI-1 wobble mutant which is resistant to inhibition by the small interfering RNA (siRNA). Conclusions/Significance These findings suggest a crucial role for BMI-1 in osteosarcoma pathogenesis. PMID:21311599

  2. Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling.

    PubMed

    Yu, Ling; Fan, Zhengfu; Fang, Shuo; Yang, Jian; Gao, Tian; Simões, Bruno M; Eyre, Rachel; Guo, Weichun; Clarke, Robert B

    2016-05-31

    Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance.

  3. Pharmacogenomics of second-line drugs used for treatment of unresponsive or relapsed osteosarcoma patients.

    PubMed

    Hattinger, Claudia M; Vella, Serena; Tavanti, Elisa; Fanelli, Marilù; Picci, Piero; Serra, Massimo

    2016-12-01

    Second-line treatment of high-grade osteosarcoma (HGOS) patients is based on different approaches and chemotherapy protocols, which are not yet standardized. Although several drugs have been used in HGOS second-line protocols, none of them has provided fully satisfactory results and the role of rescue chemotherapy is not well defined yet. This article focuses on the drugs that have most frequently been used for second-line treatment of HGOS, highlighting the present knowledge on their mechanisms of action and resistance and on gene polymorphisms with possible impact on treatment sensitivity or toxicity. In the near future, validation of the so far identified candidate genetic biomarkers may constitute the basis for tailoring treatment by taking the patients' genetic background into account.

  4. Aberrant tropoelastin secretion in MG-63 human osteosarcoma cells

    SciTech Connect

    Curtiss, S.W.

    1989-01-01

    The secretion of newly synthesized tropoelastin, the soluble precursor of the extracellular matrix protein elastin, is not well understood. MG-63 human osteosarcoma cells were found by immunoblot analysis to synthesize 62 kD and 64 kD tropoelastins. Media from 63 cells labelled for five hours with ({sup 3}H)-valine contain no detectable tropoelastin, unlike media from other tropoelastin-synthesizing cells. Immunoblots of conditioned media and 1Ox-concentrated conditioned media left on the cells for six days also show an absence of tropoelastin from the cell media. No insoluble elastin is associated with the cell layer, as determined by amino acid analysis and electron microscopy of 18-21 day cell cultures. The absence of tropoelastin from the cell medium and elastin from the extracellular matrix indicates that MG63 cells do not secrete tropoelastin as expected, but accumulate it intracellularly. This accumulation is transient: immunoblots and immunofluorescence microscopy show that cells three days after passage have the highest steady-state levels of tropoelastin per cell, that day 8 cells contain lower but still significant amounts of tropoelastin, and that by day 22 tropoelastin is no longer present in the cell cultures. Cell density is a critical factor in the observed pattern of tropoelastin expression. Cells seeded at ten fold their usual initial density have high tropoelastin levels at one day after passage, sooner than cells seeded normally. Tropoelastin also disappears from high density-seeded cells more quickly and is no longer detectable at day 10. Lysosome-like vesicles containing membranous structures appear by immunoelectron microscopy to be the primary site of intracellular tropoelastin localization.

  5. FLI-1 distinguishes Ewing sarcoma from small cell osteosarcoma and mesenchymal chondrosarcoma.

    PubMed

    Lee, Anna F; Hayes, Malcolm M; Lebrun, David; Espinosa, Inigo; Nielsen, G Petur; Rosenberg, Andrew E; Lee, Cheng-Han

    2011-05-01

    Small cell osteosarcoma and mesenchymal chondrosarcoma are 2 primary bone tumors with a small round blue cell component, which can mimic the appearance of Ewing sarcoma. Distinguishing these tumors from each other on biopsy material is important clinically, as optimal therapy differs according to the tumor type. However, separating these entities on morphology alone can be challenging. FLI-1 has been described to be a useful marker for Ewing sarcoma, particularly when hematolymphoid markers are negative. In small cell osteosarcoma and mesenchymal chondrosarcoma, the FLI-1 staining pattern has not been adequately characterized. Using a monoclonal FLI-1 antibody, nuclear immunoreactivity in tumor cells was evaluated in 10 small cell osteosarcomas, 10 mesenchymal chondrosarcomas, and 8 Ewing sarcomas, together with a number of other small, round, blue cell tumors. None of the small cell osteosarcomas or mesenchymal chondrosarcomas exhibited FLI-1 staining in the tumor cells, in contrast to the positive nuclear FLI-1 staining in the stromal endothelial cells. In comparison, 6 of the 8 Ewing sarcomas showed moderate-to-strong nuclear FLI-1 staining of the tumor cells in addition to strong staining of the stromal endothelial cell nuclei. With the exception of lymphoblastic lymphomas, FLI-1 positivity was not seen in the other small round blue cell tumors examined. These findings show that, in contrast to Ewing sarcoma, small cell osteosarcoma and mesenchymal chondrosarcoma lack FLI-1 immunoreactivity. FLI-1 is therefore useful in the differential diagnosis of small round blue cell tumors of the bone.

  6. Camptothecin Enhances Cell Death Induced by (177)Lu-EDTMP in Osteosarcoma Cells.

    PubMed

    Kumar, Chandan; Vats, Kusum; Lohar, Sharad P; Korde, Aruna; Samuel, Grace

    2014-10-01

    Lutetium-177 is an assured therapeutic radionuclide with favorable half-life and suitable β(-) energy. Radiolabeled (177)Lu-EDTMP (Ethylenediamine tetramethylene phosphonic acid) is by and large used for bone pain palliation in cancer patients. In vitro cell studies are carried out in osteosarcoma cells MG-63 to evaluate the combined effect of anticancer drug camptothecin (CPT) and (177)Lu-EDTMP. Two concentrations of (177)Lu-EDTMP (3.7 and 37 MBq) were incubated with MG63 cell line for 48 hours with and without pretreatment of CPT (10 nM) for 1 hour. After completion of incubation, the cells were harvested and cellular toxicity was estimated by LDH, MTT, and trypan blue dye. Apoptotic DNA fragmentation was estimated by ELISA kit. The expression of proteins such as bcl2, PARP, and MAPK (mitogen-activated protein kinase) that were related to apoptotic signaling pathways was assessed by western blotting. The results indicated that cellular toxicity and apoptosis were relatively higher in MG63 cells that were treated with CPT prior to treating with (177)Lu-EDTMP in comparison with the corresponding individual controls.

  7. Wnt/β-catenin Signaling Activates Expression of the Bone-related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

    PubMed

    Vega, Oscar A; Lucero, Claudia M J; Araya, Hector F; Jerez, Sofia; Tapia, Julio C; Antonelli, Marcelo; Salazar-Onfray, Flavio; Las Heras, Facundo; Thaler, Roman; Riester, Scott M; Stein, Gary S; van Wijnen, Andre J; Galindo, Mario A

    2017-03-28

    Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation and bone formation. Furthermore, WNT/β-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292 and 143B). In all six cell lines, β-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β-catenin and RUNX2 protein are enhanced in HOS, G292 and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of β-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. This article is protected by copyright. All rights reserved.

  8. Killer dendritic cells link innate and adaptive immunity against established osteosarcoma in rats.

    PubMed

    Chauvin, Camille; Philippeau, Jean-Marie; Hémont, Caroline; Hubert, Francois-Xavier; Wittrant, Yohann; Lamoureux, Francois; Trinité, Benjamin; Heymann, Dominique; Rédini, Françoise; Josien, Régis

    2008-11-15

    We have previously reported that a distinct subset of splenic CD4(-) rat dendritic cells (DC) induces a rapid and caspase-independent apoptosis-like cell death in a large number of tumor cells in vitro. The killing activity of these killer DC (KDC) was restricted to their immature state and was immediately followed by their engulfment of the apoptotic target cells, suggesting that these KDC could directly link innate and adaptive immunity to tumors. Here, we addressed this question using a transplantable model of rat osteosarcoma. First, we showed that rat KDC have an MHC II(+)CD103(+)CD11b(+)NKp46(-) phenotype and are therefore distinct from natural killer cells, which are MHC II(-)CD103(-)CD11b(-)NKp46(+). KDC numbers could be specifically and strongly (up to 10-fold) enhanced by Flt3L in vivo. The OSRGa cell line derived from the osteosarcoma tumor was killed and phagocytosed in vitro by both normal and Flt3L-induced splenic KDC. Such tumor antigen-loaded KDC were used to s.c. vaccinate progressive tumor-bearing rats. Vaccination with OSRGa-loaded KDC but not KDC loaded with irrelevant tumor cells (Jurkat) delayed tumor progression or even induced tumor regression. This vaccine effect was not observed in CD8 T cell-depleted animals and protective against tumor rechallenge. These results suggest that KDC possess the intrinsic capability not only to kill and then engulf tumor cells but also to efficiently cross-present tumor cell-derived antigen in vivo and subsequently induce an adaptive antitumor immune response.

  9. Expression of the chemokine receptor CXCR7 in CXCR4-expressing human 143B osteosarcoma cells enhances lung metastasis of intratibial xenografts in SCID mice.

    PubMed

    Brennecke, Patrick; Arlt, Matthias J E; Muff, Roman; Campanile, Carmen; Gvozdenovic, Ana; Husmann, Knut; Holzwarth, Nathalie; Cameroni, Elisabetta; Ehrensperger, Felix; Thelen, Marcus; Born, Walter; Fuchs, Bruno

    2013-01-01

    More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells co-expressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1β-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.

  10. Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

    PubMed Central

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

    2016-01-01

    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. PMID:27768062

  11. CD133(+) CD44(+) Cells Mediate in the Lung Metastasis of Osteosarcoma.

    PubMed

    He, Aina; Yang, Xiaojing; Huang, Yujing; Feng, Tao; Wang, Yonggang; Sun, Yuanjue; Shen, Zan; Yao, Yang

    2015-08-01

    CD133 and CD44 are commonly used markers of cancer stem cells (CSCs), which are characterized by their ability for self-renewal and tumorigenicity. However, the clinical value and significance of CD133 and CD44 in lung metastasis of osteosarcoma (OS) remains controversial. The purpose of this study was to investigate whether CD133(+) CD44(+) cells mediates the metastasis of OS. We identified the CD133(+) CD44(+) cells in lung metastatic lesions and OS cell lines, and next demonstrated CD133(+) CD44(+) cells were more aggressive in sphere formation, migration and invasiveness compared with CD133(+) CD44(-) , CD133(-) CD44(+) , or CD133(-) CD44(-) cells. We finally sorted the CD133(+) CD44(+) and CD133(-) CD44(-) cells from Saos-2 cell lines, after intratibial xenograft in nude mice, these cells developed primary tumors, and CD133(+) CD44(+) cells are more potential to form lung metastatic tumors. Thus we concluded that CD133(+) CD44(+) cells may mediate in the lung metastasis of OS. © 2015 Wiley Periodicals, Inc.

  12. Delocalized Claudin-1 promotes metastasis of human osteosarcoma cells

    SciTech Connect

    Jian, Yuekui; Chen, Changqiong; Li, Bo; Tian, Xiaobin

    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 and 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.

  13. Suppression of liver receptor homolog-1 by microRNA-451 represses the proliferation of osteosarcoma cells

    SciTech Connect

    Li, Zhiyong; Wu, Shuwen; Lv, Shouzheng; Wang, Huili; Wang, Yong; Guo, Qiang

    2015-06-05

    Liver receptor homolog-1 (LRH-1) plays an important role in the onset and progression of many cancer types. However, the role of LRH-1 in osteosarcoma has not been well investigated. In this study, the critical role of LRH-1 in osteosarcoma cells was described. Quantitative polymerase chain reaction and Western blot analysis results revealed that LRH-1 was highly overexpressed in osteosarcoma cells. LRH-1 was knocked down by small interfering RNA (siRNA), and this phenomenon significantly inhibited osteosarcoma cell proliferation. Bioinformatics analysis results showed that LRH-1 contained putative binding sites of microRNA-451 (miR-451); this result was further validated through a dual-luciferase activity reporter assay. miR-451 was overexpressed in osteosarcoma cells through transfection of miR-451 mimics; miR-451 overexpression then significantly inhibited LRH-1 expression and cell proliferation. The loss of LRH-1 by siRNA or miR-451 mimics significantly impaired Wnt/β-catenin activity, leading to G0/G1 cell cycle arrest. Results showed that LRH-1 is implicated in osteosarcoma. Therefore, miR-451-induced suppression of LRH-1 can be a novel therapy to treat osteosarcoma. - Highlights: • LRH-1 was highly overexpressed in osteosarcoma cells. • Knockdown of LRH-1 inhibited osteosarcoma cell proliferation. • miR-451 directly targeted and regulated LRH-1 expression. • Overexpression of miR-451 suppressed Wnt activity.

  14. The expression of P-glycoprotein is causally related to a less aggressive phenotype in human osteosarcoma cells.

    PubMed

    Scotlandi, K; Manara, M C; Serra, M; Benini, S; Maurici, D; Caputo, A; De Giovanni, C; Lollini, P L; Nanni, P; Picci, P; Campanacci, M; Baldini, N

    1999-01-21

    The relationship between P-glycoprotein expression and malignancy is controversial. We have recently found that, in osteosarcoma, multidrug resistance (MDR) is associated with a less aggressive behavior, both in vitro and in clinical settings. In this study, we evaluated whether P-glycoprotein overexpression has a cause-effect relationship with the reduced metastatic potential of MDR cells, or rather reflects a more complex phenotype. MDR1 gene-transfected osteosarcoma cell clones, showing different levels of P-glycoprotein expression, were analysed for their in vitro characteristics and their tumorigenic and metastatic ability in athymic mice. Apart from the different levels of P-glycoprotein, no significant change in the expression of surface antigens or in the differentiative features were observed in the MDR1 gene transfectants compared to the parental cell lines or control clones, obtained by transfection with neo gene alone. In contrast to controls, however, MDR1 transfectants showed a significantly lower ability to grow in semi-solid medium and were completely unable to grow and give lung metastases in athymic mice. These findings indicate that P-glycoprotein overexpression is causally associated with a low malignant potential of osteosarcoma cells, and open new insights on the role and functions of P-glycoprotein activity.

  15. Non-toxic Efficacy of the Combination of Caffeine and Valproic Acid on Human Osteosarcoma Cells In Vitro and in Orthotopic Nude-mouse Models.

    PubMed

    Igarashi, Kentaro; Yamamoto, Norio; Hayashi, Katsuhiro; Takeuchi, Akihiko; Kimura, Hiroaki; Miwa, Shinji; Hoffman, Robert M; Tsuchiya, Hiroyuki

    2016-09-01

    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. The present study aimed to investigate the efficacy of the combination of valproic acid and caffeine on human osteosarcoma cells in vitro and in orthotopic nude-mouse models. Human osteosarcoma cell lines (MG63, 143B and SaOS2) were used. Cell survival after a 72 h exposure to valproic acid and caffeine was assessed with a WST-8 assay. Half maximal inhibitory concentration (IC50) values and combination indices (CIs) were calculated. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. 143B cells were also transplanted to the tibia of nude mice and treated with these drugs. Both valproic acid and caffeine caused concentration-dependent cell death of the osteosarcoma cell lines in vitro. Apoptosis induction was observed with the Caspase 3 assay. The combination was synergistic. The combination of valproic acid and caffeine showed effective anti-tumor activity in vivo without the need for conventional anticancer drugs or any observable toxicity Conclusion: Efficacy of combination therapy with caffeine and valproic acid in osteosarcoma was observed in vitro and in vivo without toxicity, suggesting that either or both drugs can be effectively combined with appropriate chemotherapy in the future. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  16. Relative biological effectiveness in canine osteosarcoma cells irradiated with accelerated charged particles

    PubMed Central

    Maeda, Junko; Cartwright, Ian M.; Haskins, Jeremy S.; Fujii, Yoshihiro; Fujisawa, Hiroshi; Hirakawa, Hirokazu; Uesaka, Mitsuru; Kitamura, Hisashi; Fujimori, Akira; Thamm, Douglas H.; Kato, Takamitsu A.

    2016-01-01

    Heavy ions, characterized by high linear energy transfer (LET) radiation, have advantages compared with low LET protons and photons in their biological effects. The application of heavy ions within veterinary clinics requires additional background information to determine heavy ion efficacy. In the present study, comparison of the cell-killing effects of photons, protons and heavy ions was investigated in canine osteosarcoma (OSA) cells in vitro. A total of four canine OSA cell lines with various radiosensitivities were irradiated with 137Cs gamma-rays, monoenergetic proton beams, 50 keV/µm carbon ion spread out Bragg peak beams and 200 keV/µm iron ion monoenergetic beams. Clonogenic survival was examined using colony-forming as says, and relative biological effectiveness (RBE) values were calculated relative to gamma-rays using the D10 value, which is determined as the dose (Gy) resulting in 10% survival. For proton irradiation, the RBE values for all four cell lines were 1.0–1.1. For all four cell lines, exposure to carbon ions yielded a decreased cell survival compared with gamma-rays, with the RBE values ranging from 1.56–2.10. Iron ions yielded the lowest cell survival among tested radiation types, with RBE values ranging from 3.51–3.69 observed in the three radioresistant cell lines. The radiosensitive cell line investigated demonstrated similar cell survival for carbon and iron ion irradiation. The results of the present study suggest that heavy ions are more effective for killing radioresistant canine OSA cells when compared with gamma-rays and protons. This markedly increased efficiency of cell killing is an attractive reason for utilizing heavy ions for radioresistant canine OSA. PMID:27446477

  17. A polysaccharide from Agaricus blazei inhibits proliferation and promotes apoptosis of osteosarcoma cells.

    PubMed

    Wu, Bei; Cui, Juncheng; Zhang, Chaogui; Li, Zhihong

    2012-05-01

    Many reports have proved that traditional Chinese herbal medicines (TCM) have become popular used in disease prevention and as alternatives to cancer chemotherapy. In this study, we purified a polysaccharide (ABP-Ia) from the fruiting bodies of Agaricus blazei and identified its molecular weight to be 4.2×10(5)Da. ABP-Ia was a heteropolysaccharide fraction consisting of glucose, mannose, and galactose in a molar ratio of 1:1:1, along with trace of rhamnose. The effect of ABP-Ia at three concentrations of 100, 200 and 400 μg/mL on the cell growth and apoptosis was evaluated in osteosarcoma cell lines HOS and a normal human osteoblast cell line NHOst. ABP-Ia had a significant inhibitory effect against the growth of HOS cells, whereas a mild cytotoxicity to the HOS cells mediated by ABP-Ia was observed, which was in accordance with the results that ABP-Ia substantially induced apoptosis in a dose-dependent fashion in the HOS cells. However ABP-Ia had no or minor inhibitory and cytotoxic effects on the viability of NHOst cells even at the high concentration of 400 μg/mL. Base on all the observations, we could conclude that ABP-Ia had an evident inhibitory effect on the growth of HOS cells mainly through induction of apoptosis, with a minor toxicity to normal human osteoblast cell.

  18. Mechanosensitivity of human osteosarcoma cells and phospholipase C {beta}2 expression

    SciTech Connect

    Hoberg, M. . E-mail: Maik.Hoberg@med.uni-tuebingen.de; Gratz, H.-H.; Noll, M.; Jones, D.B.

    2005-07-22

    Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 {mu}strains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC {beta}1, {beta}3, {gamma}1, {gamma}2, and {delta}1 in all cells, but PLC {beta}2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC {beta}2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC {beta}1 in U-2/OS cells by 35% and PLC {beta}2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC {beta}2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

  19. Targeting CDKs with Roscovitine Increases Sensitivity to DNA Damaging Drugs of Human Osteosarcoma Cells

    PubMed Central

    Hattinger, Claudia Maria; Fanelli, Marilù; Versteeg, Rogier; Koster, Jan; Picci, Piero

    2016-01-01

    Cyclin-dependent kinase 2 (CDK2) has been reported to be essential for cell proliferation in several human tumours and it has been suggested as an appropriate target to be considered in order to enhance the efficacy of treatment regimens based on the use of DNA damaging drugs. We evaluated the clinical impact of CDK2 overexpression on a series of 21 high-grade osteosarcoma (OS) samples profiled by using cDNA microarrays. We also assessed the in vitro efficacy of the CDKs inhibitor roscovitine in a panel of drug-sensitive and drug-resistant human OS cell lines. OS tumour samples showed an inherent overexpression of CDK2, and high expression levels at diagnosis of this kinase appeared to negatively impact on clinical outcome. CDK2 expression also proved to be relevant for in vitro OS cells growth. These findings indicated CDK2 as a promising candidate therapeutic marker for OS and therefore we assessed the efficacy of the CDKs-inhibitor roscovitine in both drug-sensitive and -resistant OS cell lines. All cell lines resulted to be responsive to roscovitine, which was also able to increase the activity of cisplatin and doxorubicin, the two most active DNA damaging drugs used in OS chemotherapy. Our results indicated that combined treatment with conventional OS chemotherapeutic drugs and roscovitine may represent a new candidate intervention approach, which may be considered to enhance tumour cell sensitivity to DNA damaging drugs. PMID:27898692

  20. Targeting CDKs with Roscovitine Increases Sensitivity to DNA Damaging Drugs of Human Osteosarcoma Cells.

    PubMed

    Vella, Serena; Tavanti, Elisa; Hattinger, Claudia Maria; Fanelli, Marilù; Versteeg, Rogier; Koster, Jan; Picci, Piero; Serra, Massimo

    2016-01-01

    Cyclin-dependent kinase 2 (CDK2) has been reported to be essential for cell proliferation in several human tumours and it has been suggested as an appropriate target to be considered in order to enhance the efficacy of treatment regimens based on the use of DNA damaging drugs. We evaluated the clinical impact of CDK2 overexpression on a series of 21 high-grade osteosarcoma (OS) samples profiled by using cDNA microarrays. We also assessed the in vitro efficacy of the CDKs inhibitor roscovitine in a panel of drug-sensitive and drug-resistant human OS cell lines. OS tumour samples showed an inherent overexpression of CDK2, and high expression levels at diagnosis of this kinase appeared to negatively impact on clinical outcome. CDK2 expression also proved to be relevant for in vitro OS cells growth. These findings indicated CDK2 as a promising candidate therapeutic marker for OS and therefore we assessed the efficacy of the CDKs-inhibitor roscovitine in both drug-sensitive and -resistant OS cell lines. All cell lines resulted to be responsive to roscovitine, which was also able to increase the activity of cisplatin and doxorubicin, the two most active DNA damaging drugs used in OS chemotherapy. Our results indicated that combined treatment with conventional OS chemotherapeutic drugs and roscovitine may represent a new candidate intervention approach, which may be considered to enhance tumour cell sensitivity to DNA damaging drugs.

  1. 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.

  2. Programmed cell death 1 correlates with the occurrence and development of MG63 osteosarcoma

    PubMed Central

    Zhao, Fuyou; Wu, Qiong; Dai, Xiusong; Li, Yumei; Gan, Huaiyong; Wang, Ri; Lv, Jie; Chen, Yuqing

    2016-01-01

    The aim of the present study was to investigate the effect of programmed cell death 1 (PD-1) on osteosarcoma (OD) stem cells and T cells, and to determine their correlation. OS stem cells were sorted and identified from OS MG63 cells. Flow cytometry was used to detect the PD-1 expression of the OS tumor stem cell membrane surface. The expression of PD-1 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). MTT was used to detect the effect of PD-1 signals on T-cell proliferation. The results indicated that the cancer cells (cultured in DMEM medium containing 10% fetal bovine serum) exhibited clear proliferation within 1 week of cell culture, which showed their strong proliferation and aggressive ability. The formation of tumor cell spheres was dependent on the support of serum nutrition. The proliferation of MG63 cells in the serum culture medium was significantly higher than the number of OS cell spheres in serum-free suspension culture (P<0.05). Pluripotent stem cells in cancer cell spheres exhibited significantly higher cluster of differentiation 133 expression compared with the MG63 cells. The PD-1 expression levels of the cancer cell spheres was significantly increased compared with the MG63 cells, which is consistent with the results of the RT-PCR. In conclusion, the MG63 cell line possesses the features of OS stem cells. The MG63 cell line can express the certain cancer-associated cell markers. The expression of PD-1 in spheres was also increased significantly compared to the MG63 cells, which can reduce the immune function of patients and may be closely associated with the occurrence and development of tumors. PMID:28105229

  3. Overexpression of microRNA-495 suppresses the proliferation and invasion and induces the apoptosis of osteosarcoma cells by targeting high-mobility group nucleosome-binding domain 5.

    PubMed

    Jiang, Weibo; Zheng, Jia; Yu, Tao; Wang, Jincheng

    2017-08-01

    It has been suggested that microRNAs (miRNAs) act as critical regulators in tumorigenesis. MicroRNA-495 (miR-495) has been suggested as a cancer-associated miRNA in various types of cancers; however, the role of miR-495 in osteosarcoma is unknown. The aim of the present study was to determine whether miR-495 is involved in osteosarcoma, and to investigate the potential molecular mechanism of its involvement. We found that miR-495 was significantly downregulated in osteosarcoma tissues and cell lines, as detected by real-time quantitative polymerase chain reaction (RT-qPCR). Overexpression of miR-495 inhibited osteosarcoma cell proliferation in 3-(4,5-dimethylthiazol-2-yl)- ,5-diphenyltetrazolium bromide, colony formation and cell cycle assays. Overexpression of miR-495 induced osteosarcoma cell apoptosis. Moreover, miR-495 overexpression also inhibited osteosarcoma cell invasion. Bioinformatics and luciferase reporter assays demonstrated that miR-495 targets the 3'-untranslated region of high-mobility group nucleosome‑binding domain 5 (HMGN5), a potential oncogene in various types of cancers. Overexpression of miR-495 inhibited the expression of HMGN5, cyclin B1, Bcl-2 and matrix metalloproteinase 9. In addition, restoration of HMGN5 protein expression abrogated the miR-495-induced effects. Taken together, the present study indicated that miR-495 suppresses the proliferation and invasion and induces the apoptosis of osteosarcoma cells by targeting HMGN5, providing a novel insight into the molecular pathogenesis of osteosarcoma and suggesting a potential molecular target for the development of an miRNA-targeted therapeutic strategy for osteosarcoma.

  4. Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma

    PubMed Central

    Tavanti, E; Sero, V; Vella, S; Fanelli, M; Michelacci, F; Landuzzi, L; Magagnoli, G; Versteeg, R; Picci, P; Hattinger, C M; Serra, M

    2013-01-01

    Background: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. Methods: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines. Results: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments. Conclusion: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents. PMID:24129234

  5. Pericellular matrix formation alters the efficiency of intracellular uptake of oligonucleotides in osteosarcoma cells.

    PubMed

    Suzuki, Yoshitaka; Nishida, Yoshihiro; Naruse, Takahiro; Gemba, Takefumi; Ishiguro, Naoki

    2009-03-01

    One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-kappaB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-kappaB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-kappaB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-kappaB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.

  6. Elevated expression of serine/threonine phosphatase type 5 correlates with malignant proliferation in human osteosarcoma.

    PubMed

    Han, Kun; Gan, Zhihua; Lin, Shuchen; Hu, Haiyan; Shen, Zan; Min, Daliu

    2017-01-01

    Osteosarcoma is the most common primary malignant bone tumor in adolescents and young adults. However, the involvement of serine/threonine phosphatase type 5 (PP5) in osteosarcoma remains unclear. The aim of this study was to evaluate the functional role of PP5 in osteosarcoma cells. Firstly, we found that PP5 is widely expressed in several human osteosarcoma cell lines. Then we used lentivirus-delivered siRNA to silence PP5 expression in Saos-2 and U2OS cell lines. Knockdown of endogenous PP5 expression by shRNA-expressing lentivirus significantly decreased the viability and proliferation of the osteosarcoma cells. Moreover, FACS analysis showed that knockdown of PP5 expression induced a significant arrest in the G0/G1 phase of the cell cycle, which was associated with the inhibition of cell proliferation. Therefore, knockdown of PP5 is likely to provide a novel alternative to targeted therapy of osteosarcoma and deserves further investigation.

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

    SciTech Connect

    Zhang, Jun; Hou, Wengen; Chai, Mingxiang; Zhao, Hongxing; Jia, Jinling; Sun, Xiaohui; Zhao, Bin; Wang, Ran

    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 decreased 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.

  8. microRNA-143 is associated with the survival of ALDH1+CD133+ osteosarcoma cells and the chemoresistance of osteosarcoma

    PubMed Central

    Zhou, Jiahui; Chen, Yuxiang; Zhao, Jingfeng; Zhang, Kexiang; Wang, Jianlong; Chen, Shijie

    2015-01-01

    This study investigated the role of miR-143 in the chemoresistance of osteosarcoma tumor cells and the associated mechanisms. Real-time PCR was used to measure miR-143 levels. Western blot was used to detect protein expression. Cell proliferation was analyzed by MTT assay and Matrigel colony formation assay. Forced miR-143 expression was established by adenoviral vector infection. Cell death was detected by Hoechst33342 staining. Loss of miR-143 expression was observed in osteosarcomas, which correlated with shorter survival of patients with osteosarcomas underlying chemotherapy. In chemoresistant SAOS-2 and U2OS osteosarcomas cells, miR-143 levels were significantly downregulated and accompanied by increases in ATG2B, Bcl-2, and/or LC3-II protein levels, high rate of ALDH1+CD133+ cells, and an increase in Matrigel colony formation ability. H2O2 upregulated p53 and miR-143, but downregulated ATG2B, Bcl-2, and LC3-I expression in U2OS cells (wild-type p53) but not in SAOS-2 (p53-null) cells. Forced miR-143 expression significantly reversed chemoresistance as well as downregulation of ATG2B, LC3-I, and Bcl-2 expression in SAOS-2- and U2OS-resistant cells. Forced miR-143 expression significantly inhibited tumor growth in xenograft SAOS-2-Dox and U2OS-Dox animal models. Loss of miR-143 expression is associated with poor prognosis of patients with osteosarcoma underlying chemotherapy. The chemoresistance of osteosarcoma tumor cells to doxorubicin is associated with the downregulation of miR-143 expression, activation of ALDH1+CD133+ cells, activation of autophagy, and inhibition of cell death. miR-143 may play a crucial role in the chemoresistance of osterosarcoma tumors. PMID:25576341

  9. Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells - synergism with autophagy inhibition.

    PubMed

    Kubista, Bernd; Schoefl, Thomas; Mayr, Lisa; van Schoonhoven, Sushilla; Heffeter, Petra; Windhager, Reinhard; Keppler, Bernhard K; Berger, Walter

    2017-04-12

    Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with metastatic disease and poor response to chemotherapy still have an adverse prognosis. Hence, development of new therapeutic strategies is still of utmost importance. Anticancer activity of KP46 against osteosarcoma cell models was evaluated as single agent and in combination approaches with chemotherapeutics and Bcl-2 inhibitors using MTT assay. Underlying mechanisms were tested by cell cycle, apoptosis and autophagy assays. KP46 exerted exceptional anticancer activity at the nanomolar to low micromolar range, depending on the assay format, against all osteosarcoma cell models with minor but significant differences in IC50 values. KP46 treatment of osteosarcoma cells caused rapid loss of cell adhesion, weak cell cycle accumulation in S-phase and later signs of apoptotic cell death. Furthermore, already at sub-cytotoxic concentrations KP46 reduced the migratory potential of osteosarcoma cells and exerted synergistic effects with cisplatin, a standard osteosarcoma chemotherapeutic. Moreover, the gallium compound induced signs of autophagy in osteosarcoma cells. Accordingly, blockade of autophagy by chloroquine but also by the Bcl-2 inhibitor obatoclax increased the cytotoxic activity of KP46 treatment significantly, suggesting autophagy induction as a protective mechanism against KP46. Together, our results identify KP46 as a new promising agent to supplement standard chemotherapy and possible future targeted therapy in osteosarcoma.

  10. Selective inhibition of MG-63 osteosarcoma cell proliferation induced by curcumin-loaded self-assembled arginine-rich-RGD nanospheres

    PubMed Central

    Chang, Run; Sun, Linlin; Webster, Thomas J

    2015-01-01

    Osteosarcoma is the most frequent primary malignant form of bone cancer, comprising 30% of all bone cancer cases. The objective of this in vitro study was to develop a treatment against osteosarcoma with higher selectivity toward osteosarcoma cells and lower cytotoxicity toward normal healthy osteoblast cells. Curcumin (or diferuloylmethane) has been found to have antioxidant and anticancer effects by multiple cellular pathways. However, it has lower water solubility and a higher degradation rate in alkaline conditions. In this study, the amphiphilic peptide C18GR7RGDS was used as a curcumin carrier in aqueous solution. This peptide contains a hydrophobic aliphatic tail group leading to their self-assembly by hydrophobic interactions, as well as a hydrophilic head group composed of an arginine-rich and an arginine-glycine-aspartic acid structure. Through characterization by transmission electron microscopy, self-assembled structures of spherical amphiphilic nanoparticles (APNPs) with diameters of 10–20 nm in water and phosphate-buffered saline were observed, but this structure dissociated when the pH value was reduced to 4. Using a method of codissolution with acetic acid and dialysis tubing, the solubility of curcumin was enhanced and a homogeneous solution was formed in the presence of APNPs. Successful encapsulation of curcumin in APNPs was then confirmed by Fourier transform infrared and X-ray diffraction analyses. The cytotoxicity and cellular uptake of the APNP/curcumin complexes on both osteosarcoma and normal osteoblast cell lines were also evaluated by methyl-thiazolyl-tetrazolium assays and confocal fluorescence microscopy. The results showed that the curcumin-loaded APNPs had significant selective cytotoxicity against MG-63 osteosarcoma cells when compared with normal osteoblasts. We have demonstrated for the first time that APNPs can encapsulate hydrophobic curcumin in their hydrophobic cores, and curcumin-loaded APNPs could be an innovative treatment

  11. Hyperthermia Induces Apoptosis through Endoplasmic Reticulum and Reactive Oxygen Species in Human Osteosarcoma Cells

    PubMed Central

    Hou, Chun-Han; Lin, Feng-Ling; Hou, Sheng-Mon; Liu, Ju-Fang

    2014-01-01

    Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS. PMID:25268613

  12. MiR-193a-3p and miR-193a-5p suppress the metastasis of human osteosarcoma cells by down-regulating Rab27B and SRR, respectively.

    PubMed

    Pu, Youguang; Zhao, Fangfang; Cai, Wenjing; Meng, Xianghui; Li, Yinpeng; Cai, Shanbao

    2016-04-01

    MicroRNAs have been identified as key players in the development and progression of osteosarcoma, which is the most common primary malignancy of bone. Sequencing-based miR-omic and quantitative real-time PCR analyses suggested that the expression of miR-193a-3p and miR-193a-5p was decreased by DNA methylation at their promoter region in a highly metastatic osteosarcoma cell line (MG63.2) relative to their expression in the less metastatic MG63 cell line. Further wound-healing and invasion assays demonstrated that both miR-193a-3p and miR-193a-5p suppressed osteosarcoma cell migration and invasion. Moreover, introducing miR-193a-3p and miR-193a-5p mimics into MG63.2 cells or antagomiRs into MG63 cells confirmed their critical roles in osteosarcoma metastasis. Additionally, bioinformatics prediction along with biochemical assay results clearly suggested that the secretory small GTPase Rab27B and serine racemase (SRR) were direct targets of miR-193a-3p and miR-193a-5p, respectively. These two targets are indeed involved in the miR-193a-3p- and miR-193a-5p-induced suppression of osteosarcoma cell migration and invasion. MiR-193a-3p and miR-193a-5p play important roles in osteosarcoma metastasis through down-regulation of the Rab27B and SRR genes and therefore may serve as useful biomarkers for the diagnosis of osteosarcoma and as potential candidates for the treatment of metastatic osteosarcoma.

  13. Smoothened as a new therapeutic target for human osteosarcoma

    PubMed Central

    2010-01-01

    Background The Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition. Results To evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21cip1 wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo. Conclusions These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma. PMID:20067614

  14. MicroRNA-665 suppressed the invasion and metastasis of osteosarcoma by directly inhibiting RAB23

    PubMed Central

    Dong, Chenhui; Du, Quanyin; Wang, Zimin; Wang, Yu; Wu, Siyu; Wang, Aimin

    2016-01-01

    MicroRNAs (miRNAs) are small, short and noncoding RNAs that regulate gene expression posttranscriptionally. Increasing evidences have demonstrated that deregulated expression of miRNAs is found in osteosarcoma. In this study, we demonstrated that miR-665 was downregulated in osteosarcoma tissues compared to non-tumorous tissues. The overall survival (OS) of osteosarcoma patients with low miR-665 expression was lower than that of these patients with high miR-665 expression. Ectopic expression of miR-665 suppressed the osteosarcoma cell proliferation, EMT and invasion. We identified Rab23 as a direct target gene of miR-665. Rab23 was downregulated in osteosarcoma tissues and cell lines. The expression of miR-665 was inversely associated with the expression of Rab23 in the osteosarcoma tissues. These results suggested that miR-665 acted as a tumor suppressor gene in the development of osteosarcoma. PMID:27904698

  15. APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome

    PubMed Central

    Palumbo, Rosanna; Gogliettino, Marta; Cocca, Ennio; Iannitti, Roberta; Sandomenico, Annamaria; Ruvo, Menotti; Balestrieri, Marco; Rossi, Mosè; Palmieri, Gianna

    2016-01-01

    The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH–proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21Waf1, and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies. PMID:27669226

  16. Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1.

    PubMed

    Weekes, D; Kashima, T G; Zandueta, C; Perurena, N; Thomas, D P; Sunters, A; Vuillier, C; Bozec, A; El-Emir, E; Miletich, I; Patiño-Garcia, A; Lecanda, F; Grigoriadis, A E

    2016-06-02

    Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant owing to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signalling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 (fibroblast growth factor receptor 1) was identified as a novel c-Fos/activator protein-1(AP-1)-regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of mitogen-activated protein kinases (MAPKs), morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1-silenced osteosarcoma cells caused a marked twofold to fivefold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small-molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus deregulated FGFR signalling has an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy.

  17. Safety Concern between Autologous Fat Graft, Mesenchymal Stem Cell and Osteosarcoma Recurrence

    PubMed Central

    Perrot, Pierre; Rousseau, Julie; Bouffaut, Anne-Laure; Rédini, Françoise; Cassagnau, Elisabeth; Deschaseaux, Frédéric; Heymann, Marie-Françoise; Heymann, Dominique; Duteille, Franck; Trichet, Valérie; Gouin, François

    2010-01-01

    Background Osteosarcoma is the most common malignant primary bone tumour in young adult treated by neo adjuvant chemotherapy, surgical tumor removal and adjuvant multidrug chemotherapy. For correction of soft tissue defect consecutive to surgery and/or tumor treatment, autologous fat graft has been proposed in plastic and reconstructive surgery. Principal Findings We report here a case of a late local recurrence of osteosarcoma which occurred 13 years after the initial pathology and 18 months after a lipofilling procedure. Because such recurrence was highly unexpected, we investigated the possible relationship of tumor growth with fat injections and with mesenchymal stem/stromal cell like cells which are largely found in fatty tissue. Results obtained in osteosarcoma pre-clinical models show that fat grafts or progenitor cells promoted tumor growth. Significance These observations and results raise the question of whether autologous fat grafting is a safe reconstructive procedure in a known post neoplasic context. PMID:20544017

  18. MiR-326 is a diagnostic biomarker and regulates cell survival and apoptosis by targeting Bcl-2 in osteosarcoma.

    PubMed

    Cao, Lei; Wang, Jiandong; Wang, Prof Qiugen

    2016-12-01

    Osteosarcoma is a malignant bone tumor in which the survival rate is still low. MicroRNA-326 (miR-326) has been proved a potential diagnostic and prognostic marker for several tumors. However, the clinical value of miR-326 is still unknown. In the present study, we detected the expression of miR-326 in the serum of osteosarcoma patients and in osteosarcoma tissues using qRT-PCR. We compared the serum expression of miR-326 with the clinicopathological characteristics and survival of osteosarcoma patients. Finally, we explored the role of miR-326 of the invasion of osteosarcoma tumor cells using cell migration and invasion assays. We found that the expression of miR-326 was significantly decreased in the serum of osteosarcoma patients and osteosarcoma tumor cells compared to healthy controls (P<0.01). Moreover, a receiver operating characteristic (ROC) curve analysis is indicated that serum miR-326 is a potential diagnostic marker of osteosarcoma with an area under the ROC curve of 0.817. Importantly, patients with a lower expression of miR-326 tended to have distant metastasis (P<0.05) and a more advanced clinical stage (P<0.05). In addition, the survival time of patients with depressed miR-326 expression was significantly shorter compared to patients with high miR-326 expression (P<0.05). Further-more, we found that miR-326 could inhibit the proliferation, migration and invasion of osteosarcoma cells. Thus, we demonstrated that miR-326 might be related to the metastasis of osteosarcoma and could be used as a potential diagnostic and prognostic biomarker in osteosarcoma. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  19. Expression and prognostic relevance of PRAME in primary osteosarcoma.

    PubMed

    Tan, Pingxian; Zou, Changye; Yong, Bicheng; Han, Ju; Zhang, Longjuan; Su, Qiao; Yin, Junqiang; Wang, Jin; Huang, Gang; Peng, Tingsheng; Shen, Jingnian

    2012-03-23

    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 have 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.

  20. von Willebrand factor expression in osteosarcoma metastasis.

    PubMed

    Eppert, Kolja; Wunder, Jay S; Aneliunas, Vicky; Kandel, Rita; Andrulis, Irene L

    2005-03-01

    A number of genes are implicated in the initiation and progression of osteosarcoma; however, cytogenetic and comparative genomic hybridization studies indicate the involvement of additional unidentified genes. An examination of gene expression profiles in 22 high-grade osteosarcoma tumor specimens from 15 patients (including paired primary and metastatic samples from five patients) indicated that von Willebrand factor (vWF) mRNA expression may increase during tumor progression. vWF, a large glycoprotein previously considered to be expressed exclusively by endothelial cells and megakaryocytes, is involved in platelet aggregation and adhesion to the subendothelial matrix, processes critical to hematogenous tumor cell metastasis to the lung. Analysis of paired primary and metastatic osteosarcoma tumor samples from 10 patients revealed an increase in vWF gene expression in metastases (P=0.005). Immunohistochemistry showed that, in addition to the endothelial cells, vWF protein was also detected in osteosarcoma cells in vivo in 13 of 29 tumor specimens as well as in SAOS2, an osteosarcoma cell line. The tumor cell staining correlated positively with high vWF expression in the sample (P=0.006). Although vascular endothelial cells contribute to the vWF mRNA detected in the tumor samples, there was neither any correlation between vascular density (VD) and vWF mRNA expression nor between VD and clinical outcome. These findings suggest that vWF expression is deregulated in osteosarcoma tumors, potentially contributing to metastasis.

  1. Proflavin suppresses the growth of human osteosarcoma MG63 cells through apoptosis and autophagy

    PubMed Central

    ZHANG, MAO-SHU; NIU, FU-WEN; LI, KUN

    2015-01-01

    Proflavin is one of the novel acridine derivatives that possess various pharmacological effects. Although numerous studies have been performed to investigate proflavin, its effects have not been investigated on the human osteosarcoma MG63 cell line. The core aim of the present study was to test the effects of proflavin on the viability of MG63 cells and the induction of apoptosis and autophagy in MG63 cells. The induction of apoptosis was examined by measuring the changes in the expression of the B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein mRNA and proteins. Apoptotic cell death was identified by the proteolytic cleavage of poly (adenosine diphosphate-ribose) polymerase and caspase-3 and caspase-9. In addition, the autophagic effects of proflavin were examined by the quantitation of the mRNA expression of autophagy protein 5 and Beclin 1, in addition to the identification of the accumulation of microtubule-associated protein 1 light chain 3-II. The present results revealed that proflavin inhibited the proliferation of MG63 cells in a dose-dependent manner. Proflavin-induced cell death was attributed to apoptosis and autophagy. Overall, the present results indicated that the antiseptic agent proflavin exerts anticancer potential through the synergistic activity of apoptosis and autophagy. PMID:26171052

  2. 2-Methoxyestradiol Impacts on Amino Acids-mediated Metabolic Reprogramming in Osteosarcoma Cells by Interaction with NMDA Receptor.

    PubMed

    Gorska-Ponikowska, Magdalena; Perricone, Ugo; Kuban-Jankowska, Alicja; Lo Bosco, Giosue; Barone, Giampaolo

    2017-03-06

    Deregulation of serine and glycine metabolism, have been identified to function as metabolic regulators in supporting tumor cell growth. The role of serine and glycine in regulation of cancer cell proliferation is complicated, dependent on concentrations of amino acids and tissue-specific. D-serine and glycine are coagonists of N-methyl-D-aspartate receptor subunit GRIN1. Importantly, NMDA receptors are widely expressed in cancer cells and play an important role in regulation of cell death, proliferation and metabolism of numerous malignancies. The aim of the present work was to associate the metabolism of glycine and D-serine with the anticancer activity of 2-methoxyestradiol. 2-methoxyestradiol is a potent anticancer agent but also a physiological 17β- estradiol metabolite. In the study we have chosen two malignant cell lines expressing functional GRIN1 receptors, i.e. osteosarcoma 143B and breast cancer MCF7. We used MTS assay, migration assay, flow cytometric analyses, western blotting and immunoprecipitation techniques as well as molecular modeling studies. We have demonstrated the extensive crosstalk between the deregulated metabolic network and cancer cell signaling. Herein, we observed an anticancer effect of high concentrations of glycine and D-serine in osteosarcoma cells. In contrast, the amino acids when used at low, physiological concentrations induced the proliferation and migration of osteosarcoma and breast cancer cells. Importantly, the pro-cancergogenic effects of both glycine and D-serine where abrogated by the usage of 2-methoxyestradiol at both physiological and pharmacological relevant concentrations. The obtained data confirmed that 2-methoxyestradiol may be a physiological anticancer molecule. This article is protected by copyright. All rights reserved.

  3. The flavonoid luteolin enhances doxorubicin-induced autophagy in human osteosarcoma U2OS cells

    PubMed Central

    Zhang, Baoliang; Yu, Xin; Xia, Hong

    2015-01-01

    Luteolin (LUT), a flavone, which is universally present as constituent of medicinal plants as well as some vegetables and spices, has been demonstrated display specific anti-carcinogenic effects. However, the mechanisms by which LUT inhibits human osteosarcoma growth remain unknown. The effects of LUT on cell growth in human osteosarcoma U2OS cells were measured by MTT assay and flowcytometry. The effects of LUT on morphological markers of autophagy in U2OS were analyzed by fluorescence microscopy and electron microscopy. Autophagic markers, beclin1 and LC3 were detected by western blotting. Here, we found that LUT induced autophagy in U2OS and acted as an enhancer to sensitize doxorubicin (DOX)-mediated autophagy signaling. The combined treatment of LUT and DOX greatly decreases the growth of U2OS, showing synergistic cytotoxicity. Our results indicate that LUT in combination with DOX maybe a novel strategy for the treatment of human osteosarcoma. PMID:26629003

  4. Wnt10b Activates the Wnt, Notch and NFκB Pathways in U2OS Osteosarcoma Cells

    PubMed Central

    Mödder, Ulrike I.; Oursler, Merry Jo; Khosla, Sundeep; Monroe, David G.

    2011-01-01

    Although osteosarcoma represents the most common bone malignancy, the molecular and cellular mechanisms influencing its pathogenesis have remained elusive. Recent evidence has suggested that the Wnt signaling pathway may play a crucial role in osteosarcoma. This study employed a microarray approach to discover novel genes and pathways involved in Wnt signaling in osteosarcoma. We developed a Wnt10b-expressing cell line using the human U2OS osteosarcoma model (U2OS-Wnt10b) and performed microarray and pathway analyses using parental U2OS cells as control. Differential expression of 1003 genes encompassing 28 pathways was noted. The Wnt, NFκB and Notch pathways were chosen for further study based on their known importance in bone biology. Known Wnt-responsive genes Axin-2 (4.9-fold), CD44 (2.1-fold), endothelin-1 (4.2-fold) and sclerostin domain containing-1 (43-fold) were regulated by Wnt10b. The proinflammatory cytokines interleukin-1α and tumor necrosis factor-α, known inducers of NFκB, were upregulated both at the transcript and protein level, and NFκB reporter activity was stimulated 3.8-fold, confirming NFκB activation. Interestingly, genes involved in Notch signaling [Notch-1 (2.4-fold) and Jagged-1 (3.1-fold)] were upregulated, whereas the Notch inhibitor, lunatic fringe, was downregulated (8.2-fold). This resulted in the activation of the classic Notch-responsive genes, hairy and enhancer of split-1 (Hes-1; 2.2-fold) and hairy/enhancer-of-split related with YRPW motif-1 (Hey-1; 2.5-fold). A Hey-1 reporter construct was regulated 9.1-fold in U2OS-Wnt10b cells, confirming Notch activation. Interestingly, Wnt3a failed to induce the Notch and NFκB pathways, demonstrating Wnt-specificity. In conclusion, our data demonstrate that Wnt10b, but not Wnt3a, stimulates the NFκB and Notch pathways in U2OS osteosarcoma cells. PMID:21321991

  5. Effective Metabolic Targeting of Human Osteosarcoma Cells In Vitro and in Orthotopic Nude-mouse Models with Recombinant Methioninase.

    PubMed

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

    2017-09-01

    Methionine dependence may be the only known general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]) (recombinant methioninase [rMETase]), which was subsequently tested in mouse models of various types of human tumors. The present study aimed to investigate the efficacy of rMETase on human osteosarcoma cells in vitro and in vivo. Human osteosarcoma cell lines 143B, HOS and SOSN2 were tested in vitro for survival during a 72-h exposure to rMETase using the WST-8 assay. Half-maximal inhibitory concentrations were calculated for in vitro efficacy experiments. 143B cells were orthotopically transplanted into the tibia of nude mice. Mouse models were randomized into the following groups 1 week after transplantation: Group 1, untreated control; Group 2, cisplatinum (CDDP) [intraperitoneal (i.p.) injection at 6 mg/kg weekly, for 3 weeks], positive control; Group 3, rMETase, 100 units/mouse i.p. daily, for 21 days. Tumor sizes and body weight were measured with calipers and a digital balance once per week, respectively. rMETase significantly inhibited osteosarcoma cell growth, in a dose-dependent manner, in vitro. Both CDDP and rMETase treatment significantly inhibited tumor volume compared to untreated control mice at 5 weeks after initiation. Tumor volumes were as follows: Group 1, untreated, control: 1808.2 ± 344 mm(3); Group 2, CDDP: 1102.2 ± 316 mm(3), p=0.0008 compared to untreated control; Group 3, rMETase: 884.8 ± 361 mm(3), p=0.0001 compared to untreated control. There were no animal deaths in any group. The body weight of mice was not significantly different between any group. rMETase showed promising efficacy against osteosarcoma, a recalcitrant tumor type. Future studies will investigate the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) models of osteosarcoma as a bridge to testing rMETase in the clinic

  6. Elevated expression of matrix metalloproteinase-3 in human osteosarcoma and its association with tumor metastasis.

    PubMed

    Huang, Jie-Feng; Du, Wen-Xi; Chen, Jun-Jie

    2016-01-01

    Matrix metalloproteinase-3 (MMP-3) is one of the several MMPs that is associated with malignant tumors of breast, colon, cervix and lung, where its expression has been correlated with tumor invasion and metastasis. However, the role of MMP-3 in metastasis of osteosarcoma has not yet been explored. MMP-3 expression in 15 primary and metastatic osteosarcomas with case-matched adjacent non-tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. Further, MMP-3 mRNA and protein levels were also determined in osteoblast and osteosarcoma cell lines. Additionally, migration and invasion assays were performed in MMP-3 knockdown cells. MMP-3 was expressed in 86.6% (13/15) of the osteosarcoma patients and its expression was significantly higher in metastatic tumors as compared to the primary osteosarcoma tumor tissues. Furthermore, osteosarcoma cell lines showed higher MMP-3 expression as compared to osteoblast cell lines. siRNA mediated MMP-3 knockdown in osteosarcoma cell lines significantly inhibited their migration and invasion properties. Our results demonstrated that MMP-3 expression is deregulated in osteosarcomas and this potentially contributes to metastasis and might be a promising marker for the prognosis and therapy of metastatic osteosarcoma.

  7. Elevated expression of matrix metalloproteinase-3 in human osteosarcoma and its association with tumor metastasis.

    PubMed

    Huang, Jie-Feng; Du, Wen-Xi; Chen, Jun-Jie

    2016-01-01

    Matrix metalloproteinase-3 (MMP-3) is one of the several MMPs that is associated with malignant tumors of breast, colon, cervix and lung, where its expression has been correlated with tumor invasion and metastasis. However, the role of MMP-3 in metastasis of osteosarcoma has not yet been explored. MMP-3 expression in 15 primary and metastatic osteosarcomas with case-matched adjacent non-tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. Further, MMP-3 mRNA and protein levels were also determined in osteoblast and osteosarcoma cell lines. Additionally, migration and invasion assays were performed in MMP-3 knockdown cells. MMP-3 was expressed in 86.6% (13/15) of the osteosarcoma patients and its expression was significantly higher in metastatic tumors as compared to the primary osteosarcoma tumor tissues. Furthermore, osteosarcoma cell lines showed higher MMP-3 expression as compared to osteoblast cell lines. siRNA-mediated MMP-3 knockdown in osteosarcoma cell lines significantly inhibited their migration and invasion properties. Our results demonstrated that MMP-3 expression is deregulated in osteosarcomas and this potentially contributes to metastasis and might be a promising marker for the prognosis and therapy of metastatic osteosarcoma.

  8. Anticancer effect of thalidomide in vitro on human osteosarcoma cells.

    PubMed

    Zhu, Jianwei; Yang, Ya; Liu, Sihong; Xu, Huihua; Wu, Yong; Zhang, Guiqiang; Wang, Yuxuan; Wang, Yan; Liu, Yamin; Guo, Qifeng

    2016-12-01

    Osteosarcoma is a high‑grade malignant tumor frequently found in children and adolescents. Thalidomide has been reported for treatment of various malignancies. Thalidomide was added to osteosarcoma cells and studied by cytotoxicity assay, evaluating apoptosis, cell cycle arrest, mitochondrial membrane potential (ΔΨm), and reactive oxygen species (ROS) levels and the expression of Bcl‑2, Bax, caspase‑3 and NF‑κB. The results showed that thalidomide could inhibit the proliferation of MG‑63 and U2OS cells in a concentration‑ and time‑dependent manner. Morphological changes of apoptosis were also observed. Thalidomide increased the apoptosis rate of MG‑63 cells and induced cell cycle arrest by increasing the number of cells in the G0/G1 phase and decreasing the percentage of S phase in MG‑63 cells. Further investigation showed that a disruption of ΔΨm and upregulation of ROS were induced by thalidomide in high concentration. By western blot analysis, thalidomide resulted in the decreasing expression of Bcl‑2 and NF‑κB, and the increasing expression of Bcl‑2/Bax and caspase‑3. Here, we provide evidence that thalidomide could cause apoptosis in osteosarcoma cells. Taken together, these results indicate that thalidomide could be an antitumor drug in the therapy of osteosarcoma.

  9. Expression and prognostic relevance of PRAME in primary osteosarcoma

    SciTech Connect

    Tan, Pingxian; Zou, Changye; Yong, Bicheng; Han, Ju; Zhang, Longjuan; Su, Qiao; Yin, Junqiang; Wang, Jin; Huang, Gang; Peng, Tingsheng; Shen, Jingnian

    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 have 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.

  10. Corosolic acid inhibits the proliferation of osteosarcoma cells by inducing apoptosis

    PubMed Central

    Jia, Yong; Yuan, Hua; Shan, Shouqin; Xu, Gang; Yu, Jie; Zhao, Chenguang; Mou, Xiang

    2016-01-01

    Corosolic acid (CRA), a pentacyclic triterpene isolated from medicinal herbs, has been reported to exhibit anticancer properties in several cancers. However, the anticancer activity of CRA in osteosarcoma cells is still unclear. In the present study, the inhibitory effect of CRA in osteosarcoma MG-63 cells was investigated, and the results revealed that CRA significantly inhibited the viability of MG-63 cells in a dose- and time-dependent manner. A typical apoptotic hallmark such as DNA ladder was detected by agarose gel electrophoresis following treatment with CRA. Further experiments demonstrated that CRA induced apoptosis of MG-63 cells by flow cytometry using propidium iodide and annexin V staining. In addition, it was observed that the apoptosis of MG-63 cells induced by CRA was closely associated with activation of caspase-3 and caspase-9, loss of mitochondrial membrane potential, and release of cytochrome c from mitochondria, suggesting that CRA may trigger the activation of the mitochondria-mediated apoptosis pathway. In addition, the inhibition of caspase activity attenuated the CRA-induced apoptosis of MG-63 cells, which further confirmed the role of the mitochondrial pathway in CRA-induced apoptosis. These results indicated that CRA could induce the apoptosis of osteosarcoma cells through activating the mitochondrial pathway, which provides an evidence that CRA may be a useful chemotherapeutic agent for osteosarcoma. PMID:27895790

  11. Celastrol negatively regulates cell invasion and migration ability of human osteosarcoma via downregulation of the PI3K/Akt/NF-κB signaling pathway in vitro

    PubMed Central

    Yu, Xiaolong; Wang, Qiang; Zhou, Xin; Fu, Changlin; Cheng, Ming; Guo, Runsheng; Liu, Hucheng; Zhang, Bin; Dai, Min

    2016-01-01

    Osteosarcoma (OS) is a primary malignant tumor of the bone, with a tendency to metastasize early. Despite the advances in treatment options, more than 30% of patients develop distant metastases, and the prognosis of these patients with metastases is extremely poor. Celastrol has been demonstrated to manifest multiple pharmacological activities, including induction of apoptosis in numerous types of cancer cell lines. Our previous studies have also suggested that Celastrol is capable of inducing apoptosis of human osteosarcoma cells via the mitochondrial-dependent pathway. The purpose of this study was to investigate the effects of Celastrol on the migration and invasion of human osteosarcoma U-2OS cells in vitro. Cell migration and invasion were investigated using wound healing and Boyden chamber Transwell assays. We observed that Celastrol suppressed cell invasion and migration in human osteosarcoma U-2OS cells. Furthermore, protein expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, inhibitor of κB kinase α/β, inhibitor of κB α, nuclear factor-κB (NF-κB subunit p65) and matrix metalloproteinase (MMP)-2 and −9 were measured by western blot analysis. We observed that the PI3K/Akt/NF-κB signaling pathway was inhibited following Celastrol treatment. In addition, the expression levels of MMP-2 and −9 proteins were also reduced significantly following Celastrol treatment. Therefore, we confirmed that Celastrol suppressed osteosarcoma U-2OS cell metastasis via downregulation of the PI3K/Akt/NF-κB signaling pathway in vitro. PMID:27900015

  12. Expression and regulatory effects of microRNA-182 in osteosarcoma cells: A pilot study

    PubMed Central

    BIAN, DONG-LIN; WANG, XUE-MEI; HUANG, KUN; ZHAI, QI-XI; YU, GUI-BO; WU, CHENG-HUA

    2016-01-01

    The aim of the present study was to evaluate the expression level of microRNA-182 (miRNA-182) in human osteosarcoma (OS) MG-63 cells and OS tissues, and to elucidate the effect of miRNA-182 on the biological activity of tumors. In the present study, the expression of miRNA-182 in human OS MG-63 cells, OS tissues and normal osteoblast hFOB1.19 cells was determined using quantitative polymerase chain reaction. Subsequently, a miRNA-182 mimic and inhibitor were utilized to regulate the expression level of this miRNA in MG-63 cells. Cell viability and proliferation were examined using cell counting kit-8 assays, and cell apoptosis was detected by flow cytometry. Cell invasion and migration assays were performed using Transwell chambers to analyze the biological functions of miRNA-182 in vitro. The present study demonstrated that the expression level of miRNA-182 in MG-63 cells and OS tissues was significantly increased compared with the hFOB1.19 cell line (P<0.05). The present study successfully performed cell transfections of miRNA-182 inhibitor and miRNA-182 mimic into MG-63 cells and achieved the desired transfection efficiency. The present study confirmed that upregulation of miRNA-182 promotes cell apoptosis and inhibits cell viability, proliferation, invasion and migration. The present findings additionally demonstrated that miRNA-182 is a tumor suppressor gene in OS. Therefore, regulating the expression of miRNA-182 may affect the biological behavior of OS cells, which suggests a potential role for miRNA-182 in molecular therapy for malignant tumors. PMID:27123060

  13. A new synthetic ursolic acid derivative IUA with anti-tumor efficacy against osteosarcoma cells via inhibition of JNK signaling pathway.

    PubMed

    Chen, Jian; Fu, Haijian; Wang, Zhuoying; Yin, Fei; Li, Jianxin; Hua, Yingqi; Cai, Zhengdong

    2014-01-01

    Osteosarcoma is the most common primary malignant bone tumor in children and adolescents and is characterized by frequent metastasis and resistance to chemotherapy. Because osteosarcoma cells are not highly susceptible to current chemotherapy drugs, new alternative strategies for the treatment of osteosarcoma are needed. This study was undertaken to investigate the inhibitory effects of a new synthetic ursolic acid derivative IUA on osteosarcoma cells and to explore its molecular mechanism. We also intended to identify new therapeutic candidates. We used MTT assay to assess the effect of IUA on the proliferation of osteosarcoma cells. Western-blot analysis was performed to examine downstream molecular events. The Annexin V method was used to evaluate the effect of IUA on apoptosis of osteosarcoma cells. The cell cycle of IUA-treated cells was examined by flow cytometry, and the in vivo effects of this new ursolic acid derivative were evaluated in a mouse osteosarcoma model. The results showed that the new synthetic ursolic acid derivative IUA significantly decreased viability of osteosarcoma cells in vitro and in vivo. It could also induce apoptosis and G1 phase arrest of osteosarcoma cells. The JNK signaling pathway was significantly inhibited, and cleaved caspase-3 protein was increased. We concluded that the new synthetic ursolic acid derivative IUA induces proliferation inhibition and apoptosis of osteosarcoma cells in vitro and in vivo via the down-regulation of the JNK signaling pathway, making it a promising agent for the prevention and treatment of human osteosarcoma. © 2014 S. Karger AG, Basel.

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

    SciTech Connect

    Husmann, Knut; Ducommun, Pascal; Sabile, Adam A.; Pedersen, Else-Marie; Born, Walter; Fuchs, Bruno

    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 and 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.

  15. 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

  16. 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.

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

    SciTech Connect

    Liu, P.-S.; 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 human 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.

  18. Ezrin promotes invasion and migration of the MG63 osteosarcoma cell.

    PubMed

    Zhang, Jian; Zuo, Jianhong; Lei, Mingsheng; Wu, Song; Zang, Xiaofang; Zhang, Chaoyue

    2014-01-01

    Evidence shows that ezrin plays an important role in the development of some human malignancies. But the mechanism by which ezrin may affect tumor cell invasion and metastasis remains unclear. In this study, the expression of ezrin was verified in osteosarcoma (OS) cells and tissues by comparison with normal bone cells and tissues using Western blotting. OS-MG63 were transfected with pcDNA3.1-ezrin or pGenesil-1/shRNA-ezrin and the stably transfected cells were selected with G418 to yield the ezrin cell line. The OS-MG63 tumor cells were delivered by tail vein to female BALB/c to develop pulmonary metastasis model in vivo. Ezrin was identified as a direct target of miR-183 via a luciferase reporter carrying the 3'-untranslated region of ezrin. Migration assays and invasion assays were done with the transwells. Signaling pathway was studied by Western blotting and/or inhibitor. Ectopic overexpression of ezrin in OS cell line MG63 promoted tumor cell invasion and migration. Consistent with this, knockdown of ezrin inhibited tumor cell invasion and migration. Similar results were obtained in the experimental metastasis model in vivo. We identified ezrin as a direct target of miR-183. What is more, ectopic expression of ezrin could induce the expression of N-cadherin and enhance the activity of extracellular signal-regulated kinase (ERK) signaling. Collectively, these results suggest that ezrin as a direct target of miR-183 promotes the aggressiveness of OS via increased N-cadherin and activating ERK signaling.

  19. Construction of recombinant pEGFP-N1-hPer2 plasmid and its expression in osteosarcoma cells.

    PubMed

    Cheng, Anyuan; Zhang, Yan; Mei, Hongjun; Fang, Shuo; Ji, Peng; Yang, Jian; Yu, Ling; Guo, Weichun

    2016-04-01

    The aim of this study was to construct the eukaryotic expression vector pEGFP-N1-hPer2 and assess its expression in the human osteosarcoma cell line MG63. Total mRNA was extracted from human osteosarcoma MG63 cells, the human period 2 (hPer2) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the pEGFP-N1 vector, then the recombinant pEGFP-N1-hPer2 plasmid was constructed and transfected into MG63 cells using Lipofectamine 2000. The expression of hPer2 in MG63 cells was measured by quantitative RT-PCR and western blot analysis. The accurate construction of pEGFP-N1-hPer2 was verified by double enzyme digestion and DNA sequencing. hPer2 gene expression in the transfected cells was assessed by RT-qPCR and western blot analysis. In conclusion, the recombinant pEGFP-N1-hPer2 plasmid was constructed successfully, and expressed effectively in MG63 cells.

  20. miR-200bc/429 Inhibits Osteosarcoma Cell Proliferation and Invasion by Targeting PMP22

    PubMed Central

    Li, Xiaodong; Jiang, Han; Xiao, Lianping; Wang, Shusen; Zheng, Jinxin

    2017-01-01

    Background MicroRNAs (miRNAs) are small non-coding RNAs which play a crucial role in diverse biological processes and could contribute to cancer development and progression. MiR-200bc/429 have been found to be aberrantly expressed in osteosarcoma (OS). However, the features of miR-200bc/429 in the tumorigenesis and progress of OS remain poorly understood. Material/Methods The miR-200bc/429 expression was firstly identified in human OS clinical samples and cell lines by quantitative real-time PCR (qRT-PCR). After transfection with miR-200bc/429 mimics or negative control in U2OS or MG63 cells, cell proliferation was measured by CCK-8 assay. Following that, wound-healing assay and Transwell invasion assay were performed to evaluate cell migration and invasion, respectively. Finally, luciferase reporter assay and Western blot analysis were performed to determine if peripheral myelin protein-22 (PMP22) is a direct target of miR-200bc/429. Results Results revealed that miR-200bc/429 were significantly depressed in human OS tissues and cell lines by qRT-PCR. Then, restoration of miR-200bc/429 significantly inhibited cell proliferation (P<0.05) and invasion (P<0.05) in vitro. Luciferase reporter assay and Western blot analysis revealed that miR-200bc/429 could directly target PMP22 3′ untranslated region (UTR) and inhibit its expression in U2OS and MG63 cells. Conclusions These findings suggest that miR-200bc/429 inhibit OS cells proliferation and invasion by targeting PMP22, and function as a tumor suppressor and may be a patent molecular marker as well as a potential target for OS therapy. PMID:28234890

  1. Effects of long non-coding RNA SPRY4-IT1 on osteosarcoma cell biological behavior

    PubMed Central

    Xu, Jin; Ding, Ren; Xu, Yaozeng

    2016-01-01

    Recent findings indicate that long noncoding RNAs (lncRNAs) were dysregulated in many kinds of tumors including osteosarcoma (OS). SPRY4-IT1 has been recently revealed as oncogenic regulator in various cancers, while its clinical value and potential function in OS are still unknown. To investigate the role of SPRY4-IT1 in OS, we evaluated the expression SPRY4-IT1 in OS tissues and cell lines, and investigated the effect of SPRY4-IT1 siRNA on cell proliferation, migration and invasion of OS in vitro. Our result showed that SPRY4-IT1 was upregulated in OS tissues. Further experiments revealed that SPRY4-IT1 knockdown significantly inhibited OS cells proliferation by causing G1 arrest and promoting apoptosis. Furthermore, inhibitory effects of SPRY4-IT1 on cell migration and invasion were partly associated with EMT process. In conclusion, these data suggest that SPRY4-IT1 could be an oncogene for OS, and may be served as a candidate target for new therapies in human OS. PMID:28078006

  2. Dynamic metabolic transformation in tumor invasion and metastasis in mice with LM-8 osteosarcoma cell transplantation.

    PubMed

    Hua, Yingqi; Qiu, Yunping; Zhao, Aihua; Wang, Xiaoyan; Chen, Tianlu; Zhang, Zhiyu; Chi, Yi; Li, Quan; Sun, Wei; Li, Guodong; Cai, Zhengdong; Zhou, Zhanxiang; Jia, Wei

    2011-08-05

    While extensive evidence indicates that tumor cells shift their global metabolic programs, the molecular details of the metabolic transformation in tumor invasion, progression, and metastasis remain largely unknown. Characterization of the time-dependent metabolic shift during the tumor invasion, development, and metastasis will describe an important aspect of tumor phenotypes and potentially allow us to design therapies that inhibit tumor cell movement. In this study, a metabonomic study was performed to characterize the global metabolic changes during the process of tumor invasion and metastasis to lung in a mouse model with subcutaneous transplantation of murine osteosarcoma cell line (LM8). The serum metabolic profiling revealed that many key metabolites in glycolysis and tricarboxylic acid (TCA) cycle, as well as most of the amino acids were elevated at rapidly growing stage of tumor, presumably resulting from a high energy demand and turnover of anabolic metabolism during the tumor cell proliferation. Serum levels of succinic acid and proline significantly increased (with fold change FC = 10.75 and 4.43, relative to controls) among all the metabolites in the third week. The serum metabolic profile of lung metastasis at week 4 was different from that at week 3, in that most of previously increased serum metabolites were found decreased, except for cholesterol and several free fatty acids, suggesting lowered carbohydrate and amino acids metabolism, but an elevated lipid metabolism associated with tumor metastasis.

  3. In vitro generation of cytotoxic T lymphocyte response using dendritic cell immunotherapy in osteosarcoma

    PubMed Central

    He, Ye-Teng; Zhang, Qing-Min; Kou, Quan-Chun; Tang, Bo

    2016-01-01

    Immunotherapy with tumor lysate-pulsed dendritic cells (DCs) is one of the breakthrough strategies used in the treatment of cancer. However, DC-based immunotherapies for osteosarcoma are limited. In the present study, preclinical studies of a C3H osteosarcoma mouse model (produced by subcutaneous injection of LM8 murine osteosarcoma cells) validated the concept that LM8 cell lysate-pulsed bone marrow-derived DCs may evoke a more potent immune response compared with DCs that have been matured using polyinosinic:polycytidylic acid (poly I:C). A cytotoxic T lymphocyte (CTL) response was established using two groups of C3H mice (n=9) with osteosarcoma; the treatment group consisted of LM8 cell lysate-pulsed DCs and the control group consisted of DCs matured using poly I:C. Each group was immunized with doses of 1×106 cells twice per week for 3 weeks. No difference in the expression of cluster of differentiation markers was identified in the two groups. DCs pulsed with LM8 cell lysate were associated with the increased induction of CTL activity. Serum interferon-γ levels were increased in mice that received DCs pulsed with LM8 cell lysate compared with that in the poly I:C-matured DC group (P<0.041). Serum interleukin-4 was decreased in the treatment group vs. the control group (P<0.033). A mixed lymphocyte reaction assay confirmed that LM8-DC immunotherapy may evoke a significant antigen-specific immune response in a mouse model. The present study reveals promising data on efficacy of a DC-based immunotherapy in the treatment of osteosarcoma; however, further clinical studies are warranted. PMID:27446401

  4. Enhanced Stim1 expression is associated with acquired chemo-resistance of cisplatin in osteosarcoma cells.

    PubMed

    Sun, Xilong; Wei, Qiang; Cheng, Jie; Bian, Yanzhu; Tian, Congna; Hu, Yujing; Li, Huijie

    2017-07-01

    Osteosarcoma is the most common primary malignant bone tumor. Although cisplatin is the primary chemotherapy used in osteosarcoma treatment, the cisplatin resistance remains a big challenge for improving overall survival. The store-operated calcium (Ca(2+)) entry (SOCE) and its major mediator Stim1 have been shown to be implicated in a number of pathological processes typical for cancer. In this study, we showed that Stim1 expression was significantly increased in chemo-resistant osteosarcoma tissues compared with chemo-sensitivity tissues. Patients with Sitm1 expression exhibited poorer overall survival than Stim1-negative patients. Moreover, un-regulation of Stim1 expression and SOCE were also observed in cisplatin-resistant MG63/CDDP cells compared with their parental cells. Cisplatin treatment obviously reduced Stim1 expression and SOCE in cisplatin-sensitivity MG63 cells, but had no effects on MG63/CDDP cells. In addition, cisplatin resulted in a more pronounced increase of endoplasmic reticulum (ER) stress in MG63 cells than in their resistant variants, which was evidenced by the activation of molecular markers of ER stress, GRP78, CHOP and ATF4. Knockdown of Stim1 using siRNA remarkably enhanced cisplatin-induced apoptosis and ER stress in MG63/CDDP cells, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of Stim1 markedly reversed apoptosis and ER stress following cisplatin treatment. Taken together, these results demonstrate that Stim1 as well as Ca(2+) entry contributes cisplatin resistance via inhibition of ER stress-mediated apoptosis, and provide important clues to the mechanisms involved in cisplatin resistance for osteosarcoma treatment. Stim1 represents as a target of cisplatin and blockade of Stim1-mediated Ca(2+) entry may be a useful strategy to improve the efficacy of cisplatin to treat osteosarcoma.

  5. Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression.

    PubMed

    Cheng, Hsin-Lin; Hsieh, Ming-Ju; Yang, Jia-Sin; Lin, Chiao-Wen; Lue, Ko-Haung; Lu, Ko-Hsiu; Yang, Shun-Fa

    2016-06-07

    Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma.

  6. Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression

    PubMed Central

    Cheng, Hsin-Lin; Hsieh, Ming-Ju; Yang, Jia-Sin; Lin, Chiao-Wen; Lue, Ko-Haung; Lu, Ko-Hsiu; Yang, Shun-Fa

    2016-01-01

    Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma. PMID:27144433

  7. The HIF-1α/CXCR4 pathway supports hypoxia-induced metastasis of human osteosarcoma cells.

    PubMed

    Guan, Guofeng; Zhang, Yinglong; Lu, Yao; Liu, Lijuan; Shi, Doufei; Wen, Yanhua; Yang, Lianjia; Ma, Qiong; Liu, Tao; Zhu, Xiaodong; Qiu, Xiuchun; Zhou, Yong

    2015-02-01

    HIF-1α mediates hypoxia-induced expression of the chemokine receptor CXCR4 and contributes to metastasis in many different cancers. We have previously shown that hypoxia promotes migration of human osteosarcoma cells by activating the HIF-1α/CXCR4 pathway. Here, immunohistochemical analysis showed that unlike control osteochondroma samples, osteosarcoma specimens were characterized by elevated expression levels of HIF-1α and CXCR4. Moreover, we found that hypoxia-induced invasiveness was more pronounced in high metastatic potential F5M2 osteosarcoma cells than in low metastatic potential F4 cells, and that this induction was sensitive to treatment with the CXCR4 antagonist AMD3100 and the HIF-1α inhibitor KC7F2. Interestingly, hypoxia-induced CXCR4 expression persisted after cultured osteosarcoma cells were returned to normoxic conditions. These observations were confirmed by experiments in a mouse model of osteosarcoma lung metastasis showing that hypoxia stimulation of pulmonary metastasis was greater in F5M2 than in F4 cells, and was sensitive to treatment with AMD3100. Our study provides further evidence of the contributions of hypoxia and the HIF-1α/CXCR4 pathway to the progression of osteosarcoma, and suggests that this axis might be efficiently leveraged in the development of novel osteosarcoma therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Long non-coding RNA MALAT1 for promoting metastasis and proliferation by acting as a ceRNA of miR-144-3p in osteosarcoma cells

    PubMed Central

    Wang, Ningning; Li, Pengcheng; Zeng, Xiandong; Zhang, Weiguo

    2017-01-01

    Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases including osteosarcoma. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is overly expressed in osteosarcoma. But the function and mechanism it works on in osteosarcoma proliferation and metastasis mediated by Rho associated coiled-coil containing protein kinase 1 (ROCK1) and Rho associated coiled-coil containing protein kinase 2 (ROCK2) remain unclear. In the present study, an elevated MALAT1 was found in osteosarcoma tissues and cell lines, and the elevated MALAT1 was correlated with a poor prognosis in osteosarcoma patients. The functional experiments show that a decreased MALAT1 could remarkably inhibit osteosarcoma cell metastasis and proliferation but induce cell cycle arrest, indicating that MALAT1 functioned as an oncogene in osteosarcoma. Furthermore, we confirmed that MALAT1 and ROCK1/ROCK2 which were targeted by microRNA-144-3p (miR-144-3p) shared the same miR-144-3p combining site. Furthermore, the constructed luciferase assay verified that MALAT1 was a target of miR-144-3p. Additionally, the results of a qRT-PCR demonstrated that MALAT1 and miR-144-3p repressed each other's expression in a reciprocal manner. Finally, we affirmed that an overexpression of MALAT1 inhibited ROCK1/ROCK2 expression and its mediated metastasis and proliferation by working as a competitive endogenous RNA (ceRNA) via miR-144-3p. In summary, the findings of this study based on the ceRNA theory, combining the research foundation of miR-144-3p, ROCK1 and ROCK2, taking MALAT1 as a new point of study, provided new insights into molecular level proliferation reversal and metastasis of osteosarcoma. PMID:28938647

  9. Long non-coding RNA MALAT1 for promoting metastasis and proliferation by acting as a ceRNA of miR-144-3p in osteosarcoma cells.

    PubMed

    Wang, Yong; Zhang, Yueyang; Yang, Tao; Zhao, Wei; Wang, Ningning; Li, Pengcheng; Zeng, Xiandong; Zhang, Weiguo

    2017-08-29

    Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases including osteosarcoma. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is overly expressed in osteosarcoma. But the function and mechanism it works on in osteosarcoma proliferation and metastasis mediated by Rho associated coiled-coil containing protein kinase 1 (ROCK1) and Rho associated coiled-coil containing protein kinase 2 (ROCK2) remain unclear. In the present study, an elevated MALAT1 was found in osteosarcoma tissues and cell lines, and the elevated MALAT1 was correlated with a poor prognosis in osteosarcoma patients. The functional experiments show that a decreased MALAT1 could remarkably inhibit osteosarcoma cell metastasis and proliferation but induce cell cycle arrest, indicating that MALAT1 functioned as an oncogene in osteosarcoma. Furthermore, we confirmed that MALAT1 and ROCK1/ROCK2 which were targeted by microRNA-144-3p (miR-144-3p) shared the same miR-144-3p combining site. Furthermore, the constructed luciferase assay verified that MALAT1 was a target of miR-144-3p. Additionally, the results of a qRT-PCR demonstrated that MALAT1 and miR-144-3p repressed each other's expression in a reciprocal manner. Finally, we affirmed that an overexpression of MALAT1 inhibited ROCK1/ROCK2 expression and its mediated metastasis and proliferation by working as a competitive endogenous RNA (ceRNA) via miR-144-3p. In summary, the findings of this study based on the ceRNA theory, combining the research foundation of miR-144-3p, ROCK1 and ROCK2, taking MALAT1 as a new point of study, provided new insights into molecular level proliferation reversal and metastasis of osteosarcoma.

  10. Id-1 promotes osteosarcoma cell growth and inhibits cell apoptosis via PI3K/AKT signaling pathway

    SciTech Connect

    Hao, Liang; Liao, Qi; Tang, Qiang; Deng, Huan; Chen, Lu

    2016-02-12

    Accumulating evidence reveals that Id-1 is upregulated and functions as a potential tumor promoter in several human cancer types. However, the role of Id-1 in osteosarcoma (OS) is unknown. In present study, we found that Id-1 expression was elevated in OS tissues than adjacent normal bone tissues. More importantly, we demonstrated that overexpression of Id-1 is significantly correlated with tumor progression and poor survival in OS patients. Furthermore, increased expression of Id-1 was observed in OS cell lines and ectopic expression of Id-1 significantly enhanced in vitro cell proliferation and promoted in vivo tumor growth, whereas knockdown of Id-1 suppressed OS cells growth. Moreover, our experimental data revealed that Id-1 promotes cell proliferation by facilitating cell cycle progression and inhibits cell apoptosis. Mechanistically, the effects of Id-1 in OS cells is at least partly through activation of PI3K/Akt signaling pathway. Therefore, we identified a tumorigenic role of Id-1 in OS and suggested a potential therapeutic target for OS patients. - Highlights: • Id-1 expression is positively correlated in OS patients with poor prognosis. • Overexpression of Id-1 promotes OS cell growth in vitro and in vivo. • Id-1induces cell cycle progression and inhibits cell apoptosis. • PI3K/Akt signaling pathway contributed to the oncogenic effects of Id-1 in OS cells.

  11. Wnt inhibitory factor 1 decreases tumorigenesis and metastasis in osteosarcoma.

    PubMed

    Rubin, Elyssa M; Guo, Yi; Tu, Khoa; Xie, Jun; Zi, Xiaolin; Hoang, Bang H

    2010-03-01

    It has been reported that the progression of osteosarcoma was closely associated with the aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in NHOst and osteosarcoma cell lines was determined by real-time reverse transcription-PCR, methylation-specific PCR, and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared with normal human osteoblasts, WIF-1 mRNA and protein levels were significantly downregulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by the s.c. injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent antiosteosarcoma effect in vivo in mouse models. Therefore, the reexpression of WIF-1 in WIF-1-deficient osteosarcoma represents a potential novel treatment and preventive strategy.

  12. Tumorigenesis and spontaneous metastasis by luciferase-labeled human xenograft osteosarcoma cells in nude mice.

    PubMed

    Du, Lin; Xu, Wen-ting; Fan, Qi-ming; Tu, Bing; Shen, Yang; Yan, Wei; Tang, Ting-ting; Wang, You

    2012-11-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogenesis of this aggressive tumor. Bioluminescence technology favored tracing cancer cells in vivo. In this study, an OS model was described and evaluated using human OS cell line, Saos2, labeled with luciferase (Saos2-luc). Saos2 cells were infected by lentivirus loading a firefly luciferase gene. Luciferase expression of Saos2-luc cells was characterized both in vitro and in vivo. Specific biologic and oncologic features of Saos2-luc cells were analyzed. The OS was established as orthotopic xenografts in nude mice. Both orthotopic tumors and spontaneous lung metastasis were analyzed. Tumorigenesis and spontaneous lung metastasis in nude mice could be monitored in vivo through in vivo imaging system. The enhancement in proliferation, migration and invasion abilities and the attenuation in adhesion ability were observed in Saos2-luc cells compared with Saos2 cells. Furthermore, there were the up-regulation of Osteocalcin, CCR10, CXCR1 and ID1 and the down-regulation of ALP, collagen I, CCR1, CCR3, CXCR3, NID and N-cadherin in Saos2-luc cells compare to Saos2 cells. The rate of spontaneous lung metastasis in Saos2-luc cells was higher than that in Saos2 cells, although without significant difference. Lentivirus transfection may cause alteration of gene expression profiles and further biological functions. This model can be used in the elucidation of molecular mechanisms of tumorigenesis and the screening of new therapeutic agents.

  13. Giant cell-rich osteosarcoma of the parotid gland: An exceptionally rare entity at an unusual site.

    PubMed

    Huang, Eric C; Ghazikhanian, Varand; Qian, Xiaohua

    2016-12-01

    Giant cell-rich osteosarcoma is a rare histologic variant of conventional osteosarcoma that affects mainly the extremities. Extraskeletal giant cell-rich osteosarcoma is therefore exceedingly rare. Here, we report the first case of this uncommon tumor involving the parotid gland in a 62-year-old male who presented with initial right jaw swelling. Radiologic work-up revealed a 6.2 cm mass involving the right parotid gland. Fine-needle aspiration cytology showed numerous multinucleated giant cells in a background of dyshesive epithelioid cells and rare clusters of spindle stromal cells, suspicious for malignancy. The subsequent excisional biopsy showed histopathologic features diagnostic for giant cell-rich osteosarcoma. Diagn. Cytopathol. 2016;44:1107-1111. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Inactivation of human osteosarcoma cells in vitro by {sup 211}At-TP-3 monoclonal antibody: Comparison with astatine-211 and external-beam X rays

    SciTech Connect

    Larsen, R.H. |; Bruland, O.S.; Hoff, P.; Alstad, J.; Lindmo, T.; Rofstad, E.K.

    1994-08-01

    The potential usefulness of {alpha}-particle radioimmunotherapy in the treatment of osteosarcoma was studied in vitro by using the monoclonal antibody TP-3 and cells of three human osteosarcoma cell lines (OHS, SAOS and KPDX) differing in antigen expression. Cell survival curves were established after treatment with (a) {sup 211}At-TP-3 of different specific activities, (b) {sup 211}At-labeled bovine serum albumin (BSA), (c) free {sup 211}At and (d) external-beam X rays. The three osteosarcoma cell lines showed similar survival curves, whether treated with external-beam X rays, {sup 211}At-BSA or free {sup 211}At. The D{sub o}`s were lower for free {sup 211}At than for {sup 211}At-BSA. The survival curves for {sup 211}At-TP-3 treatment, on the other hand, differed significantly among the cell lines, suggesting that sensitivity to {sup 211}At-TP-3 treatment was governed by cellular properties other than sensitivity to external-beam X rays. The cellular property most important for sensitivity to {sup 211}At-TP-3 treatment was the antigen expression. Cell inactivation after {sup 211}At-TP-3 treatment increased substantially with increasing specific activity of the {sup 211}At-TP-3. At high specific activities, the cytotoxic effect of {sup 211}At-TP-3 was significantly higher than that of {sup 211}At-BSA. In conclusion, {sup 211}At-TP-3 has the potential to give clinically favorable therapeutic ratios in the treatment of osteosarcoma. 39 refs., 5 figs., 2 tabs.

  15. 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

  16. IR/IGF1R signaling as potential target for treatment of high-grade osteosarcoma

    PubMed Central

    2013-01-01

    Background High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment. Methods We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma – mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays. Results OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with IC50s below 100 nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines. Conclusions This study provides an in vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma. PMID:23688189

  17. Altered morphology, nuclear stability and adhesion of highly metastatic derivatives of osteoblast-like SAOS-2 osteosarcoma cells.

    PubMed

    Muff, Roman; Nigg, Natalie; Gruber, Philipp; Walters, Denise; Born, Walter; Fuchs, Bruno

    2007-01-01

    Metastasis is the leading cause of death in patients with osteosarcoma (OS). High alkaline phosphatase (ALP) activity and resistance to chemotherapy are independent predictors of poor clinical outcome of osteosarcoma. Here, the osteoblastic phenotype, cell and nuclear morphology, cell adhesion and drug resistance of the SAOS-2 cell line and two in vivo selected highly metastatic derivatives, LM5 and LM7, were compared. ALP activity and deposition of mineralized extracellular matrix were the same in the parental SAOS-2 and the LM5 and LM7 cells, but parathyroid hormone (PTH)-stimulated cAMP accumulation was lost in the LM7 cells. The LM5 and LM7 cells were smaller than the parental SAOS-2 cells, and 10% of the LM7 cells had distorted nuclei. The adhesion of LM5 and LM7 cells was decreased when compared to SAOS-2 cells. The cytotoxic responses of the SAOS-2, LM5 and LM7 cells to Cisplatin, Doxorubicin and Etoposide were indistinguishable. The increased metastatic potential of LM5 and LM7 as compared to SAOS-2 cells is not associated with a substantial change of the osteoblastic phenotype or of the cytotoxic response to current chemotherapeutic drugs. The decrease in cell size and altered cell adhesion, reflecting cytoskeletal rearrangement, together with increased nuclear instability and partial dedifferentiation, as revealed by the loss of PTH responsiveness in LM7 cells, may account for the higher metastatic potential of the LM5 and LM7 sublines as compared to the parental SAOS-2 cells.

  18. Comparative proteomic investigation of metastatic and non-metastatic osteosarcoma cells of human and canine origin.

    PubMed

    Roy, Jahnabi; Wycislo, Kathryn L; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2017-01-01

    Osteosarcoma is the most common bone cancer in dogs and people. In order to improve clinical outcomes, it is necessary to identify proteins that are differentially expressed by metastatic cells. Membrane bound proteins are responsible for multiple pro-metastatic functions. Therefore characterizing the differential expression of membranous proteins between metastatic and non-metastatic clonal variants will allow the discovery of druggable targets and consequently improve treatment methodology. The objective of this investigation was to systemically identify the membrane-associated proteomics of metastatic and non-metastatic variants of human and canine origin. Two clonal variants of divergent in vivo metastatic potential from human and canine origins were used. The plasma membranes were isolated and peptide fingerprinting was used to identify differentially expressed proteins. Selected proteins were further validated using western blotting, flow cytometry, confocal microscopy and immunohistochemistry. Over 500 proteins were identified for each cell line with nearly 40% of the proteins differentially regulated. Conserved between both species, metastatic variants demonstrated significant differences in expression of membrane proteins that are responsible for pro-metastatic functions. Additionally, CD147, CD44 and vimentin were validated using various biochemical techniques. Taken together, through a comparative proteomic approach we have identified several differentially expressed cell membrane proteins that will help in the development of future therapeutics.

  19. BMI1 Is Expressed in Canine Osteosarcoma and Contributes to Cell Growth and Chemotherapy Resistance

    PubMed Central

    Gandour-Edwards, Regina; Withers, Sita S.; Holt, Roseline; Rebhun, Robert B.

    2015-01-01

    BMI1, a stem cell factor and member of the polycomb group of genes, has been shown to contribute to growth and chemoresistance of several human malignancies including primary osteosarcoma (OSA). Naturally occurring OSA in the dog represents a large animal model of human OSA, however the potential role of BMI1 in canine primary and metastatic OSA has not been examined. Immunohistochemical staining of canine primary and metastatic OSA tumors revealed strong nuclear expression of BMI1. An identical staining pattern was found in both primary and metastatic human OSA tissues. Canine OSA cell lines (Abrams, Moresco, and D17) expressed high levels of BMI1 compared with canine osteoblasts and knockdown or inhibition of BMI1 by siRNA or by small molecule BMI1-inhibitor PTC-209 demonstrated a role for BMI1 in canine OSA cell growth and resistance to carboplatin and doxorubicin chemotherapy. These findings suggest that inhibition of BMI1 in primary or metastatic OSA may improve response to chemotherapy and that the dog may serve as a large animal model to evaluate such therapy. PMID:26110620

  20. A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro

    PubMed Central

    Rathore, Kusum; Cekanova, Maria

    2015-01-01

    Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo. PMID:26451087

  1. MiR-598: A tumor suppressor with biomarker significance in osteosarcoma.

    PubMed

    Liu, Kai; Sun, Xiaolu; Zhang, Yingang; Liu, Liang; Yuan, Qiling

    2017-11-01

    Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents. Identifying specific and sensitive biomarkers is beneficial to early detection and improvement of life qualities and overall survival rates of osteosarcoma patients. Realtime PCR was used to detect the expression of miR-598. CCK-8 assay was employed to detect the proliferation of osteosarcoma cells, while transwell assays were used to examine the migration and invasion. Tumor xenograft experiments were performed to test the in vivo malignancy of osteosarcoma cells. Co-culture experiment was used to study the relationship between osteosarcoma cells and osteoblast. Realtime PCR, Western Blotting and luciferase report assays were conducted for the target genes analysis. Using a cohort of 20 cases of osteosarcoma and paired adjacent tissue samples, we found that miR-598 expression was decreased in osteosarcoma tissues and serum, as well as the osteosarcoma cell lines. Over expression of miR-598 suppressed the proliferation, migration, and invasion of osteosarcoma cells, while inhibition of miR-598 expression stimulated the proliferation, migration, and invasion. However, MiR-598 had no effect on osteosarcoma cell apoptosis. Data from nude mice further demonstrated the inhibitory role of miR-598 in osteosarcoma progression in vivo. Mechanically, miR-598 played its role by modulating osteoblastic differentiation in the microenvironment and targeting PDGFB and MET. Our findings enrich the knowledge of miR-598 in osteosarcoma progression, and reveal miR-598 as a promising diagnostic, prognostic, therapeutic biomarker for osteosarcoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Silencing of NUF2 inhibits proliferation of human osteosarcoma Saos-2 cells.

    PubMed

    Fu, H-L; Shao, L

    2016-01-01

    NUF2 (NUF2, Ndc80 kinetochore complex component), which is essential for kinetochore-microtubule attachment in mitosis, has emerged as a critical mediator of the cell cycle in multiple tumour occurrences. In the present study, we aimed to investigate the role of NUF2 in osteosarcoma, one of the most common primary bone tumours in children and young adults. Lentivirus-mediated short-hairpin RNA (shRNA) targeting NUF2 (Lv-shNUF2) was employed for evaluation in human osteosarcoma Saos-2 cells. After NUF2 silencing, the proliferation of Saos-2 cells was significantly inhibited, as determined by the MTT assay. The colony forming ability was also significantly decreased in Saos-2 cells infected with Lv-shNUF2. Flow cytometry revealed that downregulation of NUF2 in Saos-2 cells caused a remarkable accumulation of the cell population in the S phase. Furthermore, the expression levels of cell cycle regulators cyclin A and cyclin-dependent kinase 2 (CDK2) were notably decreased, whereas those of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, were increased in response to NUF2 knockdown in Saos-2 cells. Our findings suggest that NUF2 might modulate cell proliferation via cell cycle control in Saos-2 cells. Downregulation of NUF2 by shRNA might be a novel strategy for early treatment of osteosarcoma using molecular-targeting therapy.

  3. Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma.

    PubMed

    Marko, Tracy A; Shamsan, Ghaidan A; Edwards, Elizabeth N; Hazelton, Paige E; Rathe, Susan K; Cornax, Ingrid; Overn, Paula R; Varshney, Jyotika; Diessner, Brandon J; Moriarity, Branden S; O'Sullivan, M Gerard; Odde, David J; Largaespada, David A

    2016-12-14

    Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype.

  4. Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

    PubMed Central

    Marko, Tracy A.; Shamsan, Ghaidan A.; Edwards, Elizabeth N.; Hazelton, Paige E.; Rathe, Susan K.; Cornax, Ingrid; Overn, Paula R.; Varshney, Jyotika; Diessner, Brandon J.; Moriarity, Branden S.; O’Sullivan, M. Gerard; Odde, David J.; Largaespada, David A.

    2016-01-01

    Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype. PMID:27966608

  5. The clinical significance of the Ezrin gene and circulating tumor cells in osteosarcoma

    PubMed Central

    Zhong, Guang-Xian; Feng, Shao-Dan; Shen, Rongkai; Wu, Zhao-Yang; Chen, Fei; Zhu, Xia

    2017-01-01

    Purpose The aim of this study was to investigate the clinical significance of circulating tumor cells (CTCs) in the peripheral blood of an osteosarcoma and the Ezrin gene expressed in CTCs. Patients and methods CTC enrichment was done with CanPatrol™ CTC enrichment technique in 41 patients with osteosarcoma. The characterization of CTCs was performed using a multiple messenger RNA in situ analysis (MRIA). The expression of the Ezrin gene in CTCs was detected by RNA probe technology. The correlations of CTC counts, cell type and the expression level of the Ezrin gene with clinical stage and metastasis of osteosarcoma were analyzed using SPSS 16.0 software. Results The CTC counts correlated significantly with Enneking stage (P<0.001). The ratio of mesenchymal CTCs correlated with the distant metastases (P<0.001). Ezrin gene expression in CTCs correlated significantly with distant metastases (χ2=152.51, P=0.000). Conclusion The ratio of mesenchymal CTCs in the peripheral blood of osteosarcoma correlates with distant metastases. High expression of Ezrin gene in CTCs correlates with distant metastases. PMID:28223819

  6. Resveratrol inhibits canonical Wnt signaling in human MG-63 osteosarcoma cells

    PubMed Central

    ZOU, YONGGEN; YANG, JIEXIANG; JIANG, DIANMING

    2015-01-01

    In the last 30 years, the 5-year-survival rate of patients with osteosarcoma has not improved as a result of the low prevalence and large tumor heterogeneity. Therefore, the development of novel drugs for the treatment of osteosarcoma is urgently required. The present study aimed to identify potential novel drugs for the treatment of osteosarcoma, thus used β-catenin as a target and performed high content screening. In a total of 14 botanical extracts assessed, resveratrol markedly downregulated the expression of β-catenin and significantly inhibited MG-63 cell proliferation. CCK-8 assay was used to confirm the anti-osteosarcoma effect of resveratrol and flow cytometry and western blotting were performed to analyze the underlying mechanisms of the proapoptotic effects of resveratrol. β-catenin is a vital member of the canonical Wnt signaling pathway and, therefore, the target genes of this pathway were further analyzed. The results of this analysis demonstrated that resveratrol suppressed the MG-63 cells by inhibiting the canonical Wnt signaling pathway. PMID:26398440

  7. Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Scott, D. K.; Weaver, W. R.; Clohisy, J. C.; Brakenhoff, K. D.; Kahn, A. J.; Partridge, N. C.

    1992-01-01

    The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS).

  8. Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Scott, D. K.; Weaver, W. R.; Clohisy, J. C.; Brakenhoff, K. D.; Kahn, A. J.; Partridge, N. C.

    1992-01-01

    The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS).

  9. alpha-Synuclein stimulates differentiation of osteosarcoma cells: relevance to down-regulation of proteasome activity.

    PubMed

    Fujita, Masayo; Sugama, Shuei; Nakai, Masaaki; Takenouchi, Takato; Wei, Jianshe; Urano, Tomohiko; Inoue, Satoshi; Hashimoto, Makoto

    2007-02-23

    Because a limited study previously showed that alpha-synuclein (alpha-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether alpha-syn might be involved in the regulation of tumor differentiation. For this purpose, alpha-syn and its non-amyloidogenic homologue beta-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with beta-syn-overexpressing and vector-transfected cells, alpha-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in alpha-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In alpha-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in alpha-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of alpha-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of alpha-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of alpha-syn regulation of tumor differentiation and neuropathological effects of alpha-syn may considerably overlap with each other.

  10. Antibacterial Activity of Elephant Garlic and Its Effect against U2OS Human Osteosarcoma Cells

    PubMed Central

    Huang, Zehao; Ren, Jianwu

    2013-01-01

    Objective(s): The present study was designed to investigate the antibacterial function and pharmacological effect of elephant garlic (Allium ampeloprasum var. ampeloprasum) on U2OS human osteosarcoma cells. Materials and Methods: Seven kinds of bacteria were reconstituted, inoculated and tested in this research to evaluate elephant garlic antibacterial activity. By the means of FACS analysis, cell proliferation assay, confocal laser scanning microscopy and Transwell migration assays, the effect of elephant garlic against U2OS human osteosarcoma cells was unveiled. Rerults: The antimicrobial activity of elephant garlic was stronger than ampicillin when used against Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus actinomycetes, and gray actinomycetes. Even at a very low concentration (12.5%), elephant garlic still had an antibacterial effect on common bacteria E. coli and S. aureus. The G0/G1 ratio of elephant garlic treated group cells increased while S phase decreased. Elephant garlic extract inhibited the growth of human osteosarcoma cells, U2OS, through preventing the transition from G1 phase to S phase. It reduced osteosarcoma cell, U2OS, invasion ability and significantly increased the proportion of apoptosis. It significantly affected the cytoskeleton generation. Conclusion: Elephant garlic exhibits antibacterial property and has an inhibitory effect on osteosarcoma cells (U2OS) proliferation and cell activity, suggesting the mechanism of its anticancer effects on U2OS human osteosarcoma cells. PMID:24379966

  11. High Expression of XRCC6 Promotes Human Osteosarcoma Cell Proliferation through the β-Catenin/Wnt Signaling Pathway and Is Associated with Poor Prognosis

    PubMed Central

    Zhu, Bin; Cheng, Dongdong; Li, Shijie; Zhou, Shumin; Yang, Qingcheng

    2016-01-01

    Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with the aim to reveal the expression and biological function of XRCC6 in OS and elucidate the potential mechanism. The mRNA expression level of XRCC6 was measured in osteosarcoma cells and OS samples by quantitative transcription-PCR (qRT-PCR). The expression of XRCC6 protein was measured using Western blot and immunohistochemical staining in osteosarcoma cell lines and patient samples. Cell Counting Kit 8 (CCK8), colony-forming and cell cycle assays were used to test cell survival capacity. We found that XRCC6 was overexpressed in OS cells and OS samples compared with the adjacent non-tumorous samples. High expression of XRCC6 was correlated with clinical stage and tumor size in OS. Reduced expression of XRCC6 inhibits OS cell proliferation through G2/M phase arrest. Most importantly, further experiments demonstrated that XRCC6 might regulate OS growth through the β-catenin/Wnt signaling pathway. In conclusion, these findings indicate that XRCC6 exerts tumor-promoting effects for OS through β-catenin/Wnt signaling pathway. XRCC6 may serve as a novel therapeutic target for OS patients. PMID:27455247

  12. Apigenin induces apoptosis through mitochondrial dysfunction in U-2 OS human osteosarcoma cells and inhibits osteosarcoma xenograft tumor growth in vivo.

    PubMed

    Lin, Chin-Chung; Chuang, Ya-Ju; Yu, Chien-Chih; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Jo-Hua; Lin, Jing-Pin; Tang, Nou-Ying; Huang, An-Cheng; Chung, Jing-Gung

    2012-11-14

    The cytostatic drug from natural products has acted as a chemotherapeutic agent used in treatment of a wide variety of cancers. Apigenin, a type of flavonoid, exhibits anticancer actions, but there is no report to show that apigenin induced apoptosis in osteosarcoma cells. The aim of this study was to investigate the effects of apigenin on U-2 OS human osteosarcoma cells and clarify that the apigenin-induced apoptosis-associated signals. The cytotoxic effects of apigenin were examined by culturing U-2 OS cells with or without apigenin. The percentage of viable cells via PI staining, apoptotic cells, productions of ROS and Ca²⁺, and the level of mitochondrial membrane potential (ΔΨm) were assayed by flow cytometry. The levels of apoptosis-related proteins were measured by immunoblotting. Results indicated that apigenin significantly decreased cell viability. Apigenin effectively induced apoptosis through the activations of caspase-3, -8, -9, and BAX and promoted the release of AIF in U-2 OS cells. In nude mice bearing U-2 OS xenograft tumors, apigenin inhibited tumor growth. In conclusion, apigenin has anticancer properties for induction of cell apoptosis in U-2 OS cells and suppresses the xenograft tumor growth. These findings offer novel information that apigenin possibly possesses anticancer activity in human osteosarcoma.

  13. Minnelide reduces tumor burden in preclinical models of osteosarcoma

    PubMed Central

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

    2015-01-01

    Osteosarcoma is the most common bone cancer in children and adolescents with a five-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 NF-κB pathway. PMID:23499892

  14. Clinical and biological significance of PIM1 kinase in osteosarcoma.

    PubMed

    Liao, Yunfei; Feng, Yong; Shen, Jacson; Gao, Yan; Cote, Gregory; Choy, Edwin; Harmon, David; Mankin, Henry; Hornicek, Francis; Duan, Zhenfeng

    2016-07-01

    Osteosarcoma is the most prevalent histological form of primary malignant bone tumor. The majority of osteosarcoma patients have limited alternative therapeutic options and metastatic patients generally have a poor prognosis. Proto-oncogene serine/threonine-protein kinase PIM1 is associated with growth and survival of many kinds of tumor cells. However, the role of PIM1 in osteosarcoma remains largely unknown. In this study, we investigated the functional and therapeutic relevance of PIM1 as a putative target in osteosarcoma. We found PIM1 was highly expressed in various osteosarcoma cell lines and in tumor tissues from osteosarcoma patients. Tissue microarray and immunohistochemistry analysis showed that the overall and disease-free survival rate of patients with high levels of PIM1 protein expression were significantly shorter than patients with low levels. High levels of PIM1 were also associated with present metastasis and can be considered as an independent prognostic factor in osteosarcoma patients. Knockdown of PIM1 expression by synthetic siRNA or shRNA greatly inhibited cell growth, migration, and invasion. Moreover, these changes accompanied with down-regulation of anti-apoptotic protein Bcl-2. The similar results were obtained in osteosarcoma cells treated with PIM1 specific inhibitor (SMI-4a). These results suggest that PIM1 kinase is critical for the growth and metastasis of osteosarcoma cells and can be a potential therapeutic target for osteosarcoma treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1185-1194, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  15. βig-h3 Promotes Human Osteosarcoma Cells Metastasis by Interacting with Integrin α2β1 and Activating PI3K Signaling Pathway

    PubMed Central

    Sun, Zhen; Gao, Bo; He, Shu; Han, Yue-Hu; Fan, Jing; Yang, Liu; Tang, Juan; Luo, Zhuo-Jing

    2014-01-01

    Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment. PMID:24595049

  16. βig-h3 promotes human osteosarcoma cells metastasis by interacting with integrin α2β1 and activating PI3K signaling pathway.

    PubMed

    Guo, Yun-Shan; Zhao, Rui; Ma, Jie; Cui, Wei; Sun, Zhen; Gao, Bo; He, Shu; Han, Yue-Hu; Fan, Jing; Yang, Liu; Tang, Juan; Luo, Zhuo-Jing

    2014-01-01

    Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment.

  17. 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.

  18. Molecular Mechanisms of Luteolin Induced Growth Inhibition and Apoptosis of Human Osteosarcoma Cells

    PubMed Central

    Wang, Yonghong; Kong, Daliang; Wang, Xinwei; Dong, Xiaoxiong; Tao, Yingying; Gong, Haiyang

    2015-01-01

    Luteolin is a flavone in medicinal plants as well as some vegetables and spices. It is a natural anti-oxidant with less pro-oxidant potential but apparently with a better safety profile. The purpose of this study was to investigate the molecular mechanism of luteolin-mediated apoptosis of MG-63 human osteosarcoma cells. MTT assay kit was employed to evaluate the effects of luteolin on MG-63 cells proliferation. Then, we performed Annexin V-FITC/PI to analyze the apoptotic rate of the cells. Furthermore, the inhibitory effects of luteolin on the expressions of BCL-2, BAX, Caspase-3 and Survivin were detected by Western blotting. As expected, luteolin (0.5, 2.5, 12.5 µg/mL) inhibited the growth of MG-63 cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that luteolin (0.5, 2.5, 12.5 µg/mL) inhibited the expressions of BCL-2, Caspase-3 and Survivin, and promoted the expression of BAX in MG-63 cells with a concentration dependent way. Luteolin can inhibit osteosarcoma cell proliferation and induce apoptosis effectively in a dose dependent manner through down-regulating the expression of BCL-2, Caspase-3 and Survivin proteins levels and up-regulating the expression of BAX protein level. These findings indicated that luteolin may be used as a novel herbal medicine for the treatment of osteosarcoma. PMID:25901161

  19. Kinetic effects of adriamycin and bleomycin on two osteosarcoma models.

    PubMed

    Bell, D F; Bell, R S; Mankin, H J; Gebhardt, M C; Weltie, F; O'Brien, T

    1988-01-01

    Although chemotherapeutic drugs are frequently administered to patients with osteosarcoma, there has been little research into the effect of cytotoxic drugs on osteosarcoma cell biology. The effect of two drugs (Adriamycin and bleomycin) on cell cycle kinetics was investigated in vitro in an established line of human osteosarcoma cells and in vivo using the Dunn osteosarcoma model. The cell cycle changes were consistent with G2 arrest for both drugs in vivo and in vitro. The alteration in cell cycle distribution was correlated with inhibition of 3H-thymidine incorporation in vitro. In vivo, the greater change in cell cycle distribution caused by Adriamycin was reflected in the increased inhibition of tumor growth found with this drug.

  20. Identification of DNA-PKcs as a primary resistance factor of salinomycin in osteosarcoma cells

    PubMed Central

    Wang, Xiao-dong; Zhou, Xiao-zhong; Zhu, Lun-qing

    2016-01-01

    Malignant osteosarcoma (OS) is still a deadly disease for many affected patients. The search for the novel anti-OS agent is extremely urgent and important. Our previous study has proposed that salinomycin is a novel anti-OS agent. Here we characterized DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a primary salinomycin resistance factor in OS cells. DNA-PKcs inhibitors (NU7026, NU7441 and LY294002) or DNA-PKcs shRNA knockdown dramatically potentiated salinomycin-induced death and apoptosis of OS cells (U2OS and MG-63 lines). Further, forced-expression of microRNA-101 (“miR-101”) downregulated DNA-PKcs and augmented salinomycin's cytotoxicity against OS cells. Reversely, over-expression of DNA-PKcs in OS cells inhibited salinomycin's lethality. For the mechanism study, we show that DNA-PKcs is required for salinomycin-induced pro-survival autophagy activation. DNA-PKcs inhibition (by NU7441), shRNA knockdown or miR-101 expression inhibited salinomycin-induced Beclin-1 expression and autophagy induction. Meanwhile, knockdown of Beclin-1 by shRNA significantly sensitized salinomycin-induced OS cell lethality. In vivo, salinomycin administration suppressed U2OS xenograft tumor growth in severe combined immuno-deficient (SCID) mice, and its anti-tumor activity was dramatically potentiated with co-administration of the DNA-PKcs inhibitor NU7026. Together, these results suggest that DNA-PKcs could be a primary resistance factor of salinomycin in OS cells. DNA-PKcs inhibition or silence may thus significantly increase salinomycin's sensitivity in OS cells. PMID:27765904

  1. Identification of DNA-PKcs as a primary resistance factor of salinomycin in osteosarcoma cells.

    PubMed

    Zhen, Yun-Fang; Li, Song-Tao; Zhu, Yun-Rong; Wang, Xiao-Dong; Zhou, Xiao-Zhong; Zhu, Lun-Qing

    2016-11-29

    Malignant osteosarcoma (OS) is still a deadly disease for many affected patients. The search for the novel anti-OS agent is extremely urgent and important. Our previous study has proposed that salinomycin is a novel anti-OS agent. Here we characterized DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a primary salinomycin resistance factor in OS cells. DNA-PKcs inhibitors (NU7026, NU7441 and LY294002) or DNA-PKcs shRNA knockdown dramatically potentiated salinomycin-induced death and apoptosis of OS cells (U2OS and MG-63 lines). Further, forced-expression of microRNA-101 ("miR-101") downregulated DNA-PKcs and augmented salinomycin's cytotoxicity against OS cells. Reversely, over-expression of DNA-PKcs in OS cells inhibited salinomycin's lethality. For the mechanism study, we show that DNA-PKcs is required for salinomycin-induced pro-survival autophagy activation. DNA-PKcs inhibition (by NU7441), shRNA knockdown or miR-101 expression inhibited salinomycin-induced Beclin-1 expression and autophagy induction. Meanwhile, knockdown of Beclin-1 by shRNA significantly sensitized salinomycin-induced OS cell lethality. In vivo, salinomycin administration suppressed U2OS xenograft tumor growth in severe combined immuno-deficient (SCID) mice, and its anti-tumor activity was dramatically potentiated with co-administration of the DNA-PKcs inhibitor NU7026. Together, these results suggest that DNA-PKcs could be a primary resistance factor of salinomycin in OS cells. DNA-PKcs inhibition or silence may thus significantly increase salinomycin's sensitivity in OS cells.

  2. Baicalin induces apoptosis in human osteosarcoma cell through ROS-mediated mitochondrial pathway.

    PubMed

    Wan, Daqian; Ouyang, Huoniu

    2017-09-05

    Baicalin is extracted from a traditional Chinese herb, Scutellaria baicalensis. In this study, the anticancer activity and underlying mechanisms of baicalin towards human osteosarcoma cell (HOS) were investigated. Baicalin could inhibit HOS cell proliferation in a concentration-dependent manner. Mitochondrial membrane potential decreased obviously after treated with different concentration of baicalin by flow cytometry assay and revealed that baicalin triggered a significant generation of reactive oxygen species (ROS). Western blotting assay further revealed that baicalin-induced cell apoptosis by suppressing Bcl-2 level, then activating caspase-9 and caspase-3. In vivo experiment, baicalin significantly suppressed tumour growth in female BALB/C nude mice bearing HOS tumours. In addition, baicalin did show toxicity to treated animal by comparing the body weight increase and mortality. In general, the present results demonstrated that baicalin-induced apoptosis in human osteosarcoma cell via a ROS-mediated mitochondrial pathway. The paper indicated that baicalin is a promising candidate for the treatment of HOS.

  3. p53 functions as a cell cycle control protein in osteosarcomas.

    PubMed Central

    Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

    1990-01-01

    Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae. Images PMID:2233717

  4. p53 functions as a cell cycle control protein in osteosarcomas.

    PubMed

    Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

    1990-11-01

    Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

  5. Wogonin suppresses stem cell-like traits of CD133 positive osteosarcoma cell via inhibiting matrix metallopeptidase-9 expression.

    PubMed

    Huynh, Do Luong; Kwon, Taeho; Zhang, Jiao Jiao; Sharma, Neelesh; Gera, Meeta; Ghosh, Mrinmoy; Kim, Nameun; Kim Cho, Somi; Lee, Dong Sun; Park, Yang Ho; Jeong, Dong Kee

    2017-06-12

    Several efforts have been deployed to cure osteosarcoma, a high-grade malignant bone tumour in children and adolescents. However, some challenges such as drug resistance, relapse, and tumour metastasis remain owing to the existence of cancer stem cells (CSC). There is an urgent need to develop cost-effective and safe therapies. Wogonin, an extract from the root of Scutellaria baicalensis, has long been considered as a promising natural and safe compound for anti-tumourigenesis, particularly to inhibit tumour invasion and metastasis. Hoechst 33,342 staining, wound healing assay, sphere formation assay, western blotting, and gelatin zymography assays were performed in CD133 positive osteosarcoma cell. In this study, we examined the effect of Wogonin on the mobility of human osteosarcoma CSC. Wogonin induces apoptosis of human osteosarcoma CSC, inhibits its mobility in vitro via downregulation of MMP-9 expression, and represses its renewal ability. We demonstrated that Wogonin decreases the renewal capacity of CSC. By inhibiting the formation of and reducing the size of spheres, Wogonin at a concentration of 40-80 μM effectively minimizes potential risk from CSC. Taken together, we have demonstrated a new approach for developing a potential therapy for osteosarcoma.

  6. MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    PubMed Central

    Ma, Qiong; Qiu, Xiuchun; Fan, Qingyu; Ma, Baoan

    2012-01-01

    Background MicroRNAs (miRNAs) are a class of endogenously expressed, small noncoding RNAs, which suppress its target mRNAs at the post-transcriptional level. Studies have demonstrated that miR-34a, which is a direct target of the p53 tumor suppressor gene, functions as a tumor suppressor and is associated with the tumor growth and metastasis of various human malignances. However, the role of miR-34a in osteosarcoma has not been totally elucidated. In the present study, the effects of miR-34a on osteosarcoma and the possible mechanism by which miR-34a affected the tumor growth and metastasis of osteosarcoma were investigated. Methodology/Principal Finding Over-expression of miR-34a partially inhibited proliferation, migration and invasion of osteosarcoma cells in vitro, as well as the tumor growth and pulmonary metastasis of osteosarcoma cells in vivo. c-Met is a target of miR-34a, and regulates the migration and invasion of osteosarcoma cells. Osteosarcoma cells over-expressing miR-34a exhibited a significant decrease in the expression levels of c-Met mRNA and protein simultaneously. Finally, the results from bioinformatics analysis demonstrated that there were multiple putative targets of miR-34a that may be associated with the proliferation and metastasis of osteosarcoma, including factors in Wnt and Notch signaling pathways. Conclusion/Significance The results presented in this study demonstrated that over-expression of miR-34a could inhibit the tumor growth and metastasis of osteosarcoma probably through down regulating c-Met. And there are other putative miR-34a target genes beside c-Met which could potentially be key players in the development of osteosarcoma. Since pulmonary metastases are responsible for mortality of patient carrying osteosarcoma, miR-34a may prove to be a promising gene therapeutic agent. It will be interesting to further investigate the mechanism by which miR-34a functions as a tumor suppressor gene in osteosarcoma. PMID:22457788

  7. Relationship between RFC gene expression and intracellular drug concentration in methotrexate-resistant osteosarcoma cells.

    PubMed

    Wang, J J; Li, G J

    2014-07-24

    Osteosarcoma is a primary malignant tumor in adolescents, associated with high mortality and morbidity. The high-dose methotrexate (MTX) chemotherapy used to treat this disease may induce primary or secondary drug resistance, resulting in a reduced effect of comprehensive treatment. In this study, the relationship between reduced folate carrier (RFC) gene expression and intracellular drug concentration in MTX-resistant osteosarcoma cells (Saos-2) was investigated. MTX-resistant human osteosarcoma cells (Saos-2/MTX2.2, Saos-2/MTX4.4) were prepared. The sensitivities of Saos-2 (primary cells), Saos-2/MTX2.2, and Saos-2/MTX4.4 cells to MTX, diamminedichloroplatinum (DDP), ifosfamide (IFO), epirubicine (EPI), adriamycin (ADM), theprubicin (THP), and paclitaxel (PTX) were detected by MTT. The median inhibitory concentration (IC50) and resistance index were measured. Semi-quantitative RT-PCR was used to evaluate the expression of RFC gene in cells. The intracellular (3)H-MTX concentration was determined. Results showed that IC50 of Saos-2/MTX2.2 and Saos-2/MTX4.4 was 4.87 and 12.73 times that of Saos-2, respectively. Both Saos-2/MTX2.2 and Saos-2/MTX4.4 had resistance to IFO, ADM, EPI, THP, and PTX, but not DDP. Compared to Saos-2/MTX2.2 and Saos-2/MTX4.4, the expression of RFC mRNA in Saos-2 was significantly higher. The intracellular (3)H-MTX concentration reached a peak at 50 min. After 70 min, the concentration was maintained at a plateau. During this phase, the (3)H-MTX concentration in Saos-2 cells was 2.15 times higher than the concentration in Saos-2/MTX4.4 cells. The reduced RFC mRNA expression in PTX-resistant osteosarcoma cells may be related to the decrease in intracellular (3)H-MTX concentration.

  8. Differential regulation and expression of hyaluronan synthases in human articular chondrocytes, synovial cells and osteosarcoma cells.

    PubMed Central

    Recklies, A D; White, C; Melching, L; Roughley, P J

    2001-01-01

    Recently three isoforms of hyaluronan synthase (HAS), the enzyme responsible for hyaluronate/hyaluronan (HA) biosynthesis, have been cloned, allowing us to study their expression pattern. Our objective was to determine which of the HAS isoenzymes were expressed in human articular chondrocytes, synovial fibroblasts and osteosarcoma cells, whether their expression could be modulated by growth factors (insulin-like growth factor-1, basic fibroblast growth factor and transforming growth factor (TGF-beta1) and cytokines [interleukin 1beta1 (IL-1beta)], and whether changes in the rate of HA synthesis by the cells correlated with changes in mRNA levels for one or more of the HAS isoforms. All three HAS isoforms were found to be expressed in the cultured cells analysed in this study, although the relative proportions varied for each cell type. HAS2 mRNA was usually predominant in chondrocytes, whereas synovial cells contained increased amounts of HAS1. HAS3 was always the least abundant message. The rapidly growing osteosarcoma cells contained almost exclusively HAS2 message. HAS usage in uncultured cartilage and synovial tissues was similar to that in the cultured cells, with HAS2 message being the predominant species in cartilage and HAS1 usually being the predominant species in synovium. HA synthesis was stimulated by the growth factors, but the extent of the response was cell-type specific. Synovial cells responded particularly well to IL-1beta, and showed a unique synergistic response when IL-1beta was used in combination with TGF-beta1. This response was much reduced in articular chondrocytes and absent in the osteosarcoma cells. Analysis of changes in HAS message levels indicated that there was often no correlation with the changes in HA secretion following exposure to growth factors. Although HAS-1 mRNA was increased in synovial cells after exposure to TGF-beta1/IL-1beta, the magnitude of the change was far less than the effect on HA synthesis. Our data thus

  9. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos-2 cells to cannabinoid receptor agonist WIN55,212-2.

    PubMed

    Zhang, Guodong; Bi, Haiyong; Gao, Ji; Lu, Xing; Zheng, Yanping

    2016-07-01

    WIN55,212-2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti-tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212-2 on osteosarcoma cell line Saos-2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212-2. Our results showed that the cell proliferation of Saos-2 was inhibited by WIN55,212-2 in a dose-dependent and time-dependent manner. WIN55,212-2-induced Saos-2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212-2 can induce endoplasmic reticulum stress and autophagy in Saos-2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212-2 in Saos-2 cells. These findings indicated that WIN55,212-2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. X609, a novel manassantin A derivative, exhibits antitumor activity in MG-63 human osteosarcoma cells in vitro and in vivo.

    PubMed

    Li, Ji; Zhao, Kongbo; Wang, Fu; Cai, Jinfang; Li, Zongyu; Zou, Lin

    2015-08-01

    Manassantin A has been well-established as an inhibitor of HIF-1. In the present study, a new manasantin A derivative, X609, with decreased stereochemical complexity, rendering it amenable to a simplified synthesis scheme, was synthesized and was found to increase HIF-1 inhibitory activity. X609 exhibited antiproliferative activity in a broad spectrum of tumor cell lines, via HIF-1-dependent mechanisms. X609 may induce apoptosis in MG-63 cells through activation of the mitochondrial pathway. Oral administration of X609 significantly inhibited the growth of human osteosarcomas implanted into nude mice. In light of the results of the present study, it may be possible to develop X609 for use as a novel antitumor agent, which targets human osteosarcoma.

  11. Rat Osteosarcoma Cells as a Therapeutic Target Model for Osteoregeneration via Sclerostin Knockdown.

    PubMed

    Sedaghati, Bita; Jahroomishirazi, Roomina; Starke, Annett; Hacker, Michael C; Schulz-Siegmund, Michaela

    2016-01-01

    There are various conceptually different strategies to improve bone regeneration and to treat osteoporosis, each with distinct inherent advantages and disadvantages. The use of RNA interference strategies to suppress the biological action of catabolic factors or antagonists of osteogenic proteins is promising, and such strategies can be applied locally. They are comparably inexpensive and do not suffer from stability problems as protein-based approaches. In this study, we focus on sclerostin, encoded by the SOST gene, a key regulator of bone formation and remodeling. Sclerostin is expressed by mature osteocytes but also by late osteogenically differentiated cells. Thus, it is difficult and requires long-term cultures to investigate the effects of SOST silencing on the expression of osteogenic markers using primary cells. We, therefore, selected a rat osteosarcoma cell line, UMR-106, that has been shown to express SOST and secrete sclerostin in a comparable fashion as late osteoblasts and osteocytes. We investigated the effects of differentiating supplements on SOST expression and sclerostin secretion in UMR-106 cells and found that addition of 100 ng/ml of bone morphogenetic protein (BMP)-2 strongly induced sclerostin secretion, whereas dexamethasone inhibited secretion. Effects of silencing SOST in UMR-106 cells cultured in various differentiation media including BMP-2 and/or dexamethasone were determined next with the aim to find promising test conditions for a readout system for the evaluation of future small interfering RNA release formulations for local induction of bone formation. We found a direct correlation between attenuated SOST expression and an increase in the osteogenic potential of UMR-106 cells. The combination of SOST silencing and BMP-2 could synergistically improve osteogenic factors. A lowered proliferation rate in silenced groups may indicate a faster switch to differentiation.

  12. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells

    SciTech Connect

    Komm, B.S.; Terpening, C.M.; Benz, D.J.; Graeme, K.A.; Gallegos, A.; Korc, M.; Greene, G.L.; O'Malley, B.W.; Haussler, M.R.

    1988-07-01

    High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.

  13. Pulsed Electromagnetic Field Stimulation Promotes Anti-cell Proliferative Activity in Doxorubicin-treated Mouse Osteosarcoma Cells.

    PubMed

    Muramatsu, Yoshitaka; Matsui, Takuya; Deie, Masataka; Sato, Keiji

    2017-01-02

    We aimed to investigate the synergistic effects of pulsed electromagnetic field (PEMF) and doxorubicin therapy in a mouse osteosarcoma cell line (LM8 cells) in vitro. The effects of PEMF (5 mT, 200 Hz) of different durations and doxorubicin on the proliferative activity of LM8 cells were measured by the MTT assay. Apoptotic-related factors such as cell-cycle phase, mitochondrial membrane potential, and caspase 3/7 activity were investigated using 4',6-diamidino-2-phenylindole staining and apoptosis kits. Identification of intracellular signaling molecules induced by the combination was comprehensively explored using a stress and apoptosis-related protein array kit. PEMF enhanced the inhibition of cell proliferation mediated by doxorubicin but did not affect the cell cycle, mitochondrial membrane potential, or doxorubicin-induced G2/M arrest. The combination of PEMF and doxorubicin altered a few signaling molecules. PEMF tended to reduce the doxorubicin-induced decrease of phosphorylated BAD, while reducing the increased expression of total IĸB and phosphorylated-CHK1 induced by doxorubicin. Our results indicate that combination of PEMF and doxorubicin could be a novel chemotherapeutic strategy. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  14. Pulsed Electromagnetic Field Stimulation Promotes Anti-cell Proliferative Activity in Doxorubicin-treated Mouse Osteosarcoma Cells

    PubMed Central

    MURAMATSU, YOSHITAKA; MATSUI, TAKUYA; DEIE, MASATAKA; SATO, KEIJI

    2017-01-01

    Aim: We aimed to investigate the synergistic effects of pulsed electromagnetic field (PEMF) and doxorubicin therapy in a mouse osteosarcoma cell line (LM8 cells) in vitro. Materials and Methods: The effects of PEMF (5 mT, 200 Hz) of different durations and doxorubicin on the proliferative activity of LM8 cells were measured by the MTT assay. Apoptotic-related factors such as cell-cycle phase, mitochondrial membrane potential, and caspase 3/7 activity were investigated using 4’,6-diamidino-2-phenylindole staining and apoptosis kits. Identification of intracellular signaling molecules induced by the combination was comprehensively explored using a stress and apoptosis-related protein array kit. Results: PEMF enhanced the inhibition of cell proliferation mediated by doxorubicin but did not affect the cell cycle, mitochondrial membrane potential, or doxorubicin-induced G2/M arrest. The combination of PEMF and doxorubicin altered a few signaling molecules. PEMF tended to reduce the doxorubicin-induced decrease of phosphorylated BAD, while reducing the increased expression of total IĸB and phosphorylated-CHK1 induced by doxorubicin. Conclusion: Our results indicate that combination of PEMF and doxorubicin could be a novel chemotherapeutic strategy. PMID:28064222

  15. Expression levels of insulin receptor substrate-1 modulate the osteoblastic differentiation of mesenchymal stem cells and osteosarcoma cells.

    PubMed

    Contaldo, Clara; Myers, Timothy J; Zucchini, Cinzia; Manara, Maria Cristina; Chiodoni, Claudia; Colombo, Mario P; Nicoletti, Giordano; Lollini, Pier Luigi; Li, Tieshi; Longobardi, Lara; Scotlandi, Katia; Spagnoli, Anna

    2014-02-01

    The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.

  16. Antimetastatic activity of honokiol in osteosarcoma.

    PubMed

    Steinmann, Patrick; Walters, Denise K; Arlt, Matthias J E; Banke, Ingo J; Ziegler, Urs; Langsam, Bettina; Arbiser, Jack; Muff, Roman; Born, Walter; Fuchs, Bruno

    2012-04-15

    Metastasizing osteosarcoma has a mean 5-year survival rate of only 20% to 30%. Therefore, novel chemotherapeutics for more effective treatment of this disease are required. The antineoplastic activity of honokiol, which was demonstrated previously in numerous malignancies, was studied in vivo in C3H mice subcutaneously injected with syngeneic β-galactosidase bacterial gene (lacZ)-expressing LM8 osteosarcoma (LM8-lacZ) cells. In vitro cytotoxic effects of honokiol were investigated in 8 human and 2 murine osteosarcoma cell lines with different in vivo metastatic potential. Seven days after subcutaneous flank injection of LM8-lacZ cells, daily intraperitoneal treatment of mice with 150 mg/kg honokiol reduced the number of micrometastases in the lung by 41% and reduced the number of macrometastases in the lung and liver by 69% and 80%, respectively, compared with control. Primary tumor growth was not inhibited. In osteosarcoma cell lines, honokiol inhibited the metabolic activity with a half-maximal concentration (IC(50) ) between 8.0 μg/mL and 16 μg/mL. Cyclosporin A partially reversed the inhibition of metabolic activity in LM8-lacZ cells. Cell proliferation and wound healing migration of LM8-lacZ cells were inhibited by honokiol with an IC(50) between 5.0 μg/mL and 10 μg/mL. Higher concentrations caused rapid cell death, which was distinct from necrosis, apoptosis, or autophagy but was associated with swelling of the endoplasmic reticulum, cytoplasmic vacuolation, and morphologically altered mitochondria. Honokiol exhibited prominent antimetastatic activity in experimental osteosarcoma and caused rapid cell death in vitro that was unrelated to necrosis, apoptosis, or autophagy. The authors concluded that honokiol has considerable potential for the treatment of metastasizing osteosarcoma. Copyright © 2011 American Cancer Society.

  17. Mesenchymal stem cells promote tumor engraftment and metastatic colonization in rat osteosarcoma model.

    PubMed

    Tsukamoto, Shinji; Honoki, Kanya; Fujii, Hiromasa; Tohma, Yasuaki; Kido, Akira; Mori, Toshio; Tsujiuchi, Toshifumi; Tanaka, Yasuhito

    2012-01-01

    Although mesenchymal stem cells (MSCs) are considered to be the cells of origin for most sarcomas, the role of MSCs as a source of tumor stroma is not fully understood in this tumor type. The current study investigated whether MSCs affect the tumor growth and metastatic ability in rat osteosarcoma model. Results from subcutaneous co-implantation of rat osteosarcoma COS1NR cells, established in our laboratory, with rat MSCs isolated from femur bone marrow showed that the incidence of tumor formation and tumor growth rate was higher until 5 weeks compared to COS1NR cell inoculation alone. However, no difference was observed in tumor growth afterwards and in the number of metastatic nodules at 9 weeks (0.75 vs. 1.2). Intravenous MSC injection at weeks 3 and 5 after subcutaneous inoculation of COS1NR cells significantly increased the number of lung nodules in the group with MSC injection compared to the group without MSC injection (17.33 vs. 2.0), while no difference was observed in subcutaneous tumor growth between those groups. Pathway analysis from gene expression profile identified that genes involved in focal adhesion, cytokine-cytokine receptor and extracellular matrix-receptor pathways such as CAMs (ICAM and VCAM)-integrins were highly expressed in MSCs, possibly participating in the tumor progression of osteosarcoma. These results suggest that MSCs could provide a source of microenvironments for osteosarcoma cells, and might enhance the ability of settlement and colonization which lead to early onset of growth and metastasis, possibly through their activated pathways interaction.

  18. The effect of bone morphogenetic protein-2 on osteosarcoma metastasis

    PubMed Central

    Gill, Jonathan; Connolly, Patrick; Roth, Michael; Chung, So Hak; Zhang, Wendong; Piperdi, Sajida; Hoang, Bang; Yang, Rui; Guzik, Hillary; Gorlick, Richard; Geller, David S.

    2017-01-01

    Purpose Bone Morphogenetic Protein-2 (BMP-2) may offer the potential to enhance allograft-host osseous union in limb-salvage surgery following osteosarcoma resection. However, there is concern regarding the effect of locally applied BMP-2 on tumor recurrence and metastasis. The purpose of this project was to evaluate the effect of exogenous BMP-2 on osteosarcoma migration and invasion across a panel of tumor cell lines in vitro and to characterize the effect of BMP-2 on pulmonary osteosarcoma metastasis within a xenograft model. Experimental design The effect of BMP-2 on in vitro tumor growth and development was assessed across multiple standard and patient-derived xenograft osteosarcoma cell lines. Tumor migration capacity, invasion, and cell proliferation were characterized. In addition, the effect on metastasis was measured using a xenograft model following tail-vein injection. The effect of exogenous BMP-2 on the development of metastases was measured following both single and multiple BMP-2 administrations. Results There was no significant difference in migration capacity, invasion, or cell proliferation between the BMP-2 treated and the untreated osteosarcoma cell lines. There was no significant difference in pulmonary metastases between either the single-dose or multi-dose BMP-2 treated animals and the untreated control animals. Conclusions In the model systems tested, the addition of BMP-2 does not increase osteosarcoma proliferation, migration, invasion, or metastasis to the lungs. PMID:28264040

  19. The effect of bone morphogenetic protein-2 on osteosarcoma metastasis.

    PubMed

    Gill, Jonathan; Connolly, Patrick; Roth, Michael; Chung, So Hak; Zhang, Wendong; Piperdi, Sajida; Hoang, Bang; Yang, Rui; Guzik, Hillary; Morris, Jonathan; Gorlick, Richard; Geller, David S

    2017-01-01

    Bone Morphogenetic Protein-2 (BMP-2) may offer the potential to enhance allograft-host osseous union in limb-salvage surgery following osteosarcoma resection. However, there is concern regarding the effect of locally applied BMP-2 on tumor recurrence and metastasis. The purpose of this project was to evaluate the effect of exogenous BMP-2 on osteosarcoma migration and invasion across a panel of tumor cell lines in vitro and to characterize the effect of BMP-2 on pulmonary osteosarcoma metastasis within a xenograft model. The effect of BMP-2 on in vitro tumor growth and development was assessed across multiple standard and patient-derived xenograft osteosarcoma cell lines. Tumor migration capacity, invasion, and cell proliferation were characterized. In addition, the effect on metastasis was measured using a xenograft model following tail-vein injection. The effect of exogenous BMP-2 on the development of metastases was measured following both single and multiple BMP-2 administrations. There was no significant difference in migration capacity, invasion, or cell proliferation between the BMP-2 treated and the untreated osteosarcoma cell lines. There was no significant difference in pulmonary metastases between either the single-dose or multi-dose BMP-2 treated animals and the untreated control animals. In the model systems tested, the addition of BMP-2 does not increase osteosarcoma proliferation, migration, invasion, or metastasis to the lungs.

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

    SciTech Connect

    Lin, Ping; Mobasher, Maral E.; Alawi, Faizan

    2014-04-18

    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, 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.

  1. Long Noncoding RNA miR210HG Sponges miR-503 to Facilitate Osteosarcoma Cell Invasion and Metastasis.

    PubMed

    Li, Jiang; Wu, Quan-Min; Wang, Xiao-Qing; Zhang, Cheng-Qiang

    2017-10-03

    Long noncoding RNAs (lncRNAs) have been illustrated to function as important regulator in carcinogenesis and cancer progression. However, roles of lncRNA miR210HG (miR210 host gene) in osteosarcoma remain unclear. In this study, miR210HG expression level was significantly upregulated in 55 cases of osteosarcoma tissue samples compared to adjacent normal tissue. Besides, the aberrantly enhanced miR210HG expression predicted poor prognosis and lower survival rate. In vitro, miR210HG knockdown suppressed the osteosarcoma cell proliferation, invasion, and epithelial-mesenchymal transition-related marker (N-cadherin and vimentin) expression. In vivo, miR210HG silencing decreased the tumor growth. MiR-503 was verified to be the target miRNA of miR210HG using bioinformatics online program and luciferase assay. Furthermore, miR-503 could reverse the role of miR210HG on osteosarcoma cells. In conclusion, our study indicates that miR210HG sponges miR-503 to facilitate osteosarcoma cell invasion and metastasis, revealing the oncogenic role of miR210HG on osteosarcoma cells.

  2. Decreased RECQL5 correlated with disease progression of osteosarcoma

    SciTech Connect

    Wu, Junlong; Zhi, Liqiang; Dai, Xin; Cai, Qingchun; Ma, Wei

    2015-11-27

    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 expressed 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.

  3. The prognostic value of D-dimer levels in metastatic osteosarcoma patients treated with second-line chemotherapy

    PubMed Central

    Sun, Yuanjue; Zhang, Jianjun; Yao, Yang; He, Aina

    2016-01-01

    We performed a retrospective analysis of 32 metastatic osteosarcoma cases to examine the prognostic value of the plasma D-dimer level. We assessed the D-dimer level before second-line chemotherapy (D1) and the D-dimer level after two cycles of second-line chemotherapy (D2). The change in D-dimer level (ΔD) was defined as D2 minus D1. The overall survival (OS) of patients with a high D1 was significantly shorter than those with a low D1 (median OS, 4.7 vs. 16.2 months, P=0.001). Similar results were observed for the D2 (median OS, 4.7 vs. 8.6 months, P=0.033). Multivariable analysis demonstrated that a high D1 (hazard ratio, 3.375; 95% confidence interval, 1.133–10.053; P=0.029) was an unfavorable independent prognostic factor. The mean D2 of 11 patients with stable disease decreased by 0.69 mg/mL compared to the D1 (P = 0.016). The mean D2 increased by 1.47 mg/mL compared to the D1 in 21 patients with progressive disease (P = 0.004). The data suggest that D-dimer may serve as a prognostic biomarker for metastatic osteosarcoma patients treated with second-line chemotherapy. PMID:27564105

  4. CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance

    PubMed Central

    Adhikari, Amit S.; Agarwal, Neeraj; Wood, Byron M.; Porretta, Constance; Ruiz, Bernardo; Pochampally, Radhika R.; Iwakuma, Tomoo

    2011-01-01

    Emerging evidence indicates the presence of tumor-initiating cells (TIC) or cancer stem cells (CSCs) in osteosarcoma. However, no study has demonstrated specific markers to identify osteosarcoma TICs with in vivo tumor formation ability. Additionally, there has been a lack of investigations gauging the contribution of osteosarcoma TICs to metastatic and drug-resistant properties. In this study, we have identified mouse and human osteosarcoma TICs using mesenchymal stem cell (MSC) markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. Both mouse and human cells expressing these markers were sorted and analyzed for their abilities of tumor formation with as few as 200 cells, self-renewability, multipotency, drug resistance, metastatic potential, and enrichment of a metastasis-associated marker CXCR4 and a drug-resistance marker ABCG2. CD117+Stro-1+ cells efficiently formed serially transplantable tumors, whereas CD117−Stro-1− cells rarely initiated tumors. Upon orthotopic injections, CD117+Stro-1+ cell-derived tumors metastasized at a high frequency. Further, CD117+Stro-1+ cells showed high invasive and drug-resistant properties and were efficiently enriched for CXCR4 (20–90%) and ABCG2 (60–90%). These results suggest possible mechanisms for the high metastatic and drug-resistant properties of osteosarcoma TICs. In summary, CD117 and Stro-1 identify osteosarcoma TICs associated with the most lethal characteristics of the disease - metastasis and drug resistance - and these markers offer candidates for TIC-targeted drug delivery aimed at eradicating osteosarcoma. PMID:20460510

  5. Effects of the myeloid cell nuclear differentiation antigen on the proliferation, apoptosis and migration of osteosarcoma cells.

    PubMed

    Sun, Chengliang; Liu, Chuanju; Dong, Jun; Li, Dong; Li, Wei

    2014-03-01

    Despite improvements over the past two decades, the outcome for patients with advanced osteosarcoma remains poor. Targeted therapies have emerged as promising treatment options for various malignancies. However, effective targeted cancer therapies require the identification of key molecules in the pathogenesis of cancer. The aim of this study was to evaluate the value of the myeloid cell nuclear differentiation antigen (MNDA), a member of the interferon-inducible p200 (IFI-200) family, as a therapeutic target for osteosarcoma by analyzing the baseline expression of MNDA in human osteosarcoma cells and determining the effect of MNDA overexpression on the proliferation and apoptosis profiles and migration/invasion ability in osteosarcoma cells. To this end, MNDA mRNA abundance in wild-type sarcoma osteogenic (Saos-2) cells was analyzed using reverse transcription-polymerase chain reaction, proliferation/apoptosis profiles and migration/invasion capacity in Saos-2 cells overexpressing a green fluorescence protein (GFP)-human MNDA fusion protein. Saos-2 cells found to be overexpressing GFP alone were assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis and Matrigel Transwell migration assay. The results demonstrated that MNDA mRNA was significantly less abundant in wild-type Saos-2 cells compared with human monocyte-like U-937 cells and MNDA overexpression effectively inhibited proliferation, induced apoptosis and reduced migration/invasiveness in Saos-2 cells compared with GFP overexpression alone. Preliminary observations suggested that MNDA potentially serves as a novel therapeutic target for osteosarcoma.

  6. Bufalin Inhibits Proliferation and Induces Apoptosis in Osteosarcoma Cells by Downregulating MicroRNA-221

    PubMed Central

    Han, Kun; Wang, Yaling

    2016-01-01

    Bufalin, a major component of the Chinese medicine ChanSu, which is prepared from the skin and parotid venom glands of toads, has shown cytotoxicity in several malignant tumors. Here, we reported that bufalin inhibited proliferation and induced mitochondria-dependent apoptosis in U-2OS and Saos-2 osteosarcoma cells with intracellular reactive oxygen species (ROS) production. By microRNA (miR) array analysis and quantitative reverse transcription polymerase chain reaction, we found that miR-221 was downregulated after treatment with bufalin. In accordance with TargetScan prediction and luciferase reporter assay, Bcl2 binding component 3 (BBC3) was the direct target of miR-221. Furthermore, upregulating miR-221 by its MIMIC and suppressing BBC3 by small interfering RNA (siRNA) reversed the effects of bufalin on osteosarcoma cells. Collectively, our data indicate that bufalin inhibits cell proliferation and induces mitochondria-dependent apoptosis in osteosarcoma cells through downregulating miR-221 and triggering BBC3 expression. PMID:28074104

  7. Secondary osteosarcoma developing 10 years after chemoradiotherapy for non-small-cell lung cancer.

    PubMed

    Yagishita, Shigehiro; Horinouchi, Hidehito; Yorozu, Takashi; Kitazono, Satoru; Mizugaki, Hidenori; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Mori, Taisuke; Tsuta, Koji; Sumi, Minako; Tamura, Tomohide

    2014-02-01

    A 53-year-old female patient was admitted with pain and a progressively enlarging mass in the right upper chest. Chest computed tomography revealed a mass lesion in the region of the right upper ribs. Ten years prior to this admission, the patient had undergone right lobectomy for lung adenocarcinoma. One year after the surgery, follow-up computed tomography had revealed tumor recurrence in the mediastinal and supraclavicular lymph nodes, and the patient had been treated by chemoradiotherapy. Thereafter, regular follow-up had revealed no evidence of recurrence of the non-small-cell lung cancer. Histopathological findings revealed proliferation of spindle-shaped malignant tumor cells in a background of osteoid, consistent with the diagnosis of osteosarcoma. The location of the tumor was consistent with the radiation field. Based on the clinicopathological findings, the patient was diagnosed as having secondary osteosarcoma occurring as a result of the chemoradiotherapy administered previously for the recurrent non-small-cell lung cancer. Unfortunately, the patient died of rapid progression of the osteosarcoma within a week of admission to the hospital. The autopsy revealed contiguous invasion by the tumor of the heart, with massive thrombus formation. The peripheral pulmonary arteries were diffusely occluded by metastatic tumors. Our case serves to highlight the risk of development of secondary sarcoma as a life-threatening late complication after chemoradiotherapy for locally advanced non-small-cell lung cancer, even after complete cure of the primary tumor.

  8. Characterization of the metastatic phenotype of a panel of established osteosarcoma cells

    PubMed Central

    Ren, Ling; Mendoza, Arnulfo; Zhu, Jack; Briggs, Joseph W.; Halsey, Charles; Hong, Ellen S.; Burkett, Sandra S.; Morrow, James J.; Lizardo, Michael M.; Osborne, Tanasa; Li, Samuel Q.; Luu, Hue H.; Meltzer, Paul; Khanna, Chand

    2015-01-01

    Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. In this study, we collected 18 OS cell lines, confirmed their expression of bone markers and complex karyotypes, and characterized their in vivo tumorgenicity and metastatic potential. Since prior reports included conflicting descriptions of the metastatic and in vivo phenotypes of these models, there was a need for a comparative assessment of metastatic phenotypes using identical procedures in the hands of a single investigative group. We expect that this single characterization will accelerate the study of this metastatic cancer. Using these models we evaluated the expression of six previously reported metastasis-related OS genes. Ezrin was the only gene consistently differentially expressed in all the pairs of high/low metatstatic OS cells. We then used a subtractive gene expression approach of the high and low human metastatic cells to identify novel genes that may be involved in OS metastasis. PHLDA1 (pleckstrin homology-like domain, family A) was identified as one of the genes more highly expressed in the high metastatic compared to low metastatic cells. Knocking down PHLDA1 with siRNA or shRNA resulted in down regulation of the activities of MAPKs (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs). Reducing the expression of PHLDA1 also delayed OS metastasis progression in mouse xenograft models. PMID:26320182

  9. Characterization of the metastatic phenotype of a panel of established osteosarcoma cells.

    PubMed

    Ren, Ling; Mendoza, Arnulfo; Zhu, Jack; Briggs, Joseph W; Halsey, Charles; Hong, Ellen S; Burkett, Sandra S; Morrow, James; Lizardo, Michael M; Osborne, Tanasa; Li, Samuel Q; Luu, Hue H; Meltzer, Paul; Khanna, Chand

    2015-10-06

    Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. In this study, we collected 18 OS cell lines, confirmed their expression of bone markers and complex karyotypes, and characterized their in vivo tumorgenicity and metastatic potential. Since prior reports included conflicting descriptions of the metastatic and in vivo phenotypes of these models, there was a need for a comparative assessment of metastatic phenotypes using identical procedures in the hands of a single investigative group. We expect that this single characterization will accelerate the study of this metastatic cancer. Using these models we evaluated the expression of six previously reported metastasis-related OS genes. Ezrin was the only gene consistently differentially expressed in all the pairs of high/low metastatic OS cells. We then used a subtractive gene expression approach of the high and low human metastatic cells to identify novel genes that may be involved in OS metastasis. PHLDA1 (pleckstrin homology-like domain, family A) was identified as one of the genes more highly expressed in the high metastatic compared to low metastatic cells. Knocking down PHLDA1 with siRNA or shRNA resulted in down regulation of the activities of MAPKs (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs). Reducing the expression of PHLDA1 also delayed OS metastasis progression in mouse xenograft models.

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

    PubMed

    Husmann, Knut; Ducommun, Pascal; Sabile, Adam A; Pedersen, Else-Marie; Born, Walter; Fuchs, Bruno

    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 and 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.

  11. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    PubMed Central

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma. PMID:27573585

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

    SciTech Connect

    Zhang, Shilian 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 tumorous 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.

  13. Tricetin inhibits human osteosarcoma cells metastasis by transcriptionally repressing MMP-9 via p38 and Akt pathways.

    PubMed

    Chang, Pin-Yu; Hsieh, Ming-Ju; Hsieh, Yih-Shou; Chen, Pei-Ni; Yang, Jia-Sin; Lo, Fang-Cheng; Yang, Shun-Fa; Lu, Ko-Hsiu

    2017-08-01

    Tricetin, a dietary flavonoid, has cytostatic properties and anti-metastasis activities in various cancer cells. However, the detailed impacts and underlying mechanisms of tricetin on human osteosarcoma cell metastasis are still unclear. Here, the hypothesis that tricetin possesses the anti-metastatic effects on human osteosarcoma cells was tested. The effects of tricetin on cell viability, motility, migration, and invasion in human osteosarcoma U2OS and HOS cells were investigated. Gelatin zymography, western blotting, polymerase chain reaction (PCR), and the luciferase assay were used to further explore the underlying mechanisms involved in anti-metastatic effects in U2OS cells. Their results showed that Tricetin, up to 80 μM without cytotoxicity, attenuated U2OS and HOS cells motility, invasiveness, and migration by reducing matrix metalloproteinase (MMP)-9 enzyme activities. In U2OS cells, tricetin decreased MMP-9 protein and mRNA expressions, which was confirmed by real-time PCR. Next, tricetin reduced phosphorylation of p38 and Akt, but no effect on phosphorylation of ERK1/2 and JNK. In conclusion, tricetin possesses the anti-metastatic activity of osteosarcoma cells by transcriptionally repressing MMP-9 via p38 and Akt signaling pathways. This may be potentially useful as anti-metastatic agents for osteosarcoma chemotherapy. © 2016 Wiley Periodicals, Inc.

  14. Bisphenol A Inhibits Cell Proliferation and Reduces the Motile Potential of Murine LM8 Osteosarcoma Cells.

    PubMed

    Kidani, Teruki; Yasuda, Rie; Miyawaki, Joji; Oshima, Yusuke; Miura, Hiromasa; Masuno, Hiroshi

    2017-04-01

    The aim of this study was to examine the effect of bisphenol A (BPA) on the proliferation and motility potential of murine LM8 osteosarcoma cells. LM8 cells were treated for 3 days with or without 80 μM BPA. The effect of BPA on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2'-deoxyuridine (BrdU) incorporation study. Ethanol-fixed cells were stained with hematoxylin-eosin (H&E) to visualize cell morphology. Cell motility was assayed using inserts with uncoated membranes in invasion chambers. Expression of cell division cycle 42 (CDC42) was determined by immunofluorescence staining and western blotting. BPA reduced the DNA content of cultures and the number of BrdU-positive cells. BPA induced a change in morphology from cuboidal with multiple filopodia on the cell surface to spindle-shaped with a smooth cell surface. BPA-treated cells expressed less CDC42 and were less motile than untreated cells. BPA inhibited DNA replication and cell proliferation. BPA inhibited filopodia formation and motile potential by inhibiting CDC42 expression in LM8 cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. A New Perspective for Osteosarcoma Therapy: Proteasome Inhibition by MLN9708/2238 Successfully Induces Apoptosis and Cell Cycle Arrest and Attenuates the Invasion Ability of Osteosarcoma Cells in Vitro.

    PubMed

    Liu, Renhao; Fu, Chunjiang; Sun, Jiabing; Wang, Xvming; Geng, Shuo; Wang, Xiaoyu; Zou, Jilong; Bi, Zhenggang; Yang, Chenglin

    2017-01-01

    The proteasome exists in all eukaryotic cells and provides the main route of intracellular proteins degradation involved in cell growth and apoptosis. Proteasome inhibition could block protein degradation pathways and disturb regulatory networks, possibly leading to profound effects on cell growth, particularly in cancer cells. A proteasome inhibitor with an appropriate toxicity index for malignant cells rather than normal cells would be an attractive anticancer therapy. The human osteosarcoma (OS) cell lines MG-63 and Saos-2 and normal osteoblast cells were used to study the antitumour activity of the proteasome inhibitor MLN9708/2238. MLN2238 inhibited cell growth, induced cell cycle arrest and apoptosis, and attenuated the invasion abilities of MG-63 and Saos-2 cells, with little cytotoxicity to normal cells. In addition, MLN2238 promoted antitumour mechanisms including the accumulation of E2F1, P53, P21 and other negative G2/M checkpoint proteins; up-regulated the relative expression ratio of BAX/BCL-2, APAF-1 and pro-apoptotic proteins of the BCL-2 family; triggered mitochondrial outer membrane permeabilization (MOMP); down-regulated BCL-2 and XIAP; activated caspase3/8/9; and suppressed MMP2/9 expression and secretion levels. The proteasome may be a novel biochemical target for OS treatment in vitro. Our study provides a promising mechanistic framework for MLN9708/2238 in OS treatment, supporting its clinical development. © 2017 The Author(s). Published by S. Karger AG, Basel.

  16. Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression.

    PubMed

    Tsuru, A; Setoguchi, T; Matsunoshita, Y; Nagao-Kitamoto, H; Nagano, S; Yokouchi, M; Maeda, S; Ishidou, Y; Yamamoto, T; Komiya, S

    2015-03-31

    Activation of the Notch pathway has been reported in various types of cancers. However, the role of the hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1) in osteosarcoma is unknown. We examined the function of HEY1 in osteosarcoma. Expression of HEY1 was studied in human osteosarcoma. The effects of HEY1 in osteosarcoma were evaluated in vitro and in a xenograft model. Moreover, we examined the function of matrix metallopeptidase 9 (MMP9) as a downstream effector of HEY1. HEY1 was upregulated in human osteosarcoma. Knockdown of HEY1 inhibited the invasion of osteosarcoma cell lines. In contrast, the forced expression of HEY1 increased the invasion of mesenchymal stem cell. In addition, lung metastases were significantly inhibited by the knockdown of HEY1. We found that MMP9 was a downstream effector of HEY1 that promotes the invasion of osteosarcoma cells. Knockdown of HEY1 decreased the expression of MMP9. Addition of MMP9 rescued the invasion of osteosarcoma cells that had been rendered less invasive by knockdown of HEY1 expression. Our findings suggested that HEY1 augmented the metastasis of osteosarcoma via upregulation of MMP9 expression. Therefore, inhibition of HEY1 may be a novel therapeutic strategy for preventing osteosarcoma metastasis.

  17. Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression

    PubMed Central

    Tsuru, A; Setoguchi, T; Matsunoshita, Y; Nagao-Kitamoto, H; Nagano, S; Yokouchi, M; Maeda, S; Ishidou, Y; Yamamoto, T; Komiya, S

    2015-01-01

    Background: Activation of the Notch pathway has been reported in various types of cancers. However, the role of the hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1) in osteosarcoma is unknown. We examined the function of HEY1 in osteosarcoma. Methods: Expression of HEY1 was studied in human osteosarcoma. The effects of HEY1 in osteosarcoma were evaluated in vitro and in a xenograft model. Moreover, we examined the function of matrix metallopeptidase 9 (MMP9) as a downstream effector of HEY1. Results: HEY1 was upregulated in human osteosarcoma. Knockdown of HEY1 inhibited the invasion of osteosarcoma cell lines. In contrast, the forced expression of HEY1 increased the invasion of mesenchymal stem cell. In addition, lung metastases were significantly inhibited by the knockdown of HEY1. We found that MMP9 was a downstream effector of HEY1 that promotes the invasion of osteosarcoma cells. Knockdown of HEY1 decreased the expression of MMP9. Addition of MMP9 rescued the invasion of osteosarcoma cells that had been rendered less invasive by knockdown of HEY1 expression. Conclusions: Our findings suggested that HEY1 augmented the metastasis of osteosarcoma via upregulation of MMP9 expression. Therefore, inhibition of HEY1 may be a novel therapeutic strategy for preventing osteosarcoma metastasis. PMID:25742474

  18. Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses.

    PubMed

    Takagi-Kimura, M; Yamano, T; Tagawa, M; Kubo, S

    2014-03-01

    Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.

  19. Shock wave-induced ATP release from osteosarcoma U2OS cells promotes cellular uptake and cytotoxicity of methotrexate.

    PubMed

    Qi, Baochang; Yu, Tiecheng; Wang, Chengxue; Wang, Tiejun; Yao, Jihang; Zhang, Xiaomeng; Deng, Pengfei; Xia, Yongning; Junger, Wolfgang G; Sun, Dahui

    2016-10-03

    Osteosarcoma is the most prevalent primary malignant bone tumor, but treatment is difficult and prognosis remains poor. Recently, large-dose chemotherapy has been shown to improve outcome but this approach can cause many side effects. Minimizing the dose of chemotherapeutic drugs and optimizing their curative effects is a current goal in the management of osteosarcoma patients. In our study, trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2OS cells. Cellular uptake of the fluorophores Lucifer Yellow CH dilithium salt and Calcein was measured by qualitative and quantitative methods. Human MTX ELISA Kit and MTT assay were used to assess the outcome for osteosarcoma U2OS cells in the present of shock wave and methotrexate. To explore the mechanism, P2X7 receptor in U2OS cells was detected by immunofluorescence and the extracellular ATP levels was detected by ATP assay kit. All data were analyzed using SPSS17.0 statistical software. Comparisons were made with t test between two groups. Treatment of human osteosarcoma U2OS cells with up to 450 shock wave pulses at 7 kV or up to 200 shock wave pulses at 14 kV had little effect on cell viability. However, this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2OS cells. Importantly, shock wave treatment also significantly enhanced the uptake of the chemotherapy drug methotrexate and increased the rate of methotrexate-induced apoptosis. We found that shock wave treatment increased the extracellular concentration of ATP and that KN62, an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis. Our results suggest that shock wave treatment promotes methotrexate-induced apoptosis by altering cell membrane permeability in a P2X7 receptor-dependent manner. Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.

  20. Circadian clock components RORα and Bmal1 mediate the anti-proliferative effect of MLN4924 in osteosarcoma cells.

    PubMed

    Zhang, Shuju; Zhang, Jiaming; Deng, Zhiyuan; Liu, Huadie; Mao, Wei; Jiang, Fang; Xia, Zanxian; Li, Jia-Da

    2016-10-04

    The anticancer small molecule MLN4924, a Nedd8-activating enzyme (NAE) inhibitor, triggers cell-cycle arrest, apoptosis, and senescence in cancer cells. In this study, we demonstrate that MLN4924 suppresses osteosarcoma cell proliferation by inducing G2/M cell cycle arrest and apoptosis. Our results indicate that MLN4924 stabilizes the retinoid orphan nuclear receptor alpha (RORα) by decreasing its ubiquitination. RNA interference of RORα attenuates the anti-proliferative effect of MLN4924 in U2OS osteosarcoma cells. MLN4924 up-regulates the expression of p21 and Bmal1, two transcriptional targets of RORα. However, p21 plays a minimal role in the anti-proliferative effect of MLN4924 in U2OS osteosarcoma cells. In contrast, Bmal1 suppression by siRNA attenuates the anti-proliferative effect of MLN4924 in U2OS osteosarcoma cells, indicating that the MLN4924-mediated cell growth inhibition is mediated by Bmal1. These results show MLN4924 to be a promising therapeutic agent for the treatment of osteosarcoma and suggest that MLN4924-induced tumor growth inhibition is mediated by the circadian clock components RORα and Bmal1.

  1. Circadian clock components RORα and Bmal1 mediate the anti-proliferative effect of MLN4924 in osteosarcoma cells

    PubMed Central

    Zhang, Shuju; Zhang, Jiaming; Deng, Zhiyuan; Liu, Huadie; Mao, Wei; Jiang, Fang; Xia, Zanxian; Li, Jia-Da

    2016-01-01

    The anticancer small molecule MLN4924, a Nedd8-activating enzyme (NAE) inhibitor, triggers cell-cycle arrest, apoptosis, and senescence in cancer cells. In this study, we demonstrate that MLN4924 suppresses osteosarcoma cell proliferation by inducing G2/M cell cycle arrest and apoptosis. Our results indicate that MLN4924 stabilizes the retinoid orphan nuclear receptor alpha (RORα) by decreasing its ubiquitination. RNA interference of RORα attenuates the anti-proliferative effect of MLN4924 in U2OS osteosarcoma cells. MLN4924 up-regulates the expression of p21 and Bmal1, two transcriptional targets of RORα. However, p21 plays a minimal role in the anti-proliferative effect of MLN4924 in U2OS osteosarcoma cells. In contrast, Bmal1 suppression by siRNA attenuates the anti-proliferative effect of MLN4924 in U2OS osteosarcoma cells, indicating that the MLN4924-mediated cell growth inhibition is mediated by Bmal1. These results show MLN4924 to be a promising therapeutic agent for the treatment of osteosarcoma and suggest that MLN4924-induced tumor growth inhibition is mediated by the circadian clock components RORα and Bmal1. PMID:27602774

  2. 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

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

    PubMed

    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

    2015-05-01

    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.

  4. Nasal osteosarcoma and interstitial cell tumor in a Vancouver Island marmot (Marmota vancouverensis).

    PubMed

    Dadone, Liza I; Whiteside, Douglas P; Black, Sandra R; Remedios, Audrey; Raverty, Stephen

    2011-06-01

    A 6-yr-old male Vancouver Island marmot (Marmota vancouverensis) presented for poor hibernation, weight loss, and symmetric trunk alopecia. An abdominal interstitial cell tumor was identified and surgically removed. Serum levels of estrogen were markedly elevated before surgery and decreased after tumor removal, indicating that the tumor had been functionally secretory. Nine months later, the marmot presented with respiratory stridor. A large boney nasal mass was identified radiographically and evaluated by computed tomography (CT) prior to surgical debulking. The marmot did not recover from anesthesia. Pathologic findings included a nasal osteosarcoma with lysis of the cribriform plate, and endocardial fibrosis with degenerative changes within the adjoining myocardium. This is the first known report of nasal osteosarcoma and interstitial tumor in a Vancouver Island marmot.

  5. Inhibition of casein kinase 2 prevents growth of human osteosarcoma.

    PubMed

    Takahashi, Kengo; Setoguchi, Takao; Tsuru, Arisa; Saitoh, Yoshinobu; Nagano, Satoshi; Ishidou, Yasuhiro; Maeda, Shingo; Furukawa, Tatsuhiko; Komiya, Setsuro

    2017-02-01

    High-dose chemotherapy and surgical treatment have improved the prognosis of osteosarcoma. However, more than 20% of patients with osteosarcoma still have a poor prognosis. We investigated the expression and function of casein kinase 2 (CK2) in osteosarcoma growth. We then examined the effects of CX-4945, a CK2 inhibitor, on osteosarcoma growth in vitro and in vivo to apply our findings to the clinical setting. We examined the expression of CK2α and CK2β by western blot analysis, and performed WST-1 assays using CK2α and CK2β siRNA or CX-4945. Flow cytometry and western blot analyses were performed to evaluate apoptotic cell death. Xenograft models were used to examine the effect of CX-4945 in vivo. Western blot analysis revealed upregulation of CK2α and CK2β in human osteosarcoma cell lines compared with human osteoblast cells or mesenchymal stem cells. WST assay showed that knockdown of CK2α or CK2β by siRNA inhibited the proliferation of human osteosarcoma cells. Treatment with 3 µM of CX-4945 inhibited osteosarcoma cell proliferation; however, the same concentration of CX-4945 did not affect the proliferation of human mesenchymal stem cells. Additionally, treatment with CX-4945 inhibited the proliferation of human osteosarcoma cells in a dose-dependent manner. Western blot and flow cytometry analyses showed that treatment with CX-4945 promoted apoptotic death of osteosarcoma cells. The xenograft model showed that treatment with CX-4945 significantly prevented osteosarcoma growth in vivo compared with control vehicle treatment. Our findings indicate that CK2 may be an attractive therapeutic target for treating osteosarcoma.

  6. Development of micro-contact printing of osteosarcoma cells using MeV ion beam lithography

    NASA Astrophysics Data System (ADS)

    Sangyuenyongpipat, Somjai; Marjomäki, Varpu; Ikonen, Stiina; Sajavaara, Timo; Ananda Sagari, A. R.; Gorelick, Sergey; Laitinen, Mikko; Whitlow, Harry J.

    2009-06-01

    For investigation of spatial effects in signalling between cells and also signal substances that trigger cell proliferation and behaviour we are developing a micro Contact Printing process (μCP). In order to allow printing of cells stamps with high aspect ratio are required and these have been fabricated using Programmed Proximity Aperture Lithography (PPAL) with 3 MeV 4He ions to produce PMMA masters for casting the stamps in PDMS. A simple printing device was developed and the first results using this to print human osteosarcoma cells is demonstrated.

  7. High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures

    PubMed Central

    Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

    2014-01-01

    Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49fhi/CD90lo cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49fhi/CD90lo cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. PMID:24802970

  8. Wnt Inhibitory Factor 1 (WIF-1) decreases tumorigenesis and metastasis in osteosarcoma

    PubMed Central

    Rubin, Elyssa M.; Guo, Yi; Tu, Khoa; Xie, Jun; Zi, Xiaolin; Hoang, Bang H.

    2010-01-01

    It has been reported that the progression of osteosarcoma was closely associated with aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in normal human osteoblast and osteosarcoma cell lines was determined by real-time RT-PCR, methylation-specific PCR (MSP), and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared to normal human osteoblasts, WIF-1 mRNA and protein levels were significantly down-regulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by subcutaneous injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent anti-osteosarcoma effect in vivo in mouse models. Therefore, re-expression of WIF-1 in WIF-1 deficient osteosarcoma represents a potential novel treatment and preventive strategy. PMID:20197388

  9. Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

    1994-01-01

    The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

  10. Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

    1994-01-01

    The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

  11. Transcription factor Oct4 promotes osteosarcoma by regulating lncRNA AK055347

    PubMed Central

    Fan, Hongwu; Liu, Guangyao; Zhao, Changfu; Li, Xuefeng; Yang, Xiaoyu

    2017-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents, typically presenting with a poor prognosis. Octamer-binding transcription factor 4 (Oct4) protein, encoded by the POU class 5 homeobox 1 gene, is important in maintaining self-renewal of pluripotent stem cells, and is closely associated with cancer. However, its role in osteosarcoma remains to be elucidated. The present study observed Oct4 was markedly increased in osteosarcoma cell lines and in human osteosarcoma tissue samples. Following Oct4 downregulation by small interfering RNA (siRNA) in osteosarcoma F5M2 cells, the cells exhibited significant decreases in proliferation and invasion ability, and an increase in cell apoptosis. Notably, downregulation of Oct4 decreased the expression of AK055347, a newly identified long noncoding RNA (lncRNA) in human tissues. The downregulation of AK055347 by siRNA resulted in a significant suppressive effect on proliferative and invasive ability, and promotion of cell apoptosis in osteosarcoma cells. Thus, the current study suggests Oct4 exerts a promoting effect in osteosarcoma, and identifies a novel lncRNA in osteosarcoma progression. PMID:28123573

  12. Effect of mesenchymal stem cells on hypoxia-induced desensitization of β2-adrenergic receptors in rat osteosarcoma cells

    PubMed Central

    KIDO, AKIRA; YOSHITANI, KAZUHIRO; SHIMIZU, TAKAMASA; AKAHANE, MANABU; FUJII, HIROMASA; TSUKAMOTO, SHINJI; KONDO, YUMIKO; HONOKI, KANYA; IMANO, MOTOHIRO; TANAKA, YASUHITO

    2012-01-01

    The β2-adrenergic receptor (β2AR) mediates the effects of chronic stress in several neoplasms, however, β2AR signaling is impaired by hypoxia in various tissues. While hypoxia is a common feature significant in the progression of solid tumors, little is known about the effect of hypoxia on β2AR signaling in the tumor microenvironment. Previously, it has been reported that the systemic administration of mesenchymal stem cells (MSCs) increased the engraftment and metastatic colonization of rat osteosarcoma (OS) cells. In the current study, the effect of MSCs on the hypoxia-induced desensitization of the β2AR in OS cells was investigated. Epinephrine, norepinephrine and isoproterenol increased the cellular proliferation of the rat OS cell line COS1NR and rat MSCs in a dose-dependent and β2AR antagonist-sensitive manner. While isoproterenol had significant proliferative effects on MSCs under normoxic and hypoxic conditions, COS1NR cells did not respond under hypoxic conditions. A sensitivity assay for the β2AR revealed that hypoxia impaired the sensitivity of COS1NR cells, whereas hypoxia did not affect MSCs. An immunoassay revealed no significant change in the expression of hypoxia-inducible factor-1α (HIF1α) in COS1NR cells, whilst an immunoassay demonstrated a 15% increase in MSCs following isoproterenol stimulation. In COS1NR cells co-cultured with MSCs under hypoxic conditions, isoproterenol caused a significant increase in proliferation and this effect was inhibited by an anti-interleukin (IL)-6 antibody. A tumor formation assay in syngeneic rats revealed that the systemic administration of MSCs enhances the growth of OS and the effect of MSCs was inhibited by IL-6 neutralization. In conclusion, MSCs are resistant to the hypoxia-induced desensitization to β2AR. Hypoxia caused a siginificant desensitization of the β2AR in COS1NR cells alone, whereas MSCs may support tumor progression through cellular interactions. PMID:23205094

  13. Model for Osteosarcoma-9 as a potent factor in cell survival and resistance to apoptosis.

    PubMed

    Vourvouhaki, Ekaterini; Carvalho, Carla; Aguiar, Paulo

    2007-07-01

    In this paper we use a simple model to explore the function of the gene Osteosarcoma-9 (OS-9). We are particularly interested in understanding the role of this gene as a potent anti-apoptotic factor. The theoretical description is constrained by experimental data from induction of apoptosis in cells where OS-9 is overexpressed. The data available suggest that OS-9 promotes cell viability and confers resistance to apoptosis, potentially implicating OS-9 in the survival of cancer cells. Three different apoptosis-inducing mechanisms were tested and are modeled here. A more complex and realistic model is also discussed.

  14. Model for Osteosarcoma-9 as a potent factor in cell survival and resistance to apoptosis

    NASA Astrophysics Data System (ADS)

    Vourvouhaki, Ekaterini; Carvalho, Carla; Aguiar, Paulo

    2007-07-01

    In this paper we use a simple model to explore the function of the gene Osteosarcoma-9 (OS-9). We are particularly interested in understanding the role of this gene as a potent anti-apoptotic factor. The theoretical description is constrained by experimental data from induction of apoptosis in cells where OS-9 is overexpressed. The data available suggest that OS-9 promotes cell viability and confers resistance to apoptosis, potentially implicating OS-9 in the survival of cancer cells. Three different apoptosis-inducing mechanisms were tested and are modeled here. A more complex and realistic model is also discussed.

  15. Inhibition of focal adhesion kinase induces apoptosis in human osteosarcoma SAOS-2 cells.

    PubMed

    Wang, Jialiang; Zu, Jianing; Xu, Gongping; Zhao, Wei; Jinglong, Yan

    2014-02-01

    Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, acts as an early modulator of integrin signaling cascade, regulating basic cellular functions. In transformed cells, unopposed FAK signaling has been considered to promote tumor growth, progression, and metastasis. The aim of this study was to assess the role of focal adhesion kinase in human osteosarcoma SAOS-2 cells. SAOS-2 cells were transfected with PGPU6/GFP/shNC, and PGPU6/GFP/FAK-334 (shRNA-334), respectively. Expression of FAK was detected by real-time PCR and western blots. MTT assay was used to examine changes in cell proliferation. Cell apoptosis was analyzed by flow cytometry. The expression of caspase-3,-7,-9 was measured by Western blots. The expression of FAK in SAOS-2 cells significantly decreased in shRNA-334 group contrast to the control group (P < 0.01). Cells proliferation was inhibited by shRNA-334 and shRNA-334 + cisplatin, and the effects were clearly enhanced when cells treated with the anticancer agents. The level of cell apoptosis in shRNA-334 and shRNA-334 + cisplatin group was higher than in the control group (P < 0.01). The current data support evidence that down-regulation of FAK could induce SAOS-2 apoptosis through the caspase-dependent cell death pathway. Inhibition of the kinases may be important for therapies designed to enhance the apoptosis in osteosarcoma.

  16. Mesenchymal stem cells increase proliferation but do not change quiescent state of osteosarcoma cells: Potential implications according to the tumor resection status

    PubMed Central

    Avril, Pierre; Le Nail, Louis-Romée; Brennan, Meadhbh Á.; Rosset, Philippe; De Pinieux, Gonzague; Layrolle, Pierre; Heymann, Dominique; Perrot, Pierre; Trichet, Valérie

    2015-01-01

    Conventional therapy of primary bone tumors includes surgical excision with wide resection, which leads to physical and aesthetic defects. For reconstruction of bone and joints, allografts can be supplemented with mesenchymal stem cells (MSCs). Similarly, adipose tissue transfer (ATT) is supplemented with adipose-derived stem cells (ADSCs) to improve the efficient grafting in the correction of soft tissue defects. MSC-like cells may also be used in tumor-targeted cell therapy. However, MSC may have adverse effects on sarcoma development. In the present study, human ADSCs, MSCs and pre-osteoclasts were co-injected with human MNNG-HOS osteosarcoma cells in immunodeficient mice. ADSCs and MSCs, but not the osteoclast precursors, accelerated the local proliferation of MNNG-HOS osteosarcoma cells. However, the osteolysis and the metastasis process were not exacerbated by ADSCs, MSCs, or pre-osteoclasts. In vitro proliferation of MNNG-HOS and Saos-2 osteosarcoma cells was increased up to 2-fold in the presence of ADSC-conditioned medium. In contrast, ADSC-conditioned medium did not change the dormant, quiescent state of osteosarcoma cells cultured in oncospheres. Due to the enhancing effect of ADSCs/MSCs on in vivo/in vitro proliferation of osteosarcoma cells, MSCs may not be good candidates for osteosarcoma-targeted cell therapy. Although conditioned medium of ADSCs accelerated the cell cycle of proliferating osteosarcoma cells, it did not change the quiescent state of dormant osteosarcoma cells, indicating that ADSC-secreted factors may not be involved in the risk of local recurrence. PMID:26998421

  17. Osteosarcoma cell proliferation and survival requires mGluR5 receptor activity and is blocked by Riluzole

    PubMed Central

    Gulzar, Hira; Yelskaya, Zarina; Ait Taouit, Lyes; Houssou, Murielle; Jaikaran, Trisha; Schvarts, Yuriy; Kozlitina, Kristina; Basu-Roy, Upal; Mansukhani, Alka; Mahajan, Shahana S.

    2017-01-01

    Osteosarcomas are malignant tumors of bone, most commonly seen in children and adolescents. Despite advances in modern medicine, the poor survival rate of metastatic osteosarcoma has not improved in two decades. In the present study we have investigated the effect of Riluzole on a human and mouse metastatic osteosarcoma cells. We show that LM7 cells secrete glutamate in the media and that mGluR5 receptors are required for the proliferation of LM7 cells. Riluzole, which is known to inhibit glutamate release, inhibits proliferation, induces apoptosis and prevents migration of LM7 cells. This is also seen with Fenobam, a specific blocker of mGluR5. We also show that Riluzole alters the phosphorylation status of AKT/P70 S6 kinase, ERK1/2 and JNK1/2. Thus Riluzole is an effective drug to inhibit proliferation and survival of osteosarcoma cells and has therapeutic potential for the treatment of osteosarcoma exhibiting autocrine glutamate signaling. PMID:28231291

  18. Osteosarcoma cell proliferation and survival requires mGluR5 receptor activity and is blocked by Riluzole.

    PubMed

    Liao, Sally; Ruiz, Yuleisy; Gulzar, Hira; Yelskaya, Zarina; Ait Taouit, Lyes; Houssou, Murielle; Jaikaran, Trisha; Schvarts, Yuriy; Kozlitina, Kristina; Basu-Roy, Upal; Mansukhani, Alka; Mahajan, Shahana S

    2017-01-01

    Osteosarcomas are malignant tumors of bone, most commonly seen in children and adolescents. Despite advances in modern medicine, the poor survival rate of metastatic osteosarcoma has not improved in two decades. In the present study we have investigated the effect of Riluzole on a human and mouse metastatic osteosarcoma cells. We show that LM7 cells secrete glutamate in the media and that mGluR5 receptors are required for the proliferation of LM7 cells. Riluzole, which is known to inhibit glutamate release, inhibits proliferation, induces apoptosis and prevents migration of LM7 cells. This is also seen with Fenobam, a specific blocker of mGluR5. We also show that Riluzole alters the phosphorylation status of AKT/P70 S6 kinase, ERK1/2 and JNK1/2. Thus Riluzole is an effective drug to inhibit proliferation and survival of osteosarcoma cells and has therapeutic potential for the treatment of osteosarcoma exhibiting autocrine glutamate signaling.

  19. Do Mesenchymal Stromal Cells Influence Microscopic Residual or Metastatic Osteosarcoma in a Murine Model?

    PubMed

    Aanstoos, Megan E; Regan, Daniel P; Rose, Ruth J; Chubb, Laura S; Ehrhart, Nicole P

    2016-03-01

    Mesenchymal stromal cells (MSCs) have been shown in rodent models to promote primary and pulmonary metastatic sarcoma growth when injected in the presence of gross tumor. In theory, this would limit their use in a clinical setting after limb salvage treatment for osteosarcoma. Although concerning, these models do not translate to the clinical setting wherein MSCs could be used after primary tumor resection to aid in bone healing and incorporation of tumor endoprostheses. If we can determine whether the use of MSCs in this setting is safe, it might improve our ability to augment bone healing in patients undergoing limb salvage. The purpose of this study was to determine (1) whether MSCs promote pulmonary metastatic disease progression in a murine osteosarcoma model; and/or (2) whether they affect local disease recurrence in the presence of microscopic residual osteosarcoma. An orthotopic model of luciferase-expressing osteosarcoma was developed. At 10 days, resection of the primary tumor was performed. One hundred fourteen female C3H mice were inoculated with DLM8-luc osteosarcoma in the proximal tibia. Ninety-four mice developed orthotopic osteosarcoma with luciferase expression. Mice with bioluminescent evidence of a primary tumor received either a microscopically "clean" amputation at a time when residual microscopic metastatic disease was present in the lungs (pulmonary metastasis group; n = 65) or a "dirty" amputation (local recurrence group; n = 29). Mice were randomized to receive intravenous MSCs, MSCs at the surgical site, or no MSCs. Mice were monitored for development and progression of pulmonary metastasis and local recurrence by bioluminescence imaging and daily measurements at the surgical site. The number of pulmonary nodules, time to first evidence of metastasis, and size of recurrent tumor were compared using Kruskal-Wallis, analysis of variance, Welch's, t-tests, or Mann-Whitney tests as appropriate for the specific data sets with p < 0

  20. Visfatin triggers the in vitro migration of osteosarcoma cells via activation of NF-κB/IL-6 signals.

    PubMed

    Wang, Guang-Ji; Shen, Ning-Jiang; Cheng, Liang; Yehan Fang; Huang, Hui; Li, Kang-Hua

    2016-11-15

    Pulmonary metastasis is the major challenge for clinical treatment of osteosarcoma patients. Recent studies indicated that visfatin, a 52kDa adipocytokine, can trigger the cell motility of various cancers, while its role in the progression of osteosarcoma remains not clear. Our present study revealed that visfatin can significantly promote the in vitro migration and invasion of osteosarcoma MG-63 and HOS cells and up regulate the expression of matrix metalloproteinase-2 (MMP-2) and fibronectin (FN). Furthermore, visfatin treatment also increased the expression of IL-6 in both MG-63 and HOS cells via a time dependent manner, while anti-IL-6 antibody can significantly attenuate visfatin induced cell invasion and up regulation of MMP-2 and FN. It suggested that up regulation of IL-6 mediated visfatin induced in vitro motility of osteosarcoma cells. Visfatin treatment can increase the phosphorylation of both p65 and ERK1/2 in MG-63 and HOS cells, while only the inhibitor of NF-κB, BAY 11-7082, can abolish visfatin induced up regulation of IL-6. BAY 11-7082 also attenuated visfatin induced upregulation of MMP-2 and FN in MG-63 cells. Western blot analysis revealed that visfatin treatment can significantly increase the phosphorylation of IκBα and IKKβ in MG-63 cells. ACHP, the inhibitor of IKK-β, blocked visfatin induced expression of IL-6 mRNA in both MG-63 and HOS cells. Collectively, our data suggested that visfatin can increase the motility of osteosarcoma cells via up regulation of NF-κB/IL-6 signals. It indicated that visfatin might be a potential therapeutic target of osteosarcoma treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. RhoA/ROCK pathway inhibition by fasudil suppresses the vasculogenic mimicry of U2OS osteosarcoma cells in vitro.

    PubMed

    Xia, Yun; Cai, Xianyi; Fan, Jiquan; Zhang, Liling; Li, Zhenyu; Ren, Jinghua; Wu, Gang; Zhu, Fang

    2017-02-20

    GTPase RhoA and its downstream Rho-associated coiled-coil-containing protein kinases (ROCKs) are frequently overexpressed in human cancers. Inhibition of the RhoA/ROCK pathway blocks angiogenesis mediated by the vascular endothelial growth factor, which led us to investigate the role of this pathway in vasculogenic mimicry (VM) - a process by which aggressive cancer cells form vessel-like structures that provide adequate blood supply for tumor growth. We showed that the expression of RhoA and its effector kinases ROCK1/2 was much higher in human osteosarcoma (OS) tissues and the human OS cell line U2OS than in nontumorous tissues and cell line hFOB 1.19 using western blot analysis and real-time PCR. Inhibition of the RhoA/ROCK signaling pathway by the pharmacological inhibitor fasudil reduced vascular-like channels of U2OS cells in Matrigel. Furthermore, we used rhodamine-phalloidin immunofluorescence, wound healing assay, and transwell migration assay to examine the effect of fasudil on tumor cell plasticity and motility, both of which play key roles in VM formation. Finally, we explored the underlying mechanisms of fasudil-induced VM destruction. In this context, we showed that the RhoA/ROCK signaling pathway is a novel regulator in VM of U2OS OS cells and suggest that fasudil in conjunction with established treatments may present a novel therapeutic strategy for OS.

  2. 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

  3. Hybrid liposomes inhibit tumor growth and lung metastasis of murine osteosarcoma cells.

    PubMed

    Kitajima, Hideki; Komizu, Yuji; Ichihara, Hideaki; Goto, Koichi; Ueoka, Ryuichi

    2013-06-01

    Antitumor effects of hybrid liposomes (HL) composed of l-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ether (C₁₂(EO)₂₃) on the metastatic growth of murine osteosarcoma (LM8) cells were investigated in vitro and in vivo. Remarkable inhibitory effects of HL-23 on the growth of LM8 cells were obtained through the induction of apoptotic cell death in vitro. It was also indicated that HL-23 should dramatically suppress the invasion of LM8 cells and the formation of filopodia on the cell surface in vitro. Furthermore, significantly high therapeutic effects were observed in the homograft mouse models of LM8 cells with lung metastasis after the treatment with HL-23 in vivo. That is, the histological analysis demonstrated that the primary tumor growth of LM8 cells implanted subcutaneously into the mice was inhibited along with the induction of apoptosis. In addition, it was found that HL-23 significantly decreased the lung metastasis of LM8 cells in the mouse models through the inhibition of primary tumor invasion. These results suggest that HL-23 could be a novel agent for the chemotherapy of osteosarcoma.

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

    SciTech Connect

    Bin Hafeez, Bilal; Ahmed, Salahuddin; Wang, Naizhen; Gupta, Sanjay; Zhang Ailin; Haqqi, Tariq M. . E-mail: txh5@case.edu

    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. GTP 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.

  5. Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-kappaB.

    PubMed

    Hafeez, Bilal Bin; Ahmed, Salahuddin; Wang, Naizhen; Gupta, Sanjay; Zhang, Ailin; Haqqi, Tariq M

    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-kappaB (NF-kappaB) 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 microg/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-kappaB/p65 and lowering of NF-kappaB/p65 and p50 levels in the cytoplasm and nucleus. GTP treatment of cells reduced IkappaB-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 IkappaB-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-kappaB.

  6. Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis.

    PubMed

    de Nigris, Filomena; Rossiello, Raffaele; Schiano, Concetta; Arra, Claudio; Williams-Ignarro, Sharon; Barbieri, Antonio; Lanza, Alessandro; Balestrieri, Antonio; Giuliano, Maria Teresa; Ignarro, Louis J; Napoli, Claudio

    2008-03-15

    We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth.

  7. Krüppel-like factor 4 promotes high-mobility group box 1-induced chemotherapy resistance in osteosarcoma cells.

    PubMed

    Huang, Jun; Liu, Ke; Song, Deye; Ding, Muliang; Wang, Junjie; Jin, Qingping; Ni, Jiangdong

    2016-03-01

    Osteosarcoma is the most common primary malignant bone tumor, and the frequent acquisition of chemoresistance is often an obstacle to achieving favorable outcomes during chemotherapy. Recently, Krüppel-like factor 4 (KLF4) has been shown to be associated with chemotherapy resistance in a few tumors; however, the involvement of KLF4 in chemotherapy resistance in osteosarcoma cells remains unknown. In this study, quantitative real-time PCR and western blot analysis revealed that KLF4 expression was significantly increased in response to cisplatin, methotrexate and doxorubicin treatment in osteosarcoma cells, and knockdown of KLF4 increased sensitivity to these anticancer drugs by decreasing cellular clonogenic ability and increasing apoptosis. Moreover, our data suggest that KLF4-regulated drug resistance might, at least partially, positively regulate high-mobility group box 1 (HMGB1), which was found to be a significant contributor to chemoresistance in osteosarcoma cells in our previous study. In summary, this study highlights the significance of KLF4/HMGB1 interaction in regulating chemotherapy resistance, and suggests that targeting KLF4/high-mobility group box 1 may be a therapeutic strategy for osteosarcoma chemotherapy. © 2015 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  8. Osteosarcoma: Cells-of-Origin, Cancer Stem Cells, and Targeted Therapies

    PubMed Central

    Abarrategi, Ander; Tornin, Juan; Martinez-Cruzado, Lucia; Hamilton, Ashley; Martinez-Campos, Enrique; Rodrigo, Juan P.; González, M. Victoria; Baldini, Nicola; Garcia-Castro, Javier; Rodriguez, Rene

    2016-01-01

    Osteosarcoma (OS) is the most common type of primary solid tumor that develops in bone. Although standard chemotherapy has significantly improved long-term survival over the past few decades, the outcome for those patients with metastatic or recurrent OS remains dismally poor and, therefore, novel agents and treatment regimens are urgently required. A hypothesis to explain the resistance of OS to chemotherapy is the existence of drug resistant CSCs with progenitor properties that are responsible of tumor relapses and metastasis. These subpopulations of CSCs commonly emerge during tumor evolution from the cell-of-origin, which are the normal cells that acquire the first cancer-promoting mutations to initiate tumor formation. In OS, several cell types along the osteogenic lineage have been proposed as cell-of-origin. Both the cell-of-origin and their derived CSC subpopulations are highly influenced by environmental and epigenetic factors and, therefore, targeting the OS-CSC environment and niche is the rationale for many recently postulated therapies. Likewise, some strategies for targeting CSC-associated signaling pathways have already been tested in both preclinical and clinical settings. This review recapitulates current OS cell-of-origin models, the properties of the OS-CSC and its niche, and potential new therapies able to target OS-CSCs. PMID:27366153

  9. Apigenin inhibits the proliferation and invasion of osteosarcoma cells by suppressing the Wnt/β-catenin signaling pathway.

    PubMed

    Liu, Xiaofeng; Li, Liubing; Lv, Ling; Chen, Dongmei; Shen, Liqin; Xie, Zonggang

    2015-08-01

    Osteosarcoma (OS) is the most common type of bone cancer. Even with early diagnosis and aggressive treatment, the prognosis for OS is poor. In the present study, we investigated the proliferation and invasion inhibitory effect of apigenin on human OS cells and the possible molecular mechanisms involved. The cell viability of U2OS and MG63 human OS cell lines was detected by MTT assay. Cell cycle progression and invasion were assessed by flow cytometry and the Matrigel Boyden chamber assay, respectively, and the involvement of molecular mechanisms was examined by western blot analysis. We demonstrated that apigenin inhibited proliferation and reduced invasion in human OS cells, and downregulated the expression of β-catenin in OS cells. Furthermore, the inhibitory effect of apigenin on OS cells was reversed by overexpression of β-catenin, but enhanced by knockdown of β-catenin. Collectively, our results showed that apigenin inhibits the tumor growth of OS cells by inactivating Wnt/β-catenin signaling. Therefore, apigenin is a promising chemotherapeutic agent that may be used in the treatment of human OS.

  10. Adriamycin resistance-associated prohibitin gene inhibits proliferation of human osteosarcoma MG63 cells by interacting with oncogenes and tumor suppressor genes.

    PubMed

    Du, Min-Dong; He, Kai-Yi; Qin, Gang; Chen, Jin; Li, Jin-Yi

    2016-09-01

    The resistance of cancer cells to chemotherapeutic agents is a major obstacle for successful chemotherapy, and the mechanism of chemoresistance remains unclear. The present study developed an adriamycin-resistant human osteosarcoma MG-63 sub-line (MG-63/ADR), and identified differentially expressed proteins that may be associated with adriamycin resistance. Two dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis and a protein identification assay were performed. Western blot analysis was used to examine the prohibitin (PHB) levels in the MG-63/ADR cells. Quantitative polymerase chain reaction was utilized to detect adriamycin resistant-associated genes. Laser-scanning confocal microscope was employed to examine the colocalization of PHB with v-myc avian myelocytomatosis viral oncogene homolog (c-myc), FBJ murine osteosarcoma viral oncogene homolog (c-fos), tumor protein p53 and retinoblastoma 1 (Rb). In addition, the full length of the open reading frame of human PHB was subcloned into a lentiviral vector pLVX-puro. The proliferative rate of MG-63 cells was also investigated. The overall protein expression in MG-63/ADR cells was clearly suppressed. Three notable protein regions, representing high mobility group box 1, Ras homolog gene family, member A, and PHB, were identified to be significantly altered in MG-63/ADR cells when compared with its parental cells. Therefore, PHB modulated the chemoresistance of MG-63/ADR cells by interacting with multiple oncogenes or tumor suppressor genes (c-myc, c-fos, p53 and Rb). In addition, overexpression of PHB decreases the proliferative rate of MG-63 cells. In conclusion, PHB is an adriamycin resistance-associated gene, which may inhibit the proliferation of human osteosarcoma MG-63 cells by interacting with the oncogenes or tumor suppressor genes, c-myc, c-fos, p53 and Rb.

  11. Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation

    PubMed Central

    Engel, Nadja; Adamus, Anna; Schauer, Nicolas; Kühn, Juliane; Nebe, Barbara; Seitz, Guido; Kraft, Karin

    2017-01-01

    Background Phytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts. Methods Thus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed. Results Exposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells. Conclusion From these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol

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