• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2311-2315
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• 2013

Objective: To investigate the expression level of TUG1 and one of its transcript variants (n377360) in osteosarcoma cells and assess the role of TUG1 in proliferation and apoptosis in the U2OS cell line. Methods: TUG1 and n377360 expression levels in patients with osteosarcomas and the U2OS human osteosarcoma cell line were evaluated using real-time quantitative PCR. U2OS cells were transected with TUG1 and n377360 siRNA or non-targeting siRNA. MTS was performed to assess the cell proliferation and flow cytometry was applied to analyze apoptosis. Results: We found significantly higher TUG1 and n377360 expression levels in osteosarcoma tissues compared with matched non-tumorous tissues. In line with this, suppression of TUG1 and n377360 expression by siRNA significantly impaired the cell proliferation potential of osteosarcoma cells. Furthermore, inhibition of TUG1 expression significantly promoted osteosarcoma cell apoptosis. Conclusions: The overexpression of TUG1 and n377360 in osteosarcoma specimens and the functional role of TUG1 and n377360 regarding cell proliferation and apoptosis in an osteosarcoma cell line provided evidence that the use of TUG1 or n377360 may be a viable but an as yet unexplored therapeutic strategy in tumors that over express these factors.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.1101-1106
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• 2013

Background: Osteosarcoma is the most common primary bone tumor in childhood and adolescence. Carboplatin, a platinum-derived agent, is used as neoadjuvant chemotherapy for pediatric osteosarcoma because of its anti-tumor activity and had low toxicity as compared to cisplatin. Objective: To determine demographic data, prognostic factors and outcome of childhood osteosarcoma treated with a carboplatin-based chemotherapeutic protocol at Chiang Mai University. Method: A retrospective analysis was conducted on 34 osteosarcoma patients aged less than 18 years and treated between 2003 and 2011. Results: Overall limb-salvage and amputation rates were 23.5% and 70.6%, respectively. With the mean follow-up time of 29.5 months (1.5-108.9), the Kaplan-Meier analysis for 3-year disease-free survival (DFS) and 3-year overall survival (OS) were $20.2{\pm}7.7%$ and $47.1{\pm}9.5%$ respectively. Patients who had initial pulmonary metastasis were at significantly greater risk for developing recurrence (p=0.02, OR=7; 1.2-40.1) and had a tendency to have lower 3-year OS compared to those without initial pulmonary metastasis ($28.1{\pm}13%$, $63.1{\pm}12.3%$, respectively, p=0.202). On univariate analysis, age at diagnosis >14 years and patients who were declined surgery were significantly associated with lower 3-year OS (p=0.008 and <0.05, respectively). However, age at diagnosis, sex, tumor size and histological subtypes were not found to significantly affect recurrence or survival. Conclusions: In our study, the survival rate was far lower than those reported from developed countries. These might indicate the ineffectiveness of carboplatin in combination with doxorubicin as frontline treatment of pediatric osteosarcoma, especially in those with initial pulmonary metastasis. Refinement in risk and treatment stratification and dose intensification for pediatric osteosarcoma constitutes a future challenge to improve outcomes, especially in metastatic patients who may need a more intensive regimen.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.22
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• pp.9823-9829
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• 2014

Background: High-dose methotrexate (HD-MTX) is recognized as an efficient component of therapy against pediatric osteosarcoma in combination with other drugs such as cisplatin (CDP), carboplatin (CBDCA), doxorubicin (ADM), etoposide (VP-16) and ifosfamide (IFO). Objectives: To demonstrate the feasibility and effectiveness of the HD-MTX/CDP/DOX/VP-16/IFO [MTX(+)] protocol comparable to CDP/ADM/CBDCA/IFO [MTX(-)] for treating childhood osteosarcoma at Ramathibodi Hospital (1999-2014). Materials and Methods: A retrospective analysis was conducted of osteosarcoma patients aged less than 18 years treated with two chemotherapeutic regimens between 1999 and 2014. A total of 45 patients received the MTX(-) and 21 the MTX(+) protocol. Results: Overall limb-salvage and amputation rate were 12.9% and 77.7%, respectively. Kaplan-Meier analysis results for 3-year disease free survival (DFS) and overall survival (OS) regardless of treatment regimens were $43.4{\pm}6.0%$ and $53.2{\pm}6.1%$ respectively. The 3-year DFS and OS were improved significantly with the MTX(+) protocol compared to MTX(-) protocol (p=0.010 and p=0.009, log rank test) [$69.8{\pm}10.5%$, $79.8{\pm}9.1%$ for MTX(+) and $31.1{\pm}6.9%$, $42.2{\pm}7.4%$ for MTX(-) protocol, respectively]. Patients with metastatic osteosarcoma treated with the MTX(+) protocol had statistically significant higher 3-year DFS and OS than those treated with the MTX(-) protocol ($66.7{\pm}13.6%$ and $15.0{\pm}8.0%$ for 3-year DFS, p=0.010, $73.3{\pm}13.2%$ and $20{\pm}8.9%$ for 3-year OS, p=0.006, respectively). The independent risk factors for having inferior 3-year DFS and OS were poor histological response (tumor necrosis <90%) and treatment with the MTX(-) protocol. The multivariate analysis identified only the treatment with the MTX(-) protocol as an independent predictor of inferior OS with a hazard ratio (HR) of 3.53 (95% confidence interval of 1.2-10.41, p=0.022). Conclusions: Our study demonstrated the tolerability, feasibility and efficacy of the HDMTX-based regimen improving the survival rate in pediatric osteosarcoma cases, in line with reports from developed countries.

• BMB Reports
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• v.46 no.2
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• pp.113-118
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• 2013

TTRAP is a multi-functional protein that is involved in multiple aspects of cellular functions including cell proliferation, apoptosis and the repair of DNA damage. Here, we demonstrated that the lentivirus-mediated overexpression of TTRAP significantly inhibited cell growth and induced apoptosis in osteosarcoma cells. The ectopic TTRAP suppressed the growth and colony formation capacity of two osteosarcoma cell lines, U2OS and Saos-2. Cell apoptosis was induced in U2OS cells and the cell cycle was arrested at G2/M phase in Saos-2 cells. Exogenous expression of TTRAP in serum-starved U2OS and Saos-2 cells induced an increase in caspase-3/-7 activity and a decrease in cyclin B1 expression. In comparison with wild-type TTRAP, mutations in the 5'-tyrosyl-DNA phosphodiesterase activity of TTRAP, in particular $TTRAP^{E152A}$, showed decreased inhibitory activity on cell growth. These results may aid in clarifying the physiological functions of TTRAP, especially its roles in the regulation of cell growth and tumorigenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10797-10801
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• 2015

Background: To explore the molecular mechanisms of metastatic osteosarcoma (OS) by using the microarray expression profiles of metastatic and non-metastatic OS samples. Materials and Methods: The gene expression profile GSE37552 was downloaded from Gene Expression Omnibus database, including 2 human metastatic OS cell line models and 2 two non-metastatic OS cell line models. The differentially expressed genes (DEGs) were identified by Multtest package in R language. In addition, functional enrichment analysis of the DEGs was performed by WebGestalt, and the protein-protein interaction (PPI) networks were constructed by Hitpredict, then the signal pathways of the genes involved in the networks were performed by Kyoto Encyclopaedia of Genes and Genomes (KEGG) automatic annotation server (KAAS). Results: A total of 237 genes were classified as DEGs in metastatic OS. The most significant up- and down-regulated genes were A2M (alpha-2-macroglobulin) and BCAN (brevican). The DEGs were significantly related to the response to hormone stimulus, and the PPI network of A2M contained IL1B (interleukin), LRP1 (low-density lipoprotein receptor-related protein 1) and PDGF (platelet-derived growth factor). Furthermore, the MAPK signaling pathway and focal adhesion were significantly enriched. Conclusions: A2M and its interactive proteins, such as IL1B, LRP1 and PDGF may be candidate target molecules to monitor, diagnose and treat metastatic OS. The response to hormone stimulus, MAPK signaling pathway and focal adhesion may play important roles in metastatic OS.

• Clinical and Experimental Pediatrics
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• v.62 no.2
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• pp.62-67
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• 2019

Purpose: We aimed to determine the prognostic significance of lymphocyte counts and the lymphocytemonocyte ratio (LMR) in pediatric patients with osteosarcoma. Methods: We retrospectively reviewed the medical records of 27 pediatric patients with localized extremity osteosarcoma, treated at the Korea Cancer Center Hospital between May 2002 and March 2016. Leukocyte counts and LMR before treatment and on day 14 (LMR14) of the first cisplatin-doxorubicin chemotherapy round were evaluated. Patients were dichotomized according to the median value of these parameters, and survival rates were compared. Results: The median age of the 27 patients was 9.9 years (range, 3.2-14.1 years) and tumor sites were: distal femur (n=14), proximal humerus (n=7), proximal tibia (n=2), proximal fibula (n=2), and elsewhere (n=2). Patients were followed up on for a median of 76.4 months (range, 4.5-174.7 months), and 5-year overall (OS) and event-free survival (EFS) rates were $66.0%{\pm}9.8%$ and $60.9%{\pm}9.7%$, respectively. Patients with a higher pretreatment lymphocyte count (${\geq}2,320/{\mu}L$) had better OS (90.9% vs. 46.2%, P=0.04) and EFS (83.9% vs. 38.5%, P=0.02). However, the day 14 lymphocyte count was not associated with survival. While no survival difference was observed between patients grouped according to pretreatment LMR (median value, 6.3), patients with a higher LMR14 (${\geq}5$) fared better than those with lower LMR14 (5-year OS: 83.3% vs. 46.3%, P=0.04). Conclusion: Pretreatment lymphocyte count and LMR during chemotherapy had prognostic significance in pediatric osteosarcoma patients. Further studies involving larger cohorts are necessary to validate our findings.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1817-1822
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• 2014

Objective: The aim of this study was to screen for possible biomarkers of metastatic osteosarcoma (OS) using a DNA microarray. Methods: We downloaded the gene expression profile GSE49003 from Gene Expression Omnibus database, which included 6 gene chips from metastatic and 6 from non-metastatic OS patients. The R package was used to screen and identify differentially expressed genes (DEGs) between metastatic and non-metastatic OS patients. Then we compared the expression of DEGs in the two groups and sub-grouped into up-regulated and down-regulated, followed by functional enrichment analysis using the DAVID system. Subsequently, we constructed an miRNA-DEG regulatory network with the help of WebGestalt software. Results: A total of 323 DEGs, including 134 up-regulated and 189 down-regulated, were screened out. The up-regulated DEGs were enriched in 14 subcategories and most significantly in cytoskeleton organization, while the down-regulated DEGs were prevalent in 13 subcategories, especially wound healing. In addition, we identified two important miRNAs (miR-202 and miR-9) pivotal for OS metastasis, and their relevant genes, CALD1 and STX1A. Conclusions: MiR-202 and miR-9 are potential key factors affecting the metastasis of OS and CALD1 and STX1A may be possible targets beneficial for the treatment of metastatic OS. However, further experimental studies are needed to confirm our results.

• The Journal of the Korean bone and joint tumor society
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• v.20 no.1
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• pp.1-6
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• 2014

Purpose: To examine the expression of Connective Tissue Growth Factor (CTGF) in osteosarcoma and to evaluate its role in osteosarcoma invasion and proliferation. Materials and Methods: The mRNA expression of CTGF from 23 patient-derived osteosarcoma cell lines was examined, and the role of CTGF in cell invasion and proliferation was examined using siRNA transfection. Results: The over-expression of CTGF mRNA was observed in 17 cell lines (74%). CTGF-specific siRNA transfection into SaOS-2 and MG63 cell lines resulted in efficient knockdown of CTGF expression on Western blot analysis. siRNA transfected cells showed decreased migration on Matrigel invasion assay and decreased cell proliferation on WST-1 assay. Conclusion: These results indicated that the CTGF expression may play an important role in osteosarcoma progression, and may be a therapeutic target of osteosarcoma.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.795-799
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• 2013

Ku70/80 heterodimer is a central element in the nonhomologous end joining (NHEJ) DNA repair pathway, Ku80 playing a key role in regulating the multiple functions of Ku proteins. It has been found that the Ku80 protein located at telomeres is a major contributor to radiosensitivity in some telomerase positive human cancer cells. However, in ALT human osteosarcoma cells, the precise function in radiosensitivity and telomere maintenance is still unknown. The aim of this study was to investigate the effects of Ku80 depletion in the U2OS ALT cell line cell line. Suppression of Ku80 expression was performed using a vector-based shRNA and stable Ku80 knockdown in cells was verified by Western blotting. U2OS cells treated with shRNA-Ku80 showed lower radiobiological parameters (D0, Dq and SF2) in clonogenic assays. Furthermore, shRNA-Ku80 vector transfected cells displayed shortening of the telomere length and showed less expression of TRF2 protein. These results demonstrated that down-regulation of Ku80 can sensitize ALT cells U2OS to radiation, and this radiosensitization is related to telomere length shortening.

• The Journal of the Korean bone and joint tumor society
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• v.13 no.2
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• pp.88-95
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• 2007

Purpose: The purpose of this study was to compare the proapoptotic effects of matrix metal-loproteinase inhibitor (MMPI) and doxorubicin on wild-type p53 osteosarcoma cell line, socalled U2OS cell line. Materials and Methods: U2OS cells were treated with MMP inhibitor III (MMPI III) and doxorubicin, either respectively or simultaneously. In cells treated with doxorubicin, Fas-neutralizing antibody so called ZB4 was additionally treated to examine whether the doxorubicin played a role through the Fas/FasL pathway. Cells were analysed regarding to apoptosis and cell death by flow cytometry. Results: U2OS cells incubated with doxorubicin showed significant amount of cell death in dose-dependent manner. However, those incubated with MMPI III mostly remained viable state. In addition, there is no relationship between two drugs. Cells treated with doxorubicin and ZB4 at the same time did not show down regulation of apoptosis through inhibition of Fas/FasL pathway. Conclusion: It is important to re-examine MMP inhibitor's effect on other osteosarcoma cell line with wild-type p14 as well as wild-type p53 to evaluate its proapoptotic effect.