Antimetastatic activity of honokiol in osteosarcoma

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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 (IC₅₀) 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 IC₅₀ 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.

CONCLUSIONS:

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. Cancer 2012. © 2011 American Cancer Society.     

Synergistic inhibitory effect of apomine and lovastatin on osteosarcoma cell growth

BACKGROUND:

Osteosarcoma is the most frequent malignant primary bone tumor that occurs mainly in the young, with an incidence peak observed at age 18 years. Both apomine and lovastatin have antitumor activity in a variety of cancer cell lines. Apomine, a 1,1‐bisphosphonate‐ester, increases the rate of degradation of 3‐hydroxy‐3 methylglutaryl‐coenzyme A (HMG‐CoA) reductase, the rate‐limiting enzyme in the mevalonate pathway, whereas lovastatin competitively inhibits HMG‐CoA reductase enzyme activity, thereby preventing protein prenylation and cholesterol synthesis.

METHODS:

The authors of this report investigated the effect of combined treatment with apomine and lovastatin in vitro on human and murine osteosarcoma cell lines and in vivo using a murine syngeneic model of osteosarcoma. Apomine and lovastatin synergistically decreased viability and induced apoptosis in both murine and human osteosarcoma cell lines.

RESULTS:

Combined apomine and lovastatin strongly decreased HMG‐CoA reductase enzyme levels compared with lovastatin treatment alone. Consequently, the accumulation of unprenylated ras‐related protein 1A induced by lovastatin was enhanced in the presence of apomine. All synergistic effects on cell viability, apoptosis, and protein prenylation were overcome by the addition of mevalonate or geranylgeraniol, 2 mevalonate pathway intermediates downstream from the target enzyme, HMG‐CoA reductase. This confirmed that the mechanism of synergy in osteosarcoma cells is through augmented inhibition of HMG‐CoA reductase. Finally, treatment of POS‐1 osteosarcoma‐bearing mice with a combination of apomine and lovastatin significantly reduced tumor progression in these mice compared with single treatments, which had no effect at the doses used.

CONCLUSIONS:

The results from this study revealed that combination therapy with apomine and lovastatin may be a novel treatment strategy for osteosarcoma. Cancer 2012;. © 2011 American Cancer Society.     

Cell surface receptor expression patterns in osteosarcoma

BACKGROUND:

Although the presence of numerous cell signaling receptors in osteosarcoma is known, their simultaneous characterization has not been performed to date. The current study sought to characterize and quantify the expression of cell surface receptors across a variety of osteosarcoma cell lines.

METHODS:

Standard (n = 4) and patient‐derived (n = 10) osteosarcoma cell lines were cultured and labeled with antibodies to epidermal growth factor receptor, human epidermal growth factor receptor (HER)‐2, HER‐3, HER‐4, insulin‐like growth factor 1 receptor (IGF‐1R), IGF‐2R, insulin receptor (IR), vascular endothelial growth factor receptor (VEGFR)‐1, VEGFR‐2, VEGFR‐3, c‐Met, fibroblast growth factor receptor (FGFR)‐2, FGFR‐3, and platelet‐derived growth factor receptor (PDGFR)‐β. Cell surface examination was performed using flow cytometry, and the geometric fluorescent mean for each receptor was calculated and compared against a positive control.

RESULTS:

Significant overexpression of IGF‐2R was shown in all cell lines, with an average geometric mean above the upper expression quartile. A variable expression pattern was seen for c‐Met, PDGFR‐β, IR, IGFR‐1, HER‐2, and VEGFR‐3 with expression values for the remaining receptors mainly in the lower quartile. An apparent association between the expression of IGF‐1R and HER‐2 and between the expression of PDGFR‐β and IR was demonstrated.

CONCLUSION:

IGF‐2R was consistently overexpressed on the cell surface across all tested osteosarcoma cell lines. Substantial, although variable, expression of c‐Met, HER‐2, IGF‐1R, VEGFR‐3, IR, and PDGFR‐β was demonstrated as well, suggesting that these receptors may contribute to osteosarcoma aggressiveness and biological heterogeneity and may serve as potential targets within a subset of tumors. Associated receptor expression may provide new insight into common regulatory factors or pathways. Targeting either common factors or targeting multiple specific receptors may have therapeutic relevance. Cancer 2012;. © 2011 American Cancer Society.     

Elevated expression of CXC chemokines in pediatric osteosarcoma patients

BACKGROUND:

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis.

METHODS:

The authors used an antibody microarray to identify chemokines that were elevated in the plasma samples of osteosarcoma patients. The results were validated using enzyme‐linked immunosorbent assays on an independent set of samples. The tumor expressions of 3 chemokines were examined in 2 sets of osteosarcoma tissue arrays. The authors also evaluated the proliferative effect of the chemokines in 4 osteosarcoma cell lines.

RESULTS:

The authors found that the plasma levels of CXCL4, CXCL6, and CXCL12 in the osteosarcoma patients were significantly higher than those in the controls, and the results were validated by an independent osteosarcoma cohort (P < .05). However, CXCL4 (100%) and CXCL6 (91%) were frequently expressed in osteosarcoma, whereas CXCL12 was only expressed in 4%. Survival analysis further showed that higher circulating levels of CXCL4 and CXCL6, but not CXCL12, were associated with a poorer outcome of osteosarcoma patients. Addition of exogenous chemokines significantly promoted the growth of different osteosarcoma cells (P < .05).

CONCLUSIONS:

The results demonstrate that CXCL4 and CXCL6 are frequently expressed in osteosarcoma, and that the plasma levels of these 2 chemokines are associated with patient outcomes. Further study of these circulating chemokines may provide a promising approach for prognostication of osteosarcoma. Targeting these chemokines or their receptors may also lead to a novel therapeutic invention. Cancer 2011. © 2010 American Cancer Society.     

Cancer-testis antigens expressed in osteosarcoma identified by gene microarray correlate with a poor patient prognosis

BACKGROUND:

From 30% to 40% patients with osteosarcoma eventually experience medical failure; and few biomarkers of prognostic significance have been established. High‐throughput methods like gene microarray analysis can help to identify molecular biomarkers that are useful for diagnosing osteosarcoma and targeting its treatment.

METHODS:

Oligonucleotide microarrays were used to compare expression profiles of osteosarcoma cell lines and osteoblasts. Differentially expressed genes were confirmed by real‐time polymerase chain reaction (PCR) analysis. Corresponding proteins were evaluated by flow cytometry and Western blot analysis in osteosarcoma cell lines and by immunohistochemistry in osteosarcoma tissues. The association between staining intensity and clinical outcome was analyzed further.

RESULTS:

Cancer‐testis antigens, including melanoma antigen family A (MAGEA), chondrosarcoma‐associated gene family, member 2 (CSAG2), and preferentially expressed antigen in melanoma (PRAME), were increased significantly in all osteosarcoma cell lines that were analyzed. Real‐time PCR examinations indicated that cancer‐testis antigen expression was frequent and coordinated in patients with osteosarcoma. The expression of MAGEA was confirmed by Western blot and flow cytometry analyses in osteosarcoma cell lines. Furthermore, immunohistochemical staining analysis suggested that MAGEA expression may be used to predict distant metastasis and poor survival. The adjusted relative risk for lung metastasis was 2.79 (95% confidence interval, 1.12‐6.93; P = .028) for MAGEA‐positive patients. Five‐year survival rates for patients with and without MAGEA expression were 39.6% ± 8.4% and 80% ± 8.9%, respectively (log‐rank test; P = .01).

CONCLUSIONS:

The combined use of an oligonucleotide microarray, a clinical database, and a tissue bank was useful for identifying molecular tumor markers. The frequent expression of MAGEA and other cancer‐testis antigens in osteosarcoma indicates that they may be useful as diagnostic markers and targets of immunotherapy that warrant further investigation. Cancer 2012. © 2011 American Cancer Society.     

MicroRNA cloning and sequencing in osteosarcoma cell lines: differential role of miR-93

Background

Studies show that abnormalities in non-coding genes can contribute to carcinogenesis; microRNA levels may modulate cancer growth and metastatic diffusion.

Method

MicroRNA libraries were built and sequenced from two osteosarcoma cell lines (MG-63 and 143B), which differ in proliferation and transmigration. By cloning and transfection, miR-93, expressed in both cell lines, was then investigated for its involvement in osteosarcoma progression.

Results

Six of the 19 miRNA identified were expressed in both cell lines with higher expression levels of miR-93 in 143B and in primary osteosarcoma cultures compared to normal osteoblasts. Interestingly, levels of miR-93 were significantly higher in metastases from osteosarcoma than in paired primary tumours. When 143B and MG-63 were transfected with miR-93, clones appeared to respond differently to microRNA overexpression. Ectopic expression of miR-93 more significantly increased cell proliferation and invasivity in 143B than in MG-63 clones. Furthermore, increased mRNA and protein levels of E2F1, one of the potential miR-93 targets, were seen in osteosarcoma cellular clones and its involvement in 143B cell proliferation was confirmed by E2F1 silencing.

Conclusion

Although further studies are needed to evaluate miRNA involvement in osteosarcoma progression, miR-93 overexpression seems to play an important role in osteosarcoma cell growth and invasion.     

Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma

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.     

CircFAT1 sponges miR-375 to promote the expression of Yes-associated protein 1 in osteosarcoma cells

Background

There is an urgent need to identify new molecular targets for treatment of osteosarcoma. Circular RNAs are a class of endogenous RNAs that are extensively found in mammalian cells and exert critical functions in the regulation of gene expression, but in osteosarcoma the underlying molecular mechanism of circular RNAs remain poorly understood. Here we assessed the tumorigenesis properties of a circular RNA, circFAT1 in osteosarcoma.

Methods

The effects of circFAT1/miR-375/YAP1 was evaluated on human osteosarcoma cells growth, apoptosis, migration, invasion and tumorigenesis. Signaling pathways were analyzed by western blotting, qRT-PCR, fluorescence in situ hybridization, chromogenic in situ hybridization,RNA Binding Protein Immunoprecipitation and immunofluorescence. The consequence of circFAT1 short hairpin RNA combined or not with miR-375 sponge was evaluated in mice bearing 143B xenografts on tumor growth.

Results

In this study, we observed significant upregulation of circFAT1 originating from exon 2 of the FAT1 gene in human osteosarcoma tissues and cell lines. Inhibition of circFAT1 effectively prevented the migration, invasion, and tumorigenesis of osteosarcoma cells in vitro and repressed osteosarcoma growth in vivo. Mechanistic studies revealed that circFAT1 contains a binding site for the microRNA-375 (miR-375) and can abundantly sponge miR-375 to upregulate the expression of Yes-associated protein 1. Moreover, inhibition of miR-375 reversed attenuation of cell proliferation, migration, and invasion, which was induced by circFAT1 knockdown, and therefore promoted tumorigenesis.

Conclusions

Our findings demonstrate a novel function of circFAT1 in tumorigenesis and suggest a new therapeutic target for the treatment of osteosarcoma.

     

High-throughput genotyping in osteosarcoma identifies multiple mutations in phosphoinositide-3-kinase and other oncogenes

BACKGROUND:

The identification of new genes that are mutated in osteosarcomas is critical to developing a better understanding of the molecular pathogenesis of this disease and discovering new targets for therapeutic development.

METHODS:

The authors identified somatic nonsynonymous coding mutations in oncogenes associated with human cancers and hotspot mutations from tumor suppressor genes that were either well described in the literature or observed multiple times in human cancer sequencing efforts. Then, 961 mutations in 89 genes were systematically characterized across 98 osteosarcoma tumor samples and cell lines. All identified mutations were replicated on an independent platform using homogeneous mass extend matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry.

RESULTS:

In total, 14 mutations were identified in at least 1 osteosarcoma tumor sample or cell line. Some of the genetic changes identified were in tumor suppressor genes previously identified as altered in osteosarcoma: p53 (arginine→histidine at codon 273 [R273H], R→cysteine at codon 723 [R273C], and tyrosine→C at codon 163 [Y163C]) and retinoblastoma 1 (RB1) (glutamic acid→* at codon 137 [E137*]). Notably, multiple mutations were identified in phosphoinositide‐3‐kinase (PI3K), catalytic, alpha polypeptide (PIK3CA) (H1047R, E→lysine at codon 545 [E545K], and H→proline at codon 701 [H701P]) that were not observed previously in osteosarcoma. In addition, mutations in v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) (glycine→serine at codon 12 [G12S]); cubilin (CUBN) (isolucine→valine at codon 3189 [I3189V]; observed in 2 separate tumor samples); cadherin 1, type 1, epithelial (CDH1) (alanine→threonine at codon 617 [A617T]; observed in 2 separate tumor samples); catenin (cadherin‐associated protein), beta 1, 88 kDa (CTNNB1) (asparagine→S at codon 287 [N287S]); and fibrous sheath CABYR binding protein (FSCB) (S→leucine at codon 775 [S775L]) were observed.

CONCLUSIONS:

In this largest mutational profiling of osteosarcoma to date, the authors identified for the first time several mutations involving the PI3K pathway, adding osteosarcoma to the growing list of malignancies with PI3K mutations. In addition, they initiated a mutational map detailing DNA sequence changes across a variety of osteosarcoma subtypes and offered new candidates for therapeutic targeting. Cancer 2011. © 2011 American Cancer Society.     

Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes

Background:

Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations.

Methods:

To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling.

Results:

The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes—COL1A2, KYNU, ACTG2 and NPPB—were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease.

Interpretation:

The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is the first time that expression profiles are associated with functional characteristics of osteosarcoma cell lines.     

RECK in osteosarcoma: a novel role in tumour vasculature and inhibition of tumorigenesis in an orthotopic model

BACKGROUND:

Targeted therapy in osteosarcoma (OS) is needed to improve patient outcomes. Human RECK may have a role because it inhibits cancer invasion and regulates angiogenesis. This study aimed to characterize RECK expression in human OS, to examine in vitro effects of RECK on vascular endothelium and OS cell behavior, and to analyze the effect of RECK on OS grown orthotopically in nude mice.

METHODS:

RECK was examined in human OS samples. Interactions between RECK and VEGF were studied in tissue and cells. RECK transfection was used to study its effects on vascular endothelial (HMEC‐1) and OS (SaOS‐2) cell behavior in vitro and in vivo. SaOS‐2 co‐culture with RAW 246.7‐derived osteoclasts on osteoslides was used to assess effects on osteoclast activity.

RESULTS:

RECK was absent from OS cells but was expressed in tumor vessel endothelium. Via microarray analysis, RECK mRNA was elevated in samples with low proliferative activity, a trend most evident in poorly differentiated samples. VEGF induced RECK expression in HMEC‐1. RECK transfection inhibited HMEC‐1 invasion and induced thicker, although more numerous, tube formation. RECK inhibited SaOS‐2 invasion, proliferation, colony formation, and osteoclast activity but supported SaOS‐2 adhesion to collagen I. In vivo, RECK inhibited SaOS‐2 tumor growth, bone destruction, and consequent metastasis.

CONCLUSIONS:

RECK expression is downregulated in highly proliferative OS but is present in tumor vessels and upregulated in endothelium by VEGF. RECK inhibits invasion and tumorigenic properties in SaOS‐2, as confirmed in vivo. Further testing of RECK delivery in OS is warranted. Cancer 2011. © 2011 American Cancer Society.     

Periosteal osteosarcoma: a single-institution experience

BACKGROUND:

Periosteal osteosarcoma is a rare variant of osteosarcoma. Wide surgical removal is the mainstay of treatment, but controversy remains about the role of chemotherapy. The objective of this study was to review and analyze the clinical and treatment‐related factors that influence the survival of patients with periosteal osteosarcoma who received treatment in a single institution.

METHODS:

Thirty‐three patients with periosteal osteosarcoma (19 males and 14 females) with a median age of 16 years (range, ages 6‐32 years) underwent surgery (32 patients) and received radiotherapy (1 patient). Chemotherapy was received according to different regimens for high‐grade osteosarcoma by 14 patients who had grade 3 tumors.

RESULTS:

The 10‐year overall survival rate was 84%. The only patient who did not undergo surgery died of disease after 9 months; for the remaining 32 patients the 10‐year disease‐free survival rate was 65%. Survival was not influenced by the receipt of chemotherapy. The patients who received chemotherapy had a 10‐year overall survival rate of 86%, and those who received only local treatment had an overall survival rate of 83% (P = .73).

CONCLUSIONS:

The authors' experience indicated that the treatment of periosteal osteosarcoma requires only wide surgical removal of the tumor and that adjuvant chemotherapy does not improve survival. Cancer 2011. © 2010 American Cancer Society.     

The role of angiocidin in sarcomas

BACKGROUND:

Angiocidin, first identified as a tumor‐associated thrombospondin‐1 (TSP‐1) receptor, is a key mediator of tumor progression. TSP‐1, an extracellular protein produced by stromal cells, up‐regulates gelatinases and tumor cell invasion in epithelial malignancies. The authors recently developed 2 angiocidin‐inhibitory peptides that block angiocidin–TSP‐1 binding. They hypothesized that angiocidin mediates increased gelatinase expression and tumor cell invasion in sarcomas through its interaction with TSP‐1.

METHODS:

Angiocidin, TSP‐1, and gelatinase expression was evaluated in low‐grade and high‐grade sarcoma specimens. The authors established 3 distinct cell lines from a patient with an extraskeletal osteosarcoma: EXOS‐N (normal mesenchymal), EXOS‐P (primary osteosarcoma), and EXOS‐M (lung metastasis). Each was evaluated for angiocidin, gelatinase, and gelatinase inhibitor (tissue inhibitors of metalloproteinase) expression and for invasive capacity. Their responsiveness to TSP‐1 was determined. The role of angiocidin in up‐regulating gelatinase expression and invasion was studied using the authors' angiocidin‐inhibitory peptides.

RESULTS:

Expression of angiocidin, TSP‐1, and gelatinases correlated with tumor grade. Angiocidin expression, gelatinase activity, and invasiveness in the EXOS cell lines correlated with phenotype; EXOS‐N cells did not express angiocidin or gelatinases and were not invasive; EXOS‐M cells were 5 times more invasive than EXOS‐P cells and exhibited greater angiocidin and gelatinase expression. EXOS cell gelatinase activity and invasiveness increased 4‐ to 5‐fold in response to TSP‐1. Inhibition of angiocidin with the authors' inhibitory peptides blocked TSP‐1–promoted increases in gelatinase activity and tumor cell invasion.

CONCLUSIONS:

Angiocidin promotes gelatinase up‐regulation and tumor cell invasion in sarcomas. Angiocidin‐inhibitory peptides are potent inhibitors of sarcoma cell invasion in vitro, suggesting a potential therapeutic role for these peptides in the treatment of sarcomas. Cancer 2009. © 2009 American Cancer Society.     

Genetic amplification of the vascular endothelial growth factor (VEGF) pathway genes, including VEGFA, in human osteosarcoma

BACKGROUND:

Osteosarcoma is the most common primary tumor of bone. It is a highly vascular and extremely destructive malignancy that mainly affects children and young adults. The authors conducted microarray‐based comparative genomic hybridization (aCGH) and pathway analyses to gain a systemic view of pathway alterations in the genetically altered genes.

METHODS:

Recurrent amplified and deleted genes that were detected by aCGH were subjected to an analysis based on the Kyoto Encyclopedia of Genes and Genomes to identify the altered pathways. Among the enriched pathways, vascular endothelial growth factor (VEGF) pathway genes collectively were amplified, and alterations of this pathway were validated by fluorescence in situ hybridization (FISH) and immunohistochemistry analyses in 58 formalin‐fixed, paraffin‐embedded osteosarcoma archival tissues that had clinical follow‐up information.

RESULTS:

The pathway enrichment analyses of the aCGH data revealed that VEGF pathway genes, including the VEGFA gene itself, were amplified significantly in osteosarcoma. Genetic amplification of the VEGFA gene, both focally and in larger fragment, was validated by FISH analysis. It is noteworthy that amplification of the VEGFA gene and elevated expression of the VEGFA protein were associated significantly with microvascular density and adverse tumor‐free survival in patients with osteosarcoma.

CONCLUSIONS:

The authors report for the first time that VEGF pathway genes, including the VEGFA gene, are amplified in osteosarcoma. Amplification of the VEGFA gene is not only an important mechanism for elevated VEGFA protein expression but also is a poor prognostic factor for tumor‐free survival. Combined classification of VEGFA gene amplification and positive VEGFA protein expression may provide a more accurate stratification method of selecting anti‐VEGF therapy for patients with osteosarcoma. Cancer 2011;. © 2011 American Cancer Society.     

The expression and significance of IDH1 and p53 in osteosarcoma

Background

To detect the expression of isocitrate dehydrogenase 1 (IDH1) and transformation-related protein 53 (p53) in osteosarcoma and analyze the correlation between them and the clinico-pathological features.

Methods

The expressions of IDH1 and p53 were detected in human osteosarcoma cell lines (MG-63 and U2OS) by immunocytochemistry, Real-time PCR and Western Blotting. The expressions of IDH1 and p53 in formalin-fixed paraffin-embedded tissue sections from 44 osteosarcoma patients were determined by immunohistochemistry, and the correlation between them and clinicopagthological features were analyzed. None of these patients received chemotherapy prior to surgery.

Results

IDH1 is detected in osteosarcoma cell lines and biopsies. IDH1 expresses higher in U2OS cells with wild type p53 than in MG-63 cells with mutation p53. IDH1 correlates with histological Rosen grade and metastasis negatively. P53 correlates with histological Rosen grade, metastasis and overall survival in clinical osteosarcoma biopsies. Osteosarcoma patients with High IDH1 expression have a very high p53 expression.

Conclusion

IDH1 may correlate with p53 and be a candidate biomarker for osteosarcoma correlate with histological Rosen grade and metastasis.     

低分子量肝素抑制骨肉瘤细胞Livin表达并诱导细胞凋亡的实验研究

Objective： To investigate the inhibitory effects of LMWH suppressing the expression of Livin and inducing the apoptosis of the osteosarcoma cells. Methods： Osteosarcoma cells line MG-63 was cultured in vitro. MTT assay and flow cytometry were used to study the effect of LMWH with different concentration suppressed the prolifetation and induced apoptosis in osteosarcoma cells line MG-63. The expression of Livin of osteosarcoma cells line MG-63 was analysed by the immunohistochemistrical method and PT-PCR. Results： Low molecular weight heparin could inhibit the growth of osteosarcoma cell line MG-63. With the LMWH＇s increasing, the apoptosis rate was increased significantly. Immunohistochemistrical method and PT-PCR showed that the expression of Livin of osteosarcoma cells line MG-63 declined obviously than that before medication. Conclusion： LMWH has very strong anti-tumor effect in vitro. The possible mechanisms of LMWH anti-tumor effect are associate with the effect of suppressing the expression of Livin and inducing cell apoptosis.