Relationship between receptor tyrosine kinase Axl and apoptosis relative proteins in osteosarcoma / 临床与实验病理学杂志

Purpose To identify the role of tyrosine kinase receptor Axl for anti-apoptosis which was induced by cisplatin (DDP) and methotrexate (MTX) chemotherapy and to analyze the relationship between P-Axl and apoptosis-related proteins in osteosarcoma.Methods Osteosarcoma cell lines MG63,143B and U2OS were used in apoptosis assays,Axl siRNA transfection,cytotoxicity assays,cell cycle analysis,etc.A total of 41 cases of osteosarcom patients were included for immunohistochemistry of EnVision two-step staining and clinico-pathological relative analysis.TUNEL assay was performed in ten cases for apoptosis detection.Results Among the osteosarcoma cell lines,Gas6/Axl could obviously protect tumor cells from apoptosis induced by DDP and MTX (P ＜ 0.05).Axl siRNA transfection enhanced cell apoptosis,whereas Gas6 prone to function upon previous knockdown by Axl siRNA.Among the 41 cases,the positive rate of P-Axl,BCL-2,and Bax was 85.4％,70.7％,and 36.6％,respectively.In contrast,the positive rate of them was 22.2％,11.1％,and 11.1％ in osteofibrous dysplasia,respectively.The expression levels of these apoptosisrelated factors were significantly higher in osteosarcoma than in osteofibrous dysplasia (P ＜ 0.05).Through clinico-pathological analysis,there were significant relationships between the survival status and BCL-2 or Bax expression (P ＜ 0.05).Pearson correlation analysis demonstrated that BCL-2 was positively correlated to P-Axl with statistical significance (r =0.842,P ＜ 0.0001).By Cox univariate analysis,BCL-2 or Bax was correlated with the patients' prognosis.TUNEL assay also demonstrated that P-Axl high expression inhibited apoptosis in osteosarcoma tissues.Conclusion Gas6/Axl protects osteosarcoma cells from the apoptosis induced by DDP and MTX chemotherapy and inhibits apoptosis in osteosarcoma tissue,possibly through the regulation of apoptosis-related protein BCL-2.

Comparative Study between Low-dose Chemotherapy and Surgery for Isolated Eosinophilic Granuloma Bone Lesions in Children / 中山大学学报(医学科学版)

[Objective]To comprehensively compare the feasibility of three different treatment strategies consisting of low-dose chemotherapy(LDC),surgery and surgery with adjuvant low-dose chemotherapy(SLDC)for children with solitary bone lesions of eosinophilic granuloma(SBL-EG).[Methods]We retrospectively reviewed the records of 149 pediatric patients with SBL-EG at our institutions from 2002 to 2014. Our study included 86 patients who received LDC ,33 patients who received surgery and 30 patients who received SLDC. The duration of hospital stay ,time to symptom relief,recovery time,cost,complications and relapse-free sur-vival(RFS)of each strategy were analyzed.[Results]Hospital stay,time to symptom relief,recovery time and cost in the LDC group were significantly shorter or less than those in the surgery or SLDC group (P 0.05). Chemotherapy-related adverse events in the LDC and SLDC groups included nausea(8.62%),aminotransferase elevation(7.76%),slight hair loss(4.31%), immunity decline (21.55%),growth retardation (10.34%) and moon face (7.76%). LDC and SLDC treatment resulted in a significantly longer RFS (147 months and 126 months ,respectively) than surgery alone (114 months)(P = 0.005 and 0.019 , respectively). However ,there was no statistically significant difference in RFS between the LDC and SLDC groups (P = 0.732).[Conclusions]Compared with surgery or SLDC,LDC appears to promote more rapid recovery,less invasion,increase safety and eco-nomic treatment strategy for pediatric patients with SBL-EG.

The expression level of MAGEA in osteosarcoma and its association with prognosis / 实用医学杂志

Objective To investigate the expression level and clinical significance of melanoma antigen gene A (MAGEA) in osteosarcoma patients. Methods Compare gene expression profiles in osteosarcoma cell lines and osteoblasts with gene microarrays. Validation of differentially expressed genes was carried out by real-time polymerase chain reaction analysis. Corresponding protein levels were measures by Western blot analysis in osteosarcoma cell lines and by immunohistochemistry in osteosarcoma tissues. The staining intensity of immuno-histochemistry was correlated with clinical outcome , and its prognostic significance was analyzed. Results Sev-eral genes belonging to MAGEA increased significantly in all osteosarcoma cell lines and tumor tissue , but not in normal osteoblast cell. Patients with MAGEA expression has higher risk of lung metastasis (relative risk 2.79, 95% confidence interval, 1.12-6.93; P = 0.028) and lower five-year survival rates (39.6% ± 8.4% vs. 80% ± 8.9%, P = 0.01) compared with patients without MAGEA expression. Conclusions The expression of MAGEA increased in osteosarcoma , which inversely correlating with outcome of osteosarcoma patients.

Cinobufagin-induced apoptosis in osteosarcoma cells in vitro / 中华骨科杂志

Objective To study the growth inhibition,apoptosis induction effects of cinobufagin(CB)on human osteosarcoma(OS) cell line U2OS,MG63 and SaO2 in vitro and the underlying mechanism of action of cinobufagin in OS cells.Methods Cell viability was assessed by MTT assay.Cell-cycle status,apoptosis-inducing effects were evaluated by flow cytometry,fluorescent staining and DNA fragmentation assays.Inhibitors of apoptosis proteins (IAPs) and Bcl-2 family proteins including Bax,cleaved-PARP,xIAP,cIAP-1,survivin and p65 were tested by Western blot.Results MTT assay showed that CB could inhibited the growth of U2OS,MG63 and SaO2 cells in a dose-and time-dependent manner.The 48 h IC50 of CB on U2OS,MG63 and SaO2 cells were (104.83± 16.96) nmol/L,(47.07±7.5) nmol/L,and (136.72±10.08) nmol/L respectively.The induction of G2/M cell-cycle arrest was seen in the cells treated with CB.After cells were cultured for 12 h in the presence of 100 nmol/L CB,the percentages of cells in the G0/G1 phase were decreased,while G2/M phase were increased in U2OS,MG63 and SaOS2 cells,respectively.The results showed CB inhibited the proliferation of osteosarcoma cells through blocking the cell cycle in G2/M phase.Induction of apoptosis was confirmed by Hoechst 33258 and Annexin V/PI staining.After treating with 100 nmol/L CB for 48 h,the extents of apoptosis were 33.6％±6.4％,36.4％±7.8％ and 29.3％±5.1％,respectively.These results indicate that the anti-tumor activity of cinobufagin in osteosarcoma cells was due to a G2/M cell cycle arrest and apoptosis inducing effect.Western blot showed that CB could induce the apoptosis related family proteins Bax,cleaved-PARP up-regulation,xIAP,cIAP-1,survivin and p65 downregulation in OS cells.Conclusion CB can inhibit the cell viability and induce G2/M cell cycle arrest and apoptosis in U2OS,MG63 and SaO2 cells.The apoptosis-inducing effect of CB is confirmed by the regulation of apoptosis related proteins IAPs and Bcl-2 in vitro.

Role of SCUBE3 in promoting osteosarcoma cell growth and its association with prognosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect the expression of SCUBE3 in human osteosarcoma cell lines and surgical specimens of osteosarcomas and investigate its association with the patients' prognosis.</p><p><b>METHODS</b>The expression of SCUBE3 protein was detected in 5 osteosarcoma cell lines using Western blotting. CCK8 assay was used to assess the effect of SCUBE3 suppression mediated by two specific small interfering RNAs (siRNAs) on the proliferation of U2OS osteosarcoma cells, and the cell cycle changes were detected using flow cytometry. Immunohistochemistry was performed to detect the expression of SCUBE3 in 60 osteosarcoma tissues, and Kaplan-Meier method was performed for survival analysis of the patients.</p><p><b>RESULTS</b>Compared with osteoblast hFOB1.19 cells, the osteosarcoma cell lines all showed significantly higher expressions of SCUBE3. In U2OS cells, suppression of SCUBE3 expression by siRNA significantly inhibited the cell proliferation (P<0.05). Kaplan-Meier survival analysis indicated that patients with high SCUBE3 expression had worse prognosis than those with low SCUBE3 expression (P=0.036).</p><p><b>CONCLUSION</b>SCUBE3 is up-regulated in the 5 osteosarcoma cell lines and in primary osteosarcoma tissues to promote the proliferation of osteosarcoma cells. A high SCUBE3 expression in osteosarcoma tissues is associated with a poor prognosis of the patients, suggesting that SCUBE3 can serve as a potential therapeutic target for osteosarcoma.</p>

The value of glycogen synthase kinase-3β in the target therapy of osteosarcoma / 中华骨科杂志

Objective To study the affect and the related molecular mechanism of glycogen synthase kinase-3β in the proliferation of osteosarcomaand its value in the target therapy of osteosarcoma.Methods The expression level of p-GSK-3β(Ser9)and GSK-3β were detected in human osteoblast cell and osteosarcoma cells by western blot.Observe the effect of GSK-3β inhibitors and siRNA interference on the GSK-3β regulate osteosarcoma cells using apoptosis protein chip.Evaluate the valueof GSK-3β target therapy on osteosarcoma in vivo.Results The expression level of p-GSK-3β (Ser9)was lower in osteosarcoma cells.LiCL,GSK inhibitor Ⅸ,siRNA knockdown could inhibit the cell viability and up-regulated the apoptosis-related protein cleaved-caspase3.The results of the protein array showed that downstream proteins of NF-κB downregulated significantly.The results were validated by western blot,while the downregulation of p-Iκ-Bα and nuclear NF-κB p65 were also observed after LiCL treatment.Inhibition of GSK-3β by either LiCl or specific siRNA resulted in a significant reduction of NF-κB luciferase reporter activity.Furthermore,the NF-κB luciferase reporter activity was significantly increased in CA cell lines,but not in KD cell lines.By contrast,NF-κB-luciferase reporter activity was significantly decreased in stably GSK-3β knockdown cells.GSK3β inhibitor LiCL and shRNA knock down demonstrated a strong cytotoxicity effect on osteosarcoma cells in vivo.Conclusion GSK-3β is in the state of relative active in osteosarcoma in osteosarcoma and important in cell proliferation.GSK-3β regulates cell survival partially through the NF-κB pathway.It is a promising therapeutic target in osteosarcoma.

Expression of chemokine CXCL14 in primary osteosarcoma and its association with prognosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect the expression of CXCL14 in human osteosarcoma cell lines and tissues and investigate its association with the prognosis of the patients.</p><p><b>METHODS</b>RT-PCR, enzyme-linked immunosorbent assay (ELISA) and real-time PCR were used to detect the expression of CXCL14 in 4 osteosarcoma cell lines and in 40 pairs of osteosarcoma tissues and adjacent muscular tissues. CCK8 assay and colony formation assay was used to assess the effect of CXCL14 suppression mediated by two specific siRNAs on the proliferation of U2OS osteosarcoma cells. Immunohistochemistry was performed to detect the expression of CXCL14 in 58 osteosarcoma tissues, and Kaplan-Meier method and log-rank test were performed for survival analysis of the patients.</p><p><b>RESULTS</b>Significant up-regulation of CXCL14 expression was found in the osteosarcoma cell lines and in osteosarcoma tissues compared with the adjacent muscles (P<0.01). In U2OS cell, suppression of CXCL14 expression by siRNA significantly inhibited the cell proliferation (P<0.01) and colony formation rate (P<0.05). Kaplan-Meier survival analysis indicated that patients with high CXCL14 expression had worse prognosis than those with low CXCL14 expression (P=0.02).</p><p><b>CONCLUSION</b>CXCL14 is up-regulated in both osteosarcoma cell lines and primary osteosarcoma tissues to promote the proliferation of osteosarcoma cells. A high CXCL14 expression in osteosarcoma tissues is associated with a poor prognosis, suggesting the that CXCL14 serve as a potential therapeutic target for osteosarcoma.</p>

Effect and mechanism of quercetin on proliferation and apoptosis of human osteosarcoma cell U-2OS/MTX300 / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect and mechanisms of quercetin (Qu) on proliferation and apoptosis of human methotrexate resistant osteosarcoma cell U-2OS/MTX300.</p><p><b>METHOD</b>MTT assay was used to observe cell proliferation. The apoptosis was examined by using Annexin V/PI staining. Western blot of mitochondrial membrane potential and cytochrome c were used to detect mitochondria spoptosis pathway. The protein expressions related to Akt pathway was detected by continuous activated Akt transient transfection and western blot.</p><p><b>RESULT</b>Qu can obviously inhibit the growth of human MTX resistant osteosarcoma cell U-2OS/MTX300 cells in a dose- and time-dependent manner. Annexin V/PI staining showed obvious cell apoptosis. Reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to cytosol and dephoshorylation of Akt were observed after Qu treatment.</p><p><b>CONCLUSION</b>Qu can inhibit proliferation and induce apoptosis of human MTX resistant osteosarcoma cell U-2OS/MTX300, which may be related with mitochondrial apoptosis pathway and Akt activity.</p>

Medial gastrocnemius muscle transferring reconstruction the patella tendon with long following-up / 中华显微外科杂志

Objective To evaluate the long following-up outcome of the medial gastrocnemius muscle transferring reconstruction the patella tendon after the wide resection of aggressive bone tumors in the proximal tibia. Methods With the 69 patients of the osteogenetic sarcoma in the proximal tibia were treated with the wide resection and reconstruction the patella tendon. After the long following up the knee extensor,function and complications were evaluated. Results With the 69 patients, the 45 survival patients were followed up for the average 68.6 (24-128) months. The local recurrence rate was 8.7%(6/69). The strength of knee extending was in the average of grade 4.2(3.6-5.0), the degree of knee flexion was in the average of 95°(75°-135°), the degree of knee extension was in the average of-2°(0°-12°), the knees of five patients cannot fully extension. The MSTS functional score was in the average of 77% (23.1/30). Conclusion During the limb salvage of the proximal tibial aggressive bone tumors, the medial gastrocnemius muscle transferring reconstruction the patella tendon could offer the knee extension strength; improve the soft tissue coverage and functional results.