Aim: To investigate the antiproliferative activity and apoptosis‐inducing effects of bufalin on human osteosarcoma cell lines. Methods: U‐2OS and U‐2OS methotrexate (MTX) 300‐resistant cell lines were treated with bufalin. Cell viability was assessed using the MTT assay. Cell‐cycle status, apoptosis‐inducing effects, and the expression of apoptosis‐related proteins were evaluated by flow cytometry, fluorescent staining, DNA fragmentation assays, and Western blotting. The effect of bufalin on dihydrofolate reductase (DHFR) expression was studied by RT‐PCR and Western blotting. Results: Bufalin inhibited cell growth in both U‐2OS and U‐2OS MTX300 cells. The induction of G2/M cell‐cycle arrest was also seen in the cells treated with bufalin. The induction of apoptosis by bufalin was confirmed by increased expression of the tumor suppressor protein p53 and the increased ratio of the Bax/Bcl‐2 proteins. Bufalin induced apoptosis to the same extent in both cell lines without regard to DHFR levels in the cells. Conclusion: Bufalin inhibited the growth of and induced apoptosis in both MTX‐sensitive and MTX‐resistant human osteosarcoma U‐2OS cells. The apoptosis‐inducing effect of bufalin was not influenced by the presence of high levels of the DHFR protein.

Purpose The aim of this study was to review our experiences with tibial lengthening over an intramedullary nail in comparison to the conventional Ilizarov method. Methods We performed a retrospective comparison of tibial lengthening using the conventional Ilizarov method (group A: 23 limbs in 13 patients) versus over a nail (group B: 51 limbs in 26 patients). The percentage increase in tibial length, lengthening index, external fixation index, consolidation index and complications were assessed. Results The mean gain in tibial length was 7.4 cm, which represents a mean increase of 26.0%. There was no difference in lengthening index or consolidation index; however, the patients in group A wore the external fixator longer than those in group B (281.5 versus 129.0 days), which represents a larger external fixation index (40.0 versus 17.4 day/cm). Group A had a higher complication rate (1.0 versus 0.47 per tibia) than group B. Conclusions Tibial lengthening over an intramedullary nail confers advantages over the conventional Ilizarov method, including shorter time needed for external fixation and lower complication rates.

We performed 2-D DIGE on proteins prepared from serum obtained from patients with osteosarcoma (OS) and controls, to identify differentially expressed proteins that might serve as serum biomarkers for OS prognosis. Proteins found to be differentially expressed were identified by MALDI-TOF mass spectrometric analysis, coupled with database interrogation. We compared serum samples from four individuals with OS to four age- and sex-matched healthy controls. We identified 24 protein spot-features that were significantly increased, and 34 that were significantly decreased in serum from patients with OS relative to the controls. The MS analysis revealed 18 unique proteins that were increased, and 25 unique proteins that were decreased in OS serum samples. Western blot and ELISA analysis confirmed increased levels of amyloid-related serum protein (SAA) in the OS serum samples. The increased expression levels of SAA were decreased after using MTX and cisplatin combination chemotherapy, and were further decreased after operation. Moreover, increased expression levels of sera SAA were seen in the relapsed patients. Our results suggested that the determination of serum SAA in OS patients might be utilized as a marker for relapse and in evaluation of the efficacy of therapy.

Aim: To characterize and compare the different biological behaviors of 2 novel human osteosarcoma cell lines, Zos and Zos-M, established respectively from the primary tumor and the skip metastasis of an osteosarcoma patient. Methods: In vitro studies included morphological observations, karyotype analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, and cell sensitivity to chemotherapeutic drugs. Subcutaneous and intravenous inoculations into nude mice were carried out to study the tumorigenicity and the metastatic potential. RT-PCR was performed to assess the expression of the osteoblastic markers and some metastasis-related genes. Results: Both cell lines remained stable for more than 100 passages in vitro without interruption. The RT-PCR examination indicated that they retained the molecular characteristics of an osteoblastic lineage. The karyotype analysis displayed aneuploidy and various structural abnormalities. Both cell lines are tumorigenic; Zos-M differs from Zos by the former's ability to develop lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis-related genes revealed that the decreased expression of cadherin-11 in Zos-M may correlate with a high potential of metastases. Moreover, both cell lines are less sensitive to the current chemotherapy protocols. Conclusion: The establishment of osteosarcoma cell lines, Zos and Zos-M, and related animal models provide a useful resource for studying the aggressive behavior of osteosarcoma and will be helpful for screening effective treatment strategies.

Aim: Comparative proteomics provide a powerful approach in screening for alterations in protein levels and post‐translational modifications that are associated with tumors. In the present study, we aimed to identify candidate biomarkers to distinguish osteosarcoma (OS) cells from normal osteoblastic cells. Methods: We employed 3 OS cell lines (U2OS, IOR/OS9, and SaOS‐2), and used the SV40‐immortalized normal osteoblastic cell line (hFOB1.19) as the control. The differential protein levels in OS and osteoblastic cells were identified using 2‐D gel electrophoresis followed by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry analyses. Two proteins of interest, the levels of which were significantly increased in OS cells, were further characterized by Western blot analyses. Results: Twenty‐six proteins were identified, the expression level of which was either significantly increased or decreased in the OS cells as compared to the control cells. The expression level of the activator of 90 kDa shock protein ATPase homolog 1 (AHA1), was enhanced 12.4‐, 24.1‐, and 23.8‐fold in SaOS‐2, IOR/OS9, and U2OS cells, respectively, and the level of the stomatin‐like protein 2 (SLP‐2) was increased by 10.4‐ and 7.8‐fold in IOR/OS9 and U2OS cells, respectively, as compared to normal osteoblastic cells. Those observations were confirmed by Western blot analyses. Conclusion: A differential proteomic analysis was successfully used to identify AHA1 and SLP‐2 that were significantly overproduced in OS cells as compared to normal osteoblastic cells, suggesting that those proteins among others may be effective biomarker candidates for the identification of OS cells.