Zhaoming Ye

Response of osteogenic sarcoma to neoadjuvant therapy: evaluated by 18F-FDG-PET

ObjectiveThe aim of this study was to evaluate the potential role of F-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) in assessing the chemotherapy response of osteosarcoma when compared with histologically assessed tumor necrosis.MethodsFifteen patients were examined with whole-body FDG-PET prior to and following neoadjuvant therapy. The maximum standard uptake value (SUVmax) of tumor and tumor to background ratio (TBR) prior to and following chemotherapy was used for semiquantitative PET imaging analysis. The SUVmax of prechemotherapy and post-chemotherapy was recorded as SUV1 and SUV2. TBR1 and TBR2 represented prechemotherapy and post-chemotherapy TBR. TBR was calculated by drawing an identical region of interest over the tumor and the contralateral normal limb or pelvis. Tumor necrosis was classified according to Salzer-Kuntschik’s criteria.ResultsEight patients with more than 90% tumor necrosis were classified as showing good responses and seven patients with less than 90% tumor necrosis as showing poor responses. SUV2/SUV1, TBR2/TBR1, and TBR2 were significantly correlated with the tumor necrosis degree (P < 0.01, P < 0.001, P < 0.001). TBR2/TBR1 were below 0.46 in all the patients with favorable responses, and higher than 0.49 in all the patients with unfavorable responses. However, it was difficult to distinguish good responses from poor responses by SUV2/SUV1.ConclusionsFDG-PET is a promising tool to assess the chemotherapy response of osteosarcoma noninvasively. The TBR was better than SUVmax in evaluating the chemotherapy response in this study.

Artesunate inhibits growth and induces apoptosis in human osteosarcoma HOS cell line in vitro and in vivo

This paper aims to investigate the effects of artesunate (ART) on growth and apoptosis in human osteosarcoma HOS cell line in vitro and in vivo and to explore the possible underlying mechanisms. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The induction of apoptosis was detected by light and transmission electron microscopy and flow cytometry. Western blot analysis was used to investigate the related mechanisms. Nude mice were further employed to investigate the antitumour activity of ART in vivo. MTT assay results demonstrated that ART selectively inhibits the growth of HOS cells in a dose- and time-dependent manner. Based on the findings of light and transmission electron microscopy, Hoechst 33258 staining, and fluorescein isothiocyanate (FITC)-annexin V staining, the cytotoxicity of ART in HOS cells occurs through apoptosis. With ART treatment, cytosolic cytochrome c was increased, Bax expression was gradually upregulated, Bcl-2 expression was downregulated, and caspase-9 and caspase-3 were activated. Thus, the intrinsic apoptotic pathway may be involved in ART-induced apoptosis. Cell cycle analysis by flow cytometry indicated that ART may induce cell cycle arrest at G2/M phase. In nude mice bearing HOS xenograft tumours, ART inhibited tumour growth and regulated the expressions of cleaved caspase-3 and survivin, in agreement with in vitro observations. ART has a selective antitumour activity against human osteosarcoma HOS cells, which may be related to its effects on induction of apoptosis via the intrinsic pathway. The results suggest that ART is a promising candidate for the treatment of osteosarcoma.

Piperine inhibits proliferation of human osteosarcoma cells via G2/M phase arrest and metastasis by suppressing MMP-2/-9 expression

The piperidine alkaloid piperine, a major ingredient in black pepper, inhibits the growth and metastasis of cancer cells both in vivo and in vitro, although its mechanism of action is unclear. Furthermore, its anticancer activity against osteosarcoma cells has not been reported. In this study, we show that piperine inhibited the growth of HOS and U2OS cells in dose- and time-dependent manners but had a weaker effect on the growth of normal hFOB cells. Piperine inhibited osteosarcoma cell proliferation by causing G2/M phase cell cycle arrest associated with decreased expression of cyclin B1 and increased phosphorylation of Cyclin-dependent kinase-1(CDK1) and checkpoint kinase 2 (Chk2). In addition, piperine treatment inhibited phosphorylation of Akt and activated phosphorylation of c-Jun N-terminal kinase (c-JNK) and p38 mitogen-activated protein kinase (MAPK) in HOS and U2OS cells. Piperine induced colony formation in these two cell types. We proved that piperine could suppress the metastasis of osteosarcoma cells using scratch migration assays and Transwell chamber tests. Moreover, gelatin zymography showed that piperine inhibited the activity of matrix metalloproteinase (MMP)-2/-9 and increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1/-2. Taken together, our results indicate that piperine inhibits proliferation, by inducing G2/M cell cycle arrest, and the migration and invasion of HOS and U2OS cells, via increased expression of TIMP-1/-2 and down-regulation of MMP-2/-9. These findings support further study of piperine as a promising therapeutic agent in the treatment of osteosarcoma.

Sensitization of human osteosarcoma cells to Vγ9Vδ2 T‐cell‐mediated cytotoxicity by zoledronate

Despite improvements in the treatment of osteosarcoma, there is a need for new therapeutic strategies, in particular for the treatment of recurrent tumors and metastases. Adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. We have investigated combining adoptive immunotherapy with Vγ9Vδ2 T cells and zoledronate to optimize osteosarcoma therapy. Vγ9Vδ2 T cells, from healthy volunteers and patients with osteosarcoma, cultures alone demonstrated moderate or poor cytotoxic activity against osteosarcoma cell lines, respectively. The addition of zoledronate further increased cytotoxicity in vitro. This enhancement was largely dependent on the granule exocytose and partly on TRAIL pathways, was TCR‐mediated and partly NKG2D‐mediated. These data suggest that combined treatment of human osteosarcoma with zoledronate and Vγ9Vδ2 T cells may be an effective complement to current chemotherapies.

Caspase‐8 dependent osteosarcoma cell apoptosis induced by proteasome inhibitor MG132

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG‐63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis‐inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132‐treated cells and MG132 mostly caused MG‐63 cell arrest at G2–M‐phase by cell cycle analysis. Increased activation of caspase‐8, accumulation of p27Kip1, and an increased ratio of Bax: Bcl‐2 were detected by RT—PCR and Western blot analysis. Activation of caspase‐3 and caspase‐9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase‐8 dependent, p27 and bcl‐2 family related.

Prognostic value of pathologic fracture in patients with high grade localized osteosarcoma: a systemic review and meta-analysis of cohort studies.

Consensus has not been reached regarding the ability of pathologic fracture to predict local recurrence and survival in osteosarcoma. We aim to review the available evidence to examine the association between pathologic fracture and osteosarcoma prognosis. A comprehensive literature search for relevant studies published until March 2014 was performed using PubMed, Cochrane and Web of Science. The studies investigating pathologic fracture of osteosarcoma patients were systematically analyzed. The overall relative risk (RR) was estimated using a fixed‐effect model or random‐effect model according to heterogeneity between the trials. We included nine cohort studies involving 2,187 patients (311 with pathologic fracture and 1,876 without fracture) for the analysis of survival rate and local recurrence. Studies were assessed for quality using the Newcastle–Ottawa Assessment Scale. In the fixed‐effects model, the meta‐analysis showed that pathologic fracture in osteosarcoma patients predicted poor 3‐year overall survival (OS) (RR = 1.86, 95% CI: 1.37–2.53, p < 0.001) and 5‐year OS (RR = 1.34, 95% CI: 1.06–1.70, p = 0.016). Similarly, pathologic fracture was significantly correlated with worse 3‐year event free survival (EFS) (RR = 1.52, 95% CI: 1.21–1.92, p < 0.001) and 5‐year EFS (RR = 1.24, 95% CI: 1.03–1.49, p = 0.021), whereas no significant association was noted with local recurrence (RR = 1.30, 95% CI: 0.84–2.02, p = 0.233). The meta‐analysis confirmed that pathologic fracture in osteosarcoma was a prognostic marker for both OS and EFS but not for local recurrence.

Sinomenine prevents metastasis of human osteosarcoma cells via S phase arrest and suppression of tumor-related neovascularization and osteolysis through the CXCR4-STAT3 pathway

Osteosarcoma is the most common primary malignant tumor of the bone. The long-term survivals continue to be unsatisfactory for patients with metastatic and recurrent disease. Metastasis is still a severe challenge in osteosarcoma treatment. Sinomenine, an alkaloid from traditional Chinese medicine, has been proved to possess potent antitumor and anti-invasion effect on various cancers. However, the effect of sinomenine on human osteosarcoma and the underlying mechanisms remains unknown. We report here that sinomenine inhibited proliferation by inducing S phase arrest and suppressing the clone formation. Significant inhibitory effects were found in invasion and metastasis in osteosarcoma, but little cytotoxicity was observed in tested concentrations. Exposure to sinomenine resulted in suppression of invasion and migration in osteosarcoma cells as well as tube formation ability in the human umbilical vein endothelial cells (HUVEC) and U2OS cells. Furthermore, it demonstrated that CXCR4 played a key role contributing to invasion in osteosarcoma which is considered to be a core target site in sinomenine treatment. Sinomenine inhibited invasion by suppressing CXCR4 and STAT3 phosphorylation then downregulating the expression of MMP-2, MMP-9, RANKL, VEGF downstream. In addition, then RANKL-mediated bone destruction stimulated by osteoclastogenesis and VEGF-related neovascularization were restrained. Importantly, in vivo, sinomenine suppressed proliferation, osteoclastogenesis and bone destruction. Through these various comprehensive means, sinomenine inhibits metastasis in osteosarcoma. Taken together, our results revealed that sinomenine caused S phase arrest, inhibited invasion and metastasis via suppressing the CXCR4-STAT3 pathway and then osteoclastogenesis-mediated bone destruction and neovascularization in osteosarcoma. Sinomenine is therefore a promising adjuvant agent for metastasis control in osteosarcoma.

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

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.

Multicentric Epithelioid Angiosarcoma of Bone

Bone epithelioid angiosarcoma is rare and generally shows positive immunostaining for epithelial markers. Multicentric bone epithelioid angiosarcoma is easily misdiagnosed as carcinoma, including metastatic carcinoma, multiple myeloma, and multiple lymphoma of bone. This article describes a case of multicentric bone epithelioid angiosarcoma. The patient was first misdiagnosed as having metastatic carcinoma. Examination showed osteolytic lesions in the bilateral heels and the lower left humerus. The diagnosis was confirmed postoperatively and corrected after immunohistochemical analysis of the biopsy. The immunohistochemical analysis revealed that the tumor mass was strongly positive for CD31, factor VIII, vimentin, and neuron-specific enolase. The patient refused chemotherapy and died of lung metastasis 4 months postoperatively.Most bone epithelioid angiosarcomas are immunopositive for epithelial markers (ie, keratin, cytokeratin, high-molecular-weight keratin, and epithelial membrane antigen), vascular endothelial markers (ie, CD31, CD34, and von Willebrand factor), and factor VIII-associated antigen. Bone epithelioid angiosarcoma shows a relatively high degree of malignancy. Patients often die of distant metastasis, including those found in the lung and lymph node tissue. A wide excision of epithelioid angiosarcoma should be performed during the operation of the primary tumor. A better understanding of the clinicopathologic features of this disease may help to clarify the confusion, provide better treatment, and improve the clinical prognosis.

The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it

In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 ± 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 ± 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.