Effect of chemotherapy combined with caffeine for osteosarcoma

Summary Nine patients with osteosarcoma were treated by chemotherapy combined with caffeine and surgery. All primary tumors showed complete histological response to preoperative chemotherapy consisting of three intraarterial infusions of cisplatin and caffeine without/with doxorubicin and two systemic high-dose methotrexate combined with vincristine. Limb-salvage surgery was performed in eight patients with marginal procedure, which led to the preservation of good limb function. Below-knee amputation was done in one patient with calcaneal osteosarcoma. There has been neither local recurrence nor lung metastasis in seven patients with conventional osteosarcoma during a median follow-up period of 28 months. Lung metastases leading to death were observed in one patient with small-cell osteosarcoma despite complete destruction of the primary tumor by preoperative chemotherapy. Chemotherapy combined with caffeine administration deserves further extensive and large-scale study in osteosarcoma.     

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum

Purpose Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines.Methods The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS).Results Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1.Conclusions Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.     

Let-7g reverses malignant phenotype of osteosarcoma cells by targeting Aurora-B

Accumulating studies revealed that the expression levels of several miRNAs are up or down-regulated in osteosarcoma (OS). The aim of this study was to investigate the functional significance and molecular of the let-7g in OS cells. The expression levels of let-7g was significantly down-regulated in OS cell lines U2-OS and HOS cell compared to osteoblast cell lines HOB cell. Moreover, bioinformatic prediction suggested that Aurora-B, which is overexpressed and functions as an oncogene in OS cells, is a putative target gene of let-7g. Using mRNA and protein expression analysis and luciferase assays, we further identified let-7g directly regulated Aurora-B expression in OS cells. Functional investigation revealed both restoration of let-7g and silencing Aurora-B induce cell apoptosis and suppressed cell viability, migratory and invasive ability in OS cells. Finally, we found that silencing Aurora-B in OS cells could partly dampen anti-let-7g mediated tumor promotion. Thus, our findings suggested that let-7g inhibits OS cell malignant phenotype at least partly through targeting Aurora-B. Targeting of let-7g and Aurora-B may be a novel therapeutic strategy for treating OS.     

基于骨肉瘤高表达抗原PBF CTL表位改造及鉴定

目的:观察骨肉瘤高表达抗原乳头瘤病毒结合因子(PBF)的改造表位是否有HLA-A2限制性抗肿瘤能力,开发基于骨肉瘤的多肽免疫治疗。方法:首先运用RT-PCR和Western blot方法检测PBF在骨肉瘤细胞系U2OS和Saos-2的表达情况。然后通过Net CTL 1.2、SYFPEITHI和IEDB软件预测打分来选取PBF的HLA-A2限制性表位。替换PBF抗原锚定位点氨基酸获得改造肽。候选表位肽的合成方法是标准的Fmoc化学合成法,结合力实验用于检测候选表位与T2A2细胞表面HLA-A2分子的结合能力,ELISPOT实验检测候选表位肽诱导细胞毒性T淋巴细胞(CTL)分泌干扰素γ(IFN-γ)的能力,乳酸脱氧酶(LDH)释放实验和羧基荧光素琥珀酰亚胺脂(CFSE)细胞毒实验检测候选肽诱导CTL的能力。结果:PBF在骨肉瘤细胞系U2OS和Saos-2均有表达,候选肽P75-1Y2L、P412-1Y、P416-1Y2L9V、P107-1Y和P435-1Y2L具有较好的结合力,且改造肽与HLA-A2的结合力高于原肽。ELISPOT实验结果显示表位肽P412、P412-1Y、P416、P416-1Y2L9V和P435-1Y2L诱导的CTL具有分泌IFN-γ的能力;P412-1Y和P416-1Y2L9V诱导特异性T细胞免疫分泌的IFN-γ略高于原肽。LDH释放实验和CFSE细胞毒实验结果显示表位P412、P412-1Y、P416和P416-1Y2L9V对U2OS细胞均有一定的杀伤作用,P412-1Y和P416-1Y2L9V特异性CTLs对U2OS细胞杀伤率高于原肽特异性CTLs。结论:PBF抗原改造表位P412-1Y和P416-1Y2L9V与天然表位P412和P416相比有更高的HLA-A2分子亲和力,保留了原有的免疫原性,并且改造肽抗肿瘤免疫效应强于天然表位。P412-1Y和P416-1Y2L9V是优秀的PBF抗原的HLA-A*0201限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

Distinctive Head and Neck Bone and Soft Tissue Neoplasms

Benign and malignant primary bone and soft tissue lesions of the head and neck are rare. The uncommon nature of these tumors, combined with the complex anatomy of the head and neck, pose diagnostic challenges to pathologists. This article describes the pertinent clinical, radiographic, and pathologic features of selected bone and soft tissue tumors involving the head and neck region, including angiofibroma, glomangiopericytoma, rhabdomyosarcoma, biphenotypic sinonasal sarcoma, chordoma, chondrosarcoma, and osteosarcoma. Emphasis is placed on key diagnostic pitfalls, differential diagnosis, and the importance of correlating clinical and radiographic information, particularly for tumors involving bone.     

EXPRESSION AND SIGNIFICANCE OF ONCOPROTEIN p16 AND FOS IN OSTEOSARCOMA

It is known that tumor is a gene disease both activation of oncogene and inactivity of tumor suppressor gene. In recent years the studies of molecular genetics have shown that different oncogene and tumor suppressor gene have different action in the occurrence and development of osteosarcoma. Immunohistochemistry technique was performed to detect p16 and C-fos protein expression in 41 cases of osteosarcoma so that their clinical significance were explored. MATERIALS AND METHODSClinical…     

Perspectives on cancer stem cells in osteosarcoma

Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. These mesenchymal tumors are derived from progenitor cells in the osteoblast lineage that have accumulated mutations to escape cell cycle checkpoints leading to excessive proliferation and defects in their ability to differentiate appropriately into mature bone-forming osteoblasts. Like other malignant tumors, osteosarcoma is often heterogeneous, consisting of phenotypically distinct cells with features of different stages of differentiation. The cancer stem cell hypothesis posits that tumors are maintained by stem cells and it is the incomplete eradication of a refractory population of tumor-initiating stem cells that accounts for drug resistance and tumor relapse. In this review we present our current knowledge about the biology of osteosarcoma stem cells from mouse and human tumors, highlighting new insights and unresolved issues in the identification of this elusive population. We focus on factors and pathways that are implicated in maintaining such cells, and differences from paradigms of epithelial cancers. Targeting of the cancer stem cells in osteosarcoma is a promising avenue to explore to develop new therapies for this devastating childhood cancer.     

MicroRNA-17 promotes osteosarcoma cells proliferation and migration and inhibits apoptosis by regulating SASH1 expression

MicroRNAs (miRNAs) are abnormally expressed in numerous diseases, which are intimately associated with cell proliferation, migration and invasion. Recent study indicated that miR-17 may be involved in regulating osteosarcoma (OS) occurrence and development, but its function and mechanism have not been reported. In this study, quantitative real-time PCR (qRT-PCR) was used to measure the expression of miR-17, and Western blotting assay was performed to measure the expressions of SAM and SH3 domain containing 1 (SASH1), phosphoinoinositide-3 kinase (PI3K), protein kinase B (AKT), Caspase3, Bcl-2 gene family (Bcl-2, Bax) and matrix metalloprotein (MMP-2, MMP-9) in MG-63 cells. Luciferase reporter assay was conducted to confirm the target of SASH1 by miR-17. Cell proliferation, migration, invasion and apoptosis assay was performed to investigate the role of miR-17 in OS cells. We found that the expression of miR-17 was significantly up-regulated in OS cell lines. MiR-17 inhibitor inhibited the proliferation ability, and induced apoptosis of OS cells. Besides, miR-17 inhibitor prevented the migration and invasion of OS cells. Further, we identified that SASH1 was a target gene of miR-17. In addition, knockdown of miR-17 increased the protein expression of SASH1, and regulate related genes of cell proliferation, invasion and anti-apoptosis in the downstream of OS cells. These findings indicated that miR-17 was over-expressed and promoted cell proliferation, migration and inhibited cell apoptosis by targeting SASH1 in OS cells.     

Upregulation of miR‑95-3p inhibits growth of osteosarcoma by targeting HDGF

Osteosarcoma is the most common bone malignancy and miR-95-3p plays an important role in multiple cancers. The purpose of this study was to explore the effect and potential mechanism of miR-95-3p on the growth of osteosarcoma. In vitro, the osteosarcoma cell lines, SAOS-2 and U2OS cells, were transfected with miR-95-agomir to assess the role of miR-95-3p in proliferation and apoptosis of osteosarcoma cells. We determined that overexpression of miR-95-3p significantly attenuated cell proliferation but enhanced apoptosis in SAOS-2 and U2OS cells. We also found that overexpression of miR-95-3p in osteosarcoma cells downregulated the expression of hepatoma-derived growth factor (HDGF). Next, knockdown of HDGF by siRNA targeting HDGF clearly inhibited cell proliferation and induced apoptosis in U2OS cells. In vivo, a tumor formation assay in BALB/c nude mice was conducted by injecting the pre-miR-95 or control vector lentivirus-infected U2OS cells to determine the effect of miR-95-3p on the growth of osteosarcoma. Results showed miR-95-3p overexpression inhibited the osteosarcoma growth and downregulated the HDGF expression in xenografted tumor. For mechanism study, we co-transfected HDGF/pcDNA3.1 plasmid and miR-95-agomir to U2OS cells, and we demonstrated that overexpression of HDGF could attenuate the effects of miR-95-3p on U2OS cell proliferation, apoptosis and migration. These findings indicated that miR-95-3p might act as a potential tumor suppressor in osteosarcoma by targeting HDGF. Thus, miR-95-3p may become a potential therapeutic in treatment of osteosarcoma.