A20 inhibits the release of inflammatory cytokines by suppressing the activation of the nuclear factor-kappa B pathway in osteoarthritic fibroblast-like synoviocytes.

A growing number of studies suggest that synovitis plays an important role in the pathogenesis and progression of osteoarthritis (OA). As a negative mediator of the nuclear factor-kappa B (NF-κB) signaling pathway, the zinc finger protein A20 has significant anti-inflammatory properties. In this study, the differential expression of A20 was investigated at the mRNA and protein levels in human normal OA fibroblast-like synoviocytes (FLSs) and normal FLSs pretreated with TNF-α. We then measured the activation of the NF-κB pathway and expression of pro-inflammatory cytokines in the above three groups by western blotting, a human cytokine array and ELISA. We found that TNF-α activated the NF-κB pathway, increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and A20 expression in human normal FLSs. However, the role of A20 in FLSs was unclear. To clarify this, we investigated the effect of A20 overexpression in human normal FLSs. The results indicate that A20 inhibits the NF-κB signaling pathway activation and OA-associated pro-inflammatory cytokines release. The results of this study indicate that A20 has anti-inflammatory effects in FLSs, which makes it a potential target for OA synovitis treatment.

Dual specificity phosphatase 1 has a protective role in osteoarthritis fibroblast­like synoviocytes via inhibition of the MAPK signaling pathway.

Increasing evidence indicates the important role of inflammation in the pathogenesis and progression of osteoarthritis (OA). Dual specificity phosphatase 1 (DUSP1), a negative regulator of the mitogen­activated protein kinase (MAPK) signaling pathway, has anti­inflammatory properties. In the present study, the expression of DUSP1 was investigated in human OA fibroblast­like synoviocytes (FLSs), human normal FLSs and OA FLSs pretreated with dexamethasone at the mRNA and protein levels. Then, the activation of MAPK pathway proteins and the expression of matrix metalloproteinase­13 (MMP­13) and cyclooxygenase­2 (COX­2) were measured by western blot analysis in the three groups of cells. Dexamethasone induced the expression of DUSP1 and inhibited the activation of the MAPK pathway and reduced the expression of MMP­13 and COX­2 in OA FLSs. However, the role of DUSP1 remained unclear. To clarify this, the effects of overexpression of DUSP1 in OA FLSs were determined using a DUSP1­overexpressing lentivirus. The results demonstrated that overexpression of DUSP1 in OA FLSs inhibited the activation of the MAPK pathway and expression of OA­associated mediators. The findings of the present study indicate that DUSP1 has a protective role in OA FLSs and may be a potential target in the treatment of OA.

Closed reduction and percutaneous annulated screw fixation in the treatment of comminuted proximal humeral fractures.

BACKGROUND: Displaced proximal humeral fractures remain a challenge to orthopedic surgeons. OBJECTIVES: The purpose of this study was to evaluate the functional and radiological outcomes of patients with comminuted proximal humeral fractures treated with closed reduction and percutaneous screw fixation (CRPF). MATERIAL AND METHODS: The authors retrospectively reviewed 38 cases of displaced proximal humeral fractures (2-, 3- or 4-part fractures according to the Neer classification) that were treated using the CRPF technique from May 2009 to April 2013. From this group 26 patients were followed up for a period ranging from 9 to 24 months (averaging 12.9 months) and evaluated for the functional and radiological outcomes by a series of standard questionnaires and measurements. RESULTS: The fractures in all 26 patients were healed within an average time of 14.6 weeks (ranging from 11 to 27 weeks), and the mean interval between the operation and fully functional activity was 18.6 weeks (ranging from 15 to 32 weeks). At the final follow-up visit, no patient showed shoulder instability; the mean range of abduction motion was 146.5° (ranging from 72° to 180°). For all patients, no statistically significant difference in the functional outcomes was observed between their 6-month and final follow-up visits; or in the radiological findings between their immediate post-operative and final follow-up examinations. CONCLUSIONS: The CRPF technique is a safe and effective therapeutic option for comminuted proximal humeral fractures. Good stability is obtained and aggressive impairment of the soft tissue and periosteum around the fracture is avoided, which allows for an early painless range of motion. The technique promotes bone healing, prevents ischemic osteonecrosis of the head of the humerus and leads to few complications.

Ginsenoside Rh2 enhances the antitumor immunological response of a melanoma mice model.

The treatment of malignant tumors following surgery is important in preventing relapse. Among all the post-surgery treatments, immunomodulators have demonstrated satisfactory effects on preventing recurrence according to recent studies. Ginsenoside is a compound isolated from panax ginseng, which is a famous traditional Chinese medicine. Ginsenoside aids in killing tumor cells through numerous processes, including the antitumor processes of ginsenoside Rh2 and Rg1, and also affects the inflammatory processes of the immune system. However, the role that ginsenoside serves in antitumor immunological activity remains to be elucidated. Therefore, the present study aimed to analyze the effect of ginsenoside Rh2 on the antitumor immunological response. With a melanoma mice model, ginsenoside Rh2 was demonstrated to inhibit tumor growth and improved the survival time of the mice. Ginsenoside Rh2 enhanced T-lymphocyte infiltration in the tumor and triggered cytotoxicity in spleen lymphocytes. In addition, the immunological response triggered by ginsenoside Rh2 could be transferred to other mice. In conclusion, the present study provides evidence that ginsenoside Rh2 treatment enhanced the antitumor immunological response, which may be a potential therapy for melanoma.

[CCR7 silence by siRNA inhibits proliferation, invasion and promotes apoptosis of human MG63 osteosarcoma cells].

Objective To investigate the effect of siRNA-mediated chemokine receptor 7 (CCR7) silence on the proliferation, migration, invasion and apoptosis of human MG-63 osteosarcoma cells. Methods The study designed and synthesized siRNA targeting CCR7 (CCR7-siRNA). After MG63 cells were transfected with CCR7-siRNA, the expression of CCR7 was identified by Western blotting; cell apoptosis was detected by annexinV-FITC/PI double staining combined with flow cemetery; cell proliferation was tested by MTT assay; and cell migration and invasion abilities were examined by TranswellTM migration/invasion assays. Results CCR7 expression in MG63 cells was significantly inhibited after transfected with CCR7-siRNA. At the same time, cell proliferation, migration and invasion abilities were distinctly suppressed, and cell apoptosis rate increased. Conclusion Down-regulating CCR7 expression in MG63 cells could apparently inhibit cell proliferation, migration and invasion abilities of MG63 cells, and also induce cell apoptosis.

[Estrogen exerts anti-inflammatory effects by inhibiting NF-κB pathway through binding with estrogen receptor ß on synovicytes of osteoarthritis].

Objective To investigate the mechanism of estrogen's anti-inflammatory effects on synovial cells during the pathogenic process of osteoarthritis. Methods We isolated synovicytes from synovium tissues and identified the cells with flow cytometry. Then we detected the expression level of estrogen receptor ß (ERß) in synovicytes with immunofluorescence staining. The synovicytes were divided into control group, group pretreated with 10 ng/mL IL-1ß, group pretreated with 10 ng/mL IL-1ß and 10-7 mol/L estrogen, group pretreated with 10 ng/mL IL-1ß, 10-7 mol/L estrogen and specific antagonist of ERß, 10-5 mol/L tetrahydrocannabinol (THC). Thirty-six hours later, we observed the mRNA and protein levels of IκBα, phospho-IκBα (p-IκBα) and IL-6. Results Immunofluorescence staining showed the high expression level of ERß in synovicytes. In IL-1ß treated cells, IL-6 mRNA and protein level, IκBα mRNA and p-IκBα protein levels were elevated compared with the control group, while IκBα protein level declined. In the cells pretreated with IL-1ß and estrogen, the mRNA and protein levels of IL-6, IκBα and p-IκBα were inhibited compared with IL-1ß treated cells. THC blocked the effects of estrogen on the IL-1ß and estrogen treated cells, and the mRNA and protein levels of IL-6, IκBα and p-IκBα had no significant difference compared with IL-1ß treated cells. Conclusion The estrogen can restrain the activation of NF-κB pathway in synovicytes via ERß, thus playing a vital role in anti-inflammation.

VEGF silencing inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via PI3K/AKT signaling pathway.

Vascular endothelial growth factor (VEGF) is one of the most potently angiogenic factors which promotes generation of tumor vasculature. VEGF is usually up-regulated in multiple cancers include osteosarcoma and gliomas. To further explore the potential molecular mechanism that inhibits tumor growth induced by interference of VEGF expression, we constructed an Lv-shVEGF vector and assessed the efficiency of VEGF silencing and its influence on U2OS cells. Our data demonstrated that Lv-shVEGF has high inhibition efficiency on VEGF expression, which inhibits proliferation and promotes apoptosis of U2OS cells in vitro. Our results also indicated that inhibition of VEGF expression suppresses osteosarcoma tumor growth in vivo, VEGF inhibition reduces osteosarcoma angiogenesis. We also found that the phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation was considerably reduced after osteosarcoma cells were treated with Lv-shVEGF. Taken together, our data demonstrated that VEGF silencing suppresses cells proliferation, promotes cells apoptosis and reduces osteosarcoma angiogenesis through inactivation of PI3K/AKT signaling pathway.

VEGF Silencing Inhibits Human Osteosarcoma Angiogenesis and Promotes Cell Apoptosis via PI3K/AKT Signaling Pathway.

Vascular endothelial growth factor (VEGF) is one of the most effective angiogenic factors that promote generation of tumor vasculature. VEGF is usually up-regulated in multiple cancers including osteosarcoma and glioma. To further explore the potential molecular mechanism that inhibits tumor growth induced by interference of VEGF expression, we constructed a Lv-shVEGF vector and assessed the efficiency of VEGF silencing and its influence in U2OS cells. The data demonstrate that Lv-shVEGF has high inhibition efficiency on VEGF expression, which inhibits proliferation and promotes apoptosis of U2OS cells in vitro. Our results also indicate that inhibition of VEGF expression suppresses osteosarcoma tumor growth in vivo and reduces osteosarcoma angiogenesis. We also found that the activations of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) were considerably reduced after osteosarcoma cells were treated with Lv-shVEGF. Taken together, our data demonstrate that VEGF silencing suppresses cell proliferation, promotes cell apoptosis, and reduces osteosarcoma angiogenesis through inactivation of PI3K/AKT signaling pathway.

Homologous mesenchymal stem cells promote the emergence and growth of pulmonary metastases of the rat osteosarcoma cell line UMR-106.

Osteosarcoma (OS) is the most frequent primary bone sarcoma and tends to develop pulmonary metastasis. Studies have shown that mesenchymal stem cells (MSCs) are involved in OS growth and metastasis, but the mechanism remains unclear. The aim of the present study was to identify whether homologous MSCs could promote the growth and metastasis of OS in rats with a normal immune system. The OS cell line, UMR-106, which originally derives from a Sprague-Dawley (SD) rat-transplantable osteogenic sarcoma with an osteoblastic phenotype, has a strong carcinogenic capability and a high lung metastasis. Xenotransplanted models of UMR-106 with or without MSCs injected through the tibia (IT) or caudal vein (IV) were established. SD rats were randomly divided into six groups: Control, UMR-106 (IT), MSCs (IV), UMR-106 (IT) + MSCs (IV), UMR-106 (IV) and UMR-106 (IV) + MSCs (IV). Following injection, all rats were sacrificed at week 5, and the volume and quantity of metastatic sarcoma and the serum alkaline phosphatase levels were measured. There was no metastatic sarcoma in the liver, spleen and kidney in all groups. The rats in the MSCs (IV) + UMR-106 (IV) group showed a significantly higher volume and number of pulmonary metastatic tumors than those of the UMR-106 (IV) group. In pulmonary metastatic tissues, MSCs were found in the MSCs (IV) + UMR-106 (IV) group, but not in the UMR-106 (IT) + MSCs (IV) group. Notably, the expression of vascular endothelial growth factor (VEGF) was increased in the MSCs + UMR-106 cells co-culture system. The present study indicated that MSCs can significantly promote the pulmonary metastasis of the rat OS cell line, UMR-106, with a normal immune system, and VEGF was involved in MSC-promoted UMR-106 emergence and growth of pulmonary metastasis.

Proteomic profiling of osteosarcoma cells identifies ALDOA and SULT1A3 as negative survival markers of human osteosarcoma.

Osteosarcoma (OSA) is the most common primary malignancy of bone. Molecular mechanism underlying OSA remains to be fully elucidated. It is critical to identify reliable diagnostic and prognostic markers for OSA at the molecular levels. This study is designed to investigate possible molecular mechanisms behind OSA development and to identify novel prognostic markers related to OSA survival. We conduct a comprehensive proteomic profiling analysis of human OSA cell lines with differential metastatic potential. Through comprehensive combinatorial analyses of the proteomic data and the previously obtained cDNA microarray results, we identify 37 candidate proteins which are differentially expressed in OSA sublines. Among them, ALDOA and SULT1A3 are selected for further investigation. The expressions of protein are confirmed by Western blotting analysis. We further analyze the expression levels of ALDOA and SULT1A3 from 40 clinical cases of OSA. The results demonstrate that the expression of ALDOA and/or SULT1A3 is significantly higher in patients with worse survival time than patients with better survival time. Five-year survival analysis shows there is a statistically significant difference between two patient populations. The data strongly suggest that ALDOA and/or SULT1A3 expression level in biopsy samples may predict the clinical outcomes of OSA patients. Furthermore, the biological functions of ALDOA and SULT1A3 may be implicated in OSA development and/or progression.

CXCR4-mediated osteosarcoma growth and pulmonary metastasis is promoted by mesenchymal stem cells through VEGF.

Chemokines and chemokine receptor 4 (CXCR4) play an important role in metastasis. CXCR4 is also expressed in the human osteosarcoma cell line 9607-F5M2 (F5M2), which has a high tumorigenic ability and potential for spontaneous pulmonary metastasis. Mesenchymal stem cells (MSCs) contribute to the formation of the tumor stroma and promote metastasis. However, mechanisms underlying the promotion of osteosarcoma growth and pulmonary metastasis by MSCs are still elusive. Our study co-injected the human MSCs and F5M2 cells into the caudal vein of nude mice. The total number of tumor nodules per lung was significantly increased in the F5M2+MSC group compared to the other groups (control, F5M2 cells alone and MSCs alone) at week six. Moreover, a high number of Dil-labeled MSCs was present also at the osteosarcoma metastasis sites in the lung. Using Transwell assays, we found that F5M2 cells migrate towards MSCs, while the CXCR4 inhibitor AMD3100 decreased the migration potential of F5M2 cells towards MSCs. Furthermore, upon treatment with F5M2-conditioned medium, MSCs expressed and secreted higher levels of VEGF as determined by immunohistochemistry, western blotting and ELISA, respectively. Importantly, co-cultured with F5M2 cells, MSCs expressed and secreted higher VEGF levels, while AMD3100 dramatically decreased the VEGF secretion by MSCs. However, CXCR4 expression on F5M2 cells was not significantly increased in the co-culture system. Additionally, VEGF increased the proliferation of both MSCs and F5M2 cells. These findings suggest that CXCR4-mediated osteosarcoma growth and pulmonary metastasis are promoted by MSCs through VEGF.

miR-15a and miR-16-1 downregulate CCND1 and induce apoptosis and cell cycle arrest in osteosarcoma.

Osteosarcoma, the most common primary tumor of the bones, causes many deaths due to its rapid proliferation and drug resistance. Recent studies have shown that cyclin D1 plays a key regulatory role during cell proliferation, and non-coding microRNAs (miRNAs) act as crucial modulators of cyclin D1 (CCND1). The aim of the current study was to determine the role of miRNAs in controlling CCND1 expression and inducing cell apoptosis. CCND1 has been found to be a target of miR-15a and miR-16-1 through analysis of complementary sequences between microRNAs and CCND1 mRNA. The upregulation of miR-15a and miR-16-1 in the cell line SOSP-9607 induces apoptosis and cell cycle arrest. Osteosarcoma cells transfected with miR-15a and miR-16-1 show slower proliferation curves. Moreover, the transcription of CCND1 is suppressed by miR-15a and miR-16-1 via direct binding to the CCND1 3'-untranslated region (3'-UTR). The data presented here demonstrate that the CCND1 contributes to osteosarcoma cell proliferation, suggesting that repression of CCND1 by miR-15a and miR-16-1 could be used for osteosarcoma therapy.

Treatment of complicated tibial plateau fractures with dual plating via a 2-incision technique.

The operative treatment of complicated bicondylar fractures of the tibial plateau remains a challenge to most surgeons. This retrospective study was designed to evaluate the clinical and radiological outcomes of dual plating via a 2-incision technique for the repair of complicated bicondylar tibial plateau fractures. A series of consecutive patients with bicondylar tibial plateau fractures treated by open reduction and internal fixation with a double buttress plate or a combination of locking plate and buttress plate via a 2-incision technique between March 2004 and March 2008 were retrospectively analyzed. Radiological and clinical results and complications of the 2 different fixation methods were compared. Seventy-nine patients matching the criteria of this study were followed up for at least 24 months. All of the fractures healed, with 3 cases of deep infection, 7 cases of secondary loss of reduction, 3 cases of secondary loss of alignment, and 10 cases of knee instability. At 24-month follow-up, mean Hospital for Special Surgery scores were 77.8±9.4 and 79.0±7.9 in the double buttress plate group and combination group, respectively. No significant differences in clinical or radiographic outcomes were found between the 2 groups, except that the combination group needed less bone graft. Dual plating with 2 incisions provided good exposition for the reduction and fixation of complicated bicondylar tibial plateau fractures. Using a combination of locking plate and buttress plate reduced the amount of bone graft compared with the double buttress plate technique.

[Construction and expression of human anti-HER2 scFv-9R fusion protein and identification of its activity].

AIM: To construct, express and purify a human fusion protein, which is composed of a single-chain antibody fragment scFv that recognizes HER2 protein and an oligo-9-arginine, and to analyze the binding activity of the expressed fusion protein. METHODS: Pairs of oligonucleotide primers were designed and used to amplify the scFv-9R. The fusion protein gene scFv-9R was then cloned into expression vector pQE30 and expressed in E.coli M15.Expressed protein was detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. Then, the purified protein was refolded by dialysis and concentrated by ultrafiltration. The antigen-binding activity of the scFv-9R fusion protein was confirmed by ELISA, and the siRNA binding ability was confirmed by electrophoretic mobility shift assay (EMSA). RESULTS: HER2 scFv-9R encoding sequence was correctly cloned into the expression vector. The recombinant protein was insolubly expressed in E.coli M15 induced by IPTG. ELISA confirmed that it had specific antigen binding activity; EMSA assured that it had siRNA binding activity. CONCLUSION: The scFv-9R fusion protein can specially bind with both HER2 antigen and siRNA.

BLCAP induces apoptosis in human Ewing's sarcoma cells.

Bladder cancer-associated protein (BLCAP) is a novel candidate tumor suppressor gene identified from human bladder carcinoma and highly associated with the invasion of bladder cancer. We previously reported that it also plays a key role in the tumorigenesis and metastasis of human osteosarcoma. In the present study, we constructed a recombinant encoding BLCAP cDNA. Overexpression of BLCAP resulted in growth inhibition and induced apoptosis of human TC-135 Ewing's sarcoma cells in vitro. We further investigated the caspase-3/7 activity and expressions of the fusion transcription factor Ewing's sarcoma protein-friend leukemia virus integration 1 (EWS-FLI1) and the apoptosis regulator B-cell lymphoma 2 (BCL-2). Cell apoptosis was accompanied by the down-regulated expression of EWS-FLI1 and BCL-2. Our present results suggest that BLCAP may play a role not only in regulating cell proliferation but also in coordinating apoptosis through the down-regulation of BCL-2 and EWS-FLI1 in human Ewing's sarcoma cells.

Application of locking plate in long-bone atrophic nonunion following external fixation.

The treatment of atrophic fracture nonunion continues to represent a therapeutic challenge. Large segmental osteopenia is often seen in patients who received uniplanar or hybrid external fixators as the definitive method of fixation for high-energy fractures, and this adds more difficulties to the treatment of fracture nonunion. This retrospective study was designed to assess the outcome of locking compression plating with autologous bone grafting in patients with long-bone atrophic nonunion following external fixation.From January 2004 to December 2009, a series of consecutive patients with atrophic nonunion of the long bone following external fixation were treated with this method in our institution. The clinical outcomes and complications of these patients were retrospectively analyzed. Twenty-seven patients with 28 fracture nonunions were involved in this study. Mean follow-up was 14.2±3.4 months. Bony union was achieved in all 27 patients within a mean 18.6±4.8 weeks after revision surgery. Two patients developed superficial wound infections. No deep infections were found, and no implant failure was seen. Three patients reported minor pain in the donor site of the bone graft, and no other donor site complications were found.Revision osteosynthesis of long-bone atrophic nonunion following external fixation by locking compression plating with autologous iliac crest bone grafting represents a safe and efficacious modality for the treatment of these challenging conditions.

Int7G24A variant of transforming growth factor-beta receptor 1 is associated with osteosarcoma susceptibility in a Chinese population.

The TGF-beta signaling pathway is important in the development and invasion of cancers. Int7G24A is an intronic variant of TGF-beta receptor type 1 and has been shown to be associated with the occurrence of some kinds of cancers. Nevertheless, the association of this polymorphism with osteosarcoma is unknown. In this study, we evaluated Int7G24A variant frequencies in osteosarcoma cases. The case-control study involved 168 osteosarcoma patients and 168 age- and gender-matched controls. The blood samples were obtained, and Int7G24A variant was determined by PCR amplification and DNA sequencing. The odds ratio (OR) and 95% confidence interval (95% CI) for the Int7G24A polymorphism were calculated using unconditional logistic regression adjusted for age and gender. Three analysis models, which are the dominant model, additive model and recessive model, were used to analyze the contribution of Int7G24A variant to osteosarcoma susceptibility. Heterozygotic and homozygotic Int7G24A variants were 33.93 and 6.55% in total 168 cases, while they were 28.57 and 2.98%, respectively, in total 168 controls. The ORs for homozygosity and heterozygosity of Int7G24A allele were 1.56 [95% CI, 0.98-1.83] and 2.89 [95% CI, 1.46-4.92] in additive model. The ORs of Int7G24A genotypes in dominant model and in recessive model were 1.75 [95% CI, 1.21-2.68] and 2.21 [95% CI, 1.34-4.72], respectively. There were significant increases in Int7G24A variants in osteosarcoma cases when compared to control in every three models. Further analysis showed that Int7G24A genotypes were not associated with gender and osteosarcoma location of the cases. However, Int7G24A was significantly increased in the cases less than 20 years old. Moreover, Int7G24A was significantly associated with increased distant metastasis of osteosarcoma. It is concluded that Int7G24A is a polymorphism of TGFBR1 that is associated with the susceptibility and distant metastasis of osteosarcoma.

Clinical value of signal transducers and activators of transcription 3 (STAT3) gene expression in human osteosarcoma.

The dysregulation of signal transducers and activators of transcription 3 (STAT3) has been reported to be associated with tumor progression, angiogenesis and metastasis. The purpose of this study was to analyze the clinical value of STAT3 expression in human osteosarcoma. First, semi-quantitative RT-PCR was performed to detect the expression of STAT3 mRNA in normal bone tissues, chondroma tissues and osteosarcoma tissues. Then, immunohistochemistry was performed to detect the expression of STAT3 protein in 76 osteosarcoma tissues and the relationship of STAT3 protein expression with clinicopathologic factors or prognosis of osteosarcoma patients. RNA interference (RNAi) technology was employed to inhibit STAT3 expression. MTT and flow cytometric assays were performed to analyze the effect of STAT3 inhibition on proliferation and apoptosis of osteosarcoma cells. Finally, the expression of STAT3-related target genes were also determined. Results showed that osteosarcoma tissues showed significantly higher expression levels of STAT3 mRNA than normal bone or chondroma tissues (P<0.05). Immunohistochemistry showed that the staining of STAT3 protein was mainly located in cytoplasm of osteosarcoma cells in osteosarcoma tissue samples. The high level of STAT3 protein was associated with poor tumor differentiation and presentation of metastasis (P=0.039 and 0.022). Moreover, the 5-year overall and relapse-free survival rates for osteosarcoma patients with high STAT3 expression were lower than those for patients with low STAT3 expression. In addition, the status of STAT3 protein expression was an independent prognostic factor for both disease-free survival (P=0.0235) and overall survival (P=0.0032). RNAi-mediated STAT3 inhibition could induce proliferation inhibition and apoptosis enhancement in osteosarcoma cells, which might be associated with inhibition of some anti-apoptosis genes. Overall, STAT3 plays crucial roles in osteosarcoma development and might become a potential molecular target for gene therapy of human osteosarcomas.

A novel recombinant immuno-tBid with a furin site effectively suppresses the growth of HER2-positive osteosarcoma cells in vitro.

Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-positive tumors. Previously, we constructed an immuno-carboxy terminal fragment of Bid (immuno-tBid) and reported its specific and effective destruction of HER2-positive tumor cells. In this study, in order to further reduce the immunogenicity of the previous immuno-proapoptotic protein, we constructed a novel immuno-tBid by replacing domain II of Pseudomonas exotoxin A with a short furin cleavage sequence from the diphtheria toxin. In order to explore the possible application of this novel immuno-tBid in the treatment of osteosarcoma, we first examined the expression of the HER2 protein in a subclone of a human osteosarcoma cell line with relatively high metastatic potential (SOSP-9607-E10), as well as in clinical specimens of osteosarcoma. Quantitative real-time PCR and Western blot analysis revealed that the expression of HER2 was up-regulated in the SOSP-9607-E10 cells, while immunohistochemical analysis revealed that HER2 was overexpressed in 37% of the tissue specimens examined. Both HER2-positive SOSP-9607-E10 and SKBR-3 cells, as well as HER2-negative HeLa cells were transiently transfected with the novel immuno-tBid in order to study its specific pro-apoptotic effect. We demonstrate here that this novel immuno-tBid induces the specific destruction of HER2-overexpressing SOSP-9607-E10 cells through the release of cytochrome C. These results suggest that the novel immuno-tBid with a minimized exogenous fragment could represent a competitive approach for the treatment of HER2-positive osteosarcoma.