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.

Aberrant CXCR4 and ß-catenin expression in osteosarcoma correlates with patient survival.

To determine the clinical significance of C-X-C chemokine receptor type 4 (CXCR4) and ß-catenin in osteosarcoma, their protein expression levels were assessed in 96 osteosarcoma and 20 osteochondroma cases using immunohistochemistry. Additionally, CXCR4 and ß-catenin mRNA expression levels were measured in 16 fresh osteosarcoma and 16 adjacent healthy tissue samples using fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the osteosarcoma samples, the positive CXCR4 protein expression rate was significantly higher than the rate in the osteochondroma samples (68.75 vs. 20.00%; P<0.01). Furthermore, ß-catenin protein expression was detected in 61.46% of osteosarcoma cases and 25.00% of osteochondroma cases. Similarly, the RT-qPCR data identified increased CXCR4 and ß-catenin mRNA expression levels in the osteosarcoma compared with adjacent control tissues. It was determined that CXCR4 (P<0.01) and ß-catenin (P<0.05) expression were significantly associated with the clinical Enneking stage, metastasis and survival of osteosarcoma. Furthermore, multivariate analysis identified CXCR4 and ß-catenin protein expression levels, as well as clinical stage and metastasis, as significant risk factors for survival in patients with osteosarcoma (P<0.05). In conclusion, the present study determined that CXCR4 and ß-catenin are abnormally expressed in osteosarcoma tissues, and, therefore, may be important during osteosarcoma progression.

SDF-1/CXCR4 promotes F5M2 osteosarcoma cell migration by activating the Wnt/ß-catenin signaling pathway.

Osteosarcoma (OS), the most common primary malignant bone tumor in children and adolescents, lacks an effective therapy. Stromal cell-derived factor (SDF-1) and its receptor, CXCR4, play multiple roles in migration, proliferation, and survival of different tumor cells. This study aimed to investigate whether the functional SDF-1/CXCR4 signaling mediates chemotaxis in F5M2 OS cells as well as the underlying mechanisms. Immunohistochemistry and immunofluorescence microscopy were used. RNA expression was detected by real-time quantitative polymerase chain reaction, and protein expression was examined by Western blotting. Migration assays were carried out in F5M2 cells. The results showed that the expression of CXCR4 and ß-catenin mRNA and protein was significantly higher in OS tissues compared to the surrounding non-neoplastic tissues. SDF-1 promoted F5M2 cell migration by activating the AKT and Wnt/ß-catenin signaling pathway, which was abrogated by preincubation with AMD3100 and LY294002. In conclusion, SDF-1/CXCR4 axis-promoted F5M2 cell migration was regulated by the Wnt/ß-catenin signaling pathway.

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.

A novel recombinant immunocasp-6 fusion gene specifically and efficiently suppresses HER2-overexpressing osteosarcoma.

Osteosarcoma is the most common primary malignant tumor of bone for adolescent or children. The poor prognosis of patients, due to its remote metastasis, has led to the exploration of more effective and less toxic treatments. Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing tumors. Herein, we describe experiments conducted with a fusion gene, immunocasp-6, which was generated by fusing a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A and an active caspase-6 which can directly cleave lamin A leading to nucleus damage inducing programmed cell death. We demonstrated that immunocasp-6 can specifically and efficiently recognize and induce apoptosis in HER2-overexpressing osteosarcoma cells in vitro. The immunocasp-6 was transferred into BALB/c athymic mice bearing human osteosarcoma by i.m. injection of liposome-encapsulated pCMV-immunocap-6. Expression of immunocasp-6 not only strongly inhibited tumor growth and significantly prolonged animal survival, but also greatly prevented tumor metastasis. Our data showed that the immuno-casp-6 can specifically recognize HER2-overexpressing osteosarcoma cells, can also promptly attack their nucleus and induce apoptotic death, suggesting the potential of this strategy for the treatment of human HER2-overexpressing tumors.

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.

[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.

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.

Gene expression profiles of human osteosarcoma cell sublines with different pulmonary metastatic potentials.

AIM: to screen the pulmonary metastasis-associated molecules of Osteosarcoma and evaluate their functions concerning prognosis prediction. METHODS: cDNA microarray analysis has been applied to 2 pairs of osteosarcoma cell sublines with differential metastatic potentials to the lung. Immunohistochemistry and survival analysis have been performed to clinical samples of osteosarcoma patients. RESULT: Analysis detected 484 differentially expressed genes between the high metastatic subline, F5M2, and the low metastatic subline, F4. There were 1257 genes differentially expressed between newly established high-metastatic sublines named Saos-2M2 and its parental cell line Saos-2. Furthermore, 16 commonly up-regulated genes and 5 commonly down-regulated genes were identified by clustering analysis. EREG and CHST2, two genes not previously described in osteosarcoma, were finally seen to be differentially expressed in all examined osteosarcoma cell lines and in samples between the different prognosis sample groups. Survival analysis also confirmed these two molecules could be used to predict the outcome of OSA patients. CONCLUSION: This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis. EREG and CHST2 will be likely considered as clinical molecular markers to predict the outcome of OSA.

[Pro-apoptotic effect on osteosarcoma SOSP-9607 cells by human recombinant caspase-6 fusion protein].

OBJECTIVE: To investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells. METHODS: Recombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining. RESULTS: The tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry. CONCLUSION: Immunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.