Lianhe Zheng

miR-34a inhibits the metastasis of osteosarcoma cells by repressing the expression of CD44

Osteosarcoma is the most common type of malignant bone tumor in children and adolescents and approximately 30% of patients develop lung metastasis, which is the leading cause of mortality. In this study, we investigated the role of miR-34a in the invasion and metastasis of osteosarcoma cells by examining its expression level and functional pattern in these cells. miR-34a mimics were transfected into the highly metastatic subline, F5M2, and into the F4 subline with low metastatic potential of the paired human osteosarcoma cell line, SOSP‑9607. Cell viability patterns, cell migration and alterations in gene expression levels were assessed by real-time PCR, and changes in protein levels were assessed by immunocytochemistry and western blot analysis. The ectopic overexpression of miR-34a significantly inhibited the migration and invasive ability of osteosarcoma cells by repressing the expression of CD44. These data suggest that miR-34a plays a tumor suppressor role in the metastasis of osteosarcoma cells by repressing the expression of CD44. Of note, studies have also suggested that the CD44 protein correlates with the metastatic potential of several malignant tumors. Therefore, it can be concluded that through the inhibition of CD44 expression levels, miR-34a plays a significant role in the migration and invasion of osteosarcoma cells.

Tumoricidal effects of a selenium (Se)-polysaccharide from Ziyang green tea on human osteosarcoma U-2 OS cells

Selenium(Se)-enriched green tea consumption in human diets is well-known to reduce the risk of a variety of diseases. Here, we isolated a Se-polysaccharide (Se-ZYTP) from Se-enriched Ziyang green tea and investigated its antitumor activity on human osteosarcoma U-2 OS cells in vitro and in vivo. Se-ZYTP contained 94.5% of carbohydrate and 2.1% of uronic acid, as well as 2.14 μg/g Se, revealing that Se-ZYTP was an acidic Se-conjugated polysaccharide. Monosaccharide composition analysis indicated that Se-ZYTP consisted of mannose, rhamnose and fucose in molar ratios of 2.4:1.5:1.2:0.2:0.1:0.3:0.3. In vitro, both MTT and LTH assays proved that Se-ZYTP (25, 50, 100 and 200 μg/ml) could significantly inhibit the proliferation of human osteosarcoma U-2 OS cells in a concentration-dependent fashion (P<0.05 or P<0.01). In U-2 OS cancer xenograft model in BALB/c athymic mice, Se-ZYTP oral administration at three doses of 100, 200 and 400mg/kg body weight (B.W.) daily for 28 days resulted in obvious tumor regression as compared to model control (P<0.05 or P<0.01). In addition, body weights of mice in control or Se-ZYTP treated groups did not differ significantly and no mice died during experiment, suggesting the safety of Se-ZYTP. Therefore, we postulate that Se-ZYTP might have cancer-preventive and cancer-therapeutic benefit for human osteosarcoma.

Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits

BackgroundHydroxysafflor Yellow A (HSYA), which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R) injury is still unknown.MethodsThirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6) were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining.ResultsNeurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA) level and increased superoxide dismutase (SOD) activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits.ConclusionsThese findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.

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.

FUNCTIONAL AND RADIOLOGICAL EVALUATIONS OF HIGH-ENERGY TIBIAL PLATEAU FRACTURES TREATED WITH DOUBLE- BUTTRESS PLATE FIXATION

ObjectiveThis study was designed to evaluate the functional and radiological outcomes of patients with complex tibial plateau fractures treated with double-buttress plate fixation.MethodsSixty five cases of complex (Schatzker type V and VI) tibial plateau fractures were treated with double-buttress plate fixation in our centre from September 2001 to September 2006 through two separate plate incisions. Fifty four patients were followed up for a period ranging from 12 to 48 months and evaluated for the functional and radiological outcomes by a series of standard questionnaire and measurement.ResultsDue to the good exposure without any extensive soft-tissue dissection of the double-buttress plate fixation, the fractures in all 54 patients were healed and the treatment achieved greater than 90% of satisfactory-to-excellent rates of reduction. The mean time of bone union was 15.4 weeks (range, 12-30 weeks), and the mean time of full weight-bearing was 18.7 weeks (range, 14-26 weeks). At the final follow-up visit, no patients showed knee instability; the mean range of motion was 107.6° (range, 85°-130°). For all patients, no statistically significant difference in the functional outcomes was observed between their 6-months and final follow-up visits; or in the radiological findings between their immediate postoperative and final follow-up examinations.ConclusionDouble-buttress plate fixation is a feasible treatment option for bilcondylar and complex tibial plateau fractures. Although technically demanding, it offers reliable stability without additional postoperative adjuvant external fixation, and at the same time avoids extensive soft tissue dissection, allowing the early painless range of motion.

mTOR signal transduction pathways contribute to TN-C FNIII A1 overexpression by mechanical stress in osteosarcoma cells.

Osteosarcoma is the most common primary malignant bone tumor with a very poor prognosis. Treating osteosarcoma remains a challenge due to its high transitivity. Tenascin-C, with large molecular weight variants including different combinations of its alternative spliced FNIII repeats, is specifically over expressed in tumor tissues. This study examined the expression of Tenascin-C FNIIIA1 in osteosarcoma tissues, and estimated the effect of mechanical stimulation on A1 expression in MG-63 cells. Through immunohistochemical analysis, we found that the A1 protein was expressed at a higher level in osteosarcoma tissues than in adjacent normal tissues. By cell migration assay, we observed that there was a significant correlation between A1 expression and MG-63 cell migra-tion. The relation is that Tenascin-C FNIIIA1 can promote MG-63 cell migration. According to our further study into the effect of mechanical stimulation on A1 expression in MG-63 cells, the mRNA and protein levels of A1 were significantly up-regulated under mechanical stress with the mTOR molecule proving indispensable. Meanwhile, 4E-BP1 and S6K1 (downstream molecule of mTOR) are necessary for A1 normal expression in MG-63 cells whether or not mechanical stress has been encountered. We found that Tenascin-C FNIIIA1 is over-expressed in osteosar-coma tissues and can promote MG-63 cell migration. Furthermore, mechanical stress can facilitate MG-63 cell migration though facilitating A1 overexpression with the necessary molecules (mTOR, 4E-BP1 and S6K1). In con-clusion, high expression of A1 may promote the meta-stasis of osteosarcoma by facilitating MG-63 cell migration. Tenascin-C FNIIIA1 could be used as an indicator in metastatic osteosarcoma patients.

Functional and Radiological Evaluations of Unstable Displaced Proximal Humeral Fractures Treated with Closed Reduction and Percutaneous Pinning Fixation

Objective: The purpose of this study is to evaluate the functional and radiological outcomes of patients with unstable displaced proximal humeral fractures treated with closed reduction and percutaneous pinning (CRPP) fixation. Methods: We retrospectively reviewed 87 cases of displaced (2-, 3- or 4-part fractures according to Neer classification) proximal humeral fractures treated with CRPP fixation in our center from September 2003 to September 2008. Sixty-four patients were followed up for a period ranging from 12 to 48 months (averaging 16.2 months) and evaluated for the functional and radiological outcomes by a series of standard questionnaire and measurement. Results: The fractures in all 64 patients were healed with an average time of 15.4 weeks (ranging from 12 to 43 weeks), and the mean interval between the operation and full functional exercise was 17.3 weeks (ranging from 14 to 38 weeks). At the final follow-up visit, no patients showed shoulder instability; the mean range of abduction motion was 157.1° (ranging from 70 to 180°). For all patients, no statistically significant difference in the functional outcomes was observed between their 6-month and final follow-up visits, nor in the radiological findings between their immediately postoperative and fi- nal follow-up examinations. Conclusion: CRPP fixation is a feasible treatment option for unstable displaced proximal humeral fractures, especially for 2- and 3-part fractures in elderly patients. Although technically demanding, it offers reliable stability without extensive soft tissue dissection, allowing the early painless range of motion. This technique could also promote bone healing, prevent ischemic osteonecrosis of the head of humerus and lead to few complications.

Mechanical Strain and Growth Factors Regulate Expression of Tenascin-C by OS Cells Additively

Osteosarcoma (OS) is a kind of malignancy wherein the tumor cells form malignant bone-like or bone tissue. Tenascin-C (TN-C), an important extracellular matrix (ECM) protein, plays an indispensable role in tumor development. However, its regulatory factors, expression, and function in OS pathological process have not been studied extensively. Expression of TN-C is induced by growth factors as well as mechanical strain in fibroblast. So we asked whether mechanical stain and growth factors could induce TN-C expression in OS as well as which pathways were involved in those processes. We found that when mechanical strain was applied to OS cells cultured on silicone membrane, TN-C mRNA and protein levels were increased 10-fold within 8 h compared to the resting control. Likewise, when epidermal growth factors (EGFs) and insulin-like growth factor (IGF-1) were added to cells, TN-C mRNA levels increased six fold and eightfold, respectively, within 24h compared to the control. Growth factors (EGF and IGF-1) and mechanical strain had additive effects on the induction of TN-C mRNA expression in OS. Both ROCK-I/II inhibitor and MEK-1 inhibitor inhibited TN-C induction by EGF or IGF-1, while only ROCK-I/II inhibitor had a strong subdued effect on TN-C induction by mechanical strain. Taken together, our findings suggest that growth factors and mechanical strain can induce TN-C in OS through different pathways additively.

Generation and Identification of Monoclonal Antibodies Against FNIII Domain D of Human Tenascin-C

Tenascin-C (TN-C), a key component of extracellular matrix (ECM), is strongly expressed in fetal and cancer tissues. Large-molecular-weight variants of TN-C, including different combinations of its alternative spliced FNIII repeats, are specifically expressed in tissues under certain pathological conditions. Here we report the production of monoclonal antibodies (MAbs) against FNIII domain D (FNIII D) of human TN-C. Complementary DNA encoding the FNIII D region was generated by RT-PCR from human osteosarcoma (OS) cell line, and the recombinant FNIII D-GST fusion protein was expressed and purified. Two hybridoma cell lines secreting monoclonal antibodies (MAbs) against FNIII D were obtained by routine murine hybridoma technique. The MAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). Both of them were applicable in Western blot and IHC. With our MAbs, we found TN-C was positive in OS and most of it was among the tumor stroma. To conclude, these MAbs to human FNIII D domain of TN-C may be useful for exploring OS pathogenesis and potential clinical application.

Purified dendritic cell-tumor fusion hybrids supplemented with non-adherent dendritic cells fraction are superior activators of antitumor immunity.

Background Strong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown. Methods Patient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared. Results Except for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction. Conclusions The non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy.