adrenomedullin and Osteosarcoma

Osteosarcoma is one of the most pernicious primary bone tumor characterized by high malignancy and metastasis, however its pathogenesis remain largely unknown. Our previous study showed elevated expression of adrenomedullin (ADM) is correlated with prognosis and disease severity in osteosarcoma patients. In this research, we continue to study the mechanisms of ADM-induced osteosarcoma cells proliferation in vitro and in vivo osteosarcoma models.. The Radioimmunoassay was used to test the correlations of ADM and vascular endothelial growth factor (VEGF) expressions in plasma of osteosarcoma patients. The MTT and flow cytometry analysis were accepted to monitor the proliferation of osteosarcoma cells, meanwhile the ADM and VEGF expression were detected by real-time PCR and western blot. Moreover, the relationship among ADM and VEGF expression was also assessed in the osteosarcoma nude mice models.. In this study, a significant correlation was found between ADM and VEGF expression in the plasma of osteosarcoma patients. As important stimuli in osteosarcoma proliferation, hypoxia could stimulate ADM and VEGF expression in MG-63 cells. The expressions of ADM and VEGF increased with the duration of hypoxia, which also identify the positive correlations between the expression of ADM and VEGF. The proliferation of MG-63 cells decreased when ADM was inhibited. And the proliferation increased when adding exogenous ADM, while VEGF inhibition attenuated this effect. Furthermore, ADM reduction also inhibited VEGF, CD31 expression and tumor cells growth in osteosarcoma nude mice models.. These results suggested inhibition of osteosarcoma cells proliferation might be influenced by ADM through VEGF pathway, which implied ADM-VEGF signal was a potential target for impeding the proliferation of malignant cells in osteosarcoma.

Topics: Adrenomedullin; Animals; Bone Neoplasms; Carcinogenesis; Cell Cycle; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Humans; Mice; Mice, Nude; Osteosarcoma; Platelet Endothelial Cell Adhesion Molecule-1; Prognosis; Radioimmunoassay; Vascular Endothelial Growth Factor A

Adrenomedullin (ADM), a multifunctional regulatory peptide, is potentially induced by hypoxia in physiological and pathological tissues, including many types of malignant tumors. Recent research has demonstrated that ADM expression is highly associated with the prognosis and disease severity of human osteosarcoma. However, the effect of ADM on the apoptosis of osteosarcoma cells and its possible mechanism remain to be elucidated. In the present study, we observed that mRNA and protein levels of ADM were increased in human osteosarcoma SOSP-F5M2 cells under a hypoxic microenvironment induced by cobalt chloride (CoCl2) in a time-dependent manner. Treatment with ADM significantly blunted hypoxic-induced apoptosis, evaluated by Hoechst 33342 staining and Annexin V-FITC/PI labeling. The expression of B-cell lymphoma-2 (Bcl-2) was increased by administration of ADM; meanwhile, this effect was reversed by exogenously adding U0126, a selective inhibitor of MEK or ADM22-52 (ADM-specific receptor antagonist). These results demonstrated that ADM acted as a survival factor to inhibit hypoxic-induced apoptosis via interacting with its receptors CRLR-RAMP (2,3) in osteosarcoma cells. The anti-apoptotic function of ADM was found to be mediated by upregulation of the expression of Bcl-2 partially through activation of the MEK/ERK1/2 signaling pathway. Therefore, targeting of the ADM/ADM acceptors/ERK1/2/Bcl-2 pathway may provide a potential strategy through which to induce the apoptosis of osteosarcoma cells.

Topics: Adrenomedullin; Apoptosis; Bone Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cobalt; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MAP Kinase Signaling System; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2

The treatment for osteosarcoma is a formidable challenge. Currently, treatment is not sufficiently effective, and new therapeutic targets are urgently needed. The aim of this study is to determine the expression of adrenomedullin (ADM) in human osteosarcoma tissue and to assess its effect on the proliferation of MG-63 cells and in vivo in an animal model of osteosarcoma. First, we collected clinical specimens from osteosarcoma patients and healthy controls and measured ADM expression by immunohistochemistry, RT-PCR, and radioimmunoassay. We also analyzed clinical data to investigate the relationship between ADM expression, malignancy, and tumor prognosis. Based on these data, we used RNA interference (RNAi) against ADM delivered by lentivirus vector transfected into the osteosarcoma cell line MG-63 to downregulate the expression of ADM. Finally, we observed the effect of ADM on the proliferation of MG-63 cells in vitro, and in vivo, in an animal model of osteosarcoma. We found that ADM was overexpressed in human osteosarcoma tissue, whereas expression was low in the adjacent tissue and little expression was observed in normal tissue. ADM RNAi significantly inhibited the proliferation of MG-63 cells. Therefore, the growth of osteosarcoma could be inhibited by decreasing the expression of ADM. Thus, we conclude that ADM expression is highly correlated with the degree of malignancy and metastasis of osteosarcoma.

Topics: Adolescent; Adrenomedullin; Adult; Animals; Biomarkers, Tumor; Bone Neoplasms; Child; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Osteosarcoma; Prognosis; Radioimmunoassay; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Young Adult

Hypoxia-inducible factor-1 (HIF-1) is an important tumor-selective therapeutic target for solid tumors. Icariside II was isolated from Epimedium koreanum through successive fractionation with ethyl acetate, n-butanol, chloroform and hexane, followed by gel column chromatography. Icariside II attenuated the protein level of HIF-1alpha induced by hypoxia in human osteosarcoma (HOS) cells in a concentration-dependent manner, probably by enhancing the interaction rate between von Hippel-Lindau (VHL) and HIF-1alpha. Furthermore, Icariside II down-regulated the levels of HIF-inducible genes involved in angiogenesis, metastasis, and glucose metabolism, such as vascular endothelial growth factor (VEGF), urokinase plasminogen activator receptor (uPAR), adrenomedullin (ADM), matrix metalloproteinase 2 (MMP2), aldolase A, and enolase 1 in HOS cells. Icariside II also inhibited the migration rate in HOS cells and tube formation rate in human umbilical vein endothelium cells (HUVECs). Overall, these results suggest the potential use of Icariside II as a therapeutic candidate against various diseases that involve overexpression of HIF-1alpha.

Topics: Adrenomedullin; Bone Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Epimedium; Flavonoids; Fructose-Bisphosphate Aldolase; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Matrix Metalloproteinase 2; Osteosarcoma; Phosphopyruvate Hydratase; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein