echistatin and Bone-Neoplasms

The in vitro efficacy of the disintegrin echistatin was tested on a high-metastatic variant of 143B human osteosarcoma, 143B-LM4, which over-expresses αvβ3 integrin. Echistatin is an RGD cyclic peptide and an antagonist of αvβ3 integrin. In the present study, echistatin inhibited cell proliferation, migration, invasion, and adhesion of 143B-LM4 cells. 143B-LM4 cell proliferation decreased after treatment with echistatin in a time-dependent and dose-dependent manner (P <0.01). In vitro migration and invasion of 143B-LM4 cells were also inhibited by echistatin in a dose-dependent manner (P <0.01, respectively). Cell adhesion to vitronectin of 143B-LM4 cells was also inhibited by echistatin in a dose-dependent manner (P <0.01). These results suggest that αvβ3 integrin may be an effective target for osteosarcoma.

Topics: Antineoplastic Agents; Bone Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disintegrins; Humans; Integrin alphaVbeta3; Intercellular Signaling Peptides and Proteins; Neoplasm Invasiveness; Osteosarcoma; Peptides

Echistatin, a cyclic RGD peptide, which is an antagonist of αvβ3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing αvβ3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by echistatin (P<0.05) as was overall growth. A doxorubicin (DOX)-echistatin combination inhibited orthotopic tumor growth compared to untreated control (P<0.01) or DOX alone (P<0.05) in nude mice. Tumor-bearing mice treated with the DOX-echistatin combination survived longer than those treated with DOX alone or control PBS (P<0.01 and P<0.01, respectively). Echistatin also inhibited experimental lung metastasis of 143B-LM4 cells in nude mice. These results suggest that DOX in combination with a disintegrin has potential to treat osteosarcoma and that αvβ3 integrin may be a target for osteosarcoma.

Topics: Animals; Bone Neoplasms; Chick Embryo; Doxorubicin; Humans; Integrin alphaVbeta3; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Nude; Osteosarcoma; Peptides

Expression of adhesion molecules such as alphavbeta3 integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether alphavbeta3 expression correlated with the metastatic potential of human osteosarcoma cells.. We developed a series of sublines (LM2-LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6-8 weeks. We quantified alphavbeta3 integrin expression using flow cytometry.. alphavbeta3 expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of alphavbeta3. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that alphavbeta3 was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration.. alphavbeta3 integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. Alphavbeta3 integrin may therefore be a potential new target for osteosarcoma.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Adhesion; Cell Movement; Chemotaxis; Endothelial Cells; Gene Expression Regulation, Neoplastic; Humans; Integrin alphaVbeta3; Intercellular Signaling Peptides and Proteins; Liver; Lung; Lung Neoplasms; Male; Mice; Mice, Nude; Oligopeptides; Osteosarcoma; Peptides; Platelet Aggregation Inhibitors; Receptors, Vitronectin; Tumor Cells, Cultured; Vitronectin

Synthetic Arg-Gly-Asp (RGD)-containing peptides were examined in bone resorption or attachment and detachment assays with isolated mammalian osteoclasts in an effort to elucidate the mechanistic and structural basis for the inhibition of bone resorption by s-echistatin. Bone resorption was the process most sensitive to inhibition by s-echistatin, with IC50 = 0.3 nM; inhibition of attachment to bone or detachment (lamellipodial retraction) was 30- to 70-fold less sensitive, with IC50 = 10 or 20 nM, respectively. Single amino acid substitutions within the 49-residue sequence of s-echistatin showed that although the efficacy of s-echistatin is dependent on the Arg24-Gly25-Asp26 sequence, additional residues, including Asp27, Met28, and Cys39, are also critical for potent inhibition of the resorbing activity of isolated rat osteoclasts. Because of the identification of the av beta 3 as the primary integrin on rat osteoclasts interacting the RGD peptides (Helfrich et al.), we examined the possibility of modeling bone resorption with other beta 3-mediated processes. Specifically, av beta 3 endothelial cell (human or rat) attachment to vitronectin and aIIb beta 3 platelet aggregation were compared with bone resorption for sensitivity to s-echistatin analogs, linear RGD peptides, and cyclic RGD peptides. Essentially no similarity in sensitivity to RGD peptides were observed between bone resorption, platelet aggregation, or endothelial cell attachment. Because rat osteoclasts and human giant cell tumors (osteoclastomas) shared similar sensitivity to s-echistatin and rat and human endothelial cells showed a similar sensitivity profile to RGD peptides, the dissimilarity of bone resorption to other beta 3-mediated processes cannot be explained in terms of species differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Animals; Bone Neoplasms; Bone Resorption; Cell Adhesion; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Giant Cell Tumor of Bone; Humans; Intercellular Signaling Peptides and Proteins; Mice; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Molecular Sequence Data; Oligopeptides; Organ Culture Techniques; Osteoclasts; Peptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Viper Venoms