Disintegrin targeting of an αvβ3 integrin-over-expressing high-metastatic human osteosarcoma with echistatin inhibits cell proliferation, migration, invasion and adhesion in vitro

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.


INTRODUCTION
Osteosarcoma is the most common bone cancer in children and adolescents [1][2][3]. Osteosarcoma usually arises in the distal femur and proximal tibia and proximal humerus.
Development of neo-adjuvant chemotherapy has increased the rate of overall survival and has enabled surgery, but the failure rate is still high, causing death in children and adolescents [4][5][6][7].
Novel more effective targets for osteosarcoma therapy are therefore needed. Toward this goal, α v β 3 integrin-over-expressing high-metastatic variants of the human osteosarcoma cell line 143B were previously isolated and termed 143B-LM4.
Over-expression of α v β 3 integrin in the variant selected for high-metastasis suggested that integrin maybe a promising target for osteosarcoma. The 143B-LM4 cells expressed green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, and therefore could be imaged down to the subcellular level in vitro and in vivo [8].
Lung seeding by 143B-LM4 cells was directly imaged and found to be greatly inhibited by the anti-β1 integrin monoclonal antibody, AIIB2. AIIB2 also significantly inhibited spontaneous lung metastasis and increased survival of mice with orthotopically-growing 143B-RFP [9].
In the present study, we tested echistatin, a cyclic RGD peptide antagonist of α v β 3 integrin (disintegrin) [10], as a molecular-targeting drug in human metastatic osteosarcoma in vitro on the highly metastatic 143B-LM4 cell line which over-expresses α v β 3 integrin described above.

Dual-color-labeled GFP-and RFP-expressing 143B-LM4 cells
The high-metastatic integrin-over-expressing 143B-LM4 cells have a strikingly bright GFP in the nucleus and RFP in the cytoplasm, in vitro ( Figure 1).
The effect of echistatin on the ability of 143B-LM4 cells to migrate was then tested. In vitro, migration of 143B-LM4 cells decreased to 59.4% at 1.0 μg/mL and to 8.5% at 5.0 μg/mL echistatin, compared to control (P<0.01 respectively) ( Figure 3A).
Aggressive chemotherapy of osteosarcoma in patients with metastatic or recurrent disease, most commonly in the lung [11][12][13], still results in poor prognosis with less than a 20% 5-year overall survival rate [14][15][16]. Therefore, novel targets are needed to overcome recurrence or metastasis and to improve the disease-free survival rate.
In the present study, we demonstrated that echistatin resulted in a significant decrease of cell proliferation, GFP was excited at 488 nm, RFP at 543nm. Magnification 60x. Scale bar: 50 µm. migration, invasion, and adhesion of 143B-LM4 cells in vitro. As 143B-LM4 is a high-metastatic variant that over-expresses α v β 3 integrin, these results suggest that α v β 3 integrin may be an effective target for osteosarcoma that has future clinical potential.
Previously-developed concepts and strategies of highly-selective tumor targeting can take advantage of molecular targeting of tumors, including tissue-selective therapy which focuses on unique differences between normal and tumor tissues [17][18][19][20][21][22].

Migration and invasion assay
An in vitro migratory/invasiveness assay was carried out with Corning ® (Tewksbury, MA) HTS Transwell-96 plates uncoated or coated, respectively, with a basement membrane extract (Trevigen, Gaithersburg, MD) according to manufacturer's instructions. 143B-LM4 cells (5×10 4 ) were added to the upper chamber and various concentrations of echistatin were added to the lower chamber (0.5 µg/mL, 1.0 µg/mL, 5.0 µg/m), for both the migration and invasion assays. The lower chamber had the same conditions for the migration and invasion assays. For the migration assay, an uncoated well was used for the upper chamber. For the invasion assay, a well coated with a basement membrane was used as the upper chamber. For both assays, cancer cells were seeded in the upper chamber. The plate was placed for 24 h at 37°C in a tissue culture incubator. After incubation for 24 h, 100 µl of fresh medium was gently replenished in the lower chamber and 20 µl MTS was added to the lower chamber to determine cell viability. After incubation for 1 h, the absorbance was measured using a microplate reader at 490 nm. The assays were performed in triplicate and at least twice, independently.

Adhesion assay
The adhesion assay was carried out with CultureCoat ® Vitronectin 96-well dishes (Trevigen) according to the manufacturer's instructions. 143B-LM4 cells were labeled with 2 µM calcein AM (Invitrogen, Carlsbad, CA), harvested and then re-suspended in medium to a final concentration of 1.5×10 5 cells/ml. Only live cells can absorb this agent. 143B-LM4 cells (1.5×10 4 /100 µl) were added to each well and were either left untreated or treated with echistatin (0.5 µg/ mL, 1.0 µg/mL, 5.0 µg/mL), and placed for 1 h at 37°C in a CO 2 incubator. The medium was gently removed. The wells were washed twice gently with PBS. The fluorescence intensity of the cells remaining on the bottom of the well was measured using a SPECTRAmax GEMINI Dual-Scanning Microplate Spectrofluorometer, (Molecular Devices, Sunnyvale, CA) at an excitation/ emission wavelength of 485/520 nm. The assay was performed in triplicate and at least twice independently.

Statistical analysis
The data are presented as mean ± SD or mean ± SEM. Statistical analysis was with ANOVA for multiple data sets. P-values of less than 0.05 were considered significant.

DEDICATION
This paper is dedicated to the memory of A. R. Moossa, M.D. and Sun Lee, M.D.

ACKNOWLEDGMENTS
This work was supported in part by a grant from the Nakatomi Foundation.

CONFLICTS OF INTEREST
No potential conflicts of interest were disclosed.