Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga

Abstract

Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50 μg/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2 μg/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma.

Figure 1

Cytotoxic effect of ESA on either OST cells or LM8 cells, as evaluated by means of propidium iodide staining. (a) Variation of the cell viability with increasing ESA concentration during incubation for 24 hours. (b) Time courses of the cell viabilities for [ESA] = 50 μg/mL. For both set of data, mean values and standard deviations for three separate measurements are shown.

Figure 2

Apoptotic induction in either (a) OST cells or in (b) LM8 cells after adding ESA. The cells were cultured in 10% FBS D-MEM with 50 μg/mL ESA (bottom panel). As control, only PBS (no ESA) was added (top panel). The cells were incubated with ESA for either 3 hours or 24 hours. Induction of apoptosis in these cells was detected by means of the double staining assay for annexin V-PE and 7-ADD.

Figure 3

Determination of the caspase-3 activity of OST cells treated with ESA. The OST cells were cultured during 16 hours in D-MEM containing either a solution of 10% FBS and 50 μg/mL ESA in PBS or a solution of 50 μg/mL ESA and ZVAD-FMK (as caspase inhibitor) in PBS. Caspase-3 activity was determined from the absorbance values measured at 405 nm as “activity index” by use of a spectrophotometer. The values are means and standard deviations for three separate measurements.

Figure 4

Specific binding of ESA to either OST cells or LM8 cells, as measured by using a flow cytometer. The cells were cultured with 10% FBS D-MEM containing FITC-labeled ESA at 37°C in a humidified atmosphere of 5% CO₂. After incubation for 0, 3, 6, 9, 12, and 24 hours, the cells were washed with PBS, followed by evaluation of the amount of ESA which was bound to the cells.

Figure 5

(A) Bright field image of OST cells. The diameter of the OST cells was 19.9 μm ± 1.5 μm. (B) Fluorescence microscopic observations of the binding of ESA to OST cells. The cells were pretreated for 2 hours with different glycosidases and then incubated with rhodamine 6G-labeled ESA. (a) Untreated cells (as control); (b) pretreated with α-mannosidase; (c) pretreated with β-mannosidase; and (d) pretreated with endoglycosidase H. After the pretreatment with the glycosidases, which led to a cleavage of some of the sugar chains on the surface of the OST cells, incubation of the pretreated cells with rhodamine 6G-labeled ESA occurred during 1 hour at 37°C in a humidified atmosphere of 5% CO₂. Scale bar shows approximately 20 μm. This scale was calculated from the bright field image.

Figure 6

Flow cytometric analysis of OST cells that were pretreated with a glycosidase as described in the legend of Figure 5 and then incubated with FITC-labeled ESA (balck line). Pretreatment was with either α-mannosidase (green line) or with β-mannosidase (blue line) (a), or with endoglycosidase H (green line) (b). The filled curves represent control measurements with untreated cells. PBS was added to OST cells as control (red fill).

Figure 7

Flow cytometric analysis of the interaction between OST cells and different types of Span 80 vesicles containing entrapped FITC: control vesicles (CV, black line), vesicles with immobilized ESA (EV, green line), and PEGylated vesicles with immobilized ESA (EPV, blue line). Before analysis, the OST cells were incubated with the vesicles during 15 min at 37°C in a humidified atmosphere of 5% CO₂. PBS was added to OST cells as control (red fill).

Figure 8

Cytotoxic effect of ESA in EPV on OST cells. The cell viability was evaluated with the propidium iodide staining. The OST cells were incubated during 48 hours at 37°C with EPV at the given concentration in D-MEM containing 10% FBS in a humidified atmosphere of 5% CO₂. Mean values and standard deviations for three separate measurements are plotted.

Figure 9

Graphical imaginary view indicating the binding between carbohydrate chains of high mannose type on sarcoma membrane and the ESA on the PEGylated Span 80 vesicle.