Hybrid liposomes inhibit tumor growth and lung metastasis of murine osteosarcoma cells

Abstract

Antitumor effects of hybrid liposomes (HL) composed of l-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) dodecyl ether (C₁₂(EO)₂₃) on the metastatic growth of murine osteosarcoma (LM8) cells were investigated in vitro and in vivo. Remarkable inhibitory effects of HL-23 on the growth of LM8 cells were obtained through the induction of apoptotic cell death in vitro. It was also indicated that HL-23 should dramatically suppress the invasion of LM8 cells and the formation of filopodia on the cell surface in vitro. Furthermore, significantly high therapeutic effects were observed in the homograft mouse models of LM8 cells with lung metastasis after the treatment with HL-23 in vivo. That is, the histological analysis demonstrated that the primary tumor growth of LM8 cells implanted subcutaneously into the mice was inhibited along with the induction of apoptosis. In addition, it was found that HL-23 significantly decreased the lung metastasis of LM8 cells in the mouse models through the inhibition of primary tumor invasion. These results suggest that HL-23 could be a novel agent for the chemotherapy of osteosarcoma.

Keywords: Apoptosis; hybrid liposomes; invasion; metastasis; osteosarcoma.

Figure 1

Time course of hydrodynamic diameter (d_hy) change for HL-23 at 25°C. Error bars indicate SE for four individual experiments. DMPC liposomes: [DMPC] = 10 mmol/L, HL-23: [DMPC] = 10 mmol/L, [C₁₂(EO)₂₃] = 1.1 mmol/L. HL, hybrid liposomes; SE, standard error; DMPC, l-α-dimyristoylphosphatidylcholine; C₁₂(EO)₂₃, polyoxyethylene(23) dodecyl ether.

Figure 2

Inhibitory effects of HL-23 on the growth of LM8 cells in vitro. (a) Concentration dependence of HL-23 on the growth of LM8 cells in the culture medium for 24 h. Error bars indicate SE for four individual experiments. (b) Micrographs of LM8 cells stained with Hoechst 33342 after the treatment with HL-23 for 24 h by fluorescence microscopy. Scale bars: 50 μm. (c) Percentage of apoptotic LM8 cells. Error bars indicate SE for four individual experiments. *P < 0.05 to control. Control: 5% glucose solution, DMPC liposomes: [DMPC] = 250 μmol/L, HL-23: [DMPC] = 250 μmol/L, [C₁₂(EO)₂₃] = 28 μmol/L. HL, hybrid liposomes; LM8, murine osteosarcoma; SE, standard error; DMPC, l-α-dimyristoylphosphatidylcholine; C₁₂(EO)₂₃, polyoxyethylene(23) dodecyl ether.

Figure 3

Inhibitory effects of HL-23 on the invasion of LM8 cells in vitro. (a) In vitro invasion assay. Invaded cell number of LM8 cells after the treatment of HL-23 for 24 h. Error bars indicate SE for three individual experiments. *P < 0.05 to control (5% glucose solution). (b) Micrographs of LM8 cells stained with rhodamine–phalloidin after the treatment of HL-23 for 3 h by TIRF microscopy. Scale bars: 20 μm. (c) Number of filopodia on the surface of LM8 cells. Error bars indicate SE for 8–14 individual experiments. *P < 0.05 to control. Control: 5% glucose solution, DMPC liposomes: [DMPC] = 50 μmol/L, HL-23: [DMPC] = 50 μmol/L, [C₁₂(EO)₂₃] = 5.6 μmol/L, non-FBS: LM8 cells were incubated in the serum-starved medium for 24 h. HL, hybrid liposomes; LM8, murine osteosarcoma; SE, standard error; TIRF, total internal reflection fluorescence; DMPC, l-α-dimyristoylphosphatidylcholine; C₁₂(EO)₂₃, polyoxyethylene(23) dodecyl ether; FBS, fetal bovine serum.

Figure 4

Inhibitory effects of HL-23 on the primary tumor growth of LM8 cells in homograft mouse models in vivo. (a) Weight of subcutaneous primary tumor of the mouse models treated with HL-23 for 14 days. Error bars indicate SE (n = 6 per group).*P < 0.05 to control. (b) Representative micrographs of subcutaneous primary tumor resected from the mouse models treated with HL-23 for 14 days by TUNEL method. Scale bars: 500 μm (×100), 50 μm (×400). Dotted enclosures indicate apoptotic tumor cells. Control: 5% glucose solution, DMPC liposomes: 203 mg/kg/day, HL-23: 203 mg/kg/day for DMPC, 41 mg/kg/day for C₁₂(EO)₂₃. HL, hybrid liposomes; LM8, murine osteosarcoma; SE, standard error; DMPC, l-α-dimyristoylphosphatidylcholine; C₁₂(EO)₂₃, polyoxyethylene(23) dodecyl ether.

Figure 5

Inhibitory effects of HL-23 on the lung metastasis of LM8 cells in homograft mouse models in vivo. (a) Micrographs of lung tissue resected from the mouse models treated with HL-23 for 14 days by HE staining method. Scale bars: 500 μm. (b) Dimensions of metastatic tumor in the lung tissue of mouse models treated with HL-23 for 14 days. Error bars indicate SE (n = 5 per group). *P < 0.05 to control. Control: 5% glucose solution, DMPC liposomes: 203 mg/kg/day, HL-23: 203 mg/kg/day for DMPC, 41 mg/kg/day for C₁₂(EO)₂₃. HL, hybrid liposomes; LM8, murine osteosarcoma; HE, hematoxylin and eosin; SE, standard error; DMPC, l-α-dimyristoylphosphatidylcholine; C₁₂(EO)₂₃, polyoxyethylene(23) dodecyl ether.

Figure 6

Micrographs of the subcutaneous tumor of LM8 cells resected from homograft mouse models treated with HL-23 for 7 days by HE staining method. The number of mice was three in each group. Scale bars: 500 μm. Control: 5% glucose solution, DMPC liposomes: 203 mg/kg/day, HL-23: 203 mg/kg/day for DMPC, 41 mg/kg/day for C₁₂(EO)₂₃. T, tumor; P, peritoneum; HL, hybrid liposomes; LM8, murine osteosarcoma; HE, hematoxylin and eosin; DMPC, l-α-dimyristoylphosphatidylcholine; C₁₂(EO)₂₃, polyoxyethylene(23) dodecyl ether.