The estrogen metabolite 2-methoxyestradiol regulates eukaryotic initiation factor 4E (eIF4E) and inhibits protein synthesis in MG63 osteosarcoma cells

Abstract

Osteosarcoma is a primary bone tumor that affects children and young adults. The estrogen metabolite 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. To determine whether 2-ME actions involve the control of protein synthesis, we studied the effect of 2-ME on eukaryotic initiation factor 4E (eIF4E) and eIF4E-binding protein 1 (4E-BP1) in MG63 osteosarcoma cells. Our results show that 2-ME treatment increases the association of eIF4E with 4E-BP1 in osteosarcoma cells. Also, 2-ME decreases the binding of eIF4E protein to 7-methyl-guanosine cap structure, indicating that 2-ME treatment results in the inhibition of translational initiation. These findings are further supported by the inhibition of protein synthesis in 2-ME-treated osteosarcoma cells. Taken together, our studies show that 2-ME-mediated antitumor effects in osteosarcoma cells involve the regulation of protein synthesis, and translational machinery could serve as a target in the treatment of osteosarcoma.

Keywords: 2-Methoxyestradiol; 4E-BP; Estrogen metabolite; Osteosarcoma; eIF4E.

Figure. 1

2-ME regulates eIF4E and 4E-BP1 functions in osteosarcoma cells. Cytoplasmic extracts were prepared from MG63 osteosarcoma cells (A, B) at 16 and 24 h and normal human osteoblast (HOB) cells (C, D) at 24 h following vehicle (Veh) and 2-ME treatment. The extracts were subjected to immunoprecipitation (IP) with anti-4E-BP1 antibodies (A, C) or cap-binding (B, D) and analyzed by western blot analysis using anti-eIF4E antibodies.

Figure. 2

Effect of 2-ME treatment on eIF4E and 4E-BP1 expression levels. Cytoplasmic extracts were prepared following 16 and 24 h of vehicle and 2-ME treatment from MG63 cells and analyzed by western blot using anti-eIF4E, anti-4E-BP1, antiphospho-4E-BP1 (Thr37/46), anti-nonphospho-4E-BP1 (Thr46), and anti-actin antibodies.

Figure. 3

2-ME treatment downregulates protein synthesis in osteosarcoma cells. Protein synthesis was measured following vehicle and 2-ME treatment in MG63 (A) and HOB (B) cells at 12, 16, and 24 h through pulse labeling with ³H-leucine. *P < 0.01 vs vehicle.

Figure. 4

2-ME effect on protein synthesis is decreased in cells expressing PKRmutant protein. MG63 osteosarcoma cells expressing vector (pcDNA) and PKRmutant protein were treated with vehicle and 2-ME for 24 h. The cytoplasmic extracts prepared were subjected to cap-binding (A) or IP with anti-4E-BP1 antibodies (B) and analyzed by western blot analysis using anti-eIF4E antibodies. Protein synthesis was measured through pulse labeling with ³H-leucine following 24 h of vehicle and 2-ME treatment (C). *P < 0.01 vs Veh, ^#P < 0.01 vs pcDNA 2-ME.