Anticancer effects and the mechanism underlying 2-methoxyestradiol in human osteosarcoma in vitro and in vivo

Abstract

Osteosarcoma (OS) occurs in both children and adolescents and leads to a poor prognosis. 2-methoxyestradiol (2-ME) has a strong antitumor effect and is effective against numerous types of tumor. However, 2-ME has a low level of antitumor effects in OS. The present study investigated the effects of 2-ME on the proliferation and apoptosis of human MG63 OS cells. The potential biological mechanisms by which 2-ME exerts its biological effects were also investigated in the present study. The results of the present study demonstrated that 2-ME inhibited the proliferation of OS cells in a time- and dose-dependent manner, induced G₂/M phase cell cycle arrest and early apoptosis. The expression levels of vascular endothelial growth factor (VEGF), Bcl-2 and caspase-3 were measured via western blotting and reverse transcription-quantitative PCR. As the concentration of 2-ME increased, the RNA and protein expression levels of VEGF and Bcl-2 decreased gradually, whereas the expression of caspase-3 increased gradually. In addition, tumor growth in nude mice was suppressed by 2-ME with no toxic side effects observed in the liver or kidney. Immunohistochemistry demonstrated that the expression levels of Bcl-2 and VEGF were significantly lower, and those of caspase-3 were significantly higher in test mice compared with the control group. TUNEL staining of xenograft tumors revealed that with increased 2-ME concentration, the number of apoptotic cells also gradually increased. Thus, 2-ME effectively inhibited the proliferation and induced apoptosis of MG63 OS cells in vitro and in vivo with no obvious side effects. The mechanism of the anticancer effect of 2-ME may be associated with the actions of Bcl-2, VEGF and caspase-3.

Keywords: 2-methoxyestradiol; apoptosis; osteosarcoma; proliferation.

Figure 1.

Morphological changes in MG63 cells (as observed under an inverted microscope) after exposure of cells to different concentrations of 2-ME for 24 h.

Figure 2.

Effect of 2-ME on osteosarcoma cell proliferation. MG63 cells were cultured and subsequently incubated with the indicated concentrations of 2-ME for 24, 36 or 48 h and cell proliferation was measured (n=3). The results are expressed as percentages of the control and the data were analyzed using Student's unpaired t-tests. *P<0.05 compared to negative control, 2-ME, 2-methoxyestradiol.

Figure 3.

Effect of 2-ME on MG63 cell cycle arrest and apoptosis. (A) Flow cytometry histograms of cell number vs. DNA content to investigate cell cycle arrest. Quantification of cell number is also presented. (B) Flow cytometry plots with quantitative data displaying apoptosis of MG63 cells exposed to different concentrations of 2-ME for 24 h. *P<0.05. 2-ME, 2-methoxyestradiol.

Figure 4.

Effect of 2-ME on the expression levels of VEGF, Bcl-2 and caspase-3. (A) MG63 cells were treated with 0, 10, 20 or 40 µM of 2-ME for 24 h, followed by western blotting using the appropriate antibodies. β-actin was used as the control. (B-D) Bcl-2, VEGF, and caspase-3 mRNA levels normalized to β-actin mRNA levels. The data are expressed as the mean ± SD (n=3). *P<0.05. 2-ME, 2-methoxyestradiol; VEGF, vascular endothelial growth factor; SD, standard deviation.

Figure 5.

Effects of 2-ME on MG63 ×enograft in BALB/c nude mice (n=32). (A) Tumor volume and (B) weight after implantation. *P<0.05. 2-ME, 2-methoxyestradiol.

Figure 6.

Immunohistochemistry and TUNEL staining of xenograft tumors. (A) Hematoxylin and eosin staining of xenograft tumors. (B) Immunohistochemistry with specific antibody against VEGF, Bcl-2, and caspase-3 in xenograft tumors. (C) TUNEL staining of apoptotic xenograft tumor cells in the 4 groups. Quantitative result of TUNEL assay is displayed. There are a few positive cells (brown staining) in the control group. *P<0.05. 2-ME, 2-methoxyestradiol; VEGF, vascular endothelial growth factor.