Modulation of anoikis resistance in MG63 osteosarcoma cells by sclareol via inhibiting Ezrin/Fak expression

Osteosarcoma is one primary bone tumor with high risk of blood borne metastasis, thus causing aggravation and patient death. The study of molecular mechanism underlying metastasis of osteosarcoma thus may help to relieve the threat of osteosarcoma. Sclareol has been found to have anti-tumor effects in leukemia, colorectal cancer and osteosarcoma, but with unclear functional mechanism so far. Therefore we treated MG63 osteosarcoma cells with sclareol, to observe the condition of anoikis in cells, in an attempt to investigate the modulation of osteosarcoma cell anoikis by sclareol. MG63 cells were cultured by poly HEMA approach. CCK8 reagent was used to detect the cytotoxicity of sclareol on suspended MG63 cells, in order to determine the optimal dosage of sclareol. MG63 cells were further divided into DMSO and sclareol group, in which real-time florescent quantitative PCR and Western blotting were used to detect the expression of Ezrin and FAK genes. Using RNA interference, intracellular expression of these two genes was suppressed. Moreover, flow cytometry was used to detect the cell anoikis. Compared to DMSO group, sclareol-treated cells had lowered expression of Ezrin and FAK genes, with the mRNA level decreased by 45% and 42%, respectively. The ratio of cell apoptosis was significantly elevated (P<0.05). RNA interference assay showed the enhancement of cell apoptosis by inhibiting Ezrin or FAK gene expression. Sclareol may promote the anoikis of MG63 osteosarcoma cells by inhibiting Ezrin and FAK gene expression.