Abstract:

Objective To investigate whether baicalein inhibits the proliferation, cell cycle of and pseudopod formation in A431, a skin squamous cell carcinoma cell line, by suppressing the expression of ezrin protein. Methods A431 cells were grouped to be transfected with ezrin-targeting siRNA （siRNA group）, treated with baicalein of 5, 10, 20, 40 μmol/L, respectively （baicalein group）, or remain untreated （control group）. After additional culture, wound healing assay and Transwell assay were performed to observe the migration and invasion of A431 cells, RT-PCR to detect the mRNA expression of ezrin in A431 cells, Western blot and immunofluorescence to measure the expression of ezrin protein and its phosphorylation. The pseudopod formation in A431 cells was observed by using scanning electron microscopy. Results After 24-hour culture, wound healing assay displayed that the percent wound closure was 13.3 ± 1.7, 7.6 ± 1.6 and 5.9 ± 1.3, respectively, in A431 cells treated with baicalein of 5, 10, 20 μmol/L, significantly lower than that in untreated A431 cells （16.3 ± 2.3, all P < 0.01）, and the inhibition of baicalein on the migration of A431 cells was concentration-dependent. In the Transwell assay, a significant decrease was observed in the number of A431 cells per high power field permeating through the artificial basement membrane in the groups treated with baicalein of 5, 10, 20 μmol/L for 48 hours compared with the control group （46.5 ± 3.8, 34.3 ± 3.4, 25.3 ± 2.3 vs 56.3 ± 3.8, all P < 0.01）, whereas no significant difference was noted between these baicalein-treated groups and siRNA-transfected group （28.3 ± 2.1, all P > 0.05）. RT-PCR analysis showed that the mRNA expression of ezrin in baicalein-treated A431 cells significantly decreased compared with that in untreated cells （all P < 0.01）, but showed no difference from that in siRNA group （P > 0.05）. A statistical difference was also observed in the expression of ezrin and phosphorylated ezrin protein between baicalein-treated A431 cells and untreated cells （all P < 0.05）, but not between 40 μmol/L baicalein-treated A431 cells and siRNA-transfected cells （P > 0.05）. Furthermore, the suppression of baicalein on ezrin protein and mRNA expression was concentration dependent. The number of pseudopod per cell was significantly lower in 20 μmol/L baicalein-treated A431 cells and siRNA-transfected cells than that in untreated A431 cells （5.3 ± 1.9, 4.5 ± 2.8 vs 22.6 ± 2.8, both P < 0.01）, while no significant difference was observed between the former two groups of cells （P > 0.05）; the length of pseudopodia also reduced in baicalein-treated cells. Conclusions Baicalein may inhibit the proliferation and invasion of A431 cells by directly or indirectly suppressing the expression of ezrin and phosphorylated ezrin, which in turn contributes to the effect of baicalein against tumor proliferation and metastasis.