| Objective(1) To investigate Ezrin, phosphate-Ezrin expression in normal skin(NS), seborrheic keratosis(SK), basal cell carcinoma(BCC) and squamous cell carcinoma(SCC), and the relationship between Ezrin, phosphate-Ezrin and clinic pathological parameter and prognosis.(2) To deterimine the effect of baicalein in proliferation, pseudopodia and colony formation of epithelial squamous cell carcinoma-A431 cells and find out the optimal inhibitory concentration range of baicalein in A431 cell without cytotoxicity .(3) To probe into the molecular mechanism of baicalein inhibiting the proliferation, wound-healing and pseudopodia formation and then identify whether baicalein suppressing tumor-invasive action through inhibiting the expression of Ezrin, phosphate-ezrin.Methods(1) The expression of Ezrin, phos-ezrin was detected in 36 cases of SCC, 27 cases of BCC, 20 cases of SK and 10 cases of NS using immunohistochemistry, and the relationship between ezrin, phos-ezrin and prognosis of SCC was analyzed using COX regression.(2) Proliferation-inhibitory effect of baicalein on A431 cells was determined by MTT assay. Cell growth and their Morphologic change were observed under normal-optic-microscope. The pseudopod formation of A431 cells treated by baicalein and siRNA using scanning electron microscope, and colony formation of A431 was observed after baicalein treatment. The suppression effect of baicalein was determined on creeping movement of A431 cells using wound healing assay .The invasiveness of A431 cells was detected after baicalein and siRNA treatment by Trans-well assay.(3) Cell cycle analysis was performed by flow cytometry to determine cell percentage of each stage and apoptosis rate in A431 cells treated by baicalein. RT- PCR analysis to show the expression of Ezrin-mRNA of A431 cell line treated by baicalein, Western Blotting and immunofluorescence were used to investigate Ezrin and phos - ezrin expression in A431 cells treated by baicalein and siRNA.Results1) The expression of Ezrin and phos-Ezrin in NS, SK, BCC, SCC was 20.0% and 10.0%, 25.0% and 15.0%, 66.7% and 77.7%, 91.3% and 97.2% respectively. There was significant difference between each group (P<0.01).2) Ezrin, phos-Ezrin expression was closely related to skin-tumor type, clinic grade of SCC and lymph node metastasis. (R1, r1=0.87, 0.89 ; R2, r2=0.80, 0.86; R3, r3=0.89, 0.91, P<0.01. R1, Ezrin and cancer type, r1 phos-Ezrin and cancer type; R2, Ezrin and SCC grade; r2, phos-Ezrin and SCC grade; R3, Ezrin and lymph node metastasis; r3, phos-Ezrin and lymph node metastasis ).3) Methylthiazolyl tetrazolium (MTT) data showed that the inhibitory effect of baicalein on proliferation of A431 cell increased at time-dependent and dose-dependent, and it was significance to compare with control group(P <0.01). The growth index of A431 decreased along with baicalein concentration-increasing under optic-microscope and morphous became round and curtate. The pseudopodiao of A431 cells obviously decurtated and its amount reduced under scanning electron microscope. 48 hours after being treated by baicalein, A431 cell numbers permeating the artificial basement membrane largely decreased through Transwell assay, it was significant difference compared with control group (P < 0.01). Baicalein also suppressed the wound-healing of A431 cells depending on it's dosage.4) Cell cycle results showed that cell cycle was obviously inhibited by baicalein at dosage and time dependent and apoptosis rate was also increased, it was significant compared with control (P < 0.05). Baicalein inhibited the expression of ezrin, phos-ezrin and ezrin -mRNA of A431 cells depending on it's dosage (P < 0.01).5) Baicalein restrained the expression of MMP2, MMP9 proteins in A431 cells depending on it's dosage.Conclusions1) The expression of Ezrin, phos-Ezrin in SK, BCC and SCC is closely related to skin tumor-type, clinic grade, and whether lymph node metastasis or not, both of them were expressed in a parallel way. Combined-detection of Ezrin, phos-Ezrin may be one of the important way of predicting skin malignant tumor metastasia and prognosis.2) The optimal dosage range of baicalein was 10μM to 30μM, which inhibited A431 cell proliferation and without cytotoxicity after 48 hours treated by baicalein.3) Baicalein inhibited A431 cell proliferation, invasion, wound -healing through direct or indirect inhibiting the expression of Ezrin, phos-Ezrin, so as to achieve anti-tumor hyperplasia and infiltrating-metastasis.
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