Study On The Inhibitory Mechanism Of Cutaneous Squamous Cell Carcinoma A431 In Nude Mice

Objective To study the effect of berberine on the volume,weight,proliferation and apoptosis of cutaneous squamous cell carcinoma(A431)transplanted from nude mice in vivo.By detecting the expression of apoptosis-related proteins,the inhibitory effect of berberine on cutaneous squamous cell carcinoma and its possible inhibitory mechanism was elucidated,which provided a new theoretical basis for treatment.Methods A431 cells were cultured,and the A431 cell line of cutaneous squamous cell carcinoma was injected subcutaneously into the back of nude mice.After 3 weeks of adaptive feeding,the nude mice were randomly divided into 4 groups(5 in each group)according to the size of the tumor body: control group(intraperitoneal injection of normal saline).Low dose group(10mg/kg intraperitoneally injected berberine),medium dose group(20mg/kg)and high dose group(25mg/kg).The control group was set as the negative control group.After treatment with different concentrations of berberine,the tumor volume and weight of the transplanted tumor was detected every week for 5 weeks,and the tumor inhibition rate was calculated.The cell morphology of transplanted tumor tissues stained by HE was observed by inverted microscope.The expression level of Ki67 was detected by immunohistochemistry,and the proliferation ability of transplanted tumor cells was observed.The number of brown-yellow positive apoptotic cells was detected by TUNNEL assay,and the apoptosis of transplanted tumor cells was observed.The expression levels of apoptosis-related proteins Bax,Bcl-2,Caspase-3 and Ezrin m RNA were detected by real-time quantitative PCR.The expression levels of apoptosis-related proteins Bax,Bcl-2,Caspase-3 and Ezrin were detected by Western blotting assay,which reflected the ability of cell apoptosis and invasion.Statistical software SPSS22.0 was used to analyze the data of different concentration groups and control groups.Results The growth indexes of the xenograft model of cutaneous squamous cell carcinoma in nude mice were different under different concentrations of berberine.With the increase of berberine concentration,tumor volume gradually decreased(P < 0.01),weight gradually decreased(P < 0.01),and tumor inhibition rate gradually increased,which was statistically significant.HE staining of transplanted tumor tissues showed that with the increase of berberine concentration,the area,degree and range of tumor cell necrosisincreased,suggesting that berberine can promote the necrosis of transplanted tumor cells.Immunohistochemical results showed that with the increase of berberine concentration,the expression level of Ki67 decreased significantly,suggesting that berberine could inhibit the proliferation of A431 transplanted tumor cells in nude mice,which was statistically significant compared with the control group(P < 0.01).TUNNEL experiment showed that brown-yellow apoptotic cells in tissues increased with the increase of berberine concentration compared with the control group,suggesting that berberine could promote the apoptosis of A431 transplanted tumor in nude mice(P < 0.05 in the low dose group,P < 0.01 in the medium dose group and high dose group).According to the real-time quantitative PCR method,the relative expression of apoptotic protein Bax m RNA was not statistically significant at low dose and medium dose with the increase of drug dose compared with the control group(P>0.05),while the expression of apoptotic protein Bax m RNA was significantly increased at high dose(P<0.01).Compared with the control group,the relative expression of Bcl-2 m RNA gradually decreased(P < 0.01).Compared with the control group,the relative expression of Caspase-3 m RNA was not statistically significant in the low dose group(P>0.05),but increased in the medium dose group and high dose group(P < 0.05 in the medium dose group,P < 0.01 in the high dose group).Compared with the control group,the relative expression levels of Ezrin m RNA were not statistically significant at low dose and medium dose(P>0.05),but at the high dose(P < 0.01).Western blotting experimental results show that,compared with control group,the concentration of berberine gradually increased,Bax protein expression increased(P < 0.01),the Bcl-2 protein low dose group of no statistical significance(P > 0.05),in the medium dose group and high dose group were gradually reduced(P < 0.01),Caspase 3 expression increased(P < 0.01),Ezrin protein gradually(P < 0.01).Conclusion Within a certain concentration range,berberine by inhibiting cutaneous squamous cell carcinoma A431 xenograft from nude mice proliferation and promote cutaneous squamous cell carcinoma A431 xenograft from nude mice cells apoptosis,and effectively restrain cutaneous squamous cell carcinoma A431 xenograft from nude mice growth and inhibit the transplanted tumor cells survive,its mechanism may be related to berberine increases the expression of Bax,Caspase-3,while lowering the Bcl-2,Ezrin expression.