Expressions Of Livin And Caspase-3 Proteins In Human Cervical Cancer And In Vitro Study Of Antitumor Mechanism Of Ursolic Acid On SiHa Cell Line

Objective: The expressions of Livin and Caspase-3 proteins were analyzed in cervical squamous cell carcinoma(CSCC). The mechanism of Ursolic Acid(UA) inhibiting SiHa cell line was investigated by detecting Livin and Caspase-3 mRNA levels.Methods: A total of 89 CSCC tissue blocks and 19 normal cervical epithelium(NCE) tissue blocks were collected. All of them were fixed in 10% formalin, embedded in paraffin, and serial section at the thickness of 4μm. The immunohistochemistry was performed and expressions of Livin and Caspase-3 proteins were analyzed. The method of MTT was used to explore the effect of UA on SiHa cells at concentration of 20μM,30μM,40μM,50μM,60μM in turn. The morphologic changes of apoptosis were observed by Hoechst 33258 fluorescent staining and transmission electron microscopy(TEM). Flow cytometer(FCM) was used to detect the changes of cell cycle and apoptosis rate. RT-PCR was used to detect the changes of apoptosis related genes(Livin,Caspase-3,Bcl-2,Bax) and cell cycle regulator genes(p53,p21).Results: The expression rate of Livin protein in CSCC tissues(60.67 %) was higher than that in NCE tissues(21.05 %). The expression rate was associated with clinical stage,pathological grade,infiltration depth and lymph node metastasis(p﹤0.05). The expression rate of Caspase-3 protein in CSCC tissues(30.34 %) were lower than those in NCE tissues(63.16 %). The expression rate was associated with pathological grade and infiltration depth(p﹤0.05). The expressions of Livin protein were negatively related to those of Caspase-3 protein in CSCC tissues(r=?0.469, p﹤0.01). UA had an anti-proliferation effect on SiHa cells in dose-dependant and time-dependant manner in vitro. By Hoechst 33258 staining, fluorescence microscopy showed dense nucleus or broken bits. Morphological changes of apoptosis, such as nucleus shrinkage and chromatin margination were observed by TEM. FCM showed that the apoptosis rate and the percent of G0/G1 were increased by time. RT-PCR revealed that the mRNA lever of p53,p21,Bax and Caspase-3 were increased, whereas that of Livin and Bcl-2 were decreased.Conclusions: The Livin and Caspase-3 proteins may act as a marker to determine the malignant degree of CSCC. UA could inhibit the proliferation, induce apoptosis and block cell cycle of SiHa cells in vitro. These effects were in a dose-dependant and time-dependant manner. The mechanism of inhibiting the proliferation of SiHa cells was mainly by upregulating p53,p21,Bax,Caspase-3 mRNA and downregulating Livin,Bcl-2 mRNA levers.