Anti-Tumor Effect And Its Mechanisms Of Ursolic Acid On Human Esophageal Carcinoma Cell Eca-109 In Vitro And In Vivo

Objective:Ursolic acid (UA) is one of pentacyclic triterpenoids which is extracted from the natural herb. In recent years, some studies have shown that UA exhibits growth inhibition properties against many human cancer cell lines, but its anti-tumor mechanisms have not been completely clarified. To date, however, rare study has been reported about the anti-tumor effects of ursolic acid against human esophageal carcinoma cells. In this study, we investigated the effects of UA on inhibiting proliferation, inducing apoptosis, the expression of apoptosis-related protein and COX-2 on Eca-109 cells cultured in vitro and transplanted human esophageal carcinoma in nude mice, further clarified its anti-tumor mechanisms.Methods:1. The study of UA against Eca-109 cells cultured in vitro: The cell proliferation of Eca-109 cells treated with different concentrations of UA (0, 10, 20, 30, 40, 50μmol / L) after 24, 48, 72h was determined by MTT assay. Electron microscope was used to observe the ultrastructural changes of Eca-l09 induced by UA. The morphological changes of apoptosis were observed by fluorescence microscopy. Cell cycle and apoptosis rate were analysed by flow cytometry (FCM), while the expression of P27kip1, Bcl-2 and Bax by Western blot. 2. The study of UA against transplanted human esophageal carcinoma in nude mice: A transplanted tumor model by injecting Eca-109 cells into subcutaneous tissue of BALB/c nude mice was established. 12 nude mice with tumors were randomly divided into 4 groups and 0.2 ml saline or 0.2 ml ursolic acid(25, 50, 100mg/Kg/d) was injected into abdominal cavity respectively once everyday and lasted for fourteen days. The morphological changes of tumor tissue and the vital organs (liver, kidney, heart, lung, spleen and so on.) of nude mice in each group were observed by HE staining .The changes of tumor volume were measured continuously and tumor inhibition rate was calculated. The morphological changes of apoptosis were observed by electron microscope, while the apoptosis rate were analysed by TUNEL assay. The expressions of COX-2, Bcl-2 and Bax protein in transplanted tumors were detected by immunohistochemistry.Results: 1. UA(20～50μmol/L)could significantly inhibit the growth of Eca-109 cells(P<0.01). The cell apoptosis of Eca-109 cells treated with UA (18.32～36.65μmol/L)were observed by electron microscop. Marked morphological changes of cell apoptosis were found clearly using Hoechst 33258 staining. With UA concentration of a gradual increase, the apoptotic rate of Eca-109 cells had increased, the S-phase cells gradually decreased, but the G0/G1 cells gradually increased. Western blot test results indicated that the expression of Bcl-2 of Eca-109 cells were down-regulated, while the expression of Bax and P27kip1 up-regulated. 2. Treatment of nude mice with 50, 100mg/kg/day of ursolic acid significantly inhibited the growth of the human esophageal carcinoma cell tumor in nude mice. HE staining results suggest that tumor tissue treated with UA grew at a poor state, while vital organs of nude mice did not find obvious pathological changes.The cell apoptosis of tumor tissues were observed by electron microscope.The apoptotic index in each group(25,50,100mg/Kg/d UA) detected by TUNEL were respectively 1.70%±1.05,3.53%±0.60,13.47%±1.07,29.60%±2.89. The expressions of COX-2 and Bcl-2 in the transplanted tumors were decreased in ursolic acid groups, while the Bax increased.Conlusion : Ursolic acid has a significant effect of inhibiting proliferation , inducing apoptosis on Eca-109 cells cultured in vitro. Blocking Eca-109 cells in the G0/G1 phase by enhancing the expression of P27kip1 protein, reducing the expression of Bcl-2 and increasing the expression of Bax may be its mechanism. Meanwhile, UA has the anti-tumor effect against human esophageal carcinoma cells in vivo, which are likely mediated via induction of tumor cell apoptosis and inhibition of COX-2.