Research Of Anti-Proliferation And Apoptosis Induction Of Ursolic Acid On Human Adenoid Cystic Carcinoma 83 Cells In Vitro

ObjectiveUrsolic 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 adenoid cystic carcinoma cells. To assess the inhibition of proliferation and the induction of apoptosis of UA on human adenoid cystic carcinoma 83 cells (ACC-83). we investigated the effect of COX-2, Bcl-2, Bax and Caspase-3 and the possible mechanism in UA-induced Adenoid Cystic Carcinoma 83 cells apoptosis.Methods(1) MTT was used to detect optical density(OD) of ACC-83 by intervention of UA in five different concentration groups in 24h, 48h and 72h. The effect of Ursolic Acid on ACC-83 cell proliferation was measured using MTT assay, calculating 50% inhibitory concentration(IC50) in every group. (2) Propidium iodide (PI) single-staining flow cytometry assay method was used to analyze cell cycles of ACC-83 by intervention of UA in three different concentration groups. To observe cell percentage distribution in G0/G₁, G₂/M and S phases by intervention of different UA concentration groups after 24h. Cell apoptosis inspection method of Annexin V-FITC and PI double labeling was used to detect the cell apoptosis effect of UA on ACC-83 in three different concentration groups after 24h. (3) Morphological analysis was performed by cytospin preparation with Wright-Giemsa stain and the slides were observed under a light microscope. (4) Western blot was used to detect cells expression changes of Bcl-2, Bax, COX-2 and Caspase-3.Results(1) MTT assay showed that UA inhibited ACC-83 cell proliferation in time- and concentration-dependent manner. The inhibiting concentration of 50% cell growth (IC50) at 24h, 48h and 72h was (57.14±6.62)μmol/L, (16.57±4.68)μmol/L and (7.29±4.26)μmol/L, respectively (P﹤0.05). (2) As compared with normal control group in the same phase, cell percentage of S phase in ACC-83 cell were increased significantly in three different UA concentration groups (25μmol/L, 50μmol/L, 100μmol/L) after administration for 24h, with statistical signifieance(P﹤0.05). Higher the UA concentration, lower the cell percentage of G₀/G₁ phase, the cell percentage of G₂/M phase was not changed. A concentration-dependent manner made ACC-83 cell cycle arrest in S phase. Apoptosis of ACC-83 could be efficiently induced by UA, and early apoptosis rate were inereased gradually in three different UA concentration groups. The percentage of early apoptosis cells were (3.93±0.67)%, (5.86±1.26)% and (8.34±1.85)% after the cells exposed to UA for 24h, compare with the normal control group (P<0.01). (3) The morphological analysis demonstrated that the apoptotic cells became rounded in shape and their nuclei exhibited a fragmented morphology, forming apoptotic bodies. (4) COX-2 protein level was decerased by UA in a concentration-dependent manner, Bcl-2 portein level was decreased in a concentration-dependent manner, but Bax was not changed. Caspase-3 protein was activated during UA-induced apoptosis in ACC-83 cell (P﹤0.05).ConclusionsUrsolic acid can inhibit ACC-83 cell and to a large number of cells ac-cumulated in S phase, ursolic acid may be by downregulated expression of COX-2 and Bcl-2 and reduce the Bcl-2/Bax ratio to induce apoptosis, a-ctivation of Caspase-3 may be involved in its mechanism.