Mechanism Of Apoptosis In Human Hepatoma SMMC-7721 Cells Induced By Oleanolic Acid Derivatives

Objective:Many study has found that pentacyclic triterpene compounds showed a good antitumor activity, oleanolic acid derivatives is a new type of pentacyclic triterpene compound in this article, this paper is intended to investigate the anti-cancer mechanism of oleanolic acid derivatives in human hepatocellular carcinoma cells SMMC-7721.Method:(1) Human hepatocellular carcinoma cells SMMC-7721 and human liver cells HL-7702 were treated with different concentrations of oleanolic acid derivatives, by detecting the cell growth curve, the observion of cell lesion, the pharmacodynamics effects of oleanolic acid derivatives was identified.(2) By detecting the cell membrane permeability with annexin V-FITC/propidium iodide, the loss of mitochondrial membrane potential (??m) using the JC-1 assay, the content of cytochrome c (Cyt-C) in the cytoplasm and mitochondria respectively using ELISA, and the protein expression level of apoptosis related proteins (Bcl-2, Bax, caspase-9, caspase-3), the apoptotic mechanism of oleanolic acid derivatives was identified.(3) We found that the abnormal activation in the Wnt/?-catenin signaling pathway in many kinds of tumor cells (including HCC) through referring to literature review, so we detect the key molecular ?-catenin, c-myc and cyclinD1 gene and protein evels of Wnt/p-catenin signaling pathway using the fluorescent quantitative PCR technique and immunoblot method after different concentrations of oleanolic acid derivatives act on SMMC-7721 cell at different time points.(4) In order to further explore the mechanism of oleanolic acid derivatives on down-regulating the gene and protein level of key molecular in Wnt/?-catenin signaling pathway, therefore, we detected the nuclear transfer and the phosphorylation level of ?-catenin, then, the inhibition mechanism on Wnt/p-catenin signaling pathway of oleanolic acid derivatives was identified.Results:(1) Oleanolic acid derivatives could obviously influence the cell status of SMMC-7721 cells, inhibit growth and proliferation of SMMC-7721 cells with a time and dose dependent style.(2) Oleanolic acid derivatives destroyed the SMMC-7721 cell membrane permeability, green fluorescent and red fluorescence have increased with a time and dose dependent style when Annexin V-FITC/PI double staining in SMMC-7721 cells. Antiapoptotic protein Bcl-2 was down-regulated and pro-apoptotic protein Bax was up-regulated after drug act on cell for 48h, what's more, we detected that the pyrolysis fragments of caspase-9 and caspase-3, so, it can proved that SMMC-7721 cell apoptosis.(3) Cell ATP levels and mitochondrial membrane potential (??m) were decreased, and the release of Cyt-c was increased after oleanolic acid derivatives act on SMMC-7721.(4) Oleanolic acid derivatives can down-regulate the gene and protein level of ?-catenin, c-myc and cyclin D1, further study found that oleanolic acid derivatives can inhibit the nuclear transfer and increase the phosphorylation(Ser33/37/Thr41) of ?-catenin in intracytoplasmic.Conclusion:(1) Oleanolic acid derivatives can obviously inhibit human hepatocellular carcinoma SMMC-7721 cells proliferation and induce cell apoptosis though destroying cells mitochondrial function.(2) Oleanolic acid derivatives promote human hepatocellular carcinoma SMMC-7721 cells apoptosis by inhibiting the nuclear transfer of ?-catenin as well as increasing the phosphorylation(Ser33/37/Thr41) level.In summary, oleanolic acid derivatives is expected to become a new type and effective traditional Chinese medicine monomer on inhibiting the cell proliferation and inducing the apoptosis in liver cancer.