Effect Of Baicalin-Copper On The Induction Of Apoptosis In Human Hepatoblastoma Cancer HepG-2 Cell Based On Western Blotting Analysis

The present study was designed to investigate the effect and possible mechanism of apoptosis induced by baicalin-copper in human hepatoblastoma cancer cell (HepG-2). And the effects of baicalin-copper on HepG-2 cell xenografts in nude mice were also investigated.The nude mice with HepG-2 cell xenografts were divided into four groups:control group,50 mg/kg baicalin,50mg/kg baicalin-copper and 100mg/kg baicalin-copper. The nude mice with HepG-2 cell xenografts were treated with drugs via muscle injection daily. After treatment with baiclin-copper for 32 d, the changes of HepG-2 cell xenografts’weight and histology were used to evaluate the effects of baicalin-copper on xenografts growth in nude mice.Cell viability was assessed by MTT assay. Typical apoptotic nuclei were shown by acridine orange staining and fluorescence microscope with different concentrations of baicalin-copper for 48 h. Cell apoptosis rate was detected by flow cytometry with Annexin-V/PI staining. Cell cycle is also analyzed following PI staining by flow cytometry. The expression of PI3K, p-PI3K、Akt、p-Akt、 mTOR、p-mTOR, casepase-3, Bax, Bcl-2 and p38 proteins were analyzed by Western blot analysis.Intraperitoneal (i.p.) injection of baicalin-copper resulted in a significant decrease in tumor growth in xenografts in nude mice. HepG-2 cells treated by different concentration of baicalin-copper after 48h, baicalin-copper can inhibit proliferation of HepG-2 in vitro and the inhibition in significant does-dependent manner. After treation of baicalin-copper, karyorrhexis, caryotin and karyopyknosis were observed by AO fluorescein stain and fluorescence microscope after treatment with baicalin-copper. Flow cytometric analysis showed that compared to control apoptosis rate increased remarkably after treatment with baicalin-copper. The result of FCM demonstrated that baicalin-copper cause G2/M phase cell cycle arrest, and the proportion of cells in G2/M was increased, and the percentage of cells at G0/G1 phase decreased the proportion of cells in G2/M was increased, and the percentage of S phase cells no significanted varied. Baicalin-copper treatment significantly increased the Bax/Bcl-2 ratio and p38 levels, as well as decreased the expression of caspase-3, p-PI3K, p-Akt and p-mTOR (p<0.01).In summary, baicalin-copper could inhibit growth and induce cell apoptosis of HepG-2 cell xenografts in nude mice. In addition, baicalin-copper can inhibit prolifration of HepG-2, induce apoptosis and arrest cell cycle. In addition, the mechanism of baicalin-copper inducing apoptosis probably is related to down-regulate the PI3K/Akt/mTOR signaling pathway and down regulation Bcl-2 expression and the up-regulation Bax expression.