In Vitro Study On Antitumor Mechanism Of Aspidin PB Towards Human Hepatocarcinoma HepG2Cells

Many cancer-related signaling pathways have been elucidated and there are considerable efforts to develop new treatment strategies, which target specific signal transduction molecules. Among the downstream survival signals, PI3K/Akt pathway plays a crucial role. Thus, a better understanding of this pathway may facilitate to develop novel therapeutic agents.Aspidin PB, a phloroglucinol derivative isolated from Dryopteris fragrans (L.) Schott, has been previously reported to exert high biological activities. However, the mechanism of aspidin PB-induced apoptosis has not been reported in depth. Preliminary experiments in our laboratory showed that HepG2cells were the most sensitive towards aspidin PB among a panel of cell lines of different tumor types. Therefore, we investigated the anti-proliferative effect and molecular mechanisms of aspidin PB on HepG2cells in more detail. The results were as follows:1. The anti-proliferative activity of aspidin PB was determined using the MTT assay. The results showed that HepG2cells were the most sensitive towards aspidin PB, and the IC50value of HepG2was10.59±0.67μM after72h treatment. Further research showed that aspidin PB inhibited the growth of HepG2cells in a time and dose-dependent manner. However, the IC50values on two kinds of normal cells, mouse macrophage cell line RAW264.7and mouse MC3T3-E1cells were42.50μM and38.19μM after72h treatment, respectively, much bigger than HepG2cells, which indicated that aspidin PB was less cytotoxicity to normal cells. Moreover, after treatment of HepG2cells with aspidin PB (10μM and20μM) for48h, chromatin gradually became condensed and marginalized. The nucleus broke into fragments and formed apoptotic bodies.2. Aspidin PB could induce apoptosis of HepG2cells. Firstly, the integrity of the inner mitochondrial membrane can be assessed by monitoring the potential gradient across the membrane using the fluorescent dye, Rh123. Representative How cytometric results showed that aspidin PB led to a dose-dependent depolarization of mitochondria. Secondly, characteristic DNA ladder was observed apparently in HepG2cells incubated with aspidin PB at5,10or20μM for48h. Finally, as detected by annexin V(+), PI(-)%cells using flow cytometry, aspidin BB led to a concentration-dependent externalization of phosphatidylserine (PS), a hallmark of the apoptosis.3. Aspidin PB induces apoptosis in human hepatocarcinoma HepG2cells by modulating PI3K/Akt/GSK-3βpathway.(1) Aspidin PB inhibited PI3K expression, phosphorylation of Ser473Akt and Ser9GSK-3β followed by up-regulation of nonsteroidal anti-inflammatory drugs activated gene-1 (NAG-1) expression. And the effects of aspidin PB on PI3K, Akt, GSK-3β,NAG-1expression were similar to that of PI3K inhibitor, wortmannin. This part suggested that the PI3K/Akt/GSK-3β signal pathway might represent one of the major mechanisms of the effects of aspidin PB on human hepatocarcinoma cells.(2) Aspidin PB evoked caspase-3activation and PARP cleavage. Notably, pretreatment with the broad spectrum caspase inhibitor Z-VAD-FMK inhibited the activation of caspase-3and cleavage of PARP, suggesting that aspidin PB-induced apoptosis indeed occurred via a caspase-dependent pathway.In conclusion, the present study demonstrated that aspidin PB exhibited potential anti-cancer activity in hepatocarcinoma HepG2cells through induction of apoptosis and it had low cytotoxicity to normal cells. In a word, aspidin PB induced apoptosis in hepatocellular carcinoma cells via a caspase-dependent pathway. The Akt/GSK-3β/NAG-1signal pathway may represent one of the major mechanisms of aspidin PB-mediated apoptosis in HepG2cells. The results above might provide the theoretical basis for future clinical application of aspidin PB treatment in hepatocarcinoma.