In Vitro Study On Antitumor Activity Mechanism Of Dryofragin

Apoptosis is important in chemotherapy-induced tumor cell killing, and many anticancer drugs induce restoration of apoptosis. Intracellular reactive oxygen species (ROS) is considered to be a death signal in apoptosis. ROS induces disruption of the mitochondrial membrane potential (MMP) and release of cell death signals to trig cell death. Accumulation of excessive ROS leads to oxidative DNA damage, and induces cell apoptosis signals transduction. Therefore, ROS is a hot point for anti-cancer drug research.Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. To the best of our knowledge, this is the first report demonstrating the anticancer activity of dryofragin. Preliminary experiments in our1ab showed that MCF-7cells were the most sensitive towards dryofragin among a panel of cell lines of different tumor types. Taking into account the above, the aim of the present investigation was to explore the cytotoxic activity of dryofragin towards MCF-7cells and the underlying mechanisms in more details. And the results are as follows:1. The anti-proliferative activity of dryofragin was determined using the MTT assay. The results show that MCF-7cells were the most sensitive towards dryofragin, and the IC50value of MCF-7was27.26±0.89μM after72h treatment. Further research shows that dryofragin inhibited the growth of MCF-7cells in a time and concentration-dependent manner. Meanwhile, the IC50value on African green monkey kindney cell Vero is52.68±1.33μM, which indicates that dryofragin is less cytotoxicity to normal cells.2. Dryofragin can inhibit cell proliferation by inducing apoptosis in MCF-7cells. We observed the apparent morphology, DNA fragmentation as well as detection of cell cycle arrest and the Annexin V-FITC/PI double staining. After stained with acridine orange, MCF-7cells were visualized by a laser scanning confocal microscope. Cells treated with dryofragin for48h showed increased percentages of cell shrinkage, condensed and fragmented chromatin, and apoptotic bodies. Increasing concentration of dryofragin increased DNA cleavage appearing as typical ladder pattern in agarose gel electrophoresis. Apoptosis-induced DNA fragmentation was visible after treatment with20and30μM dryofragin. The results of flow cytometry show that a significant number of cells arrested at G2/M phase. Meanwhile, the percentage of apoptotic cells increased along with the concentration of dryofragin.3. Dryofragin induces apoptosis through ROS-mediated mitochondrial pathway(1)Dryofragin-treatment MCF-7cells had a significantly accumulation of reactive oxygen species, as well as an increased percentage of cells with mitochondrial membrane potential disruption. To investigate the effect of dryofragin on cellular ROS level in cancer cells, we analyzed it by flow cytometry. There was markedly increase in DCFH-DA fluorescence after treatment with dryofragin in a dose-and time-dependent manner. After treatment with various concentrations of dryofragin, the MMP level was decreased. These phenomena were blocked by pretreatment for2h of MCF-7cells with the antioxidant compound NAC.(2) Dryofragin induces apoptosis through ROS-mediated mitochondrial pathway. To study whether dryofragin induced apoptosis via activation of mitochondria signaling pathway, we examined the release of cytochrome C and the key protein about apoptosis. After treatment with various concentrations of dryofragin, the ROS level was increased and the MMP level was decreased, the combination of Bcl-2and Bax was inhibited, which lead to pro-apoptosis protein Bax increased and the expression of cytochrome C in mitochondrial downregulated. Caspases-9and-3play critical role in the execution of apoptosis and concomitant PARP cleavage occurs in a caspase-dependent manner. Therefore, our results suggest that dryofragin activated the mitochondrial apoptosis pathway.In conclusion, the results of this research indicated that dryofragin exhibited potential anti-cancer activity in human breast cancer MCF-7cells through induction of apoptosis and had low cytotoxicity to normal cells. Mitochondrial dysfunction was involved in dryofragin induced apoptosis, which was tightly associated with ROS production. Our results might provide the basis for future clinical application of dryofragin treatment in breast cancer.