05 level of signifi cant difference. Statistical analyses were performed using SPSS 13. 0 statistics software Results Bostrycin inhibited the proliferation of A549 cells First, we used the MTT assay to detect effect of bostrycin on A549 cell proliferation. There was a dose dependent and time dependent inhibition of A549 cell proliferation by bostrycin with an optimal linear relation ship seen between 10 30 uU of bostrycin. This indicated that bostrycin could significantly inhibit A549 cell prolif eration in vitro. Bostrycin induced cell cycle arrest and apoptosis in A549 cells Then, we used flow cytometry to determine cell cycle distribution and apoptosis in A549 cells exposed to dif ferent concentrations of bostrycin for 24, 48, and 72 hours.

We showed a significant increase in the num ber of G0/G1 phase cells and a decrease in the number of S and G2/M phase cells after 72 hours of bostrycin treatment. We also used propidium iodide staining to show that bostrycin induced apoptosis of A549 cells in a dose dependent and time dependent manner. Figure 2C shows the flow cytome try data of cells treated with different concentrations of bostrycin for 24 h, 48 h and 72 h. Analysis of microRNA expression in A549 cells by microarrays and real time RT PCR We used a gene chip probe techniques to detect changes in microRNA expression in bostrycin treated A549 cells when compared with untreated cells. We found a statistically significant difference in the expres sion of fifty four microRNAs. We selected microRNA 638 and microRNA 923 for further validation with real time RT PCR since these two microRNAs showed the most significant difference.

We used RT PCR to demonstrate a significant upregulation in the levels of microRNA 638 and microRNA 923 in bostrycin treated A549 cells. These data were consistent with our microarray analysis. Detection of p110a, p Akt, and p27 levels in bostrycin treated cells Finally, we detected the possible signal pathway involved in the effects of bostrycin on A549 cells. We showed by western blots that there was a decrease in the expression of p110a protein over time in bostrycin treated A549 cells. Although there was an increase in the expression of p Akt protein in cells treated with bostrycin for 12 hours, when compared with cells at the 0 hour time point, we showed a gradual decrease in p Akt Entinostat levels over time, with the most obvious reduction at 48 hours.

We also showed a time dependent increase in the levels of p27 protein in bostrycin treated cells. Discussion In this study, we demonstrated that bostrycin, a novel compound isolated from marine fungi in the South China Sea, inhibited cell proliferation, blocked cell cycle progression, and promoted apoptosis of lung cancer A549 cells. Our data also suggested that the PI3K/AKT signaling pathway may play a role in bostrycin mediated inhibition of cell proliferation.