Isoaaptamine Induces t-47D Cells Apoptosis and Autophagy via Oxidative Stress

Bioactivity-guided fractionation of the marine sponge Aaptos sp. extract led to the isolation of aaptamine (1), demethyloxyaaptamine (2) and isoaaptamine (3). The cytotoxic activity of the isolated compounds (1-3) was evaluated revealing that compound 3 was the most potent cytotoxic against breast cancer T47D cells. In a dose dependent manner, 3 inhibited the growth of T47D cells as indicated by the short- (MTT) and long-term (colony formation) assays. The cytotoxic effect of 3 was mediated through apoptosis which was suggested by DNA ladder formation, caspase-7 activation, XIAP inhibition and PARP cleavage. Furthermore, TEM and flow cytometric analysis using acridine orange dye indicated that that 3 treatment could induce t-47D cells autophagy. Immunoblot assays demonstrated that 3 treatment significantly activated autophagy marker proteins such as the increase in Type II LC-3. In addition, 3 treatment enhanced the activation of DNA damage (γH2AX) and ER stress-related proteins (IRE1αand BiP). Moreover, the use of 3 resulted in a significant increase in the generation of reactive oxygen species (ROS) as well as in the disruption of mitochondrial membrane potential (MMP). The pretreatment of t-47D cells with an ROS scavenger, NAC, attenuated apoptosis- and MMP disruption-induced by 3 up to 90%.Taken together, these findings suggest that the anticancer effect of 3 is associated with the induction of apoptosis and autophagy through oxidative stress. Accordingly, our data indicated that 3 exhibited as an outstanding lead for the development of marine derived anti-breast cancer agent.

Fig. 1