Probing the cell death signaling pathway of HepG2 cell line induced by copper-1,10-phenanthroline complex

Copper-1,10-phenanthroline (phen) complex Cu(phen)₂] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)₂. HepG2 cells were more sensitive to Cu(phen)₂ with an IC50 of 4.03 μM than other three kinds of cell lines. After treated by Cu(phen)₂, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder’s occurence and Annexin V-positive increased cells indicated that Cu(phen)₂ induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)₂ treatment resulted in significant G₂/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)₂-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-x_L and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca²⁺ was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)₂ induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.