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-xL 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. Ca(2+) 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.