A novel copper(I) induces ER stress-mediated apoptosis in leukemia cell lines

Recently, a novel phosphine copper(I) complex [Cu(thp)4][PF6] (CP), was identified as an efficient, in vitro antitumoral agents. It has also been demonstrated that it induces in colon cancer cells a programmed nonapototic cell death called paraptosis or type III cell death. In this study we evaluated CP antiproliferative activity on a panel of leukemia cell lines and it significantly inhibited cancer cells growth at micro and submicromolar concentration, especially against SEM and RS4;11 cell lines. Flow cytometric analysis demonstrated that CP did not affected cell cycle in RS4;11 and SEM cell line but we observed a concentration-dependent increase of the cell population with a hypodiploid DNA content peak (subG1), suggesting that CP may induce apoptosis. To better characterize the mode of cell death induced by CP, a biparametric cytofluorimetric analysis was performed using propidium iodide (PI), which stains DNA and is permeable only to dead cells, and fluorescent immunolabeling of the protein annexin-V, which binds to PS in a highly selective manner. We found a concentration-dependent increase in annexin-V positive cells in well agreement with the appearance of hypodipolid peak. Western blot analysis demonstrated that the activation of the apical caspase-9 and the two effector caspase-3 and 7 occur after treatment with CP, while caspase-8 was not affected by the treatment. Interestingly we did not observed mitochondrial depolarization or cytochrome c release into the cytoplasm, suggesting that mitochondria was not involved in the process of cell death. Previous observations indicated that CP may induces functional suppression of the ubiquitin–proteasome pathway thus triggering endoplasmic reticulum stress in solid tumor cells. We also evaluated if CP exerts ER stress in leukemia cell lines. Western blot analysis showed a remarkable increase of GRP78 in RS4;11 and SEM, a well known marker of ER stress. Moreover the protein synthesis inhibitor cicloheximide significantly protected the cells from CP-induced cell death suggesting that protein synthesis machinery is involved in the mechanism of action. To evaluate if CP directly inhibits the proteasome, semipurified proteasome from cell extracts were incubated with increasing concentrations of CP and the chymotrypsin-like activity was measured. The results showed a decreased activity in a concentration-dependent way, with an IC50 of approximately 12 μM. It is well known that copper represent an excellent catalyst of redox cycle and therefore it can stimulate ROS production. Interestingly the CP-induced cell death was significantly reduced in presence of ROS scavenger such as tocopherol, nacetyl cysteine (NAC) and butylated hydroxyanisole (BHA) suggesting that also ROS may contribute to the process of cell death. Further experiments are in progress to elucidate the mechanism of action of this copper complex and the results will be discussed.