Role of p53 and reactive oxygen species in apoptotic response to copper and zinc in epithelial breast cancer cells

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Abstract
Previous studies revealed that cells may differ in their response to metal stress depending on their p53 status; however, the sequence of events leading to copper-induced apoptosis is still unclear. Exposure of copper (10 and 25 μM) and zinc (10 and 25 μ M) caused activation of p53 in ER+/p53+ human epithelial breast cancer MCF7 cells and resulted in up-regulation of p21. Transactivation of p53 in MCF7 cells also led to increase in expression of Bax, proapototic Bcl-2 family member, triggering mitochondrial pore opening, and PIG3 (p53-induced gene 3 product), and also generation of intracellular reactive oxygen species (ROS). The treatment of MCF7 cells with either copper or zinc for 4 h also caused decrease in mitochondrial membrane potential (Δ ψ m ), accompanied by an elevation in the ROS production and redistribution of p53 into mitochondria. The loss of Δ ψ m was correlated with accumulation of Annexin V positive apoptotic cells. However, the release of apoptosis inducing factor (AIF) and its translocation into nucleus was observed only in MCF7 cells treated with copper. In MDA-MB-231 (ER−/p53−) and MCF7-E6 (ER+/p53−) cells, both p53 and p21 protein levels were not altered in the presence of metals. These cells were resistant to metals, and there was no alteration in Δ ψ m . Copper treatment did not result in accumulation of ROS in these cell lines with an inactive p53 even after exposure to 50 μM of copper for 6 h, indicating a key role for p53 in the ROS generation. Pretreatment of MCF7 cells with p53 inhibitor, pifithrin-α, resulted in decrease of copper and zinc induced ROS production to the control level, suppression of both Bax expression and AIF release. Therefore, the activation of p53 seems to play a crucial role in copper and zinc induced generation of ROS in epithelial breast cancer cells, and expression of downstream targets of p53, such as PIG3 and Bax, responsible for increased generation of the intracellular ROS, as well as disruption of mitochondrial integrity. Our data suggest that copper induces apoptosis in MCF-7 cells with no caspases through the depolarization of mitochondrial membrane with release of AIF and its translocation into the nucleus. The results demonstrate that a functional p53 is required for the execution of apoptosis in epithelial cells.