1. ¹Department of Medicine, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA 23298, USA
2. ²Department of Radiation Oncology, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA 23298, USA
3. ³Department of Biochemistry, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA 23298, USA
4. ⁴Department of Pharmacology, Virginia Commonwealth University/Medical College of Virginia, Richmond, VA 23298, USA

Correspondence: S Grant, Division of Hematology/Oncology, Virginia Commonwealth University/Medical College of Virginia, MCV Station Box 230, Richmond, VA 23298, USA. E-mail: [email protected]

Received 12 October 2004; Revised 06 January 2005; Accepted 06 January 2005; Published online 25 April 2005.

Abstract

The effects of 2-Methoxyestradiol (2ME)-induced apoptosis was examined in human leukemia cells (U937 and Jurkat) in relation to mitochondrial injury, oxidative damage, and perturbations in signaling pathways. 2ME induced apoptosis in these cells in a dose-dependent manner associated with release of mitochondrial proteins (cytochrome c, AIF), generation of reactive oxygen species (ROS), downregulation of Mcl-1 and XIAP, and inactivation (dephosphorylation) of Akt accompanied by activation of JNK. In these cells, enforced activation of Akt by a constitutively active myristolated Akt construct prevented 2ME-mediated mitochondrial injury, XIAP and Mcl-1 downregulation, JNK activation, and apoptosis, but not ROS generation. Conversely, 2ME lethality was potentiated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Furthermore, in U937 cells, the hydrogen peroxide scavenger catalase and a superoxide dismutase (SOD) mimetic, TBAP, blocked these events, as well as Akt inactivation. Interruption of the JNK pathway by pharmacologic or genetic (e.g. siRNA) means attenuated 2ME-induced mitochondrial injury, XIAP and Mcl-1 downregulation, and apoptosis. Collectively, these findings suggest a hierarchical model of 2ME-related apoptosis induction in human leukemia cells in which 2ME-induced oxidative injury represents a primary event resulting in Akt inactivation, leading, in turn, to JNK activation, and culminating in XIAP and Mcl-1 downregulation, mitochondrial injury, and apoptosis. They also suggest that in human leukemia cells, the Akt pathway plays a critical role in mediating the response to oxidative stress induced by 2ME.