Abstract 2109: Activation of multiple cellular stresses by the NO generating antineoplastic agent JS-K.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K is active against acute myeloid leukemia (AML), multiple myeloma, hepatocellular carcinoma, prostate cancer, glioma and non-small lung cancer. We have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic® P123 polymers to stabilize and solubilize the drug. In the present study, the mechanism of action of JS-K was investigated. We speculated that, being a NO-releasing drug, JS-K causes nitrosative/oxidative stress. We therefore hypothesized that JS-K causes multiple stress responses that ultimately trigger apoptosis in the cell. We also hypothesized that JS-K affects the “intrinsic component” (defined as cell cycle, survival and stress pathways in the cell) of leukemic cells. In this work, using human myeloid leukemia HL-60 cells, we studied the effect of JS-K on two major cellular stresses, namely ER stress and nitrosative/oxidative stress. On treating HL-60 cells with 5μM JS-K formulated in DMSO (JS-K) or P123/JS-K for 4-8 hours, activation of both GRP-78 and CHOP (markers of ER stress) was observed using western blot analysis. ER stress could also be triggered by phosphorylation of JNK protein. Again, using western blot analysis, JNK phosphorylation was observed when HL60 cells were treated with JS-K or P123/JS-K for 4-6 hours. Oxidative stress was studied by quantifying intracellular levels of reactive oxygen/reactive nitrogen species (ROS/RNS) using the ROS/RNS sensitive fluorophore 5-(and-6)-chloromethyl-2’-7’-dichlorodihydrofluorescein diacetate. Treatment of HL-60 cells with 5μM of JS-K or P123/JS-K for 30, 60 or 120 minutes lead to increased levels of ROS/RNS. These experiments show that JS-K activates the intrinsic component of cell death by producing ER stress and oxidative stress. JS-K is at an advanced stage of pre-clinical development. Its novel and multiple mechanisms of action make it an attractive therapy for the treatment of AML and other malignancies. Citation Format: Imit Kaur, Moises Terrazas, Ken M. Kosak, Paul J. Shami. Activation of multiple cellular stresses by the NO generating antineoplastic agent JS-K. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2109. doi:10.1158/1538-7445.AM2013-2109