Downregulation of the c-myc target gene, peroxiredoxin III, contributes to Arsenic Trioxide-induced apoptosis in Acute Promyelocytic Leukemia (APL)

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1 Downregulation of the c-myc target gene, peroxiredoxin III, contributes to Arsenic Trioxide-induced apoptosis in Acute Promyelocytic Leukemia (APL) Pablo E. Vivas-Mejía, Ph.D. Research Scientist, University of Puerto Rico Visiting Scientist, Experimental Therapeutics The University of Texas M. D. Anderson Cancer Center

2 All trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in APL cells APL is a subtype of acute myeloid leukemia (AML), accounting for 1% of cases of AML (27: 14, new cases). APL is cytogenetically characterized by the translocation of chromosomes 15 and 17. ATRA ATO PML-RARα 1. μm <.4 μm >.5 μm PML-RARα Apoptosis Differentiation Differentiation

3 ATO has been used in Cancers other Than APL Clinical trials with ATO have been conducted in leukemias others than APL, as well as in solid tumors. ATO can eliminate cancer initiating stem cells (Ito, et al. Nature, May 28). ATO use in other malignancies has been hampered by safety concerns.

4 Arsenic Trioxide Inhibits Mitochondrial Gluthatione Peroxidase (GPx) O 2 O 2 SOD catalase H H 2 O 2 O 2 As 2 O 3 GPx Prxs H 2 O As(GS) 3 OH + OH - Mitochondrial Membrane potential (ΔΨM) Cytochrome c release Apoptosis

5 Peroxiredoxins (Prxs) Prxs is a family of antioxidant thioredoxin-dependent enzymes catalyzing the reduction of hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO-), and organic hydroperoxides (ROOH). Prxs are located in different cell compartments. Prx III is located in the mitochondria. Prxs expression is increased in several human cancers. White blood cells express high levels of Prx-III.

6 Hypothesis Prx-III is a target of ATO resulting in the induction of apoptosis in APL.

7 To examine: Experimental Design Whether ATO treatment of NB4 cells affects the expression levels of Prx-III. ATO effects on reactive oxygen species (ROS), ΔΨm, and apoptosis. Effect of Prx-III depletion on ROS, ΔΨm, and apoptosis. Effect of Prx-III overexpression on apoptosis. The role of c-myc in the ATO-mediated regulation of Prx III.

8 ATO decreased the protein and mrna levels of Prx-III in NB4 cells Time (hr) [ATO] μm NT Prx-III Prx-III Time (hours) NT Prx III

9 ATO induced accumulation of ROS, dissipation of the ΔΨm and apoptosis in NB4 cells 2 8 hr Untreated 2. μm ATO 2 Untreated 2. μm ATO Cell number Events 2 24 hr Cell number Events 2 Events 8 hr Events 24 hr Log fluorescence intensity of DCFDA Log TMRM Fluorescence Untreated 9% 8 hr 7% 24 hr 32%

10 ATO induced Prx-III downregulation prior to significant accumulation of ROS, dissipation of the ΔΨm or apoptosis Percentage of cells ROS accumulation Low ΔΨm Apoptosis * ** * Incubation time with 2. μm ATO (hr)

11 Depletion of Prx-III enhanced ROS accumulation and damage of mitochondrial membrane function C-siRNA C-siRNA+ ATO SiRNA- Prx III sirna-prx III + ATO Prx III Relative decrease (%) C-siRNA C-siRNA + ATO sirna-prx III sirna-prx III +ATO C-siRNA sirna-prx III * ** cyt-c C-siRNA sirna-prx III * ** Relative change in DCFDA fluorescence Relative change in TMRM fluorescence - ATO + ATO - ATO + ATO

12 Depletion of Prx-III in NB4 cells promoted mitochondrial-dependent apoptotic events caspase-9 caspase-3 PARP C-siRNA C-siRNA + ATO sirna-prx III sirna-prx III +ATO Full Length 47 KDa Cleaved p37/p35 KDa Full Length 32 KDa Cleaved 17 KDa Full length 112 kda Cleaved 85 KDa % of Annexin-V positive cells C-siRNA sirna-prx III ** -ATO +ATO

13 Overexpression of Prx-III inhibited ATO-induced apoptosis U937-NT U937-EV U U U937-NT U937-EV U U % of Annexin-V positive cells Prx-III -ATO 4 μm ATO * **

14 c-myc inactivation correlated with downregulation of Prx III during ATO-induced apoptosis in NB4 cells Time (hr) NT C-siRNA sirna c-myc c-myc c-myc Prx-III Time (hr) NT c-myc

15 ATO treatment of NB4 cells led to reduced c-myc binding to its canonical E-box in the Prxd-3 gene CACGTG CATGCG CGCGTG A NT 4-hr 24-hr B IP/c-MYC Input Primers A Primers A IP/c-MYC Primers B Input Primers B IP/Pol-II GAPDH IP/IgG Primers A

16 Conclusion ATO-induced downregulation of c-myc leads to the downregulation of Prx III, contributing to the mitochondrial-apoptotic events triggered by the accumulation of H2O2 in APL-derived NB4 cells.

17 Acknowledgments Center to Reduce Cancer Health Disparities U54 (U54 CA 963, U54 CA 96297) Partnership for Excellence in Cancer Research Gabriel Lopez-Berestein, MD