Zinc Depletion by TPEN Induces Apoptosis in Human Acute Promyelocytic NB4 Cells

Abstract

Background/Aims: The effects of zinc signaling on proliferation or apoptosis of leukemia cells remain elusive. In the present study, we used N, N, N’, N’-tetrakis-(2-pyridylmethyl)-ethylene-diamine (TPEN), a membrane-permeable zinc chelator, to evaluate the effect of zinc depletion on survival and apoptosis of NB4 acute promyelocytic leukemia (APL) cells. Methods: The pro-apoptotic effects of TPEN on NB4 cells were examined by flow cytometry, and observed using an optical microscope. Intracellular labile zinc, nitric oxide (NO) or reactive oxygen species (ROS) changes caused by TPEN were measured by flow cytometry. We then explored possible roles of the crosstalk between intracellular labile zinc signaling and nitric oxide signaling in TPEN-triggered apoptosis. Results: we found that TPEN induced apoptosis in NB4 APL cells in a dosage-dependent manner. We further demonstrated that TPEN triggered apoptosis by attenuating intracellular zinc and nitric oxide signaling in NB4 cells. Both exogenous zinc supplement and the nitric donor sodium nitroprusside (SNP) pre-incubation reversed TPEN-mediated inhibition of intracellular NO and Zn²⁺ signaling, and rescued NB4 cells from apoptosis. Conclusion: These results suggest for the first time that crosstalk between zinc signaling and nitric oxide pathway is essential for the survival of NB4 cells. TPEN induces apoptosis in NB4 cells via negatively regulating intracellular NO and Zn²⁺ signaling. Our in vitro data suggest that zinc depletion by TPEN may be a potential therapeutic strategy for APL.

Single Cell Analysis Reveals Multiple Requirements for Zinc in the Mammalian Cell Cycle

“…When noted, an even milder ZD 25 condition of 2 µM TPA was used and this condition resulted in only ~ 1% cell death 26 (Supplementary Figure S2). Our results are consistent with several studies that found TPEN 27 induces apoptosis (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). 28 To determine how defined Zn 2+ media conditions influenced intracellular labile Zn 2+ 29 levels in the cytosol, we created an MCF10A cell line stably expressing a genetically encoded 30 FRET-based sensor for Zn 2+ (ZapCV2 (41)), grew the cells in ZD, MM, and ZR media, and measured the resting FRET ratio in individual cells.…”

“…Our results are consistent with several studies that found TPEN 27 induces apoptosis (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). 28 To determine how defined Zn 2+ media conditions influenced intracellular labile Zn 2+ 29 levels in the cytosol, we created an MCF10A cell line stably expressing a genetically encoded 30 FRET-based sensor for Zn 2+ (ZapCV2 (41)), grew the cells in ZD, MM, and ZR media, and measured the resting FRET ratio in individual cells. Cells grown in ZD had a significantly lower 1 average FRET ratio than cells grown in MM, while those grown in ZR conditions had 2 significantly higher FRET ratios ( Figure 1A).…”

“…However, in ZD media the number of mitosis 26 events ceased after about 20 hrs, the CDK2 activity either stayed low or rose to an intermediate 27 level, and cells rarely underwent multiple rounds of the cell cycle ( Figure 2B). Computational 28 alignment to mitosis and cell classification revealed that few cells were born with CDK2 activity 29 sufficient to commit to the next cell cycle (9% CDK2 inc ), a significant decrease compared to MM 30 and ZR, and there was a substantial increase in the percentage of cells with low CDK2 activity following mitosis CDK2 low (41%). One of the most striking differences between ZD and MM or 1 ZR conditions was the fate of cells that attempted to re-enter the cell cycle (i.e.…”

“…Zn 2+ was first reported to be 24 essential for growth of Aspergillus niger in 1869 and subsequently demonstrated for plants, 25 animals, and humans (2) with the first cases of human Zn 2+ deficiency and the associated growth 26 and developmental disorders described in 1961 (3). Zn 2+ deficiency has since been recognized 27 as a global health problem, and the World Health Organization (WHO) estimates a staggering 28 one third of the world's population does not consume adequate Zn 2+ and is therefore at risk for 29 associated side effects and comorbidities 30 (https://www.who.int/whr/2002/chapter4/en/index3.html) (4). While the clinical manifestations resupply stimulates synchronized cell cycle reentry.…”

Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-κB and Induction of Antiviral Response in Epithelial Cells

“…We sought to mimic zinc deficiency in vitro using TPEN and determine the effect of Zn depletion on DENV infection. Many previous studies have shown around 70-80% depletion in labile Zn levels using high concentrations of TPEN for short duration (37,38). We first measured the effect of increasing concentrations of TPEN on cell proliferation.…”