TPEN Induces Apoptosis Independently of Zinc Chelator Activity in a Model of Acute Lymphoblastic Leukemia and Ex Vivo Acute Leukemia Cells through Oxidative Stress and Mitochondria Caspase-3- and AIF-Dependent Pathways

Table 2

Signaling inhibitors, ZnSO₄ and NAC rescue Jurkat cells from TPEN-induced apoptosis. Jurkat cells (1 × 10⁶ cells/mL) were preexposed to TPEN (3 μM) for 6 hours. After this time, either PDTC (10 nM), pifithrin-α (PFT, 50 nM), SP600125 (1 μM), NSCI (10 μM), ZnSO₄ (10 μM) or NAC (1 mM) were supplemented for upto 24 h at the same experimental conditions, as mentioned above. Cells were then evaluated for apoptotic nuclei morphology, plasma membrane integrity, and mitochondrial membrane potential, as described in Material and Methods section. The percentage of positive AO/EB/Hoechst staining, AV⁺/7-AAD^−/+ and PI^−/+/DiOC₆(3)⁻ of Jurkat cells treated with or without TPEN are expressed as a mean percentage ± S.D. from three independent experiments. ^∗∗𝑃<0.05.

Treatment/assay	AO/EB/Hoechst (%)	AV^+/7-AAD^−/+ (%)	PI^−/+/DiOC6(3)^− (%)
Untreated	<1 ± 0	4±1	3±1
TPEN (3 μM)	100	82±2	80±1
PDTC (10 nM)	<1 ± 0	2±1	3±2
PDTC (10 nM) + TPEN (3 μM)	21±1^∗∗	31±1^∗∗	17±1^∗∗
SP600125 (1 μM)	<1 ± 1	3±1	2±1
SP600125 (1 μM) + TPEN (3 μM)	11±1^∗∗	9±1^∗∗	16±1^∗∗
PFT (50 nM)	<1 ± 0	2±1	3±1
PFT (50 nM) + TPEN (3 μM)	30±1^∗∗	39±1^∗∗	38±2^∗∗
NSCI (10 μM)	<1 ± 0	3±1	2±2
NSCI (10 μM) + TPEN (3 μM)	32±1^∗∗	45±2^∗∗	30±1^∗∗
ZnSO4 (10 μM)	<1 ± 0	1±1	1±1
ZnSO4 (10 μM) + TPEN (3 μM)	12±2^∗∗	29±2^∗∗	15±1^∗∗
Zn (10 μM)/TPEN (3 μM) complex	10±2^∗∗	21±2^∗∗	9±1^∗∗
NAC (1 mM)	<1 ± 0	3±2	2±2
NAC (1 mM) + TPEN (3 μM)	5±1^∗∗	7±3^∗∗	20±2^∗∗