Requirement of caspase and p38^MAPK activation in zinc‐induced apoptosis in human leukemia HL‐60 cells

Abstract

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL‐60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL‐60 cells with Zn for 6 h in the presence of Py (1 µm) exhibited cytotoxicity in a Zn dose‐dependent manner (25–200 µm). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose‐dependent fashion (50–100 µm), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase‐mediated dUTP‐biotin nick end‐labeling method, peaked at 25 µm, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N′,N′‐tetrakis (2‐pyridylmethyl)ethylenediamine, a cell‐permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 µm Py and 25 µm Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP‐ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen‐activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z‐Asp‐CH2‐DCB, a broad‐spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK‐specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z‐Asp‐CH2‐DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.

Activation of Caspase-3 in HL-60 Cells Treated with Pyrithione and Zinc

“…17) In this study, we speculated that this acute increase in Zn levels may induce apoptosis partly via activation of caspase-3.…”

“…Indeed, we indicated that activation of p38 mitogen-activated protein kinase (p38MAPK) may be located upstream of the activation of caspases in Py/Zn-treated HL-60 cells. 17) SB203680, an inhibitor of p38MAPK, partly decreased caspase-3 activity in Py/Zn-treated HL-60 cells (Vehicle, OD400 nmϭ0.070Ϯ0.001; Py/Zn OD400 nmϭ 0.225Ϯ0.004; Py/Zn plus SB203580 OD400 nmϭ0.137Ϯ 0.003), suggesting that Py/Zn may activate p38MAPK following activation of caspase-3. Taken together, these results indicate that Py/Zn-induced apoptosis may be triggered by activation of caspase-3.…”

“…[14][15][16] Previously, we found that an influx of Zn into the cell, which was mediated by a Zn ionophore (Py, pyrithione) and Zn, induced apoptosis via activation of caspases in human leukemia HL-60 cells. 17) Although a broad-spectrum inhibitor of caspases attenuated Py/Zn-induced apoptosis, the involvement of caspase-3 in Py/Zn-induced apoptosis is obscure. In the present study, we investigated the activation of caspase-3 in Py/Zn-treated cells and estimated the involvement of caspase-3 in Py/Zn-induced apoptosis.…”

A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells

“…Previously, we found THP-1 cells display an inverse relationship between loss of cellular zinc concentration and cell viability (18). TPEN can result in apoptosis (35), the sequelae of which usually involves cellular redox perturbation, reduction in ⌬⌿m, and then caspase-3 activation for commitment to apoptosis. Our data show the effects of TPEN-induced reduction in cellular zinc, but without altered cell viability.…”

Metal ionophores – An emerging class of anticancer drugs

“…Its anticancer activity was first reported in the 1980s, though it was thought to be functioning as an iron chelator (35,36). When added to cells along with zinc it induced apoptosis (37)(38)(39). Although the sodium salt of pyrithione is water soluble, the zinc salt is not, prompting the synthesis and description of several water soluble derivatives designed to be used as anticancer drugs (23).…”