Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes

Abstract

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis

“…Expression array analysis indicated massive upregulation of the MT2A gene encoding metallothionein-2A (Fig. 4d; Table 1), shown earlier to be induced in the context of a cellular zinc stress response observed in Hep-2 cervical tumor cells exposed to ZnPT (Rudolf and Cervinka 2010). Further expression array analysis indicated upregulation of a wide range of stress response genes encoding heat shock proteins, antioxidants, and inflammatory factors (Fig.…”

“…3a–b) and ICP-MS (Fig. 3c), building on earlier ICP-MS data that determined total zinc ion accumulation in cultured cancer cell lines exposed to ZnPT (Kondo et al 2002; Rudolf and Cervinka 2010). ZnPT-induced impairment of viability (Fig.…”

“…ZnSO 4 , ZnCl 2 , and Zn-acetate) is well tolerated at concentrations up to 200 μM without reduction of viability as also observed in this study (Figs. 1, 2) (Hatayama et al 1993; Jourdan et al 2002; Cortese et al 2008), but potentiation of zinc effects and induction of cytotoxicity by zinc-specific ionophores including pyrithione has been documented earlier in non-cutaneous mammalian cells (Kim et al 1999; Magda et al 2008; Mann and Fraker 2005; Klein et al 2006; Rudolf and Cervinka 2010). Recently, we have demonstrated that primary keratinocytes and melanocytes exposed to nanomolar concentrations of ZnPT undergo rapid loss of genomic integrity with PARP activation and heat shock response gene expression followed by non-apoptotic cell death (Lamore et al 2010a).…”

“…Published ICP-MS protocols detecting total intracellular zinc were adapted as follows (Kondo et al 2002; Rudolf and Cervinka 2010): After treatment, NHEKs (1 × 10 6 ) or EpiDerm ™ tissue were rinsed with PBS and dried, followed by dissolution in concentrated nitric acid (0.5 ml, 85°C, 3 h). Solutions were analyzed using an ELAN DRC-II ICP-MS (Perkin Elmer).…”

Zinc-mediated Allosteric Inhibition of Caspase-6

“…Extremely high zinc concentrations (Ͼ500 M) trigger apoptosis, but at physiologically relevant concentrations, zinc is a suppressor of apoptosis (24,25). Depletion of zinc also leads to apoptosis.…”