Mediating of caspase‐independent apoptosis by cadmium through the mitochondria‐ROS pathway in MRC‐5 fibroblasts

Abstract

Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.

Effects of cadmium chloride on some mitochondria-related activity and gene expression of human MDA-MB231 breast tumor cells

“…Noteworthy, in marine invertebrates Cd-triggered apoptosis can take place in the absence of significant alterations of mitochondrial membrane permeability [52]. On the other hand, the prolonged production of ROS can cause the disruption of the mitochondrial electron transport and strongly interfere with mitochondrial permeability transition pores, as reported in other model systems [48,50]. Interestingly, Rip-1, member of a kinase family acting as sensor of cellular stress and crucial regulator of cell viability [53], whose gene expression is prominently switched-on after CdCl 2 exposure of MDA-MB231 cells [25], is known to be involved in ROS formation by activation of cytosolic phospholipase A2 and/or enhanced electron flow through the mitochondrial electron transport chain [54].…”

“…This result suggests that CdCl 2 induced an increased rate of respiration when accumulated in MDA-MB231 cells. In different tissues, in fact, Cd was proven to affect the activity of complexes II and III of the electron transfer chain, also depleting glutathione and other free radical scavengers, thereby generating respiratory dysfunctions and production of ROS, and, in some cases, inducing some of the known variants of apoptosis death [48][49][50]. An equivalent oxidative damage was also reported recently for Cd-intoxicated plant cells indicating that such Cd-dependent effect is shared by different model systems [51].…”

The heavy metal cadmium induces valosin-containing protein (VCP)-mediated aggresome formation

“…Cadmium (Cd 2+ ) poisoning is a serious health threat due to an increased level of industrial pollution and lack of an effective therapy (Shih et al, 2004). Cd 2+ has an extremely long halflife in the human body (Elinder et al, 1976), and causes disorders of the renal, skeletal, vascular, and respiratory systems (Nordberg, 2004).…”

Cadmium affects osmotic phase and regulatory volume decrease in cultured human embryonic kidney cells

“…Investigations on Cd 2+ -induced ROS also reveal an increase in both superoxide anion (O2 -) and H2O2 levels in HeLa and bovine endothelial cells, under 1-20 µM Cd 2+ concentrations. 43 Shih and co-authors 45 confirmed these results and assessed that in normal human lung fibroblasts (MRC5) H2O2 is 2.9-fold elevated after 3 h of Cd 2+ treatment.…”