Involvement of caspase‐3 and p38 mitogen‐activated protein kinase in cobalt chloride‐induced apoptosis in PC12 cells

Abstract

Our previous study showed that cobalt chloride (CoCl2) could induce PC12 cell apoptosis and that the CoCl2-treated PC12 cells may serve as a simple in vitro model for the study of the mechanism of hypoxia-linked neuronal disorders. The aim of this study is to elucidate the mechanism of CoCl2-induced apoptosis in PC12 cells. Caspases are known to be involved in the apoptosis induced by various stimuli in many cell types. To investigate the involvement of caspases in CoCl2-induced apoptosis in PC12 cells, we generated PC12 cells that stably express the viral caspases inhibitor gene p35 and analyzed the effect of p35 on the process of apoptosis induced by CoCl2. We also examined the effect of cell-permeable peptide inhibitors of caspases. The results showed that the baculovirus p35 gene and the general caspases inhibitor Z-VAD-FMK significantly block apoptosis induced by CoCl2, confirming that caspase is involved in CoCl2-induced apoptosis. Further investigation showed that in this process the caspase-3-like activity is increased, as indicated by the cells' ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-AMC and to degrade the DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. At the same time, caspase-3-specific inhibitors, namely, the peptide Ac-DEVD-CHO, Ac-DEVD-FMK, partially inhibit CoCl2-induced apoptosis. These findings suggested that caspase-3 or caspase-3-like proteases are involved in the apoptosis induced by CoCl2 in PC12 cells. Additionally, we have observed that another apoptotic marker, p38 mitogen-activated protein kinase (MAPK), is significantly activated in this process in a time-dependent manner and that a selective p38 MAPK inhibitor, SB203580, partially inhibits this cell death. The addition of SB203580 also partially suppresses caspase-3-like activity. All these results confirm that the CoCl2-treated PC12 cell is a useful in vitro model with which to study hypoxia-linked neuronal disorders. Furthermore, the results showing that the baculovirus p35 gene and caspase inhibitors possess a remarkable ability to rescue PC12 cells from CoCl2-induced cell death may have implications for future neuroprotective therapeutic approaches for the hypoxia-associated disorders.

Antiapoptotic action of hypoxia-inducible factor-1α in human endothelial cells

“…26 Caution should, however, be exercised when interpreting these studies since they were based on indirect evidence obtained from mutant cell lines, 24 from studies of downstream target proteins such as VEGF, 26 or from the use of chemical inducers of HIF-1a such as CoCl 2 25 that, by themselves, can induce apoptosis. 27,28 In the current study, we used the technique of RNAi to evaluate the role of HIF-1a in modulating anoxia-induced apoptotic injury in primary cultured HUVECs. For this purpose, HUVECs were transfected with siRNA duplexes targeting HIF-1a mRNA prior to being subjected to conditions of A/R.…”

E2F6 Inhibits Cobalt Chloride-Mimetic Hypoxia-induced Apoptosis through E2F1

“…Cobalt chloride (CoCl 2 ) can mimic hypoxic responses in many respects, including the induction of apoptosis in different types of cells (Zou et al, 2002;Jung and Kim 2004;Guo et al, 2006). Therefore, in the present study, we used CoCl 2 -treated human embryonic kidney (HEK) 293 cells as a model to investigate the biological role of E2F6 in regulation of hypoxia-induced apoptosis and molecular pathways involved.…”

In vitro profiling of epigenetic modifications underlying heavy metal toxicity of tungsten-alloy and its components

“…The confounding factors may include among others the existence of several ionic/metallic forms of the same element, different metal mixtures present in the environment, cell-type/species specificity in mechanisms of action and targeting of interconnected signaling pathways (Florea and Busselberg, 2006;Riley et al, 2005). Heavy metals can induce cell death by apoptosis and necrosis through DNA fragmentation, elevated production of reactive oxygen species (ROSs), triggering hypoxia like-effects, elevating levels of p53, activating caspases, and mitogen activated protein kinases (MAPKs) or other signaling pathways (for reviews see: Akita et al, 2007;Valko, et al, 2005;Zou et al, 2002). Heavy metals are also known to affect calcium homeostasis by blocking voltage-dependent calcium channels and inducing calcium release from intracellular calcium stores, thereby, interfering with multiple downstream signaling cascades (McNulty and Taylor, 1999;Miledi et al, 1989;Platt and Busselberg, 1994;Smith et al, 1989).…”