Activation of alternative apoptosis pathways in HepG2 cells after caspase arrest

Aims: Actinomycin D-sensitized HepG2 cells are highly susceptible to induction of apoptosis via stimulation of death receptors (TNF-R1, CD95 and TRAIL receptor) [1]. We and others showed that the pan-caspase inhibitor zVAD-fmk conferred complete protection of HepG2 cells against apoptosis induced by TNFα or agonistic anti-CD95 antibody [2]. Here we provide evidence that in presence of zVAD-fmk HepG2 cells can switch to caspase-independent apoptotic signaling pathways. Results: HepG2 cells were incubated with different apoptosis-inducing stimuli in presence or absence of various inhibitors. Apoptosis was characterized by morphological criteria, caspase activation and secondary necrosis, i.e. LDH release or with the Alamar Blue assay. Incubation of HepG2 cells with high concentrations of the pan-caspase inhibitor zVAD-fmk conferred complete protection against death receptor agonists. While it was assumed that this protection was due to caspase arrest, we noticed that an approximately 200 fold lower concentration of the inhibitor is sufficient to completely block caspase activity. Morphological analysis confirmed that cell death under caspase arrest was still of apoptotic nature. Similar results were obtained when apoptosis was induced by direct activation of the intrinsic death signaling pathway (UV radiation or staurosporine). In contrast, in different apoptosis models – either being induced by death receptor agonists or by UV radiation – hepatocyte death was strictly dependent on caspase activation, since inhibition of caspases and prevention of cell death strictly correlated. This dicrepancy suggest the existence of alternative intracellular death-inducing proteases. We tested this hypothesis by incubating HepG2 cells on top of very low but completely caspase-inhibiting concentration of zVAD-fmk with various other serine protease inhibitors. In fact, the established serine protease inhibitors TLCK and TPCK conferred protection against TRAIL under such conditions, i.e. only in presence of very low zVAD-fmk. These data suggest a switch to a serine protease-dependent pathway in HepG2 cells when caspases are inhibited. Conclusion: From our data we conclude that caspases are activated in, but dispensable for apoptosis of HepG2 cells. The existence of a fundamentally different apoptotic mechanism in this transformed tumor cell line as compared to primary hepatocytes has general bearings for potential anti-neoplastic approaches in the liver.

Literatur: 1. Leist M et al. Cytokine-mediated hepatic apoptosis. Rev. Physiol. Biochem. Pharmacol. 1998; 133:109-155. 2. Künstle et al. ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-alpha. Immunol. Lett. 1997; 55:5-10.

Schlüsselwörter

HepG2 - TNF - apoptosis - death receptors