Dependence of Reactive Oxygen Species and FLICE Inhibitory Protein on Lipofectamine-Induced Apoptosis in Human Lung Epithelial Cells

Abstract

Cationic liposomes such as Lipofectamine (LF) are widely used as nonviral gene delivery vectors; however, their clinical application is limited by their cytotoxicity. These agents have been shown to induce apoptosis as the primary mode of cell death, but their mechanism of action is not well understood. The present study investigated the mechanism of LF-induced apoptosis and examined the role of reactive oxygen species (ROS) in this process. We found that LF induced apoptosis of human epithelial H460 cells through a mechanism that involves caspase activation and ROS generation. Inhibition of caspase activity by pan-caspase inhibitor (z-VAD-fmk) or by specific caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (z-LEHD-fmk) inhibited the apoptotic effect of LF. Overexpression of FLICEinhibitory protein (FLIP) or B-cell lymphoma-2, which are known inhibitors of the extrinsic and intrinsic death pathways, respectively, similarly inhibited apoptosis by LF. Induction of apoptosis by LF was shown to require ROS generation because its inhibition by ROS scavengers or by ectopic expression of antioxidant enzyme superoxide dismutase and glutathione peroxidase strongly inhibited the apoptotic effect of LF. Electron spin resonance studies showed that LF induced multiple ROS; however, superoxide was found to be the primary ROS responsible for LF-induced apoptosis. The mechanism by which ROS mediate the apoptotic effect of LF involves down-regulation of FLIP through the ubiquitination pathway. In demonstrating the role of FLIP and ROS in LF death signaling, we document a novel mechanism of apoptosis regulation that may be exploited to decrease cytotoxicity and increase gene transfection efficiency of cationic liposomes.

Insights Into Sepsis Therapeutic Design Based on the Apoptotic Death Pathway

“…Some in vitro studies have reported on the cytotoxicity of cationic liposomes (104,105). Additionally, a recent report provides evidence that Lipofectamine induces rapid generation of reactive oxygen species that is required for cFLIP down-regulation and subsequent activation of apoptosis in human lung epithelial cells (106). However, our survival studies showed a marginal influence of Lipofectamine alone on mortality in mice after CLP (52).…”

Growth Factors Polymerized Within Fibrin Hydrogel Promote Amylase Production in Parotid Cells

“…19 This might be due to the interaction of cells with fibrin (possibly through integrin a v b 3 ), which may suppress caspase activation and reactive oxygen species generation. 18 Fibrin, a major protein component of blood clots, is able to form a gel at physiological temperatures. It has been used extensively in biomedical engineering applications ranging from cardiovascular tissue engineering 19,20 to wound-healing experiments (due to its biocompatibility and its ability to biodegrade in vivo).…”

A comparative study on nonviral genetic modifications in cord blood and bone marrow mesenchymal stem cells

“…Remarkably cells exposed to Lipofectamine showed reduced adherences which led to spherical morphology and may result in early apoptosis. A recent study has reported similar changes in morphology and consequent apoptosis of cultured human lung epithelial cells which were treated with Lipofectamine for 6 h (Kongkaneramit et al 2008). The dependency of the production of reactive oxygen species on Lipofectamine-induced apoptosis has also been described in the mentioned paper.…”