Inhibition Of Late-stage Autophagic Flux Contributes To The Cytotoxic Effect Of Cationic Liposomes

Cationic liposomes(CLs) are nonviral vectors widely used in gene therapy, whereas its application is hampered by their toxicity. The mechanism underlying the toxicity of CLs remains largely unknown. In this study we investigated the mechanism of CLs-induced toxicity. We showed here that CLs induced dose-dependent cytotoxic effect on liver cells, accompanied with mitochondrial dysfunction and increased reactive oxygen species(ROS) production, whereas the pretreatment of liver cells with the antioxidant N-acetyl cysteine reduced the CLs-induced cytotoxic effect. Our findings further revealed that CLs initially induced autophagic flux in a mTOR-independent manner, but eventually inhibited late-stage autophagic flux. The autophagic flux inhibition by CLs was evidenced by increased levels of LC3-II and P62, GFP-LC3 staining, Cyto-ID staining and a RFP-GFP-LC3 vector. Furthermore, the autophagosome-lysosome fusion was inhibited by CLs, as evidenced by acridine orange and LysoTracker-Red staining. Accordingly, the effect of CLs on lysosomal volume and function was investigated, and the results showed that CLs caused lysosomal damage, as evidenced by enlarged lysosomes, impairment of lysosome degradation capacity and lysosome alkalinization. Importantly, pharmacological and genetic inhibition of autophagic flux almost completely reiterated the effect of CLs, including cytotoxic effect, mitochondrial dysfunction and ROS production. Taken together, our results reveal a novel mechanism whereby CLs induced cytotoxic effect through autophagic flux inhibition, with the subsequent mitochondrial dysfunction and ROS production as key events that mediated the cytotoxic effect of CLs. Our study has important implications for evaluating the biocompatibility of nanoparticles and designing better and safer gene delivery systems.Gene therapy is the insertion, alteration, or removal of genes with the cells to treat diseases, and it holds great promise for the treatment of various diseases [1,2]. Efficient and safe gene vectors is crucial for gene therapy. The two main types of gene vectors are viral or non-viral vectors [3]. Compared to viral vectors, nonviral vectors have the advantages of relative safety and simplicity [3]. A variety of nonviral vectors, including cationic liposomes(CLs), peptide and polymers, have been developed over the years [1,4]. Among them, CLs represent one of the most widely used nonviral vectors. Although CLs are far less toxic than the viral vectors, they are not without overt toxicity [5]. The application of CLs in both in vitro transfection and in vivo gene therapy is hampered by their cytotoxic effect [6,7]. So far, only one recent study has suggested that CLs induced cell necrosis through the impairment of Na+/K+-ATPase in lung cells [61]. Thus, the potential mechanism underlying the cytotoxic effect of CLs is still needed to be elucidated.Autophagy is a self-degradation process whereby cytosolic components and organelles are sequestered in double membrane-bound vesicles and delivered to lysosomes for degradation and recycling [8]. Autophagy has recently regarded as an essential regulator of the cell death pathway [8-10]. Autophagy functions as a protective or survival mechanism in cancer cells against cellular stress(e.g., nutrient deprivation), and hence promotes tumorigenesis and drug resistance [10]. As autophagy functions as an important cell survival mechanism, it can be expected that autophagy inhibition may induce toxicity towards cells [10]. Recently, a variety of nanoparticles, including CLs, have been shown to play variable roles in regulating autophagy [11-13]. Generally, most nanoparticles are regarded as a novel class of autophagy activators [11,14], and elevated autophagy after nanoparticle treatment induces cell death in most studies, accompanied with significant autophagosome accumulation [15-18].However, the rule that nanoparticles as sole autophagy activators is being challenged recently [18,19]. First, autophagosome accumulation may indicate either autophagy activation or inhibition, and thus mere evaluation of autophagosome number and LC3 processing is insufficient to estimate autophagy [20]. The term “autophagic flux”, used to represent the dynamic process of autophagy(autophagosome formation, maturation, fusion with lysosomes, subsequent breakdown), is a more reliable indicator to estimate autophagy activation or inhibition [20]. The possibility of autophagic flux inhibition was most often not investigated in nanoparticles, thus the activated autophagic flux in many cases is uncertain [18]. In fact, in cases where autophagic flux has been examined, autophagic flux inhibition is more often seen upon exposure to nanoparticles, such as gold nanoparticles, fullerene and fullerene derivative fullerenol [19,21,22]. Second, the toxicity of various nanoparticles, including CLs, have been well demonstrated in various studies [6,7]. In contrast, most studies support autophagy as a pro-survival pathway rather than a cell death pathway, and there is not sufficient evidence of autophagy as a true cell death inducer [18]. Evidence for a direct role in cell death only comes from artificial systems in which apoptosis is chemically or genetically suppressed, thus the role of autophagy in “programmed cell death” is debatable [22]. Thus, the toxicity resulting from the inhibition of the pro-survival mechanisms of autophagy by nanoparticles would appear the more likely scenario.Based on the above rationales, we hypothesize that the toxicity of CLs may be attributed to its inhibition of autophagic flux. The impact of CLs on autophagic flux has been discussed in only two studies, in which Roberts R et al. suggested that CLs induced autophagy [13], and Man N et al. showed that cationic lipids induced autophagic flux in a mTOR-independent manner [12]. The critical point to keep in mind is that autophagic flux is a highly dynamic process, and autophagic flux activation and inhibition may be reversed during a certain time period, suggesting that autophagic flux should be evaluated at different time points [24]. Thus, we hypothesize that CLs may play an alterable role in regulating autophagic flux: CLs increased autophagic flux at early stages but inhibited autophagic flux at late stages, resulting in toxicity derived from inhibiting the pro-survival mechanisms of autophagic flux.To elucidate the potential mechanism underlying the cytotoxic effect of CLs, we investigated the cytotoxic effect of CLs, and the effect of CLs on the autophagic flux of human liver epithelial cell lines(since most nanoparticles including CLs tend to accumulate in the liver). Our results reveal a novel mechanism whereby CLs induced cytotoxic effect through autophagic flux inhibition, with the subsequent mitochondrial dysfunction and ROS production as key event that mediated the cytotoxic effect of CLs. Our data clarify the mechanism underlying the cytotoxic effect of CLs and might have important implications for developing safe gene delivery systems.