FRET based high-throughput screen for caspase activation
To analyze the effects of nanoparticles on apoptosis, we decided to adapt two FRET-based molecular assays to detect caspase activation [8] using high-throughput microscopy. The indicator molecules for caspase activation use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (ECFP, the donor) linked to an improved yellow fluorescent protein (Venus, the acceptor). The caspase-3 indicator (SCAT3) contains a caspase-3 cleavage site, i.e., the amino acid sequence DEVD (Asp-Glu-Val-Asp), which is cleaved by caspase-3 in stress-induced apoptosis. The caspase-9 indicator (SCAT9) contains a caspase-9 cleavage site, i.e., the amino acid sequence LEHD (Leu-Glu-His-Asp). Whether the linkage chain remains intact or not depends on caspase-3 or caspase-9 activity. If apoptosis is not induced when the compound in question is added to our seeded cells, the amino acid linkage chain remains intact. Venus will absorb the light emitted by ECFP and it will emit its own 530 nm light. If apoptosis is induced, caspase-3 and/or caspase-9 will be activated and their respective amino acid linkage chains will be cleaved. This severs the linkages between the fluorophores. The 475 nm light emitted by ECFP will not be absorbed by Venus and will instead be visible; consequently, Venus' 530 nm wavelength light will not be observed. The FRET signal is measured as a ratio of 530 nm light emitted to 475 nm light emitted (Venus:ECFP).
As displayed in Figures 1, after our treatment of HT29-SCAT3 and HT29-SCAT9 cells with zinc oxide, titanium dioxide, and iron oxide nanoparticles for 18 hrs, no caspase activation was observed. No significant amount of caspase activation was observed with longer incubation either (data not shown). The positive control, staurosporine, induced caspase activation as indicated by the loss of FRET (Figure 1); this indicated that the assay was working. This result differs from relevant research as [4, 5] had reported that zinc oxide and titanium dioxide nanoparticles would induce apoptosis.
Next, we examined the morphologies of the treated cells. Although cells treated for 18 hours with up to 100 μg/ml of titanium dioxide and iron oxide nanoparticles appeared largely normal (data not shown), the cells treated with zinc oxide nanoparticles rounded up and died after 18 hours of incubation with 5-20 μg/ml zinc oxide (Figure 2). Furthermore, cell death induced by zinc oxide nanoparticles could not be inhibited by IDN6556, a potent and specific caspase inhibitor that has been shown to inhibit all caspases [10]. This is consistent with our FRET assay results, which indicate that zinc oxide nanoparticles cannot induce caspase activation. Our results suggest that zinc oxide nanoparticles induce cell death through a non-apoptosis pathway. Existing evidence suggests that the cells have multiple regulated pathways to mediate their death [11]. The alternative mechanisms of regulated cell death are currently a subject of intensive study. This part of our study demonstrates the feasibility of using FRET-based caspase activation assay in a high-throughput format to examine the pro-apoptotic activities of nanoparticles as well as other commonly used compounds.
LC3-GFP-based high-throughput screen for autophagy activation
Because autophagy is often activated in stressed cells [9], we considered the possibility that nanoparticles would induce autophagy. Induction of autophagy would not necessarily result in the death of the cell, but it would nevertheless indicate a stress response. To determine the effects of nanoparticles on autophagy induction, we used human neuroblastoma H4 cells stably expressing the LC3-GFP reporter [12]. LC3 is an important signaling molecule involved in mediating autophagy. When LC3 is activated, it is tagged with a small lipid, PE (phosphatidylethanolamine), that allows it to be translocated onto an autophagosomal membrane. In H4-LC3-GFP cells, LC3 has been tagged with a green fluorescent protein (GFP) to allow LC3 to be easily detected using fluorescent microscopy. Under normal conditions, LC3 is mostly present in the cytosol of a cell. When autophagy is activated, cytosolic LC3 (LC3 I) is conjugated into PE to form LC3 II which then translocates to the preautophagosomal membrane. We first tested using rapamycin as a positive control, as rapamycin is known to strongly induce autophagy [13]. Figure 3A shows that treatment of the cells with rapamycin, our positive control, strongly increased levels of observed autophagy. This indicates that our assay was working.
On the other hand, although treatment with zinc oxide nanoparticles also induced death in H4-LC3-GFP cells as it did in HT29 cells, zinc oxide nanoparticles had no effect on autophagy (the number of green LC3-GFP dots did not increase) (Figure 3B & 4). Thus, although zinc oxide nanoparticles induced cell death, it did not induce autophagy in H4 cells. Treatment with iron oxide also did not induce autophagy (data not shown). In contrast, we found that the treatment of the cells with titanium dioxide nanoparticles in concentrations that had no effect on cell morphology clearly led to significant increases in the levels of autophagy (Figures 4 and 5).
The accumulation of free radicals often plays a role in mediating autophagy [14]. Our next step was to investigate the involvement of free radicals in titanium dioxide induced autophagy. We soon found, however, that the addition of N-acetylcysteine (NAC), an antioxidant, did not inhibit the increases in autophagy induced by titanium dioxide nanoparticles (Figure 5). This suggests that the increases in autophagy induced by titanium dioxide nanoparticles may not be mediated by an increase in free radicals.
Because autophagosomes eventally fuse with lysosomes to degrade the contents of the autophagosome via hydrolytic enzymes, we had to consider the possibility that the apparent increases in the levels of autophagy may be due to a block in the lysosomes induced by titanium dioxide nanoparticles. The standard method to test this possibility is to use a blocker of lysosomes and to see if the combination of the lysosomal inhibitor with the compound of interest can lead to an additive increase in the level of autophagy [15]. We used E64d ([2S, 3S]-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester), a cysteine protease inhibitor commonly used to inhibit lysosomal degradation [15]. We found that the combination of E64d and titanium dioxide led to a level of autophagy greater than that observed when E64d was applied alone, suggesting that the treatment of titanium dioxide led to increases in the flux of autophagy.