Professor Rice University Houston, Texas, United States
Introduction: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein that specifically induces apoptosis in a broad spectrum of cancer cell lines while sparing most healthy ones. TRAIL activates the extrinsic apoptotic pathway upon binding to its cognate death receptors 4 and 5 (DR4 and DR5). Like other members of the TNF family, TRAIL must form a homotrimer to achieve its cancer cell-specific toxicity. DR4/5 receptors bind to the gaps between two neighboring monomers in the TRAIL trimer, one receptor to each of the three gaps. This binding results in receptor trimerization that leads to conformational changes in the TRAIL receptors. This conformational change alone is not always sufficient to transduce the intracellular signal. Previous studies have shown that DR5 requires further cross-linking or oligomerization to form high-order clusters on the cell surface to correctly initiate the apoptotic signaling, while DR4 does not require such additional cross-linking. This discovery is supported by the fact that TRAIL is much more potent to certain cell lines as a naturally expressed transmembrane protein, likely in the form of clusters, than in its free, soluble form. We expect that it is possible to increase the apoptotic activity of recombinant soluble TRAIL proteins by allow them to form clusters on artificial surfaces. In this study, we demonstrated the ability to control the extent of TRAIL clustering on the outer surface of nanoscale liposomes and thus their apoptotic ability using the technology of lipid phase separation.
Materials and
Methods: It is challenging to directly observe the phase separation on nanoscale liposomes using optical microscopy. We used giant unilamellar vesicles (GUVs), which are microsized vesicles as an alternative to liposomes to visualize the phase separation. GUVs were prepared by formation of a lipid/sugar film, followed by hydration with PBS. The three lipids for the GUVs included DSPC, DOPC and cholesterol at a molar ratio of 3:1:1.2. These lipids were chosen because the phase separation behavior of GUVs and liposomes composed of them have been well studied. Nanoscale liposomes were prepared using the same lipid formulation. The film was hydrated before the mixture was extruded through a 100 nm polycarbonate membrane. The number concentration of liposomes was estimated using an equation from www.liposomes.org. A calculated amount of his-tagged TRAIL was added to a liposome suspension with 100, 200 and 400 copies of TRAIL on average on an individual liposome, which were then designated as 100T-, 200T- and 400T-Lipo, respectively. The size distribution before and after TRAIL conjugation was measured using a ZetaSizer instrument. For long term storage, trehalose was added at 10x the molar concentration of the total lipids and the liposomes were flash frozen. The liposomes were stored at -80°C before use. Human prostate cancer PC3 or DU145 cells and T lymphocyte immortalized Jurkat cells were used to analyze the apoptotic abilities of the TRAIL liposomes. Cell apoptosis was analyzed at 24 h after treatment by an Annexin V/Propidium Iodide (PI) apoptosis assay using a Guava EasyCyte flow cytometer.
Results, Conclusions, and Discussions: The GUVs were directly visualized with confocal microscopy as the DSPC phase was labelled in green while DOPC was labelled in red (Figure 1A). The size of liposomes becomes larger with more TRAIL conjugated, suggesting success of TRAIL association with the liposome surface (Figure 1B). In PC3 and DU145 cell lines, all three TRAIL liposome formulations showed higher cytotoxic effects than free TRAIL (Figure 1C,D). More importantly, the liposomes conjugated with more TRAIL molecules are more potent that those conjugated with fewer TRAIL molecules. In the treatment of DU145 cells, 400T-Lipo achieved more than twice the apoptotic activity of free TRAIL. Therefore, these results are consistent with the idea that more TRAIL on the liposome surface, potentially forming larger TRAIL clusters, can more potently engage DR5 to initiate the apoptotic signaling. Jurkat cells are known to exhibit a high expression of DR5 and are highly sensitive to TRAIL-induced apoptosis. However, the TRAIL liposomes with fewer copies of TRAIL were found to be more apoptotic to Jurkat cells than those with more TRAIL, which is opposite to our expectation (Figure 1E). One possible reason for this is that the density of TRAIL clusters of 100T-Lipo is more consistent with the distribution density of DR5 on the surface of Jurkat cells than that of 200T-Lipo or 400T-Lipo, on which a greater number of TRAIL molecules lacked access to the receptor and thus did not result in signal transduction. We then used confocal microscopy to analyze the binding of Jurkat cells with TRAIL liposomes. The TRAIL liposomes were found to surround the membrane of Jurkat cells (Figure 1F). The merging of the two colors labeling DSPC and DOPC phases, respectively, indicates an intact structure of the liposomes. No binding of naked liposomes to the cell surface or internalization was observed within the first hour of incubation. We demonstrated the potential to control the clustering of TRAIL on the liposome surface with phase separation. This new technology helps to achieve optimal apoptotic capability of TRAIL to specific tumors, and thus could expedite the translation of TRAIL-based therapies into the clinic.
