High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer

Abstract

During the last years, a great effort has been invested into developing new TRAIL formulations with increased bioactivity, trying to overcome the resistance to conventional soluble TRAIL (sTRAIL) exhibited by many primary tumours. In our group, we have generated artificial lipid nanoparticles decorated with sTRAIL (LUV-TRAIL), emulating the physiological TRAIL-containing exosomes by which T-cells release TRAIL upon activation. We already demonstrated that LUV-TRAIL has greater cytotoxicity against both chemoresistant haematologic tumour cells and epithelial carcinoma cells compared to a form of sTRAIL similar to that used in clinical trials. In this study we have tested LUV-TRAIL in several human colon cancer cell lines with different sensitivity to sTRAIL. LUV-TRAIL significantly improved sTRAIL cytotoxicity in all colon cancer cell lines tested. Trying to ascertain the molecular mechanism by which LUV-TRAIL exhibited improved cytotoxicity, we demonstrated that TRAIL-coated lipid nanoparticles were able to activate DR5 more efficiently than sTRAIL, and this relied on LUV-TRAIL ability to promote DR5 clustering on the cell surface. Moreover, we show that TRAIL molecules are arranged in higher order oligomers only in LUV-TRAIL, which may explain their enhanced DR5 clustering ability. Finally, LUV-TRAIL showed significantly better antitumour activity than sTRAIL in an in vivo model using HCT-116 xenograft tumours in nude mice, validating its potential clinical application.

Double-Edged Lipid Nanoparticles Combining Liposome-Bound TRAIL and Encapsulated Doxorubicin Showing an Extraordinary Synergistic Pro-Apoptotic Potential

“…In this line, silencing caspase-8 in SKBR3 cells completely abrogated LDT-induced cell death (Supplementary Figure S2b). Finally, given the double-edged nature of LDT, being a priori able to trigger both the extrinsic and the intrinsic apoptotic pathways, and considering the strong activation of caspase-9 observed in Figure 3c, we also checked the activation of the intrinsic apoptotic pathway by using modified HCT-116 cells lacking both Bax and Bak (HCT-116 BB), therefore lacking a functional mitochondrial apoptotic pathway [49] (Supplementary Figure S2c). HTC-116 BB cells were completely resistant to LDT, suggesting an important role for the mitochondrial apoptotic pathway in LDT-induced cell death.…”

“…Western blot was performed for protein expression analysis, as previously described [47][48][49]. The antibodies used to analyze the expression of the main proteins involved in the apoptotic pathway were: anti-caspase-8 (BD Biosciences), anti-caspase-3 (Cell Signaling), anti-Bid (BD Biosciences), anti-PARP-1 (BD Biosciences), and anti-caspase-9 (MBL).…”

“…For Western blot analysis, cells (5 × 10 6 ) were lysed at 4 • C with 100 µL of a buffer containing 1% Triton X-100 and protease and phosphatase inhibitors, as previously described [46][47][48][49]. Then, protein from lysed cells were separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with TBS-T buffer (10 mM Tris/HCl, pH 8.0, 0.12 M NaCl, 0.1% Tween-20, 0.05% sodium azide) containing 5% skimmed milk.…”

“…In this line, our group generated an original formulation of TRAIL based on immobilizing TRAIL molecules on the surface of artificial lipid nanoparticles (large unilamellar vesicle (LUV)-TRAIL), resembling the natural TRAIL-containing exosomes secreted by activated human T-cells [43,44]. We already demonstrated that LUV-TRAIL exhibited an enhanced anti-tumor activity when compared to soluble TRAIL (sTRAIL) against leukemic cells resistant to TRAIL or to chemotherapeutic drugs [37,45,46], and on epithelial carcinoma cells both in vitro and in vivo [47][48][49]. The increased cytotoxicity of LUV-TRAIL relied on its ability to induce an improved DR5 clustering leading to an enhanced DISC recruitment in comparison to sTRAIL [45,49].…”

“…We already demonstrated that LUV-TRAIL exhibited an enhanced anti-tumor activity when compared to soluble TRAIL (sTRAIL) against leukemic cells resistant to TRAIL or to chemotherapeutic drugs [37,45,46], and on epithelial carcinoma cells both in vitro and in vivo [47][48][49]. The increased cytotoxicity of LUV-TRAIL relied on its ability to induce an improved DR5 clustering leading to an enhanced DISC recruitment in comparison to sTRAIL [45,49]. However, although LUV-TRAIL has proven more efficient than sTRAIL in all tumor cell lines tested, some cancer cell lines remain somewhat resistant to its pro-apoptotic activity.…”