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

Although TRAIL (TNF-related apoptosis-inducing ligand, also known as Apo2L) was described as capable of inducing apoptosis in transformed cells while sparing normal cells, limited results obtained in clinical trials has limited its use as an anti-tumor agent. Consequently, novel TRAIL formulations with enhanced bioactivity are necessary for overcoming resistance to conventional soluble TRAIL (sTRAIL) exhibited by many primary tumors. Our group has generated artificial liposomes with sTRAIL anchored on their surface (large unilamellar vesicle (LUV)-TRAIL), which have shown a greater cytotoxic activity both in vitro and in vivo when compared to sTRAIL against distinct hematologic and epithelial carcinoma cells. In this study, we have improved LUV-TRAIL by loading doxorubicin (DOX) in its liposomal lumen (LUVDOX-TRAIL) in order to improve their cytotoxic potential. LUVDOX-TRAIL killed not only to a higher extent, but also with a much faster kinetic than LUV-TRAIL. In addition, the concerted action of the liposomal DOX and TRAIL was specific of the liposomal DOX and was not observed when with soluble DOX. The cytotoxicity induced by LUVDOX-TRAIL was proven to rely on two processes due to different molecular mechanisms: a dynamin-mediated internalization of the doxorubicin-loaded particle, and the strong activation of caspase-8 exerted by the liposomal TRAIL. Finally, greater cytotoxic activity of LUVDOX-TRAIL was also observed in vivo in a tumor xenograft model. Therefore, we developed a novel double-edged nanoparticle combining the cytotoxic potential of DOX and TRAIL, showing an exceptional and remarkable synergistic effect between both agents.

Importance of TRAIL Molecular Anatomy in Receptor Oligomerization and Signaling. Implications for Cancer Therapy.

(TNF)-related apoptosis-inducing ligand (TRAIL) is able to activate the extrinsic apoptotic pathway upon binding to DR4/TRAIL-R1 and/or DR5/TRAIL-R2 receptors. Structural data indicate that TRAIL functions as a trimer that can engage three receptor molecules simultaneously, resulting in receptor trimerization and leading to conformational changes in TRAIL receptors. However, receptor conformational changes induced by the binding of TRAIL depend on the molecular form of this death ligand, and not always properly trigger the apoptotic cascade. In fact, TRAIL exhibits a much stronger pro-apoptotic activity when is found as a transmembrane protein than when it occurs as a soluble form and this enhanced biological activity is directly linked to its ability to cluster TRAIL receptors in supra-molecular structures. In this regard, cells involved in tumor immunosurveillance, such as activated human T cells, secrete endogenous TRAIL as a transmembrane protein associated with lipid microvesicles called exosomes upon T-cell reactivation. Consequently, it seems clear that a proper oligomerization of TRAIL receptors, which leads to a strong apoptotic signaling, is crucial for inducing apoptosis in cancer cells upon TRAIL treatment. In this review, the current knowledge of oligomerization status of TRAIL receptors is discussed as well as the implications for cancer treatment when using TRAIL-based therapies.

Role of Exosomes in the Regulation of T-cell Mediated Immune Responses and in Autoimmune Disease.

: T-cell mediated immune responses should be regulated to avoid the development of autoimmune or chronic inflammatory diseases. Several mechanisms have been described to regulate this process, namely death of overactivated T cells by cytokine deprivation, suppression by T regulatory cells (Treg), induction of expression of immune checkpoint molecules such as CTLA-4 and PD-1, or activation-induced cell death (AICD). In addition, activated T cells release membrane microvesicles called exosomes during these regulatory processes. In this review, we revise the role of exosome secretion in the different pathways of immune regulation described to date and its importance in the prevention or development of autoimmune disease. The expression of membrane-bound death ligands on the surface of exosomes during AICD or the more recently described transfer of miRNA or even DNA inside T-cell exosomes is a molecular mechanism that will be analyzed.

Lipid Nanoparticles Decorated with TNF-Related Aptosis-Inducing Ligand (TRAIL) Are More Cytotoxic than Soluble Recombinant TRAIL in Sarcoma.

Sarcomas are rare and heterogeneous cancers classically associated with a poor outcome. Sarcomas are 1% of the cancer but recent estimations indicate that sarcomas account for 2% of the estimated cancer-related deaths. Traditional treatment with surgery, radiotherapy, and chemotherapy has improved the outcome for some types of sarcomas. However, novel therapeutic strategies to treat sarcomas are necessary. TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand initially described as capable of inducing apoptosis on tumor cell while sparing normal cells. Only few clinical trials have used TRAIL-based treatments in sarcoma, but they show only low or moderate efficacy of TRAIL. Consequently, novel TRAIL formulations with an improved TRAIL bioactivity are necessary. Our group has developed a novel TRAIL formulation based on tethering this death ligand on a lipid nanoparticle surface (LUV-TRAIL) resembling the physiological secretion of TRAIL as a trasmembrane protein inserted into the membrane of exosomes. We have already demonstrated that LUV-TRAIL shows an improved cytotoxic activity when compared to soluble recombinant TRAIL both in hematological malignancies and epithelial-derived cancers. In the present study, we have tested LUV-TRAIL in several human sarcoma tumor cell lines with different sensitivity to soluble recombinant TRAIL, finding that LUV-TRAIL was more efficient than soluble recombinant TRAIL. Moreover, combined treatment of LUV-TRAIL with distinct drugs proved to be especially effective, sensitizing even more resistant cell lines to TRAIL.

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

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.

Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein.

We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells.

PURPOSE: Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. METHODS/PATIENTS: LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. RESULTS: LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. CONCLUSION: The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.

Improved Anti-Tumor Activity of Novel Highly Bioactive Liposome-Bound TRAIL in Breast Cancer Cells.

BACKGROUND: Apo2-ligand/TRAIL, a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells, is a promising anti-tumor agent. However, about 50% of human cancer are TRAIL resistant. Consequently, future TRAIL-based therapies will require the use of novel highly bioactive forms of TRAIL and/or the addition of sensitizing agents to TRAIL-induced apoptosis. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive TRAIL (LUV-TRAIL) greatly improved TRAIL activity and were able to induce apoptosis in chemoresistant hematological tumor cells. OBJECTIVE: In this study, we have tested LUV-TRAIL-pro-apoptotic potential in human breast cancer. METHOD: Comparative analysis of cytotoxicity induced by sTRAIL and LUV-TRAIL was performed using several human breast tumor cell lines with different sensitivity to TRAIL. In vivo anti-tumor activity of LUV-TRAIL was also studied using a xeno-graft tumor model. RESULTS: LUV-TRAIL improved not only sTRAIL in vitro cytotoxicity in all breast tumor cell lines tested but also showed more anti-tumor activity than sTRAIL in an in vivo xeno-graft tumor model. On the other hand, the concomitant treatment of LUV-TRAIL with the sensitizing agent flavopiridol (FVP) induced a higher level of cell death in TRAIL-resistant cell lines. TRAIL-sensitization induced by FVP was mediated by DR5 up-regulation, and interestingly TRAIL-apoptotic signaling was completely shifted towards DR5 upon FVP treatment. LUV-TRAIL could especially take advantage of this DR5 up-regulation, while sTRAIL was not able. CONCLUSION: To date, no special attention had been paid to this aspect of FVP-induced TRAIL-sensitization, may be because sTRAIL used were not able to take advantage of this DR5 up-regulation. Hence, LUV-TRAIL could be a better choice than sTRAIL to be used in combination with anti-tumor drugs inducing DR5 over-expression, since LUV-TRAIL is especially effective activating this death-receptor.

Death ligands and granulysin: mechanisms of tumor cell death induction and therapeutic opportunities.

The immune system plays a key role in cancer immune surveillance to control tumor development. The final goal is recognizing and killing transformed cells and consequently the elimination of the tumor. The main effector cell types exerting cytotoxicity against tumors are natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although the mechanism of activation of NK cells and CTLs are quite different, both cell types share common antitumor effector mechanisms of cytotoxicity which lead to induction of cell death of tumor cells by apoptosis. Among these mechanisms are the death ligand- and granulysin-mediated cell deaths. In this review, we summarize the main concepts of these effector cytotoxic mechanisms against cancer cells, how NK cells and CTLs use them to control tumor development and the therapeutic approaches currently developed based on these molecules.

Liposome-bound TRAIL induces superior DR5 clustering and enhanced DISC recruitment in histiocytic lymphoma U937 cells.

Human Apo2-Ligand/TRAIL is a promising antitumor agent. Our group demonstrated that TRAIL was physiologically released to the extracellular medium inserted in lipid vesicles, known as exosomes. Recently we demonstrated that artificial lipid nanoparticles coated with bioactive TRAIL (LUV-TRAIL), which resemble the natural exosomes, greatly improved TRAIL activity compared with the soluble form of this death ligand and were able to induce apoptosis in hematological malignancies. In this study we have deepened the underlying mechanism of action of LUV-TRAIL in hematologic cells. Using histiocytic lymphoma U937 cells, we demonstrated that TRAIL signaling almost exclusively depends on DR5 despite these cells expressing high amounts of DR4, and proved that LUV-TRAIL's higher pro-apoptotic effect relies on its superior ability to induce DR5 clustering on cell surface, therefore enhancing DISC recruitment and triggering caspase activation more efficiently than the soluble form of TRAIL.