Lipid nanoparticles decorated with TNF-related aptosis-inducing ligand (TRAIL) are more cytotoxic than soluble recombinant TRAIL in sarcoma

International Journal of Molecular Sciences Article Lipid Nanoparticles Decorated with TNF-Related Aptosis-Inducing Ligand (TRAIL) Are More Cytotoxic than Soluble Recombinant TRAIL in Sarcoma Ana Gallego-Lleyda 1,2, Diego De Miguel 1,3, Alberto Anel 1,2 and Luis Martinez-Lostao 2,4,5,6,* 1Departamento de Bioquímica, Biología Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain; [email protected] (A.G.-L.); [email protected] (D.D.M.); [email protected] (A.A.) 2Instituto de Investigación Sanitaria de Aragón (ISS), 50009 Zaragoza, Spain 3Cell Death, Cancer and Inflammation, University College of London, London WC1E 6BT, UK 4Servicio de Inmunología, Hospital Clínico Universitario Lozano Blesa, 50009 Zaragoza, Spain 5Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, 50009 Zaragoza, Spain 6Instituto de Nanociencia de Aragón, 50009 Zaragoza, Spain *Correspondence: [email protected]; Tel.: +34-976-76-88-59 Received: 15 April 2018; Accepted: 11 May 2018; Published: 13 May 2018   Abstract: 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. Keywords: sarcoma; TRAIL; flavopiridol; immunotherapy; lipid nanoparticles 1. Introduction Sarcomas include a heterogeneous group of complex cancers which derive from mesenchymal transformed cells. Sarcomas are rare, making up for about 1% of all cancers [ 1 ]. It is well known that prognosis of sarcomas is poor, with a 5-year survival rate of less than 15% when metastasis happens [ 2 ]. Over the last years, a great effort for improving treatment outcome has been made, but the complexity of sarcomas makes that advancements in developing new therapies are extremely slow [ 3 ]. Therefore, there is an urgent need to develop and test novel therapeutic strategies in order to improve prognosis of sarcoma patients [ 4 ]. Among the novel anti-tumor agents used, apoptosis ligand 2/TNF-related Int. J. Mol. Sci. 2018,19, 1449; doi:10.3390/ijms19051449 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018,19, 1449 2 of 18 apoptosis-inducing ligand (Apo2L/TRAIL) has been tested both in pre-clinical studies and clinical trials in some types of sarcoma [5]. Apoptosis ligand 2/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) is a TNF family member described as capable of inducing apoptosis in a wide variety of transformed cells while sparing normal cells [ 6 , 7 ]. This fact made TRAIL to be considered as a promising anti-tumor agent. In fact, TRAIL-based therapeutic strategies were developed and used in several phase I/II clinical trials on a wide variety of human cancers [ 8 – 10 ]. However, although TRAIL-based therapies were proven safe, about 50% of human cancers were intrinsically resistant to TRAIL since the clinical studies using TRAIL showed limited therapeutic activity of this death ligand in different types of cancer [ 9 , 11 , 12 ] including sarcoma [ 5 ]. Therefore, novel formulations of TRAIL with improved bioactivity and combination of TRAIL with sensitizing agents could be plausible strategies to overcome TRAIL resistance [13–16]. Our group generated artificial lipid nanoparticles containing membrane-bound TRAIL (LUV-TRAIL), resembling the natural TRAIL-containing exosomes physiologically released by activated human T cells upon re-stimulation [ 17 , 18 ]. We demonstrated that LUV-TRAIL are more effective than soluble recombinant TRAIL (sTRAIL), inducing apoptosis both in vitro and in vivo in hematological malignancies [19–21] as well as in epithelial-derived cancers [22–24]. In the present work, we have tested LUV-TRAIL in several human sarcoma cell lines of distinct origin with different sensitivity to sTRAIL. LUV-TRAIL were more efficient inducing apoptosis in all sarcoma cells tested than sTRAIL. However, although LUV-TRAIL showed a greater pro-apoptotic potential compared to sTRAIL, some sarcoma cell lines still remained fairly resistant to LUV-TRAIL. In this line, a wide range of sensitizing strategies have been described to overcome TRAIL-resistance in tumor cells. Here, we have tested the therapeutic combination of LUV-TRAIL with several drugs previously described as sensitizing agents [ 23 – 30 ], in order to improve the pro-apoptotic ability of LUV-TRAIL. Our results showed that, among all the combinations tested, flavopiridol induced the strongest sensitizing effect in all sarcoma cell lines tested, by inducing a down-regulation of the anti-apoptotic protein FLIP. In summary, LUV-TRAIL showed an improved cytotoxicity against sarcoma cells, overcoming the intrinsic resistance of these cells to sTRAIL. This was further potentiated when they were combined with sensitizing agents such as flavopiridol (FVP), opening the door to future clinical applications as anti-tumor therapy in sarcoma. 2. Results 2.1. LUV-TRAIL Showed an Enhanced In Vitro Cytotoxic Activity Compared to Soluble Recombinant TRAIL in Human Sarcoma Cells First, dose-response assays were carried out to compare the in vitro bioactivity of sTRAIL and LUV-TRAIL, and cell viability was analyzed by the MTT assay (Figure 1a). Whereas A673 cells showed a great sensitivity both to sTRAIL and LUV-TRAIL, HT-1080 and RD cell lines showed a moderate sensitivity to sTRAIL However, LUV-TRAIL was capable of inducing a significant decrease of cell viability both in HT-1080 and RD cells in comparison with sTRAIL. To assess whether the decrease in cell viability observed after treatment with both forms of TRAIL (sTRAIL and LUV-TRAIL) was due to the onset of cell death, annexin-V staining was performed (Figure 1b). In all sarcoma cell lines tested, LUV-TRAIL induced a remarkable increase of cell death at higher dose used (1000 ng/mL) when compared to sTRAIL. Importantly, the cell death observed in all sarcoma cell lines was specifically attributable to TRAIL-receptor activation by TRAIL, as cell death was completely inhibited when cells were pre-incubated with the TRAIL neutralizing antibody RIK2 before treatment with sTRAIL and LUV-TRAIL (Figure 1b). In this line, LUVs alone (without TRAIL anchoring on their surface), did not exert any cytotoxic effect in any sarcoma cell lines tested (see black bars on control points in Figure 1b). Int. J. Mol. Sci. 2018,19, 1449 3 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 18 Figure 1. (a) Analysis of cell viability after treatment with LUV-TRAIL on human sarcoma cell lines. Dose-response assays using the indicated doses of sTRAIL (ST) and LUV-TRAIL (LT) were performed on A673 cells, HT-1080 cells, and RD cells. Cells were treated with ST or LT for 24 h. Then, cell viability was measured by the MTT assay method. Graphs show the mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; (b) Cytotoxicity assays on human sarcoma cell lines. Cells were treated with indicated doses of sTRAIL (ST) or LUV-TRAIL (LT) for 24 h and annexin V positive cells were quantified by flow cytometry. When cells were treated with 1000 ng/mL, they were previously pre-incubated in presence or absence of the anti-TRAIL blocking mAb, RIK2 (500 ng/mL). Graphics show the percentage of annexin-V positive cells analyzed expressed as the mean ± SD of at least three experiments. * p < 0.05. (ST versus LT). # p < 0.05, ## p < 0.01 (ST versus ST + RIK2 and, LT versus LT + RIK2). TRAIL, TNF-related apoptosis-inducing ligand; LUV-TRAIL, TRAIL on a lipid nanoparticle surface; sTRAIL, soluble recombinant TRAIL. Figure 1. ( a ) Analysis of cell viability after treatment with LUV-TRAIL on human sarcoma cell lines. Dose-response assays using the indicated doses of sTRAIL (ST) and LUV-TRAIL (LT) were performed on A673 cells, HT-1080 cells, and RD cells. Cells were treated with ST or LT for 24 h. Then, cell viability was measured by the MTT assay method. Graphs show the mean ± SD of at least three independent experiments. * p< 0.05, ** p< 0.01, *** p< 0.001; ( b ) Cytotoxicity assays on human sarcoma cell lines. Cells were treated with indicated doses of sTRAIL (ST) or LUV-TRAIL (LT) for 24 h and annexin V positive cells were quantified by flow cytometry. When cells were treated with 1000 ng/mL, they were previously pre-incubated in presence or absence of the anti-TRAIL blocking mAb, RIK2 (500 ng/mL). Graphics show the percentage of annexin-V positive cells analyzed expressed as the mean ± SD of at least three experiments. * p< 0.05. (ST versus LT). # p< 0.05, ## p< 0.01 (ST versus ST + RIK2 and, LT versus LT + RIK2). TRAIL, TNF-related apoptosis-inducing ligand; LUV-TRAIL, TRAIL on a lipid nanoparticle surface; sTRAIL, soluble recombinant TRAIL. Int. J. Mol. Sci. 2018,19, 1449 4 of 18 2.2. LUV-TRAIL Activated the Caspase Cascade More Efficiently than sTRAIL in Human Sarcoma Cells Next, the implication of caspases in the cytotoxicity induced by LUV-TRAIL in sarcoma cells was assessed. For that purpose, sarcoma cells were incubated with sTRAIL or LUV-TRAIL and activation of the main caspases involved in the extrinsic apoptotic pathway was analyzed by Western blot. Activation of both caspase-8 and caspase-3 was clearly increased when sarcoma cells were treated with LUV-TRAIL compared to sTRAIL, as evidenced by the disappearance of the pro-forms of both caspases (Figure 2a). Moreover, cleavage of the specific caspase-3 substrate, PARP-1, and the specific caspase-8 substrate, Bid, correlated with the activation of both caspases -3 and -8, respectively, indicating a fully functional activation of the extrinsic apoptotic pathway upon LUV-TRAIL treatment. When time course assays were performed (Figure 2b), caspase activation was faster in A673 cells when they were treated with LUV-TRAIL, although, as seen previously, both formulations of TRAIL present similar cytotoxicity at 24 h. In HT-1080 cells, similar kinetics was observed at shorter times when they were treated both with sTRAIL and LUV-TRAIL. However, as shown in Figure 2a, caspase activation was greater when HT-1080 cells were treated with LUV-TRAIL in comparison with sTRAIL after 24 h of treatment. These data reflect that LUV-TRAIL required longer time of incubation to induce a greater caspase activation and, hence, a greater cytotoxicity than sTRAIL in HT-1080 cells. In case of RD cells, although no obvious differences could be observed in caspase activation after treatment with sTRAIL or LUV-TRAIL, Bid and PARP-1 degradation was faster when cells were treated with LUV-TRAIL. Finally, to fully assess and characterize the role of caspases in LUV-TRAIL induced cell death, cell death-inhibition assays were performed using the general caspase inhibitor z-VAD-fmk (Figure 2c). As expected, caspase inhibition fully abrogated cell death induced not only by sTRAIL but also by LUV-TRAIL. Moreover, when cells were pre-incubated with the specific caspase-8 inhibitor IETD-fmk, cell death induced by LUV-TRAIL was also fully abrogated, proving that cell death was fully dependent on the activation of the canonical extrinsic apoptotic pathway, ruling out any other form of cell death that could be triggered by TRAIL, such as necroptosis. 2.3. Combination of LUV-TRAIL with Anti-Cancer Agents Improved its Cytotoxicity against Human Sarcoma Cells Although LUV-TRAIL showed a greater cytotoxic effect than sTRAIL and was able to overcome sTRAIL resistance in HT-1080 and RD cells, LUV-TRAIL was only capable of inducing a decrease of cell viability of around 50% in both sarcoma cell lines at the 1000 ng/mL dose. Therefore, we decided to combine LUV-TRAIL with several anti-cancer agents such as flavopiridol (FVP), doxorubicin (DOX), trabectedin (TRAB), and bortezomib (BORT) in order to improve LUV-TRAIL-induced cytotoxicity. First of all, dose-response assays were carried out to analyze the cytotoxic effect of all anti-cancer agents used alone (Figure 3). Aiming for a sensitizing effect rather than an additive effect, sub-toxic doses of the different drugs were selected by choosing the maximum concentrations not inducing a decrease of cell viability greater than 20% (marker doses for each anti-cancer drug in Figure 3). After that, dose-response assays were carried out by pre-incubating sarcoma cells with the selected doses of each anti-cancer drug for 2 h before treating with either sTRAIL or LUV-TRAIL. In the case of A673 cells, although both sTRAIL and LUV-TRAIL induced a great decrease of cell viability at the highest dose used (1000 ng/mL), all different anti-cancer drugs were able to sensitize to LUV-TRAIL at intermediate doses (Figure 4a). In HT-1080 cells, although FVP greatly sensitized to both sTRAIL and LUV-TRAIL, the combination with LUV-TRAIL was the most cytotoxic (Figure 4b). Combination of TRAIL with DOX sensitized to both sTRAIL and LUV-TRAIL to the same extent. Similarly, BORT equally sensitized the cells to both sTRAIL and LUV-TRAIL, whereas TRAB did not show any sensitizing effect whatsoever (Figure 4b). Finally, in RD cells, combination of both sTRAIL and LUV-TRAIL with FVP or TRAB induced similar cytotoxocity levels, whereas DOX and BORT did not sensitize to neither sTRAIL nor LUV-TRAIL (Figure 4c). Int. J. Mol. Sci. 2018,19, 1449 5 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 18 Figure 2. (a) Analysis of caspase activation in human sarcoma cells. Cells were untreated (Control, designed as C), or treated with LUVs without TRAIL (LUV), sTRAIL (ST), and LUV-TRAIL (LT) at 1000 ng/mL for 24 h. After that, cells were lysed, and lysates were subjected to SDS-PAGE and to Western blot analysis. Levels of caspase-8, caspase-3, Bid, and PARP-1 were analyzed using specific antibodies. Level of actin levels was used as a control for equal protein loading. Cell death was measured in parallel by flow cytometry after annexin-V staining (bottom graphs); (b) Analysis of time-course caspase activation in human sarcoma cells. Cells were treated with sTRAIL or LUVTRAIL at 1000 ng/mL at the indicated times. After that, cells were lysed, and lysates were subjected to SDS-PAGE and to Western blot analysis. Levels of caspase-8, caspase-3, Bid, and PARP-1 were analyzed using specific antibodies. Level of actin levels was used as a control for equal protein loading. Caspase activation was evidenced by the disappearance of the pro-forms shown in the Western blot; (c) Analysis of cell death inhibition by caspase inhibition. Human sarcoma cells were treated with 1000 ng/mL of both sTRAIL (ST) and LUV-TRAIL (LT) for 24 h previously incubated in presence or absence of the pan-caspase inhibitor z-VAD-fmk (30 µM) and of the specific caspase-8 inhibitor IETD-fmk (30 µM). Graphics show the mean ± SD of the cell death of treated cells expressed as percentage of at least three experiments. * p < 0.05. (ST versus LT). ## p < 0.01 (ST versus ST + caspase inhibitors and, LT versus LT + caspase inhibitors). Figure 2. ( a ) Analysis of caspase activation in human sarcoma cells. Cells were untreated (Control, designed as C), or treated with LUVs without TRAIL (LUV), sTRAIL (ST), and LUV-TRAIL (LT) at 1000 ng/mL for 24 h. After that, cells were lysed, and lysates were subjected to SDS-PAGE and to Western blot analysis. Levels of caspase-8, caspase-3, Bid, and PARP-1 were analyzed using specific antibodies. Level of actin levels was used as a control for equal protein loading. Cell death was measured in parallel by flow cytometry after annexin-V staining (bottom graphs); ( b ) Analysis of time-course caspase activation in human sarcoma cells. Cells were treated with sTRAIL or LUV-TRAIL at 1000 ng/mL at the indicated times. After that, cells were lysed, and lysates were subjected to SDS-PAGE and to Western blot analysis. Levels of caspase-8, caspase-3, Bid, and PARP-1 were analyzed using specific antibodies. Level of actin levels was used as a control for equal protein loading. Caspase activation was evidenced by the disappearance of the pro-forms shown in the Western blot; ( c ) Analysis of cell death inhibition by caspase inhibition. Human sarcoma cells were treated with 1000 ng/mL of both sTRAIL (ST) and LUV-TRAIL (LT) for 24 h previously incubated in presence or absence of the pan-caspase inhibitor z-VAD-fmk (30 µ M) and of the specific caspase-8 inhibitor IETD-fmk (30 µ M). Graphics show the mean ± SD of the cell death of treated cells expressed as percentage of at least three experiments. * p< 0.05. (ST versus LT). ## p< 0.01 (ST versus ST + caspase inhibitors and, LT versus LT + caspase inhibitors). Int. J. Mol. Sci. 2018,19, 1449 6 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 18 Figure 3. Analysis of the effect of sensitizer agents in cell viability of human sarcoma cells. Doseresponse assays using the sensitizer agents: flavopiridol (FVP), doxorubicin (DOX), trabectedin (TRAB), and bortezomib (BORT) were performed on A673 cells (a), HT-1080 cells (b), and RD cells (c). Cell viability was assessed by MTT assay after 24 h. The results were expressed as the mean ± SD of at least three experiments. Marked doses of each drugs were selected for further experiments of sensitization. After that, dose-response assays were carried out by pre-incubating sarcoma cells with the selected doses of each anti-cancer drug for 2 h before treating with either sTRAIL or LUV-TRAIL. In the case of A673 cells, although both sTRAIL and LUV-TRAIL induced a great decrease of cell viability at the highest dose used (1000 ng/mL), all different anti-cancer drugs were able to sensitize to LUV-TRAIL at intermediate doses (Figure 4a). Figure 3. Analysis of the effect of sensitizer agents in cell viability of human sarcoma cells. Dose-response assays using the sensitizer agents: flavopiridol (FVP), doxorubicin (DOX), trabectedin (TRAB), and bortezomib (BORT) were performed on A673 cells ( a ), HT-1080 cells ( b ), and RD cells ( c ). Cell viability was assessed by MTT assay after 24 h. The results were expressed as the mean ± SD of at least three experiments. Marked doses of each drugs were selected for further experiments of sensitization. Int. J. Mol. Sci. 2018,19, 1449 7 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 18 In HT-1080 cells, although FVP greatly sensitized to both sTRAIL and LUV-TRAIL, the combination with LUV-TRAIL was the most cytotoxic (Figure 4b). Combination of TRAIL with DOX sensitized to both sTRAIL and LUV-TRAIL to the same extent. Similarly, BORT equally sensitized the cells to both sTRAIL and LUV-TRAIL, whereas TRAB did not show any sensitizing effect whatsoever (Figure 4b). Finally, in RD cells, combination of both sTRAIL and LUV-TRAIL with FVP or TRAB induced similar cytotoxocity levels, whereas DOX and BORT did not sensitize to neither sTRAIL nor LUVTRAIL (Figure 4c). Figure 4. Analysis of cell viability of human sarcoma cell lines after treatment with sTRAIL or LUV-TRAIL in combination with sensitizer agents. Cells were pre-incubated for 2 h with sensitizer drugs: flavopiridol (FVP), doxorubicin (DOX), trabectedin (TRAB), and bortezomib (BORT) at indicated doses. Afterwards, sTRAIL (ST) or LUV-TRAIL (LT) were added at the indicated concentrations and left overnight. The following day, cell viability was measured by the MTT assay. In every graph, the results from treatments with ST and LT in the absence of drugs are superposed in light grey to show the sensitizing effect more clearly. Graphs show the mean ± SD of at least independent experiments. Asterisk signs indicate significance between LT alone with LT in combination with a given drug ( *p< 0.05 , ** p< 0.005, *** p< 0.001). Pound signs indicate significance between ST alone with ST in combination with a given drug (# p< 0.05, ### p< 0.005, ### p< 0.001). A673 cells ( a ), HT-1080 cells ( b ), and RD cells ( c ). Ampersand signs indicate significance between ST in combination with a given drug with LT in combination with same drug. (& p< 0.05, && p< 0.005, &&& p< 0.001). Int. J. Mol. Sci. 2018,19, 1449 8 of 18 2.4. Combination of FVP with LUV-TRAIL Induced Apoptotic Cell Death and Decreased Long-Term Clonogenic Survival of Human Sarcoma Cells According to the results obtained, combination of LUV-TRAIL with FVP greatly enhanced their cytotoxic ability in all sarcoma cell lines tested. Next, we sought to confirm whether the decrease of cell viability observed in Figure 4was due to the induction of apoptosis or to a cytostatic effect. First, appearance of typical apoptotic features was assessed by microscopy (Figure 5a). In all cases, cells treated with sTRAIL and LUV-TRAIL alone or in combination with FVP exhibited the typical apoptotic nuclear morphological changes such as blebbing, nuclear fragmentation, and chromatin condensation (Figure 5a). Moreover, these nuclear apoptotic morphological changes were more pronounced when both forms of TRAIL were combined with FVP compared with treatments with TRAIL alone (both sTRAIL and LUV-TRAIL). Finally, apoptotic morphological changes correlated in all cases with induction of apoptosis carried out in parallel using annexin V staining (Figure 5a, showed as percentage of annexin V positive cells below each picture). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 18 Figure 4. Analysis of cell viability of human sarcoma cell lines after treatment with sTRAIL or LUVTRAIL in combination with sensitizer agents. Cells were pre-incubated for 2 h with sensitizer drugs: flavopiridol (FVP), doxorubicin (DOX), trabectedin (TRAB), and bortezomib (BORT) at indicated doses. Afterwards, sTRAIL (ST) or LUV-TRAIL (LT) were added at the indicated concentrations and left overnight. The following day, cell viability was measured by the MTT assay. In every graph, the results from treatments with ST and LT in the absence of drugs are superposed in light grey to show the sensitizing effect more clearly. Graphs show the mean ± SD of at least independent experiments. Asterisk signs indicate significance between LT alone with LT in combination with a given drug (* p < 0.05, ** p < 0.005, *** p < 0.001). Pound signs indicate significance between ST alone with ST in combination with a given drug (# p < 0.05, ### p < 0.005, ### p < 0.001). A673 cells (a), HT-1080 cells (b), and RD cells (c). Ampersand signs indicate significance between ST in combination with a given drug with LT in combination with same drug. (& p < 0.05, && p < 0.005, &&& p < 0.001). 2.4. Combination of FVP with LUV-TRAIL Induced Apoptotic Cell Death and Decreased Long-Term Clonogenic Survival of Human Sarcoma Cells According to the results obtained, combination of LUV-TRAIL with FVP greatly enhanced their cytotoxic ability in all sarcoma cell lines tested. Next, we sought to confirm whether the decrease of cell viability observed in Figure 4 was due to the induction of apoptosis or to a cytostatic effect. First, appearance of typical apoptotic features was assessed by microscopy (Figure 5a). In all cases, cells treated with sTRAIL and LUV-TRAIL alone or in combination with FVP exhibited the typical apoptotic nuclear morphological changes such as blebbing, nuclear fragmentation, and chromatin condensation (Figure 5a). Moreover, these nuclear apoptotic morphological changes were more pronounced when both forms of TRAIL were combined with FVP compared with treatments with TRAIL alone (both sTRAIL and LUV-TRAIL). Finally, apoptotic morphological changes correlated in all cases with induction of apoptosis carried out in parallel using annexin V staining (Figure 5a, showed as percentage of annexin V positive cells below each picture). Figure 5. ( a ) Analysis of nuclear morphological changes after combination of LUV-TRAIL with FVP. Human sarcoma cells were pre-incubated for 1 h with flavopiridol (FVP, 200 nM for A673 and RD cells, 50 nM for HT-1080 cells). Afterwards, sTRAIL (ST) or LUV-TRAIL (LT) were added (300 ng/mL for A673 cells and 1000 ng/mL for HT-1080 and RD cells) and left overnight. The following day, nuclear staining was performing by using Hoechst 33342. As control, cells were untreated (CONTROL) or treated only with FVP (FVP) at above indicated doses. Original magnification at 400 × . Scale bar = 20 µ M. Int. J. Mol. Sci. 2018,19, 1449 9 of 18 Cell death was measured in parallel by flow cytometry after annexin-V staining (bottom graphs); ( b ) Analysis of clonogenic survival after combination of LUV-TRAIL with FVP. Clonogenic assay was performed to analyze long-term survival in human sarcoma cells after treatment with sTRAIL (ST) or LUV-TRAIL (LT) for 24 h (300 ng/mL for A673 cells and 1000 ng/mL for HT-1080 and RD cells). As indicated, cells were pre-incubated with FVP (FVP, 200 nM for A673 and RD cells, 50 nM for HT-1080 cells) for 1 h. Upper panels show 6-well plates seeded with human sarcoma cells and stained with crystal violet after 11 days. Bottom panels show the quantification of crystal violet absorbance after solubilizing in DMSO and measuring absorbance at 550 nm. Graphic shows the mean ± SD of the absorbance of treated cells expressed as percentage with respect to the untreated cells (control). Asterisk signs indicate significance between LT and LT (* p< 0.05, ** p< 0.005, *** p< 0.001 ). Pound signs indicate significance between TRAIL (ST or LT) alone with TRAIL in combination with FVP (# p< 0.05). Next, we sought to analyze the long-term cytotoxic effect of the combinations of both forms of TRAIL alone or in combination with FVP. For this purpose, clonogenic assays were carried out. (Figure 5b). Notably, liposomes alone (without TRAIL anchored on their surface) were added as controls to rule out any long-term cytotoxic effect of the lipid nanoparticles (see controls for LUV-TRAIL in Figure 5b). Treatment with LUV-TRAIL for 24 h significantly delayed clonogenic survival of sarcoma cells when compared to sTRAIL, indicating that LUV-TRAIL not only showed a greater cytotoxic short-term effect than sTRAIL but also showed a long-term effect affecting to survival and proliferation of sarcoma cells. Remarkably, when both forms of TRAIL were combined with FVP, the clonogenic suvival of sarcoma cells was enhanced compared to TRAIL alone, being again more pronounced in the case of LUV-TRAIL. 2.5. Combination of FVP with LUV-TRAIL Activated the Caspase Cascade More Efficiently than with sTRAIL in Human Sarcoma Cells After assessing the greater cytotoxic effect of the combination of LUV-TRAIL with FVP at both short and long term, activation of the main caspases involved in the extrinsic apoptotic pathway (caspase-8 and caspase-3) was analyzed by Western blot. As aforementioned (Figure 2), both sTRAIL and LUV-TRAIL induced the activation of the extrinsic apoptotic pathway, as shown by the clear decrease of the pro-forms of both caspases-8 and -3, as well as of Bid and PARP-1, in the three sarcoma cell lines tested (Figure 6a). Moreover, caspase activation correlated in all cases with induction of apoptosis as indicated by annexin V staining performed in parallel (Figure 6a, lower panels). Pretreatment with FVP greatly increased the disappearance of all proteins, proving that FVP sensitization to both sTRAIL and LUV-TRAIL relies on an enhanced activation of the extrinsic apoptotic pathway. Importantly, FVP did not induce any detectable caspase activation in any sarcoma cell tested (Figure 6a, lower panels). On the other hand, cell death induced by combination of either sTRAIL or LUV-TRAIL with FVP was fully inhibited by the general caspase inhibitor z-VAD-fmk and the specific caspase inhibitor IETD-fmk. These data demonstrated that cell death induced by the combination of FVP with TRAIL (sTRAIL and LUV-TRAIL) was a caspase-dependent apoptotic process through the activation of the extrinsic apoptotic pathway by caspase-8 (Figure 6b). 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