BT12 and BT16 cell lines are epithelial AT/RT cells isolated from a 2 month old female and a 2-year-old male, respectively. These cell lines were generously donated by Peter Houghton, St. Jude Children's Research Hospital (Memphis, TN, USA). G401 cells (American Type Culture Collection, Manassas, VA, USA) are epithelial kidney MRT cells isolated from a 3 month old male. BT12 and BT16 cell lines were cultured in Gibco RPMI-1640 GlutaMAX (Biosciences, Dublin, Ireland). G401 cells were cultured in Gibco Dulbecco's modified Eagle's medium (DMEM) GlutaMAX (Biosciences). All media used was supplemented with 10% (v/v) foetal bovine serum (FBS; Biosciences), 100 U/ml penicillin and 100 μg/ml streptomycin (Biosciences). Cultured cells were incubated at 37°C with 5% CO₂. Cells were screened for mycoplasma using PCR and were consistently found to be negative for infection. A 1 mg/ml stock of recombinant human TRAIL (Merck KGaA, Darmstadt, Germany) was prepared in sterile dH₂O and stored at -80°C. Dilutions were prepared fresh on the day of treatment. A 10 mM stock of embelin (Sigma-aldrich; Merck KGaA) was prepared in ethanol and stored at -20°C. Stocks (50 mM) of both the general caspase inhibitor (ZVAD-FMK; Calbiochem; Merck KGaA) and caspase-8 Inhibitor II [Z-IE(OMe) TD(OMe)-FMK; Calbiochem; Merck KGaA] were prepared in DMSO and ethanol respectively. Dilutions of both inhibitors were prepared fresh on the day of treatments in sterile dH₂O.

BT12, BT16 and G401 cell lines (200 μl) were seeded at densities of 10x10³, 10x10³ and 5x10³ cells/well, respectively, in 96-well plates. Cells were left overnight to adhere and then treated with a range of concentrations of TRAIL or embelin for 72 h. Viability was assessed via the alamar blue assay. Briefly, 20 μl of AlamarBlue (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA) was added to each well. The plate was incubated in the dark at 37°C for 5 h. Fluorescence was measured on a SpectraMax plate reader at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. All alamar blue assays were performed in triplicate.

For cell death analysis via propidium iodide (PI) staining, cells were collected by centrifugation and fixed in 70% ethanol overnight. The following day, 5 μl of FBS and 1 ml of PBS were added to each sample. Cells were collected by centrifugation and resuspended in BD FACSflow sheath fluid (BD Biosciences, San Jose, CA, USA) supplemented with 10 μg/ml of RNase A (Sigma-Aldrich; Merck KGaA) and 100 μg/ml of PI (Sigma-Aldrich; Merck KGaA). The samples were then incubated in the dark at 37°C for 30 min. Analysis was performed with a BD Accuri C6 flow cytometer using BD Accuri C6 software (BD Biosciences). Samples were first gated on vehicle controls. A total of 1x10⁵ cells from each sample were counted and results were visualised on histograms. PI was detected using a 585/40 bandpass filter. The sub G1/G0 population (<2n) was assigned as apoptotic cells.

For cell death analysis via Annexin V-fluorescein isothiocyanate (FITC) and PI staining, cells were collected by centrifugation and washed with 0.5 ml Annexin V binding buffer (5 mM HEPES, 70 mM NaCl, 1.25 mM CaCl₂ pH 7.4). The pellet from this wash was collected and stained with Annexin V-FITC (iQ Corporation, Groningen, Netherlands) for 30 min in the dark on ice. Cells were washed with 0.5 ml of Annexin V binding buffer and collected by centrifugation. The pellet was resuspended in 500 μl PI (0.5 μg/ml). The samples were then analysed immediately on BD Accuri C6 flow cytometer using BD Accuri C6 software. Samples were first gated on vehicle controls. These gates were then analysed on a FITC-Annexin V (535 nm; FL1 channel using a 530/30 bandpass filter) vs PI (488 nm; FL2 using a 585/40 bandpass filter) dot plot.

For whole cell lysates, cell pellets were resuspended in ice-cold RIPA buffer (Sigma-Aldrich; Merck KGaA) supplemented with protease inhibitor cocktail II and III (Sigma-Aldrich; Merck KGaA) and complete ultra protease inhibitor (Roche Applied Science, Penzberg, Germany) and kept on ice for 30 min. Lysates were centrifuged at 10,000 x g for 10 min and the pellet was discarded. For the cytosolic lysates, the cell pellet was resuspended in 75 μl subcellular fractionation buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCL, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitor cocktail) and kept agitated at 4°C for 30 min. The suspension was centrifuged at 720 x g for 5 min and the resultant supernatant was centrifuged further at 10,000 x g for 10 min. The final resultant supernatant was the cytosolic lysate. For all lysates, protein concentration was determined using the BCA assay (Pierce; Thermo Fisher Scientific, Inc.) and normalised. Cell lysates were boiled with Laemmli sample buffer supplemented with 50 μM DTT for 10 min at 90°C. Equal concentrations of denatured samples were separated using polyacrylamide gel electrophoresis and transferred to an immobilion-P PVDF membrane (Merck KGaA). The membrane was blocked for 1 h in 5% non-fat dried milk and then incubated overnight at 4°C with the relevant primary antibody. The following primary antibodies were purchased form Cell Signaling Technology, Inc., (Danvers, MA, USA): XIAP (1:2,500; cat no. 2045), cIAP1 (1:1,000; cat no. 7065), cIAP2 (1:1,000; cat no. 3130), livin (1:1,000; cat no. 5471), survivin (1:1,000; cat no. 2808), caspase-3 (1:1,000; cat no. 9662), cleaved caspase-3 (1:1,000; cat no. 9664), caspase-8 (1:1,000; cat no. 9746), cleaved caspase-8 (1:1,000; cat no. 9496), caspase-9 (1:1,000; cat no. 9508) Bid (1:1,000; cat no. 2002) and Mcl-1 (1:1,000; cat no. 4572). The following primary antibodies were also used: second mitochondria-derived activator of caspases (SMAC; 1:1,000; cat no. 612246; BD Biosciences), FLIP_L (1:2,000; cat no. MABC148; Merck KGaA) GAPDH (1:10,000; cat no. CB1001; Calbiochem), and α tubulin (1:1,000; cat no. CP0; Calbiochem). The following day the membrane was washed three times for 10 min each with TBST, probed with the relevant anti-rabbit (1:2,500; cat no. W402B; Promega Corporation, Madison, WI, U.S.A.) or anti-mouse (1:2,500; cat no. W401B; Promega Corporation) horseradish peroxidase (HRP)-conjugated secondary antibody, and then washed again as aforementioned. The membrane was soaked in enhanced chemiluminescence detection reagent (Merck KGaA). The western blot results were visualised using the BioRad Gel Doc™ XR+System with Image Lab software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

On-targetplus human XIAP siRNA SMARTpool (GE Healthcare Dharmacon, Inc., Lafayette, CO, USA) consisted of four siRNA sequences targeting XIAP, as follows: GUAGAUAGAUGGCAAUAUG, GAACUGGGCAGGUUGUAGA, GAAAGAGAUUAGUACUGAA and GGACUCUACUACACAGGUA. The negative control On-targetplus non-targeting pool (GE Healthcare Dharmacon, Inc.) consisted of four non-targeting siRNA sequences: UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, and UGGUUUACAUGUUUUCCUA. BT12 cells were seeded at 10x10⁴ cells/ml in antibiotic-free media in a 6-well plate. The following day, XIAP siRNA or non-targeting siRNA was transfected into cells using Lipofectamine (Thermo Fisher Scientific, Inc.) at a final concentration of 25 nM, as per manufacturer's instructions. Cell death was assessed 48 h post-treatment by flow cytometric analysis of PI-stained cells and subsequent quantification of the sub G1/G0 peak, as described above. Western blot analysis confirmed XIAP knockdown at 0 and 48 h of TRAIL treatment.

TRAIL and embelin combination treatments were assessed for synergism based upon the apoptosis they elicited, both as single drugs and in combination with one another, as determined by flow cytometric analysis of PI-stained cells, as described above. The computer software program Compusyn (ComboSyn, Inc., New Jersey, U.S.A.) was used to study the interactions between the drugs. This program uses the Chou-Talalay method, based upon the median effect principle, to determine Combination Index (CI) values for each drug combination. CI values <1, =1 and >1 indicate synergistic, additive and antagonistic effects respectively.

GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis of experimental data. Results are displayed as the mean ± standard error of the mean. To determine statistical significance, Student's t-test was used for comparisons between two groups, while a one-way analysis of variance followed by Tukey's multiple comparison test was performed for comparisons of more than two groups. P<0.05 was considered to indicate a statistically significant difference.

BT12, BT16 and G401 cell lines were firstly analysed for expression of XIAP, cIAP1, cIAP2, livin and survivin via western blotting (Fig. 1A). Densitometric analysis was then performed (Fig. 1B-G). All three cell lines were demonstrated to express a range of IAPs, with XIAP, cIAP1 and survivin universally expressed. Low levels of cIAP2 were detected in BT12 cells only. Livin exists as two splice variants; livin α and livin β. Both splice variants were detected in BT12 and G401 cells, while neither was detected in the BT16 cell line.

Yoshida et al (55) have previously reported a decreased viability of MRT cell lines upon TRAIL treatment. Given this, and combined with the XIAP expression demonstrated in Fig. 1, the viability of BT12, BT16 and G401 cell lines was assessed following treatment with TRAIL or the XIAP inhibitor, embelin, for 72 h via the alamarBlue assay (Fig. 2A). Viability dropped to only 60, 71 and 89% for G401, BT12 and BT16 cells, respectively, at 1,000 ng/ml TRAIL. Embelin reduced the viability of all three cell lines in a dose dependent manner. However, even at 50 μM dose, the viability did not decrease below 33% for any cell line. BT12 and BT16 cells had similar IC₅₀ values of 13.06 and 14.52 μM, respectively, with viability plateauing approximately 44 and 37%, respectively. The IC₅₀ value for the G401 cell line was considerably higher at 47.14 μM.

Having shown the expression of XIAP in a range of MRT cell lines, and their resistance to TRAIL treatment, the present study next assessed if embelin could sensitise MRT cell lines to TRAIL. BT12, BT16 and G401 cell lines were treated with a range of concentrations of TRAIL, both alone and in combination with 10 μM embelin, for 24, 48 and 72 h. This concentration of embelin is at the lowest end of the range (10-50 μM) previously demonstrated to enhance susceptibility to TRAIL (47-54). It was selected for the present study, as lower doses are preferable in cancer therapeutics in order to minimise potential side effects. Cell death was assessed via PI staining of permeabilised cells and subsequent quantification of the sub G1/G0 peak. The computer program Compusyn was used to determine synergism. This program computes combination index (CI) values for each drug combination. CI values <1, =1 and >1 indicate synergistic, additive and antagonistic effects respectively, with lower CI values indicating greater synergism. In BT12 cells, treatment with TRAIL or embelin alone did not cause significant cell death compared with vehicle, at any concentration tested. Combination treatment resulted in a dose and time-dependent increase in cell death, which was significant and synergistic at all concentrations and at several timepoints (Fig. 2B; Table I). Treatment with TRAIL at 50 ng/ml in combination with 10 μM embelin was determined to give the greatest synergism with a CI value at 0.169 (Table I). Annexin V-PI staining, was then employed to confirm the increased cell death upon combination treatment with concurring results obtained (Fig. 2C). Similarly, in BT16 cells, embelin synergistically enhanced TRAIL-mediated cell death across a range of timepoints and concentrations, with CI values <1 reported for all concentrations tested at 48 and 72 h (Fig. 2B; Table I). In the G401 cell line, combination treatment did not result in significantly higher cell death compared with TRAIL treatment alone, at any concentration tested (Fig. 2B), possibly owing to differences between MRTs arising in the kidney and the brain.

To determine the mode of cell death initiated upon combination treatment, BT12 cells were pretreated with an inhibitor of necroptosis, necrostatin 1 or the general caspase inhibitor, Z-VAD-FMK. Pretreatment with necrostatin 1 had no effect on cell death (Fig. 3A). By contrast, pretreatment with Z-VAD-FMK prevented cell death (Fig. 3B), indicating apoptosis was occurring. In the literature, TRAIL-induced cell death has mostly been attributed to activation of the extrinsic apoptotic pathway, resulting firstly in caspase-8 activation, followed by downstream activation of caspase-3, caspase-7 and in certain cell types caspase-9 (12). Pretreatment of BT12 cells with a specific caspase-8 inhibitor, Z-IETD-FMK, abrogated cell death (Fig. 3C), suggesting that the extrinsic apoptotic pathway was induced upon TRAIL and embelin treatment. Caspase inhibition was further confirmed via western blot analysis (Fig. 3D).

BT12 and BT16 cells were treated with TRAIL and embelin, both alone and in combination, from 1 to 48 h. In both cell lines, TRAIL treatment resulted in an initial burst of cell death from 4 h (Fig. 4A and B). However, the cells recovered from TRAIL treatment with cell death decreasing steadily from 16 to 48 h in BT12 cells (Fig. 4A), and from 4 to 48 h in BT16 cells (Fig. 4B). By contrast, cell death increased upon combination treatment in a time-dependent manner in both cell lines. This suggests that embelin prevented the recovery of MRT cell lines from TRAIL treatment over time. Given the aforementioned findings that TRAIL and embelin-induced cell death was dependent on caspase-8 activation, the extrinsic apoptotic pathway was examined over a range of timepoints. Treatment with TRAIL alone resulted in a decrease in full length caspase-8 and generation of active caspase-8 from 4 h (Fig. 4D). However, in agreement with the cell death results, caspase-8 activation decreased from 4 to 48 h. By contrast, combination treatment resulted in enhanced caspase-8 cleavage compared with treatment with TRAIL alone from 4 h onwards (Fig. 4D). Bid, a target of caspase-8, was determined to be cleaved at 24 h in TRAIL-treated cells, with cleavage enhanced upon combination treatment (Fig. 4E). To determine if the generation of tBid was sufficient for MOMP, SMAC release into the cytosol was assessed from 1 to 24 h. SMAC is a pro-apoptotic protein usually resident within the mitochondria. However, upon MOMP, SMAC is released into the cytosol where it is free to carry out its proapoptotic functions by binding and inhibiting XIAP (33,56,57). Both TRAIL alone and combination treatment resulted in SMAC release, with levels slightly enhanced at 24 h in combination-treated cells (Fig. 4F). Despite SMAC release into the cytosol, indicative of MOMP, caspase-9 cleavage fragments, which would be formed upon caspase-9 activation, were not detected using an antibody capable of detecting both full length and cleaved fragments (Fig. 4G). A further target of active caspase-8 is caspase-3. Cleavage of caspase-3 results in the generation of an inactive cleavage product of 19 kDa and active cleavage products of 17 and 15 kDa. Treatment with TRAIL alone resulted in generation of both active and inactive cleavage fragments from 4 h onwards (Fig. 4H). However, at later timepoints it was the inactive fragment which predominated. By contrast, combination treatment resulted in enhanced generation of active cleavage fragments over time, with active caspase-3 levels being higher compared with the TRAIL alone-treated cells from 4 h onwards (Fig. 4H). Caspase-3 activation was also assessed in BT16 cells at 24 and 48 h. As per the BT12 cells, combination treatment resulted in enhanced generation of active caspase-3 over time (Fig. 4C).

BT12 cells were treated with TRAIL and embelin, either alone or in combination, from 1 to 48 h and protein expression was analysed via western blotting. The expression levels of proteins which were implicated in sensitising the cells to apoptosis in the BT12 cells were then assessed in BT16 cells at 24 and 48 h. No differences in XIAP expression levels were observed following TRAIL or embelin treatment at any timepoint (Fig. 5A). Combination treatment resulted in slightly reduced expression at 24 and 48 h. XIAP expression in BT16 cells was similar (Fig. 6A). cIAP1 expression levels in BT12 cells were unaffected by any treatment at any timepoint (Fig. 5B). cIAP2 levels in BT12 cells were examined at 48 h only, due to technical limitations with the cIAP2 antibody, and were found to be slightly decreased following embelin or combination treatment (Fig. 5C). As aforementioned, livin exists as two splice variants, both of which can be cleaved by caspase-3, with the cleaved fragments suggested to be proapoptotic. In line with the cleaved caspase-3 data, treatment with TRAIL resulted in livin cleavage from 4 h in BT12 cells (Fig. 5D). However, by 48 h livin levels were comparable to vehicle, in accordance with the recovery of cells from treatment. By contrast, combination treatment resulted in increased livin cleavage compared with treatment with TRAIL alone from 4 h, with cleavage increasing in a time-dependent manner (Fig. 5D). In BT12 cells, survivin levels were increased upon TRAIL treatment from 24 h (Fig. 5E). By contrast, treatment with embelin either alone or in combination with TRAIL resulted in reduced survivin expression from 16 h (Fig. 5E). Survivin levels in BT16 cells at 24 and 48 h were also decreased upon embelin or combination treatment (Fig. 6B). Expression levels of FLIP_L, an analog and inhibitor of caspase-8, were unaffected by TRAIL or embelin single treatment (Fig. 5F). However, combination treatment resulted in reduced FLIP_L expression from 16 h. By contrast, in BT16 cells, FLIP_L levels were unchanged (Fig. 6A). The anti-apoptotic Bcl-2 family member Mcl-1, has previously been demonstrated to be overexpressed in MRT. Mcl-1 levels were unaffected by TRAIL or embelin single treatment, but were found to be decreased from 4 h in combination-treated cells (Fig. 5G). Similarly, in BT16 cells, embelin as a single agent or in combination with TRAIL resulted in reduced Mcl-1 expression at 24 and 48 h (Fig. 6B). Densitometric analysis of the proteins examined in Figs. 5 and 6 have been included as supplemental material (Figs. S1 and S2).

As described above, TRAIL and embelin co-treatment did not result in downregulation of XIAP until 24 h in MRT cell lines, despite enhanced caspase activity from 4 h. However, the initial screening studies which identified embelin as an inhibitor of XIAP did not suggest degradation was necessary for inhibition (37). Given this and combined with previous reports of enhanced susceptibility to TRAIL upon XIAP knockdown, we next assessed if XIAP inhibition could account for the sensitisation of MRT cell lines to TRAIL upon embelin co-treatment. BT12 cells were treated with TRAIL in combination with XIAP siRNA or non-targeting siRNA and XIAP knockdown was confirmed via western blotting (Fig. 7A). While significant knockdown was achieved, low levels of XIAP expression were still detected (Fig. 7A). Despite this, cells treated with XIAP siRNA exhibited significantly enhanced TRAIL-mediated cell death, in contrast to cells treated with non-targeting siRNA (Fig. 7B). It should be noted that this enhanced cell death was not as pronounced as with the embelin cotreatment (Figs. 2B and 4A). While the incomplete knockdown of XIAP may account for this, it is likely that XIAP inhibition via embelin only partially mediates the enhanced cell death. A complex multifactorial mechanism of cell death is supported by the reduced survivin, FLIP and Mcl-1 expression demonstrated upon cotreatment.