Sensitization of renal carcinoma cells to TRAIL-induced apoptosis by rocaglamide and analogs

Rocaglamide has been reported to sensitize several cell types to TRAIL-induced apoptosis. In recent years, advances in synthetic techniques have led to generation of novel rocaglamide analogs. However, these have not been extensively analyzed as TRAIL sensitizers, particularly in TRAIL-resistant renal cell carcinoma cells. Evaluation of rocaglamide and analogs identified 29 compounds that are able to sensitize TRAIL-resistant ACHN cells to TRAIL-induced, caspase-dependent apoptosis with sub-µM potency which correlated with their potency as protein synthesis inhibitors and with loss of cFLIP protein in the same cells. Rocaglamide alone induced cell cycle arrest, but not apoptosis. Rocaglates averaged 4–5-fold higher potency as TRAIL sensitizers than as protein synthesis inhibitors suggesting a potential window for maximizing TRAIL sensitization while minimizing effects of general protein synthesis inhibition. A wide range of other rocaglate effects (e.g. on JNK or RAF-MEK-ERK signaling, death receptor levels, ROS, ER stress, eIF4E phosphorylation) were assessed, but did not contribute to TRAIL sensitization. Other than a rapid loss of MCL-1, rocaglates had minimal effects on mitochondrial apoptotic pathway proteins. The identification of structurally diverse/mechanistically similar TRAIL sensitizing rocaglates provides insights into both rocaglate structure and function and potential further development for use in RCC-directed combination therapy.


SUPPLEMENTARY FIGURE S1: Effects of rocaglates on ACHN renal carcinoma cell growth/survival:
ACHN cells were pre-treated for 4 h in the presence of variable concentrations of rocaglates followed by 18-24 h in the presence or absence of TRAIL (40 ng/ml final). Cell numbers were assessed using the XTT assay and normalized to untreated control cells on the same assay plate. Error bars (n = 2-6) not included for clarity. A: Cells were treated for 4 h with the indicated rocaglate at 100 nM followed by 4 h TRAIL and assessment of caspase 8 activity or 12 h TRAIL and assessment of caspase 3 activity. Results were normalized to untreated (DMSO, -TRAIL) controls on the same plate. Error bars represent ± sd (n = 3). B: Cells were pretreated for 2 h with or without 100 µM Z-VADFMK followed by 4 h ± rocaglate (100 nM final concentration), then ± TRAIL for 18 h and assessment of cell survival by XTT. Error bars represent ± sd (n = 4).

SUPPLEMENTARY FIGURE S3: Effects of the rocaglates on cFLIP protein expression:
Cells were treated for 4 h with the indicated rocaglates at 10, 100, or 1000 nM followed by quantitative western blot analysis of cFLIP isoforms. Values were normalized to loading control (GAPDH) and expressed as a % of signal for untreated cells run on the same gel. Results are sorted by IC 50 from most to least potent for TRAIL sensitization (see Supplementary Table 1 for values). Cells were pretreated for 1 h with either the MEK inhibitor UO126 (20 µM), the JNK inhibitor SP600125 (50 µM), or N-acetyl cysteine (NAC, 10 mM) followed by the indicated rocaglate (1000 nM final) for 4 h, then ± TRAIL for 24 h. Cell survival was estimated using the XTT assay. However, NAC interferes with this assay, so for that experiment, cell survival was estimated by assessment of total protein using an SRB assay. Cell numbers were normalized to untreated (vehicle) control and reported as % of control values. ACHN cells were treated for 24 h with ROC followed by flow cytometric analysis of surface expression of TRAIL receptors DR4, DR5, and "decoy" receptors TR3 (DcR1) and TR4 (DcR2).

Top panel shows vehicle control cells (left) and ROC-treated cells (right) while the bottom panel shows vehicle control vs. ROC treated cells for each receptor.
PE-conjugated antibodies were obtained from ThermoFisher and used according to the manufacturers recommendations. Antibodies: DR4 (clone DJR1), DR5 (clone DJR2-4), TR3 (clone DJR3), TR4 (clone TRAIL-R4-01) Data were acquired on an Accuri C6 flow cytometer and analyzed using the instrument's CFLOW software (Accuri Cytometers, Ann Arbor, MI, USA).

SUPPLEMENTARY FIGURE S7: Effect of rocaglamide and analogs on CAKI-1 and SN12C RCC cells:
CAKI-1 and SN12C cells were pre-treated for 4 h in the presence of variable concentrations of rocaglates followed by 18-24 h in the presence or absence of TRAIL (40 ng/ml final). Cell numbers were assessed using the XTT assay and normalized to untreated control cells on the same assay plate (left panels below). In parallel, cells were treated for 4 h with rocaglates followed by analysis of protein synthesis (right panels below). Error bars represent sd, n = 3-4. HRE cells were pre-treated for 4 h in the presence of variable concentrations of rocaglates followed by 18-24 h in the presence or absence of TRAIL (40 ng/ml final). Cell numbers were assessed using the XTT assay and normalized to untreated control cells on the same assay plate. In parallel, cells were treated for 4 h with rocaglates followed by analysis of protein synthesis (combined in last graph below). Error bars represent sd, n = 4. Vertical bar represents ~IC 50 value for sensitization of ACHN cells. ACHN cells were pretreated for 4 h or 8h with 1 µM rocaglamide followed by total RNA extraction using Qiagen RNeasy Mini Kit. 2 µg of RNA was used to make cDNA using ThermoFisher High-Capacity reverse transcription kit in a 25 µL reaction. 5 µL of cDNA was used for a 50 µL PCR reaction using Qiagen PCR Master Mix kit. PCR conditions were 95 degrees 10 min, 95 degrees 45 sec, 55 degrees 45 sec, 72 degrees 45 sec for 25 cycles followed by a final extension at 72 degrees for 7 min. The following custom primers from IDT were used for detection and quantification of mRNA for cFLIP short and long form: cFLIPL_Forward GGA CCT TGT GGT TGA GTT GG cFLIPL_Reverse ATC AGG ACA ATG GGC ATA GG cFLIPS_Forward GGC TCC CAG AGT GTG TAT GG cFLIPS_Reverse AGC TTC TCG GTG AAC TGT GC 28 SUPPLEMENTARY FIGURE S10: Original uncropped blots -data for Figures 3, 4, and 6.
For all original uncropped blots, GREEN: rabbit primary antibody, detection with goat anti-rabbit (800) secondary RED: mouse primary antibody, detection with goat anti-mouse (680) secondary Analysis on LiCor Odyssey -simultaneous analysis of red and green signals. For clarity, each individual signal is shown separately (side-by-side for same blot). In some cases 3 blots appear because the blot was stripped and re-probed.
NOTE: unlabeled lanes include extracts from cell extracts irrelevant to this manuscript.
EFFECT OF ROCAGLAMIDE ON cFLIP PROTEIN (main text Figure 3C).