HepG2 and Huh-7 cells (1×10⁴/well) are seeded in 96-well plates in complete culture medium and incubated for 24 h. The cells are then exposed to 100 nM Rocaglamide and/or 100 ng/mL TRAIL for 24 h. The control cells are treated with DMSO at a concentration equal to that used for the drug-treated cells. The complete culture medium is then removed and MTT (200 μL, 0.5 mg/mL in 10% FBS-containing DMEM) is added to each well and the plate is incubated for 2 h at 37°C in a humidified incubator. The solution is then removed from the wells and 200 μL DMSO is added to each well prior to agitation. The absorbance at 570 nm is read using a microplate reader (Bio-Tek ELx800). The value for the vehicle-treated cells is considered to indicate 100% viability. Furthermore, a crystal violet assay is carried out. Briefly, the cells (1×10⁵/mL) are seeded in a 12 well plate for 12 h, and treated with TRAIL (0-100 ng/mL) and/or RocA(1-100 nM) for 12 h. The treated cells are washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and stained using crystal violet for a further 30 min^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
The Huh-7 cells (3×10⁶), suspended in 100 μL mix (equal volumes of DMEM and Matrigel), are implanted subcutaneously into the right flank of 10 female SCID mice (6-week-old) and then randomly divided into two equal groups, one of which received an intraperitoneal injection of Rocaglamide (2.5 mg/kg in 80 μL olive oil; n=5) and the other, used as a vehicle control, received olive oil alone (n=5). These treatments are performed once daily for 32 days and the tumor volumes and body weights of the animals are measured twice a week. The tumor volumes (mm³) are calculated using the following formula: Tumor volume=LS²/2, where L is the longest diameter and S is the shortest. At the end of the experiments, the mice are sacrificed and tumor samples are harvested, fixed in formalin and embedded in paraffin as tissue sections for immunohistochemical analysis.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Santagata S, et al. Tight coordination of protein translation and heat shock factor 1 activation supports the anabolic malignant state. Science. 2013 Jul 19; 341(6143): 1238303.  [Content Brief]

  [2]. Luan Z, et al. Rocaglamide overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells by attenuating the inhibition of caspase-8 through cellular FLICE-like-inhibitory protein downregulation. Mol Med Rep  [Content Brief]

  [3]. Baumann B, et al. Rocaglamide derivatives are potent inhibitors of NF-kappa B activation in T-cells. J Biol Chem. 2002 Nov 22;277(47):44791-800.  [Content Brief]