Crocetin

Cells were treated with different doses of crocetin (MP Biomedical, India) at different time intervals to select the optimum dose (100 μm) and time (24 h) for cancer cell death which was used in all experiments unless stated otherwise. HCT116 and HT29 cells were pre-treated with 20 μM each of the specific caspase-2 (Z-VDVAD-FMK), caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK), caspase-3 (Z-DEVD-FMK) and Pan-caspase (Z-VAD-FMK) inhibitor for 2 h, (Calbiochem, ED chemicals, NJ) or for 1 h with 25 μM mitochondrial pore blocker CsA (Merck, Germany) prior to treatment with crocetin.

The radiation procedure was performed according to our previously described protocols [4 (link)]. Briefly, IEC-6 cells were exposed to 10 Gy doses of radiation using a linear accelerator (Siemens PRIMUS) at a dose-rate of 300 cGy/min. IEC-6 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and grown to 70%~80% confluence prior to experiment. After 10 Gy radiation, IEC-6 cells were replaced with serum-free DMEM-F12 medium and subsequently treated with different doses of crocetin (0, 0.1, 1, 10, and 100 μM, MP Biomedicals, Santa Ana, CA, USA; CAS no.: 27876-94-4), then incubated for 24 h at 37°C. After 24 h incubation, the culture medium was collected for biochemical assay and ELISA and then replaced with new fresh serum-free medium for subsequent condition of IEC-6 cells. To determine the most effective concentration of crocetin in the following experiments, cell viability was assessed daily for the next 7 days after radiation. Further studies were performed at the most effective concentration to improve cell viability.