MTT assay

The HCT-116 (wild-type p53) and HT-29 (mutant p53) cells were plated into the 96 well plates at a density of 5×10³ in 200 μL of medium per well and the cells were incubated and allowed to attach overnight. The attached cells in the plates were treated with a series of drug concentrations: pramlintide (0–102.4 μg/mL), 5-FU (0–200 μM), OXA (0–300 μM), or IRN (0–160 μM) alone or in combination with three different concentrations of pramlintide (5, 10, and 20 μg/mL) that correspond to 0.5×IC50, IC50, and 2×IC50 in HT-29. Cells grown in medium alone (for treatment with pramlintide only) or containing an equivalent amount of DMSO served as control (for other treatment conditions).

Cells were incubated with the drugs at the indicated concentrations for 72 hours. All measurements were done in triplicate. After that, cell proliferation assay was performed per the manufacturer’s protocol. Briefly, MTT dye was added to the treated cells at a final concentration of 0.5 mg/mL in PBS. Then, the plates were incubated at 37°C for 3 hours and the MTT was discarded and the formazan product was dissolved by adding 100 μL of DMSO to each well, followed by shaking for 5 minutes. Then, the plates were read using an enzyme-linked immunosorbent assay plate reader at 570 nm with a reference wavelength of 690 nm. Cell viability was calculated as follows: absorbance of the experimental group/absorbance of the control group. The IC50 value was defined as the concentration needed for a 50% reduction in cell viability. Dose–effect analyses and IC50 calculations were performed using Compusyn software 1.0 (Combosyn Inc., Paramus, NJ, USA).