Cytotoxicity assay

The cytotoxic effect of MTX, [Dlys⁶]-LHRH, and [Dlys⁶]-LHRH-MTX was evaluated using the methylthiazol tetrazolium (MTT) assay (Sigma Aldrich, MO), which determines the number of viable cells from the formazan crystals produced by metabolic activity^26,33,34. The cells were plated in 96-well plates at 5 × 10³ cells/well in triplicate, and were allowed to reattach overnight. Culture media with various concentrations of MTX or [Dlys⁶]-LHRH or [Dlys⁶]-LHRH-MTX were then added in triplicate, and the incubation was continued for an additional 24 h. After removing the supernatant, DMSO was added (150 μL per well) to dissolve the formazane of MTT. The absorption at 570 nm was recorded with an ELISA plate reader (Bio-Rad, Microplate Reader 550 and the growth inhibitory (GI) rate was calculated according to the following equation: GI(%) = 100 − [T − B)/(C − B) × 100].

In the equation, T represents the absorption value of the treatment group; C is the absorption value of the control (untreated) group; and B refers to the absorption value of the culture medium. IC₅₀ values (μg/mL) were calculated with SPSS software. In a competition assay, Prostate cancer cells (5,000 per well) were separately incubated in 96-well plates in triplicate with [DLys⁶]-LHRH–MTX alone and [DLys⁶]-LHRH free peptide and [DLys⁶]-LHRH–MTX together, and the cells were incubated for 24 hr. The plates were then analyzed with the MTT method.