MTT and LDH Cytotoxicity Assays in Prostate Cancer

DU145 and LNCaP cells were maintained at low passages (below 30) and cultured in RPMI 1640 (Wisent Bioproducts, QC) with 5 and 10% fetal bovine serum, respectively. For MTT proliferation assays 3500 LNCaP (on poly-L-lysine coated wells) or 1500 DU145 cells were seeded in 96-well plates, and after 24 h, the cells were treated with various concentrations of inhibitors (from 300 to 1 μM). Next, the cells were incubated for 72 h with peptides prior to addition of MTT reagent (Sigma–Aldrich, Canada) at a final concentration of 1 mg/mL. Formazan salt was solubilized with 50 μL DMSO and the metabolic activity was normalized relatively to vehicle-treated cells (sterile bi-distilled water). IC₅₀ values were determined using Prism 5.0 (GraphPad Software, USA), as previously described^{12 (link)}. For LDH assay, the LDH Cytotoxicity Detection Kit (Promega, USA) was used to quantify the release of lactate dehydrogenase following membrane disruption. As described in our previous work^{15 (link)}, DU145 cells were seeded at a density of 3000 cells/well and allowed to incubate for 24 h. Then, the media was subsequently replaced and cells were incubated with compounds at the selected concentration. Cells treated with 1% SDS were used as a control for maximal LDH release and further relative quantification.