Drug Resistance Induction in Prostate Cancer Cell Lines

The Pca cell lines 22RV1 (CRL-2505), LNCaP (CRL-1740), DU145 (HTB-81) and PC3 (CRL-1435) and 293 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or Dulbecco's modified Eagle's medium (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) that were supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO₂. All drugs were purchased from Selleck Chemicals (Houston, TX, USA), and Pca cells were treated at the following concentrations: Nintedanib (3 µM for 4 weeks to develop resistance), PI3K inhibitor buparlisib (1.5 µM for 4 weeks) (17 (link)), protein kinase B (Akt) inhibitor MK2206 (10 µM for 4 weeks) (18 (link)), extracellular-signal-regulated kinase (ERK)1/2 inhibitor SCH772984 (3 µM for 4 weeks) (19 (link)), CDC42 inhibitor ML141 (2 µM for 4 weeks) (20 (link)), or ROCK1/2 inhibitor Y-27632 (10 µM for 1 week) (21 (link)). For drug treatment experiments, all the cells were seeded at a density of 1×10⁵ cells/well in 6-well culture plates in the corresponding medium. Following 24 h of incubation at 37°C for attachment, the cells were treated with drugs.