Chronic IL-1 exposure drives LNCaP cells to evolve androgen and AR independence
Fig 5
LNas1 and LNbs1 IL-1 sublines have active AR signaling but constitutively low PSA levels.
LNCaP, LNas1, LNbs1 and C4-2B cells were transfected with 70 nM AR siRNA or siRNA control in DMEM/2.5% FBE or treated with 10 nM R1881 or vehicle control in DMEM/0% FBE and analyzed on day 4 for (A, B) RNA levels by RT-qPCR or (C, D) protein accumulation by western blot. AR siRNA reduced and R1881 increased the mRNA and/or protein accumulation of AR target genes PSA, KLK2 and NKX3.1 in LNCaP, LNas1 and LNbs1 cells. (E) LNCaP cells were treated with 25 ng/ml IL-1α or IL-1β for 3 days and LNas1 and LNbs1 cells were left untreated. Conditioned medium from the LNCaP, LNas1 and LNbs1 cells was analyzed for secreted PSA by western blot. LNCaP cells secreted PSA and IL-1 treatment reduced PSA secretion in LNCaP cells. LNas1 and LNbs1 secreted low levels of PSA compared to LNCaP cells. * indicates BSA on the ponceau stain. (F) LNCaP, LNas1 and LNbs1 were grown in DMEM/10% serum for 1 month and analyzed for PSA protein accumulation. PSA levels remain repressed in LNas1 and LNbs1 cells. Error bars, ± STDEV of 3 biological replicates; p-value, *≤0.05, **≤0.005, ***≤0.0005. For each individual cell line, fold mRNA levels are normalized to the cell line vehicle control; basal levels are not compared between the cell lines. Western blot band densitometry is normalized to β-actin and fold change normalized to LNCaP vehicle control.