Apalutamide (ARN-509) is an antiandrogen that binds selectively to androgen 
receptors (AR) and does not show antagonist-to-agonist switch like bicalutamide. 
We compared the activity of ARN versus bicalutamide on prostate cancer cell 
lines. The 22Rv1, PC3, and DU145 cell lines were used to study the effect of ARN 
and bicalutamide on the expression cytoplasmic/nuclear kinetics of AR, AR-V7 
variant, phosphorylated AR, as well as the levels of the AR downstream proteins 
prostate-specific antigen and TMPRSS2, under exposure to testosterone and/or 
hypoxia. The effects on autophagic flux (LC3A, p62, TFEB, LAMP2a, cathepsin D) 
and cell metabolism-related enzymes (hypoxia-inducible factor 1α/2α, BNIP3, 
carbonic anhydrase 9, LDHA, PDH, PDH-kinase) were also studied. The 22Rv1 cell 
line responded to testosterone by increasing the nuclear entry of AR, AR-V7, and 
phosphorylated AR and by increasing the levels of prostate-specific antigen and 
TMPRSS2. This effect was strongly abrogated by ARN and to a clearly lower extent 
by bicalutamide at 10 μmol/l, both in normoxia and in hypoxia. ARN had a 
stronger antiproliferative effect than bicalutamide, which was prominent in the 
22Rv1 hormone-responsive cell line, and completely repressed cell proliferation 
at a concentration of 100 μmol/l. No effect of testosterone or of antiandrogens 
on autophagy flux, hypoxia-related proteins, or metabolism enzyme levels was 
noted. The PC3 and DU145 cell lines showed poor expression of the proteins and 
were not responsive to testosterone. On the basis of in-vitro studies, evidence 
has been reported that ARN is more potent than bicalutamide in blocking the AR 
pathway in normoxia and in hypoxia. This reflects a more robust, dose-dependent, 
repressive effect on cell proliferation.