Fig. 1.

Chronic-ADT in hypoxia induces resistance to targeted disruption of androgen/AR-axis. a Schematic representation of repeated-ADT treatment of LNCaP and LAPC4 cells in hypoxia (1% O₂) or normoxia (20% O₂). Cells were cultured in charcoal-stripped serum (CSS) media with or without 1 nM synthetic androgen R1881 (R1881 or ADT). Each round/cycle of treatment lasted 48 h, and the survived cells were recovered and re-plated for the same treatment in the following cycle. b Dose-dependent sensitivity of LNCaP clones to enzalutamide. Cells derived from a were cultured with CSS + R1881, and treated by increasing doses of enzalutamide in normoxia or hypoxia for 96 h. Afterwards, cell growth and viability were determined based on viable cells quantitated with SYTO 60 fluorescence staining and imaging. The AdtHs is represented by purple line. All values were mean ± s.d. relative to solvent-treated controls, n = 3. *P < 0.01 two-sided t-test. c Time-dependent sensitivity of LNCaP clones to AR-siRNA. Cells derived from a were transfected with a cocktail of siRNA constructs silencing AR (siAR) or non-target control (siNT), and then cultured in media with CSS + R1881 in normoxia or hypoxia for 96 h. Viable cells were quantitated with SYTO 60 every 48 h. The AdtHs treated by siNT or siAR is represented by solid purple or dotted purple line, respectively. All values were mean ± s.d. relative to the beginning of the siRNA at day 1, n = 3 *P < 0.01 two-sided t-test. d LNCaP-AdtHs or AdtNs cells, cultured in media with CSS + R1881, were treated with negative control (R1881), 10 uM enzalutamide (Enza), or siAR for 48 h in normoxia or hypoxia. The cell cycle distribution was determined by flow cytometry, mean ± s.d. n = 3. *P < 0.05, NSD (no significant difference), two-sided t-test. e LNCaP-AdtHs cells were treated as in d. The AR-target gene expression was determined by qRT-PCR, mean ± s.d. relative to R1881 in normoxia, n = 3, ^#P < 0.01 two-sided t-test. f The growth/viability and TMPRSS2 gene expression of LAPC4-AdtHs cells, cultured with CSS + R1881, after being treated by negative control (R1881), ADT, 10 µM enzalutamide or siAR for 96 h in normoxia or hypoxia. All values are mean ± s.d. n = 3, *P < 0.05, **P < 0.01, NSD, two-sided t-test. g The antitumor activity of enzalutamide in nude mice against subcutaneous xenografts established with AdtHs or AdtNs cells. Equal numbers of indicated cells (~5 million) were injected into male nude mice. Enzalutamide (Enza) or vehicle (V) treatment started when the tumors were ~100 mm³. All values are mean ± s.d. n = 6, *P < 0.05, two-sided t-test