We determined protein levels and subcellular distribution of thioredoxin 1 (Trx1) in human prostate tissues using tissue microarrays and analyzed redox changes of Trx1 in the nucleus and cytoplasm in cell culture models with redox western blot technique. We demonstrated increased nuclear Trx1 levels in high- versus low-grade human prostate cancers. Despite increased protein levels, the oxidized forms of nuclear Trx1 were higher in prostate cancer cell lines compared to their benign counterparts, suggesting that nuclear redox imbalance occurred selectively in cancer cells. A growth-stimulating dose of androgen caused transient oxidation of Trx1 in androgen-responsive prostate cancer cells only, suggesting a loss of both androgen- and redox-signaling mechanism during cancer progression. Androgen-independent PC3 cells showed a significant increase in nuclear and cytoplasmic Trx1 protein levels, but a significant decrease in total Trx activity. Trx1 redox states and activity, correlated with the sensitivity of prostate cancer cells to prooxidant agents, and downregulation of Trx1 sensitized cancer cells to these agents. Our findings suggest that loss of Trx function due to oxidation and corresponding redox imbalance may play important roles in prostate cancer progression and response to therapies; and Trx1 may serve as a biomarker of subcellular redox imbalance in prostate cancer.
Cell culture and treatment Benign human prostate epithelial cells (PrEC) were purchased from Lonza Walkersville Inc. (Walkersville, MA). LNCaP (ATCC CRL-1740) and PC3 (ATCC CRL-1435) cell lines were obtained from the American Type Culture Collection (Manassas, VA). Recent cultures of PC3 and LNCaP cells were analyzed using single-locus short tandem repeats (STR) DNA typing by Biosynthesis Inc. (Lewisville, TX) to authenticate cell identity. No contamination or misidentification was detected (data not shown). C4-2B cells were purchased from ViroMed Laboratories (Minnetonka, MN). PrEC cells were grown in serum-free prostate epithelial growth media (PrEGM) (Lonza/Cambrex). LNCaP, C4-2B and PC3 cells were maintained in RPMI 1640 supplemented with 5% fetal bovine serum and 1% antibiotic-antimycotic (Life Technologies, Inc., Rockville, MD) at 37°C in a humidified atmosphere of 95% air and 5% CO2, and cells grown less than 30 passages were used. In experiments designed to assess the effects of androgen treatment, cells were seeded in androgen-depleted media containing 5% Charcoal/Dextran treated FBS and 1% antibiotic-antimycotic for 24 h before R1881 treatment. R1881 concentration used in this study was 1 nM, an optimal dose stimulating growth of LNCaP cells. Vehicle was 0.001% ethanol.