LNCap cells treated with 3-diol; No_Tmt and 1, 3 and 6hrs after treatment

5α-androstane-3α,17β-diol (3α-diol) is reduced from the potent androgen, 5α-dihydrotestosterone (5α-DHT), by reductive 3α-hydroxysteroid dehydrogenases (3α-HSDs). 3α-diol is believed to be a weak androgen, and has to be oxidized to 5α-DHT before it can exert its androgenic activity on androgen target tissues including the prostate. However, we repeatedly demonstrated that 3α-diol can be a potent androgen, activates cytoplasmic signaling pathway, and may be responsible for androgen-independent prostate cancer growth. A cancer-specific, cDNA-based membrane array was used to determine 3α-diol-mediated gene expressions in prostate cancer progression. Several canonical pathways appeared to be affected by 3α-diol-regulated signaling in LNCaP cells; among them are apoptosis signaling, PI3K/Akt signaling, and death receptor signaling pathways. Biological analysis of 3α-diol-activated signaling confirmed that 3α-diol augmented PI3K/Akt activation can be independent from the classical androgen receptor (AR) signaling. These observations sustained our previous report that 3α-diol continues to supported prostate cell survival and proliferation regardless the status of the AR. We provided the first global analysis of 3α-diol-activated gene expressions and identified cytoplasmic signaling pathways as important components of this response in human prostate cells. 3α-diol may play, therefore, a significant role for transition from androgen-dependent to androgen-independent prostate cancer progression in the presence of androgen blockade. Keywords: array, time course, androgen, prostate, cancer Human prostate cancer cell line LNCaP were maintained in the complete medium consisting of RPMI 1640 supplemented with 10% FBS, 5 units/ml penicillin, and 5 µg/ml streptomycin at 37C and 5% CO2. Cells were passaged every 3-5 days or whenever cells reach 70-80% confluence. To perform androgen stimulation followed by total RNA and protein extraction, 1 x 106 LNCaP cells were first seeded in 60 mm tissue culture plates, subjected to serum starvation by culturing the cells with OPTI-MEM supplemented with 1% CD FBS for 24 hours followed by 3α-diol stimulation. To determine the involvement of AR in 3α-diol regulated cytoplasmic signaling, AR was suppressed by transfecting LNCaP cells with the plasmid vector-based siRNA constructs. Briefly, 2×106 LNCaP cells were first mixed with 3 µg AR specific or control siRNA plasmid in 100 µl of Nucleofector solution R; and transfection was accomplished using the Nucleofector transfection device (Amaxa Biosystems; Gaithersburg, MD). Cells were then returned to pre-warmed completed medium for 24 hours followed by serum deprivation for another 24 hours and 3α-diol stimulation. To identify transcription profiling in LNCaP cells following 3α-diol stimulation, cDNA-based membrane arrays were used. There were a total of 1,186 genes present in the Atlas Human Cancer 1.2 Expression Array. Following the washing steps, array membranes were exposed to phosphor-imaging screens (Packard BioScience; Meriden, CT); and images were captured by a Cyclone storage phosphorimager system (Packard BioScience). Results from the phosphor-imaging system were presented as digital light units (DLU) and interpreted using OptiQuant image analysis software (Packard BioScience). The background of the array membranes was determined by the average and standard deviation (SD) of all 276 background spots for each array consisting of spots between probes areas and spots on the rims indicated in the array membranes. Total system noise around the true zero point was normally distributed with right-tail positively skewed. Points exceeding 3 SD above the mean were eliminated until the true normal distribution was successively observed; usually this procedure involved the removal of no more than 5-10 points. The mean of the remaining background measurements was subtracted from all expression measurements. Genes whose expression levels exceeded above 2 SD of the background levels were considered to be regulated genes. In contrast, genes whose expression levels did not exceed the 2 SD thresholds were marked as below noise threshold (BNT). 3α-diol-regulated genes were identified if their expression levels changed from BNT to exceed the 2 SD thresholds (off-on genes) or the opposite (on-off genes) following 3α-diol stimulation. The collective responsive genes were used to identify underlying pathways regulated by 3α-diol in prostate cells. The responsive genes were analyzed as one set by Ingenuity Pathway Analysis, a web-based bioinformatics application, to identify canonical pathways over-represented in the data set. The identified pathways were then evaluated against the full expression data to identify each gene’s potential participation in 3α-diol-regulated cellular responses. The responsive genes were not ranked by expression levels. Human prostate cancer cell line LNCaP was obtained from ATCC (CRL-1740; Manassas, VA). 3α-diol and mouse anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). [α-32P]dATP (3,000 Ci/mmol, 10 µCi/µl) was acquired from Amersham Pharmacia (Piscataway, NJ). RPMI 1640 medium, OPTI-MEM, penicillin-streptomycin, fetal bovine serum (FBS), and Lipofectamine 2000 were purchased from Invitrogen (Grand Island, NY). Charcoal-dextran treated (CD) FBS was obtained from HyClone (Logan, UT). Atlas nylon Human Cancer 1.2 cDNA Expression Array was obtained from Clontech Laboratories (Palo Alto, CA). Rabbit anti-phospho Akt Ser(473) antibody was purchased from Cell Signaling Technology (Danvers, MA). For Western blot analysis total cellular proteins were harvested with RIPA buffer supplemented with proteinase inhibitors. Protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Pierce; Rockford, IL). Total proteins separation and transfer were also performed. Phospho-Akt and b-actin expressions were detected by incubating protein membranes with appropriate primary antibodies against these molecules followed by peroxidase-conjugated secondary antibodies incubation. Immunoreactive proteins were detected using the enhanced chemiluminescent (ECL) reagent (Pierce).