Cell lines and culture conditions
The androgen independent PC-3 prostate cancer cell line, derived from a bone metastasis of a grade IV human prostatic adenocarcinoma displaying epithelial morphology, and the androgen sensitive LNCaP prostate cancer cells, derived from a left supraclavicular lymph node of human prostate carcinoma, were purchased from the American Type Culture Collection (Manassas, VA), and maintained as recommended by the supplier. Briefly, the cells were grown in RPMI1640 supplemented with 10% FBS, and penicillin/streptomycin (Life Technologies, Grand Island, NY). RWPE-1 primary prostate epithelial cells were purchased from Lonza Bioscience (Basel, Switzerland) and grown as instructed, in keratinocyte SFM medium supplemented with growth factors (Fisher Scientific, Waltham, MA). For cytotoxicity and metabolism activity assays, the cells were seeded on 96 well plates at a density of 5x103 cells/well. For immunoblotting experiments, the cells were seeded on 60 mm plates at a density of at 5x105 cells/cm2.
Reagents
GT3 (98% pure), was purchased from Cayman Chemical (Ann Arbor, MI).The concentration of GT3 in ethanol was determined using an HP-8542A diode array spectrophotometer with the following maximum wavelengths (λmax) and molar extinction coefficients (ε): GT3 λmax 298 nm, ε = 4230. AT (97% pure) was purchased from Tama Biochemical (Tokyo, Japan), its concentration was determined in the same fashion as for GT3.
MTT assay
LNCaP, PC-3, and RWPE-1 cells were dosed with GT3 or AT at concentrations of 10, 20, 40, 60, and 80 µM. After treatment, 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added and incubated for 3 hours (Invitrogen, Carlsbad, CA). Formazan products were solubilized with acidified SDS overnight. Optical density was measured at 570 nm using a Spectramax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). All experiments were done at least three times.
Cell viability assay by measuring the presence of ATP
Cells on tissue culture 96 well plates were treated with either GT3 or AT in concentrations of 10, 20, 40, 60, and 80 µM; dissolution vehicle was ethanol. Following 6, 12 and 24 hours of treatment, cells were allowed to come to room temperature before adding 100 µL/well of CellTiter Glo® reagent from Promega (Madison, WI, USA) that measures ATP via a luciferase reaction. Luminescence indicative of the presence of ATP was measured on a luminometer (Promega).
Western blot analysis
LNCaP, PC-3, and RWPE-1 cells were treated with indicated concentrations of GT3, AT, or vehicle as a control for 6 or 12 hours. Harvested cells were lysed with RIPA Buffer (150 mM NaCl, 1% sodium deoxycholate, 1% Triton, 0.1% SDS, 10mM Tris, 100µM sodium orthovanadate, 50mM sodium fluoride) containing phosphatase and protease inhibitors (Sigma, St. Louis, MO). The protein concentrations of the cell lysates were determined by the BCA protein assay (Cytoskeleton, Denver, CO).The samples were run in SDS-PAGE and blotted onto nitrocellulose or PVDF membranes (Pall Life Sciences, Ann Arbor, MI). Blotted membranes were processed according to recommended protocols for each antibody against total and phosphorylated forms of ERK (9101 and 9102), c-JUN (9165 and 9164), BAD (5284), and mitochondrial marker COX IV, clone 3E11(Cell Signaling, Danvers, MA, USA). Antibodies against AKT, clone SK703 (EMD Millipore, Billerica, MA), cytochrome C clone 7H8.2C12 (BD Biosciences, San Jose, CA), and actin from Sigma (St. Louis, MO). Caspase activation was analyzed with anti- cleaved caspase 3, and cleaved caspase 9 from Cell Signaling (clones 5A1E, D2D4), Abcam and R&D systems. The signal of primary monoclonal or polyclonal antibodies was detected using affinity–purified secondary antibodies with no cross-reactivity with other species, coupled to peroxidase (Pierce and Promega, Fitchburg, WI, USA) and analyzed by a chemiluminescent system. The intensities of the bands on x-ray film were estimated by digitizing the image using Image J, and were compared against a control.
Statistical analysis
Data are represented as the mean ± SE. Comparisons were done relative to the control, and analyses were run by Student's t test when comparing against a control, or ANOVA followed by Bonferroni test when comparing two treatment groups. p < 0.05, p< 0.01 and p< 0.001 indicate statistical significance. (GraphPad Prism 7, La Jolla, CA). The data are shown with error bars representing standard deviation.