The chemicals used for cell culture were acquired from Gibco® (Gaithersburg, MD, USA). Aminopolycarboxylic acid/acetoxy methyl (fura-2/AM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM) were purchased from Molecular Probes® (Eugene, OR, USA). All other chemicals were purchased from Sigma-Aldrich® (St. Louis, MO, USA).
PC3 human prostate cancer cells were purchased from Bioresource Collection and Research Center (Taiwan). Cells were cultured in RPMI-1640 medium. The medium had penicillin (100 units/mL)-streptomycin (100 μg/mL) and fetal bovine serum (10%) kept at 37°C in a humidified 5% CO2 atmosphere.
Solutions used in [Ca2+]i measurements.
Ca2+-containing medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 5 mM glucose. Ca2+-free medium contained similar chemicals as Ca2+-containing medium except that CaCl2 was replaced with 0.3 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM MgCl2. Esculetin was dissolved in ethanol as a 0.1 M stock solution. The other chemicals were dissolved in water, ethanol or dimethyl sulfoxide (DMSO). The concentration of solvents in the experimental solutions did not exceed 0.1%, and did not affect viability or basal [Ca2+]i.
Confluent cells grew on 6 cm dishes were trypsinized and made into a suspension in culture medium at a concentration of 106mL-1. Trypan blue exclusion was used to determine cell viability. After the treatment, the viability was greater than 95%. Then cells were loaded with 2 mM fura-2/AM for 30 min at 25oC in the same medium. Afterwards, cells were washed with Ca2+-containing medium twice and were made into a suspension in Ca2+-containing medium at a concentration of 107mL-1. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25oC) with continuous stirring; the cuvette had 1 mL of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer immediately after 0.1 mL cell suspension was added to 0.9 mL Ca2+-containing or Ca2+-free medium, by recording excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1-s intervals. During the recording, reagents (PMA (1 nM); GF109203X (2 mM); econazole (0.5 mM), nifedipine (1 mM), SKF96365 (5 mM), thapsigargin (1 mM), U73122 (2 mM), or ATP (4 mM )) were administered to the cuvette by pausing the recording for 2 s to open and close the cuvette. After completion of the experiments, the detergent Triton X-100 (0.1%) and CaCl2 (5 mM) were added to the cuvette to obtain the maximal fura-2 fluorescence; then the Ca2+ chelator EGTA (10 mM) was added to chelate Ca2+ in the cuvette to obtain the minimal fura-2 fluorescence for calibration of [Ca2+]i after 20 min of fluorescence measurements. Control experiments showed that cells bathed in a cuvette had a viability of 95%. [Ca2+]i was calculated as described previously17. Mn2+ smothering of fura-2 fluorescence was performed in Ca2+-containing medium containing 50 mM MnCl2. MnCl2 was added to cell suspension in the cuvette 30 s before the fluorescence recording was started. Data were recorded at excitation signal at 360 nm (Ca2+-insensitive) and emission signal at 510 nm at 1-s intervals as described previously18.
Cell viability assays.
Cell viability measurements were based on the ability of cells to cleave tetrazolium salts by dehydrogenases. Increases in the intensity of color correlated with the number of live cells. Assays were conducted based on manufacturer’s instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at a concentration of 104 cells/well in culture medium for 24 h in the presence of 0-70 mM esculetin. The cell viability detecting tetrazolium reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1; 10 mL pure solution) was added to samples after esculetin treatment, and cells were incubated for 30 min in a humidified atmosphere. In experiments using BAPTA/AM to chelate cytosolic Ca2+, cells were treated with 5 mM BAPTA/AM for 1 h before esculetin (0-70 mM) incubation. The cells were washed once with Ca2+-containing medium and incubated with or without esculetin for 24 h. The absorbance of samples (A450) was analyzed using an enzyme-linked immunosorbent assay (ELISA) reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value.
Data are reported as mean ± SEM (standard error of the mean) of three separate assays and were analyzed by one-way analysis of variances (ANOVA) using the Statistical Analysis System (SASâ, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post-hoc analysis using the Tukey’s HSD (honestly significantly difference) procedure. A p-value less than 0.05 were considered significant.