Osthole Induces Apoptosis, Suppresses Cell-Cycle Progression and Proliferation of Cancer Cells

Materials and Methods

Apparatus. A Spectrum HPCCC apparatus (Dynamic Extractions, Slough, UK) was employed in the present study. The instrument was equipped with two multilayer PTFE coils: analytical and semipreparative (0.8 mm ID, 22 ml volume and 1.6 mm ID, 136 ml volume), respectively and working with the optimal speed of 1,600 rpm. The effluent was monitored at 320 nm by an ECOM Sapphire UV detector (Prague, Czech Republic). Identification of separated fractions was elucidated on an Agilent 1100 HPLC chromatograph coupled with 250 mm × 4.6 mm stainless steel column packed with 5 μm C₁₈ (Zorbax Eclipse XDB-C18, Agilent Technologies, address) with a diode array detector (DAD) detector.

Reagents. Dichloromethane, n-heptane and ethyl acetate used for preparation of the extract from plant material was of analytical grade and purchased from Polish Reagents (POCH, Gliwice, Poland). Water was purified using a Millipore laboratory ultra pure water system (Simplicity™ system, Millipore, Molsheim, France). Methanol used for HPLC was of chromatographic grade (J.T. Baker Inc., city, The Netherlands).

Plant material. The plant material was collected in the Medicinal Plants Garden, Medical University in Lublin in June 2011. Aerial parts (herb) of Mutellina purpurea were dried at room temperature (RT), powdered and left for further extraction. Voucher specimens No ES17/18-19/2011 were deposited in the Department of Pharmacognosy with Medicinal Plant Unit, Medical University, Lublin. Fruits were air-dried at RT, powdered and 50 g was extracted with 500 ml of petroleum ether under reflux in 30 min. The procedure was repeated three times. The filtrates were combined and concentrated with a rotary evaporator to remove the solvent. The dried crude extract (4.9 g) was stored in a refrigerator for subsequent HPCCC separation.

Selection and preparation of two-phase solvent system. Two-phase solvent systems made of n-heptane, ethyl acetate, methanol and water (HEMWat) in different volume ratios were tested. Partition coefficient K, as a ratio of target substance in stationary to mobile phase, was determined by high-performance liquid chromatography (HPLC) analysis (comparing the peak area of the osthole both in upper and lower phases). A volume ratio 3:2:3:2 was chosen as the most proper for purification of target compounds (K=1.8). Then, the analytical column was first entirely filled with the upper stationary phase. The apparatus was rotated at 1,600 rpm and the lower mobile phase was pumped into the column at a flow rate of 1.0 ml/min. After hydrodynamic equilibrium was reached, 60 mg of extract dissolved in 1 ml of two-phase solvent system was loaded onto the column through a 1-ml injection valve. The solid phase retention was 70%. The effluent from the column was continuously monitored with a UV detector at 320 nm. Each one minute fraction was manually collected into test tubes. At the end of the run, the fractions were evaporated under reduced pressure and redissolved in methanol for subsequent HPLC analysis. Osthole was detected in fractions 40-45 with amount of 0.2 mg. The separation procedure was repeated in order to obtain the desirable amount. The crude methanol extract and each fraction obtained from the HPCCC separations were analyzed by HPLC. During HPLC analysis the flow rate was 1 ml/min, the column temperature was 25°C. A stepwise mobile phase gradient was prepared from methanol (A) and water (B). The gradient was: 0-5 min 50-60% A; 5-25 min 60-80% A; 25-30 min isocratic 80% A; 30-40 min 80-100% A.

The identification of isolated compound was carried out by comparison of retention time and UV-DAD spectra with those obtained by standards under the same conditions. The purity of osthole was 99% (established by the HPLC-DAD method).

Cell lines. Human medulloblastoma (TE671) was obtained from the Department of Medical Biology of Institute of Agricultural Medicine in Lublin. Human larynx carcinoma (RK33) was derived from a patient with diagnosed larynx squamous cell carcinoma. Cancer tissue was removed from the larynx after total laryngectomy and established as stable cell line, as previously described (35). Cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) culture medium (Sigma, address) and 1:1 mixture of DMEM and Nutrient mixture F-12 Ham (Sigma) for RK33 and TE671 cells, respectively. All media were supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Karlsruhe, Germany), penicillin (100 U/ml) (Sigma) and streptomycin (100 μg/ml) (Sigma). Cultures were kept at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Cell viability assessment. Tumor cells were placed on 96-well microplates (Nunc, Langenselbold, Germany) at a density of 2×10⁴/ml (RK33), 1x10⁴/ml (A549, TE671). The folloing day the culture medium was removed and cells were exposed to serial dilutions of osthole in a fresh culture medium. Cell proliferation was assessed after 72 h by means of the methylthiazolyldiphenyl-tetrazolium bromide (MTT) method in which the yellow tetrazolium salt (MTT) is metabolized by viable cells to purple formazan crystals. Tumor cells were incubated for 3 h with MTT solution (5 mg/ml, Sigma). Formazan crystals were solubilized overnight in SDS buffer (10% SDS in 0.01 N HCl) and the product was determined spectrophotometrically by measuring absorbance at a wavelength of 570 nm using an Infinite M200 Pro microplate reader (Tecan, Männedorf, Switzerland).

Cell proliferation assay. The Cell Proliferation ELISA, 5-bromo-2-deoxyuridine (BrdU) Kit (Roche Diagnostics, Mannheim, Germany), was applied. Optimized cell amount (2×10⁴) was applied on the 96-well plate (100 μl/well). The cells were treated with 100 μM concentrations of osthole for 72 h, followed by incubation with BrdU (100 μM). Cells were than fixed in FixDenat solution (30 min, RT). Monoclonal anti-BrdU antibodies coupled with horseradish peroxidase were subsequently added (90 min, RT) and detected using TMB (tetramethylobenzidine) substrate (30 min, RT). 1M sulphuric acid was added to stop enzymatic reaction and quantitation was performed spectrophotometrically at 450 nm using the Infinite M200 Pro microplate reader (Tecan).

Assessment of cell death. Measurement of cell death was performed using the Cell Death Detection ELISAPLUS kit (Roche). RK33 and TE671 cells growing on 96-well microplates were subjected to osthole (100 μM) for 24 h, whereupon supernatants were removed and cells lysed with 200 μl of lysis buffer for 30 min on ice. Subsequently, cell lysates were centrifuged at 200 × g for 10 min. and 20 μl of the sample were carefully transferred into the streptavidin-coated 96-well microplate. The immunoreagent (80 μl) containing anti-histone-biotin and anti-DNA-POD mouse monoclonal antibody was added and incubated under gentle shaking (300 rpm) for 2 h at 20°C. The solution was removed by tapping, each plate well was rinsed three times with 250 μl of incubation buffer and, finally, 100 μl per well of substrate, 2,2’-azino-bis(3-ethylbenzthiazoline-sulfonic acid) solution (ABTS), was applied and incubated at RT for 15 min on a plate shaker (250 rpm). Absorbance was measured at a wavelength of 405 nm using an Infinite M200 Pro microplate reader (Tecan).

Flow cytometry analysis. Experiments were performed using the FACSCalibur™ flow cytometer (BD Biosciences, San Diego, CA, USA), equipped with a 488-nm argon-ion laser. For cell-cycle analysis, cells were fixed in 70% ethanol at −20°C. After fixation, the cells were stained with propidium iodide utilizing the PI/RNase Staining Buffer (BD Biosciences) according to the manufacturer's instructions. Acquisition rate was at least 60 events per second in low acquisition mode and at least 10,000 events were measured. Cell-cycle analysis was performed by using a non-commercial flow cytometry analyzing software - Cylchred Version 1.0.2 for Windows (source: University of Wales, Cardiff, Wales, UK) and WinMDI 2.9 for Windows (source: facs.scripps.edu/software.html). The cells were acquired and gated by using dot plot FL-2 Width (X) versus FL-2 Area (Y)-gate to exclude aggregates and analyzed in histograms displaying fluorescence 2-area (yellow-orange fluorescence: 585 nm).

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Figure 1.

Osthole suppress proliferation of TE671 and RK33 cells. The cells were exposed to either culture medium-alone (Control), or osthole (10-100 μM) for 72 h. Normalized cell viability, measured by the MTT assay (A), and BrdU incorporation (B) is presented as mean±SEM at each concentration. Student's t-test revealed significant effect (*p<0.05, **p<0.01, ***p<0.001) of treatment with osthole compared to vehicle-treated cultures. This effect was also concentration-dependent, as judged by analysis of variance ANOVA test (***p<0.001). n=24 per concentration from three independent experiments.

RNA isolation and cDNA synthesis. Tumor cell lines RK33, A549 and TE671 were incubated on the 6-well microplates with the 100 uM concentration of osthole for 24 hours. Total RNA from the cells was isolated and digested with DNase using the High Pure RNA Isolation Kit (Roche) following the manufacturer's instructions. The RNA concentration was determined spectrophotometrically with a UV-VIS Genesys 10S spectrophotometer at 260/280 nm (Thermo Fisher Scientific, Madison, WI, USA). Three μg of total RNA was reverse transcribed for 30 min at 50°C using an oligo(dT) primer and the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) followed by 5 min enzyme inactivation at 85°C according to manufacturer's instructions.

Quantitative polymerase chain reaction (qPCR). Quantitative real-time expression analysis was performed using a LightCycler® 480 II instrument (Roche). Analysis was performed utilizing Universal ProbeLibrary (UPL) hydrolysis probes specific for the TP53 and CDKN1A genes labeled with FAM in duplex with probe for reference gene GAPD labeled with Yellow 555 (Roche). The primers and probe sets were as following: TP53 (For 5’-CCCCAGCC AAAGAAGAAAC-3’, Rev 5’-AACATCTCGAAGCGCTCAC-3’, Probe 5’-GGATGGAG-3’); CDKN1A (For 5’-TCACTGTCTTGTACCCTTGTGC-3’, Rev 5’-GGCGTTTGGAGTGGTAGAAA-3’, Probe 5’-CCTGGAGA-3’); GAPD (For 5’-CTCTGCTCCT CCTGTTCGAC-3’, Rev 5’-GCCCAATACGACCAAATCC-3’, Probe 5’-CTTTTGCGTCGC-3’). Amplification was performed in 10 μl of reaction mixture containing cDNA amount corresponding to 12.5 ng of total RNA, 1 x LightCycler® 480 Probes Master (Roche) and appropriate set of 0.4 μM primers and 0.2 μM UPL hydrolysis probes for each target and reference duplex. After 10 min of initial incubation at 95°C, cDNA was amplified in 45-55 cycles consisting of 10 s denaturation at 95°C, 30 s annealing at 60°C and 10 s elongation at 72°C. Obtained fluorescence data was calculated using a relative quantification method with efficiency correction.

Statistics. The calculations were done by means of the Student's t-test and analysis of variance (ANOVA) test for multiple comparisons. Data are expressed as the mean±standard error of the mean (SEM) (*p<0.05, **p<0.01, ***p<0.001).