Cell Cycle Arrest and Apoptosis Induced by Kinamycin F in Human Osteosarcoma Cells

Materials and Methods

Materials and cell culture. The sample of kinamycin F that was used in this study was provided by Professor G. I. Dmitrienko at the University of Waterloo, Waterloo, Ontario, Canada and was prepared semi-synthetically from kinamycin D that was isolated from S. murayamaensis as described previously (7). Control and kinamycin F treatments were dissolved in DMSO and the final DMSO concentration was always 0.5% (v/v). In this study, we analyzed three cell lines derived from human osteosarcomas. The HOS, U-2 OS, and MG-63 human osteosarcoma cell lines were purchased from the American Type Culture Collection (Manassas, VA). Cell lines were routinely cultured in Iscove's modified Dulbecco's medium supplemented with 10% inactivated fetal calf serum (FCS; EuroClone, Wetherby, UK) and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere.

MTT assay. To test the effects of kinamycin F, osteosarcoma cell lines were cultured for 24 or 48 h in the presence of the vehicle (DMSO 0.1%) or increasing drug concentrations, and cell growth was determined using the MTT (3-(4,5-Dimethylthythiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation kit (Roche Diagnostic, Basel, Switzerland), according to the manufacturer's instructions. Briefly, 0.5 mg/ml of MTT labeling reagent was added to each well and incubated for 4 h. Purple formazan crystals were solubilized by adding 100 μl of the solubilization solution (0.01M HCl and 10% SDS) overnight. The plate was subsequently read on an Infinite M200 photometer (Tecan Group Ltd., Männedorf, Switzerland) at a wavelength of 570 nm. Colorimetric readings were normalized against plates of non-treated cells under identical culture conditions. The experiment was performed 3 times, each time in triplicate.

Trypan blue dye exclusion assay. The viability effects of kinamycin F on osteosarcoma cells were determined using trypan blue exclusion and manually counting in microscope chambers. Briefly, cells were washed and suspended (1.0×10⁵ cells/ml) in a buffer solution containing PBS 1X, EDTA 0.5 mM (Life technologies, Burlington, ON, Canada), and BSA 0.2% (Sigma-Aldrich, St. Louis, MO, USA). Fifty (50) μl of cell suspension was taken and mixed with an equal volume of 0.4% Trypan blue. The solution was mixed thoroughly and allowed to stand for 5 min at room temperature. 10 μl of the solution was transferred to a hemocytometer and viable cells were counted as clear cells and dead cells as blue ones. The number of live cells in both treated and control flasks was used for calculating the percent viability.

Cell cycle analysis. Cell cycle analysis was performed using the Muse™ Cell Analyzer (Merck Millipore, Milan, Italy). In brief, after 24 h of treatment, cells were harvested, centrifuged at 300 × g for 5 min and washed once with 1X PBS. After fixing them with 70% ethanol for at least 3h at −20°C, cells were centrifuged at 300 × g for 5 min, washed once with 1X PBS and then 200 μl of Muse™ Cell Cycle reagent was added to each tube with an incubation of 30 min at room temperature in the dark. Samples were then analyzed according to the manufacturer's protocol.

Western blot analysis. Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (ThermoFisher Scientific, Waltham, MA, USA), and 25 U/ml benzonase. The protein concentration was determined by the Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's indications, and 80 μg of cellular lysate was separated by SDS-PAGE on 4-20% gradient gels (ThermoFisher Scientific, Waltham, MA, USA). Gels were transferred onto nitrocellulose membranes and blocked in 5% BSA in 1× TBS containing 0.1% Tween-20 (TBST) and then incubated in primary antibody in TBST. Membranes were washed in 1× PBST and incubated for 1 h in the appropriate secondary antibody in TBST. Primary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA): α-cyclin A (sc-596), α-actin (sc-1616); from Cell Signaling Technologies (Danvers, MA, USA): α-cyclin D3 (2936), α-Cdk2 (2546), α-phospho-Cdk2(Thr160) (2561). The incubation was performed overnight at 4°C. The blots were washed 3 times with 1× TBST, detected using SuperSignal West Pico Reagent (Pierce, Rockford, IL, USA), and visualized in a ChemiDoc digital imaging station (Bio-Rad, CA, USA).

Fluorometric caspase-3 enzyme activity assay. Caspase-3 activity in total cell lysates was determined by a fluorometric EnzChek caspase-3 assay kit (Molecular Probes, Eugene, OR, USA) using 7-amino-4-methylcoumarin-derived substrate Z-DEVD-AMC, according to the manufacturer's instructions. Briefly, kinamycin F and DMSO were administered to the wells (1,5×10⁵ cells/well in a 6-well plate). After 48 h, the cells were harvested using trypsinization and cell lysates were prepared. The protein concentration of the samples was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) and measured against bovine serum albumin standards. Samples of the cell lysates were seeded into standard black 96-well plates as each sample contained 50 μg protein per well. Samples were mixed with reaction buffer and the plate was incubated for 30 min at room temperature avoiding direct light. Then, 200 μmol/l substrate of caspase-3, Z-DEVD-AMC, was added to each well and incubated for 1 h at 37°C in the dark. The assay conditions were standardized in order that the products of the reaction remained in the linear range of detection. The plate was detected by a fluorescence spectrophotometer using 496/520 nm excitation/emission wavelengths and the sample readings were calculated by subtracting the absorbance of blank samples.

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Figure 1.

Cell viability rates and IC₅₀s. MTT assays of HOS, MG-63 and U-2 OS treated for 24 and 48 h with increasing concentrations of kinamycin F (1a). IC₅₀s were calculated after 24 and 48 h of exposure by using the software CalcuSyn (1b). Cells were washed and suspended in a buffer solution containing PBS 1X, EDTA 0.5 mM, and BSA 0.2%. The influence of kinamycin F treatment on the detachment of viable cells grown in culture was studied by comparing the fraction of floating cells between kinamycin F-treated and control cells. Counting and viability were achieved with Trypan blue 0.4% using a hemocytometer chamber (1c). Results are the mean of three different experiments±s.d. Ctrl, Untreated cells.