Interaction of Radiation and Pemetrexed on a Human Malignant Mesothelioma Cell Line In Vitro

Materials and Methods

Cell line and culture. The cell line used was derived from a human malignant pleural mesothelioma (NCI-H28 cells) and was obtained from the American Type Tissue Culture Collection (Manassas, VA, USA). The NCI-H28 cells were maintained in RPMI-1640 (Invitrogen Corporation, Carlsbad, CA, USA) medium supplemented with 10% heat-inactivated fetal calf serum (BioWest, Nuaillé, France). For experimental use, 5ml of complete medium containing 500-10000 of exponentially growing NCI-H28 cells were seeded into 60 mm diameter Petri dishes at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Radiosensitivity. Irradiation was started 12 h after incubation at 37°C to ensure sufficient time for any cell damage induced by the trypsinization procedure to disappear. Radiation was performed using an X-ray machine (RX-650, Faxitron X-ray Corporation, Lincolnshire, IL, USA) operated at 130 kV and 5 mA with a 0.5 mm Al filter. The dose rate, which was quantified by thermoluminescent dosimetry at the irradiation distance (30 cm), was 87 cGy/min. Graded doses of radiation ranging from 1 to 7 Gy were used. The cells were irradiated in air at room temperature. The effect of irradiation on the cells NCI-H28 was assessed by clonogenic survival.

Pemetrexed sensitivity. PEM was provided by Eli Lilly and Company (Indianapolis, IN, USA), at a concentration of 25 mg/ml. The drug was then divided into aliquots and stored at −80°C and was freshly diluted in culture medium before each experiment. The experimental cultures were exposed to PEM for 2 h at 37°C using graded doses of PEM ranging from 50 to 250 ng/ml. Then, the PEM containing medium was removed and new pre-warmed complete RPMI-1640 medium containing serum was added to the dishes after the dishes had been washed with PBS. The effect of PEM treatment was assessed by clonogenic assay.

Effect of pemetrexed on radiation survival responses. The concentration of PEM required to reduce colony formation by 50% after 2 h treatment (the IC₅₀ of PEM) was used to evaluate the sensitizing effect of PEM in combination with irradiation. The IC₅₀ of PEM was 100 ng/ml (see Results). The experiments were started 12 h after the cell inoculation using the procedures described above. The NCI-H28 cells were irradiated with graded doses of radiation ranging from 1 to 7 Gy. After the irradiation, the NCI-H28 cells were immediately exposed to PEM for 2 h. After the PEM treatment, the PEM containing medium was changed under the conditions described above and the clonogenic assay was performed.

Clonogenic survival. After the treatment, the dishes were incubated in a CO₂ incubator at 37°C for 14 days to allow colony growth. The colonies were then fixed and stained with crystal violet solution and those with 50 or more cells were counted. The survival response/fraction was calculated as the colony survival after a given treatment relative to the mean value for the control Petri dishes. The plating efficiency of the cells was also determined for the non-irradiated cells. The D0 definition was calculated from the reciprocal of the slope of the radiation dose response curve. All the experiments were performed in triplicate.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The NCI-H28 cells were treated with 3 Gy irradiation alone, PEM alone (IC₅₀ dose), or a combination of irradiation and PEM. Each treatment was performed according to the method described above. The TUNEL assay was performed using an ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA), as described previously (8). The cells were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) 6, 12 or 24 h after each treatment. The fixed cells were then permeabilized with ethanol: acetic acid in a 2:1 ratio for 15 min at −20°C, before being washed twice in PBS for 5 min and incubated with ApopTag equilibration buffer for 5 minutes prior to terminal deoxynucleotidyl transferase enzyme linkage of dUTP-digoxigenin to the 3’-OH end of their DNA at 37°C for 60 min. The reaction was then terminated in stop/wash buffer at 37°C for 30 min. After washing the cells, the slides were treated with anti-digoxigenin-peroxidase for 30 min and mounted with Vectashield and 4’, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). All the samples were assessed without any previous knowledge of the experimental details. All fluorescent cell images were obtained using a Keyence BZ-9000 microscope (Keyence, Osaka, Japan) equipped with a 20 × objective lens and a cooled Charge Coupled Device camera. The cells in four randomly chosen microscopic fields of each slide were examined. TUNEL-positive cells were considered to be undergoing apoptosis. The percentage of apoptotic cells (apoptotic index: AI) was determined by dividing the number of TUNEL-positive cells by the total number of DAPI stained cells. All the fluorescent images were analyzed with the BZ-II analysis application (Keyence). Both the TUNEL-positive cells and DAPI-positive cells were counted automatically using the BZ-II analysis application after modifying the application by haze reduction. All the data were reviewed by human observation. All the experiments were performed in triplicate.

Analysis of TS protein expression. The NCI-H28 cells were treated with 3 Gy irradiation alone, PEM alone (IC₅₀ dose), or a combination of irradiation and PEM. Each treatment was performed as described above. From 1-24 h after each treatment, the total cellular protein was isolated from the cells in lysis buffer containing proteinase inhibitors and equal quantities (50g) of protein were separated by SDS-PAGE (8-16% Tris/glycine gels purchased from Novex, Frankfurt, Germany) and electroblotted onto a nitrocellulose membrane. Immunoblotting was performed using the primary antibodies, anti-human TS (Abcam, Cambridge, UK) and anti-beta actin (Santa Cruz Biotechnology, SA, USA), followed by appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Life Sciences, Bucks, UK). The proteins were detected using enhanced chemiluminescence according to the manufacturer's instructions (Amersham Life Sciences).

Statistical analyses. Multiple-comparisons analysis was performed by one-way analysis of variance, using the Tukey-Kramer method. All the analyses were performed using SPSS software (version 11.0, SPSS Institute, IL, USA). The level of significance was set at p=0.01.