Ro 90-7501 Is a Novel Radiosensitizer for Cervical Cancer Cells that Inhibits ATM Phosphorylation

Materials and Methods

Cell culture. The human cervical cancer cell lines HeLa and ME-180 were purchased from American Type Culture Collection (VA, USA) and were cultured in Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Ro 90-7501 was purchased from Sigma (MO, USA) and dissolved in DMSO.

Irradiation. Cells were irradiated under ambient conditions using a ceasium-137 gamma-ray irradiator, Gammacell® 40 Exactor (MDS Nordion, Ottawa, Canada) at the Osaka University Graduate School of Medicine with a delivering dose rate of approximately 0.85 Gy/min. The mice were irradiated using an orthovoltage X-ray irradiator (Rigaku Denki, Tokyo, Japan) under conditions of 180 kV and 15 mA at the Institute of Experimental Animal Sciences Faculty of Medicine, Osaka University.

WST-1 assay. A total of 500 cells were plated per well in 96-well plates. Cells in each well were treated with several concentrations of Ro 90-7501. WST-1 solution (10 μl) was added to each well 48 h later and the cells were then incubated for 1 h. After incubation, absorbance was measured at 450 nm using a microplate reader (Bio-Rad, CA, USA).

Clonogenic survival assay. Cells were plated into 60 mm-diameter dishes, treated with 10 μM Ro 90-7501, and irradiated 4 h later. The medium was replaced with fresh medium 24 h after irradiation. The cells were fixed and stained with crystal violet, 10–14 days after irradiation. Colonies of more than 50 cells were counted and normalized according to the nonirradiated controls for each treatment.

Apoptosis assay. Apoptotic cells were analyzed using Annexin V-FITC Apoptosis Detection Kit (Nacalai tesque, Kyoto, Japan). In brief, 24 h after 8 Gy irradiation, HeLa cells were washed in PBS and suspended in annexin V binding solution (1×). Annexin V-FITC solution and propidium iodide (PI) solution were then added to the cells. After incubation in the dark for 15 min, cells were analyzed with BD FACS Canto II (BD Biosciences, NJ, USA).

Tumor growth delay assay. Eight-week-old female BALB/c nude mice were purchased from Charles River Laboratories Japan (Kanagawa, Japan). Mice were maintained under clean conventional conditions at Osaka University Graduate School of Medicine's Institute for Experimental Animal Sciences. All animal experiments were performed with the approval of Animal Experiments Committee of the Osaka University. A total of 1.5×10⁷ HeLa cells in 100 μl PBS were inoculated into the legs of mice. Treatments were applied when tumors grew to 10 mm. The mice were divided into four groups: control group (1% DMSO/PBS i.p., 0 Gy), Ro 90-7501 group (5 μg/g of Ro 90-7501 i.p., 0 Gy), irradiation group (1% DMSO/PBS i.p., 10 Gy), and combination group (5 μg/g of Ro 90-7501 i.p., 10 Gy). Tumor diameters were measured using digital calipers, and tumor volume was calculated using the formula:

Western blotting. Western blotting was performed using standard protocols. After irradiation, cells were harvested using trypsin, washed twice with PBS, and lysed in a radioimmunoprecipitation assay lysis buffer (89900, Thermo Fisher Scientific; 25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail and phosphatase inhibitors. Equal amounts of protein (30 μg) were loaded onto 10% SDS polyacrylamide gel. After electrophoresis, polyvinylidene difluoride membranes were blocked for 1 h at room temperature with 5% skim milk in tris-buffered saline with Tween 20 (TBST), followed by incubation for 16 h at 4°C with primary antibodies. The membranes were then incubated for 1 h at 23°C with 1:100,000 HRP conjugated secondary antibodies (111-035-144 for anti-rabbit, Jackson ImmunoResearch, PA, USA). After three additional washes with TBST for 10 min, the signal was developed using the Amersham ECL Prime Western blotting detection reagent (RPN2232, GE Healthcare, Little Chalfont, UK) through incubation at 23°C for 5 min. Finally, the signal was detected using ChemiDoc Touch Imaging System (Bio-Rad, CA, USA). The primary antibodies used were: 1:1000 anti-ATM (#2873, CST), 1:1,000 anti-phospho-ATM(Ser1981) (#5883, CST), 1:500 anti-phospho-ATR(Ser428) (#2853, CST), 1:1,000 anti-phospho-Histon H2A.X(Ser139) (#9718, CST), 1:1,000 anti-phospho-Chk1(Ser345) (#2348, CST), 1:1,000 anti-phospho-Chk2(Thr68) (#2197, CST), and 1:1,000 beta-actin (#4870, CST).

Cell cycle analysis. Cells were harvested using trypsin, washed with cold PBS, and fixed with 70% ethanol at −20°C overnight. Subsequently, the cells were resuspended in PI and RNase in PBS at room temperature in the dark for 30 min. The stained cells were analyzed using FACS canto II (BD Biosciences).

Statistical analysis. The results were expressed as mean±standard deviations. Statistical significance was analyzed using the Student's t-test. A p-value of <0.05 was considered statistically significant.