Everolimus was obtained from Novartis (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) as a concentrated stock solution. DMSO was used as the vehicle solution. As an internal control, medium only was used. DMSO and anti-actin antibody were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Ras, phospho-eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1), 4EBP1, phospho-ribosomal protein S6 kinase 1 (p-S6K1), S6K1 and phospho-Akt (p-Akt; Ser-473) were from Upstate Biotechnology (Lake Placid, NY, USA). Antibodies against phospho-mTOR (p-mTOR), mTOR, Akt and eukaryotic translation initiation factor 4E (eIF4E) were from BD Biosciences (San Diego, CA, USA). Beclin 1 small interfering RNA (siRNA) was from Thermo Fisher Scientific (Carlsbad, CA, USA).

RK3E/tv-a cells were obtained from the laboratory of Shu-Ling Fu, who established a non-transformed rat kidney epithelial RK3E cell line that constitutively expresses tv-a (receptor for subgroup A avian leukosis virus, ALV) for the delivery of foreign genes via avian retroviral infection. The RK3E/tv-a cells were infected with retrovirus carrying the oncogene, H-ras, or the control gene puromycin N-acetyltransferase (puro) as previously described (10). Ras-expressing cells, but not puro-expressing cells, underwent malignant transformation in vitro and tumorigenesis in vivo. These cells were maintained as previously described (10).

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assays were carried out to measure the cytotoxic and radiosensitizing effects of everolimus. Ras-transformed cells (5×10³) were seeded into 96-well plates, incubated overnight, treated with everolimus and/or radiation and cultured for 4 days. The medium was removed and the cells were incubated with MTT solution (0.5 mg/ml, 4 h, overnight, 5% CO₂ incubator, 37°C) and lysed with solubilization solution [12.5% sodium dodecyl sulfate (SDS), 45% dimethylformamide (DMF)]. The absorbance of converted dye was measured at 570 nm using a microplate reader. (Multiskan RC, Labsystems, Stockholm, Sweden). The absorbance of the untreated cells measured at 570 nm was defined as 100%. The relative viability was calculated by dividing the A570 nm of the treated cells by that of the untreated cells.

The Ras-transformed cells (4×10⁵) were seeded into a 6-cm dish, cultured overnight, and transfected at 40% confluency with the indicated specific constructs (Beclin 1 siRNA, or non-targeted control siRNA) for 24 h according to the manufacturer’s instructions (Thermo Scientific Dharmacon Inc., Lafayette, CO, USA). These sequences were 5′-GGACAGUUUGGCACAAUCA-3′ (951–969). For each transfection, 2 solutions (one of 3 μl of siRNA diluted in 100 μl transfection medium and another of 3 μl of transfection reagents diluted in 100 μl transfection medium) were combined at room temperature for 20 min and added to each well, and the wells were incubated for 24 h. The transfection efficiency was determined at 24 h by western blot analysis. MTT assays were used to measure the effects of everolimus-mediated radiosensitization.

An Annexin V-FITC kit was used according to the manufacturer’s instructions (R&D Systems Inc., Minneapolis, MN, USA). In brief, the cells (10⁵) were grown on 6-well plates, treated with the vehicle (DMSO) or everolimus (10 nM) with or without 6 Gy radiation, cultured for 96 h and stained with Annexin V-FITC and propidium iodide (PI) (5 μg/ml). The stained cells were analyzed using a flow cytometer (FACScalibur; Becton-Dickinson, San Jose, CA, USA).

A (TUNEL) assay was performed to measure cell death according to the manufacturer’s instructions (Roche, Mannheim, Germany). The cells (10⁵) were seeded into 6-well plates, incubated overnight and subsequently treated with the vehicle, everolimus and/or radiation under various conditions. Following treatment, the floating and adherent cells were collected and fixed with 4% paraformaldehyde in PBS, permeablized with 0.1% Triton X-100, stained with TUNEL reaction mixture for 60 min and analyzed by flow cytometry using a FACScan flow cytometer (Becton-Dickinson).

Cells (10⁵) were grown on 6-well plates, treated under various conditions, incubated for 96 h at 37°C, then trypsinized, washed, fixed in 80% cold ethanol for 30 min, stained with 50 μg/ml propium iodide in PBS containing 50 μg/ml of RNase A for 30 min, and immediately analyzed by flow cytometry using a FACScan flow cytometer.

The cells (10⁶) were seeded into 100-mm plates, incubated overnight, treated with the vehicle, everolimus or radiation under various conditions, and collected by scraping and centrifugation at 3,000 rpm for 5 min. The cell pellets were then lysed in radioimmune precipitation assay buffer (RIPA) in the presence of general protease inhibitors. Total protein (50 μg of lysate proteins) was analyzed by 10% sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After blocking with 5% non-fat milk/TBS-Tween, the membranes were incubated with primary antibodies, including anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR, anti-phospho-S6K (p-S6K), anti-S6K, anti-p-4EBP1, anti-4EBP1, anti-eIF4E and anti-LC3 antibodies overnight at 4°C, reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, treated with Immobilon™ Western Chemiluminescent HRP Substrate (ECL) and exposed to X-ray film to detect the signals.

Data are expressed as the means ± SD of 3independent experiments. Statistical analyses were performed using the unpaired two-tailed Student’s t-test to compare 2 groups of data. A value of P<0.05 was considered to indicate a statistically signficant difference.

We first evaluated the cytotoxicity of everolimus to Ras-transformed cells by MTT assay. The cytotoxic effects were found to increase with the higher everolimus concentrations. The survival fraction of the cells treated with everolimus was (10, 30 and 50 nM) was 0.91±0.03, 0.84±0.04 and 0.8±0.02, respectively. Thus, everolimus (30 nM; 96 h of incubation) was used in the subsequent radiosensitivity experiments. As shown in Fig. 1, treatment with everolimus (30 nM) sensitized the Ras-transformed cells to radiation (6 Gy). The survival fractions of the irradiated cells in the medium, vehicle and everolimus solution were 0.45±0.04, 0.42±0.04 and 0.12±0.04, respectively.

We wished to determine whether everolimus, with or without radiation, induces apoptosis. Thus, we performed Annexin V staining and TUNEL assay. The percentage of Annexin V-stained cells was increased following radiation, but not following treatment with everolimus (Fig. 2). Consistent with the above observations, DNA fragmentation (measured by TUNEL assay) was mainly caused by radiation (Fig. 3). By contrast, combination treatment with everolimus and radiation decreased the percentage of dead/apoptotic cells. Taken together, these data indicate that apoptosis is mainly induced by radiation and not is promoted by everolimus. The decreased percentage of apoptotic cell death suggests that everolimus-mediated radiosensitization leading to cell death may occur through a mechanism other than apoptosis.

We wished to determine whether everolimus, with or without radiation, affects cell cycle distribution. Thus, we used PI staining followed by flow cytometry using a FACScan flow cytometer. As shown in Table I, radiation significantly increased the size of the sub-G₁ and G₂/M fractions (P<0.05), and radiation in combination with everolimus (compared to radiation alone) only slightly decreased the size of the sub-G₁ fraction.

We wished to determine whether everolimus, with or without radiation, induces autophagy. Thus, we performed western blot analysis. Fig. 4 shows the protein expression of LC3-II under various conditions. Everolimus in combination with radiation significantly increased LC3-II expression. We then wished to determine whether the everolimus-mediated radiosensitizing effects were dependent on autophagy. Thus, we transfected the cells with Beclin 1 siRNA. As shown in Fig. 1, the radiosensitization effects of everolimus were partially reversed following transfection with Beclin 1 siRNA.

Since the PI3K/Akt pathway is a target of everolimus, the possible association of the radiosensitizing activity of everolimus with the PI3K/Akt signaling pathway was assessed. Western blot analysis revealed that the expression or activation of Akt protein was not significantly altered by everolimus or radiation (Fig. 5).

We wished to determine whether everolimus, with or without radiation, regulates the PI3K/AKT/mTOR pathway. Thus, we performed western blot analysis in order to determine the protein expression of p-mTOR, mTOR, p-S6K1, S6K1, p-4EBP1, 4BEP1 and eIF4E. There was no significant change observed in eIF4E expression (data not shown). However, treatment with everolimus alone attenuated the phosphorylation of mTOR and S6K1 (Figs. 6 and 7), and everolimus in combination with radiation markedly suppressed the phosphorylation of 4EBP1 (Fig. 8).