The human uterine cervical cancer cell line (HeLa) was used. HeLa cells were kindly provided by Dr. Ruey Min Lee (Huntsman Cancer Institute, Salt Lake, UT, USA). Cells were maintained in RPMI-1640 (Sigma, St. Louis, MO, USA) supplemented with 10% FCS and penicillin-streptomycin. These cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
The cells were irradiated at doses between 2 and 14 Gy at room temperature using an MBR-1505R2 X-ray generator (Hitachi Medical Co.) delivering 0.8 Gy/min.
Enzyme activity assay
Cell surface APN/CD13 activity in HeLa cells was detected spectrophotometrically, as reported by Amoscato et al
]. After incubating 4 × 105
cells in a 60 × 15 mm culture dish for 24 h at 37°C, each dish was irradiated with doses of 16 Gy. Twelve hours following, aspirating off the medium and washing with PBS, 1 mM alanine-p-nitroanilido (Peptide Institute, Inc.) was added to each dish as a substrate. Each dish was then incubated at 37°C for 60 min. The supernatant was subsequently collected, and 200 μl of the solution was added to 96-well microtiter plates. APN/CD13 enzyme activity was measured by a microplate reader (Tecan; λexc
of 405 nm and λem
495 nm) (Labsystems, Multiskan Bichromatic, Helsinki, Finland).
For colony formation assays, cells (from 100 to 1 × 105 cells) in a 60 × 15 mm culture dish were seeded in triplicate according to treatment conditions and cell lines. After allowing cells to attach to the dishes, a single dose of radiation and/or Ubenimex was given. Cells were always pretreated with Ubenimex for 24 h before radiation when the two treatments were combined. Cells were cultured for up to 14 days. Colonies were then fixed, stained with crystal violet, and counted. The surviving fraction was estimated as follows: (number of colonies formed)/(number of cells seeded) × [plating efficiency for the no-treatment or Ubenimex group (control group)].
Western blot analysis
Cells were grown to 70–80% subconfluence and treated with lysis buffer containing 1% Triton ×-100 in PBS and protease inhibitor mixture tablets (Roche, Barcelona, Spain). Ten μg of total cell lysate were electrophoresed on a 10% SDS-polyacrylamide gel and transferred electrophoretically to Immobilon membranes (Millipore, Bedford, MA, USA). After adding blocking solution (5% nonfat dry milk/0.1% Tween-20/PBS), the membranes were incubated overnight with a recommended dilution of primary antibodies. We used anti-Bcl-xL (Cell Signaling BD, 2760), anti-Bcl-2 (Cell Signaling BD, 2872), anti-caspase-3 (Santa Cruz, sc-7272), anti-cleaved caspase-3 (Cell Signaling, 7190), anti-PARP (Cell Signaling, 9542), anti-cleaved PARP (Cell Signaling, 9541), and anti-β-actin antibodies (Sigma, AC-74). The primary antibodies were washed in 0.05% Tween-20/PBS and then incubated with horseradish peroxidase-conjugated secondary antibody. Proteins were visualized using enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) followed by exposure to X-ray film.
Flow cytometric analysis for apoptosis
To quantify the apoptotic death of HeLa cells, annexin V and PI (propidium iodide) staining was performed, followed by flow cytometry. Cells were plated at 4 × 105 cells in a 60 × 15 mm culture dish for 24 h, and treated with 16 Gy irradiation. After treatment for 72 h, attached cells were collected by brief trypsinization, washed twice with PBS, and then subjected to annexin V and PI staining using a MEBCYTO apoptosis kit (MBL). After staining, quantitative analysis for apoptosis was performed by flow cytometry.
Tumor model and treatment conditions
Female nude mice (BALB/c) at 6 weeks were provided from Chubu Kagaku (Nagoya, Japan). HeLa cells (1 × 107 cells/0.5 ml of medium/mouse) were injected into the left flank of animals. Before initiating the study, 20 mice were assigned to four groups.
Each group contained an equal number of large and intermediate-sized tumors, and mice were stratified into groups so that the mean tumor volume in each group was comparable.
(a) Control group (no radiation, sterile PBS injections),
(b) Radiation alone group (locally radiated at a dose of 4 Gy, sterile PBS injections),
(c) Ubenimex alone group (treatment with Ubenimex for 7 days at 10 mg/kg/day), and
(d) Combination treatment group (concomitant radiation plus Ubenimex for 7 days at 10 mg/kg/day scheduling: started 24 hours before 4 Gy radiation).
For radiation treatment, mice were immobilized in a customized harness facilitating exposure of the left flank, whereas the remainder of the body was shielded by lead.
4 Gy radiation was delivered locally in one fraction on day 7 using an MBR-1505R2 X-ray generator (Hitachi Medical Co.) delivering 0.8 Gy/min.
Intratumoral injections of Ubenimex began 24 h before radiation and were continued daily for 7 days. The tumor volume was estimated from two-dimensional tumor measurements by the formula: V = length (mm) × width2 (mm2)/2.
The unpaired Student's t-test was used to determine the significance of the relative tumor volumes and for comparisons between groups.
The TUNEL assay was carried out using MEBSTAIN Apoptosis KitII(MBL CO., LTD., Nagoya, Japan). DNA fragmentation was detected by this assay. Paraffin-embedded sections (4 μm thick) of mice were cut and mounted on precoated glass slides. Sections were deparaffinized prior to digestion with 20 μg/mL proteinase K for 15 min at room temperature. The slides were washed 4 times in distilled water for 2 min and covered with 2% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min at room temperature to inactivate endogenous peroxidase. The slides were rinsed with PBS twice and immersed in TdT-containing buffer (30 mmol/L Triazma base, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride) for 15 minutes to prepare digoxigenin-binding sites. An antidigoxigenin antibody fragment carried a conjugated reporter enzyme (peroxidase) to the reaction sites, and then localized peroxidase generated an intense signal from the chromogenic substrate diaminobenzidine. The counterstain was made by methyl green. Apoptosis was defined as the number of stained positive cells under a 200 × fluorescence microscope.
Data were obtained from three individual experiments performed in triplicate. Statistical analysis was performed using the unpaired Student's t-test. Differences were considered significant at p < 0.05.