Standard cloning techniques were used to establish the pC1-tk-IRES-luc vector with dual reporter genes in the present study, as previously described (21). In brief, murine CT26 colorectal adenocarcinoma cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA). A plasmid vector carrying HSV-1 thymidine kinase (tk) and luciferase (luc) genes was transfected into parental CT26 cells using Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the cells were renamed CT26/tk-luc cells, as previously described (22). The CT26/tk-luc stable clones were cultured under the same condition as the parental cells. G418 (600 µg/ml) was added to the medium to maintain the stable expression of tk-luc genes.

For the in vitro study, TET (cat. no. 365629; Sigma-Aldrich; Merck, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) to 10 mM, sterilized by filtration through a 0.22-µm filter as a stock solution, then stored at −20°C. For the working solution, the stock solution was diluted to desired concentrations with serum-free medium immediately before each experiment. The final concentration of DMSO was <0.5%. For the in vivo study, TET was dissolved in a drop of 1 N HCl and then adjusted to pH 7.0 with 1 N NaOH, then further diluted with 0.9% NaCl solution to desired concentrations. Finally, it was sterilized by filtration through a 0.22-µm filter and stored at 4°C.

For the in vitro study, cultured monolayer cells were irradiated with a Co-60 AECL Eldorado-78 irradiator at a dose rate of 32 cGy/min at room temperature, 80 cm source-to-surface distance (SSD) and a field size of 30×30 cm. Before and immediately after irradiation, cells were maintained on ice to arrest the cell cycle. For the tumor-bearing animal model, 6 mice per group were placed in an acrylic restraint and irradiated with 18 Gy (field size of 30×5 cm) administered in 6 fractions, one fraction a day, three times a week.

CT26/tk-luc cells were seeded in T-25 flasks (1×10⁶ cells/flask) overnight, then treated with 0, 4, 8, 16, 24 and 32 µM of TET for 3 h, different doses of radiation, or 20 µM TET for 3 h followed by irradiation. The plating efficiency (PE) was defined as the number of colonies divided by the number of cells plated in the control group. The surviving fraction (SF) under various treatments was the number of counted colonies divided by the number of colonies plated and corrected for PE. The mean lethal dose (D₀) was calculated from the radiation survival curve using D₁₀ = 2.3×D₀. The experiments were repeated more than three times.

CT26/tk-luc cells were cultured in 60 mm-diameter dishes (1×10⁶ cells/dish) overnight. When the cell growth reached ~80% confluence, the cells were treated with different concentrations of TET for 3 h, or TET for 3 h followed by 4 Gy irradiation. Cells were washed twice with phosphate-buffered saline (PBS), then cultured in fresh medium containing 10% FBS for a further 24 h prior to analysis with a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cells in the sub-G1 phase (i.e. apoptotic cells) could be distinguished from cells with the normal diploid DNA peak (G₀/G₁ phase) on the fluorescence profiles of propidium iodide-stained cells. The percentage of cells in the sub-G1 phase was estimated with CellQuest software (BD Biosciences). CT26/tk-luc cells treated with 50 µM camptothecin (CAM; cat. no. 7689-03-4; Sigma-Aldrich; Merck KGaA) for 24 h were used as the positive control.

CT26/tk-luc cells were cultured in 60 mm-diameter dishes (1×10⁶ cells/dish) and incubated at 37°C overnight, then treated with 0, 10, 20 and 50 µM TET for 3 h. The combination group was irradiated with 4 Gy immediately after TET treatment. Cells were harvested and centrifuged at 80 × g for 5 min and supernatants were aspirated. DNA fragmentation was performed using Suicide Track™ DNA Ladder Isolation kit [cat. no. AM41; Calbiochem; EMD Millipore (Billerica, MA, USA)]. Total DNA was extracted using the kit, then dissolved in 50 µl resuspension buffer. A volume of 21 µl DNA sample was transferred to a clean centrifuge tube and 4 µl of 6× gel loading buffer was added. Then, it was loaded onto a 1.5% agarose gel and stained with ethidium bromide for electrophoresis in 0.5 M Tris/EDTA buffer.

CT26/tk-luc cells were harvested at 24 h after TET alone and combined with 4 Gy irradiation, then washed twice with PBS and centrifuged at 80 × g for 3 min. The cell pellet was resuspended in 100 µl protein lysis buffer (for 1.5×10⁶−2×10⁷). Suspended cells were transferred to an Eppendorf tube on ice, then centrifuged at 10,956 × g for 10 min at 4°C. The supernatant was transferred to a new tube on ice and the pellet was discarded. The total protein concentration was determined using bovine serum albumin (BSA) as the standard by measuring the absorbance at 595 nm. Samples were stored at −20°C for western blot analysis and caspase-3 activity assay.

Lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40 and 1 mM phenylmethanesulfonyl fluoride) was used for the extraction of total proteins from cells of each group at 4°C. The proteins (40 µg/lane) were separated via 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk in TBST buffer solution (10 mM Tris-base, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature, followed by incubation with the appropriate primary antibodies against caspase-3 (1:300; cat. no. IMG-144A; Imgenex, Novus Biologicals, LLC, Littleton, CO, USA), β-actin (1:3,000; cat. no. MAB1501; Chemicon International, Inc., Billerica, MA, USA) overnight at 4°C. The membranes was further incubated with anti-mouse IgG-horseradish peroxidase (HRP) for 1 h at room temperature (1:5,000; cat. no. AP124P; Chemicon International, Inc.), then detected using ECL Western Blot Chemiluminescence Reagent Plus (cat. nos. NEL104 and NEL105; PerkinElmer Life Science, Inc., Waltham, MA, USA). Membranes were dried by dabbing with tissue, then exposed to FUJI Medical X-ray film (cat. no. 50407; Fujifilm, Tokyo, Japan) and quantified with Scion Image software (version 4.0.3.2; Scion Corp., Frederick, MD, USA).

Caspase-3 plays an important role in triggering the apoptotic process, and its activity has been suggested to be an index of apoptosis (23). To determine whether caspase activation was involved in TET-induced cell death, CT26/tk-luc cells were exposed to TET for 3 h with or without radiation, and incubated for a further 3, 9 and 21 h at 37°C. The protein expression of caspase-3 was assayed by western blotting. Total protein (50 µg) was added to the buffer to a final volume of 500 µl. Ac-DEVD-AFC (20 µM), the substrate for caspase-3, was added to the sample and incubated at 37°C for 30 min. After cleavage by activated caspase-3, the substrate released a yellow-green fluorescent compound, AFC, which could be detected with a spectrophotometer (Hitachi F-4500; Hitachi, Ltd., Tokyo, Japan), with excitation and emission at 380 and 508 nm, respectively. Cells treated with 50 µM CAM for 24 h were used as the positive control. The results were expressed as the percentage change in activity compared with the untreated control. The experiments were performed three times independently.

Six-week-old male BALB/c mice (number of mice, 85; body weight, 20±2 g) were housed in the following conditions: 22±1°C, 55–60% relative humidity, 12-h light/dark cycle control and food and water with ad libitum. The mice were anesthetized with an i.p. injection of 45 mg/kg ketamine and 15 mg/kg xylazine, and 2×10⁵ CT26/tk-luc cells in 0.2 ml serum-free medium were subcutaneously injected into the right hind legs. Perpendicular tumor diameters were measured using a digital caliper and the tumor volume was calculated using the formula: 0.523 × (length × width × thickness) (24) Treatment was initiated when the tumor volume reached 100 mm³. Mice were randomly separated into six groups (6 mice for the control group, 7 mice per group for the rest groups): Normal saline, 5 mg/kg TET for 12 consecutive days, 10 mg/kg TET (three times a week for 2 weeks), 20 mg/kg TET (three times in 2 weeks), radiation alone (3 Gy/fraction, three fractions per week for 2 weeks) and concurrent 10 mg/kg TET and 3 Gy radiation. Normal saline and TET were i.p. injected. Tumor growth inhibition was measured by a digital caliper, non-invasive BLI and gamma scintigraphy. All animal experiments were repeated twice.

Body weight and tumor volume were assayed twice per week throughout the experimental period. The time required to reach 8-fold of the initial tumor volume (i.e. 100 mm³) was used as the biological endpoint. Tumor growth delay (TGD) was calculated as follows: TGD = TGT_treated- TGT_control, where TGT represents tumor growth time (25). The enhancement ratio (ER) was calculated as follows: ER = (TGD_TET+IR - TGD_IR)/TGD_TET, where TGD_TET+IR stands for the tumor growth delay of the combination group; TGD_IR stands for the tumor growth delay of the radiation alone group and TGD_TET represents the tumor growth delay of the TET alone group.

Survival curves were determined by the Kaplan-Meier method. Mice were considered expired when the tumor volume reached 2,500 mm³ post-treatment in accordance with the Institutional Animal Care and Use Committee regulations (26). IACUC approval no. for the animal experiments in the present study was 950501 issued by the National Yang-Ming University.

CT26/tk-luc murine CRC cells (2×10⁵ cells) were inoculated into the flank of BALB/c mice to monitor tumor growth and the therapeutic efficacy of TET with or without radiation, assayed by direct measurement with calipers and BLI using the Xenogen IVIS50 Imaging system (Xenogen Corp., Alameda, CA, USA). Images and measurements of bioluminescent signals were acquired and analyzed using Living Image software (Xenogen Corp.). Mice were i.p. injected with D-luciferin 150 mg/kg in PBS and anesthetized using 1–3% isoflurane 15 min prior to imaging. Mice were placed on a warmed platform inside the camera box and received continuous exposure to 1–2% isoflurane to sustain sedation during imaging. The image acquisition time ranged from 30 sec to 5 min depending on the bioluminescence of the tumors. Regions of interest (ROIs) from displayed images were drawn around the tumor and quantified as photons/second (ph/s) using the Living Image software (version 3.1; Information Technologies LLC, St. Louis, MO, USA). The serial bioluminescent signals were quantified and displayed over time.

Planar imaging was performed on BALB/c mice bearing CT26/tk-luc tumors derived from 2×10⁵ cells as aforementioned. After mice were injected with 100 µCi [¹³¹I] FIAU via the tail vein, static imaging was obtained from anaesthetized animals at 24 h post-injection with e.Cam Multiangle Cardiac camera (Siemens AG, Munich, Germany) equipped with a pinhole collimator. ROIs were selected over the tumor area and the reference organs.

All data are presented as the mean ± standard error (SE). Student's t-test was used for comparison between two groups. The differences among multiple groups was analyzed using ANOVA and the Tukey-Kramer method for the post hoc test. A difference between the means was considered significant at P<0.05.

The cell growth curves of CT26 and CT26/tk-luc in vitro are shown in Fig. 1A. The cell doubling time of CT26 and CT26/tk-luc were 14.6 and 15.3 h, respectively. Fig. 1B shows the tumor growth curve of C26/tk-luc in vivo. The tumor doubling time of CT26/tk-luc-bearing mice was 4.07 days. To evaluate the cytotoxicities of TET and radiation, surviving fractions of CT26/tk-luc cells were determined by colony formation assay. Cells were treated with various concentrations of TET for 3 h. The growth inhibition by TET was found to occur in a dose-dependent manner. The IC₅₀ (50% inhibition concentration) was ~10 µM, as shown in Fig. 1C. The radiation response of CT26/tk-luc cells was examined following single doses of Co-60 gamma irradiation. The radiation survival curve is shown in Fig. 1D. The D₀ was ~ 2.0 Gy, as calculated from the formula D₁₀=2.3 D₀ using D₁₀=4.7 Gy. In addition, CT26/tk-luc cells were treated with 20 µM TET for 3 h, and then combined with different doses of radiation. The survival curve for the combination of TET and radiation shifted the radiation survival curve downward without changing the curve shape, suggesting that the combined effect of TET and radiation on CT26/tk-luc cells was additive.

Apoptosis induced by TET with or without radiation in CT26/tk-luc cells was also determined via the sub-G1 cell population by flow cytometry. Fig. 2A reveals the DNA histograms of CT26/tk-luc cells post-treatment. The sub-G1 populations of CT26/tk-luc cells were 0.93, 0.95, 8.09 and 55.76% for 0, 10, 20 and 50 µM, respectively, following TET treatment for 3 h. The sub-G1 populations of CT26/tk-luc cells were 0.85, 1.06, 11.72 and 61.59% for 0, 10, 20 and 50 µM TET treatment for 3 h combined with 4 Gy, respectively. Notably, significant G1 phase arrest was found in all TET-treated groups. CAM (50 µM) was used as the positive control. The mean sub-G1 populations in CT26/tk-luc cells induced by TET with or without radiation determined by flow cytometric assay are displayed in Fig. 2B. The decrease in cell viability due to apoptosis rather than necrosis was detected by DNA ladder assay. To evaluate the nature of TET-induced apoptosis, CT26/tk-luc cells were treated with the indicated TET concentrations or treated with TET in combination with radiation. Internucleosomal DNA fragmentation was observed, as revealed in Fig. 2C. The results indicated that TET alone or in combination with radiation induced apoptosis in CT26/tk-luc cells.

Activation of caspase-3 can be demonstrated from the cleavage of pro-caspase-3 into cleaved caspase-3. Cleaved caspase-3 was detected in CT26/tk-luc cells after 20 mM TET treatment for 6, 12 or 24 h with or without 4 Gy irradiation, respectively, as shown in Fig. 3A. In addition, CT26/tk-luc cells were treated with various concentrations of TET for 24 h with or without 4 Gy. The expression levels of cleaved caspase-3 quantified by Scion imaging software were higher when concurrently treated with TET and radiation, as shown in Fig. 3B. These results unambiguously indicated that activation of caspase-3 contributed to TET-induced apoptosis in murine colorectal CT26/tk-luc cancer cells.

Antitumor activity of TET combined with radiation was assessed in a CT26/tk-luc tumor-bearing Balb/c mouse model. The experimental protocol is presented in Fig. 4A. The monitoring of CT26/tk-luc tumor volumes was initiated on day 12 post-tumor cell inoculation, when bulges were observed. Treatment was administered when the tumor volume reached ~100 mm³. The tumor growth inhibition caused by TET with or without irradiation is shown in Fig. 4B. No significant differences were observed among the groups treated with TET alone in any fractionation protocol. However, radiation alone and combination therapy exhibited a significant decrease in tumor volume as compared with either TET alone or the control group (P<0.01). To determine whether TET could enhance tumor growth inhibition in irradiated tumors, mice were i.p. injected with 10 mg/kg TET 24 h before 3 Gy irradiation for a total of six administrations. The time required to reach 800 mm³ tumor volume for the control, 5, 10 and 20 mg/kg of TET, radiation alone (3 Gyx6) and combination therapy (TET 10 mg/kg+3 Gy) groups was 8.0±1.8, 10.7±1.6, 10.7±1.0, 11.3±1.8, 18.4±2.1 and 23.1±1.3 days, respectively. These results are presented in Table I. The TGD times were 2.7±2.4, 2.7±2.1, 3.3±2.5, 10.4±2.8 and 15.1±2.2 for 5, 10 and 20 mg/kg of TET, radiation alone and combination therapy, respectively. The calculated ER for the combination group compared with the radiation alone group based on the TGD time was 1.45, indicating that TET combined with IR for the treatment of CT26/tk-luc cancer in vivo was synergistic.

CT26/tk-luc cells (2×10⁵) were inoculated into the right flank of BALB/c mice to monitor subcutaneous tumor growth and the therapeutic efficacy of TET with or without radiation by non-invasive molecular imaging modalities: BLI using the Xenogen IVIS50 system and gamma scintigraphy. Fig. 5A shows the longitudinal monitoring of the tumor growth by BLI. In addition, the mice were also monitored by gamma scintigraphy at 24 h after ¹³¹I-FIAU intravenous administrations. The representative images of the CT26/tk-luc tumor-bearing animals are shown in Fig. 5B. ROIs of the tumor exhibited higher radioactivity compared with normal soft tissue. The radioactive counts over the tumor area were normalized against the tumor size (pixels). The ROI was also created over a non-tumor area (muscle) to determine the radioactivity of the background. Notably, the tumor/muscle (T/M) ratios were decreased as the therapeutic time increased. The tumor/muscle (T/M) ratios were 90.52, 74.51, 69.37, 66.25, 64.86 and 41.95 for the control, 5, 10 or 20 mg/kg TET, radiation alone and combination therapy, respectively.

The general toxicity of TET with or without radiation was monitored via body weight changes during the experimental period, as shown in Fig. 6A. No significant body weight changes among the six groups were found. Additionally, no other adverse effects, such as skin changes or hair loss, were observed in the mice post-radiation or with combination therapy. The survival curves in vivo were calculated using the Kaplan-Meier method, as presented in Fig. 6B. Mice were considered expired when the tumor volume reached 2,500 mm³.