293 cells were obtained from Microbix Biosystems, Inc. (Mississauga, ONT, Canada). The human colorectal cancer cell lines HT-29, SW620 and SW480, and the human normal lung/bronchial normal epithelial cells Beas-2B, were acquired from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 293 cells and Beas-2B cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The other cell lines, HT-29, SW620 and SW480, were cultured in DMEM supplemented with 5% FBS. All cells were cultured in a 5% CO₂ humidified incubator at 37°C.

The OA CD55-TRAIL was constructed in a previous study (11). The CD55-TRAIL was amplified by infecting 293 cell cultures. The virus was collected by CsCl gradient centrifugation, followed by dialysis. Viral titer was determined by 50% tissue culture infective dose (TICD50) analysis in 293 cells (11).

Cells (5,000) were seeded into 96-well plates and cultured for 24 h. The culture medium was subsequently replaced with medium containing CD55-TRAIL [5 multiplicity of infection (MOI), 10 MOI, 15 MOI], luteolin (25 µM) (Beyotime Institute of Biotechnology, Haimen, China), or a combination of CD55-TRAIL and luteolin. Following incubation for approximately 24, 48, 72 and 96 h, MTT (0.5 mg/ml) was added to each well. Following 4 h incubation, the cell supernatant was completely removed and 150 ml dimethyl sulfoxide was added to each well. Following mixing thoroughly, the 96-well plates were read by a microplate reader at a wavelength of 490 nm (Tecan Group, Ltd., Mannedorf, Switzerland). The effect of combination treatment was evaluated using CalcuSyn version 2.1 (Biosoft, Cambridge, UK).

A total of 5×10⁴ cells were seeded into 6-well plates and were left to attach overnight. The cells were treated with CD55-EGFP (15 MOI) or a combination of CD55-TRAIL and luteolin (25 µM). Following 48 h incubation, the cells were subsequently observed under a fluorescence microscope (0.2 mm fields; magnification, ×200; IX71-22FL/PH, Olympus Corporation, Japan).

A total of 5×10⁴ cells were seeded into 6-well plates and were left to attach overnight. The cells were treated with CD55-TRAIL (15 MOI), luteolin (25 µM), or a combination of CD55-TRAIL and luteolin. Following 48 h incubation, the cells were incubated in Hoechst 33342 (Beyotime Institute of Biotechnology) for 10 min, and washed twice with PBS. The cells were subsequently observed under a fluorescence microscope (0.2 mm fields; magnification, ×200; IX71-22FL/PH, Olympus Corporation, Japan).

A total of 5×10⁵ cells were seeded into 6-well plates and were left to attach overnight. The cells were treated with CD55-TRAIL (15 MOI), luteolin (25 µM), or a combination of CD55-TRAIL and luteolin. After 72 h, the cells were removed and lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibiters to collect protein. The concentration of proteins collected was determined using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Subsequently, the protein (20 µg) was separated using 6% SDS-PAGE to detect cleaved poly-ADP-ribose polymerase (PARP; cat. no. 5625) and PARP and the protein (20 µg) were separated using 12% SDS-PAGE to detect GAPDH (cat. no. 51332), procaspase-9 (cat. no. 9508), cleaved caspase-9 (cat. no. 7237), E3 ubiquitin-protein ligase XIAP (XIAP; cat. no. 14334), E1A (cat. no. sc374663) and TRAIL (cat. no. 3219) and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat dried milk at room temperature for 2 h and incubated in primary antibodies [GAPDH, procaspase-9, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (PARP), PARP, E3 ubiquitin-protein ligase XIAP (XIAP), E1Aand TRAIL] overnight at 4°C. The working dilution of all primary antibodies was 1:1,000. The membranes of GAPDH, procaspase-9 and E1A were subsequently incubated in the corresponding mouse secondary antibody (cat. no. 10230269) and the membranes of cleaved caspase-9 cleaved PARP, PARP and TRAIL were subsequently incubated in corresponding rabbit secondary antibody (cat. no. 10245169). the working dilution of all secondary are the ratio of 1:5,000 and the all membranes are incubated at room temperature for 2 h. Protein expression was detected using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). All antibodies, except the E1A antibodies, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The E1A antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The mouse and rabbit secondary antibodies were obtained from PerkinElmer, Inc. (Waltham, MA, USA).

A total of 5×10⁵ cells were seeded into 6-well plates and were left to attach overnight. The cells were treated with CD55-TRAIL (15 MOI), luteolin (25 µM), or a combination of CD55-TRAIL and luteolin. After 48 h, the cells were trypsinized and harvested. Aliquots of cells were resuspended in 500 ml binding buffer and stained with annexin V fluorescein isothiocyanate/propidium iodide (BD Biosciences, San Jose, CA, USA), according to the manufacturer's protocol. The cells were immediately identified using fluorescence-activated cell sorting (BD Biosciences).

Animal studies followed the regulations and standards set by the US Department of Agriculture and the National Institutes of Health. A total of 24 female BALB/c nude mice (4 weeks old; mean body weight 18 g) were purchased from the Zhejiang Chinese Medical University (Hangzhou, China) and housed at 28°C, 40–60% humidity and a 12-h light/dark cycle. The experimental protocol was approved by the Animal Care and Welfare Committee of Zhejiang Chinese Medical University (Hangzhou, China).

A total of 8×10⁶ HT-29 cells were injected subcutaneously into the right flank of nude mice. When the tumor had grown to a diameter of 400–600 mm³, the mice were divided randomly into 4 groups and were injected with PBS (vehicle), luteolin (50 mg/kg) alone, CD55-TRAIL (2×10⁹ plaque-forming units) alone or CD55-TRAIL and luteolin. Subsequent to the injection, the tumor size was measured using a Vernier calliper every 5 days.

The liver, kidney, spleen and tumor tissues were harvested and fixed in 5% paraformaldehyde at room temperature for 48 h, dehydrated with gradient increasing ethanol concentrations and embedded in paraffin wax, and were cut into 5-mm sections. The sections were stained with hematoxylin and eosin at room temperature for 5 min for histological analysis. The sections were blocked with 5% BSA at room temperature for 20 min and subsequently incubated in anti-TRAIL antibodies (3219; Cell Signaling Technology, Inc.) overnight at 4°C, followed by avidin-biotin-peroxidase complex reagents (Vector Laboratories, Inc., Burlingame, CA, USA) for the IHC analysis. The working dilution of anti-TRAIL antibodies was 1:300. Hematoxylin was used as a counterstain at room temperature for 5 min. The in-situ apoptosis detection kit (Sino-American Biotechnology Co., Luoyang, China) was used to stain apoptotic cell tumor tissue sections, according to the manufacturer's protocol, for the TUNEL assay. All sections were counterstained with hematoxylin at room temperature for 5 min. All sections were subsequently observed under a microscope (0.2 mm fields; magnification, ×200; IX71-22FL/PH, Olympus Corporation, Japan).

All experiments were repeated three times and data are presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 6 version is 6.01 (GraphPad Software, Inc., La Jolla, CA, USA) for a Student's t test. P<0.05 was considered to indicate a statistically significant difference.

The oncolytic virus CD55-TRAIL was constructed by inserting a TRAIL gene-expressing cassette into CD55. TRAIL gene expression was regulated by the human cytomegalovirus immediate-early promoter. CEA protein expression in HT-29 and SW620 cells was detected via western blotting to ensure the replication of CD55-TRAIL.

In order to evaluate the antitumor effect of CD55-TRAIL and luteolin in vitro, CRC cell lines SW620, SW480 and HT-29 were infected with CD55-TRAIL (at various concentrations) and luteolin (25 µM) (Fig. 1A) for 48 h. It was observed that, following treatment, cell viability significantly decreased in a time-dependent manner. At 96 h post-infection, cell viability was <30%. Treatment with CD55-TRAIL and luteolin was similar to luteolin alone, illustrating the decreased toxicity of CD55-TRAIL in normal BEAS-2B cells (Fig. 1B) at 48 h. An increased concentration of luteolin (80 µM), and the combination of CD55-TRAIL (15 MOI) with luteolin (25 µM), was used to treat HT-29 and BEAS-2B cells; in HT-29 CRC cells, the same cytotoxic effects were observed leading to a viability of <35%, although the combination treatment induced less toxicity in normal Beas-2B cells (>65% viability) (Fig. 1C). The results of the present study suggested that the combination of CD55-TRAIL with luteolin suppressed CRC cell proliferation with minimal toxic impact to normal cells compared with luteolin alone.

In order to detect whether the luteolin was able to enhance oncolytic adenovirus CD55-mediated gene expression, HT-29 cells were treated with CD55-enhanced green fluorescent protein (EGFP) (15 MOI) or CD55-EGFP (15 MOI) and luteolin (25 µM). After 24 h, it was observed that the percentage of EGFP fluorescent cells was increased in HT-29 cells treated with CD55-EGFP and luteolin compared with CD55-EGFP alone (Fig. 2A), suggesting that luteolin promoted CD55-EGFP replication and EGFP expression. In addition, E1A and TRAIL expression was detected in CD55-TRAIL-infected HT-29 cells. The results demonstrated that combination treatment with CD55-TRAIL and luteolin led to increased E1A and TRAIL expression compared with CD55-TRAIL alone or luteolin alone (Fig. 2B), indicating that luteolin was able to enhance CD55-mediated associated protein expression.

In order to determine whether the antitumor effect of the combination therapy was synergistic or additive, the effect of combination treatment was evaluated using CalcuSyn. HT-29 cells were treated with CD55-TRAIL alone at various concentrations, luteolin alone or a combination at the constant ratio of 3:5 for 48 h. The X-marks on the graph (Fig. 2C) were used as the combination index (CI) values. All the experimental CI values were <1, illustrating that the combination treatment had a synergistic effect (Fig. 2C).

In order to determine whether the antitumor effects of the combination therapy were induced by an enhancement of apoptosis in cancer cells, Hoechst 33342 analysis was used to examine morphological alterations indicative of apoptosis in HT-29 cells. The results indicated that compared with CD55-TRAIL or luteolin alone, the combination-treated cells displayed greater chromatin condensation, nuclear fragmentation and apoptotic body formation (Fig. 3A). Notably, normal Beas-2B cells exhibited few signs of apoptosis (Fig. 3A). The examination of cell death by flow cytometry of treated HT-29 cells revealed that compared with 1.7% for luteolin and 3.8% for CD55-TRAIL, treatment with CD55-TRAIL with luteolin resulted in a significantly increased percentage of apoptotic cells at 9.8% (Fig. 3B). Therefore, combination treatment induced apoptosis to a greater degree compared with each treatment alone.

In order to further elucidate the underlying mechanism of cellular apoptosis, expression of the caspase signaling pathway proteins was detected by western blot analysis. Procaspase-9, cleaved caspase-9, PARP, cleaved PARP and XIAP were detected in HT-29 cells treated for 48 h (Fig. 3C). There was a significant decrease in the expression of anti-apoptosis proteins procaspase-9 and PARP, and an increase in the pro-apoptosis proteins cleaved caspase-9 and cleaved PARP, in cells treated with the combination therapy compared with the other treatments. The decreased expression of XIAP with the combination treatment compared with other apoptosis treatment significantly increased the effect of apoptosis. The results of the present study suggested that the combination treatment enhanced cellular apoptosis via activation of the caspase apoptosis pathway.

In order to further examine their antitumor efficacy in vivo, the HT-29 xenograft model was established in nude mice. Treatment with luteolin, CD55-TRAIL or a combination of the two for 48 h was applied subcutaneously to the right flank of the mice. As solid tumors may not exceed 20 mm diameter for ethical reasons, animals were sacrificed at day 30. The results demonstrated that the tumor volume in the groups receiving PBS, luteolin or CD55-TRAIL reached ~3,761 mm³, ~3,415 mm³ and ~3,072 mm³, respectively, within 30 days; the combination of CD55-TRAIL with luteolin significantly impeded the growth of the tumor, with a tumor volume of ~1,780 mm³ representing a 58% inhibition of tumor growth (Fig. 4A). Additionally, after 25 days, the tumor volume in the combination treatment was decreased, suggesting that the combination treatment has better anti-tumor effect.

Examination of TRAIL and cell death-associated protein TUNEL expression by IHC analysis of tumor tissues 20 days subsequent to drug treatment revealed that the expression of TUNEL was increased in animals receiving combination treated cells, compared with other mice (Fig. 4B). Hematoxylin and eosin staining demonstrated that the combination of CD55-TRAIL with luteolin caused more severe cytopathic effects in tumor tissues compared with each therapy alone (Fig. 4B). In addition, the liver, kidney and spleen tissues without toxic effects were indicated by hematoxylin and eosin staining, implying that little to no cell damage or apoptosis had occurred in these tissues (Fig. 4C). IHC staining demonstrated that the TRAIL protein was highly expressed in tumor tissue following treatment with the combination of CD55-TRAIL and luteolin (Fig. 4B).