The cervical cancer HeLa cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in media consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine (GE Healthcare Life Sciences, Logan, UT, USA), plus 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China), and 10% fetal bovine serum (FBS; GE Healthcare Life Sciences).

A DNA fragment encoding CXCL10 was cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) between the restriction sites for EcoRI and XhoI. The plasmid was purified by two rounds of passaging over Endo-free columns (Qiagen, Valencia, CA, USA), as previously described (15).

Total RNA was isolated from HeLa cells transfected with the pcDNA3.1-CXCL10 plasmid using TRIzol reagent (Invitrogen), according to the manufacturer's instructions. Total RNA was reverse transcribed to cDNA using the reverse transcription system from Promega (Madison, WI, USA). PCR was subsequently performed with an initial denaturation step that was performed at 94°C for 4 min, followed by 28 cycles at 94°C for 20 s, 60°C for 20 s and 72°C for 30 s, with a final extension of 72°C for 4 min. The primers used for PCR were as follows: CXCL10 forward, 5′-CCTTATCTTTCTGACTCT AAGTGGC-3′ and reverse, 5′-ACGTGGACAAAATTG GCTTG-3′; GAPDH forward, 5′-TCATCTCTGCCCCCT CTG-3′ and reverse, 5′-CCTGCTTCACCACCTTCTTG-3′.

To establish the cervical cancer tumors, eight-week-old female nude mice (Beijing HFK Bioscience Co., Ltd, Beijing, China) were inoculated with 1×10⁶ HeLa cells subcutaneously in the right flank. Tumor dimensions were measured with calipers every 3 days, and tumor volumes were calculated as follows (16):

Tumor volume (mm³) = 0.5 × length × width²

Animal experiments were conducted according to institutional guidelines concerning animal use and care, and the study was approved by the ethics committee of Luzhou Medical College, (Luzhou, China).

The mice were randomly divided into five groups when the tumors were ~15 mm³ in volume, which occurred ~12 days subsequent to HeLa cell inoculation, and each group consisted of 10 mice. The recombinant CXCL10 or pcDNA3.1 plasmids were gently combined with a liposomal transfection agent in a 1:3 ratio (50:150 µg for each mouse), and incubated at room temperature for ~30 min. The mixture was then administered intravenously 5 times on days 12, 15, 18, 21 and 24. Mice from the phosphate-buffered saline (PBS) group were injected with PBS as the control agent and acted as the control group.

Radiotherapy was performed when the tumors were ~300 mm³ in volume, which occurred ~27 days subsequent to inoculation of the mice with HeLa cells. The tumors were radiated with 6-MV X-rays, and each mouse was administered with a total radiation dose of 20 Gy, with 4 Gy being administered for each dose on 5 days, at the rate of 200 cGy/min.

The mice were sacrificed 39 days subsequent to inoculation with the HeLa cells and the tumors were removed and embedded in paraffin. Tissue sections were immunostained with rat anti-mouse monoclonal antibody against cluster of differentiation 31 (CD31; catalog no. 553369; BD Biosciences, Franklin Lakes, NJ, USA; dilution, 1:200) or rabbit anti-human polyclonal antibody against Ki-67 (catalog no. 19972-1-AP; Proteintech Group, Inc., Wuhan, Hubei, China; dilution, 1:100). Vessel density was calculated by counting the number of microvessels per high-power field (magnification, x200; Leica DM3000, Leica Microsystems GmbH, Wetzlar, Germany) in the tumor sections, as previously described (17). The rate of cell proliferation was calculated for five random fields by dividing the number of Ki-67-positive cells by the total number of cells. TUNEL was performed using an in situ cell-death detection kit (Roche, Mannheim, Germany). In total, five fields were randomly selected and analyzed. The apoptotic index was calculated as the ratio of the apoptotic cell number to the total tumor cell number in each field.

The data were assessed by a one-way analysis of variance and Student's t-test, and a post-hoc test was conducted according to the Student-Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

The recombinant plasmid encoding CXCL10 was cloned, and the transcript of CXCL10 mRNA was confirmed in transfected cells, as revealed by RT-PCR.

The effects of CXCL10 gene therapy and radiotherapy alone and in combination on xenograft tumor models of cervical cancer were assessed in nude mice. The tumor volumes were measured every 3 days subsequent to inoculation with HeLa cells. The tumor volume in the PBS and pcDNA3.1 control groups increased rapidly. By contrast, the tumor growth rate in nude mice treated with CXCL10 gene therapy alone decreased compared with the control group, and this decline was more evident in the group treated with radiotherapy. In addition, the combined treatment of CXCL10 gene therapy and radiotherapy inhibited tumor growth more effectively compared with all other groups (P<0.05) (Fig. 2A). Similarly, the treatment of tumors demonstrated corresponding effects on tumor weight (Fig. 2B). Overall, CXCL10 gene therapy in combination with radiotherapy significantly inhibited cervical cancer tumor growth.

Immunohistochemical labeling of microvessels by CD31 in tumor sections revealed a decrease in tumor vessel density in the CXCL10 group and radiotherapy group compared with those in the control group. In combination, treatment with CXCL10 plus radiotherapy appeared more effective at reducing microvessel density compared with the other groups (P<0.05; Fig. 3).

Cell proliferation was analyzed by immunohistochemical staining of Ki-67 in tumor sections. The number of Ki-67-positive cells were calculated and divided by the total number of cells. A slight but significant decrease in the number of Ki-67-positive cells was observed in the CXCL10 and radiotherapy groups compared with the control group (P<0.05). Additionally, a greater reduction in the number of Ki-67-positive cells was identified in the combination group (P<0.05; Fig. 4).

The apoptotic rate was analyzed by TUNEL assay in the five groups. As shown in Fig. 5, the cellular apoptosis rate in the CXCL10 group and radiotherapy group was found to be ~10% higher compared with the control, while the rate was even higher in the combination group compared with the CXCL10 and radiotherapy groups (P<0.05) (Fig. 5).