The LoVo cell line purchased from the Cell Bank, Chinese Academy of Science (Shanghai, China) was grown in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37°C. The empty plasmid vector and the plasmid vector containing CXCL8 cDNA were purchased from GeneChem Co. (Shanghai, China) and were transfected into LoVo cells using Lipofectamine 2000 reagent and Opti-MEM^® reduced serum media (Invitrogen, Carlsbad, CA, USA). Multiple clones were selected in the presence of 2 µg/ml puromycin.

The LoVo cell suspension was seeded into a 96-well plate (200 µl/well) with the adjusted concentration of 10×10³ cells/ml, and immediately incubated overnight at 37°C with 5% CO₂ and saturated humidity. Groups included the control, empty vector and CXCL8 groups, in four duplications. Cell growth was assessed 12, 24, 48, 72 and 96 h later. Thirty microliters of 5 mg/ml MTT solution (Sigma, St. Louis, MO, USA) was added to each well, and the plate was further incubated for 4 h. After this incubation period the bulk of the medium was removed using a Pasteur pipette fitted to a vacuum line, taking care to leave the formazan crystals behind. After removing the medium, dimethyl sulfoxide (DMSO) was supplemented with oscillation for 10 min. The optical density (OD) was measured with a spectrophotometer (Olympus, Tokyo, Japan) at 450 nm. The graph of the OD value of the cells was drawn with time as the x-axis and absorbance as the y-axis. Proliferation inhibition rate = (1 - OD of experiment group/OD of control group) × 100%.

The LoVo cell suspension (control, empty vector and CXCL8-transfected groups) was seeded into a 6-well plate with the adjusted concentration of 4×10⁵ cells/ml and incubated overnight until cells reached 90% confluency. A 20-µl pipette tip was used to create a scratch in the cell monolayer, and the plate was washed with phosphate-buffered saline (PBS). The cells were cultured in RPMI-1640 medium with 5% FBS for 48 h before being observed under a microscope.

Cell invasion assay. Transwell invasion experiments were performed with 6-well Matrigel-coated chambers from BD Biosciences (Bedford, MA, USA). Briefly, the LoVo cells (control, empty vector and CXCL8 transfected groups) were allowed to grow to subconfluency and were serum-starved for 24 h. After detachment with trypsin, the cells were washed with PBS, resuspended in serum-free medium and 6×10⁴ cells were added to the upper chamber. Complete medium was added to the bottom wells of the chambers. After 24 h, the cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated were fixed and stained with Giemsa stain. Images of three different ×10 fields of view were captured from each membrane, and the number of migratory cells was counted using Fujifilm v1.1 software.

CXCL8-transfected clones were screened for CXCL8 expression. The RNA was collected with TRIzol^® reagent (Roche, Mannheim, Germany) and the mRNA was reverse transcribed to cDNA using the PrimeScript RT reagent kit using the gDNA Eraser kit. Cycling conditions were 16°C for 30 min, 42°C for 30 min and 85°C for 10 min. Approximately 3 µl of total cDNA of each sample was amplified using real-time PCR in a final volume of 20 µl of reaction mixture using the reagent kit purchased from Toyobo Co., Ltd. (Osaka, Japan). Cycling conditions consisted of denaturation at 95°C for 3 min, 40 cycles at 95°C for 12 sec, and 62°C for 40 sec. CXCL8 mRNA expression level was determined by normalizing against GAPDH gene expression using the 2^−ΔΔCt method.

Total cellular proteins (40 µg/lane) prepared from the cultured cells were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were blocked in 5% skim milk and sequentially immunostained with primary and secondary horseradish peroxidase-conjugated antibodies at 4°C overnight and at room temperature for 1 h, respectively. All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system with exposure to X-ray film. The images were analyzed by Quantity One (Bio-Rad).

CXCL8 siRNAs (Santa Cruz Biotechnology, Inc., TX, USA) and Lipofectamine 2000 (Invitrogen) were separately diluted according to the manufacturer's protocol, mixed gently and incubated for 20 min at room temperature. The siRNA-Lipofectamine 2000 mixture was added to LoVo cells grown in 6-well plates to 80–90% confluence. The transfection reagent was replaced after 6 h, and the cells were then treated with chemokines as described above. Scrambled control siRNA was used as the blank control.

BALB/c nude mice (Guangdong Medical Laboratory Animal Center, Guangdong, China) aged 6–8 weeks were housed in laminar flow cabinets under specific pathogen-free conditions in our animal laboratory at Kunming Medical University. All animal experiments were conducted with the approval from the Ethics Committee for Animal Research of the Kunming Medical University. Subcutaneous tumors were generated by subcutaneous injection of 1×10⁶/0.2 ml of LoVo parental cells or CXCL8-transfected LoVo cells in the right flank of the mice (three mice/group). The subcutaneous tumors were measured using Vernier gauge, and tumor volumes were calculated according to the equation: Tumor volume = (short diameter)² × (long diameter) × 0.5. After one week of inoculation, the tumor volume was measured every four days up to 28 days. The growth rate of the tumors was compared between the parental cells and CXCL8-transfected cell groups.

All statistical analyses were performed using GraphPad Prism 5.0. Results are expressed as mean ± SD. Between-group comparisons were performed using analyses of variance. All analyses were two-tailed and P<0.05 was considered to indicate a statistically significant result.

We quantified the expression of CXCL8 in the transfected LoVo cancer cell lines (Fig. 1). Real-time PCR showed that the CXCL8-transfected LoVo cells had significantly elevated CXCL8 mRNA expression when compared with that noted in the control and empty-vector cells (P<0.05). Western blotting showed a significantly increased level of CXCL8 protein in the CXCL8-transfected cells than the level noted in the control and empty-vector cells (P<0.05).

Fig. 2 shows the changes in OD values for the control, empty-vector and CXCL8-transfected LoVo cells using MTT assay. Overexpression of CXCL8 increased proliferation of the LoVo cells and its growth promotion effect was positively related to time. Significant differences in cell viability were observed 48 h after transfection (P<0.05) and remained significant at 72 and 96 h.

The role of CXCL8 expression in the migratory potential of LoVo cells was investigated by scratch test. Fig. 3 shows that 12 and 24 h after transfection, the CXCL8-transfected cells had a significantly (P<0.01) higher migration rate than that observed in the control and empty-vector cells. Invasive potential was measured using Transwell invasion assay (Fig. 4). The CXCL8-transfected LoVo cells exhibited a 2-fold increased invasion than that observed in the control and empty-vector cells (P<0.001).

Western blotting showed that CXCL8-transfected LoVo cells exhibited EMT-like phenotype compared with control and empty-vector cells, with a decreased expression of E-cadherin accompanied by increased expression of N-cadherin, vimentin and α-SMA (Fig. 5).

Compared with the control and empty-vector cells, overexpression of CXCL8 activated the PI3K/Akt/NF-κB pathway by promoting phosphorylation of PI3K, Akt and NF-κB (Fig. 6). The efficacy of the silencing of CXCL8 on the PI3K/Akt/NF-κB pathway was determined by western blot analysis (Fig. 7). Compared to the cells transfected with non-specific siRNA, silencing of CXCL8 lowered the phosphorylation of PI3K and NF-κB. Phosphorylation of Akt appeared to be unaffected.

Subcutaneous tumors were generated by subcutaneous injection of LoVo parental cells or CXCL8-transfected LoVo cells in BALB/c nude mice. Fig. 8 shows the growth of tumor in the two groups. Tumor growth was more rapid in the CXCL8-transfected group than the growth observed in the parental cell group. At day 28, the tumor volume was significantly larger in the transfected cell group (P<0.05).