The poorly differentiated gastric carcinoma cell line MKN45 was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (DMEM; (Gibco-BRL, Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified cell culture incubator at 37°C and in 5% CO₂.

The level of soluble IL-8 secreted into the culture medium of MKN45 cells was performed using enzyme linked-immunosorbent assay (ELISA; Sigma-Aldrich, St. Louis, CA, USA) according to the manufacturer's instructions. In brief, related reagents and samples were prepared, and the samples containing IL-8 were added to each well at 100 μl. After incubation for 90 min, plates were incubated with a biotinylated antibody. Immunoreactivity was determined using the avidin-HRP-TMB detection system. The reactions were stopped by addition of TMB stop buffer, and absorbance was measured at 450 nm using a microplate reader (Bio-Rad 680). A curve of the absorbance vs. concentrations of standard wells was plotted. The concentration of human IL-8 in the tested samples was determined by comparing the absorbance of the samples to the standard curve.

Cells were seeded in a 96-well plate at a density of 5,000 cells/well for MTT assays. Treatments with different concentrations of repertaxin (dissolved in 0.9% saline) alone (0.025, 0.25, 2.5, 25 and 50 μg/ml) (Sigma-Aldrich), 5-FU alone (0.01, 0.1, 1, 10 and 20 μg/ml) (Shanghai Xudong Haipu Pharmaceutical, Co., Ltd., Shanghai, China), and repertaxin (0.025, 0.25, 2.5, 25 and 50 μg/ml) in combination with 5-FU (0.01, 0.1, 1, 10 and 20 μg/ml) were administered 12 h after seeding. Cell viability was determined on days 1, 2, 3, 4 and 5 after treatment by addition of 20 μl MTT solution (5 mg/ml in PBS) (Sigma-Aldrich). Cells were incubated in MTT solution for 4 h at 37°C. Next, the culture medium was removed and replaced with 150 μl DMSO to dissolve the crystals. Samples were incubated at room temperature for 10 min, and then absorbance was measured at 568 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Cells were seeded in a 6-well plate at a density of 200 cells/well and cultured for two weeks. After the medium was removed, cells were washed three times with PBS and fixed for 15 min in pure methanol, and then stained for 25 min with crystal violet (0.5% w/v) (Sigma). Colonies of more than 50 cells were identified using an inverted microscope (Olympus IX70; Olympus Corp., Tokyo, Japan). Colony efficiency (CE) and the rate of colony inhibition (CI) were calculated as follow: CE = (colonies formed/cells seeded) × 100% and CI = (CE in experimental group/CE in control group) × 100%, respectively.

MKN45 cells were plated in 60-mm dishes and grown to 50% confluence. Complete medium was then removed and replaced with medium containing repertaxin and/or 5-FU. After 48 h, treated cells were harvested and fixed overnight with cold 70% ethanol at −20°C. After washing with PBS, the samples were incubated with 50 μg/ml propidium iodide (PI; Sigma-Aldrich), 100 μg/ml RNase A and 0.2% Triton X-100 in the dark for 30 min. Flow cytometric analysis was performed using flow cytometry system and ModFit software (B&D Biosciences, San Diego, CA, USA) (38). Cell cycle distribution, including the percentage of cells in G0/G1, S and G2/M was obtained.

Cells were treated with repertaxin and/or 5-FU and then incubated in buffer containing Annexin V-fluorescein isothiocyanate (0.5 μg/ml; Annexin V-FITC apoptosis detection kit; Sigma-Aldrich) and PI (0.6 μg/ml) in the dark, at room temperature for 15 min. Samples were then measured by flow cytometry (B&D Biosciences), and the percentage of apoptotic cells was calculated using CellQuest software.

Cell migration and invasion were performed using Transwell inserts (Corning Costar) with polycarbonate membranes (8 μm pore size) in 24-well plates. For invasion assays, the filter was coated with 20 μg/ml Matrigel (Becton-Dickinson) overnight at 4°C. A total of 100 μl cells at 1.0×10⁵/ml in serum-free medium containing the indicated compounds, were seeded in the upper chamber. The lower chamber was also treated with compounds in 800 μl culture medium containing 10% FBS. After 24-h incubation at 37°C in 5% CO₂, non-invading cells were removed from the upper surface of the membrane by gently scrubbing with a cotton swab. Cells that had successfully invaded the lower surface of the membrane were fixed with methanol, stained with crystal violet, rinsed with water and then air dried. The invading cells were photographed and counted using an inverted microscope in ten random fields (magnification, ×200). Experimental conditions for migration assays were similar to those used for invasion assays with the exception of Matrigel-coated chambers.

Wound-healing assays were performed to assess cancer cell migration. Briefly, MKN45 cells were seeded at a density of 10⁵ cells/well in a 6-well plate and grown to near confluent monolayers in DMEM containing 10% FBS. Wounds were made by scratching the cell monolayer with a sterile 200 μl pipette tip. The cells were then washed twice with PBS. The scratched areas were photographed at ×10 magnification using computer-assisted microscopy (Olympus IX70; Olympus Corp.). Images were captured at 0, 24, 48 and 72 h after the scratch was made. Images were analyzed using CellProfiler 2.0 cell image analysis software (http://www.cellprofiler.org) (39).

Total RNA from cells was isolated using TRIzol reagent (Invitrogen) and quantified spectrophotometrically. Single-stranded cDNA was synthesized using SuperScript First-Strand Synthesis System (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Sequences of all primers used are listed in Table I. All real-time PCR reactions were performed with SYBR-Green Master Mix kit (all-in-One™ qPCR SYBR-Green I Mix from GeneCopoeia, Rockville, MD, USA) using an ABI PRISM 7500 sequence detection system. GADPH was used as an internal loading control. The reactions were performed as follows: denaturation for 10 min at 95°C, 40 cycles of 95°C for 10 sec, and 60°C for 20 sec. The 2^−ΔΔCt method was performed to calculate the relative mRNA expression of target genes.

Cells were lysed in buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA and 1% Triton X-100) containing protease inhibitor, and then samples were centrifuged at 12,000 rpm for 12 min at 4°C. Equal amounts of protein (50 μg), quantified by BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA), were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 8–12%). After electrophoresis, separated proteins were transferred from the gel to polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were then incubated in blocking solution containing 5% non-fat milk for 1.5 h. After blocking, membranes were incubated with primary antibody. Phosphorylated antibodies (p-AKT-Ser⁴⁷³, anti-AKT, p-ERK-Thr²⁰²/Tyr²⁰⁴ and anti-ERK1/2) were obtained from Anbo Biotechnology Co., Ltd. (San Francisco, CA, USA). Additionally, primary antibodies detecting CXCR1, CXCR2, Bcl-2, Bax, cyclin D1, EGFR, Ki-67, VEGF, MMP-9, MMP-2, TIMP-2 and E-cadherin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); each of these antibodies were prepared at a 1:500 dilution in TBST overnight at 4°C. Following three washes, the blots were incubated with the appropriate horseradish peroxidase conjugated secondary antibody (1:2,000 dilution in TBST) for 1 h at room temperature. GADPH was used as a loading control. Proteins were visualized using enhanced chemiluminescence (Pierce Biotechnology).

Four to six-week old athymic female Balb/c-nu/nu nude mice with a body weight of 15–20 g were obtained from the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). Mice were housed in individually ventilated cages with isolated ventilation at the Animal Care Center of the Third Xiang-ya Hospital, Central South University under specific pathogen-free conditions. Animals were allowed to adapt to their environment for 1 week prior to experimentation.

Twenty-four mice were randomly divided into four groups, and 0.2 ml 1.0×10⁷ MKN45 cells were implanted subcutaneously (s.c.) into the right back side of each mouse. Once the tumor reached ~5 mm in size (14 days after cell inoculation), we initiated treatment with repertaxin alone (30 mg/kg, s.c. next to the tumor, once every two days for 3 weeks), 5-FU alone (10 mg/kg, s.c. once every two days for 3 weeks), repertaxin plus 5-FU in combination, or saline (s.c. once every two days for 3 weeks), which was used as a control. Tumor volume was measured using digital calipers every other day, and calculated according to the following formula: [length × (width)²]/2. After three weeks of therapy, all mice were euthanized with disoprofol according to the institutional guidelines and local tumors were resected and analyzed. A portion of the tumor tissue was fixed and processed for H&E, immunohistochemistry for PCNA and CD34 and another portion was used for TUNEL staining.

All experimental procedures were conducted in conformity with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Ethics Committee of Third Xiang-ya Hospital, Central South University (no. 2012–10). All procedures were in line with the most recent specifications of the National Research Council (US) Committee for the Care and Use of Laboratory Animals (2011) Guide for the Care and Use of Laboratory Animals (National Academies Press, Washington, DC), 8th edition.

Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis. Tumor tissue sections were stained according to the manufacturer's instructions (In situ Cell Death Detection kit, fluorescein; Roche Diagnostics, Indianapolis, IN, USA; cat. no. 11684795910). Sections were detected with a fluorescent microscope (Olympus Corp.) and apoptotic cell nuclei were stained in green.

At least 10 serial, thin (4 μm) sections were cut from each paraffin block. Sections were deparaffinized and then hydrated for hematoxylin and eosin staining and immunohistochemical detection of PCNA and CD34. For immunohistochemistry, endogenous peroxidase activity was inhibited, and then slides were treated with antigen retrieval buffer (citrate buffer, pH 6.0, at 100°C, 2 min or EDTA buffer, pH 8.0, at 100°C, 2 min in a pressure cooker). Samples were then blocked in normal goat serum and stained with primary antibodies including PCNA (Santa Cruz Biotechnology) and CD34 (Santa Cruz Biotechnology) at 4°C overnight. PBS, instead of primary antibody, was applied as a negative control. Slides were washed and then incubated with the appropriate horseradish peroxidase conjugated secondary antibody for 1 h. DAB was used as a chromogen and sections were counterstained with hematoxylin. CD34-positive microvessels were counted under a light microscope (Olympus Corp.) with 10 high magnification fields each group.

The SPSS 13.0 software system (SPSS, Inc., Chicago, IL, USA) was used for statistical evaluation. Data are expressed as mean ± standard deviation (SD) for at least 3 independent experiments. Statistical significance was evaluated by analysis of variance (ANOVA) with Tukey's for post-hoc test. A probability value of <0.05 (P<0.05) was considered to indicate a statistically significant result.

Previously it was established that some tumor cells secrete IL-8 as an autocrine growth factor, which can bind CXCR1/2 receptors and promote tumor growth, invasion and metastatic spread (40). We performed ELISA analysis and found that the gastric cancer cell line MKN45 secretes significant levels of IL-8 over time (Fig. 1).

We investigated the effects of repertaxin both alone and in combination with 5-FU on the proliferation of MKN45 gastric cancer cells. Cells were treated with multiple concentrations of repertaxin alone (50, 25, 2.5 0.25 and 0.025 μg/ml) and 5-FU alone (20, 10, 1, 0.1 and 0.01 μg/ml). Both compounds inhibited proliferation in a dose-dependent and time-dependent manner (Fig. 2A and B). Based on these initial experiments, we selected 25 μg/ml repertaxin and 10 μg/ml 5-FU for follow-up experiments.

As shown in Fig. 2C, the anti-proliferative effect of repertaxin in combination with 5-FU was better than that of repertaxin or 5-FU alone. Similar effects were observed in colony formation assays. That is, the combined treatment of repertaxin and 5-FU more significantly (P<0.05) inhibited colony formation compared to either compound administered individually (Fig. 2D).

We next examined the effect of repertaxin and 5-FU on cell cycle progression by performing flow cytometric analysis. Compared to control, repertaxin increased the percentage of cells in G0/G1 and S phases (96.33 compared to 85.07% in controls) and decreased the percentage of cells in G2/M phase (3.67 compared to 14.93% in controls) (Fig. 3A). These observations were made 24 h after repertaxin (25 μg/ml) treatment. Additionally, the combination of repertaxin and 5-FU performed better than either compound alone (Fig. 3A). We also performed Annexin V/PI staining to assess the effect of repertaxin and 5-FU on apoptosis of MKN45 cells. Repertaxin alone (25 μg/ml) increased the percentage of cells undergoing early (Annexin V⁺/PI⁻) apoptosis (Fig. 3B). Importantly, the early apoptotic rates of repertaxin alone, 5-FU alone, and repertaxin plus 5-FU were 6.43±1.14, 2.21±0.33 and 9.63±0.78%, respectively. Although all treatment groups were significantly different from controls (1.00±0.32%) (P<0.05), the combination performed significantly better than either agent alone (P<0.05).

In addition to effects on cellular proliferation, CXCR1/2 can also influence the migration and invasion of certain tumor cells (41). In the present study, we examined the effect of repertaxin and 5-FU on the migratory and invasive behavior of MKN45 cells. Repertaxin (25 μg/ml) significantly attenuated (P<0.05) chemotaxis migration of MKN45 cells compared to controls (Fig. 4A and C). Similarly, the number of invading cells was also decreased by repertaxin in an in vitro Matrigel invasion assay (Fig. 4A and C). Even further inhibition was observed when repertaxin was combined with 5-FU (10 μg/ml) (Fig. 4A and C). In agreement with results from the Transwell assay, data from the wound healing assay also showed significantly improved inhibition of wound closure in the repertaxin-5-FU combination treatment group compared to either the control group or the individual treatments alone (P<0.05) (Fig. 4B and D).

To characterize the in vivo effects of repertaxin alone and in combination with 5-FU, we established in vivo MKN45 xenograft models in nude mice. Mice treated with either repertaxin (30 mg/kg) or 5-FU (10 mg/kg) alone showed significant reduction in tumor volume and weight compared to control-treated mice (Fig. 5A and B). Importantly, combined administration of repertaxin and 5-FU performed better at reducing xenograft tumor growth compared to either agent alone (both P<0.05) (Fig. 5A and B). All treatments were well tolerated, and we did not observe any signs of general toxicity or body weight loss during therapy. Taken together, our findings suggest that combination therapy of repertaxin and 5-FU may cooperate to effectively reduce gastric cancer tumor growth in vivo.

This ability of combination treatment to repress tumor growth was followed up with immunohistochemical staining of tumor sections isolated from each of the treatment groups. Thirty-five days following treatment, tumors were excised and subject to molecular analysis. H&E staining showed large number of tumor cells, more heterogeneity and pathological mitosis in the controls, but repertaxin and 5-FU alone or in combination significantly reduced the number of tumor cells, showing less heterogeneity and pathological mitosis (Fig. 5C).

TUNEL staining showed that repertaxin and 5-FU were both individually capable of inducing apoptosis (Fig. 5D); importantly, this effect was further increased when both compounds were administered together.

We next performed PCNA staining to assess the effects on proliferation in vivo (42). Repertaxin alone significantly reduced the number of PCNA-positive cells, and combined treatment with 5-FU may have even further decreased the number of proliferating cells (Fig. 5E).

Analysis of apoptosis and proliferation was complemented by examination of angiogenesis, a critical component of gastric cancer growth and metastasis (43,44). Furthermore, the relationship between CXCR1/2 and tumor angiogenesis is well-established (45). The extent of neovascularization of transplanted tumor in nude mice was examined by staining tumor sections with an anti-CD34 antibody and determining microvessel density (MVD) (Fig. 5F). Treatment with repertaxin or 5-FU alone decreased MVD, and the combination of these two compounds may have further decreased the number of MVD/each high-power field (MVD: repertaxin +5-FU: 3.1±1.7; repertaxin: 3.7±1.6; 5-FU: 4.1±1.4 and controls: 6.1±1.9) (Table II).

We next sought to determine which signaling pathways were activated downstream of CXCR1/2. Since AKT and ERK1/2 are well characterized regulators of cell growth, survival and invasion (46,47), we examined the effect of the CXCR1/2 inhibitor repertaxin on phosphorylation of these kinases. Treatment of gastric cancer MKN45 cells with either repertaxin alone or in combination with 5-FU for 48 h significantly downregulated phosphorylation of both AKT and ERK1/2 compared to control cells (Fig. 6E). These findings suggest that AKT and ERK1/2 signaling may be key downstream mediators of CXCR1/2 signaling in gastric cancer cells, and that inhibition of these kinases may be responsible, at least in part, for the anti-proliferative, anti-invasive and anti-angiogenic activity of repertaxin.

We previously showed that repertaxin in combination with 5-FU induces apoptosis of MKN45 cells. To gain insight into this regulation, we examined the effect of these compounds on expression of key apoptosis regulators Bax and Bcl-2 (48). We found that repertaxin significantly decreased expression of the anti-apoptotic molecule Bcl-2 at both the mRNA and protein levels (Fig. 6B and E). In contrast, repertaxin increased expression of the pro-apoptotic molecule Bax (Fig. 6B and E). These findings suggest that repertaxin may influence apoptosis of gastric cancer cells by regulating the Bax/Bcl-2 ratio.

To better understand how repertaxin regulates proliferation of MKN45 cells, we examined expression of cyclin D1, EGFR and Ki-67. While repertaxin significantly decreased mRNA expression of EGFR (Fig. 6C), repertaxin alone had no effect on transcript levels of Ki-67 or cyclin D1 (Fig. 6C); in contrast, repertaxin was able to decrease expression of all three molecules at the protein level (Fig. 6E). Notably, repertaxin plus 5-FU significantly decreased expression of EGFR, cyclin D1, and Ki-67 at both the mRNA and protein levels (Fig. 6C and E). These results suggest that repertaxin alone or in combination with 5-FU may induce cell cycle arrest and inhibit proliferation by reducing levels of cyclin D1, EGFR and Ki-67.

MMP-9, MMP-2, TIMP-2 and E-cadherin are all regulators of cellular migration and invasion. Repertaxin alone significantly decreased mRNA expression of MMP-2 and increased transcript levels of E-cadherin (Fig. 6D). Combination treatment with repertaxin and 5-FU significantly altered mRNA levels of MMP-2, MMP-9, TIMP-2 and E-cadherin (Fig. 6D). Consistent with these findings, protein levels of the molecules were similarly regulated by combined administration of repertaxin and 5-FU (Fig. 6E). Taken together, our data suggest that repertaxin and 5-FU may regulate gastric cancer cell migration and invasion via regulation of MMP-2, MMP-9, TIMP-2 and E-cadherin.

Finally, we examined the effect of repertaxin and 5-FU on expression of the key angiogenesis regulator VEGF. Expression of VEGF at both the mRNA (Fig. 6A) and protein (Fig. 6E) levels was most significantly decreased by combined treatment of repertaxin and 5-FU, suggesting that these compounds regulate angiogenesis by altering levels of VEGF in these cells.