Chinese Medical Journal 2007;120(20):1766-1772
Role of interleukin-8 in the progression of estrogen receptor-negative breast cancer

YAO Chen,  LIN Ying,  YE Cai-sheng,  BI Jiong,  ZHU Yi-fan,  WANG Shen-ming

YAO Chen (Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China)

LIN Ying (Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China)

YE Cai-sheng (Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China)

BI Jiong (Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China)

ZHU Yi-fan (Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China)

WANG Shen-ming (Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China)

Correspondence to:WANG Shen-ming,Division of Breast Surgery, Department of Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China (Tel: 86-20-87755766 ext. 8198. Fax:86-20-87755766 ext. 8198. E-mail:shenmingwang@sohu.com)
Keywords
breast cancer; interleukin-8; estrogen receptor; RNA interference
Abstract

Background  Estrogen receptor (ER) is a very important biomarker of breast cancer. ER deletion has been consistently associated with tumor progression, recurrence, metastasis and poor prognosis, but the biological mechanism is still unclear. ER negative breast cancer expresses high levels of interleukin-8 (IL-8). ER expression can downregulate IL-8 promotor activity. As a multifunctional cytokine, IL-8 has many important biological activities in tumor genesis and development. With the goal of investigating the role of IL-8 in ER-negative breast cancer progression, we applied RNA interference technology to specifically knockdown the IL-8 expression in ER-negative breast cancer cell line MDA-MB-231.
Methods  Interfering pRNA-IL-8 and the control was transfected into ER(-) MDA-MB-231. The proliferation, cell apotosis, and invasive ability were recorded in transfected, untransfected and negative transfected cells. These cells were injected into nude mice to assess tumorigenicity, proliferation, metastasis and microvessel density (MVD).
Results  In vitro, decreased expression of IL-8 was associated with reduced cell invasion (P<0.001), but had no effect on cell proliferation (P>0.05). In vivo, neutrophils infiltration was significantly inhibited in pRNA-IL-8 transfected cells compared with untransfected and negatively transfected cells (P=0.001, P<0.001). Less metastasis was found in transfected cells compared with negatively transfected cells (0% vs 80%, P=0.048). Nevertheless, we observed less MVD in transfected cells compared with control in nude mice (P<0.001).
Conclusions  IL-8 inhibits ER-negative breast cancer cell growth and promotes its metastasis in vivo, which may be correlated with neutrophils infiltration induced by IL-8.

Breast cancer is the most common malignant tumor in women. Estrogen receptor (ER) is a very important biomarker of breast cancer. ER has been consistently associated with tumor progression, recurrence, metastasis, and poor prognosis,1,2 but the biological mechanism is still unclear. Recent efforts have focused on exploring the role and prognostic value of tumor cell derived cytokines in human breast cancer. We reported expression of interleukin-8 (IL-8) is linked to status of ER, metastais, and vimentin in breast cancer cells. Tumor biological experiments suggest that IL-8 is involved in breast cancer cell invasion and angiogenesis.3 IL-8 is closely correlated with ER. ER expressiones can downregulate IL-8 promotor activity.3-5

With the goal of investigating the role of IL-8 in ER-negative breast cancer progression, we applied RNA interference technology to specifically inhibit the expression of IL-8 in ER-negative breast cancer cell line MDA-MB-231 that expresses high level of IL-8.

METHODS

Cell culture
The breast cancer cell line MDA-MB-231 cells were cultured with Leibovitz’s L-15 medium, containing 15% fetal calf serum (FCS). Cells were cultured adhered to the flask. Culture conditions were 37°C, 5% CO2, and 95% humidity.

Plasmids
The IL-8 siRNA expression plasmid (pRNA-IL-8) and the ‘empty’ plasmid (pRNA H1.1) were provided by Prof. Ruo-Pan Huang (Emory University, USA). Vector pRNA H1.1 from Genescript (Piscataway, NJ, USA) was used to construct IL-8 siRNA expression vectors. The sequences of corresponding IL-8 siRNA (65 bp) were as follows: sense: 5’-GATCCCGATGCCAGTGAAACTTCAATTG-ATATCCGTTGAAGTTTCACTGGCATCTTTTTTCCAAA-3’, and anti-sense: 5’-AGCTTTTGGAAAAAAGAT- GCCAGTGAAACTTCAACGGATATCAATTGAAGTTTCACTGGCATCGG-3’. The sequences were designed using Genescript software through the company website, synthesized (Qiagen, Valencia, CA), annealed to be sticky dsDNA with Bam H1 and Hind III clone sites and cloned into Bam H1 and Hind III sites of psiRNA. The sequence cloned into pRNA H1.1 was verified by DNA sequence.

DNA transfection
Human breast cancer cells (MDA-MB-231) were plated onto 100-mm dishes at a density of 2 × 105/dish and grown in complete media until they reached 70%–80% of confluence. The IL-8 siRNA vector (pRNA-IL-8) and vector alone (pRNA H1.1) were individually transfected into the cells by lipofection (LipofectamineTM 2000, Invitrogen, USA). After 48 hours, G418 (800 µg/ml) was added to the cells. Medium was changed every 3 days. Ninety-nine percent of cells in the control plate died whereas transfected cells produced colonies, which survived 3 weeks of G418 treatment. G418-resistant clones were randomly selected, isolated, and expanded individually in order to test the level of the transfected genes.

Reverse-transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated with Trizol (Invitrogen) according to the protocol providing by the manufacturer. After washing twice with 70% ethanol, the RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water. Four μg of total RNA was reverse transcribed with the MuLV reverse transcriptase (Fermentas, Lithuanian) at 42°C for 1 hour, and the reaction was terminated by incubation in 72°C for 10 minutes. One microliter out of the 20 μl resulting cDNA was used in each PCR. Intron-spanning primers were designed with the Primer3 output program (http://www.genome.wi.mit.edu/cig-bin/ primer/primer3.cgi, Whitehead Institute, Cambridge, MA, USA) and identified by the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). The primers used to amplify IL-8 mRNA from cDNA were forward: 5’-ATGACTTCCAAGCTGGCCGTGGCT-3’ and reverse: 5’-TCTCAGCCCTCTTCAAAAACTTCTC-3’. PCR was performed in a 25-ml reaction volume for 35 cycles under the following conditions: initial denaturation at 94°C for 10 minutes, then 35 amplification cycles were run, consisting of 94°C for 60 seconds, 60°C for 60 seconds, 72°C for 60 seconds, and a final extension at 72°C for 10 minutes. The product was 293 bp. β-actin was used as an internal control; the forward primer was: 5´-GGAACCGCTCATTGCCAA-3´ and reverse: 5´-GG- ATTCCTATGTGGGCGACG-3’. PCR was performed in a 25 ml reaction volume for 30 cycles under the following conditions: initial denaturation at 94°C for 5 minutes, then 30 amplification cycles were run, consisting of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. The product was 602 bp. PCR products were examined by agarose gel electrophoresis.

Enzyme linked immunosorbent assay (ELISA)
The IL-8 concentration in culture supernatants was determined by ELISA as recommended by the manufacturer. Basically, 96 well ELISA plates were coated with 100 μl of 4 μg/ml monoclonal anti-IL-8 antibody (MAB-208, R&D System, Minneapolis, MN, USA) overnight at 4°C. All wells were then blocked with 1% bovine serum albumin (BSA, Genview, USA). Samples and standard IL-8 (100 μl) were added to the wells. The plates were incubated for 3 hours at room temperature, or overnight at 4°C. After removing unbound materials by washing with phosphate buffer solution/0.05% Tween, 100 μl of 1 μg/ml of the appropriate biotinylated anti-IL-8 antibody (BAF-208, 20 ng/ml, R&D System) were added to each well. The plates were incubated for 1 hour at room temperature. After washing, 100 μl of streptavidin-horseradish peroxidase conjugate (RPN1231, Amersham-Pharmacia, Bucking- hamshire, UK) was added to the wells and incubation was continued for 30 minutes at room temperature. Plates were washed again and chromogen substrate (Sigma Fast OPD, Sigma, St. Louis, MO, USA) added. The plates were read at OD 450 nm in a microplate reader.

Proliferation assays
Assays were done with a cell titer 96 non-radioactive (3-(4,5-dimethylthiazol-2-yle) 2,5-diphenyltetrazolium bromide) assay kit (MTT assays) according to the manufacture’s instruction (Promega). Briefly, cells were plated in 96-well plates with complete culture medium. The experiment was stopped every 24 hours for 7 consecutive days. On the given days, cells were treated with MTT for 4 hours, followed by incubation with lysis buffer at room temperature overnight. OD at 570 mm was determined. The OD values were expressed as relative viable cell number.

Flow cytometry
Cell cycle state and apoptosis were assessed by flow cytometry. Approximately 5×106 cells in the logarithmic phase were collected from each group and were centrifuged (1000 r/min, 5 minutes). The supernatant was removed. The cells were fixed for 24 hours at 4°C in pre-cooled 70% ethanol, washed with PBS 3 times, and re-suspended in PBS (0.4 ml). RNaseA was then added (10.15 g/L) and incubated for 1 hour at 37°C. Cells were stained with propidium iodide (1 g/ml) at 4°C overnight. Cell cycle state and apoptosis were assessed by flow cytometry; data were analyzed using the ModFit software.

Invasion assays
Invasion assays were performed using 12-well polycarbonate filter (12-μm pore size) Transwell (Corning-Costar, Corning, NY, USA) coated with matrigel (BD Pharmingen, USA). After 24 hours of incubation, cells that migrated to lower side of the filter were fixed in 4% formaldehyde for 30 minutes and stained with hematoxylin solution (DAKO, USA) for 90 seconds. The number of matrigel-invading cells was counted under microscope. Each experiment was carried out 3 times.

Animal studies
Nude mice (n=15, BALB/c-nu/nu mice, female, 4–6 weeks old, weighing 14–21 g) were purchased from the Laboratory Animal Center of Sun Yat-Sen University, Guangzhou, China. They were maintained under pathogen-free conditions in the facilities of Laboratory Animal Center at Sun Yat-Sen University. Cells from the three groups were cultured in the logarithmic phase for injection. Viable cells were >90%. Cells were centrifuged (100 r/min, 5 minutes) and the supernatant was removed. Cell suspension was prepared with a viable cell concentration of 2.5×107/ml.

The nude mice were divided randomly into three groups, five in each. Each nude mouse was injected with 0.2 ml (i.e., 5×106 cells) cell suspension in the subcutaneous layer in the right lower back using a 1-ml syringe. After injection, the mice were observed for time of tumor appearance, change in tumor size, and determining the tumor’s growth curve. Tumor volume (mm3) was calculated by the formula: turmor size= ab2/2. The symbol ‘a’ is the larger and ‘b’ is the smaller of the two demesions.

Histological analysis
After a 6-week observation, all mice were sacrificed by cervical dislocation. The tumors were removed and tumor tissues were peeled off. Examinations were conducted to determine whether cancer cells had moved to the lymph nodes, brain, lung, heart, liver, spleen, pancreas, or kidneys. The peeled tissues were fixed with 10% formaldehyde for 24 hours and subsequently embedded with paraffin. Five micron sections were stained with hematoxylin and eosin (HE). Graded ethanol solutions were used for gradual dehydration. Two changes of xylene were then used to clear the tissues before embedding them in paraffin wax. Sections (4–6 µm) were cut, stained with HE, and were examined under a microscope.

Immunohistochemical staining
After antigen recovery by microwave boiling, sections were put into PBS, to which 3% H2O2 was added to block endogenous peroxidase activity, and incubated for 10 minutes. They were then washed with distilled water, put in the PBS for 10 minutes, and incubated at room temperature for 1 hour with polyclonal anti-CD34 antibody (Rabbit anti-mouse, Boster, China). Sections were washed in PBS for 10 minutes, before the EnVisionTM reagent (DAKO, Denmark) was added for a 30-minute incubation. Sections were again washed in PBS for 10 minutes before the color was developed with 3-3’ diaminobenzidine tetrachloride (DAKO, Denmark). Sections were observed by light microscopy. Microvessel density (MVD) was quantified by counting yellow-brown-stained single endothelial cells or endothelial cell clusters, as long as the cells were clearly separated from the surrounding microvessel, tumor cell, or other connected tissues.

Statistical analysis
All data were expressed as mean ± standard deviation (SD). Data were analyzed with SPSS 13.0. A P value less than 0.05 was considered statistically significant.

RESULTS

Inhibition of IL-8 expression by IL-8 siRNA in MDA-MB-231 cells
To test the role of IL-8 in ER-negative breast cancer progression, we applied RNA interference technology to knock down the endogenous IL-8 expression. The expression of IL-8 mRNA in breast cancer cells was examined by RT-PCR analysis. As shown in Fig. 1, all 3 cell lines expressed β-actin without difference. The expressions of IL-8 were further confirmed by ELISA. As shown in Table 1, the expression level of IL-8 siRNA transfected cell was significantly lower than those in control groups.
 

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Fig. 1. RT-PCR was used to measure the levels of IL-8 mRNA (A: pRNA-IL-8 transfected cells, B: pRNA H1.1 transfected cells, C: untransfected cells, M: DNA marker).


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Table 1. IL-8 concentration in culture supernatants

Influence of IL-8 on cell proliferation
Growth curves for the three groups are shown in Fig. 2. There was no significant difference between the OD540 values for the three groups (P>0.05). There was no effect of IL-8 on proliferation of the ER-negative breast cancer cell line in vitro.
 

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Fig. 2. Cell proliferation rates

Influence of IL-8 on cell cycle state and apoptosis
All three cell lines had no significant differences in cell cycles and apoptosis rates, although they had different expression levels of IL-8 (Table 2, Figs. 3 and 4). This result suggests that IL-8 did not affect cell cycle state or apoptosis of these ER-negative breast cancer cells.
 

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Table 2. Influence of IL-8 on cell cycle state


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Fig. 3. Flow cytometry labeling of nuclear DNA (A: pRNA-IL-8 transfected cells, B: pRNA H1.1 transfected cells, C: untransfected cells).


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Fig. 4. Flow cytometry labeling apoptosis (A: pRNA-IL-8 transfected cells, B: pRNA H1.1 transfected cells, C: untransfected cells).

Influence of IL-8 on tumor cell invasion
As shown in Fig. 5, cells expressing high amounts of IL-8 tend to be more potent to invade through matrigel. Only 23.4±7.8 cells were found invading in pRNA-IL-8 transfected cells, while 66.9±11.5 in control transfected cells and 75.5±17.6 in untransfected cells were found (P<0.001). This result suggests that high IL-8 expression is correlated with ER-negative breast cancer invasion and metastasis potential.
 

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Fig. 5. The invasive ability through metrigel (Hematoxylin staining, original magnification ×200) (A: pRNA-IL-8 transfected cells, B: pRNA H1.1 transfected cells, C: untransfected cells).

Influence of IL-8 on the growth of inoculated tumor
Tumors appeared in all nude mice of the three groups 6 to 7 days after inoculation (100% incidence). Thus, it appears that IL-8 had no influence on the tumorigenicity of these ER-negative breast cancer cells. Growth curves of transplanted tumors and samples excised from the nude mice of the three groups are shown in Figs. 6 and 7. The tumor growth rate in the mice of the pRNA-IL-8 transfected group was significantly higher than those of the two control groups. This result suggests that IL-8 may suppress the growth of ER-negative breast cancers in vivo.


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Fig. 6. Tumor sizes and growth curves for each cell lines.


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Fig. 7. Tumor gross appearance. A1–5: pRNA-IL-8 transfected cells, B1–5: pRNA H1.1 transfected cells, C1–5: untransfected cells.

Histological analysis
Tumor samples of the three groups were stained with HE. Low differentiated adenocarcinomas were seen under the microscope. Much infiltration of inflammatory cells was seen around the tumor tissues of the control groups (mainly lymphocytes, monocytes, and neutrophils). However, the infiltration of inflammatory cell around the tumor tissues of the pRNA-IL-8 transfected group was much less (Fig. 8). Under low magnification (×100), the three most densely infiltrated areas were selected and then the number of inflammatory cells in these areas was counted under higher magnification (×400). There was a significant difference between the pRNA-IL-8 transfected group (44.0±9.1) and control transfected group (127.4±25.9) (P=0.001), and also untransfected group (140.0±34.3)(P<0.001), though there was no significant difference between the control groups (P=1.000).


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Fig. 8. Tumor sections with HE staining (Original magnification ×100) (A: pRNA-IL-8 transfected cells, B: pRNA H1.1 transfected cells, C: untransfected cells. Arrow shows infiltration of inflammatory cell around the tumor tissues).

Tumor metastasis
No metastasis was seen in the pRNA-IL-8 transfected group. However, three metastasis cases (lymph node of the armpit) were found in negative control group and four metastasis cases (again, lymph node of the armpit) were found in the untransfected control group; one of these also had a liver metastasis. This metastasis rate was significantly different between the pRNA-IL-8 transfected group and untransfected control group (P=0.048), but there was no significant difference between the pRNA-IL-8 transfected group and the negative control group (P=0.167) or between the untransfected group and negative control group (P=1.000).

MVD of the tumors
As shown in Fig. 9, the MVD of the tumor tissue in the pRNA-IL-8 transfected group (10.8±2.2) was significantly lower than those in the two control groups (28.6±2.3 and 31.2±3.5) (P<0.001), though there was no significant difference between the control groups (P=0.470). These results suggest that IL-8 may promote angiogenesis in ER-negative breast cancer in vivo.


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Fig. 9. Tumor sections with CD34 antibody staining for MVD (Original magnification ×200) A: pRNA-IL-8 transfected cells, B: pRNA H1.1 transfected cells, C: untransfected cells.

DISCUSSION

IL-8 was the first chemotactic factor identified by Yoshimura et al6 in 1987. IL-8 is a member of the CXC family and is the most powerful neutrophil chemotactic factor and promoter yet known.7 It is also a chemoattractant for eosinophils and T lymphocytes.8 Recent research has identified many properties of IL-8, including important biological activities in the development and progression of various tumors. However, its detailed mechanism in the development of ER-negative breast cancer remained unknown. We established an IL-8-knockdown breast cancer cell line so that we could examine changes in the malignant phenotype in vitro and in vivo associated with specific suppression of IL-8 expression (by RNA interference) in the ER-negative breast cancer line MDA-MB-231.

IL-8 inhibits the growth of tumors in vivo, but not in vitro
The influence of IL-8 on the proliferation of tumors remained unsettled. Wang et al9 reported that IL-8 might inhibit the proliferation activity of non-small cell carcinoma cell through autocrine and paracrine secretion. On the other hand, other reports have suggested that IL-8 might induce proliferation of lung cancer cells.10 Hirose et al5 reported that transfection of a human IL-8 expression plasmid into the ovarian cells of Chinese hamsters did not affect the proliferation rate of the cells in vitro, but that the proliferation of the cells was significantly suppressed when they were transplanted into nude mice. Lee et al11 reported that IL-8 did not change the growing characteristics of ovary cancer cells in vitro, but it was found that IL-8 could suppress the growth of ovarian cancer in vivo. Our study showed that decreased IL-8 expression did not significantly affect the proliferation activity of ER-negative breast cancer cell in vitro. Furthermore, we found no impact of IL-8 on the cell cycle state or apoptosis, as assessed by flow cytometry. In fact, our results suggest that IL-8 doesn’t participate in the growth and proliferation of ER-negative breast cancer cell in vitro.

In contrast, the results of our in vivo experiments were quite different. High level of IL-8 expression apparently inhibited tumor growth in nude mice of a transplanted tumor of ER-negative breast cancer cells. The tumors growth after IL-8 knock-down in the group with siRNA was significantly faster than that in the group with high level of IL-8 expression. This result is consistent with those of Hirose et al5 and Lee et al.11

Previous reports and our study showed that the infiltration of inflammatory cells, such as neutrophils, around the tumor increased significantly. We believe that the inhibitory effect of IL-8 on the tumor growth in vivo was due to its powerful chemotactic effects on neutrophils. It has been suggested that there are direct and indirect mechanisms whereby IL-8-activated nentrophils can exhibit their anti-tumor effects. Direct mechanisms may involve IL-8-activated neutriphils inducing shape change, granular release, and production of oxygen free radicals and proteases, which may lead to tumor cell death.12 Indirect mechanism may involve the production of mediators by neutrophils in response to IL-8, e.g., TNFα, IL-1, and interferons (IFNs).13

IL-8 may promote the invasiveness and metastasis of ER-negative breast cancer
De Larco et al14 discovered that IL-8 had a positive correlation with the invasive ability of breast cancer cells. Specifically, the more powerful the invasive ability of the cell, the higher the IL-8 expression was. In vitro studies of malignant melanoma and prostate cancer showed that IL-8 could promote invasiveness and metastasis of tumor cells.15-17 However, Balbay et al18 showed no influence of IL-8 on the metastasis of prostate cancer cells and Lee et al11 reported that IL-8 did not promote metastasis of ovarian cancer cells. Our study showed that decreased IL-8 expression reduced significantly the metastatic and invasive ability of ER-negative breast cancer cells in vitro. The number of cells with IL-8 knocked down passing through the matrigel reconstructed basement membrane was only one-third to one-quarter of the control groups. The in vivo experiment showed that the rate of tumor metastasis of the IL-8 knockdown group (0%) was significantly lower than that of untransfected control group (80%), suggesting that IL-8 may promote metastasis of ER-negative breast cancer cells in vivo. Metastasis of ER-negative breast cancer cells promoted by IL-8 may be the result of direct promotion of invasion and metastasis of ER-negative breast cancer cells, as well as by neutrophil infiltration induced by IL-8. Some reports have indicated that neutrophils attracted by IL-8 can release proteases, heparin, and enzymes in the process of moving to the region of a tumor, causing degradation of the extracellular matrix, resulting in metastasis of the tumor.19

IL-8 appears to promote angiogenesis in ER-negative breast cancer in vivo. Substantial tumor growth and metastasis depend on angiogenesis.20 When the size of a tumor exceeds 12 mm3, sustained angiogenesis is required. The MVD of tumor tissue is an important indicator of angiogenesis. Our study showed that MVD in the pRNA-IL-8 transfected group was significantly lower than those of the control groups. This suggests that IL-8 may promote angiogenesis in ER-negative breast cancers. Other reports have also indicated that IL-8 may induce angiogenesis21,22 and that inhibition of IL-8 expression could attenuate angiogenesis in bronchogenic carcinoma.23

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  1. Guangdong National Science Foundation,No. 06300809;