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10.1245/ASO.2005.07.015
Annals of Surgical Oncology 12:800-807 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Interleukin 8 in Human Hepatocellular Carcinoma Correlates With Cancer Cell Invasion of Vessels But Not With Tumor Angiogenesis

Fumitake Kubo, MD, Shinichi Ueno, MD, PhD, Kiyokazu Hiwatashi, MD, Masahiko Sakoda, MD, Koichi Kawaida, MD, Kensuke Nuruki, MD and Takashi Aikou, MD, PhD

Course of Advanced Therapeutics, Field of Oncology, Department of Surgical Oncology and Digestive Surgery, Kagoshima University Graduate School of Medicine and Dental Sciences, 8-35-1 Sakuragaoka Kagoshima, 890-8520, Japan

Correspondence: Address correspondence and reprint requests to: Fumitake Kubo, MD; E-mail: f-kubo{at}m3.kufm.kagoshima-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Angiogenic factor seems necessary for the development of hepatocellular carcinoma (HCC), which is a hypervascular malignancy. This study examined the expression of interleukin (IL)-8, a potent angiogenic factor, in HCC samples.

Methods: We measured IL–8 expression by using reverse transcriptase-polymerase chain reaction in clinical HCC tissues from 45 patients who underwent surgical resection. We then assessed correlations between IL–8 expression and microvessel growth or clinicopathologic factors. We also elucidated the in vitro effect of IL–8 on HepG2 development by using fluorometric assays of proliferation, chemotaxis, and invasion.

Results: The expression of IL–8 did not significantly correlate with the microvessel count in HCC tissues, but the incidence of microscopic vessel invasion was significantly higher in IL–8–positive than in IL–8–negative tissues. Thus, more IL–8 was expressed in HCCs at pathologic stage III/IV than in those at stage I/II. Assays in vitro showed that IL–8 stimulates HepG2 chemotactic and invasive activities rather than cell proliferation.

Conclusions: The expression of IL–8 in human HCC has more relevance to metastatic potential, such as vessel invasion, than to angiogenesis or cell proliferation.

Key Words: Interleukin 8 • Hepatocellular carcinoma • Tumor progression • Angiogenesis • Chemotaxis • Vessel invasion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular carcinoma (HCC) is usually hypervascular, and it is a common malignancy.1,2 With respect to host- and tumor-related factors, we have applied surgical resection, percutaneous ethanol injection, transarterial chemoembolic therapy, microwave coagulation therapy, or radiofrequency ablation. The 5-year survival rate is only approximately 60% even among patients with stage I/II tumors.3 Thus, understanding the biological behavior of HCC is important for improving the clinical treatment of HCC patients.

Without the ability to recruit new vessels, the growth of most tumors would be limited.4–6 Tumor angiogenesis is significantly correlated with tumor progression, invasion, and metastasis and is recognized as an important step in the pathogenesis involved in a poor prognosis.7 Changes in angiogenic and angiostatic factors might precede the conversion of tumor cells to an angiogenic phenotype. Furthermore, the initiation and maintenance of tumor angiogenesis depends on a balance between these factors.

Originally discovered as a chemotactic factor for leukocytes, multifunctional interleukin (IL)–8 belongs to the superfamily of CXC chemokines and is potently angiogenic both in vivo and in vitro.8–10 Stimulation with various factors, including lipopolysaccharide, IL-1, and tumor necrosis factor {alpha}, results in rapid IL-8 transcription and the production of IL-8 protein.11–14 IL-8 contributes to human cancer progression through potential mitogenic, angiogenic, and motogenic functions. Potently angiogenic, IL-8 is constitutively and frequently produced in various carcinomas cell lines, including those of HCC. One study found that 29 of 30 carcinoma cell lines expressed IL-8 and that 19 of those were high producers.15 We reported that IL-8 messenger RNA (mRNA) is constitutively expressed in HepG2 cells and in human HCC and that polyomavirus enhancer A–binding protein 3 might play an important role in IL-8 expression rather than nuclear factor-{kappa}B, which is a known IL-8 transcription factor.16

This study investigated IL-8 mRNA expression, the microvessel count (MVC) in clinical HCC samples, and the correlation between IL-8 mRNA expression and MVC and clinicopathologic factors. We also explored the in vitro effect of IL-8 on HepG2 development by using reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorometric assays of proliferation, chemotaxis, and invasion.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
HCC (HepG2) cells that produce IL-8 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/mL each of penicillin and streptomycin.

Clinical Samples
We obtained HCC tissue samples from 45 patients with primary HCC (36 men and 9 women; mean age, 63 ± 11 years; range, 21–78 years) who underwent surgical resection as a primary treatment at our department between January 1995 and December 1998. Samples for RT-PCR were frozen and stored at –80°C. Samples of HCC were fixed in 10% formalin and embedded in paraffin for immunohistochemical analysis. This clinical study protocol was approved by the Kagoshima University Graduate School of Medicine and Dental Sciences Institutional Review Board.

Immunohistochemical Staining for CD34
We counted microvessels after staining with a primary mouse monoclonal immunoglobulin G antibody for CD34 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; diluted 1/100). Formalin-fixed, paraffin-embedded tissue samples cut in 4-µm-thick sections were mounted on slides coated with poly-L-lysine and then deparaffinized and rehydrated. The slides were autoclaved at 120°C for 10 minutes in citrate buffer (10 µmol/L) at pH 6.0 and cooled to room temperature for 2 hours. Endogenous peroxidase was blocked with.3% H2O2 in absolute methanol for 30 minutes, and then nonspecific binding was blocked with 1% bovine serum albumin for 2 hours. The sections were incubated overnight with the primary specific antibody for CD34, followed by 1 hour each with biotinylated anti-mouse immunoglobulin (immunoglobulin G; Vector Laboratories Inc., Burlingame, CA) and avidin-biotin-peroxidase complex. Antibody binding was visualized with 3,3'-diaminobenzidine tetrahydrochloride, and the sections were lightly counterstained with Mayer’s hematoxylin.

Microvessel Quantitation
We counted vessels in the three most highly vascularized areas visualized by CD34 immunostaining under x40 and x200 fields. We considered single endothelial cells or clusters with or without lumen as neovascularization.17,18 Finally, the average of three x200 field counts by four observers (F.K., S.U., K.H., and K.K.) was evaluated as MVC. Tumors with MVC >40 or ≤40 were classified as having high-grade (Fig. 1aGo) or low-grade (Fig. 1bGo) vascularity, respectively.



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  		FIG. 1. Immunohistochemical staining of human hepatocellular carcinoma tissue with anti-human CD34 antibody. Slides were immunostained with avidin-biotin-peroxidase complex. Intratumoral microvessels or de novo capillaries were detected as single or clustered endothelial cells with or without lumen. (a) High-grade vascularity. (b) Low-grade vascularity (counterstain, Mayer’s hematoxylin; original magnification, x200).

 
Reverse Transcriptase-Polymerase Chain Reaction
We detected IL-8 mRNA expression by using RT-PCR of frozen HCC tissues obtained from patients with HCC who had undergone surgical hepatectomy. Total RNA (.5 µg) was extracted by using the Total RNA Extraction Miniprep system (Viogene, Sunnyvale, CA). Pelleted RNA was resuspended in diethylpyrocarbonate-treated water and stored at –80°C. Reverse transcription proceeded with oligo-dT primers and the Advantage RT-for-PCR kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. First-strand complementary DNA synthesized at 42°C for 1 hour in a thermal cycler was amplified by 30 PCR cycles (95°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute). For detection of matrix metalloproteinase (MMP) mRNA expression, the same method was used (94°C for 30 seconds, 58°C for 1 minute, and 72°C for 1 minute). The oligonucleotide primer pairs were as follows:

ß-Actin sense, 5'-AAGAGAGGCATCCTCACCCT-3'

ß-Actin antisense, 5'TACATGGCTGGGGTGTT GAA-3'

IL-8 sense, 5'-ATGACTTCCAAGCTGGCCCG-3'

IL-8 antisense, 5'-CTCAGCCCTCTTCAAAAACT T-3'

MMP-2 sense, 5'-GTGCTGAAGGACACACTAAA GAAGA-3'

MMP-2 sense, 5'-TTGCCATCCTTCTCAAAGTTG TAGG-3'

MMP-7 sense, 5'-GGTCACCTACAGGATCGTAT CATAT-3'

MMP-7 sense, 5'-CATCACTGCATTAGGATCAG AGGAA-3'

MMP-9 sense, 5'-CACTGTCCACCCCTCAGAGC-3'

MMP-9 sense, 5'-GCCACTTGTCGGCGATAAGG-3'

The predicted PCR products for ß-actin, IL-8, MMP-2, MMP-7, and MMP-9 were 218, 278, 605, 373, and 263 base pairs long, respectively (Figs. 2Go and 3Go).



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  		FIG. 2. Analysis of human interleukin (IL)-8 and ß-actin messenger RNAs in hepatocellular carcinomas (HCC) and HepG2 cells by reverse transcriptase-polymerase chain reaction. Lane 1, HepG2 cells; lanes 2–8, clinical HCC samples. bp, base pairs.

 


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  		FIG. 3. Expression of matrix metalloproteinase (MMP) messenger RNA in HepG2 cells treated with recombinant interleukin (IL)-8. After 12 hours of incubation with various concentrations, RNAs were served for reverse transcriptase-polymerase chain reaction as described. Left lane, untreated; middle lane, IL-8 (1 ng/mL); right lane, IL-8 (10 ng/mL). bp, base pairs.

 
HepG2 Proliferation Assay
HepG2 cells (1 mL per well; 1 x 105 cells/mL) in DMEM containing 10% FBS were placed in 24-well plates and stimulated with various concentrations of IL-8. Plates were incubated at 37°C in 5% CO2 for 72 hours, and then cell proliferation was determined by using a fluorometric method, as described below. Plates were centrifuged at 1500 rpm for 15 minutes; the supernatant was then aspirated, and 1 mL of calcein AM (Dojindo Lab, Kumamoto, Japan) diluted 2 µg/mL in phosphate-buffered saline (PBS) was added to each well. After incubation for 60 minutes, the calcein AM was replaced with PBS. Fluorescence was immediately measured from the bottom of the plates by using a CytoFluor 4000 TC fluorescent multiwell plate reader (PerSeptive Biosystems, Framingham, MA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm (gain, 55). Each sample was tested in triplicate, and each experiment was performed at least three times.

HepG2 Chemotaxis Assay
We used a fluorometric chemotaxis assay as described previously.19 HepG2 cell chemotaxis was investigated in Falcon 24-well cell-culture plates (fluorescent immunoassay [FIA] chamber) with 8-µm-pore Falcon HTS FluoroBlok inserts (Becton Dickinson, Franklin Lakes, NJ) that block fluorescence. The chemoattractant in the lower wells was DMEM containing 10% FBS (700 µL). Growth medium in the upper wells was DMEM containing 1% FBS (200 µL) with or without various concentrations of recombinant IL-8. Cell suspensions (100 µL of culture medium containing 1 x 106 cells per milliliter) were added to the upper wells. Various concentrations of anti-human IL-8 antibody were added to the cells to assay inhibition. After incubation at 37°C for 12 hours, the FIA chamber was gently rinsed to remove remaining serum. Subsequently, 700 and 300 µL of calcein AM (2 µg/mL in PBS) were added to the outer and inner chambers, respectively. After incubation for 60 minutes, calcein AM was replaced with PBS to reduce the background. We measured fluorescence as described for the HepG2 proliferation assay.

HepG2 Invasion Assay
We assayed the effects of IL-8 on the invasive activity of HepG2 cells in vitro by using Matrigel-coated Falcon HTS FluoroBlok inserts. We measured fluorescence in the FIA chamber immediately after a 72-hour incubation as described for the HepG2 chemotaxis assay.

Statistical Analysis
Differences between subgroups with respect to IL-8 expression and clinicopathologic findings or MVC were evaluated by the {chi}2 test or Fisher’s exact test. P values <.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relationship Between IL-8 Expression in HCC Tissue and Clinicopathologic Factors
Of the 45 HCCs, 35 (77.7%) expressed IL-8. Table 1Go shows the correlation between IL-8 expression and clinicopathologic factors. The expression of IL-8 in HCC did not significantly differ with respect to age, sex, viral infection, liver function (data not shown), liver damage,20 gross classification of tumors,20 distinct tumor size, number of tumors, capsule formation, capsule invasion, or histological differentiation. However, the incidence of microscopic vessel invasion (including both portal vein and hepatic venous invasion) was significantly higher in the IL-8–positive than in the IL-8–negative group. Furthermore, HCCs at pathologic stage III/ IV expressed more IL-8 than those at pathologic stage I/II.


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  		TABLE 1. Correlation between IL-8 expression and clinicopathologic characteristics of patients with hepatocellular carcinoma
 
Relationship Between MVC and IL-8 Expression in HCC Tissue
Microvessels were heterogeneously distributed in tumors, and density was generally maximal at boundaries or near the capsules of capsulated tumors. Of the 45 HCCs, 24 (53.3%) and 21 (46.7%) were of high-grade (MVC >40) and low-grade (MVC ≤40) vascularity. The expression of IL-8 did not correlate with the MVC in HCC tissue (Table 2Go).


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  		TABLE 2. Relationship between IL-8 expression and microvessel count in hepatocellular carcinoma tissue
 
Effects of IL-8 and Anti–IL-8 Antibody on HepG2 Proliferation
Because IL-8 is mitogenic for some cell types and because HepG2 cells express IL-8, we examined whether HepG2 cells can use IL-8 for proliferation. Exogenous IL-8 in the range of 1 to 10 ng/mL did not accelerate HepG2 proliferation (Fig. 4aGo), and the neutralizing antibody did not significantly influence proliferation (Fig. 4bGo).



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  		FIG. 4. Effect of human recombinant interleukin (IL)-8 (a) or anti-human IL-8 antibody (b) on HepG2 cell proliferation. IL-8 did not remarkably accelerate cell proliferation, and neutralizing antibody added to culture medium did not remarkably inhibit cell proliferation. n.s, not significant.

 
Effects of IL-8 and Anti–IL-8 Antibody on HepG2 Chemotaxis
Chemotaxis assays in vitro showed that HepG2 migrated in response to human recombinant IL-8 in a dose-dependent manner (Fig. 5Go) within the range of 1 to 10 ng/mL. Exogenous anti–IL-8 antibody (10 µg/ mL) significantly inhibited this chemotactic response (Fig. 6Go).



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  		FIG. 5. Effect of human recombinant interleukin (IL)-8 on invasion of HepG2 cells. IL-8 remarkably accelerated cell invasion. *P < .05.

 


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  		FIG. 6. Effect of human recombinant interleukin (IL)-8 or anti-human IL-8 antibody on HepG2 cell migration. IL-8 induced remarkable acceleration of cell migration, and neutralizing antibody (10 µg/mL) added to culture medium containing 1 to 10 ng/mL of recombinant IL-8 remarkably inhibited cell migration. *P < .05.

 
Effects of IL-8 on the Invasion of HepG2
Recombinant IL-8 within the range of 1 to 10 ng/ mL significantly accelerated the invasion of HepG2 cells. To establish the mechanism of acceleration of cell-invasive ability, we examined MMP-2, -7, and -9 expressions of HepG2 cells by RT-PCR. Only MMP-7 seemed to be correlated with IL-8 administration at the concentration of 10 ng/mL. In contrast, MMP-2 and MMP-9 did not change (Fig. 3Go). Thus, IL-8 induces MMP-7 and promotes invasion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and platelet-derived endothelial cell growth factor, have been characterized. Several reports have identified the importance of VEGF or b-FGF expression in human HCC.21,22 IL-8 expression is closely correlated with the clinicopathologic factors of solid tumors such as gastric,23 non–small-cell lung,17 and prostate24 cancers. However, little is known about the correlation between IL-8 expression and clinicopathologic factors in HCC.

Although the precise mechanism of IL-8 regulation in tumor cells remains unknown, experimental studies have shown that IL-8 derived from tumor cells is associated with tumor angiogenesis and metastatic potential. Several experimental studies have demonstrated that IL-8 mRNA increases the activity of some MMPs and collagenase, thus increasing the invasiveness of tumor cells.25,26 In this study, we examined representative MMPs (MMP-2, MMP-7, and MMP-9) in HepG2 cell lines stimulated with various IL-8 concentrations (1–10 ng/mL). As in previous reports, it was shown that IL-8 induced MMP-7 in our study.

This study analyzed IL-8 mRNA expression in clinical HCC samples to assess the correlation between IL-8 expression and clinicopathologic features. The results showed that 77% of clinical HCCs expressed IL-8 mRNA. Akiba et al.27 found IL-8 expression in 100% of clinical cases and in four of seven HCC cell lines. We previously detected IL-8 expression in 8 of 13 clinical samples, in 5 of 10 HCCs <2 cm, and in all of 3 HCCs >2 cm.16 We showed here that the incidence of microscopic vessel invasion was significantly higher in the IL-8–positive than in the IL-8–negative group. Furthermore, advanced pathologic stage III/IV tumors expressed more IL-8 than those at pathologic stage I/II. Akiba et al.27 reported that the incidence of portal and venous invasion was significantly higher in patients who expressed more IL-8 at tumor sites than in the normal liver. These results show that IL-8 is expressed in clinical HCCs at a high frequency and that it might have an important correlation with metastatic potential, such as vessel invasion.

The expression of IL-8 is significantly correlated with angiogenesis, as evaluated by MVC in malignant solid tumors such as gastric cancer,23 non–small-cell lung cancer,17 prostate cancer,24 and uterine endometrial cancers.28 These findings indicate that IL-8 expression significantly correlates with MVC in HCC tissue. However, this study did not distinguish a significant relationship between IL-8 expression and MVC. Although IL-8 expression significantly correlates with angiogenesis in several solid malignant tumors, this study found that it was not necessarily correlated with angiogenesis in HCC.

A previous report27 described similar results—that vessel density measured by double immunohistochemical staining of muscle vessels was not related to the IL-8 level in HCC tissue. With respect to clinical HCC, factors other than IL-8, such as VEGF, b-FGF,21,22 and platelet-derived endothelial cell growth factor, might have more influence on tumor angio-genesis.

Our examination of the influence of IL-8 on HepG2 cell proliferation showed that IL-8 does not have a notable effect. These results support the notion that tumor size is not significantly associated with IL-8 expression. However, this study showed that IL-8 accelerated the chemotactic and invasive activities of HepG2 cells and that anti–IL-8 antibody significantly diminished these events. These results support the fact that the incidence of microscopic vessel invasion was significantly higher in the IL-8–positive than in the IL-8–negative group; anti–IL-8 therapy might be a novel option for HCCs through the inhibition of chemotactic or invasive activity.

In conclusion, IL-8 expression plays a more critical role in the metastatic potential of human HCC (such as vessel invasion) than in angiogenesis or tumor proliferation. Anti–IL-8 might be an alternative treatment strategy against the progression of human HCC through inhibiting vessel invasion.


   ACKNOWLEDGMENTS
 
Supported by Grant-in-Aid 14370395 for Scientific Research from the Japan Society of the Promotion of Science (S.U. and T.A.).

Received for publication July 14, 2004. Accepted for publication April 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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