Abstract
The growth of tumor cells can be regulated by a variety of cytokines. To investigate the pathogenesis of head and neck cancer and explore a new therapeutic approach for the carcinoma, the role of interleukin-6 (IL-6) in the growth of a human head and neck squamous cell carcinoma (HNSCC) cell line was examined. Whether or not IL-6 is increased in HNSCC and whether or not IL-6 antisense oligonucleotide treatment could decrease proliferation and angiogenic activity of HNSCC cell lines, was determined. Established human HNSCC cell lines were screened for IL-6 expression at both mRNA and protein levels. By using a 15-mer antisense phosphorothioate oligonucleotide targeting a sequence in the second exon of the IL-6 gene, modulation of IL-6 and vascular endothelial growth factor (VEGF) expression was examined in UMSCC IIA in cell supernatants by capture enzyme-linked immunosorbent assay (ELISA), and in cell lysates by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, cell growth was determined by cell count. Endothelial cell migration was measured using a modified Boyden chamber. IL-6 was identified in the supernatant of the cell culture medium, indicating that these cells secreted IL-6, and the mRNAs of IL-6 were shown to be present in the cell lysates. IL-6 antisense oligonucleotide treatment resulted in a significant reduction of IL-6 protein expression compared to the sense control. The antisense oligonucleotides targeting IL-6 mRNA, also, inhibited cell growth and IL-6 production as well as VEGF expression. The addition of conditioned medium from IL-6 antisense-treated tumor cells resulted in decreased endothelial cell migration and tubule formation. Taken together, these findings indicate that endogenous IL-6 plays an important role in the growth of HNSCC and exerts its action by an autocrine growth mechanism, and that therapeutic trials with antisense oligonucleotides targeted to IL-6 mRNA may have some value for the treatment of HNSCC due to a decrease of neovascularization.
Head and neck squamous cell carcinoma (HNSCC) is the most common neoplasm arising in the upper aerodigestive tract. Currently, more than 500, 000 new cases of HNSCC are reported world-wide every year with prevalence increasing (1, 2). Due to improved therapeutic concepts locoregional disease control and organ preservation are achievable even in patients with advanced HNSCC. However they are associated with severe treatment-related toxic effects, furthermore, improved survival is hampered by relatively high incidence of distant metastasis, second primary tumors and comorbidity (3). Thus, it is imperative that new treatment modalities are developed to increase the long-term survival of patients with HNSCC.
A variety of cytokines have been identified, as playing potentially important roles in tumorigenesis (4). Among these, interleukin-6 (IL-6), a proinflammatory cytokine, produces multifunctional effects. Besides regulation of immune reaction, hematopoiesis and inflammatory state, it has been shown to be associated with tumor progression by means of inhibition of cancer cell apoptosis, tumor growth, development of distant metastasis and stimulation of angiogenesis (5). Elevated serum level IL-6 concentrations were observed in various cancer types, e.g. esophageal squamous cell carcinoma, renal cell carcinoma, glioblastoma, and small cell lung cancer (5-8). Serum IL-6 levels correlate with tumor stage, and survival of patients (9). Additionally, IL-6 has been found to be involved in tumorigenesis of oral cancer, being a valuable biomarker for predicting recurrence and overall survival among HNSCC patients (10, 11). In a previous study, our group found significantly higher IL-6 serum levels in HNSCC patients than in healthy individuals and significant correlation of the IL-6 serum with the tumor stage (12).
Angiogenesis plays a key role in the survival of cancer cells, in local tumor growth and in the development of distant metastasis and has been shown to be increased in HNSCC (13-15). Vascular endothelial growth factor (VEGF) is one of the most potent cytokines stimulating endothelial cell proliferation, preventing regression of newly formed vessels, and increasing microvascular permeability (13, 16), and elevated VEGF levels have been demonstrated in HNSCC patients (14, 15, 17).
Anti-neoplastic therapy using neutralising antibodies against IL-6 has demonstrated inhibition of tumor growth and vascularization in vivo (18, 19). In this study, whether or not decreased IL-6 expression in HNSCC cells by the use of antisense oligonucleotides specifically inhibiting the expression of IL-6 gene product might affect proliferation and angiogenic activity in vitro, was investigated.
Materials and Methods
Cell culture. The five different UMSCC cell lines used are well-described human HNSCC cell lines and were obtained from T. Carey (The University of Michigan, Ann Arbor, Michigan, USA). Cell cultures were carried out in Falcon petri dishes at 37°C in a 5% CO2 fully humidified atmosphere using Dulbecco´s modified minimum essential medium (DMEM) (Fisher Scientific Co., Pittsburgh, PA, USA) supplemented with 10% fetal calf serum (FCS) and antibiotics (Life Technologies, Inc. [Gibco BRL], Gaithersburg, MD, USA). For antisense treatment, the medium from the cultures was aspirated and replaced with DMEM containing 5% FCS and antibiotics followed by the addition of oligo-deoxynucleotides (see below). Human umbilical vein endothelial cells (HUVEC-p, PromoCell, Heidelberg, Germany) were used for the in vitro angiogenesis analysis. The cells were grown in Endothel Cell Growth Medium (PromoCell) supplemented with 2% fetal bovine serum (FBS).
Oligodeoxynucleotides. Phosphorothioated 15-mer oligodeoxynucleotides were synthesised on an Applied Biosystem 394 DNA synthesiser (Aplied Biosystems, Darmstadt, Germany) by means of B-cyanothylphosphoramidite chemistry to minimise degradation by endogenous nucleases. The antisense oligonucleotide (5’-TCCTGGGGGTACTGG-3’) was directed against a sequence in the second exon of the IL-6 gene, as described by others previously (26). Oligonucleotides in scrambled order were used as a control. All the experiments were performed with 12.5 μM oligodeoxynucleotides.
Human IL-6/VEGF ELISA. To quantitate IL-6 and VEGF secretion to the supernatant of the HNSCC cells were treated with medium (control) or medium containing IL-6 antisense or sense oligonucleotides for 48 hours. The cell culture supernatants were collected in sterile test tubes and stored at −20°C until used. Then, the IL-6 and VEGF concentrations in 100 μl supernatant samples were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique (R&D Systems, Wiesbaden, Germany), using a solid-phase monoclonal antibody and an enzyme-linked polyclonal antibody raised against recombinant IL-6 or VEGF, according to the manufacturer's directions. All the analyses and calibrations were carried out in duplicate. The calibrations on each microtiter plate included recombinant human IL-6 or VEGF standards. Optical density was determined using a microtiter plate reader (Dynatech) at 450 nm. Wavelength correction was set to 540 nm and the concentrations were reported as pg/ml.
Human IL-6/VEGF RT-PCR. To determine the effect of the oligonucleotides on the expression of IL-6 mRNA or VEGF mRNA, the cells were plated at a density of 2,5×105 cells / well in 24 well polystrene plates (Falcon). After 24 h the cells were rinsed twice with medium and then 500 μl/well fresh medium alone or oligo medium containing antisense or scrambled oligodeoxynucleotides was added, followed by an incubation period of 48 h. To isolate RNA from the cells grown in monolayer, the cells were directly lysed in the culture dish by the addition of 1 ml RNA-Clean (RNA-Clean System, AGS, Heidelberg, Germany). After addition of 0.2 ml chloroform per 2 ml of homogenate and centrifugation for 15 minutes at 12.000 g (4°C), the aqueous phase was transferred to a fresh tube. After the addition of an equal volume of isopropanol and centrifugation for 15 minutes at 12,000 g (4°C), the supernatant was removed from the RNA precipitate. The RNA pellet was washed twice with 70% ethanol by vortexing and subsequent centrifugation for 8 minutes at 7,500 g (4°C). After drying the RNA pellet, it was dissolved in diethylpyrocarbonate water. The RNA was reverse transcribed (StrataScript First-Strand Synthesis System; Stratagene, La Jolla, CA, USA) into cDNA using random-oligonucleotide primers. IL-6 and VEGF mRNA levels were measured using RT-PCR (Interleukin-CytoXpress Multiplex PCR Kit, BioSource, San Francisco, CA, USA) according to the manufacturer's instructions. To fractionate the multiplex-PCR DNA products, these products were mixed with 6× loading buffer and separated on a 2% agarose gel containing 0.5 mg/ml ethidium bromide, visualised with UV light and recorded using a charged-coupled device camera. To test the quality of the cDNA, the kit includes primers for Glycerinealdehyde-3-phosphate-dehydrogenase. Results were obtained from two independent experiments.
Proliferation assay. To determine the effect of the oligonucleotides on growth rates of the tumor cells, the HNSCC cells were plated in DMEM at a density of 2,5×105 cells/well in 24 well polystrene plates (Falcon). After 24 h the cells were rinsed twice with medium and then 500 μl fresh medium alone or oligo medium containing sense or antisense oligodeoxynucleotides was added. The cell counts were determined using a hemocytometer in duplicate samples at each time point. The viability of the cells was analysed by trypan blue exclusion.
Migration assay. Human umbilical vein endothelial cells were grown on gelatin-coated dishes until confluence in Endothel Cell Growth Medium (PromoCell) supplemented with 2% FBS. Migration assays were performed in transwell-chambers (Corning-Costar Corp., Cambridge, MA, USA). Conditioned medium from the tumor cells was placed in the lower chambers, which were covered with polycarbonate filters (8-mm pore size). Then, 0.5 ml of 1×105 cells/ml of endothelial cells were placed in the upper chamber. After 4 h of incubation at 37°C, the medium in the upper chamber was aspirated, and cells on the upper surface of the filter were removed with a cotton swab. The cells on the lower surface were fixed, stained with Diff Quick (Dade International Inc., Miami, FL, USA) as previously described (20). The number of stained nuclei were counted consecutively in five high-power fields per each chamber.
IL-6 in 5 HNSCC cell lines, A: measured in the supernatant by ELISA assay after the cells were cultured for 48 h, B: IL-6 mRNA measured by RT-PCR.
Results
Characterization of the cell lines. IL-6 protein was detectable in the supernatant of all the HNSCC lines, as summarised in Figure 1A. The values are reported as the means per 106 cells of duplicate experiments. RT-PCR for IL-6 mRNA exhibited IL-6 expression in all the HNSCC cell lines tested (Figure 1B). Among the cell lines, a relatively high level was noted in the UMSCC 11A cells. This cell line was chosen for further study.
Effect of antisense oligonucleotides on IL-6 and growth rate. IL-6 levels were significantly decreased by the antisense IL-6 oligonucleotide treatment (Figure 2A). The IL-6 mRNA expression was notably decreased by the IL-6 antisense oligonucleotide treatment (Figure 2B). In line with the reduction of IL-6 protein secretion in the tumor cells treated with IL-6 antisense oligonucleotides, we did observe a a pronounced reduction of the growth rate of the tumor cells was also demonstrated (Figure 3).
Effect of the antisense oligonucleotides on VEGF. Analysis of the supernatant by ELISA assay showed a decrease in the concentration of VEGF after IL-6 targeting (Figure 4A). The expression level of VEGF mRNA was notably decreased by IL-6 antisense treatment (Figure 4B).
IL-6 in UMSCC 11A cells treated with medium (C=control) or medium containing IL-6 antisense (AS) or scrambled (SC) oligonucleotides for 48 h, A: concentration in the supernatant measured by ELISA assay, B: m-RNA expression measured by RT-PCR.
Effect of antisense IL-6 oligonucleotides on endothelial cell migration. The addition of non-concentrated conditioned medium from the UMSCC 11A cells treated with IL-6 antisense oligonucleotides resulted in a distinctive decrease of HUVEC migration compared with the addition of conditioned medium from the untreated or sense-oligonucleotide-treated cells (Figure 5). The growth pattern of endothelial cells in response to conditioned medium from UMSCC 11A cells treated with IL-6 antisense or sense oligonucleotides was markedly affected showing a decrease of tubule formation in the presence of conditioned medium taken from the IL-6 antisense-treated tumor cells (Figure 6).
Discussion
An autocrine mechanism is hypothesized for IL-6 mediated tumor growth, with secretion of IL-6 by tumor cells and specific membrane receptors on the very same tumor cell surface to exhibit a biological effect such as proliferation (4, 21, 22).
The detection of elevated IL-6 levels was consistent with similar findings by others (6, 10). IL-6 has already been recognized as a potential target for anti-neoplastic therapy in previous studies. The possible mechanism of action of antisense oligonucleotides includes inhibition of transcription or translation and mRNA degradation through an RNase H cleavage mechanism (23).
Growth of UMSCC 11A cells treated with medium (control) or medium containing IL-6 antisense or scrambled oligonucleotides.
In this study the treatment of HNSCC cells with IL-6 antisense oligonucleotides in vitro efficiently down-regulated IL-6 expression at both the pre- and posttranslational levels. The IL-6 antisense treatment also decreased the tumor cell growth rate. These results were in accordance with other in vitro studies showing down-regulation of IL-6 expression by IL-6 antisense oligonucleotides in normal human keratinocytes and in human glioma cells (24, 25). Thus endogenous IL-6 does play a role in the growth of the HNSCC cells and IL-6 appears to act in an intracellular autocrine fashion (26). Kong et al. using recombinant IL-6, monoclonal antibodies against IL-6, or IL-6 antisense oligonucleotides to determine an autocrine control mechanism in a choriocarcinoma cell line showed the greatest effect on cell growth and IL-6 expression with the antisense oligonucleotides (27).
IL-6 has been shown to be elevated in tissues undergoing active angiogenesis, but does not directly induce the proliferation of endothelial cells. Treatment with IL-6 resulted in a significant induction of VEGF mRNA in various cell lines (28). In the present study IL-6 appeared to play a role in VEGF expression in the HNSCC cells since notable decrease of VEGF expression was demonstrated with the IL-6 oriented antisense oligonucleotides.
Chen et al. observed elevated serum concentrations of both cytokines in patients with head and neck cancer (29). However, they did not observe a correlation between serum cytokine levels and the degree of tumor differentiation, or lymph node metastasis in the HNSCC patients. Thus HNSCC cells may not be the only source of elevated cytokine levels in patients, and high levels of VEGF may not only correlate with increased concentrations of IL-6, but some subsets of HNSCC cells may produce high levels of VEGF in response to factors other than IL-6 (e.g. hypoxia). However, at the in vitro level the present results showed an impact of IL-6 abrogation on VEGF expression. Furthermore, endothelial cells grown in conditioned medium from the antisense-treated tumor cells exhibited a reduction of cell migration. These data suggested that down-regulation of angiogenesis by targeting the IL-6 signaling pathways might contribute to an anti-tumor activity in addition to the antiproliferative effects of IL-6 system inhibition.
VEGF in UMSCC 11A cells treated with medium (C=control) or medium containing IL-6 antisense (AS) or scrambled (SC) oligonucleotides for 48 h, A: concentration in the supernatant measured by ELISA assay, B: mRNA expression measured by RT-PCR.
In HNSCC new formation of malignant vasculature is mainly stimulated by VEGF. IL-6 acts in three major signalling pathways by second messengers, including signal transducer and activator of transcription 3 (STAT 3), mitogen activated protein kinase (MAP kinase), and phosphoinositide 3 kinase (PI3 K)/Akt (30). All of these second messengers have been shown to mediate VEGF expression in a variety of cells (30). Although the exact pathways need further examination, the present evidence suggests that IL-6 exerts its action by influencing VEGF expression and proliferation of malignant vessel formation.
Endothelial cell migration assay in response to conditioned medium from UMSCC 11A cells treated with IL-6 antisense or scrambled oligonucleotides. In vitro migration was measured in Boyden chambers.
The present results suggested that IL-6 does play an important role in HNSCC cells and might function in an intracellular autocrine fashion. Therapeutic strategies targeting IL-6 signalling might have an anti-tumor effect mediated in part by inhibition of tumor angiogenesis.
Antisense therapeutics have already been successfully tested in phase I and II clinical trials leading to promising results which warrant further clinical development (31, 32). The future of antisense oligonucleotides depends on better elucidating their mechanisms and also their effectiveness. Improvement of binding affinity or intracellular uptake by combination of therapeutic oligonucleotides with other gene therapy techniques, appear to be promising. Further studies are needed in order to establish effective methods of treatment. The benefit of already existing techniques, as a useful tool for analysing the function of a gene product by inhibiting its gene expression and analyzing biomolecular downstream effects, was highlighted in the present study.
Representative example of endothelial cell tubule formation in response to conditioned medium from UMSCC 11A cells treated with medium containing IL-6 scrambled (A) or antisense oligonucleotides (B).
- Received July 29, 2010.
- Revision received March 11, 2011.
- Accepted March 15, 2011.
- Copyright © 2011 The Author(s). Published by the International Institute of Anticancer Research.
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