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Interleukin-1 beta in unstimulated whole saliva is a potential biomarker for oral squamous cell carcinoma
Cytokine 64 (2013) 497–502
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Interleukin-1 beta in unstimulated whole saliva is a potential biomarker for oral squamous cell carcinoma T. Kamatani ⇑, S. Shiogama, Y. Yoshihama, S. Kondo, T. Shirota, S. Shintani Department of Oral & Maxillofacial Surgery, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-City, Tokyo 145-8515, Japan
a r t i c l e
i n f o
Article history: Received 5 October 2012 Received in revised form 6 July 2013 Accepted 16 August 2013 Available online 21 September 2013 Keywords: Cytokines Unstimulated whole saliva Oral squamous cell carcinoma Interleukin-1 beta
a b s t r a c t The objective of this study was to evaluate cytokines in unstimulated whole saliva (UWS) of oral squamous cell carcinoma (OSCC) patients as compared to those with pre- and post-operation for evaluation as markers of OSCC. Sixteen OSCC patients were included in this study. Cytokine concentrations in resting saliva were measured using a Bio-Plex suspension array system. Only interleukin-1 (IL-1) beta showed significantly different cytokine concentration in saliva between pre- and post-operation. IL-1 beta was released from cultured OSCC cells confirmed by ELISA and immunohistochemistry. From this study, IL-1 beta in UWS may be useful for detection of early stage OSCC. More studies are needed to accept the utility of IL-1 beta in UWS for predicting, diagnosis and evaluation of OSCC. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Oral squamous cell carcinoma (OSCC) is the 6th most common cancer globally. Its annual estimated incidence is approximately 275,000, but unlike many other cancers, its incidence is increasing [1]. OSCC is usually detected at late stages when the cancer has advanced and therefore results in poor prognosis and survival. Early disease detection is imperative because it can result in more effective treatment with superior results. Salivary proteins have been studied as potential diagnostic markers for various diseases such as breast cancer [2], ovarian cancer [3], hepatocellular carcinoma [4], leukoplakia and oral cancer [5]. Saliva can be a promising and valuable diagnostic fluid because it contains measurable proteins at differential levels that can discriminate disease process [6]. Proinflammatory, proangiogenic cytokines can be detected in the saliva of subjects with oral preneoplastic lesions and are also elevated when compared to controls [5]. Increased concentrations of salivary cytokines in OSCC patients compare to healthy controls were also reported [6–9]. Roles of cytokines have been mentioned in the process of malignant transformation, question arises whether salivary cytokines could be used as markers for predicting, diagnosis and evaluation of OSCC. The purpose of this study was to evaluate cytokines in unstimulated whole saliva of OSCC patients as compared to those with pre- and post-operation for evaluation as markers of OSCC using multianalyte profiling technology.
2.1. Patients
⇑ Corresponding author. Tel.: +81 3 3787 1151; fax: +81 3 5498 1543. E-mail address: [email protected] (T. Kamatani). 1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.08.011
Sixteen stage I OSCC patients were participated in this study. No patient had a history of prior malignancy, immunodeficiency, autoimmune disorders, hepatitis or human immunodeficiency virus infection as well as prior treatment in the form of chemotherapy, radio-therapy, surgery or alternative medicine. All the patients were diagnosed with early stage OSCC (Table 1). Healthy periodontal tissues having sulcus depths <3 mm, no bleeding on gentle probing, and with no clinical signs of inflammation were also selected. The study was undertaken with approval from the university ethics committee. Written informed consent was obtained from each of the patient. The patients received periodontal treatment after consent of this research. 2.2. Sample collection and analysis Samples of unstimulated whole saliva (UWS) were collected 3 weeks of both pre- and post-operation between 9 AM and 10 AM. The patients were asked to refrain from eating, drinking and smoking from 7 AM. Before collection of saliva, oral hygiene procedures were performed 1 h ago. Samples were collected by asking subjects to first rinse their mouth with water and then expectorate saliva into a 50 mL centrifuge tube that kept on ice at least 10 min. Samples were visually inspected before being immediately frozen and stored at 80 °C until analysis. Samples which contained visible traces of blood were discarded from this study. Protease inhibitor cocktail (Sigma–Aldrich, USA) was added
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T. Kamatani et al. / Cytokine 64 (2013) 497–502
Table 1 Clinical characteristics of patients. Gender Male Female Age (y)
10 6 56.3 ± 5.6
Site of location Tongue Lower gingiva Buccal mucosa
10 4 2
Biosciences INC., Japan) was used following primary antibody incubations, and reaction products were visualized by immersing the sections for 3–5 min in 0.03% diaminobenzidine solution containing 2 mM hydrogen peroxide. Finally, the sections were lightly counterstained with hematoxylin. Normal squamous epithelium of the tongue was processed for negative control identical to experimental sections except that the primary antibody was omitted and replaced with buffer. 2.7. Reverse transcription-polymerase chain reaction (RT-PCR)
immediately soon after sample collection to minimize protein degradation. Two ml of clear whole saliva was obtained from the patients after centrifugation at 2600 g for 15 min to remove cell pellets and debris. Concentrations of salivary cytokines were determined using a Bio-Plex suspension array system (Bio-Rad Laboratories, Inc., USA) and commercially available kits according to the manufacturer’s instructions. A series of 8 standards ranging in concentration from 0.21 to 3369.4 pg/mL were included in each assay. 2.3. Statistical analysis Wilcoxon signed-rank test was employed for the comparison of data from Bio-Plex suspension array system and the critical alpha level of 0.05 was defined for statistical significance. 2.4. Cell culture Human OSCC cell lines (HSC2, 3, 4 and SAS) derived from a human OSCC and oral mucosa fibroblast (HOMF) derived from a human gingiva were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Immortalized normal salivary gland (NS–SV–AC) cell lines were cultured in keratinocyte basal medium-2 (KBM-2, Cambrex Bio Science Inc., Walkersville, MD) [10]. 2.5. Enzyme-linked immunosorbent assay The IL-1 beta levels in the cultured OSCC cells and HOMF cells were determined using enzyme-linked immunosorbent assay kit (Pierce Biotechnology, Inc., USA). Samples were diluted 1:8 in sample diluents and 50 ll was loaded, in duplicate, onto a 96-microwell plate coated with anti-human IL-1 beta antibody. After incubation for 1 h on a rotator (2.5 g), the microwell strips were washed for 3 times with approximately 300 ll of washing buffer, followed by the addition of 50 ll of biotinylated antibody reagent to each well. The plate was incubated again for 1 h followed by wash it 3 times with the washing buffer. Subsequently, 100 ll of streptavidin–horseradish peroxidase solution was added to all wells and incubated for 30 min. After another wash of the plate 3 times, 100 ll of premixed 3,30 ,5,50 -tetra-methylbenzidine substrate solution was added to each well and incubated in the dark for 30 min. Finally, 100 ll of stop solution was added to each well and the absorbance was measured. 2.6. Immunohistological examination The specimens were fixed with 10% buffered formalin for a few days and then embedded in paraffin wax. Three lm thick serial sections, taken from the tissue blocks, were put on silane-coated glass slides and prepared for light microscopy using routine procedures. The initial sections were stained with hematoxylin and eosin, with subsequent immunohistochemical staining using primary monoclonal antibodies to IL-1 beta (SC7884, SantaCruz, USA; diluted 1:100). Histofine Simple Stain MAX-PO (M) (Nichirei
Total RNA was isolated from cells using the RNAzol (Biotecx laboratories, Houston, TX) according to the manufacturer’s instructions and quantitated by spectrophotometry. One microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega Co., Madison, WI). The PCR reaction was carried out under the conditions recommended by the manufacturer (Takara Co., Otsu, Japan). Briefly, 50 ll of a reaction mixture including 2.5 units of Taq polymerase (Takara Co.), 5 ll of 10 buffer, 1.5 mM MgCl2, 200uM dNTPs, 1 ll of first-stand cDNA, and 25 pmol of each primer, was subjected to 28 PCR cycles (denaturation at 94 °C for 1.5 min, annealing at 58 °C for 1 min, and polymerization at 72 °C for 1 min). The PCR products were analyzed on 1.5% agarose gel. The primer sequences and product sizes were as follows: GAPDH (forward, 50 -CGT CTT CAC CAC CAT GGA GA-30 ; reverse, 50 -CGG CCA TCA CGC CAC AGT TT-30 ), 300 base pair (bp); IL-1 beta (forward, 50 -AAA AGC TTG GTG ATG TCT GG-3; reverse, 50 -TTT CAA CAC GCA GGA CAG G-30 ; reverse, 50 -TTT CAA CAC GCA GGACAG G-30 ), 179 bp; IL-6 (forward, 50 -GTG TGA AAG CAG CAA AGA GGC-30 ; reverse, 50 CTG GAG GTA CTC TAG GTA TAC-30 ), 159 bp; TNF-alpha (forward, 50 -CAA AGT AGA CCT GCC CAG AC-30 ; reverse, 50 -GAC CTC TCT CTA ATC AGC CC-30 ), 490 bp; VEGF (forward, 50 -TGT TTG TAC AAG ATC CGC AGA CGT G-30 ; reverse, 50 -TCA CCG CCT CGG CTT GTC ACA TCT GCA AGT ACG TT-30 ), VEGF121 367 bp, VEGF 165 499 bp. 2.8. Zymographic analyses and measurement of matrix metalloproteinase (MMP)-2 and -9 in cultured oral squamous cell carcinoma cells MMP-2 and 9 activities were assayed using 0.05% gelatin zymography. The protein concentration of each sample was measured using a BCA protein assay kit (BioTeke, Beijing, China). Same amount of total proteins from different samples were separated I 10% SDS–PAGE gel copolymerized with 0.05% gelatin. To regain enzymatic activity SDS was removed from the gels by washing in 2.5% Triton-X-100 three times for 1.5 h at room temperature (RT). Washed gels were then bathed in proteolysis buffer (50 mM CaCl2, 0.5 M NaCl, 50 mM Tris, pH 7.8), and incubated at 37 C for 15 h. Following incubation, gels were rinsed again in 2.5% TritonX-100 solution and stained at RT with coomassie blue (45% methanol, 44.75% H2O, 10% acetic acid, and 0.25% coomassie blue R-250) for 1 h on a rotator. Gels were destained (40% methanol, 7.5% acetic acid, and 52.5% H2O) for a couple hours until the bands became clear. The gelatin inside the gel binds to the coomassie blue, dyeing the gel blue and leaving the MMP digested regions white in color. Bands were scanned using a densitometer (GS-800; Bio-Rad, CA), and quantification was performed using Quantity One 4.6.3 software (Bio-Rad). 3. Results 3.1. Concentrations of cytokines in saliva Concentrations of cytokine values in UWS of arithmetic mean and standard deviation of pre- and post-operation are shown in
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Table 2. Only interleukin-1 (IL-1) beta shows significantly different between pre- (169.2 ± 45.1 pg/mL) and post- (98.4 ± 29.6 pg/mL) operation of cytokine concentration in UWS (Fig. 1).
3.2. Secretion of IL-1 beta from cultured oral squamous carcinoma cells ELISA analysis of cell lysate from the cultured cells revealed a significant increase in the levels of IL-1 beta in OSCC cells (HSC2, HSC3, HSC4 and SAS) compared to those of fibroblasts (HOMF) (Fig. 2). IL-1 beta was detected from cell lysate of oral squamous cell carcinoma cells. Same results were obtained in cultured medium. In cell lysate and medium of HOMF, no IL-1 beta was detected. These results indicate that IL-1 beta in UWS may be released from OSCC cells.
Table 2 Cytokine values of arithmetic mean and standard deviation (SD) of salivary samples of pre- and post-operation. Pre-operation
a
Post-operation
Cytokine
Mean
S.D.
Mean
S.D.
CTACK Eotaxin FGF basic G-CSF GM-CSF GRO-alpha HGF ICAM-1 IFN-alpha2 IFN-gamma IL-1alpha IL-1 beta IL-1ra IL-2 IL-2ra IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-12 IL-12 IL-13 IL-15 IL-16 IL-17 IL-18 IP-10 LIF MCP-1 MCP-3 M-CSF MIF MIG MIP-1alpha MIP-1 beta NGF PDGF bb RANTES SCF SCGF-bb SDF-1alpha TNF-alpha TNF-bb TRAIL VCAM-1 VEGF
–a –a –a 25.4 5.0 324.5 336.6 853.9 –a 50.0 476.8 169.2 7480.2 –a –a –a –a –a 28.6 2.6 124.4 –a 8.4 –a 5.2 12.6 –a 174.2 –a 28.2 582.9 –a 33.8 –a 50.1 333.8 547.7 3.8 23.2 –a –a –a 2.6 –a –a –a 2.1 590.6 225.5 913.0
–a –a –a 34.6 2.9 694.0 877.9 898.9 –a 59.7 339.4 45.1 9360.1 –a –a –a –a –a 41.6 3.0 168.3 –a 8.5 –a 5.6 13.6 –a 385.0 –a 42.3 1331.8 –a 41.7 –a 56.0 642.0 375.1 4.1 29.6 –a –a –a 4.0 –a –a –a 1.5 952.6 406.6 1080.4
–a –a –a 21.3 5.8 117.4 444.1 685.7 –a 42.6 532.6 98.4 4073.6 –a –a –a –a –a 38.1 2.2 92.7 –a 7.2 –a 4.3 11.3 –a 73.9 –a 40.7 151.8 –a 10.2 –a 40.5 208.7 775.2 2.8 9.2 –a –a –a 1.9 –a –a –a 2.1 551.8 164.3 1287.8
–a –a –a 33.9 6.0 255.2 1345.1 1294.4 –a 71.2 320.4 29.6 6509.9 –a –a –a –a –a 53.6 2.1 168.1 –a 8.9 –a 4.6 11.8 –a 156.4 –a 101.5 167.3 –a 12.9 –a 36.8 467.9 811.9 2.2 19.3 –a –a –a 1.7 –a –a –a 1.7 617.0 316.6 1951.5
Concentrations were below level of detection.
Fig. 1. Concentration of interleukin-1 beta in unstimulated whole saliva of pre- and post-operation were plotted. Same patient data were lined each other.
3.3. Immunohistochemical staining using antibody of IL-1 beta OSCC cells were strongly positive for staining with IL-1 beta. There was no reaction in cells of oral dysplasia (Fig. 3). These results indicate that IL-1 beta was released from OSCC cells not from normal and dysplasia cells. 3.4. Effect of IL-1 beta on expression of proinflammatory cytokines mRNA in cultured human oral squamous cell carcinoma (HSC) cells To study the effect of IL-1 beta treatment on the expression of IL-1 beta, IL-6 and TNF-alpha, HSC cells were exposed to IL-1 beta at doses ranging from 1 to 100 ng/ml, and then cells were harvested 24 h after treatment for RT-PCR analysis. IL-1 beta, IL-6, and TNF-alpha expression was dramatically increased in a dosedependent manner. Increased expression of IL-1 beta, IL-6 and TNF-alpha was clearly visualized following the sustained increased expression after IL-1 beta treatment (Fig. 4). These results indicate that IL-1 beta induces the up-regulation of IL-1 beta, IL-6 and TNFalpha in HSC cells. 3.5. IL-1 beta enhanced VEGF mRNA expression in cultured oral squamous cells carcinoma cells To study the effect of IL-1 beta treatment on the expression of VEGF, HSC cells were exposed to IL-1 beta at doses ranging from 1 to 100 ng/ml, and then cells were harvested 24 h after treatment for RT-PCR analysis. VEGF expression was expressed without in a dose-dependent manner (Fig. 4). These results indicate that IL-1 beta does not induce the up-regulation of VEGF in HSC cells. 3.6. Expression of MMP-2 and MMP-9 in cultured oral squamous cells stimulated by IL-1 beta The gelatin zymography demonstrated that the major proteinase present in the conditioned media migrated at approximately 72 kDa and 92 kDa, which corresponds to the latent form of
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Fig. 2. Concentration of interleukin-1 beta in cell lysate and medium of oral squamous cell carcinoma cells (HSC2, HSC3, HSC4 and SAS) and fibroblasts (HOMF) by enzyme linked immunosorbent assay.
Fig. 3. Immunohistochemical staining of epithelial dysplasia and squamous cell carcinoma of oral mucosa using antibody of interleukin-1 beta. Immunohistochemistry showing interleukin-1 beta expression in oral squamous cell carcinoma only. Lack of interleukin-1 beta expression in oral epithelial dysplasia.
IL 1 IL-1 -
1
10 100
IL-1 -
1
10 100
IL-1 -
1
10 100
IL-1 -
1
10 100 ng/ml
IL-1 IL-6 TNF-a VEGF 165 VEGF 121
GAPDH Fig. 4. Concentration dependency analysis of the effects of pro-inflammatory cytokines on the levels of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha mRNA in NS–SV–AC cells and HSC cells. Up-regulation of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha mRNA expression in IL-1 beta treated NS–SV–AC cells and HSC cells. The cells were cultured to 90% confluence in DMEEM supplemented with 10% fetal bovine serum at 37 °C and in 5% CO2. After interleukin-1 beta treatment (1, 10 and 100 ng/ml), cells were harvested and prepared for RT-PCR analysis. Similar results were observed in two different experiments.
MMP-2 and MMP-9, respectively. IL-1 beta slightly elevated a partially activated form of MMP-2 and MMP-9 (Fig. 5).
4. Discussion The identification of molecular markers in body fluids for screening patients that would predict the development of cancer is needed [11]. To date, no reliable or clinically applicable marker has been shown to universally identify OSCC [12,13]. In human saliva, cytokines are one of the most low-abundant proteins. The
results in this study suggest that only IL-1 beta is elevated in UWS of OSCC patients which may be useful for diagnostic and/or prognostic significance. Cytokines are intercellular signaling proteins which play a role in regulating growth, cellular proliferation, angiogenesis and tissue repair. They also function in immune responses to infection, injury and inflammation [14,15]. Previous studies of in vitro OSCC cells have demonstrated that certain proinflammatory, proangiogenic NF-kB dependent cytokines levels are increased [5]. The promoter regions for the proinflammatory, proangiogenic cytokines (IL-1, IL-6, and IL-8) are all found to contain in common the nuclear
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IL-1
IL-1 M -
1
10
100
-
1
10
IL-1 100
-
1
10
IL-1 100
-
1
10
100 ng/ml
proMMP9 MMP9 proMMP2 MMP2
Fig. 5. Gelatin zymography of conditioned media from oral squamous cell carcinoma cells incubated for 15 h in the absence (control) and in the presence of 1, 10 and 100 ng/ ml of interleukin-1 beta.
transcription factor, NF-kB binding sequence [5]. IL-1 beta is a proinflammatory cytokine that stimulates the induction of adhesion molecules and other mediators that facilitate and amplify the inflammatory response [16]. IL-1 beta is detected in gingival crevicular fluid (GCF). A strong relationship between the severity of adult periodontitis and the increasing GCF levels of IL-1 beta was suggested [17]. IL-1 beta in samples from healthy GCF was not presented [18]. All the patients received periodontal therapy during this research to reduce the cytokine levels from GCF by periodontitis. VEGF is a glycoprotein which induces the microvascular permeability and angiogenesis during the stage of proliferation [19]. IL-1 beta significantly induced VEGF in a time-dependent manner [20]. IL-1 beta induced VEGF gene expression is mediated through the activation of two distinct SAPKs, p38 MAPK and JUNK [21]. SAPKs are strongly activated by a wide variety of chemical and environmental stresses [22]. In this study, IL-1 beta induced VEGF from oral cancer. The effects of IL-1 beta on MMP expression and extracellular matrix degradation of OSCC are in agreement with other studies examining different cell types. The effects of IL-1 beta in this study may be explained, in part, by the fact that the cells were seeded on collagen during these experiments. Accordingly, the high expression of IL-1 beta, as well as proinflammatory cytokines, may induce excessive tissue degradation [23]. There is controversy between the results of cytokine levels in saliva by investigations, therefore, the result of these investigations could not express definitely about usefulness for biomarkers [24]. Further studies with larger sample size are needed in predicting, diagnosis and evaluation of OSCC. 5. Conclusion From the results of the presents study, it can be concluded that cytokines are important and are detectable in saliva of patients with OSCC. IL-1 beta increase the pathogenicity of OSCC and prove useful as potential biomarker for diagnosis. Funding None. References [1] Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45:309–16.
[2] Streckfus C, Bigler L, Tucci M, Thigpen JT. A preliminary study of CA15-3, cerbB-2, epidermal growth factor receptor, cathepsin-D, and p53 in saliva among women with breast carcinoma. Cancer Invest 2000;18:101–9. [3] Gorelik E, Landsittel DP, Marrangoni AM, Modugno F, Velikokhatnaya L, Winans MT, et al. Multiplexed immunobead-based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol Biomarkers Prev 2005;14:981–7. [4] Yio XY, Jiang J, Yin FZ, Ruan KH. Highly sensitive sandwich enzyme immunoassay for alpha-fetoprotein in human saliva. Ann Clin Biochem 1992;29:519–22. [5] Rhodus NL, Ho V, Miller CS, Myers S, Ondrey F. NF-kappaB dependent cytokine levels in saliva of patients with oral preneoplastic lesions and oral squamous cell carcinoma. Cancer Detect Prev 2005;29:42–5. [6] Arellano-Garcia ME, Hu S, Wang J, Henson B, Zhou H, Chia D, et al. Multiplexed immunobead-based assay for detection of oral cancer protein biomarkers in saliva. Oral Dis 2008;14:705–12. [7] Katakura A, Kamiyama I, Takano N, Shibahara T, Muramatsu T, Ishihara K, et al. Comparison of salivary cytokine levels in oral cancer patients and healthy subjects. Bull Tokyo Dent Coll 2007;48:199–203. [8] Wong HL, Pfeiffer RM, Fears TR, Vermeulen R, Ji S, Rabkin CS. Reproducibility and correlations of multiplex cytokine levels in asymptomatic persons. Cancer Epidemiol Biomarkers Prev 2008;17:3450–6. [9] Jablonska E, Piotrowski L, Grabowska Z. Serum levels of IL-1b, IL-6, TNF-a, sTNF-RI and CRP in patients with oral cavity cancer. Pathol Oncol Res 1997;3:126–9. [10] Azuma M, Tamatani T, Kasai Y, Sato M. Immortalization of normal human salivary gland cells with duct-, myoepithelial-, acinar-, or squamous phenotype by transfection with SV40 ori-mutant deoxyribonucleic acid. Lab Invest 1993;69:24–42. [11] Brinkmann O, Wong DT. Salivary transcriptome biomarkers in oral squamous cell cancer detection. Adv Clin Chem 2011;55:21–34. [12] St John MA, Li Y, Zhou X, Denny P, Ho CM, Montemagno C, et al. Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2004;130:929–35. [13] Marcus B, Arenberg D, Lee J, Kleer C, Chepeha DB, Schmalbach CE, et al. Prognostic factors in oral cavity and oropharyngeal squamous cell carcinoma. Cancer 2004;101:2779–87. [14] Ray CA, Bowsher RR, Smith WC, Devanarayan V, Willey MB, Brandt JT, et al. Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum. J Pharm Biomed Anal 2005;36:1037–44. [15] de Jager W, Rijkers GT. Solid-phase and bead-based cytokine immunoassay: a comparison. Methods 2006;38:294–303. [16] Miller CS, King Jr CP, Langub MC, Kryscio RJ, Thomas MV. Salivary biomarkers of existing periodontal disease: a cross-sectional study. J Am Dent Assoc 2006;137:322–9. [17] Rawlinson A, Dalati MH, Rahman S, Walsh TF, Fairclough AL. Interleukin-1 and IL-1 receptor antagonist in gingival crevicular fluid. J Clin Periodontol 2000;27:738–43. [18] Ishihara Y, Nishihara T, Kuroyanagi T, Shirozu N, Yamagishi E, Ohguchi M, et al. Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites. J Periodontal Res 1997;32:524–9. [19] König JE, Tolnay E, Wiethege T, Müller KM. Expression of vascular endothelial growth factor in diffuse malignant pleural mesothelioma. Virchows Arch 1999;435:8–12. [20] Solà-Villà D, Camacho M, Solà R, Soler M, Diaz JM, Vila L. IL-1 beta induces VEGF, independently of PGE2 induction, mainly through the PI3-K/mTOR pathway in renal mesangial cells. Kidney Int 2006;70:1935–41. [21] Tanaka T, Kanai H, Sekiguchi K, Aihara Y, Yokoyama T, Arai M, et al. Induction of VEGF gene transcription by IL-1 beta is mediated through stress-activated
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MAP kinases and Sp1 sites in cardiac myocytes. J Mol Cell Cardiol 2000;32:1955–67. [22] Kyriakis JM, Avruch J. Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 1996;271:24313–6. [23] Kobayashi M, Squires GR, Mousa A, Tanzer M, Zukor DJ, Antoniou J, et al. Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum 2005;52:128–35.
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Interleukin-1 beta in unstimulated whole saliva is a potential biomarker for oral squamous cell carcinoma T. Kamatani ⇑, S. Shiogama, Y. Yoshihama, S. Kondo, T. Shirota, S. Shintani Department of Oral & Maxillofacial Surgery, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-City, Tokyo 145-8515, Japan
a r t i c l e
i n f o
Article history: Received 5 October 2012 Received in revised form 6 July 2013 Accepted 16 August 2013 Available online 21 September 2013 Keywords: Cytokines Unstimulated whole saliva Oral squamous cell carcinoma Interleukin-1 beta
a b s t r a c t The objective of this study was to evaluate cytokines in unstimulated whole saliva (UWS) of oral squamous cell carcinoma (OSCC) patients as compared to those with pre- and post-operation for evaluation as markers of OSCC. Sixteen OSCC patients were included in this study. Cytokine concentrations in resting saliva were measured using a Bio-Plex suspension array system. Only interleukin-1 (IL-1) beta showed significantly different cytokine concentration in saliva between pre- and post-operation. IL-1 beta was released from cultured OSCC cells confirmed by ELISA and immunohistochemistry. From this study, IL-1 beta in UWS may be useful for detection of early stage OSCC. More studies are needed to accept the utility of IL-1 beta in UWS for predicting, diagnosis and evaluation of OSCC. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Oral squamous cell carcinoma (OSCC) is the 6th most common cancer globally. Its annual estimated incidence is approximately 275,000, but unlike many other cancers, its incidence is increasing [1]. OSCC is usually detected at late stages when the cancer has advanced and therefore results in poor prognosis and survival. Early disease detection is imperative because it can result in more effective treatment with superior results. Salivary proteins have been studied as potential diagnostic markers for various diseases such as breast cancer [2], ovarian cancer [3], hepatocellular carcinoma [4], leukoplakia and oral cancer [5]. Saliva can be a promising and valuable diagnostic fluid because it contains measurable proteins at differential levels that can discriminate disease process [6]. Proinflammatory, proangiogenic cytokines can be detected in the saliva of subjects with oral preneoplastic lesions and are also elevated when compared to controls [5]. Increased concentrations of salivary cytokines in OSCC patients compare to healthy controls were also reported [6–9]. Roles of cytokines have been mentioned in the process of malignant transformation, question arises whether salivary cytokines could be used as markers for predicting, diagnosis and evaluation of OSCC. The purpose of this study was to evaluate cytokines in unstimulated whole saliva of OSCC patients as compared to those with pre- and post-operation for evaluation as markers of OSCC using multianalyte profiling technology.
2.1. Patients
⇑ Corresponding author. Tel.: +81 3 3787 1151; fax: +81 3 5498 1543. E-mail address: [email protected] (T. Kamatani). 1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.08.011
Sixteen stage I OSCC patients were participated in this study. No patient had a history of prior malignancy, immunodeficiency, autoimmune disorders, hepatitis or human immunodeficiency virus infection as well as prior treatment in the form of chemotherapy, radio-therapy, surgery or alternative medicine. All the patients were diagnosed with early stage OSCC (Table 1). Healthy periodontal tissues having sulcus depths <3 mm, no bleeding on gentle probing, and with no clinical signs of inflammation were also selected. The study was undertaken with approval from the university ethics committee. Written informed consent was obtained from each of the patient. The patients received periodontal treatment after consent of this research. 2.2. Sample collection and analysis Samples of unstimulated whole saliva (UWS) were collected 3 weeks of both pre- and post-operation between 9 AM and 10 AM. The patients were asked to refrain from eating, drinking and smoking from 7 AM. Before collection of saliva, oral hygiene procedures were performed 1 h ago. Samples were collected by asking subjects to first rinse their mouth with water and then expectorate saliva into a 50 mL centrifuge tube that kept on ice at least 10 min. Samples were visually inspected before being immediately frozen and stored at 80 °C until analysis. Samples which contained visible traces of blood were discarded from this study. Protease inhibitor cocktail (Sigma–Aldrich, USA) was added
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Table 1 Clinical characteristics of patients. Gender Male Female Age (y)
10 6 56.3 ± 5.6
Site of location Tongue Lower gingiva Buccal mucosa
10 4 2
Biosciences INC., Japan) was used following primary antibody incubations, and reaction products were visualized by immersing the sections for 3–5 min in 0.03% diaminobenzidine solution containing 2 mM hydrogen peroxide. Finally, the sections were lightly counterstained with hematoxylin. Normal squamous epithelium of the tongue was processed for negative control identical to experimental sections except that the primary antibody was omitted and replaced with buffer. 2.7. Reverse transcription-polymerase chain reaction (RT-PCR)
immediately soon after sample collection to minimize protein degradation. Two ml of clear whole saliva was obtained from the patients after centrifugation at 2600 g for 15 min to remove cell pellets and debris. Concentrations of salivary cytokines were determined using a Bio-Plex suspension array system (Bio-Rad Laboratories, Inc., USA) and commercially available kits according to the manufacturer’s instructions. A series of 8 standards ranging in concentration from 0.21 to 3369.4 pg/mL were included in each assay. 2.3. Statistical analysis Wilcoxon signed-rank test was employed for the comparison of data from Bio-Plex suspension array system and the critical alpha level of 0.05 was defined for statistical significance. 2.4. Cell culture Human OSCC cell lines (HSC2, 3, 4 and SAS) derived from a human OSCC and oral mucosa fibroblast (HOMF) derived from a human gingiva were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Immortalized normal salivary gland (NS–SV–AC) cell lines were cultured in keratinocyte basal medium-2 (KBM-2, Cambrex Bio Science Inc., Walkersville, MD) [10]. 2.5. Enzyme-linked immunosorbent assay The IL-1 beta levels in the cultured OSCC cells and HOMF cells were determined using enzyme-linked immunosorbent assay kit (Pierce Biotechnology, Inc., USA). Samples were diluted 1:8 in sample diluents and 50 ll was loaded, in duplicate, onto a 96-microwell plate coated with anti-human IL-1 beta antibody. After incubation for 1 h on a rotator (2.5 g), the microwell strips were washed for 3 times with approximately 300 ll of washing buffer, followed by the addition of 50 ll of biotinylated antibody reagent to each well. The plate was incubated again for 1 h followed by wash it 3 times with the washing buffer. Subsequently, 100 ll of streptavidin–horseradish peroxidase solution was added to all wells and incubated for 30 min. After another wash of the plate 3 times, 100 ll of premixed 3,30 ,5,50 -tetra-methylbenzidine substrate solution was added to each well and incubated in the dark for 30 min. Finally, 100 ll of stop solution was added to each well and the absorbance was measured. 2.6. Immunohistological examination The specimens were fixed with 10% buffered formalin for a few days and then embedded in paraffin wax. Three lm thick serial sections, taken from the tissue blocks, were put on silane-coated glass slides and prepared for light microscopy using routine procedures. The initial sections were stained with hematoxylin and eosin, with subsequent immunohistochemical staining using primary monoclonal antibodies to IL-1 beta (SC7884, SantaCruz, USA; diluted 1:100). Histofine Simple Stain MAX-PO (M) (Nichirei
Total RNA was isolated from cells using the RNAzol (Biotecx laboratories, Houston, TX) according to the manufacturer’s instructions and quantitated by spectrophotometry. One microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega Co., Madison, WI). The PCR reaction was carried out under the conditions recommended by the manufacturer (Takara Co., Otsu, Japan). Briefly, 50 ll of a reaction mixture including 2.5 units of Taq polymerase (Takara Co.), 5 ll of 10 buffer, 1.5 mM MgCl2, 200uM dNTPs, 1 ll of first-stand cDNA, and 25 pmol of each primer, was subjected to 28 PCR cycles (denaturation at 94 °C for 1.5 min, annealing at 58 °C for 1 min, and polymerization at 72 °C for 1 min). The PCR products were analyzed on 1.5% agarose gel. The primer sequences and product sizes were as follows: GAPDH (forward, 50 -CGT CTT CAC CAC CAT GGA GA-30 ; reverse, 50 -CGG CCA TCA CGC CAC AGT TT-30 ), 300 base pair (bp); IL-1 beta (forward, 50 -AAA AGC TTG GTG ATG TCT GG-3; reverse, 50 -TTT CAA CAC GCA GGA CAG G-30 ; reverse, 50 -TTT CAA CAC GCA GGACAG G-30 ), 179 bp; IL-6 (forward, 50 -GTG TGA AAG CAG CAA AGA GGC-30 ; reverse, 50 CTG GAG GTA CTC TAG GTA TAC-30 ), 159 bp; TNF-alpha (forward, 50 -CAA AGT AGA CCT GCC CAG AC-30 ; reverse, 50 -GAC CTC TCT CTA ATC AGC CC-30 ), 490 bp; VEGF (forward, 50 -TGT TTG TAC AAG ATC CGC AGA CGT G-30 ; reverse, 50 -TCA CCG CCT CGG CTT GTC ACA TCT GCA AGT ACG TT-30 ), VEGF121 367 bp, VEGF 165 499 bp. 2.8. Zymographic analyses and measurement of matrix metalloproteinase (MMP)-2 and -9 in cultured oral squamous cell carcinoma cells MMP-2 and 9 activities were assayed using 0.05% gelatin zymography. The protein concentration of each sample was measured using a BCA protein assay kit (BioTeke, Beijing, China). Same amount of total proteins from different samples were separated I 10% SDS–PAGE gel copolymerized with 0.05% gelatin. To regain enzymatic activity SDS was removed from the gels by washing in 2.5% Triton-X-100 three times for 1.5 h at room temperature (RT). Washed gels were then bathed in proteolysis buffer (50 mM CaCl2, 0.5 M NaCl, 50 mM Tris, pH 7.8), and incubated at 37 C for 15 h. Following incubation, gels were rinsed again in 2.5% TritonX-100 solution and stained at RT with coomassie blue (45% methanol, 44.75% H2O, 10% acetic acid, and 0.25% coomassie blue R-250) for 1 h on a rotator. Gels were destained (40% methanol, 7.5% acetic acid, and 52.5% H2O) for a couple hours until the bands became clear. The gelatin inside the gel binds to the coomassie blue, dyeing the gel blue and leaving the MMP digested regions white in color. Bands were scanned using a densitometer (GS-800; Bio-Rad, CA), and quantification was performed using Quantity One 4.6.3 software (Bio-Rad). 3. Results 3.1. Concentrations of cytokines in saliva Concentrations of cytokine values in UWS of arithmetic mean and standard deviation of pre- and post-operation are shown in
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Table 2. Only interleukin-1 (IL-1) beta shows significantly different between pre- (169.2 ± 45.1 pg/mL) and post- (98.4 ± 29.6 pg/mL) operation of cytokine concentration in UWS (Fig. 1).
3.2. Secretion of IL-1 beta from cultured oral squamous carcinoma cells ELISA analysis of cell lysate from the cultured cells revealed a significant increase in the levels of IL-1 beta in OSCC cells (HSC2, HSC3, HSC4 and SAS) compared to those of fibroblasts (HOMF) (Fig. 2). IL-1 beta was detected from cell lysate of oral squamous cell carcinoma cells. Same results were obtained in cultured medium. In cell lysate and medium of HOMF, no IL-1 beta was detected. These results indicate that IL-1 beta in UWS may be released from OSCC cells.
Table 2 Cytokine values of arithmetic mean and standard deviation (SD) of salivary samples of pre- and post-operation. Pre-operation
a
Post-operation
Cytokine
Mean
S.D.
Mean
S.D.
CTACK Eotaxin FGF basic G-CSF GM-CSF GRO-alpha HGF ICAM-1 IFN-alpha2 IFN-gamma IL-1alpha IL-1 beta IL-1ra IL-2 IL-2ra IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-12 IL-12 IL-13 IL-15 IL-16 IL-17 IL-18 IP-10 LIF MCP-1 MCP-3 M-CSF MIF MIG MIP-1alpha MIP-1 beta NGF PDGF bb RANTES SCF SCGF-bb SDF-1alpha TNF-alpha TNF-bb TRAIL VCAM-1 VEGF
–a –a –a 25.4 5.0 324.5 336.6 853.9 –a 50.0 476.8 169.2 7480.2 –a –a –a –a –a 28.6 2.6 124.4 –a 8.4 –a 5.2 12.6 –a 174.2 –a 28.2 582.9 –a 33.8 –a 50.1 333.8 547.7 3.8 23.2 –a –a –a 2.6 –a –a –a 2.1 590.6 225.5 913.0
–a –a –a 34.6 2.9 694.0 877.9 898.9 –a 59.7 339.4 45.1 9360.1 –a –a –a –a –a 41.6 3.0 168.3 –a 8.5 –a 5.6 13.6 –a 385.0 –a 42.3 1331.8 –a 41.7 –a 56.0 642.0 375.1 4.1 29.6 –a –a –a 4.0 –a –a –a 1.5 952.6 406.6 1080.4
–a –a –a 21.3 5.8 117.4 444.1 685.7 –a 42.6 532.6 98.4 4073.6 –a –a –a –a –a 38.1 2.2 92.7 –a 7.2 –a 4.3 11.3 –a 73.9 –a 40.7 151.8 –a 10.2 –a 40.5 208.7 775.2 2.8 9.2 –a –a –a 1.9 –a –a –a 2.1 551.8 164.3 1287.8
–a –a –a 33.9 6.0 255.2 1345.1 1294.4 –a 71.2 320.4 29.6 6509.9 –a –a –a –a –a 53.6 2.1 168.1 –a 8.9 –a 4.6 11.8 –a 156.4 –a 101.5 167.3 –a 12.9 –a 36.8 467.9 811.9 2.2 19.3 –a –a –a 1.7 –a –a –a 1.7 617.0 316.6 1951.5
Concentrations were below level of detection.
Fig. 1. Concentration of interleukin-1 beta in unstimulated whole saliva of pre- and post-operation were plotted. Same patient data were lined each other.
3.3. Immunohistochemical staining using antibody of IL-1 beta OSCC cells were strongly positive for staining with IL-1 beta. There was no reaction in cells of oral dysplasia (Fig. 3). These results indicate that IL-1 beta was released from OSCC cells not from normal and dysplasia cells. 3.4. Effect of IL-1 beta on expression of proinflammatory cytokines mRNA in cultured human oral squamous cell carcinoma (HSC) cells To study the effect of IL-1 beta treatment on the expression of IL-1 beta, IL-6 and TNF-alpha, HSC cells were exposed to IL-1 beta at doses ranging from 1 to 100 ng/ml, and then cells were harvested 24 h after treatment for RT-PCR analysis. IL-1 beta, IL-6, and TNF-alpha expression was dramatically increased in a dosedependent manner. Increased expression of IL-1 beta, IL-6 and TNF-alpha was clearly visualized following the sustained increased expression after IL-1 beta treatment (Fig. 4). These results indicate that IL-1 beta induces the up-regulation of IL-1 beta, IL-6 and TNFalpha in HSC cells. 3.5. IL-1 beta enhanced VEGF mRNA expression in cultured oral squamous cells carcinoma cells To study the effect of IL-1 beta treatment on the expression of VEGF, HSC cells were exposed to IL-1 beta at doses ranging from 1 to 100 ng/ml, and then cells were harvested 24 h after treatment for RT-PCR analysis. VEGF expression was expressed without in a dose-dependent manner (Fig. 4). These results indicate that IL-1 beta does not induce the up-regulation of VEGF in HSC cells. 3.6. Expression of MMP-2 and MMP-9 in cultured oral squamous cells stimulated by IL-1 beta The gelatin zymography demonstrated that the major proteinase present in the conditioned media migrated at approximately 72 kDa and 92 kDa, which corresponds to the latent form of
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Fig. 2. Concentration of interleukin-1 beta in cell lysate and medium of oral squamous cell carcinoma cells (HSC2, HSC3, HSC4 and SAS) and fibroblasts (HOMF) by enzyme linked immunosorbent assay.
Fig. 3. Immunohistochemical staining of epithelial dysplasia and squamous cell carcinoma of oral mucosa using antibody of interleukin-1 beta. Immunohistochemistry showing interleukin-1 beta expression in oral squamous cell carcinoma only. Lack of interleukin-1 beta expression in oral epithelial dysplasia.
IL 1 IL-1 -
1
10 100
IL-1 -
1
10 100
IL-1 -
1
10 100
IL-1 -
1
10 100 ng/ml
IL-1 IL-6 TNF-a VEGF 165 VEGF 121
GAPDH Fig. 4. Concentration dependency analysis of the effects of pro-inflammatory cytokines on the levels of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha mRNA in NS–SV–AC cells and HSC cells. Up-regulation of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha mRNA expression in IL-1 beta treated NS–SV–AC cells and HSC cells. The cells were cultured to 90% confluence in DMEEM supplemented with 10% fetal bovine serum at 37 °C and in 5% CO2. After interleukin-1 beta treatment (1, 10 and 100 ng/ml), cells were harvested and prepared for RT-PCR analysis. Similar results were observed in two different experiments.
MMP-2 and MMP-9, respectively. IL-1 beta slightly elevated a partially activated form of MMP-2 and MMP-9 (Fig. 5).
4. Discussion The identification of molecular markers in body fluids for screening patients that would predict the development of cancer is needed [11]. To date, no reliable or clinically applicable marker has been shown to universally identify OSCC [12,13]. In human saliva, cytokines are one of the most low-abundant proteins. The
results in this study suggest that only IL-1 beta is elevated in UWS of OSCC patients which may be useful for diagnostic and/or prognostic significance. Cytokines are intercellular signaling proteins which play a role in regulating growth, cellular proliferation, angiogenesis and tissue repair. They also function in immune responses to infection, injury and inflammation [14,15]. Previous studies of in vitro OSCC cells have demonstrated that certain proinflammatory, proangiogenic NF-kB dependent cytokines levels are increased [5]. The promoter regions for the proinflammatory, proangiogenic cytokines (IL-1, IL-6, and IL-8) are all found to contain in common the nuclear
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IL-1
IL-1 M -
1
10
100
-
1
10
IL-1 100
-
1
10
IL-1 100
-
1
10
100 ng/ml
proMMP9 MMP9 proMMP2 MMP2
Fig. 5. Gelatin zymography of conditioned media from oral squamous cell carcinoma cells incubated for 15 h in the absence (control) and in the presence of 1, 10 and 100 ng/ ml of interleukin-1 beta.
transcription factor, NF-kB binding sequence [5]. IL-1 beta is a proinflammatory cytokine that stimulates the induction of adhesion molecules and other mediators that facilitate and amplify the inflammatory response [16]. IL-1 beta is detected in gingival crevicular fluid (GCF). A strong relationship between the severity of adult periodontitis and the increasing GCF levels of IL-1 beta was suggested [17]. IL-1 beta in samples from healthy GCF was not presented [18]. All the patients received periodontal therapy during this research to reduce the cytokine levels from GCF by periodontitis. VEGF is a glycoprotein which induces the microvascular permeability and angiogenesis during the stage of proliferation [19]. IL-1 beta significantly induced VEGF in a time-dependent manner [20]. IL-1 beta induced VEGF gene expression is mediated through the activation of two distinct SAPKs, p38 MAPK and JUNK [21]. SAPKs are strongly activated by a wide variety of chemical and environmental stresses [22]. In this study, IL-1 beta induced VEGF from oral cancer. The effects of IL-1 beta on MMP expression and extracellular matrix degradation of OSCC are in agreement with other studies examining different cell types. The effects of IL-1 beta in this study may be explained, in part, by the fact that the cells were seeded on collagen during these experiments. Accordingly, the high expression of IL-1 beta, as well as proinflammatory cytokines, may induce excessive tissue degradation [23]. There is controversy between the results of cytokine levels in saliva by investigations, therefore, the result of these investigations could not express definitely about usefulness for biomarkers [24]. Further studies with larger sample size are needed in predicting, diagnosis and evaluation of OSCC. 5. Conclusion From the results of the presents study, it can be concluded that cytokines are important and are detectable in saliva of patients with OSCC. IL-1 beta increase the pathogenicity of OSCC and prove useful as potential biomarker for diagnosis. Funding None. References [1] Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45:309–16.
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