Background
The prognosis of human oral squamous cell carcinoma (OSCC) is usually poor with a 5-year survival rate of approximately 50 ~ 60%, which has generally been attributed to the insensitivity of most patients to chemotherapy [
1,
2]. Recently, there has been a growing recognition of interest in anti-tumor functions initiated by the innate immune response. The role of toll-like receptors (TLRs) and their signaling in tumor immune escape and resistance to apoptosis, for example, is among the frontiers of exploration [
3,
4].
TLRs are type I transmembrane proteins with extracellular domains comprised largely of leucine-rich repeats and intracellular signaling domains that play a crucial role in inflammation and host defense against invading microorganisms through the recognition of pathogen-associated molecular patterns (PAMPs) such as LPS, lipopeptides, dsRNA, and bacterial DNA [
4]. After binding with ligands, TLRs and Toll/interleukin-1 receptor-like domain (TIR) are dimerized and undergo a conformational change that is required to recruit downstream signaling molecules, such as MyD88. In mammals, the TLR family consists of at least 12 members expressed predominantly on the surface of macrophages and various immune cells [
5]. LPS is specifically recognized by TLR4 [
6,
7]. More recent studies have demonstrated TLRs expression in a broad variety of tumor tissues and tumor cell lines [
8‐
11]. Their activated signaling pathways in cancer cells could have profound consequences for tumor growth by promoting cancer progression, anti-apoptotic activity, and resistance to host immune responses [
12]. Although there have been reports of TLRs in various tumor types, the function of these receptors in OSCC prognosis and resistance to chemotherapy remains unknown [
13].
OSCC is sensitive to cisplatin-based chemotherapy in patients with an initial response to treatment. However, chemoresistance is observed in the treatment of recurrent patients because of acquired or intrinsic resistance. The main mechanisms and pathways that lead to clinical resistance to cisplatin in OSCC have not been well defined. So far, little is known about the function and biological importance of TLR4 on OSCC chemoresistance. Thus, it is important to address the pattern of TLR4 expression on OSCC cell lines and to determine the pathophysiological significance of TLR4 signaling in the immune escape and chemoresistance of OSCC. The purpose of this study was to explore the expression and role of TLR4 in OSCC and how TLR4 signaling correlated with cisplatin resistance in OSCC.
Discussion
We found that TLR4 was expressed in human OSCC cell lines but not on normal human oral epithelial cells. The expression levels of TLR4 and MyD88 in situ were correlated with tumor differentiation. TLR4 and MyD88 were highly expressed in well-differentiated and moderately-differentiated tumors, but weakly expressed in poorly differentiated tumors. Cisplatin is widely used for chemotherapy of many malignancies, especially for OSCC. However, the efficiency of cisplatin in the treatment of recurrent tumors is limited because of acquired or intrinsic resistance. In our study, ligation of LPS to TLR4 can protect cisplatin-induced apoptosis in OSCC cell lines but not in HIOEC cells. LPS activated NF-κB and p38 MAPK pathways and triggered target gene transcription. IL-6, IL-8 and VEGF were found to be elevated in OSCC cell lines upon LPS stimulation. Therefore, OSCC cell lines could respond to oral bacteria via the derived LPS, which may lead to apoptotic resistance to cisplatin.
It is well known that IL-6 production and increased circulating level have been emerged as biomarkers of poor prognosis in many human cancers. In ovarian cancer, increased levels of IL-6 in patients’ sera are linked to tumor progression, resistance to apoptosis and chemoresistance [
14]. IL-6 is also able to promote tumor angiogenesis and invasion [
15‐
17]. In clinical investigations, high IL-6 levels in the sera of patients with colon carcinoma correlate with tumor size [
18]. The effect of IL-6 on hepatocyte proliferation depends on the balance between its pro- and anti-proliferative arms, after the integration of the effects of other transcription factors acting on the same genes as IL-6 [
19]. IL-8 is a proangiogenic cytokine/chemokine and anti-apoptotic molecule that can promote tumor metastasis and death resistance. IL-8 and VEGF are involved in the malignant transformation process. It has been reported that IL-8 is overexpressed in ovarian cancer and its level is associated with decreased patient survival and poor clinical outcome [
20,
21]. VEGF, in addition to inducing angiogenesis, is also an immunosuppressive cytokine that promotes ascite formation through stimulation of vascular permeability [
20,
22]. The expression of VEGF is negatively correlated with DC numbers in the tumor tissue and peripheral blood of cancer patients [
23,
24]. Also, VEGF has an inhibitory effect on DC differentiation in patients with non-small-cell lung cancer [
25]. Thus, our results show that the elevated production of IL-6, IL-8 and VEGF in OSCC cell lines by LPS stimulation may lead to the development of resistance to cisplatin in human OSCC.
It is well established that NF-κB is an anti-apoptotic transcriptional factor upon tumor cell stimulation with LPS, radiation, and some chemotherapeutic agents. NF-κB has also been previously linked to upregulation of anti-apoptotic protein expression and an increase in cell proliferation [
26]. Due to the anti-apoptotic properties of activated NF-κB, its high expression levels in tumor cells are associated with tumor progression and induction of chronic inflammation in the tumor microenvironment [
27]. Activation of NF-κB has been shown to induce resistance through the expression of the MDR1 gene [
28]. It was also reported that LPS could induce NF-κB activation in colon cancer cells and pancreatic cancer cells [
29,
30]. Silencing of TLR4 expression in SKOV3 cells resulted in sensitization of SKOV3 cells to PTX-induced apoptosis, and this sensitization was accompanied by the inhibition of cytokines production in response to PTX and LPS [
11]. Inhibition of LPS-induced TLR4 signaling could improve therapeutic outcomes by preventing cancer metastasis during the perioperative period of colorectal cancer resection [
31]. In our study, OSCC cell lines were shown to be resistant to cisplatin-induced apoptosis upon LPS pretreatment. Moreover, cisplatin-induced apoptosis decreased by LPS was actually mediated through TLR4. TLR4 induced NF-κB pathway activation and nuclear translocation may be associated with the increased transcription of a number of different genes and the production of proinflammatory cytokines such as IL-6, IL-8 and VEGF.
LPS-triggered TLR4 signaling usually activates two different pathways: the NF-κB signal pathway and the p38 MAPK signal pathway. In the cancer cells, Szajnik et al. demonstrated that activation of NF-κB induced by LPS was responsible for the production of cytokines such as IL-6, IL-8 and VEGF in SKOV3 cells [
11]. Triggering of TLR4 by LPS induced tumor promotion by the induction of proliferation, activation of NF-κB, p65 binding to DNA, and resistance to NK cell-mediated cytotoxicity accompanied by the increased production of proinflammatory cytokines (IL-6 and IL-8), VEGF [
10]. The p38 MAPK pathway has also been shown to be involved in LPS-induced IL-6 secretion in pituitary adenomas and bladder cancer cells [
29,
32]. However, up to now, the mechanisms by which TLR4 activation with LPS induces resistance to chemotherapy have not been fully understood. One report showed that inhibition of the NF-κB pathway can significantly attenuate LPS-induced apoptosis resistance [
9]. In our present study, we found that TLR4 ligation can activate both p38 MAPK and NF-κB pathways in human OSCC cell lines, and inhibition of TLR4 by siRNA can significantly attenuate LPS-induced NF-κB and p38 MAPK activation. We also found that the secretion of cytokines was significantly abrogated by p38 MAPK specific inhibitor, which proved that p38 MAPK was responsible for LPS induced production of IL-6, IL-8 and VEGF in OSCC cells. LPS pretreatment can decrease cisplatin-induced cell death and apoptosis through TLR4 signaling pathway. On the contrary, HIOEC cells, which have very low expression levels of TLR4 and MyD88, did not show these effects. These findings strongly suggest that LPS can provide a survival benefit to OSCC cell lines and alter their sensitivity to cisplatin through activation of p38 MAPK and NF-κB signaling pathway via TLR4.
Methods
Cell lines and reagents
The human OSCC cell line CAL-27 used in this study was obtained from the American Type Culture Collection (ATCC), which was established in 1982 by J. Gioanni (Centre Antoine Lacassagne, Nice Cedex, France) from tissue taken prior to treatment from a 56 year old Caucasian male with a lesion of the middle of the tongue. CAL-27 cells are epithelial, polygonal with a highly granular cytoplasm. The cell line WSU-HN6, a human OSCC cell line from tongue, was obtained from the National Institutes of Health (NIH). HIOEC and HB cells were obtained from the Laboratory of Oral Oncology, Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University, School of Medicine [
33,
34]. HIOEC is human immortalized oral epithelial cells, which had been obtained from normal oral mucosa immortalized by transfection of HPV16 E6/E7 gene. HB cell was establish from the HIOEC by induction with benzo(a)pyrene [
34]. HIOEC cells were maintained in defined keratinocyte-SFM (Gibco, NY, USA) medium and other cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Gibco). All cells were cultured in a humidified atmosphere of 5% CO
2 at 37°C. LPS was obtained from Sigma (MO, USA), and p38 specific inhibitor PD169316 was from Abcam (MA, USA).
Specimens and immunohistochemistry
Thirty human OSCC specimens were collected from patients who had undergone surgery between 2007 and 2009 in the Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital. All experimental procedures received ethical approval by the Independent Ethics Committee of Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University, School of Medicine (Number 200926). Pathological characterization of the OSCC patients included in this study is summarized in Table
1. For immunohistochemical examination, OSCC tissues were fixed with 4% paraformaldehyde and embedded with paraffin. Sections of the samples were blocked with 10% goat serum in PBS and incubated overnight at 4°C with either anti-TLR4 antibody (Imgenex, CA, USA) or with anti-MyD88 antibody (Abcam) at a dilution of 1:100. After three washes with PBS, the sections were incubated with peroxidase-conjugated goat anti-mouse/rabbit antibody for 1 h, followed by incubation with 3, 3’-Diaminobenzidine (DAB) substrate for 3 min. Counter-staining was performed with hematoxylin, and then dehydration was performed with ethanol and dimethyl benzene. Slides were mounted with Permount (Santa Cruz, CA, USA) and visualized by Axio Imager (Zeiss, Oberkonchen, Germany). The IHC results in tissues were scored by two independent investigators based on the level of staining intensity as follows: none (−): 0% of stained cells; weak (+): 1-25% of stained cells; moderate (++): 26-50% of stained cells; strong (+++): >50% of stained cells.
Reverse transcription-PCR (RT-PCR)
To determine TLR expression at the mRNA level, RT-PCR was performed. Tumor cells (2 × 106) were harvested, after cell washing, and total RNA was isolated using Trizol reagent (Invitrogen, CA, USA). RT-PCR was performed according to the manufacturer’s instructions. Reverse transcription was performed with random primers using an Omniscript RT kit (Promega, WI, USA). The sequences of primers for TLR4 were 5’-CAGAGTTGCTTTCAATGGCATC-3’ (forward) and 5’-AGACT GTAATCAAGAACCTGGAGG-3’ (reverse); and 5’-CTCCTCCACATCCCTTCC-3’ (forward) and 5’-CCGCACGTTCAAGAACAGAGA-3’ (reverse) for MyD88. PCR was performed using an Expand High Fidelity PCR System (TaKaRa, Shiga, Japan).
Flow cytometry
TLR4 expression in cells was evaluated by flow cytometry as follows: cells were collected and then labeled with the APC-labeled mouse anti-human TLR4 antibody (eBioscience, CA, USA) for 30 min at 4°C. The cells were analyzed using Cell Quest Software. Apoptosis was measured by flow cytometry as follows: cells were harvested and washed in PBS, resuspended in pre-diluted binding buffer, and stained with AnnexinV-FITC (BD Biosciences, CA, USA) for 30 min at room temperature. After being washed and resuspended in PI binding buffer, the cells were immediately subjected to apoptosis analyses by flow cytometry using Cell Quest Software.
Elisa
Cells (5 × 105 ml-1) were stimulated with 1 μg/ml LPS for 24 h. Culture supernatants were collected. Cytokines in the supernatants were measured by ELISA (R&D, MN, USA) according to the manufacturer’s instructions.
Immunofluorescence microscope
Tumor cells deposited on glass slides were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 20 min. The cells were further permeabilized with 0.1% TritonX in PBS for 8 min, washed and blocked with 5% bovine serum albumin in PBS for 30 min, then treated with monoclonal mouse anti-p65 (Santa Cruz) antibody overnight. FITC-labeled (1:100) anti-mouse IgG served as the secondary antibody. Sections were then mounted in a medium containing Hoechst33342 for 5 min to visualize cell nuclei. Slides were evaluated with a laser scanning confocal microscope TCS SP2 (Leica, Wetzlar, Germany), and Adobe Photoshop 7.0 was used for the digital image analysis.
Luciferase reporter gene assay used to indicate NF-κB activity
Cal-27 cells were cotransfected with the mixture of 200 ng pNF-κB-Luc and 10 ng pRenilla using the LipofectamineTM 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. 24 h after transfection, the cells were either left untreated or stimulated with 1 μg/ml of LPS. Cell lysates were assayed for expression of luciferase using a dual luciferase assay kit (Promega). Chemiluminescence, representing the expression of luciferase, was measured in a Junior LB9505 luminometer (Berthold, Wildbad, Germany). Relative luciferase activity (RLA) was obtained by normalizing the luciferase activity with the pRenilla activity. The extent of NF-κB activation was represented by the relative increase in the level of RLA. All transfection experiments were performed in two wells and repeated independently three times. The activity of controls was set at 1.0.
Western blotting analysis
OSCC cells were stimulated with LPS for various time periods at 37°C and then lysed with M-PER® Mammalian Protein Extraction Reagent. Protein was quantified by the BCA Protein Assay Kit according to the manufacturer’s instruction, separated by SDS-polyacrylamide gel electrophoresis, and then electrotransferred onto a polyvinylidene difluoride membrane. Essential component detection in the cells was performed with the following antibodies via overnight incubation at 4°C: anti-MyD88 (Abcam), anti-I-κBa (Santa Cruz), anti-phosph-p38 (Cell Signaling Technology, MA, USA), and anti-TLR4 (Imgenex). HRP-conjugated secondary Antibody (Pierce Chemical, MO, USA; 1:5000 dilution) was added for 1 h at room temperature, followed by development of reactions in a chemiluminescent detection system. β-actin antibody was used as a control.
Effects of TLR4-specific siRNA
Expression of TLR4 in the cell lines was temporarily silenced using small interfering RNA (siRNA) of TLR4. The sense and antisense strands of siRNA were: 5-GUCUAGUGGCUAAUUCCUA-3 and 5-UAGGAAUUAGCCACUAGAC-3. Briefly, 2 × 105 tumor cells were seeded in wells of a six-well plate and cultured in DMEM. The next day, the cells were transfected with human TLR4 siRNA (GenePharma Co., Shanghai, China) at different concentrations according to the manufacturer’s instructions. The negative control consisted of siRNA with no homology to known sequences from humans. Cells were incubated in complete DMEM medium at 37°C in an atmosphere of 5% CO2. Western blot was used to test TLR4 expression and further experiments were carried out.
Statistical analysis
Table
1 data were analysed by the statistical software SPSS10.0 for windows (SPSS10.0 Inc., USA). Other statistical analysis was performed using the Student’s
t-test. Values of
P <0.05 were considered significant.
Competing interests
The authors declare that they have no competing interests.
Author’s contributions
CFX designed the study and interpreted the data and amended the manuscript; CWT provided all the cell lines and gave instructions; SZJ performed the experiments and drafted the manuscript; LQQ participated in immunohistochemistry and flow cytometry analysis; and YDX helped to do the immunofluorescence microscope analysis. All of the authors read and approved the final version of this manuscript.