interleukin-8 and Mouth-Neoplasms

Oral squamous cell carcinoma (OSCC) is gradually increasing its incidence in our society. Unfortunately, this entity is diagnosed at an advanced stage in most patients, a fact that implies greater difficulty in its treatment and a worse prognosis. This systematic review aims to assess whether the cytokines IL-6, IL-8 and TNF-α are potential salivary biomarkers that allow early diagnosis of cancer.. An electronic search was performed in three databases (Pubmed, Scopus and Web of Science). We used the following keywords: "salivary cytokines", "saliva cytokines", "salivary interleukins", "biomarkers", "oral squamous cell carcinoma" and "diagnosis", combined with the Boolean operators "AND" and "OR".. 128 publications were found and finally 23 articles were included in the review and 15 in the meta-analysis. It has been observed that the majority of OSCC patients express higher salivary concentrations of IL-6, IL-8 and TNF-α compared to the control (CL) and premalignant lesion (OPML) groups. It has also been observed that the different premalignant lesions do not have statistically significant differences in the salivary concentration of the cytokines, and on the other hand, differences have been observed between the different TNM stages. The meta-analysis has shown that the difference in concentration of IL-6, IL-8 and TNF-α is statistically significant between the CL group and the OSCC, and also between the CL group and OPML.. There is sufficient evidence to affirm that IL-6, IL-8 and TNF-α are useful salivary cytokines in the early diagnosis and prognosis of OSCC. Although future studies are necessary to establish greater reliability of these biomarkers and thus be able to develop a valid diagnostic test.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cytokines; Head and Neck Neoplasms; Humans; Interleukin-6; Interleukin-8; Mouth Neoplasms; Prognosis; Reproducibility of Results; Saliva; Squamous Cell Carcinoma of Head and Neck; Tumor Necrosis Factor-alpha

Inflammation and cell-mediated immunity can have significant roles in different stages of carcinogenesis. The present meta-analysis aimed to evaluate the association between the polymorphisms of. PubMed/MEDLINE, Web of Science, Cochrane Library, and Scopus databases were searched until December 18, 2020 without any restrictions. RevMan 5.3 software was used to calculate the results of forest plots (odds ratios (ORs) and 95% confidence intervals (CIs)); CMA 2.0 software was used to calculate funnel plots (Begg's and Egger's tests), and SPSS 22.0 was used for the meta-regression analysis. Moreover, trial sequential analysis was conducted to estimate the robustness of the results.. Eleven articles including twelve studies were selected for the meta-analysis. The pooled ORs for the association between. The meta-analysis confirmed that there was no association between the polymorphisms of

Topics: Genetic Predisposition to Disease; Humans; Interleukin-6; Interleukin-8; Mouth Neoplasms; Polymorphism, Genetic; Risk Factors

This meta-analysis aimed to assess the salivary and serum concentrations of IL-6 and IL-8 in oral squamous cell carcinoma (OSCC) patients compared to the controls. Four electronic databases (Scopus, PubMed, Cochrane Library, and Web of Science) were searched up to January 2019. The study quality was checked according to the Newcastle-Ottawa Scale. The mean difference (MD) plus 95% confidence interval (95%CI) were calculated using RevMan 5.3 software. The publication bias and sensitivity analysis were done using CMA 2.0 software. Out of 309 studies retrieved from the 4 databases, 26 studies were analyzed in the present meta-analysis. In this meta-analysis, the pooled MD in the OSCC patients compared to the controls was 19.06 pg/mL (95%CI: 14.78-23.33) for the serum IL-6 level, 199.14 pg/mL (95%CI: 47.39-350.89) for the serum IL-8 level, 122 pg/mL (95%CI: 64-179) for the salivary IL-6 level, and 958 pg/dL (95%CI: 718-1197) for the salivary IL-8 level. All values in this meta-analysis were statistically significant. In conclusion, according to the meta-analysis results, the serum and salivary IL-6 and IL-8 levels in OSCC patients were significantly elevated compared to the controls, and both cytokines can be useful as potential biomarkers in early OSCC diagnosis.

Topics: Carcinoma, Squamous Cell; Humans; Interleukin-6; Interleukin-8; Mouth Neoplasms; Saliva

The findings of associations between interleukin-8 (IL-8) polymorphisms and risk of oral cancer are controversial. We conducted a meta-analysis on the basis of data from all published studies to provide evidence of the current understanding of the genetic association with oral cancer. Eligible studies were identified by means of an electronic search of PubMed, Elsevier, ScienceDirect, EMBASE, EBSCO, and CBM databases for studies published up to March 2013. In accordance with the inclusion and exclusion criteria, a total of six eligible studies were included in the pooled analyses. In the overall analysis, we did not observe any significant associations between the IL-8-251A>T polymorphism and oral cancer risk under any of the genetic models (all P > 0.05). In the stratified analysis by ethnicity, Caucasian individuals with genotype AA had a higher risk of oral cancer under the dominant model (OR = 1.35, 95 % CI 1.09-1.67, P = 0.006). This meta-analysis indicated that the IL-8-251A>T polymorphism was not associated with the susceptibility of oral cancer, while individuals in the Caucasian population with genotype AA had a higher risk of oral cancer under the dominant model.

Topics: Asian People; Case-Control Studies; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Mouth Neoplasms; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors; White People

Emerging evidence showed that the most common functional polymorphism (-251A>T, rs4073) in the promoter region of the interleukin-8 (IL-8) gene is involved in the regulation of the activities of interleukin-8, thus increasing an individual's susceptibility to oral cancer; but individually published results are inconclusive. The aim of this meta-analysis was to investigate the associations between IL-8 -251A>T polymorphism and oral cancer risk.. The PubMed, Embase, Web of Science and CBM databases were searched for all articles published up to October 1st, 2012 that addressed IL-8 -251A>T polymorphism and oral cancer risk. Statistical analyses were performed using STATA 12.0 software. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations.. Six case-control studies were included with a total of 1324 oral cancer cases and 1879 healthy controls. When all available studies were pooled into the meta-analysis, the results showed that the AA and AT genotypes of IL-8 -251A>T polymorphism were associated with increased risk of oral cancer (OR=1.23, 95% CI: 1.03-1.46, P=0.025; OR=1.25, 95% CI: 1.07-1.47, P=0.006; respectively). In the subgroup analysis by ethnicity, significant associations were observed between the AA and AT genotypes of IL-8 -251A>T polymorphism and increased risk of oral cancer among Caucasian populations (OR=1.40, 95% CI: 1.14-1.72, P=0.001; OR=1.29, 95% CI: 1.06-1.57, P=0.011; respectively). However, no statistically significant associations were found between IL-8 -251A>T polymorphism and oral cancer risk among Asian populations.. Results from the current meta-analysis indicate that the AA and AT genotypes of IL-8 -251A>T polymorphism might increase the risk of oral cancer, especially among Caucasian populations.

Topics: Asian People; Case-Control Studies; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Mouth Neoplasms; Polymorphism, Single Nucleotide; Risk; White People

Cancer-related inflammation (CRI) significantly increases the difficulty of treating oral squamous cell carcinoma (OSCC) and remains a major treatment challenge. Our objective was to determine whether tumor ALDH3A1 could attenuate OSCC tumorigenesis by inhibiting tumor-associated macrophages (TAMs) that promoted CRI.. ALDH3A1 in Cal27 cells was overexpressed, and the tumor-conditioned medium (TCM) was collected. We induced THP-1 cells with TCM and recombinant human IL-6. The phosphorylation of STAT3 and the TLR4/TRAF6/TBK1 cascade reaction in TAMs was analyzed using Western blotting, and mitochondrial ROS (mtROS) production was measured using a MitoSox kit. A tumorigenicity assay was performed to examine the tumor volume and weight, and the expression of CD68, CD11b, IL-6, Ki67, and CD31 was analyzed via immunohistochemistry.. ALDH3A1 attenuated STAT3 phosphorylation at Ser727 rapidly and mtROS production earlier in TAMs via inhibiting TLR4/TRAF6/TBK1 cascade reaction. MtROS reduction inhibited IL-1β and IL-8 secretions by NLRP3/caspase-1/IL-1β/IL-8 pathway. Meanwhile, the inhibition of pro-tumor phenotypes of TAMs, tumor proliferation, and tumor angiogenesis during the process was proved in vivo.. ALDH3A1 was associated closely with CRI and inhibited CRI regulated by TAMs. This finding may achieve clinical transformation and open new therapeutic options for targeting CRI regulated by TAMs.

Topics: Aldehyde Dehydrogenase; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Head and Neck Neoplasms; Humans; Inflammation; Interleukin-6; Interleukin-8; Macrophages; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4; Tumor-Associated Macrophages

The tumor microenvironment plays a significant role in tumor growth, metastasis and chemoresistance via dysregulated signaling pathways. Toward this, an inflammatory chemokine, interleukin-8 (IL-8), is overexpressed in various cancers and is involved in tumor progression and chemoresistance. However, the mechanistic role of IL-8 in mediating metastasis and chemoresistance in oral squamous cell carcinoma (OSCC) is not known.. In the present study, we evaluated the effect of IL-8 in regulating metastasis as well as chemoresistance in OSCC cell lines. For this, IL-8 was blocked exogenously using neutralizing IL-8 monoclonal antibody and IL-8 levels were enhanced by exogenous supply of recombinant human IL-8 (rhIL-8) to OSCC cells. The epithelial-to-mesenchymal transition (EMT) was evaluated using qPCR, migration by scratch/wound healing assay and invasion ability using transwell assay. rIL-8 induced chemoresistance was studied by apoptosis assay and the nuclear localization of NFκB using immunocytochemistry. IL-8 was significantly overexpressed in OSCC patients and cell lines. While exogenous blockade of IL-8 significantly reduced EMT, migration and invasion potential in OSCC cells, IL-8 overexpression upregulated these cellular traits thereby confirming the role of IL-8 in OSCC metastasis. Exogenous blockade of IL-8 also reversed chemoresistance in cisplatin resistant OSCC subline via NFκB signaling.. IL-8 plays a crucial role in OSCC metastasis and its targeted blockade can help in management of cisplatin resistance.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Humans; Interleukin-8; Mouth Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-akt; Tumor Microenvironment

To study whether damaged epithelial cells and gingival fibroblast could affect the expression of inflammatory cytokines in healthy cells.. Cell suspensions were submitted to different treatments to obtain the lysates: no treatment (supernatant control), sonication, and freeze/thawing. All treatments were centrifuged, and the supernatants of the lysates were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB p65 were applied to verify the inflammatory crosstalk of damaged cells over healthy plated cells. Furthermore, titanium discs and collagen membranes were treated with lysates and checked for IL8 expression by RT-qPCR.. Lysates obtained upon sonication or freeze/thawing of oral squamous carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by IL6 immunoassays. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous carcinoma cells. Additionally, oral squamous carcinoma cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65. Finally, oral squamous carcinoma cell lysates adhered to the titanium and collagen membrane surfaces and increased IL8 expression by gingival fibroblasts growing in these materials.. Injured oral epithelial cells can release factors that incite gingival fibroblasts to become pro-inflammatory.. Injuries affecting the oral mucosa generate epithelial fragments that may reach the underlying connective tissue and provoke inflammation. These injuries are routinely caused by mastication, sonication for teeth cleaning, teeth preparation, prostheses maladaptation, and implant drilling.

Topics: Carcinoma, Squamous Cell; Collagen; Cytokines; Fibroblasts; Gingiva; Humans; Interleukin-6; Interleukin-8; Mouth Neoplasms; NF-kappa B; Titanium

The purpose of this pilot study was to detect the presence of interleukin-8 (IL-8) and the potential downstream effects of IL-8 receptor activation in 2 previously characterized feline oral squamous cell carcinoma cell lines (SCCF1 and SCCF2). Interleukin-8 messenger RNA (mRNA) was initially detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A previously validated and commercially available enzyme-linked immunosorbent assay (ELISA) test was used to measure IL-8 production in the supernatant of the 2 cell lines. Western blot was used to detect phosphorylation of proteins (AKT, ERK1/2, JAK2, STAT3, and Src), known to be downstream of interleukin-8 receptor activation. The IL-8 receptor-specific antagonists, Reparixin and SCH527123, were used to identify effects on phosphorylation of these proteins. Interleukin-8 mRNA and protein were detected in both SCCF1 and SCCF2 by RT-PCR and ELISA, respectively. Phosphorylation of ERK1/2, STAT3, and Src was detected in both cell lines. Inhibition of the IL-8 receptor led to a decrease in phosphorylation of Src, but not ERK1/2 or STAT3. In conclusion, feline squamous cell carcinoma cell lines can produce IL-8. Phosphorylation of Src seems, at least in part, a consequence of IL-8 receptor activation. The phosphorylation of ERK1/2 and STAT3, although present, seems independent of IL-8 receptor activation. Due to its potential effects on the tumor microenvironment, in addition to its autocrine effects on Src phosphorylation, the inhibition of the IL-8 receptor may become a beneficial therapeutic tool. Evaluation of the presence of both IL-8 and Src in many cases should elucidate their importance.. Le but de cette étude pilote était de détecter la présence d’interleukine-8 (IL-8) et les effets potentiels en aval de l’activation du récepteur IL-8 dans deux lignées cellulaires de carcinome épidermoïde oral félin (SCCF1 et SCCF2) précédemment caractérisées. L’ARN messager de l’interleukine-8 (ARNm) a été initialement détecté par amplification en chaîne par la polymérase à transcription inverse quantitative (qRT-PCR). Un test immuno-enzymatique ELISA précédemment validé et disponible dans le commerce a été utilisé pour mesurer la production d’IL-8 dans le surnageant des deux lignées cellulaires. L’immunobuvardage a été utilisé pour détecter la phosphorylation des protéines (AKT, ERK1/2, JAK2, STAT3 et Src), connues pour être en aval de l’activation du récepteur de l’interleukine-8. Les antagonistes spécifiques du récepteur IL-8, Reparixin et SCH527123, ont été utilisés pour identifier les effets sur la phosphorylation de ces protéines. L’ARNm et la protéine de l’interleukine-8 ont été détectés dans SCCF1 et SCCF2 par RT-PCR et ELISA, respectivement. La phosphorylation de ERK1/2, STAT3 et Src a été détectée dans les deux lignées cellulaires. L’inhibition du récepteur IL-8 a conduit à une diminution de la phosphorylation de Src, mais pas ERK1/2 ou STAT3. En conclusion, les lignées cellulaires de carcinome épidermoïde félin sont capables de produire de l’IL-8. La phosphorylation de Src semble, au moins en partie, une conséquence de l’activation du récepteur IL-8. La phosphorylation de ERK1/2 et STAT3, bien que présente, semble indépendante de l’activation du récepteur IL-8. En raison de ses effets potentiels sur le micro-environnement tumoral, en plus de ses effets autocrines sur la phosphorylation de Src, l’inhibition du récepteur IL-8 peut devenir un outil thérapeutique bénéfique. L’évaluation de la présence à la fois d’IL-8 et de Src dans un grand nombre de cas devrait élucider leur importance.(Traduit par Docteur Serge Messier).

Topics: Animals; Carcinoma, Squamous Cell; Cat Diseases; Cats; Cell Line, Tumor; Interleukin-8; Mouth Neoplasms; Pilot Projects; RNA, Messenger; Signal Transduction; Tumor Microenvironment

Vascular adhesion protein-1 (VAP-1) is believed to play a role in inflammation. Studies have suggested that VAP-1-mediated activation of inflammation is dependent on NF-κB, leading to secretion of the interleukin (IL)-8; however, no reports have addressed the association between VAP-1 and NF-κB/IL-8 signaling in oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of VAP-1 in OSCC and further explore whether VAP-1 is involved in the regulation of neutrophil infiltration in the tumor microenvironment (TME).. Immunochemistry staining was used to observe VAP-1 expression. CCK-8 and Transwell assays were used to measure cell proliferation, migration, and invasion. OSCC xenograft mouse models were used for in vivo verification of the VAP-1 function. The expression of NF-κB and IL-8 were determined by qRT-PCR and western blot. ELISA for IL-8 was also conducted. The relationship between VAP-1 expression and neutrophil infiltration was analyzed by immunofluorescence.. VAP-1 was overexpressed in human OSCC tissues. Downregulation of VAP-1 suppressed OSCC cells proliferation, migration, and invasion in vitro and inhibited tumor proliferation and metastasis in vivo. Additionally, downregulation of VAP-1 inhibited NF-κB/IL-8 signaling in vitro and in vivo. VAP-1 expression was positively correlated with neutrophil infiltration in human OSCC tissues. Moreover, blocking VAP-1 decreased neutrophil infiltration by reducing IL-8 production.. VAP-1 downregulation in OSCC suppresses tumor growth and metastasis by inhibiting NF-κB/IL-8 signaling and reducing neutrophil infiltration in the TME, suggesting that VAP-1 may be a potential therapeutic target for OSCC.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Head and Neck Neoplasms; Humans; Inflammation; Interleukin-8; Mice; Mouth Neoplasms; Neutrophil Infiltration; NF-kappa B; Squamous Cell Carcinoma of Head and Neck; Tumor Microenvironment

Lymph node metastasis is associated with poor prognosis of oral squamous cell carcinoma (OSCC), and few studies have explored the relevance of postoperative lymphatic drainage (PLD) in metastatic OSCC. Alpha-enolase (ENO1) is a metabolic enzyme, which is related to lymphatic metastasis of OSCC. However, the role of ENO1 in PLD in metastatic OSCC has not been elucidated. Herein, we collected lymphatic drainage after lymphadenectomy between metastatic and non-metastatic lymph nodes in OSCC patients to investigate the relationship between ENO1 expression and metastasis, and to identify the proteins which interacted with ENO1 in PLD of patients with metastatic OSCC by MS/GST pulldown assay. Results revealed that the metabolic protein apolipoprotein C-III (ApoC3) was a novel partner of ENO1. The ENO1 bound to ApoC3 in OSCC cells and elicited the production of interleukin (IL)-8, as demonstrated through a cytokine antibody assay. We also studied the function of IL-8 on Jurkat T cells co-cultured with OSCC cells in vitro. Western blot analysis was applied to quantitate STAT3 (signal transducer and activator of transcription 3) and p-STAT3 levels. Mechanistically, OSCC cells activated the STAT3 signaling pathway on Jurkat T cells through IL-8 secretion, promoted apoptosis, and inhibited the proliferation of Jurkat T cells. Collectively, these findings illuminate the molecular mechanisms underlying the function of ENO1 in metastasis OSCC and provide new strategies for targeting ENO1 for OSCC treatment.

Topics: Apolipoprotein C-III; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Interleukin-8; Lymphatic Metastasis; Mouth Neoplasms; Phosphopyruvate Hydratase; Squamous Cell Carcinoma of Head and Neck; STAT3 Transcription Factor

Cancer stem cells (CSCs) drive tumor initiation and progression and participate in tumor chemoresistance. We recently discovered that oral squamous cell carcinoma (OSCC) cells that highly express CD10 (CD10H cells) present cancer stem cells (CSC)-associated characteristics, which, in turn, affect the tumor growth, epithelial-mesenchymal transition (EMT), and resistance to cisplatin. In this study, we further investigated this mechanism in vitro and in vivo. We hypothesized that IL8 might regulate migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells through the ERK pathway.. CD10 MicroBead Kit was used to select HN6 cells with high and low expression of CD10. The target protein IL8 was screened via protein chip assay. Lentiviral transduction and specific inhibitor were applied to investigate the signaling pathway. Real-time PCR, Western blot, and immunohistochemistry were used to analyze the mRNA and protein expression; transwell assay, spheroid formation assay, and cell viability assay were used to study the cell biological behavior in vitro; xenograft animal model was used to evaluate the tumor formation rate in vivo.. Overexpression of CD10 promoted CSC-related genes expression and enhanced migration, invasion, spheroid formation, and chemoresistance in HN6 cells. Moreover, the overexpression of IL8 was detected in OSCC tumor tissue and cell lines (HN6 and CAL27) overexpressing CD10. IL8 secreted by CD10H HN6 promoted migration and invasion and restored tumor chemosensitivity via the p-ERK signaling pathway, while the inhibition of IL8 increased the chemosensitivity to cisplatin.. IL8 secretion by CD10 positive cells promotes migration, invasion, and cisplatin resistance of OSCC via the p-ERK signaling pathway.

Topics: Animals; Antineoplastic Agents; Cell Communication; Cell Movement; Cisplatin; Drug Resistance, Neoplasm; eIF-2 Kinase; Female; Gene Expression; Heterografts; Humans; Interleukin-8; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Transplantation; Neoplastic Stem Cells; Neprilysin; Signal Transduction; Spheroids, Cellular; Squamous Cell Carcinoma of Head and Neck

Proteasome inhibitor MG132 was shown to enhance the secretion of interleukin 8 (IL-8) by various cells. The enhancement is regulated by the transcription factor activator protein-1 (AP-1) at the transcriptional level. AP-1 is a dimer formed by AP-1 family proteins. The purpose of the present study was to explore the combinations of the AP-1 family proteins that contribute to MG132-driven IL-8 secretion. Oral squamous cell carcinoma-derived cell lines, Ca9-22 and HSC3, were used to demonstrate their response to MG132. IL-8 secretion was augmented by MG132 in both cell lines. c-Jun expression was detected in both the cell lines, whereas c-Fos expression was detected only in the HSC3. The influence of MG132 stimulation on c-Jun and c-Fos expression was further examined by western blot analysis. c-Jun expression was increased by MG132 stimulation, whereas c-Fos expression was not detected even after MG132 stimulation. As JunB is reported to inhibit the transcriptional activity of the AP-1 complex, we speculated that the c-Jun homodimer should contribute to IL-8 enhancement. Expression vectors encoding wild type and c-Jun mutants, M17 and M22-23, respectively, were constructed and transfected into the Ca9-22 cells. In contrast to our expectations, MG132-induced IL-8 secretion was significantly reduced in all the transfectants suggesting that other c-Jun members might form homodimers with c-Jun and contribute to IL-8 enhancement. Transfection of the cells with c-Jun or JunB small hairpin RNA (shRNA) reduced IL-8 secretion up to 50% and 65% of the control shRNA transfectant. Furthermore, cotransfection of both shRNA almost completely inhibited the IL-8 secretion. These results indicate that JunB not only inhibits but also enhances the transcription of c-Jun targets in combination with c-Jun.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Humans; Interleukin-8; Mouth Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Messenger; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection

To evaluate the effects of allicin on mediators of pain secreted by oral cancer cells in vitro, single-cell suspensions were prepared by enzymatic method from oral squamous cell carcinoma (OSCC). Cancer stem cells were isolated by the CD133+ selection method with magnetic cell sorting. Stemness markers were checked in both cancer cells and cancer stem cells by RT-PCR. Comparative analysis of pain mediators TNF-alpha, IL-8, and endothelin at both RNA and protein levels for normal epithelial cells, cancer cells, and cancer stem cells was carried out with and without allicin treatment. CD133 and CD44 expression levels were checked in cancer cells and cancer stem cells flow cytometrically. Allicin inhibited both gene and protein expression of TNF-alpha, IL-8, and endothelin in both cancer cells and cancer stem cells. Allicin is more likely to be a promising treatment in alleviating the levels of pain and inflammation in OSCCs.

Topics: AC133 Antigen; Antioxidants; Cancer Pain; Disulfides; Endothelins; Humans; Interleukin-8; Mouth Neoplasms; Neoplastic Stem Cells; Primary Cell Culture; Sulfinic Acids; Tumor Necrosis Factor-alpha

Salivary biomarkers can be used as diagnostic and predictive aids in early detection of oral cancer or potentially malignant disorders. A cross-sectional comparative study was conducted over a period of one year from August 2016 to August 2017 in a multicentre setting in Islamabad. A total of 60 patients were recruited and divided into two equal groups of naswar users and non-users. Un-stimulated saliva samples were collected and analysed by using an enzyme-linked immunosorbent assay (ELISA) technique. Data was entered in SPSS version 22.0. The results were then analysed by using independent t-test. Statistically significant difference was found regarding the levels of salivary IL-8 between the naswar users and non-users (p <0.001). The levels of salivary IL-8 in non-users were 33.39 ±22.44 pg/ml, while the increased levels of salivary IL-8 in naswar users were found to be 173.48 ±46.52 pg/ml.

Topics: Adult; Biomarkers; Carcinoma, Squamous Cell; Cross-Sectional Studies; Humans; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Pakistan; Predictive Value of Tests; Saliva; Tobacco, Smokeless; Young Adult

Recent studies reveal a great value of interleukin-8 (IL-8), a pro-inflammatory cytokine, as a potent biomarker for early diagnosis of oral cancer. Herein, a new electrochemical method is proposed to detect IL-8 by facilely incorporating DNA-templated quantum dots (QDs). In principle, target IL-8 is first treated with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to yield active thiols and then captured by antibody-functionalized magnetic beads (MBs). Thereafter, via the Michael addition reaction between the active thiol and maleimide group, a maleimide-modified DNA probe is linked to the surface of MBs, which can initiate a process of rolling circle amplification. In this way, long-range DNA strands are generated on the MB surface, subsequently recruiting DNA-templated CdTe/CdS QDs (DNA-QDs) to act as electrochemical reporters. By tracing the responses of DNA-QDs, the method allows IL-8 detection in a linear range from 5 to 5000 fg/mL with a detection limit of 3.36 fg/mL. The selectivity, reproducibility, and applicability in complex serum samples are also demonstrated to be favorable, indicating that the method may have a great potential in the future. More importantly, the use of TCEP treatment in the method not only provides a facile way to incorporate DNA-QDs, avoiding the complicated and time-consuming preparation process of antibody-DNA conjugates or functional nanomaterials; but also makes the method capable of being extended to detect other protein biomarkers in view of widespread presence of disulfides, which may hold a broad potential to facilitate efficient biosensing designs.

Topics: Antibodies, Immobilized; Biosensing Techniques; Cadmium Compounds; Electrochemical Techniques; Humans; Immobilized Nucleic Acids; Interleukin-8; Limit of Detection; Mouth Neoplasms; Quantum Dots; Sulfides; Tellurium

Early detection and easier follow-up of oral squamous cell carcinoma (OSCC) would significantly improve the morbidity and mortality associated with it. With newer technologies, it has become possible to validate cancer biomarkers in saliva with high sensitivity and specificity. There is however a need to further validate these biomarkers in cohorts of different ethnic groups. Our objective was to validate previously evaluated salivary biomarkers in Indian population. The study enrolled 117 patients. These were grouped into subcatergories of 31 early (TNMstage I-II) and 27 late-stage OSCC (TNM stage III-IV), 30 PMOD and 29 post-treatment patients. There were 42 control subjects. We evaluated 3 protein markers, IL-1β, IL-8 and LGALS3BP using ELISA, from unstimulated saliva samples. Statistical analysis was done to calculate p-value, ROC, AUC, sensitivity, and specificity. Protein markers IL-1β and IL-8 were significantly elevated (p < 0.05) in OSCC patients. Though the markers could not discriminate PMOD and post-treatment subjects from controls, they proved to be significantly discriminatory between OSCC and controls. Both these markers were especially strong discriminators of late stage OSCC (stage III-IV). IL-1β had the most statistically significant discriminative power (AUC = 0.9017) in late-stage OSCC followed by IL-8 (AUC = 0.7619). Although LGALS3BP was not found to be significantly elevated in late stage OSCC patients, but it was a significant discriminator of early stage OSCC (stage I-II) with p-value = 0.0008 and AUC = 0.7296. These salivary biomarkers have been discovered and validated in other ethnic groups earlier. Hence, the fact that these markers were discriminatory in Indian population too, strengthens the possibility of using these salivary biomarkers as screening tools in different ethnic cohorts. Such trials would potentiate use of a non-invasive tool, like saliva for diagnosis and follow-up of oral cancer.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Squamous Cell; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-1beta; Interleukin-8; Mouth Neoplasms; Saliva

Bone mesenchymal stem cells (BMSCs) play critical roles in tumour microenvironment. However, molecular mechanisms of how BMSCs to be recruited and effect subsequent tumour progression are poorly understood in oral squamous cell carcinoma (OSCC).. The distribution of CXCL8 was detected by immunohistochemical staining in OSCC tissues. The chemotaxis of conditioned media from different epithelial cells to BMSCs was examined by trans-well assay. Real-time quantitative PCR (qPCR) and ELISA were used to detect the expression of related cytokines and chemokine receptors. The migration of BMSCs was observed in BALB/c nude mice. The roles of BMSCs in proliferation, migration and invasion of OSCC were detected by CCK-8, flow cytometry and trans-well assay. Epithelial-mesenchymal transition (EMT)-related markers were analysed by qPCR and Western blot in vitro, and growth was evaluated in BALB/c nude mice using subcutaneously implanted OSCC in nude mouse model in vivo.. Using OSCC, we show CXCL8, secreted by OSCC, binds to exclusively CXCR2 in BMSCs to facilitate migration of BMSCs to OSCC. TGF-β secreted by BMSCs subsequently induces EMT of OSCC to promote their proliferation, migration and infiltration. We also showed that the Ras/Raf/Erk axis plays a critical role in tumour progression.. Our results provide the molecular basis for BMSC recruitment into tumours, and how this process leads to tumour progression and leads us to develop a novel OSCC treatment target.

Topics: Animals; Cadherins; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Interleukin-8; Male; Mesenchymal Stem Cells; Mice, Nude; Mouth Neoplasms; Receptors, Interleukin-8B; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

This study investigated the risk and prognostic value of single nucleotide polymorphisms (SNP) inIL-8, MMP-1 and MMP-13 in oral and oropharyngeal squamous cell carcinomas (SCCs).. SNPs rs2227532 and rs4073 inIL-8, rs2071230 and rs470558 in MMP-1, and rs2252070 in MMP-13 were genotyped in 125 oral and oropharyngeal SCC patients and 130 healthy controls, using TaqMan allelic discrimination assays. Multiple logistic regression models were used to explore the association between SNPs and cancer development, as well as SNP-SNP interaction and gene-environmental factor (GxE) interaction. Univariate and multivariate methods were applied for survival analyses.. With exception of rs2227532, all the SNPs were in Hardy-Weinberg equilibrium in the control. No associations between rs4073 in IL-8 and rs2071230 and rs470558 in MMP-1 were observed, but rs2252070 in MMP-13, in the dominant model, was associated in a protective manner to oral and oropharyngeal SCC (OR: 0.20, 95% CI: 0.06-0.71, p = 0.007). All SNPs interact significantly with cigarette smoking and alcohol consumption on susceptibility to oral and oropharyngeal SCC, but they showed no influence on survival of the patients.. Our results show that rs2252070 inMMP-13 may confer protection effect against oral and oropharyngeal SCC. In addition, the combined effects of IL-8 (rs4073), MMP-1 (rs2071230 and rs470558) and MMP-13 (rs2252070) with environmental carcinogens, such as tobacco and alcohol, are related to increased risk for oral and oropharyngeal SCC development.

Topics: Carcinogens; Carcinoma, Squamous Cell; Genetic Predisposition to Disease; Humans; Interleukin-8; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Mouth Neoplasms; Polymorphism, Single Nucleotide; Prognosis

The development of pharmaceutical agents possessing anti‑invasive and anti‑metastatic abilities, as well as apoptotic activity, is important in decreasing the incidence and recurrence of oral cancer. Cancer cells are known to acquire invasiveness not only through epigenetic changes, but also from inflammatory stimuli within the tumor microenvironment. Accordingly, the identification of agents that can suppress the inflammation‑promoted invasiveness of cancer cells may be important in treating cancer and improving the prognosis of patients with cancer. Acetylshikonin, a flavonoid with anti‑inflammatory activity, inhibits proliferation and induces apoptosis of oral cancer cells. In the present study, the anti‑invasive effect of acetylshikonin on YD10B oral cancer cells infected with Porphyromonas gingivalis, a major pathogen of chronic periodontitis, and the mechanisms involved were investigated. Firstly, we examined whether P. gingivalis infection increased the invasiveness of YD10B cells. Results suggested that YD10B oral cancer cells become more aggressive when they are infected with P. gingivalis. Secondly, acetylshikonin significantly inhibited the invasion of P. gingivalis‑infected YD10B cells by suppressing IL‑8 release and IL‑8‑dependent MMP release. These data suggest that acetylshikonin may be a useful preventive and therapeutic candidate for oral cancer that is chronically infected with periodontal pathogens.

Topics: Anthraquinones; Bacteroidaceae Infections; Cell Line, Tumor; Cells, Cultured; Epithelial-Mesenchymal Transition; Gene Expression; Humans; Interleukin-8; Matrix Metalloproteinases; Mouth Neoplasms; Porphyromonas gingivalis

The let-7 family of microRNAs has been considered as tumor suppressors in various cancers; however, the role of let-7c in oral squamous cell carcinoma has not been determined yet.. In this study, phenotypical behaviors and the radio/chemoresistance were examined subsequent to overexpression of let-7c. In addition, the expression of let-7c in cancer stem cells (CSCs) was evaluated and the effect of let-7c on stemness characteristics was assessed. Also, luciferase activity assays were performed to test whether interleukin (IL)-8 was a putative target of let-7c.. Our results confirmed that the expression of let-7c in CSCs was reduced, while overexpression of let-7c attenuated the oncogenicity. Moreover, ectopic expression of let-7c in CSCs downregulated the stemness hallmarks and the radio/chemoresistance. Expression and secretion of IL-8 in oral CSCs were both reduced following overexpression of let-7c. Besides, the inhibitory effect of let-7c on various stemness phenotypes was reverted by IL-8, indicating that lower expression of let-7c may confer higher cancer stemness through a failure to downregulate IL-8.. These findings revealed the significance of let-7c in the contribution of oral cancer stemness and radio/chemoresistance. Targeting let-7c and its downstream IL-8 may be beneficial to prevent cancer recurrence and metastasis of oral squamous cell carcinoma.

Topics: Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Humans; Interleukin-8; MicroRNAs; Mouth Neoplasms; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Phenotype; Radiation Tolerance; Squamous Cell Carcinoma of Head and Neck; Survival Rate; Tumor Cells, Cultured

Oral cancer is at rise in our population due to increasing use of areca nut (Betel nut) with or without tobacco. It is the second frequent malignant tumour for both the gender in Pakistan. This non-interventional case control study was carried out with the aim to explore saliva as diagnostic medium for detecting interleukins (IL) 6 and 8 as biomarkers of pre-malignant lesions (PML) and oral carcinoma. Total 105 subjects were recruited and were divided into three groups "A", "B" and "C" each comprising of 35 subjects. Group "A" comprised of cases with strong clinical evidence of oral PML. Group "B" constitute clinical and histologically proven OSCC and group "C" include disease free subjects as controls. Saliva from all the recruited subjects was procured by drooling method and stored at-200C before further process. All the collected samples were centrifuged at 4500 rpm for 15 minutes at 4oC. Supernatant fluid was used in ELISA for detection and quantification of IL-6 & IL-8. Data was analysed by using Chi-square test and multivariate analysis was done by non-parametric test. P-value of 0.05 was taken as standard reference. Significant co-relation was found for qualitative salivary detection of IL-6 and IL-8 among the groups (P<0.001 and <0.0001 respectively). Regarding quantitative salivary concentration of leukotrienes, no significant co-relation was found in levels of IL-6 among the groups while there was significant association of IL-8 levels between the groups (P<0.0001).On post Hoc multiple comparison, significant co-relation was found among oral PML group and controls (P=0.001) and OSCC group and control (P=<0.0001). In conclusion salivary detection of IL-6 & IL-8 could be used as probable biomarker for early detection of oral PML & OSCC in etiologically distinct population of Pakistan.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Pakistan; Saliva; Young Adult

An efficient electrochemical transducer matrix for biosensing devices requires specific characteristics, such as fast electron transfer, stability, high surface area, biocompatibility, and presence of specific functional groups, to facilitate biomolecule attachment. We demonstrate the fabrication of an electrochemical immunosensor based on a highly stable gold nanoparticles-reduced graphene oxide (AuNPs-rGO) composite material as a transducer matrix for label-free and noninvasive detection of salivary oral cancer biomarker interleukin-8 (IL8). The synergy between rGO and AuNPs allowed the immunosensor to exhibit fast response and high sensitivity due to the improved electron transfer behavior of the composite. The immunosensor shows very fast detection (9 min) of IL8 and high sensitivity with an experimental linear dynamic range of 500 fg mL

Topics: Biosensing Techniques; Electrochemical Techniques; Gold; Graphite; Humans; Immunoassay; Interleukin-8; Limit of Detection; Metal Nanoparticles; Mouth Neoplasms

Cancer associated fibroblasts (CAFs) are known to be involved in initiation, progression and metastasis of various cancers. However, the molecular mechanism of how CAFs affects the biological function of oral cancer (OC) has not been fully-addressed. In this study, we demonstrated that miR-124 was downregulated in oral CAFs and oral cancer cells (OCCs) when compared with matched normal fibroblasts (NFs). Hypermethylation in the promoter region of miR-124 genes was accounted for its downregulation. Interestingly, CAFs but not NFs exerted promotion effect on OCCs cell proliferation, migration and tumor growth in CAFs/NFs-OCCs co-culture. Furthermore, we identified Chemokine (C-C motif) ligand 2 (CCL2) and Interleukin 8 (IL-8) as two direct targets of miR-124. Over-expression of miR-124 in CAFs-OCCs co-culture abrogated CAFs-promoted OCCs cell growth and migration, and this inhibitory effect can be rescued by addition of CCL2 and IL-8. Finally, we showed that restoration of miR-124 expression by lentiviral infection or formulated miR-124 injection inhibited oral tumor growth in vivo suggesting miR-124 rescue could be a potential rationale for therapeutic applications in oral cancer in the future.

Topics: Carcinoma; Cell Line, Tumor; Chemokine CCL2; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; MicroRNAs; Mouth Neoplasms; Promoter Regions, Genetic

Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells.. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death.. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis.. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Diaminopimelic Acid; Head and Neck Neoplasms; Humans; Immunity, Innate; Interleukin-8; Mitogen-Activated Protein Kinases; Mouth Neoplasms; Nod1 Signaling Adaptor Protein; Nod2 Signaling Adaptor Protein; Oligopeptides; RNA, Messenger; Squamous Cell Carcinoma of Head and Neck

The development of electrochemical magnetobiosensors for the simultaneous determination of two biomarkers associated with salivary oral cancer, protein IL-8 and its messenger RNA (IL-8 mRNA) associated, in undiluted human saliva samples is reported in this work. The implemented methodology involves the use of functionalized magnetic beads, specific antibodies against IL-8 protein, a specific hairpin DNA sequence for IL-8 mRNA and amperometric detection at disposable dual screen printed carbon electrodes. This methodology exhibits high sensitivity and selectivity for the target analytes providing detection limits of 0.21 nM for IL-8 mRNA and 72.4 pgmL(-1) (far below the clinical established cut-off of 600 pgmL(-1)) for IL-8 protein in undiluted saliva samples. The dual amperometric magnetobiosensor was applied to the direct determination of both biomarkers in spiked raw saliva samples and to determine the endogenous content of IL-8 protein in saliva samples from 7 healthy individuals. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.

Topics: Biomarkers, Tumor; Complex Mixtures; Conductometry; Equipment Design; Equipment Failure Analysis; Humans; Interleukin-8; Mouth Neoplasms; Reproducibility of Results; RNA, Messenger; Saliva; Sensitivity and Specificity

Oral cancer is a multifactorial disease process and involves complex interactions between gene to gene and gene to environmental factors. Interleukin 8 (IL-8), a pro-inflammatory cytokine, having angiogenic activity with elevated expression in tumor cells, is reported to play an essential role in oral cancer development. This study was conducted with the aim to investigate the role of IL-8 (-A251T) gene polymorphism in susceptibility, progression, and self-reporting pain in oral cancer. The single nucleotide polymorphisms of the IL-8 (-A251T) gene were screened in 300 patients with oral cancer and 300 healthy controls, by polymerase chain reaction-restriction fragment length polymorphism. Genotype and allele frequencies were evaluated by chi-square test and odds ratio (OR) with 95% confidence intervals (CIs) were used to evaluate the strength of associations. The results of the study demonstrated that IL-8 (-A251T) gene polymorphism was significantly associated with susceptibility of oral cancer, whereas its correlation with clinico-pathological status or pain due to oral cancer could not be established. The AT heterozygous (OR 5.31; CI 3.38-8.34; p 0.0001) and AA homozygous (OR 2.89; CI 1.76-4.75; p 0.0001) had a greater risk for oral cancer compared to TT homozygous. Furthermore, significantly increased values of A allele frequencies compared to T allele were observed in all patients (OR 1.56; CI 1.24-1.96; p 0.0002). Tobacco chewing and smoking were also found to influence the development of oral cancer and increased the incidence of pain in oral cancer patients. The findings of this study suggest that the IL-8 (-A251T) gene polymorphism may be associated with increased risk of oral cancer.

Topics: Adult; Case-Control Studies; Female; Humans; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Pain; Polymorphism, Single Nucleotide

This study evaluated the discriminatory power of salivary transcriptomic and proteomic biomarkers in distinguishing oral squamous cell carcinoma cases from controls and potentially malignant oral disorders (PMOD).. A total of 180 samples (60 OSCC patients, 60 controls, and 60 PMOD patients) were used in the study. Seven transcriptomic markers (IL8, IL1β, SAT1, OAZ1, DUSP1, S100P, and H3F3A) were measured using qPCR, and two proteomic markers (IL8 and IL1β) were evaluated by ELISA.. Among 7 transcriptomic markers, transcript level of DUSP1 was significantly lower in OSCC patients than in controls and PMOD patients. Between the proteomic markers, the protein concentration of IL8 and IL1β was significantly higher in OSCC patients than controls and dysplasia patients. Univariate fractional polynomial (FP) models revealed that salivary IL8 protein (IL8p) has the highest AUC value between OSCC patients and controls (0.74) and between OSCC and PMOD patients (0.72). Applying a 2-marker FP model, salivary IL8p combined with IL1β gave the best AUC value for discrimination between OSCC patients and controls, as well as the IL8p combined with H3F3A mRNA, which gave the best AUC value for discrimination between OSCC and PMOD patients. Multivariate models analysis combining salivary analytes and risk factor exposure related to oral carcinogenesis formed the best combinatory variables for differentiation between OSCC versus PMOL (AUC = 0.80), OSCC versus controls (AUC = 0.87), and PMOD versus controls (AUC = 0.78).. The combination of transcriptomic and proteomic salivary markers is of great value for oral cancer detection and differentiation from PMOD patients and controls. Clin Cancer Res; 22(13); 3340-7. ©2016 AACR.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Dual Specificity Phosphatase 1; Early Detection of Cancer; Enzyme-Linked Immunosorbent Assay; Female; Head and Neck Neoplasms; Humans; Interleukin-1beta; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Proteomics; Risk Factors; Saliva; Squamous Cell Carcinoma of Head and Neck; Taiwan

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR-424-5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR-424-5p. The miR-424-5p-induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR-424-5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR-424-5p expression could be induced by the cytokine IL-8 primarily through enhancing STAT5 transcriptional activity rather than NF-κB signaling. Antagomir-mediated inactivation of miR-424-5p prevented the IL-8-induced cell migration and invasion, indicating that miR-424-5p is required for IL-8-induced cellular invasiveness. Taken together, these data indicate that STAT5-dependent expression of miR-424-5p plays an important role in mediating IL-8/STAT5/SOCS2 feedback loop, and scavenging miR-424-5p function using antagomir may have therapeutic potential for the treatment of OSCC.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; MicroRNAs; Mouth; Mouth Neoplasms; Neoplasm Invasiveness; Signal Transduction; STAT5 Transcription Factor; Suppressor of Cytokine Signaling Proteins

This study aimed to evaluate the prognostic implications of insulin-like growth factor-II mRNA binding protein-3 (IMP3) and podoplanin (PDPN) as therapeutic targets against oral squamous cell carcinoma (OSCC) with bone invasion.. We elucidated the correlation of IMP3 and PDPN expression with bone invasion in 160 OSCC tissue specimens, and assessed a mouse calvarium xenograft model using an IMP3- and PDPN-depleted OSCC cell line.. The retrospective analysis revealed that the expression of IMP3 and PDPN is significantly correlated with T stage, lymph node metastasis, and the overall survival of OSCC patients. In addition, the dual expression of IMP3 and PDPN but not the single expression of either IMP3 or PDPN was associated with bone invasion and the number of osteoclasts in patients with OSCC. In support of these findings, IMP3 or PDPN depletion inhibited the invasive capacity of OSCC cells in a three-dimensional culture system, tumorigenesis, and regional bone destruction in a xenograft mouse model. In addition, IMP3 or PDPN depletion inhibited the expression of interleukin (IL)-6 and IL-8 in OSCC cells, and decreased the expression of receptor activator of NF-κB ligand (RANKL) in xenograft tumor tissues of OSCC.. These results suggest that IMP3 and PDPN may have strong influence on the pathogenesis of OSCC, especially in bone invasion, and may serve as novel therapeutic targets with prognostic implications for bone-invasive OSCC.

Topics: Adult; Aged; Aged, 80 and over; Animals; Bone Neoplasms; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Female; Head and Neck Neoplasms; Heterografts; Humans; Interleukin-11; Interleukin-6; Interleukin-8; Lymphatic Metastasis; Male; Membrane Glycoproteins; Mice; Middle Aged; Mouth Neoplasms; Osteoclasts; Prognosis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Retrospective Studies; RNA-Binding Proteins; RNA, Messenger; Squamous Cell Carcinoma of Head and Neck

Recent studies indicate that chronic inflammation promotes the aggressiveness of cancers. However, the direct molecular mechanisms underlying a functional link between chronic periodontitis, the most common form of oral inflammatory diseases, and the malignancy of oral cancer remain unknown. To elucidate the role of chronic periodontitis in progression of oral cancer, we examined the effect of Porphyromonas gingivalis (P. gingivalis), a major pathogen that causes chronic periodontitis, on the invasiveness of oral squamous cell carcinoma (OSCC) cells, including SCC-25, OSC-20 and SAS cells. Exposures to P. gingivalis promoted the invasive ability of OSC-20 and SAS cells via the upregulation of matrix metalloproteinases (MMPs), specifically MMP-1 and MMP-2. However, P. gingivalis-infected SCC-25 cells did not exhibit changes in their invasive properties or the low expression levels of MMPs. In an effort to delineate the molecular players that control the invasiveness, we first assessed the level of interleukin-8 (IL-8), a well-known inflammatory cytokine, in P. gingivalis-infected OSCC cells. IL-8 secretion was substantially increased in the OSC-20 and SAS cells, but not in the SCC-25 cells, following P. gingivalis infection. When IL-8 was directly applied to SCC-25 cells, their invasive ability and MMP level were significantly increased. Furthermore, the downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells attenuated their invasive potentials and MMP levels. Taken together, our findings strongly suggest that P. gingivalis infection plays an important role in the promotion of the invasive potential of OSCC cells via the upregulation of IL-8 and MMPs.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Humans; Interleukin-8; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Mouth Neoplasms; Neoplasm Invasiveness; Porphyromonas gingivalis; Real-Time Polymerase Chain Reaction; Up-Regulation

Late diagnosis, low therapeutic response, and metastasis are accountable for poor 5-year survival rate of OSCC. These failures are attributed to the existence of "cancer stem cell (CSC)" subpopulation. Hence, it is necessary to identify and understand the mechanism of CSCs in tumor development, metastasis, and chemotherapeutic response. Propelling evidences suggest that microRNA (miRNA)-mediated regulation and cytokines of tumor microenvironment have the ability to modulate CSC signalling pathway; however, their exact mechanism needs to be elucidated. Thus, in this study, we characterized CSC markers and highlighted the miRNA dynamics and cytokine profile regulating these CSCs in a pathway-dependent manner. Our results demonstrated CD44+ subpopulation as tumor-initiating cells with self-renewal capability, tumorigenic growth potential and intrinsic chemoresistance. These tumors exhibited increased expression of CSC markers (CD44v3, CD44v6, Nanog, and Bmi1) and significantly reduced expression of PTEN and ATM in OSCC patients. Pathway analysis of these CSC markers demonstrated a prospective pathway regulated by miRNA and cytokine network. On analyzing these modulators, we observed decreased expression of miRNA542-3p, miRNA34a and miRNA9, and significant upregulation of miRNA21, thus forming an unexplored axis. Cytokine profiling revealed significantly increased levels of IL-6 and IL-8 compared to normals and demonstrated their strong association with CD44v6. Collectively, this study indicates that miR5423p and miR34a targets the CD44v6-Nanog-PTEN axis, thus playing a vital role in regulating the CSC properties. Furthermore, we speculate an impinging role of cytokines IL-6 and IL-8 in regulating this CSC-mediated pathway which can have prognostic and therapeutic implications.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Humans; Hyaluronan Receptors; Interleukin-6; Interleukin-8; MicroRNAs; Mouth Neoplasms; Nanog Homeobox Protein; Neoplastic Stem Cells; PTEN Phosphohydrolase; Spheroids, Cellular; Tumor Cells, Cultured; Tumor Microenvironment

Silicon nanowire (SiNW) field-effect transistor (FET) biosensors have already been used as powerful sensors for the direct detection of disease-related biomarkers. However, the multiplexed detection of biomarkers in real samples is still challenging. Interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) are two typical biomarkers of oral squamous cell carcinoma (OSCC). In this study, we developed a multiplexed detection methodology for IL-8 and TNF-α detection in saliva using SiNW FET biosensors. We fabricated the SiNW FET sensors using a top-down lithography fabrication technique. Subsequently, we achieved the multiplexed detection of two biomarkers in saliva by specific recognition of the two biomarkers with their corresponding antibodies, which were modified on the SiNW. The established method was found to have a limit of detection as low as 10 fg/mL in 1 × PBS as well as 100 fg/mL in artificial saliva. Because of its advantages, including label-free and multiplexed detection, non-invasive analysis, highly sensitive and specific determination, the proposed method is expected to be widely used for the early diagnosis of OSCC.

Topics: Animals; Biomarkers, Tumor; Biosensing Techniques; Buffers; Carcinoma, Squamous Cell; Early Detection of Cancer; Humans; Interleukin-8; Mouth Neoplasms; Nanowires; Saliva; Silicon; Transistors, Electronic; Tumor Necrosis Factor-alpha

Surgical-site infection (SSI) after cervical neck dissection (CND) for head and neck squamous cell carcinoma (HNSCC) increases morbidity and delays adjuvant treatment. This study assessed changes in cytokines levels in postsurgical drainage fluid after CND and examined their predictive value for the early diagnosis of SSI.. An observational prospective pilot study was conducted in 39 consecutively recruited patients with HNSCC undergoing CND who were treated at the authors' service within the past 2 years. Patients met the following inclusion criteria: no previous chemotherapy or radiotherapy, closed-suction drainage, 30-day follow-up, prophylactic treatment with amoxicillin plus clavulanic acid and dexamethasone, no chronic inflammatory disease, and no previous neck surgery. Drainage samples were collected at postoperative days +1 and +3. Sample size was estimated based on SSI incidence after HNSCC surgery (∼15%; α risk, 0.05; β risk, 0.2; 2-sided test). Interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) levels were measured. Patients were followed to detect SSI. Sensitivity, specificity, and prognostic values were calculated for each cytokine at days +1 and +3 to diagnose SSI.. SSI was diagnosed in 6 of 39 patients. Bilateral CND, tracheostomy, surgery duration longer than 7 hours, HNSCC stage T3 or T4, and reconstruction with pedicled flaps versus microvascular flaps for advanced-stage tumors were considered risk factors for SSI. All cytokines except IL-10 showed statistical differences between patients with SSI and those without SSI. The best receiver operating characteristic curves yielded cutoff values at day +1 (TNF-α >14.5 pg/mL; sensitivity, 100%; specificity, 87.88%) and day +3 (IL-1β >115 pg/mL; sensitivity, 83.33%; specificity, 78.79%). Also, IL-2 levels higher than 6.5 pg/mL at day +1 (sensitivity, 83.33%; specificity, 69.7%) and day +3 (sensitivity, 100%; specificity, 69.7%) and IL-6 levels higher than 3,300 pg/mL at day +3 (sensitivity, 100%; specificity, 60.61%) yielded adequate diagnostic profitability.. The results of this study suggest that the assessment of cytokine levels in drainage fluid soon after CND could provide a novel method for the early detection of SSI.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Drainage; Exudates and Transudates; Female; Follow-Up Studies; Forecasting; Humans; Interleukin-1beta; Interleukin-2; Interleukin-6; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Neck Dissection; Neoplasm Staging; Operative Time; Pilot Projects; Prospective Studies; Risk Factors; Sensitivity and Specificity; Surgical Flaps; Surgical Wound Infection; Tracheostomy; Tumor Necrosis Factor-alpha

Aberrant activity of transcription factors in oral squamous cell carcinoma (OSCC) results in the spontaneous secretion of various cytokines and chemokines. Among them, IL-8, owing to its angiogenic activity, promotes the growth of OSCCs. In the present study, we examined the role of IL-8 secreted by OSCCs, on the angiogenic activity of monocytic THP1 cells. Culture supernatant (Ca-sup) augmented IL-8 secretion by THP1 cells, which was found to be significantly reduced following the removal Ca9-22-derived IL-8 from the Ca-sup. IL-8 induction was regulated at the transcriptional level, because real-time PCR demonstrated the augmented IL-8 messenger RNA (mRNA) expression. We further performed the luciferase assay using the 5'-untranslated region of IL-8 gene. Contradictory to our speculations, luciferase activity was not augmented by Ca-sup stimulation. NF-κB-independent IL-8 induction was further confirmed by pre-treating THP1 cells with NF-κB-specific inhibitors. To elucidate the signaling pathway, THP1 was pre-treated with MEK inhibitors. The results demonstrated that pre-treatment of cells with MEK inhibitor drastically reduced IL-8 levels, suggesting the role of MEK. Moreover, Ca-sup was found to increase ERK1/2 phosphorylation in a time-dependent manner. These results indicated that OSCC-derived IL-8 appears to activate angiogenic activity in monocytes within the tumor microenvironment via the mitogen-activated protein kinase (MAPK) pathway.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Macrophages; Mitogen-Activated Protein Kinases; Monocytes; Mouth Neoplasms; NF-kappa B; RNA, Messenger; Signal Transduction

Neutrophils have recently been shown to promote invasion and correlate with a poor prognosis in different cancers, including head and neck squamous cell carcinomas. In this study, we analyze the effects of neutrophils in the invasion of oral squamous cell carcinoma (OSCC) using a combination of conditioned media, direct and indirect coculture of human peripheral blood neutrophils, and UMSCC47 cells (OSCC cell line). Invasion and matrix degradation were determined using a modified in vitro invasion assay and an invadopodia assay, respectively. UMSCC47 and neutrophil cocultures or conditioned media from cocultures increased UMSCC47 invasion, invadopodia formation, and matrix degradation. Further analysis revealed an increase in TNFα and IL8 in supernatants of cocultures compared with neutrophil or UMSCC47 cultures alone and that inhibition of TNFα and IL8 significantly decreased OSCC invasion. Our results show that neutrophils increase the invasiveness of OSCC through the activation of invadopodia and matrix degradation, suggesting a paracrine activation loop between the two cells. Importantly, the presence of neutrophils in the oral environment may modulate the clinical behavior of OSCC.

Topics: Carcinoma, Squamous Cell; Coculture Techniques; Culture Media, Conditioned; Extracellular Matrix; Humans; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Neutrophils; Podosomes; Prognosis; Signal Transduction; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Periodontitis is the most common chronic inflammatory condition occurring in the human oral cavity, but our knowledge on its contribution to oral cancer is rather limited. To define crosstalk between chronic periodontitis and oral cancer, we investigated whether Porphyromonas gingivalis, a major pathogen of chronic periodontitis, plays a role in oral cancer progression. To mimic chronic irritation by P. gingivalis in the oral cavity, oral squamous cell carcinoma (OSCC) cells were infected with P. gingivalis twice a week for 5 weeks. Repeated infection of oral cancer cells by P. gingivalis resulted in morphological changes of host cancer cells into an elongated shape, along with the decreased expression of epithelial cell markers, suggesting acquisition of an epithelial-to-mesenchymal transition (EMT) phenotype. The prolonged exposure to P. gingivalis also promoted migratory and invasive properties of OSCC cells and provided resistance against a chemotherapeutic agent, all of which are described as cellular characteristics undergoing EMT. Importantly, long-term infection by P. gingivalis induced an increase in the expression level of CD44 and CD133, well-known cancer stem cell markers, and promoted the tumorigenic properties of infected cancer cells compared to non-infected controls. Furthermore, increased invasiveness of P. gingivalis-infected OSCC cells was correlated with enhanced production of matrix metalloproteinase (MMP)-1 and MMP-10 that was stimulated by interleukin-8 (IL-8) release. This is the first report demonstrating that P. gingivalis can increase the aggressiveness of oral cancer cells via epithelial-mesenchymal transition-like changes and the acquisition of stemness, implicating P. gingivalis as a potential bacterial risk modifier.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Matrix Metalloproteinase 1; Mouth Neoplasms; Neoplastic Stem Cells; Periodontitis; Porphyromonas gingivalis

Long non-coding RNAs (lncRNAs) have recently attracted more attention about the role in a broad range of biological processes and complex cancers. We aimed to identify differentially expressed lncRNAs that play an important role in the pathogenesis of oral squamous cell carcinoma (OSCC). Microarray data GSE25099 consisting of 57 samples from patients with OSCC and 22 normal samples were downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) and lncRNAs were identified between OSCC samples and control using samr package in R and noncoder software. Co-expression network was constructed for lncRNAs and candidate target DEGs, followed by functional and pathway enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery online tool. OSCC-related genes were screened by Genetic-Association-DB-Database analysis, and then protein-protein interaction (PPI) network construction of OSCC-related and co-expressed genes. Bioinformatic analysis revealed that there were 998 DEGs and 160 differentially expressed lncRNAs between OSCC and normal control. We found LOC100130547, FTH1P3, PDIA3F and GTF2IRD2P1 targeted most of DEGs. Predicted targets-related functional annotation showed significant changes in inflammation-related functions and Toll-like receptor signaling pathway. By further conducting PPI network with lncRNA co-expressed DEGs, we found that OSCC-associated genes including MMP1 (matrix metallopeptidase), MMP3, MMP9, PLAU (plasminogen activator, urokinase) and IL8 (interleukin 8) were targeted by FTH1P3, PDIA3F and GTF2IRD2P1. Our results indicate that lncRNAs FTH1P3, PDIA3F and GTF2IRD2P1 may responsible for progression and metastasis of OSCC via targeting MMP1, MMP3, MMP9, PLAU and IL8 which are key regulators of tumorigenesis.

Topics: Carcinoma, Squamous Cell; Computational Biology; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Head and Neck Neoplasms; Humans; Interleukin-8; Mouth Neoplasms; Oligonucleotide Array Sequence Analysis; RNA, Long Noncoding; RNA, Messenger; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Up-Regulation

Previous research has indicated that salivary interleukin (IL)-6 and IL-8 are potential biomarkers for oral squamous cell carcinoma (OSCC). However, their levels have been found to be significantly elevated in patients with chronic periodontitis (CP) or oral lichen planus (OLP). The data also showed wide variations in levels among the different studies, and no standardization procedure was ever performed. Therefore, the objective of this study is to determine whether CP or OLP confounds the use of IL-6 or IL-8 for OSCC detection.. Saliva samples were collected from five groups: OSCC before treatment (n = 18); CP (n = 21); disease-active OLP (n = 21); disease-inactive OLP (n = 20); and healthy controls (n = 21). IL-6 and IL-8 concentrations (determined by enzyme-linked immunosorbent assays) were compared, using total salivary protein-standardized levels to validate the data. The Kruskal-Wallis test (α = 0.05) followed by pairwise Mann-Whitney U (post hoc) tests with Bonferroni adjustments (α = 0.00625) were used for statistical analysis.. Salivary IL-6 levels were significantly higher in patients with OSCC than in patients with CP (P <0.001), disease-active OLP (P = 0.001), disease-inactive OLP (P <0.001), and healthy controls (P <0.001). Salivary IL-8 levels were significantly higher in patients with OSCC than in patients with CP (P <0.001), but only marginally significantly higher than in healthy controls (P = 0.014). Statistical results of standardized IL-6 and IL-8 levels were consistent with the non-standardized levels in all pairs except one.. Salivary IL-6 may be a useful biomarker in the detection of OSCC, unconfounded by CP or OLP.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Biomarkers, Tumor; Carcinoma, Squamous Cell; Chronic Periodontitis; Female; Humans; Interleukin-6; Interleukin-8; Lichen Planus, Oral; Male; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Saliva; Salivary Proteins and Peptides

To evaluate the clinical utility of salivary interleukin 8 (IL-8) in the differential diagnosis of potentially malignant lesions (PMLs) and oral squamous cell carcinoma (OSCC) in a region with high oral cancer prevalence.. Saliva and blood samples were collected from 100 participants in each group (OSCC, PMLs, and healthy controls). Serum and salivary IL-8 levels were measured by enzyme-linked immunosorbent assay. The data were subjected to appropriate statistical analysis.. A significant increase in levels of serum and salivary IL-8 was found in OSCC compared with PMLs and healthy controls. Receiver operating characteristic curve analysis found salivary IL-8 to have superior sensitivity in detecting OSCC. A significant increase in IL-8 levels based on the histologic grading of OSCC was also observed.. This study confirms that salivary IL-8 can be a potent marker that can be used as a tool in the differential diagnosis of PMLs and OSCC.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Precancerous Conditions; Risk Factors; Saliva; Sensitivity and Specificity

Chemokines regulate physiological and pathological leucocyte trafficking, and chemokine receptors play a role in tumorigenesis. Expression of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 has been shown in oral squamous cell carcinoma (OSCC) but remains poorly characterised. This aim of this study was to investigate CXCR1 and CXCR2 expression on normal oral keratinocytes (NOKs) and oral cancer cell lines (OCCL) and their relative response when exposed to IL-8 and growth-related oncogene-α (which selectively binds CXCR2).. mRNA and protein expression was studied using RT-PCR, immunocytochemistry and flow cytometry. ELISAs were used to investigate ERK1/2 phosphorylation and MMP production, whereas a MTS-based assay was employed to study proliferation. Migration assays were carried out using modified Boyden chambers with a matrigel coating used for invasion assays.. mRNA expression of CXCR1 and CXCR2 was seen in both NOKs and OCCL with significantly higher protein expression in OCCL. Exposure to IL-8 and GROα increased intracellular ERK phosphorylation, proliferation, migration and invasion with OCCL showing a greater response than NOKs. These effects were mediated through CXCR1 and CXCR2 (for IL-8) and CXCR2 (for GROα) as receptor-blocking antibodies significantly inhibited the responses. IL-8 and GROα also increased MMP-9 release from NOKs and OCCL with significantly higher amounts released by OCCL. However, an increase in MMP-7 production was only seen in OCCL.. Functional CXCR1 and CXCR2 exist on normal and cancerous oral epithelial cells, and our data suggests a role for these receptors in oral cancer biology.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL1; Fibroblasts; Gingiva; Humans; Interleukin-8; Keratinocytes; Matrix Metalloproteinase 2; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; Neoplasm Invasiveness; Phosphorylation; Receptors, Interleukin-8A; Receptors, Interleukin-8B

We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC).. Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens.. We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10.. These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.

Topics: Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Carcinoma, Squamous Cell; Disease-Free Survival; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-10; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Multivariate Analysis; Prognosis; Receptors, Cell Surface; Treatment Outcome

Oral squamous cell carcinoma (OSCC) ranks among the top ten most prevalent cancers worldwide. Like most head and neck squamous cell carcinomas (HNSCCs), OSCC is highly inflammatory and aggressive. However, the signaling pathways triggering the activation of its inflammatory processes remain elusive. G protein-coupled receptor signaling regulates the inflammatory response and invasiveness of cancers, but it remains unclear whether Gα12 is a critical player in the inflammatory cytokine pathway during the tumorigenesis of OSCC. This study was undertaken to determine the role of Gα12 signaling in the regulation of proinflammatory cytokines in their mediation of OSCC invasion. We found that both the transcription and protein levels of Gα12 are up-regulated in OSCC tumors. The elevated Gα12 expressions in OSCC patients also correlated with extra-capsular spread, an indicator of tumor invasiveness in HNSCCs. This clinical finding was supported by the studies of overexpression and RNAi knockdown of Gα12 in OSCC cells, which demonstrated that Gα12 promoted tumor cell migration and invasion. To understand how Gα12 modulates OSCC invasiveness, we analyzed key biological processes in microarray data upon depletion of Gα12 and found that cytokine- and other immune-related pathways were severely impaired. Importantly, the mRNA levels of IL-6 and IL-8 proinflammatory cytokines in clinical samples were found to be significantly correlated with the increased Gα12 levels, suggesting a potential role of Gα12 in modulating the IL-6 and IL-8 expressions. Supporting this hypothesis, overexpression or RNAi knockdown of Gα12 in OSCC cell lines both showed that Gα12 positively regulated the mRNA and protein levels of IL-6 and IL-8. Finally, we demonstrated that the Gα12 promotion of tumor cell invasiveness was suppressed by the neutralization of IL-6 and IL-8 in OSCC cells. Together, these findings suggest that Gα12 drives OSCC invasion through the up-regulation of IL-6 and IL-8 cytokines.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; GTP-Binding Protein alpha Subunits, G12-G13; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Mouth Neoplasms; Neoplasm Invasiveness; Up-Regulation

The spontaneous IL-8 secretion observed in OSCC is partially dependent on the disregulated activity of transcription factor NF-κB. Nickel compounds are well established human carcinogens, however, little is known about the influence of nickel on the spontaneous secretion of IL-8 in oral squamous cell carcinoma (OSCC) cells. The aim of the present study was to investigate whether Ni(2+) ions can influence on IL-8 secretion by OSCC.. The IL-8 secretion was measured by ELISA. The expression of IL-8 mRNA was examined by real-time PCR. The NF-κB activity was measured by luciferase assay. The phosphorylation status and nuclear localization of NF-κB subunits were examined by Western blotting or Transfactor kit and immunofluorescence staining, respectively. The interaction of NF-κB p50 subunit and Ni(2+) ions was examined by Ni(2+)-column pull down assay. The site-directed mutagenesis was used to generate a series of p50 mutants. Scratch motility assay was used to monitor the cell mobility. Our results demonstrated that, on the contrary to our expectations, Ni(2+) ions inhibited the spontaneous secretion of IL-8. As IL-8 reduction was observed in a transcriptional level, we performed the luciferase assay and the data indicated that Ni(2+) ions reduced the NF-κB activity. Measurement of p50 subunit in the nucleus and the immunofluorescence staining revealed that the inhibitory effect of Ni(2+) ions was attributed to the prevention of p50 subunit accumulation to the nucleus. By Ni(2+)-column pull down assay, Ni(2+) ions were shown to interact directly with His cluster in the N-terminus of p50 subunit. The inhibitory effect of Ni(2+) ions was reverted in the transfectant expressing the His cluster-deleted p50 mutant. Moreover, Ni(2+) ions inhibited the OSCC mobility in a dose dependent fashion.. Taken together, inhibition of NF-κB activity by Ni(2+) ion might be a novel therapeutic strategy for the treatment of oral cancer.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Enzyme Activation; Gene Expression; Heavy Ions; Humans; Interleukin-8; Mouth Neoplasms; NF-kappa B; NF-kappa B p50 Subunit; Nickel; Protein Binding; Protein Transport; Toll-Like Receptor 4

Interleukin 8 (IL-8) is a pro-angiogenic, pro-inflammatory mediator that belongs to the family of chemokines. Due to its pro-angiogenic characteristic, it may play a vital role in tumour angiogenesis and progression.. This study was designed to estimate the levels of salivary IL-8 in oral precancer and oral squamous cell carcinoma (OSCC) patients and compare them with healthy controls. The aim was to evaluate its efficacy as a potential biomarker for these diseases.. Each group comprised 25 individuals. The salivary IL-8 levels were determined by enzyme-linked immunosorbent assay.. The levels of salivary IL-8 were found to be significantly elevated in patients with OSCC as compared to the precancer group (p < 0.0001) and healthy controls (p < 0.0001). However, the difference in salivary IL-8 concentrations among the precancer group and controls was statistically non-significant (p = 0.738).. Our results suggested that salivary IL-8 can be utilised as a potential biomarker for OSCC. Salivary IL-8 was found to be non-conclusive for oral premalignancy in this preliminary study. Hence, its possible role in transition from premalignancy to malignancy needs further research with larger sample sizes.. Saliva as a diagnostic biofluid offers a number of advantages over blood-based testing. The role of IL-8 in oral cancer if validated further by future research can provide an easy diagnostic test as well as a prognostic indicator for patients undergoing treatment. Therefore, if it's role in tumourigenesis can be sufficiently assessed, it could open up new avenues to find out novel treatment modalities for oral cancer.

Topics: Adolescent; Adult; Aged; Angiogenesis Inducing Agents; Areca; Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Female; Humans; Inflammation Mediators; Interleukin-8; Leukoplakia, Oral; Male; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Oral Submucous Fibrosis; Precancerous Conditions; Saliva; Salivary Proteins and Peptides; Smoking; Tobacco Use; Young Adult

Drosomycin-like defensin (DLD) is a recently discovered antimicrobial peptide mainly active against filamentous fungi. The present study investigated the expression profile of DLD in oral epithelium and oral squamous cell carcinoma (SCC) cell lines.. Tissue sections of human oral mucosa, keratinocytes derived from oral mucosa (NOK) and eight kinds of SCC cell lines were used. In situ hybridization was performed on tissue sections of oral mucosa. Expression levels of DLD in the cells were observed by reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR assays. The cells were treated with IL-1β, IL-8 and TNF-α, and agonists for TLR2, TLR4 and β-glucan. DLD expression in cells was increased and decreased by the DLD gene and its siRNA transfection, respectively. The proliferation rates were assessed by cell counting.. By means of in situ hybridization, DLD mRNA positive staining was detected in the epithelial layer of the oral mucosa. An RT-PCR assay confirmed the expression of DLD mRNA in keratinocytes derived from oral epithelium. Expression of DLD in two out of eight cell lines was significantly lower than in NOK cells. The expression levels of DLD mRNA were not significantly changed in the cells stimulated with any cytokines or agonists. The cell proliferation rate where there was decreased expression of DLD was significantly lower than in the control.. DLD may be partially involved in the defence against filamentous fungal infection in the oral mucosa, and may also serve other functions, such as contribution to cell growth.

Topics: Antifungal Agents; beta-Glucans; Carcinoma, Squamous Cell; Cell Count; Cell Line; Cell Line, Tumor; Cell Proliferation; Defensins; Drosophila Proteins; Epithelium; Humans; In Situ Hybridization; Interleukin-1beta; Interleukin-8; Keratinocytes; Mouth Mucosa; Mouth Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Structural Homology, Protein; Toll-Like Receptor 2; Toll-Like Receptor 4; Transfection; Tumor Necrosis Factor-alpha

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Proliferation; Female; Humans; I-kappa B Proteins; Interleukin-6; Interleukin-8; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; Poly(ADP-ribose) Polymerases; Pyrazines; Radiation-Sensitizing Agents; Transcription Factor RelA; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Lysophosphatidic acid (LPA) is a bioactive lipid with a growth factor-like activity on a large range of cell types. Several pieces of evidence raise the possibility that LPA may play an important role in bone metastasis. Bone is a frequent metastatic site for oral cancer. However, the role of LPA in the progression of oral cancer metastasis to the bone is poorly understood. Here, we provide evidence for the role of LPA in the progression of oral cancer bone metastases and its regulatory mechanism. LPA induced the secretion of IL-6 and IL-8 in oral squamous cell carcinoma (OSCC). LPA-stimulated secretion of IL-6 and IL-8 is partly dependent on the LPA and EGF receptor (EGFR) pathways. ERK1/2 and Akt-mediated NF-κB and AP-1 were responsible for the LPA-induced IL-6 and IL-8 secretion. Moreover, conditioned medium (CM) derived from the LPA-stimulated OSCC supported osteoclast formation in bone marrow-derived macrophages (BMMs). Neutralization against both human IL-6 and IL-8 suppressed osteoclast formation induced by CM derived from the LPA-stimulated OSCC. Direct treatment with recombinant IL-6 (rIL-6) and/or soluble IL-6 receptor (sIL-6R), or IL-8 (rIL-8) reproduced the effect of the CM derived from the LPA-stimulated OSCC on osteoclast formation. In addition, CM derived from the LPA-stimulated OSCC induced receptor activator of nuclear factor (NF)-κB ligand (RANKL) expression in human osteoblasts and direct treatment with rIL-6 and/or sIL-6R or rIL-8 mimicked the effect of the CM derived from the LPA-stimulated OSCC for RANKL expression. Taken together, LPA may be a potent inducer of osteolytic factor IL-6 and IL-8 in OSCC. LPA-induced IL-6 and IL-8 exerted propound effects on RANKL expression in osteoblast and thereby promoted osteoclast formation from osteoclast precursors.

Topics: Animals; Biological Assay; Blotting, Western; Bone Neoplasms; Bone Resorption; Carcinoma, Squamous Cell; Humans; Interleukin-6; Interleukin-8; Lysophospholipids; Mice; Mouth Neoplasms; Osteoclasts; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction

Interleukin-8 (IL-8), which is an angiogenic chemokine with a high expression level in tumor tissues, plays important roles in developing many human malignancies including oral squamous cell carcinoma (OSCC). This study was designed to examine the association of IL-8 gene polymorphisms with the susceptibility and clinicopathological characteristics of OSCC.. A total of 270 patients with OSCC and 350 healthy control subjects were recruited. Four single nucleotide polymorphisms (SNPs) of IL-8 genes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping analysis.. Results showed that four IL-8 SNPs (-251 T/A, +781 C/T, +1633 C/T, and +2767 A/T) were not associated with oral cancer susceptibility as well as clinicopathological parameters. But among 345 smokers, IL-8 polymorphisms carriers with betel quid chewing were found to have a 17.41- to 23.14-fold risk to have oral cancer compared to IL-8 wild-type carriers without betel quid chewing. Among 262 betel quid chewers, IL-8 polymorphisms carriers with smoking have a 10.54- to 20.44-fold risk to have oral cancer compared to those who carried wild type without smoking.. Our results suggest that the combination of IL-8 gene polymorphisms and environmental carcinogens might be highly related to the risk of oral cancer.

Topics: 3' Untranslated Regions; Adenine; Areca; Carcinogens; Carcinoma, Squamous Cell; Case-Control Studies; Cytosine; Female; Gene-Environment Interaction; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Introns; Lymphatic Metastasis; Male; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Neovascularization, Pathologic; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Risk Factors; Smoking; Thymine

Oral cancer is the sixth most common cancer with a 5-year survival rate of approximately 60%. Presently, there are no scientifically credible early detection techniques beyond conventional clinical oral examination. The goal of this study is to validate whether the seven mRNAs and three proteins previously reported as biomarkers are capable of discriminating patients with oral squamous cell carcinomas (OSCC) from healthy subjects in independent cohorts and by a National Cancer Institute (NCI)-Early Detection Research Network (EDRN)-Biomarker Reference Laboratory (BRL).. Three hundred and ninety-five subjects from five independent cohorts based on case controlled design were investigated by two independent laboratories, University of California, Los Angeles (Los Angeles, CA) discovery laboratory and NCI-EDRN-BRL.. Expression of all seven mRNA and three protein markers was increased in OSCC versus controls in all five cohorts. With respect to individual marker performance across the five cohorts, the increase in interleukin (IL)-8 and subcutaneous adipose tissue (SAT) was statistically significant and they remained top performers across different cohorts in terms of sensitivity and specificity. A previously identified multiple marker model showed an area under the receiver operating characteristic (ROC) curve for prediction of OSCC status ranging from 0.74 to 0.86 across the cohorts.. The validation of these biomarkers showed their feasibility in the discrimination of OSCCs from healthy controls. Established assay technologies are robust enough to perform independently. Individual cutoff values for each of these markers and for the combined predictive model need to be further defined in large clinical studies.. Salivary proteomic and transcriptomic biomarkers can discriminate oral cancer from control subjects.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Feasibility Studies; Female; Follow-Up Studies; Genotype; Humans; Interleukin-8; Los Angeles; Male; Middle Aged; Mouth Neoplasms; Neoplasm Proteins; Prognosis; Real-Time Polymerase Chain Reaction; Retrospective Studies; RNA, Messenger; Saliva

Toll-like receptor 4 (TLR4) is expressed on immune cells as a sensor that recognizes lipopolysaccharide (LPS), a microbial conserved component. It has recently been determined that the expression of TLR4 is also found in various types of tumor cells. Cisplatin is a widely used chemotherapeutic agent for oral squamous cell carcinoma (OSCC) treatment. However, the mechanisms responsible for cisplatin resistance are not well understood.. The present study was designed to elucidate the role of TLR4 expression in human OSCC regarding immune escape and apoptotic resistance to cisplatin. TLR4 and the myeloid differentiation primary response gene 88 (MyD88) were highly expressed in OSCC cell lines. Upon LPS stimulation both NF-κB and p38 MAPK pathways were activated in OSCC cell lines, followed by the production of large quantities of IL-6, IL-8 and VEGF compared with human immortalized oral epithelia cells (HIOECs). OSCC cell lines were found to be resistant to cisplatin-mediated apoptosis after pretreatment with LPS.. Our results suggested that TLR4 was functionally expressed in human OSCC cells and development of resistance to cisplatin in human OSCC might occur through the mechanism involving TLR4 and its signaling pathway. Suppression of TLR4 and its signaling pathway might thus elevate sensitivity to cisplatin and potentially help improve the prognosis of patients with OSCC.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mouth Neoplasms; Myeloid Differentiation Factor 88; NF-kappa B; p38 Mitogen-Activated Protein Kinases; RNA Interference; Signal Transduction; Toll-Like Receptor 4; Tumor Escape; Vascular Endothelial Growth Factor A

To explore the clinical significance of serum interleukin-8 (IL-8) level in patients with oral squamous cell carcinoma (OSCC).. Twenty-seven serum specimens pathologically confirmed as OSCC were tested,10 healthy serum specimens were used as control. The expression of serum IL-8 was measured by ELISA. Data was presented as mean ± standard error. Statistical analysis was performed by SPSS 13.0 software package and two-tailed independent sample t test was used to determine the difference between the two groups.. The level of serum IL-8 in OSCC patients was significantly higher than that in the control (P < 0.01). The high expression of IL-8 correlated with clinical pathologic stage (P < 0.01) and lymph node metastasis (P < 0.05).. The expression of serum IL-8 correlates significantly with the biological behavior of OSCC, and it can be used as a prognostic molecular marker for OSCC. Supported by the Third Session of Excellent Youth Foundation of Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Humans; Interleukin-8; Lymphatic Metastasis; Mouth Neoplasms; Prognosis

Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression.. We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines.. More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia.. Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.

Topics: Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Cell Nucleus; Decorin; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Interleukin-8; Male; Mouth Neoplasms; Neoplasm Invasiveness; Precancerous Conditions; RNA, Small Interfering

Experimental data and limited patient experience suggest that rhBMP-2 can be used to regenerate bone in acquired segmental defects of the mandible. Most of these defects are caused by resection of oral squamous cell carcinoma (OSCC) and the biologic effects of rhBMP-2 on these carcinoma cells are unknown. The objective of this study was to determine whether rhBMP-2 produces adverse effects on proliferation and angiogenesis in OSCC, two biologic processes critical to tumor formation. In vitro studies included treating OSCC cells with rhBMP-2 or an adenoviral vector containing the cDNA for BMP-2. In vivo studies involved co-transplantation of OSCC cells with bone marrow stromal cells genetically modified to over express BMP-2, to mimic a clinically relevant scenario for regenerating bone using cell-based therapy in a wound containing microscopic residual disease. Proliferation, as measured by a MTT assay in vitro and tumor growth in vivo was not affected by treatment with BMP-2. Angiogenesis, measured by secretion of the proangiogenic molecules VEGF and IL-8 in vitro and microvessel density in vivo, was not affected. Exposure of OSCC cells to BMP-2 does not stimulate proliferation or angiogenesis. Further studies are needed before using rhBMP-2 for bone tissue engineering in oral cancer-related defects.

Topics: Adenoviridae; Animals; Bone Marrow Transplantation; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA, Complementary; Female; Genetic Vectors; Humans; Interleukin-8; Mice; Mice, Mutant Strains; Mice, Nude; Microvessels; Mouth Neoplasms; Neoplasm Transplantation; Neovascularization, Pathologic; Recombinant Proteins; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; von Willebrand Factor

Toll-like receptors (TLRs) signaling has been found to promote cell proliferation, invasiveness, and angiogenesis in a variety of cancers. This study was performed to examine whether TLR signaling is involved in tumor progression of an oral squamous cell carcinoma, YD-10B cells.. TLRs expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) in YD-10B cells. Interleukin (IL)-6 and IL-8 production by YD-10B cells in response to various TLR agonists was examined by ELISA. Cell viability and proliferation was determined by colorimetric MTT and Bromodeoxyuridine (BrdU) assay. The effect of TLR agonists on invasiveness was determined by migration and invasion assay using commercial kits. mRNA expression of vascular endothelial growth factor (VEGF) was also evaluated by RT-PCR.. All tested TLRs including TLR2, 3, 4, 5, 7, and 9 were expressed in YD-10B cells. IL-6 and IL-8 production was increased by Pam(3) CSK(4) , flagellin, Poly I:C, and imiquimod, but not lipopolysaccharide (LPS). Porphyromonas gingivalis LPS (Pg LPS) also led to increase of IL-8 production. However, Pam(3) CSK(4,) flagellin, and Pg LPS did not affect cell proliferation, migration, invasion, and gene expression of VEGF in YD-10B cells.. These findings indicated that TLR activation by bacterial molecules may not affect tumor progression of YD-10B cells.

Topics: Bacterial Proteins; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Humans; Interleukin-6; Interleukin-8; Mouth Neoplasms; Neoplasm Invasiveness; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 5; Toll-Like Receptors; Tumor Cells, Cultured

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-gamma were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFkappaB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-gamma. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.

Topics: Animals; Blotting, Western; Cell Differentiation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Humans; Interferon-gamma; Interleukin-2; Interleukin-6; Interleukin-8; Killer Cells, Natural; Mouth Neoplasms; Neoplasms, Squamous Cell; Neoplastic Stem Cells; Stem Cells

Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco, and pathogenic agents are known risk factors and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-κB activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis, and growth regulation. In particular IL-8, CCL5, STAT1, and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines as compared with normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells, indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-κB pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-κB pathway shows promise in OSCCs.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Aspirin; Biomarkers; Boronic Acids; Bortezomib; Carcinoma, Squamous Cell; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Profiling; Humans; Inflammation; Interleukin-8; Microarray Analysis; Mouth Neoplasms; NF-kappa B; Pyrazines; RNA, Small Interfering

Cepharanthine is a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata, which is widely used for the treatment of many acute and chronic diseases, and can exert antitumor effects on several human cancer cell lines. However, little is known about the detailed mechanisms of the antitumor activity of Cepharanthine. In the present study, we determined whether Cepharanthine could suppress angiogenesis and growth of human oral squamous cell carcinoma (OSCC) cells in vitro and in vivo. Cepharanthine significantly inhibited expression of two major pro-angiogenic molecules, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), in cultured cells and in cells implanted into the subcutaneous tissue of nude mice. Also, Cepharanthine inhibited the nuclear factor-kappaB (NF-kappaB) activity in human OSCC cells in vitro and in vivo. The decreased expression of VEGF and IL-8 correlated with decreased tumor cell growth and decreased vascularization in vitro and in vivo. These findings suggest that Cepharanthine can suppress angiogenesis and growth of OSCC cells by inhibiting expression of VEGF and IL-8 involved in the blockade of NF-kappaB activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Benzylisoquinolines; Blotting, Western; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Interleukin-8; Mice; Mice, Nude; Mouth Neoplasms; Neovascularization, Pathologic; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

We used suppression subtractive hybridization (SSH) and oligonucleotide microarray to differentiate expression profiles of metastasis-related genes and to evaluate their clinical significance in patients with invasive oral cancer (OCa). Overexpression of the specific genes was confirmed by reverse transcription-PCR (RT-PCR). Cells expressing the gene were identified by immunohistochemistry in pathology specimens. Clinical correlation and significance were analyzed statistically. Using these methods, we detected increased expressions of MMP-1, -3, -7, -9, -10 and interleukin (IL)-8 in invasive OCa. Moreover, our data showed that overexpressions of MMP-1, -3, -7, -10 and IL-8 were associated with reduced survival.

Topics: Adult; Aged; Cell Survival; Cluster Analysis; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-8; Kaplan-Meier Estimate; Male; Matrix Metalloproteinases, Secreted; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Treatment Outcome; Up-Regulation

For clinical applications of biomarkers, there is a need for multiplex assays using high throughput platforms. The objective of this study was to determine the efficacy of Luminex Multianalyte Profiling (xMAP) technology for measurement of salivary proteins and to evaluate whether multiplex assays are as effective as single-plex assays and enzyme-linked immunosorbent assay (ELISA).. The average levels of interleukin-8 (IL-8) from the single-plex assay were 3313.2 +/- 3759.8 pg ml(-1) [oral squamous cell carcinoma (OSCC), n = 20] and 1061.7 +/- 1978.8 pg ml(-1) (control, n = 20). The IL-1beta average levels from the single-plex assay were 945.2 +/- 1134.8 pg ml(-1) (OSCC, n = 20) and 314.2 +/- 444.8 pg ml(-1) (control, n = 20). The average levels of IL-8 from the multiplex assay were 2834.9 +/- 3385.6 pg ml(-1) (OSCC, n = 20) and 947.3 +/- 2036.8 pg ml(-1) (control, n = 20). The IL-1beta average levels from the multiplex assay were 1013.5 +/- 1221.1 pg ml(-1) (OSCC, n = 20) and 376.3 +/- 576.3 pg ml(-1) (control, n = 20). The correlation coefficient between Luminex and ELISA assay for IL-8 (n = 19) and IL-1beta (n = 19) was 0.91 and 0.84, respectively.. Luminex xMAP single-plex and multiplex assays are as effective as ELISA assays for quantification of proteins in saliva. Both IL-8 and IL-1beta were expressed at significantly higher levels in OSCC subjects than in the matched healthy control subjects.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Chromogenic Compounds; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunoassay; Interleukin-1beta; Interleukin-8; Middle Aged; Mouth Neoplasms; Neoplasm Proteins; Periodontitis; Protein Array Analysis; Saliva; Salivary Proteins and Peptides; Sensitivity and Specificity; Young Adult

The aim of this study is to identify the phenotype of resistant oral tumors, and to delineate the contribution of immune effectors to resistance of oral tumors. UCLA-1 oral tumors which were resistant to NK cell mediated cytotoxicity secreted increased amounts of IL-6, IL-1beta, GM-CSF, and IL-8 when cultured with or without immune effectors. In addition, the levels of vascular endothelial growth factor (VEGF) secretion in the co-cultures of naïve immune effectors with UCLA-1 rose significantly when compared to tumor cells alone. IL-2 activated NK cells decreased VEGF secretion in all tumor cells. However, NK cells which were induced to undergo cell death with anti-CD16 antibody were not only unable to decrease VEGF secretion, but they also contributed further to the increase in VEGF secretion by oral tumors. Overall, we show in this paper that naïve as well as non-viable immune effectors may contribute to the growth and resistance of oral tumors by triggering the secretion of key tumor cell growth factors.

Topics: Cell Death; Cell Line, Tumor; Coculture Techniques; Cytotoxicity, Immunologic; Drug Synergism; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1beta; Interleukin-2; Interleukin-6; Interleukin-8; Killer Cells, Natural; Mouth Neoplasms; Phenotype; Sensitivity and Specificity; Tumor Escape; Vascular Endothelial Growth Factor A

Functional DNA polymorphisms affecting gene expression and serum or saliva levels of interleukins IL-1 beta,-4,-6,-8,-10 and tumor necrosis factors TNF-alpha,-beta have been associated with increased risk for the development of oral squamous cell carcinoma (OSCC). The present retrospective case-control study examines possible interactions between seven cytokine genotype polymorphisms and their combinatory effect in predicting the occurrence of OSCC in Caucasians.. Three hundred and thirty Greeks and Germans were studied, consisting of 162 OSCC cases and 168 healthy controls of comparable age, gender, and ethnicity. A series of multivariate logistic regression models, adjusted for age and gender, was constructed in order to assess the contribution of homozygous or heterozygous variant genotypes of polymorphisms IL-1 beta (+3953C/T), IL-4 (-590C/T), IL-6 (-174G/C), IL-8 (-251A/T), IL-10 (-1082A/G), TNF-alpha (-308G/A) and TNF-beta (+252G/A) upon overall, early and advanced stages of OSCC development.. The contribution of TNF-alpha and IL-6 was consistent and robust in almost all models constructed. Furthermore, when the mode of inheritance of each variant allele was taken into account in a "biological" multivariate logistic regression model, four polymorphisms emerged as primary predictors for overall stages of OSCC: TNF-alpha (OR = 15.27; 95% CI = 7.30-31.96), IL-6 (OR = 8.33; 95% CI = 3.95-17.58), IL-8 (OR = 3.54; 95% CI = 1.69-7.43) and IL-10 (OR = 2.65; 95% CI = 1.28-5.46). Finally, IL-1 beta, IL-4 and TNF-beta polymorphisms were not primary predictors of OSCC development in all constructed models.. This study revealed the highly significant contributions of two out of seven studied cytokines (IL-6 and TNF-alpha) in the occurrence of OSCC. Based on these findings and previous reports, possible stoichiometrical interactions of cytokines leading to OSCC development are discussed.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Female; Gene Expression; Genetic Predisposition to Disease; Humans; Interleukin-1; Interleukin-10; Interleukin-1beta; Interleukin-4; Interleukin-6; Interleukin-8; Interleukins; Lymphotoxin-alpha; Male; Middle Aged; Mouth Neoplasms; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Regression Analysis; Retrospective Studies; Tumor Necrosis Factor-alpha

The aim of this study was to compare the concentration of tumor necrosis factor alpha, interleukin 1 alpha, 6, and 8 in the saliva of oral squamous cell carcinoma patients with control group.. In this study 18 subjects were involved, nine patients with oral squamous cell carcinomas and nine age-sex-matched healthy individuals that were matched for gingival conditions too. Active dental abscesses, collagen vascular diseases, and infectious diseases during one month before saliva sampling were considered as exclusion criteria. Unstimulated whole saliva was collected and after processing the samples were analyzed by Enzyme Linked Immune Assay.. The concentration of salivary interleukin 6 in oral squamous cell carcinoma patients was higher than control group and it was statistically significant (p < 0.05). The concentration of salivary tumor necrosis factor alpha, interleukin 1 alpha and 8 in case group was higher than control group but it was not statistically significant (p > 0.05).. These results shows that more studies are needed to accept the utility of these cytokines in predicting or diagnosis of oral squamous cell carcinoma or evaluation of treatment.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Female; Humans; Interleukin-1alpha; Interleukin-6; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Saliva; Tumor Necrosis Factor-alpha

In light of recently found contribution of angiogenic and inflammation-related factors to malignancies, this study investigated the possible association of interleukin-8 gene (IL-8) to increased risk of oral cancer.. The IL-8 (-251 A/T) polymorphism, which influences IL-8 gene expression, was evaluated by restriction fragment length polymorphism analysis in DNA samples of 158 German and Greek patients with oral squamous cell carcinoma and 156 healthy controls of equivalent sex, ethnicity and age.. Significant increase of mutant (A-251) allele, which results in higher IL-8 gene expression, was observed in all patients in comparison to normal controls (P<0.001). The A/T heterozygotes had a two-fold greater risk (odds ratio 1.76, CI 1.11-2.79) for developing oral cancer compared to normal TT homozygotes. Furthermore, significantly increased values of mutant allele frequencies compared to controls were observed in all patients as well as in subgroups of patients with or without positive history of cancer (P<0.05 and P<0.001, respectively) and with or without positive history of thrombophilia (P<0.05 and P<0.001, respectively).. In light to known observations of elevated plasma levels of IL-8 in several types of cancer including oral squamous cell carcinoma, the findings of this study suggest that the mutant allele of the (-251 A/T) polymorphism may be a major contributing genetic factor to risk for oral cancer.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Case-Control Studies; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Germany; Greece; Humans; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Polymorphism, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Risk

We have already demonstrated that human head and neck cancer cells have significantly enhanced levels of transcription factor nuclear factor (NF)-kappaB activity compared to their normal counterparts, suggesting that NF-kappaB plays an important role in the development of head and neck cancer. However, it has been reported that chemotherapeutic agents and radiation activate NF-kappaB activity in cancer cells, thus making the cells radioresistant and chemoresistant. In addition, we have shown that the suppression of NF-kappaB activity enhanced apoptosis in oral squamous cell carcinoma cells. In this study, we examined whether cepharanthin-induced inhibition of NF-kappaB activity enhances radiosensitivity in human oral carcinoma cells. Cepharanthin is a biscoclaurine alkaloid extracted from the roots of Stephania cepharantha hayata, and is widely used in Japan for the treatment of patients with leucopenia, nasal allergy, and venomous snakebites. gamma-irradiation (IR) induces NF-kappaB activity in oral carcinoma cells through the activation of upstream molecules, including Akt and IkappaB kinase. However, a luciferase assay revealed that cepharanthin suppresses IR-induced NF-kappaB activity in oral squamous cell carcinoma cells, thereby enhancing the radio-sensitivity. Western blot analysis showed an enhanced cleavage of poly-(ADP-ribose) polymerase protein in carcinoma cells by both cepharanthin treatment and IR exposure compared to IR or cepharanthin alone. In an in vivo study, B88 cells were s.c. inoculated into the backs of nude mice. Tumor-bearing nude mice received either cepharanthin, IR alone, or a combination of cepharanthin and IR. The combined treatment suppressed tumor growth significantly more than either cepharanthin or IR alone. Cepharanthin inhibited the production of IR-induced IL-6 and IL-8, which are downstream targets of NF-kappaB. In quantitative real-time RT-PCR, IR also induced the expression of anti-apoptotic proteins [cellular inhibitor of apoptosis protein (cIAP)-1 and -2] in carcinoma cells. Treatment of cancer cells with cepharanthin combined with exposure to IR decreased cIAP-1 and -2 mRNA expression. These findings suggested that the combination of radiotherapy and cepharanthin could enhance radiosensitivity in the treatment of human oral cancer.

Topics: Alkaloids; Animals; Apoptosis; Benzylisoquinolines; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Fluorescent Antibody Technique; Gamma Rays; Humans; I-kappa B Kinase; Inhibitor of Apoptosis Proteins; Interleukin-6; Interleukin-8; Luciferases; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; NF-kappa B; Radiation-Protective Agents; Radiation, Ionizing; RNA, Messenger

Interleukin-8 (IL-8) is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK) and malignant human oral keratinocytes (HN12) cells with deferoxamine (DFO).. IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Electrophoretic mobility shift assays was used to determine NF-kappaB binding activity. Phosphorylation and degradation of the I-kappaB were analyized by Western blot.. IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1beta to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IkappaB, and activation of NF-kappaB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione), and by pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(P)H oxidase.. This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions.

Topics: Cells, Cultured; Deferoxamine; Humans; Interleukin-8; Iron Chelating Agents; Keratinocytes; Mitogen-Activated Protein Kinases; Mouth Neoplasms; NF-kappa B; Siderophores; Signal Transduction

In order to find informative salivary biomarkers specific to oral cancer we examined expression of 4 kinds of cytokine in saliva. Levels of interleukins (IL-1beta, -6, -8) and osteopontin were measured by ELISA using whole saliva samples collected from 19 patients with oral cancer (9 men, 10 women; mean age, 60.9 years) and 20 healthy persons (15 men, 5 women; mean age, 32 years). Expression of the 4 cytokines was higher in patients with oral cancer than in healthy controls. The difference was significant in IL-6, in particular. The results suggest that saliva offers a potential target for a screening test aimed at detection of precancerous lesions.

Topics: Adult; Biomarkers, Tumor; Cytokines; Female; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Middle Aged; Mouth Neoplasms; Osteopontin; Saliva; Salivary Proteins and Peptides

Researchers at UCLA have discovered that the levels of interleukin-8 (IL-8) protein in the saliva of healthy individuals and patients with oropharyngeal squamous cell carcinoma (OSCC) are 30 pM and 86 pM, respectively. In this study, we present the development of the first immunoassay for the quantification of picomolar IL-8 concentrations in human saliva using Biacore surface plasmon resonance (SPR) in a microfluidic channel. A sandwich assay using two monoclonal antibodies, which recognize different epitopes on the antigen (IL-8), was used. Only 13 minutes were required to determine the quantity of pure IL-8 added to just 100 microL of either buffer or saliva-based samples. The limit of detection (LOD) of this immunoassay in buffer was 2.5 pM, and the precision of the response for each concentration was <3% of the coefficient of variation. When first analyzing the saliva supernatants, non-specific binding to the surface was observed. By adding carboxymethyl dextran sodium salt (10 mg mL(-1)) to compete with the surface dextran and primary antibody for non-specific interactions, the signal to noise ratio was greatly improved. The LOD of this immunoassay in saliva was 184 pM. A minimum concentration of 250 pM of exogenous IL-8 could then be consistently detected in a salivary environment. The precision of the response for each IL-8 concentration tested was <7% of the coefficient of variation. Diagnostic sensitivity for oral cancer can be achieved by pre-concentrating the saliva samples 10 fold prior to SPR analysis, making the target levels of IL-8 300 pM for healthy individuals and 860 pM for oral cancer patients.

Topics: Animals; Antibodies, Monoclonal; Biosensing Techniques; Calibration; Carcinoma, Squamous Cell; Dextrans; Epitopes; Humans; Immunoassay; Interleukin-8; Kinetics; Mice; Mouth Neoplasms; Reproducibility of Results; Saliva; Sensitivity and Specificity; Surface Plasmon Resonance

Vesnarinone is a synthesized positive oral inotropic agent that has multiple biological activities on mammalian cells both in vitro and in vivo. This agent has been reported in relation to its antitumor effect with apoptosis-inducing activity. In the present study, we determined whether vesnarinone could suppress angiogenesis and growth of human oral squamous cell carcinoma cells in vitro and in vivo. Vesnarinone significantly inhibited the in vitro and in vivo expression of two major proangiogenic molecules, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), in cultured cells and in cells implanted into the subcutaneous tissue of nude mice. Also, vesnarinone inhibited the nuclear factor-kappa B (NF-kappaB) activity in human oral squamous cell carcinoma cells in vitro. The decreased expression of VEGF and IL-8 correlated with decreased tumorigenicity and decreased vascularization of lesions in vivo. These findings suggest that vesnarinone can suppress the angiogenesis and growth of oral squamous cell carcinoma cells by inhibiting the expression of VEGF and IL-8 involved in blockade of NF-kappaB activity.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-8; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neovascularization, Pathologic; NF-kappa B; Protein Binding; Pyrazines; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to gamma-irradiation (IR) and 5-Fluorouracil (5-FU) in human oral carcinoma cells (B88) in which NF-kappaB activity was constitutively suppressed. Three super-repressor form of IkappaBalpha cDNA-transfected cell (B88mI) clones and 1 empty vector-transfected cell clone (B88neo) have been established. We found that the tumor-forming ability in nude mice of B88mI clones was significantly lower than that of B88 or B88neo. This suppressed ability in tumorigenicity was attributed to the down-regulation of the expression of interleukin (IL)-1alpha, IL-6, IL-8, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 in B88mI cell clones as compared to that in B88 or B88neo. IR and 5-FU induced a much greater degree of apoptosis, as evidenced by flow cytometry analysis and annexin V staining, in B88mI cell clones than in B88 or B88neo. When tumor-bearing nude mice were treated with IR or 5-FU, the suppression of tumor growth was significantly augmented in B88mI cell clones as compared to that in B88 or B88neo. ELISA analysis indicated that although a remarkable increase in production of IL-6 and IL-8 was observed in B88 and B88neo after in vitro exposure to IR or treatment with 5-FU, radiotherapy and chemotherapy-induced production of these cytokines was significantly suppressed in B88mI cell clones. These findings suggest that production of angiogenic factors and growth factors in response to radiotherapy and chemotherapy is a principal mechanism of inducible radioresistance and chemoresistance in human oral cancers, and establish the inhibition of NF-kappaB as a rational approach to improve conventional radiotherapy and chemotherapy outcomes.

Topics: Animals; Annexin A5; Antimetabolites, Antineoplastic; Apoptosis; Blotting, Western; Carcinoma; Cell Division; Cell Nucleus; Cell Separation; Culture Media, Conditioned; Cytosol; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorouracil; Gamma Rays; Genetic Vectors; Humans; I-kappa B Proteins; Immunohistochemistry; Interleukin-6; Interleukin-8; Luciferases; Matrix Metalloproteinase 9; Mice; Mice, Nude; Mouth Neoplasms; Mutation; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; Oligonucleotides; Time Factors; Transfection; Vascular Endothelial Growth Factor A

Since morbidity and mortality rates due to oral cavity and oropharyngeal squamous cell carcinoma (OSCC) have improved little in the past 30 years, early detection or prevention of this disease is likely to be most effective. Using laser-capture microdissection, we have identified the expression of 2 cellular genes that are uniquely associated with OSCC: interleukin (IL) 6 and IL-8. These cytokines may contribute to the pathogenesis of this disease, and have been linked with increased tumor growth and metastasis.. To investigate whether IL-6 and/or IL-8 could serve as informative biomarkers for OSCC in saliva and/or serum and to determine if there is a role for saliva as a diagnostic medium for OSCC.. Patients with newly diagnosed T1 or T2 oral cavity or oropharyngeal histologically confirmed squamous cell carcinoma were recruited for the study. Age and sex-matched disease-free subjects were used as controls. Using quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, we respectively assessed the expression of IL-6 and IL-8 in serum (controls, n = 32; patients with OSCC, n = 19) and saliva (controls, n = 32; patients with OSCC, n = 32) at the messenger RNA (mRNA) and protein levels.. Specificity and sensitivity of these biomarkers for OSCC and their predictive value.. Interleukin 8 was detected at higher concentrations in saliva (P<.01) and IL-6 was detected at higher concentrations in serum of patients with OSCC (P<.01). We confirmed these results at both the mRNA and the protein levels, and the results were concordant. The concentration of IL-8 in saliva and IL-6 in serum did not appear to be associated with sex, age, or alcohol or tobacco use (P>.75). Using statistical analysis, we were able to determine the threshold value, sensitivity, and specificity of each biomarker, as well as a combination of biomarkers, for detecting OSCC.. Our findings indicate that IL-8 in saliva and IL-6 in serum hold promise as biomarkers for OSCC. A saliva-based test could be a cost-effective adjunctive tool in the diagnosis and follow-up of patients with OSCC.

Topics: Adult; Biomarkers, Tumor; California; Carcinoma, Squamous Cell; Female; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Mouth; Mouth Neoplasms; Oropharyngeal Neoplasms; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Saliva; Sensitivity and Specificity

A number of studies have implicated the fibrin-coagulation-fibrinolysis system in human tumour progression. Interleukin-8 (IL-8) mediates most of the angiogenic activity induced by human oral squamous cell carcinoma (OSCC) cells. We have recently demonstrated that: (1) fibrin is present in association with IL-8 expressing human OSCC cells in vivo and (2) in situ fibrin polymerisation induces a specific, dose and time-dependent upregulation of IL-8 expression from human OSCC cells in vitro. Our present studies extend this observation by demonstrating that in addition to fibrin formed in situ, both fibrin-derived liquid expressates (soluble fibrin) and preformed fibrin clots induced an over eight-fold stimulation of IL-8 expression from human OSCC cells as compared to media controls. IL-8 upregulation by soluble fibrin was dose-dependent. A monoclonal antibody against the N terminal region of the beta chain of human fibrin (Bbeta15-42) inhibited 67% of soluble fibrin-induced IL-8 expression from human OSCC cells. A peptide (GHRP), representing the sequence at the N terminus of this region, induced a dose-dependent stimulation of IL-8 expression, further confirming the role of this region. These studies directly support our hypothesis that fibrin induces protumourigenic factor expression from tumour cells, thus promoting tumour progression. Future studies to further characterise the role of the Bbeta15-42 region in tumour cell activation may lead to the design of peptide antagonists with important therapeutic potential.

Topics: Aged; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Interleukin-8; Macromolecular Substances; Male; Mouth Neoplasms; Oligopeptides; Peptide Fragments; Structure-Activity Relationship; Tumor Cells, Cultured; Up-Regulation

We have recently demonstrated that fibrin induces a specific, dose- and time-dependent upregulation of the angiogenic factor interleukin 8 (IL-8) from human oral squamous cell carcinoma (OSCC) cells in vitro. In this study we begin to test the hypothesis that fibrin induces IL-8 expression from tumor cells in vivo by studying their in vivo association in OSCC.. The presence of fibrin(ogen) was initially evaluated in 20 archival human OSCCs by means of immunohistochemistry with a polyclonal antibody. The presence of fibrin and IL-8 was then studied in 19 sections from 8 different patients' head and neck tumors (including 6 OSCCs) by means of immunohistochemistry with a monoclonal antibody against fibrin. These 8 tumors had been treated with inhibitors of new fibrin formation and degradation immediately after surgical removal.. Fibrin staining was found in 100% of the tumor sections tested. IL-8 staining was found in the cytoplasm of tumor cells in 100% of the studied tumors, including areas adjacent to fibrin.. These data demonstrate an in vivo association between fibrin and IL-8 in OSCC. These studies support our hypothesis that fibrin induces expression of protumorigenic factors such as IL-8 from tumor cells in vivo.

Topics: Antibodies; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Coloring Agents; Cytoplasm; Fibrin; Fibrinogen; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-8; Mouth Neoplasms; Tumor Cells, Cultured; Up-Regulation

Interleukin-8 (IL-8) is an important cytokine involved in tumor growth and angiogenesis in a variety of malignancies. Furthermore, matrix metalloptoteinases (MMPs) also play important roles in the invasion and metastasis of carcinomas including oral squamous cell carcinoma (OSCC). We studied whether IL-8 and MMPs participate in tumorigenesis and metastasis of OSCC. First, we investigated the gene and protein expressions of IL-8 and IL-8 receptor (IL-8R), and the effect of IL-8 on proliferation, migration and invasion of OSCC. Second, we thus also investigated the effect of IL-8 on MMP release in OSCC cells. OSCC cell lines NA and HSC-4 constitutively expressed IL-8 mRNA and secreted its protein in vitro. The production of IL-8 was significantly enhanced by the addition of tumor necrosis factor (TNF)-alpha and IL-beta, but not interferon (IFN)-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-2. Flow cytometric analysis revealed the constitutive expression of both receptors of IL-8, IL-8RA and IL-8RB, in OSCC cell lines. The expression of IL-8 receptors in HSC-4 cells was stronger than that in NA cells. The intensity of IL-8RA expression was stronger than that of IL-8RB expression in each cell line. The expression of IL-8 receptors was not altered by the addition of cytokines such as TNF-alpha and IL-1beta. The conditioned medium containing IL-8 from OSCC cell lines induced migration and invasion of OSCC cells, but did not change cell proliferation. The differences in migrational and invasive ability between NA cells and HSC-4 cells were correlated with the expression intensity of IL-8 receptors in each cell line. Neutralizing antibodies to IL-8, IL-8RA and IL-8RB partially inhibited the chemotactic activity induced by conditioned medium. The expression of MMP-2, -7 and -9 was detected in culture supernatants from these OSCC cell lines. The expressions of MMP-7 protein and mRNA were enhanced by the addition of rIL-8, but that of other MMPs was not observed in a similar manner. These results suggest that IL-8 secreted from OSCC may contribute to the invasion of OSCC through the regulation of MMP-7 expression.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cytokines; Flow Cytometry; Humans; Interleukin-8; Metalloendopeptidases; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Tumor Cells, Cultured

Vascular endothelial growth factor (VEGF) has been shown to be a potent mediator of angiogenesis that functions as a survival factor for endothelial cells by up-regulating Bcl-2 expression. We have recently reported that human dermal microvascular endothelial cells (HDMECs) seeded in biodegradable sponges and implanted into severe combined immunodeficient (SCID) mice organize into functional human microvessels that transport mouse blood cells. In this study, we implanted sponges seeded with OSCC-3 (oral squamous cell carcinoma) or SLK (Kaposi's sarcoma) together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels. This model system was used to examine the role of both endothelial cell Bcl-2 and the proangiogenic chemokine interleukin-8 (IL-8) on tumor growth and intratumoral microvascular density. Coimplantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) and tumor cells resulted in a 3-fold enhancement of tumor growth when compared with the coimplantation of control HDMECs and tumor cells. This was associated with increased intratumoral microvascular density and enhanced endothelial cell survival. To determine whether the enhanced neovascularization mediated by Bcl-2 overexpression in endothelial cells was influenced by the synthesis of endogenous mediators of angiogenesis, we screened these cells for expression of VEGF, basic fibroblast growth factor (bFGF), and IL-8 by ELISA. HDMEC-Bcl-2 cells and VEGF-treated HDMECs exhibited a 15-fold and 4-fold increase, respectively, in the expression of the proangiogenic chemokine IL-8 in vitro, whereas the expression of VEGF and bFGF remained unchanged. Transfection of antisense Bcl-2 into HDMECs blocked VEGF-mediated induction of IL-8. Conditioned media from HDMEC-Bcl-2 induced proliferation and sprouting of endothelial cells in vitro and neovascularization in rat corneas. Anti-IL-8 antibody added to HDMEC-Bcl-2 conditioned media markedly reduced the potency of these responses. SCID mice bearing VEGF-producing tumor implants that were treated with anti-lL-8 antibody exhibited a 43% reduction in microvessel density and a 50% reduction in tumor weight compared with treatment with a nonspecific antibody. These results demonstrate that the up-regulation of Bcl-2 expression in endothelial cells that constitute tumor microvessels enhances intratumoral microvascular survival and density and accelerates tumor growth. Furthermore, endothelial cells that overexpress Bcl-2 h

Topics: Animals; Antibodies; Carcinoma, Squamous Cell; Cell Division; Cell Transplantation; Disease Models, Animal; Endothelium, Vascular; Gene Expression Regulation; Genes, bcl-2; Humans; Interleukin-8; Mice; Mice, SCID; Mouth Neoplasms; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Proto-Oncogene Proteins c-bcl-2; Rats; Sarcoma, Kaposi; Transplantation, Heterologous; Up-Regulation

In recent studies, we have demonstrated that fibrin is present in association with tumor cells in oral squamous cell carcinoma (OSCC) in vivo. We hypothesized that this fibrin can directly induce the expression of known angiogenic factors from oral tumor cells. Since IL-8 is known to be the major inducer of angiogenesis caused by these cells, we examined the ability of fibrin to stimulate IL-8 expression from OSCC cells in vitro. A physiologically relevant concentration of fibrin was found to cause a dose and time-dependent stimulation of IL-8 expression from oral and pharyngeal tumor cells but not from a non-tumorigenic oral cell line. Fibrinogen, thrombin and collagen were all unable to induce significant IL-8 expression, establishing the specificity of fibrin in causing this response. Gel filtration chromatography confirmed the molecular identity of the IL-8 antigen detected in the ELISA system used. These results suggest that fibrin may promote angiogenesis in oral tumors in vivo by directly upregulating the expression of IL-8 from tumor cells.

Topics: Analysis of Variance; Carcinoma, Squamous Cell; Cell Line; Dose-Response Relationship, Drug; Epithelium; Fibrin; Humans; Interleukin-8; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; Neovascularization, Pathologic; Pharyngeal Neoplasms; Stimulation, Chemical; Time Factors; Tumor Cells, Cultured

The anti-tumour effect of the angiogenic inhibitor TNP470, sigma-(chloro-acetyl-carbamoyl) fumagillol, a synthetic analogue of fumagillin, was studied in vitro and in vivo using KB cells, one of the human head and neck carcinoma cell lines that produce interleukin(IL)-8. In the in vitro study, the combination treatment of TNP470 and anti-IL-8 antibody significantly reduced the proliferation of KB cells. In the in vivo studies, TNP470 administration by any route (intratumoral: i.t., intraperitoneal: i.p., intravenous: i.v.) reduced the tumour volume significantly, compared to the control group. Among the groups administered TNP470, the anti-tumour effect was strongest in the it group. Furthermore, the concurrent treatment of anti-IL-8 antibody and TNP470 also maximally reduced the tumour volume. The combination therapy of TNP470 and anti-IL-8 antibody was very effective. These results suggest that combination therapy of TNP470 and anti-IL-8 antibody could be beneficial for solid tumours, such as head and neck cancer.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Combined Modality Therapy; Cyclohexanes; Depression, Chemical; Flow Cytometry; Humans; Injections, Intralesional; Interleukin-8; Male; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

Interleukin-8 (IL-8) is one of the multifunctional cytokines that can play a role on immune and inflammatory activities. Other in vitro observations indicated that IL-8 is a growth factor for keratinocytes. However, as the role of IL-8 in oral cancer cells is unclear, this study is thus designed to examine IL-8 secretion in cultured oral epidermoid carcinoma KB CCL17 cells treated with nicotine and/or arecoline. The cultures were treated with nicotine (1 or 100 microM) and arecoline (1 or 100 microM), alone or both, for 72 hrs. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-8 concentrations in culture supernatants. A repeated measure analysis of variance was used to identify differences among the treatments. Nicotine and arecoline, single or combined treatment, increased IL-8 secretion in KB CCL17 cells. When monoclonal 1 microgram/ml of antibody was added against IL-1 alpha or IL-1 beta in the treatment, IL-8 concentration significantly decreased compared with the non-added one. Exposure of cells to antibody against IL-1 alpha or IL-1 beta showed no significant increase in cell growth as compared with the control (medium alone). However, incubation of cells for 72 hrs in the presence of nicotine and/or arecoline treatments and antibody against IL-1 alpha or IL-1 beta significantly increased cell growth as compared with the antibody free one. It was concluded that IL-8 secretion by KB CCL17 cells may be partially mediated by IL-1 which could inhibit the KB CCL17 cell growth. Thus, IL-8 may be a vital participant in the cascade of interacting cytokines during smoking and areca quid chewing, inducing inflammation in oral cancer.

Topics: Antibodies, Monoclonal; Arecoline; Carcinoma, Squamous Cell; Cell Division; Humans; Interleukin-1; Interleukin-8; Mouth Neoplasms; Nicotine; Tumor Cells, Cultured

In order to gain further understanding of the role of chemokines in healthy oral mucosa, we analyzed mRNA expression of the alpha (CXC)-family chemokines IL-8 and GROgamma as well as of the beta (CC)-family chemokines MIP-1alpha, MIP-1beta and MCP-1 in twenty young and healthy subjects with good oral hygiene. Twenty biopsies were taken from clinically healthy oral mucosa before surgical removal of impacted wisdom teeth. In addition, five biopsies from patients presenting with specific oral lesions were studied. RNA was purified, quantitated and utilized as substrate for competitive reverse transcription-polymerase chain reaction (RT-PCR). In healthy tissue, IL-8 and MCP-1 mRNA was constitutively expressed in all biopsies, whereas GROgamma, MIP-1alpha, and MIP-1beta were significantly lower. These findings suggest that IL8 and MCP-1 play a significant role in oral tissue homeostasis. The few samples from pathological conditions encourage exploring diseased tissue in more detail.

Topics: Adolescent; Adult; Analysis of Variance; Biopsy; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokine CXCL1; Chemokines, CC; Chemokines, CXC; Chemotactic Factors; Female; Fibroma; Gene Expression Regulation; Gingival Neoplasms; Growth Inhibitors; Growth Substances; Hemostasis; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lichen Planus, Oral; Lymphoma, AIDS-Related; Macrophage Inflammatory Proteins; Male; Mouth Diseases; Mouth Mucosa; Mouth Neoplasms; Polymerase Chain Reaction; RNA, Messenger