Salivary IL-8, IL-6 and TNF-α as Potential Diagnostic Biomarkers for Oral Cancer

Saliva has been useful as a liquid biopsy for the diagnosis of various oral or systemic diseases, and oral squamous cell carcinoma (OSCC) is no exception. While its early detection and prevention is important, salivary cytokines expression, specifically of Interleukin-8 (IL-8), Interleukin-6 (IL-6) and Tumor necrosis factor (TNF-α), does contribute to the pathogenesis of cancer and these cytokines serve as potential biomarkers. Their excessive production plays a role in cancer progression and establishment of angiogenesis. However, other inflammatory or immunological conditions may affect the levels of cytokines in saliva. This article reviews the expression of levels of specific cytokines i.e., IL-8, IL-6 and TNF-α, their signaling pathways in the development of oral cancer, and how they are essential for the diagnosis of OSCC and updates related to it. Apart from serum, the saliva-based test can be a cost-effective tool in the follow-up and diagnosis of OSCC. Moreover, large-scale investigations are still needed for the validation of salivary cytokines.


Introduction
Oral cancer was previously rated as the sixth most common cancer worldwide, and has now acquired the fifth position [1]. Living in 21st century, with various scientific advancements being explored and discovered each day, the outcome of oral cancer worldwide has yet a prognosis which has deteriorated rather than improved. It is an established public health care problem with high mortality and morbidity rate [2]. Biopsy was known as the ultimate gold standard' for the diagnosis of oral cancer [3]. Different techniques are being implemented to predict the development of oral interact not with one another but with other cellular targets, and cytokine crossover, i.e., a single cellular target can respond to multiple cytokines. They are chemotactic in nature, i.e., they attract particular cells via a concentration gradient. The doses of cytokines are generally maintained within a specified range and time. If not properly maintained, they can lead to induction of tissue damage. Cytokines can be classified according to the division of labour, on a structural basis and based on receptors (see Figure 1). Systemic conditions including obesity, psoriasis, anaphylactic shock, Steven-Johnson syndrome and other acute inflammatory conditions are known to increase the levels of cytokines several times over. Oral conditions, specifically oral aphthous ulcers, oral dysplastic lesions, oral and pharyngeal carcinomas and periodontitis, are known to contribute to elevated levels of cytokines as well.
Diagnostics 2017, 7, 21 3 of 10 cytokines. They are chemotactic in nature, i.e., they attract particular cells via a concentration gradient. The doses of cytokines are generally maintained within a specified range and time. If not properly maintained, they can lead to induction of tissue damage. Cytokines can be classified according to the division of labour, on a structural basis and based on receptors (see Figure 1). Systemic conditions including obesity, psoriasis, anaphylactic shock, Steven-Johnson syndrome and other acute inflammatory conditions are known to increase the levels of cytokines several times over. Oral conditions, specifically oral aphthous ulcers, oral dysplastic lesions, oral and pharyngeal carcinomas and periodontitis, are known to contribute to elevated levels of cytokines as well. IL-8 is regarded as the prototype of the chemokine family. It is pro-inflammatory in nature and is released by the neutrophils and macrophages in response to various stimuli, including chemical environment, steroids, inflammatory signals and environmental stresses. These stimuli activate the nuclear factor-kappa-B (NF-κB) pathway and this, in turn, activates the expression of IL-8 production. The IL-8 produced acts on two structurally similar but antigenically different receptors, namely CRCX-1 and CRCX-2. These receptors are located on tumor-associated macrophages, neutrophils and cancer cells. The presence of the receptors on cancer cells strongly suggests that the levels of IL-8 are an important chemokine for cancer cells microenvironment [22]. The pathogenicity in cancer cells is derived from neutrophil recruitment, angiogenic potential, proliferation, survival, migration of vascular endothelial cells, protection from apoptosis and, ultimately, metastasis [23]. The role in cancer can also be postulated from the fact that treatment of cancerous lesions with chemotherapeutic agents like 5-fluorouracil, among many others, reduces the expression of IL-8 [18,24]. The stimulating agents, if removed or decreased, also alter the IL-8 expression and levels, which further provide confirmation. Moreover, Selvam et al. suggested salivary 1L-6 as a diagnostic marker for leukoplakia and OSCC by determining its concentration by enzyme-linked immunosorbent assay (ELISA), the results of which proved its high concentration and production by tumor cells [25]. Juretic et al. suggested and confirmed through ELISA the high levels of IL-6 and TNF-α, proving their diagnostic and prognostic significance in potentially IL-8 is regarded as the prototype of the chemokine family. It is pro-inflammatory in nature and is released by the neutrophils and macrophages in response to various stimuli, including chemical environment, steroids, inflammatory signals and environmental stresses. These stimuli activate the nuclear factor-kappa-B (NF-κB) pathway and this, in turn, activates the expression of IL-8 production. The IL-8 produced acts on two structurally similar but antigenically different receptors, namely CRCX-1 and CRCX-2. These receptors are located on tumor-associated macrophages, neutrophils and cancer cells. The presence of the receptors on cancer cells strongly suggests that the levels of IL-8 are an important chemokine for cancer cells microenvironment [22]. The pathogenicity in cancer cells is derived from neutrophil recruitment, angiogenic potential, proliferation, survival, migration of vascular endothelial cells, protection from apoptosis and, ultimately, metastasis [23]. The role in cancer can also be postulated from the fact that treatment of cancerous lesions with chemotherapeutic agents like 5-fluorouracil, among many others, reduces the expression of IL-8 [18,24]. The stimulating agents, if removed or decreased, also alter the IL-8 expression and levels, which further provide confirmation. Moreover, Selvam et al. suggested salivary 1L-6 as a diagnostic marker for leukoplakia and OSCC by determining its concentration by enzyme-linked immunosorbent assay (ELISA), the results of which proved its high concentration and production by tumor cells [25]. Juretic et al. suggested and confirmed through ELISA the high levels of IL-6 and TNF-α, proving their diagnostic and prognostic significance in potentially premalignant lesions and conditions and in OSCC [26]. Nelson et al. suggested the elevation of IL-6, IL-8, IL-1 and TNF-α in oral neoplastic lesions and OSCC because of their characteristic feature involvement in pro-angiogenesis and pro-inflammation which, in turn, has diagnostic value [27].
IL-6, apart from being a pro-inflammatory cytokine, also regulates regenerative, metabolic and neural processes. It follows the ras/raf/Mitogen-activated protein (MAP) kinase (MAPK) pathway [28]. TNF-α involves caspase cascades, transcription factors, nuclear factor kappa B (NF-κB) and activating protein 1 (AP-1), which are involved in inflammation, signal regulation, cell growth and death [29]. IL-8, IL-6 and TNF-α proved to be overexpressed in OSCC, as investigated by the University of California, Los Angeles (UCLA) Collaborative Oral Fluid Diagnostic Research Centre, the pioneer center for the studies conducted in this field [30]. Keeping in view the above, various researchers have been adamant on concluding the proportionality and relationship of IL-8, IL-6 and TNF-α with oral cancer. Analysis of saliva and serum both were carried out with significant changes noted (p-value < 0.05). Additionally, Figure 2 explains how the activation of IL-8, IL-6 and TNF-α takes place through activated macrophages. Figure 2 also illustrates the range of their signaling pathways in tumor microenvironments, which are activated by, for example, IL-8 via chemokine receptors CXCR1 and CXCR2, which activate signal transducers and activators of transcription-3 (STAT3) and β-catenin, which has been shown to promote cell proliferation and angiogenesis. IL-6 receptor leads to the activation of the Janus kinases (JAK) and signal transducers and activators of transcription (STATs), which then stimulate pathways involving mitogen-activated protein kinase (MAPK), which in turn supports cancer development. While tumor necrosis factor receptor type 1-associated DEATH domain protein in humans is encoded by the TRADD gene, which recruits TNF receptor-associated factor-2, a protein in humans is encoded by the TRAF2 gene TRAF2 and signaling molecule RIP which activates and induces Nuclear Factor Kappa-light-chain-enhancer of activated B Cells (NF-κB) pathway, which gets involved in cell survival and proliferation and anti-apoptotic factors, which explains the major role in the development of oral cancer. premalignant lesions and conditions and in OSCC [26]. Nelson et al. suggested the elevation of IL-6, IL-8, IL-1 and TNF-α in oral neoplastic lesions and OSCC because of their characteristic feature involvement in pro-angiogenesis and pro-inflammation which, in turn, has diagnostic value [27]. IL-6, apart from being a pro-inflammatory cytokine, also regulates regenerative, metabolic and neural processes. It follows the ras/raf/Mitogen-activated protein (MAP) kinase (MAPK) pathway [28]. TNF-α involves caspase cascades, transcription factors, nuclear factor kappa B (NF-κB) and activating protein 1 (AP-1), which are involved in inflammation, signal regulation, cell growth and death [29]. IL-8, IL-6 and TNF-α proved to be overexpressed in OSCC, as investigated by the University of California, Los Angeles (UCLA) Collaborative Oral Fluid Diagnostic Research Centre, the pioneer center for the studies conducted in this field [30]. Keeping in view the above, various researchers have been adamant on concluding the proportionality and relationship of IL-8, IL-6 and TNF-α with oral cancer. Analysis of saliva and serum both were carried out with significant changes noted (p-value < 0.05). Additionally, Figure 2 explains how the activation of IL-8, IL-6 and TNF-α takes place through activated macrophages. Figure 2 also illustrates the range of their signaling pathways in tumor microenvironments, which are activated by, for example, IL-8 via chemokine receptors CXCR1 and CXCR2, which activate signal transducers and activators of transcription-3 (STAT3) and β-catenin, which has been shown to promote cell proliferation and angiogenesis. IL-6 receptor leads to the activation of the Janus kinases (JAK) and signal transducers and activators of transcription (STATs), which then stimulate pathways involving mitogen-activated protein kinase (MAPK), which in turn supports cancer development. While tumor necrosis factor receptor type 1-associated DEATH domain protein in humans is encoded by the TRADD gene, which recruits TNF receptor-associated factor-2, a protein in humans is encoded by the TRAF2 gene TRAF2 and signaling molecule RIP which activates and induces Nuclear Factor Kappa-light-chainenhancer of activated B Cells (NF-κB) pathway, which gets involved in cell survival and proliferation and anti-apoptotic factors, which explains the major role in the development of oral cancer.  Since 1863, Rudolf Virchow had claimed cancer to be a progressive, untreated form of chronic inflammation. Currently, it is widely accepted that chronic inflammation leads to approximately a quarter of the total malignancies diagnosed [31]. This establishes the link between cytokines, which pro-inflammatory in nature, and cancer. Typically, the cytokine group would induce tissue repair and healing. However, in cancerous cells, they induced DNA damage, inhibition of DNA repair, inactivation of tumor suppressor genes functionality, vascular permeability, extravasation of fibrin, tissue remodeling, tumor cell migration, leukocyte infiltration, alteration of cell to cell adhesion molecules, decreased the immune response, and angiogenesis [20].

Salivary IL-8, IL-6 and TNF-α Role in Oral Cancer Diagnosis
In the past decade, saliva has emerged as a medium for disease analysis, including local and systemic conditions. Many investigators use saliva collected by simple drool technique. The cytokines were then analyzed quantitatively and qualitatively by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), respectively. Other tests utilized are Western blotting, migration assay, immuno-histochemical staining, spectrophotometer and neutrophil count, assessed either by Giemsa staining or culture. A comparison of the IL-8, IL-6 and TNF-α levels in serum and saliva have also been carried out, in which the results obtained showed equal values of IL-8 in both mediums. This highlights the use of saliva rather than serum. A comparative table showing the details of the research conducted, patients evaluated, results and p-values is listed below ( Table 1). Khyani et al. [32] evaluated the salivary levels of IL-6 and IL-8 in patients diagnosed with histologically proven OSCC, potentially malignant lesions (PML), and a control group. The results revealed that in the OSCC group both biomarkers were found to be statistically elevated when compared with the control group. On the other hand, in the PML group, IL-8 was only found to be elevated [32]. However, it has been noted that the levels of IL-8 were found to be similar in both serum and salivary samples, whereas IL-6 was found to be higher in serum when compared to salivary samples of patients diagnosed with OSCC [33]. In another study conducted by Saheb et al., TNF-α, IL-8 and IL-6 levels were compared between patients diagnosed with OSCC, and age and sex matched controls. Only IL-6 levels were found to be statistically elevated. The other biomarkers, although raised compared to the control group, showed no statistically significant difference [34]. On the contrary, the levels of the same cytokines were evaluated in a study by Kaur et al. in salivary samples obtained from histologically proven patients of lichen planus, leukoplakia and oral submucous fibrosis (OSF), all three representing PML. The results obtained showed statistically significant, higher levels of IL-8 when compared to the control group. Also, the serum and the salivary samples, when compared, revealed a strong correlation amongst the groups [35]. In oral leukoplakia patients, salivary samples were studied, and the levels of salivary interleukin-6 and TNF-α were found to be significant as clinical diagnostic markers [36].
The levels obtained in samples from OSCC patients have been noted to be several times higher when compared to a control group, outweighing any other potential cause of the increase in levels. IL-8 levels may alter with lifestyle, geographical distribution, ethnic differences, genetic differences, and peculiar habits of individuals. Also, the levels may be found to be raised in mere gingivitis and periodontitis. Yet, none of these reasons can raise the levels to the extent seen in OSCC cases. Punyani et al. [12] discussed the pro-inflammatory and pro-angiogenic features of IL-8 through performing experiments with samples from 25 patients of OSCC, and hence found increased levels of IL-8 that confirmed the role of IL-8 in tumor angiogenesis and progression [12]. Furthermore, another reported work identified salivary and serum levels of IL-8, IL-6 and TNF-α in patients of oral leukoplakia, oral lichen planus, oral submucous fibrosis, and their results showed high concentrations of all cytokines, which again proved to be diagnostic markers for oral precancerous lesions [35]. A definitive alteration in the levels of IL-8 is noted from this; we can easily derive that IL-8 has a major potential to be used as a single salivary biomarker. This can open a new horizon for treatment plans of targeted leukotriene therapy [21,22]. The pathogenesis of the biomarker is elaborated upon in Figure 3, below. The concentration of salivary tumor necrosis factor α, interleukin-1α and 8 in case group was higher than control group, but it was not statistically significant (p > 0.05).

Conclusions
The detection of cytokines in the saliva of cancerous patients has led to the conclusion that chemokines are held responsible, amongst the many other pro-inflammatory cytokines, for inducing oral cancer. Using saliva as a liquid media for evaluation has its unique advantages, including ease of handling and performing, compliance of patients and, in short, being a cost-effective tool for diagnosing, screening and assessment of OSCC treatment. These biomarkers can be utilized as a major asset for early detection as their role in tumorigenesis is exceedingly evident. Unfortunately, the results from various studies are still incomplete; numerous studies need to be conducted to form an accurate statement.

Acknowledgments:
We are acknowledging Pakistan Human Saliva Research Group (PakHSRG) for providing us guidelines, technical data and funds for completing this review paper.

Conclusions
The detection of cytokines in the saliva of cancerous patients has led to the conclusion that chemokines are held responsible, amongst the many other pro-inflammatory cytokines, for inducing oral cancer. Using saliva as a liquid media for evaluation has its unique advantages, including ease of handling and performing, compliance of patients and, in short, being a cost-effective tool for diagnosing, screening and assessment of OSCC treatment. These biomarkers can be utilized as a major asset for early detection as their role in tumorigenesis is exceedingly evident. Unfortunately, the results from various studies are still incomplete; numerous studies need to be conducted to form an accurate statement.