NLRP3 Inflammasome Inhibitor BAY-117082 Reduces Oral Squamous Cell Carcinoma Progression

Oral cancer is one of the most common human malignancies, and its incidence is increasing worldwide. In particular, oral squamous cell carcinoma (OSCC) is characterized by high rates of proliferation, invasiveness, and metastasis. Currently, standard treatment for OSCC includes surgical removal, chemotherapy, and radiotherapy; however, the survival rate of patients with OSCC remains low, thus new therapies are needed. It has been proven that excessive NLRP3 inflammasome activation and apoptosis alteration may contribute to oral cancer progression. This study aimed to investigate the effect of BAY-117082, an NLRP3 inflammasome inhibitor, in an in vitro and in vivo xenograft model of oral cancer. In vitro results revealed that BAY-117082 at concentrations of 5, 10, and 30 µM was able to reduce OSCC cell viability. BAY-117082 at higher concentrations significantly reduced NLRP3, ASC, caspase-1, IL-1β, and IL-18 expression. Moreover, Bax, Bad, and p53 expression were increased, whereas Bcl-2 expression was reduced. Furthermore, the in vivo study demonstrated that BAY-117082 at doses of 2.5 and 5 mg/kg significantly decreased subcutaneous tumor mass, and also reduced NLRP3 inflammasome pathway activation. Therefore, based on these results, the use of BAY-117082 could be considered a promising strategy to counteract oral cancer progression, thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways.


Introduction
Oral cancer is one of the most common malignancies in the world [1]. Oral cancer commonly occurs in older individuals, although a troubling number of these malignancies have also been documented in younger adults [1]. Oral squamous cell carcinoma (OSCC) represents more than 90% of all oral cancers [2]. Smoking and excessive alcohol consumption are the major risk factors for the development of OSCC [2]. The clinical manifestations of OSCC can negatively influence the quality of life of patients, causing dysfunction in talking and swallowing, sensory impairment, and chronic pain [3]. Currently, the treatment for oral cancer includes surgical resection of the tumor followed by postoperative adjuvant therapy [3]. However, the survival rate for patients with OSCC still remains low [1]; consequently, identifying new therapeutic targets and new molecules able to reduce or arrest the progression of oral cancer is an important goal in cancer research. Evidence demonstrates that excessive innate immune system activation may contribute to various human malignancies, including oral cancer [4,5]. The nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) are a family of intracellular innate immune receptors that play a key role in the inflammatory response and the innate immune Our results show that BAY-117082 treatment only at concentrations of 5, 10, and 30 µM significantly reduced CAL27, HSC-2, and SCC-4 cell viability in the same way, as shown in Figure 1.
Based on the MTT results, we decided to investigate in another analysis only BAY-117082 at concentrations of 5, 10, and 30 µM, because these represented the most cytotoxic concentrations.
Furthermore, since BAY-117082 showed similar effects on cell viability in all three cell lines, we decided to continue to investigate its effect on only the CAL27 cell line, because it represented a frequently used cell line in the field of OSCC [18,19]. Based on the MTT results, we decided to investigate in another analysis only BAY-117082 at concentrations of 5, 10, and 30 µM, because these represented the most cytotoxic concentrations.
Furthermore, since BAY-117082 showed similar effects on cell viability in all three cell lines, we decided to continue to investigate its effect on only the CAL27 cell line, because it represented a frequently used cell line in the field of OSCC [18,19].

BAY-117082 Reduced NLRP3 Inflammasome Pathway Activation
Inflammasome is a cytoplasmic multi-protein complex that regulates innate immunity response through ASC and caspase-1 activation [6]. NLRP3 inflammasome is reported to be abnormally expressed and activated in malignancies such as cancer, favoring its progression [20]. Therefore, in this study we decided to investigate the NLRP3 inflammasome pathway by Western blot analysis in CAL27 cell lysates. Our results show that the control group was characterized by elevated NLRP3, ASC, and caspase-1 expression, while BAY-117082 treatment only at concentrations of 10 and 30 µM significantly reduced their expression compared to control (Figures 2A,B and 3A). The data for caspase-1 were also confirmed by immunofluorescence staining, showing that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly reduced its expression compared to control (Figure 3B-E; also see caspase-1 positive cell score, Figure 3F).

BAY-117082 Reduced NLRP3 Inflammasome Pathway Activation
Inflammasome is a cytoplasmic multi-protein complex that regulates innate immunity response through ASC and caspase-1 activation [6]. NLRP3 inflammasome is reported to be abnormally expressed and activated in malignancies such as cancer, favoring its progression [20]. Therefore, in this study we decided to investigate the NLRP3 inflammasome pathway by Western blot analysis in CAL27 cell lysates. Our results show that the control group was characterized by elevated NLRP3, ASC, and caspase-1 expression, while BAY-117082 treatment only at concentrations of 10 and 30 µM significantly reduced their expression compared to control (Figure 2A,B and Figure 3A). The data for caspase-1 were also confirmed by immunofluorescence staining, showing that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly reduced its expression compared to control (Figure 3B-E; also see caspase-1 positive cell score, Figure 3F).
Once activated, NLRP3 inflammasome induces the release of pro-inflammatory cytokines IL-1β and IL-18, which can contribute to cancer progression [21,22]. Thus, in this study we evaluated IL-1β and IL-18 expression by Western blot analysis in CAL27 cell lysates. Our results show that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly reduced their expression compared to the control group ( Figure 4).  Once activated, NLRP3 inflammasome induces the release of pro-inflammatory cytokines IL-1β and IL-18, which can contribute to cancer progression [21,22]. Thus, in this study we evaluated IL-1β and IL-18 expression by Western blot analysis in CAL27 cell lysates. Our results show that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly reduced their expression compared to the control group ( Figure 4).  Once activated, NLRP3 inflammasome induces the release of pro-inflammatory cytokines IL-1β and IL-18, which can contribute to cancer progression [21,22]. Thus, in this study we evaluated IL-1β and IL-18 expression by Western blot analysis in CAL27 cell lysates. Our results show that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly reduced their expression compared to the control group ( Figure 4).

BAY-117082 Modulated Apoptosis Pathway
Apoptosis is a physiological process of programmed cell death that is essential for normal tissue development and cell hemostasis [9]. Dysregulation in the apoptotic process has been associated with several diseases, including oral cancer [9]. Therefore, we decided to investigate the apoptotic markers Bax, Bad, and Bcl2 by Western blot analysis in CAL27 cell lysates. Our results show that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly increased pro-apoptotic Bax and Bad expression compared to the control group ( Figure 5A,B), while anti-apoptotic Bcl2 expression was significantly reduced following BAY-117082 treatment compared to control ( Figure 5C).

BAY-117082 Modulated Apoptosis Pathway
Apoptosis is a physiological process of programmed cell death that is essential for normal tissue development and cell hemostasis [9]. Dysregulation in the apoptotic process has been associated with several diseases, including oral cancer [9]. Therefore, we decided to investigate the apoptotic markers Bax, Bad, and Bcl2 by Western blot analysis in CAL27 cell lysates. Our results show that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly increased pro-apoptotic Bax and Bad expression compared to the control group ( Figure 5A,B), while anti-apoptotic Bcl2 expression was significantly reduced following BAY-117082 treatment compared to control ( Figure 5C).

BAY-117082 Modulated Apoptosis Pathway
Apoptosis is a physiological process of programmed cell death that is essential for normal tissue development and cell hemostasis [9]. Dysregulation in the apoptotic process has been associated with several diseases, including oral cancer [9]. Therefore, we decided to investigate the apoptotic markers Bax, Bad, and Bcl2 by Western blot analysis in CAL27 cell lysates. Our results show that treatment with BAY-117082 only at concentrations of 10 and 30 µM significantly increased pro-apoptotic Bax and Bad expression compared to the control group ( Figure 5A,B), while anti-apoptotic Bcl2 expression was significantly reduced following BAY-117082 treatment compared to control ( Figure 5C).  Recent studies highlighted the role of pro-apoptotic p53 protein in modulating various cellular processes such as metabolism, metastasis, and communication within the tumor microenvironment [23]. p53 deficiency and altered apoptosis can enhance the initiation or progression of cancer [23]. Thus, in this study we decided to investigate p53 expression by immunofluorescence assay in CAL27 cells.
Our results demonstrate that the control group was characterized by low p53 expression, while treatment with BAY-117082 at concentrations of 10 and 30 µM significantly increased pro-apoptotic p53 expression ( Figure 6A-D; also see p53 positive cell score, Figure 6E).
Recent studies highlighted the role of pro-apoptotic p53 protein in modulating v ous cellular processes such as metabolism, metastasis, and communication within the mor microenvironment [23]. p53 deficiency and altered apoptosis can enhance the in tion or progression of cancer [23]. Thus, in this study we decided to investigate p53 pression by immunofluorescence assay in CAL27 cells.
Our results demonstrate that the control group was characterized by low p53 exp sion, while treatment with BAY-117082 at concentrations of 10 and 30 µM significa increased pro-apoptotic p53 expression (Figure 6A-D; also see p53 positive cell score, ure 6E).

BAY-117082 Reduced Tumor Growth
To evaluate the effect of BAY-117082 on the growth of OSCC cells in vivo, the CA xenograft model was established in nude mice. In this context, our results show that tr ment with BAY-117082 at doses of 2.5 and 5 mg/kg significantly reduced subcutane tumor mass and neutrophilic infiltration in a dose-dependent manner ( Figure 7A Moreover, BAY-117082 at these two doses significantly reduced tumor burden and tum weight in a dose-dependent manner compared to the control group ( Figure 7D,E). Dur the course of treatment, no important change in the animals' weight was seen (Figure

BAY-117082 Reduced Tumor Growth
To evaluate the effect of BAY-117082 on the growth of OSCC cells in vivo, the CAL27 xenograft model was established in nude mice. In this context, our results show that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg significantly reduced subcutaneous tumor mass and neutrophilic infiltration in a dose-dependent manner ( Figure 7A-C). Moreover, BAY-117082 at these two doses significantly reduced tumor burden and tumor weight in a dose-dependent manner compared to the control group ( Figure 7D,E). During the course of treatment, no important change in the animals' weight was seen ( Figure 7F).

BAY-117082 Reduced NLRP3 Inflammasome Activation in CAL27 Xenograft Model
To confirm the effect of BAY-117082 on the NLRP3 inflammasome pathway, we decided to also investigate this pathway in the xenograft model by Western blot analysis. The results show that the control group was characterized by high expression of NLRP3, ASC, and caspase-1; however, treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce their expression ( Figure 8).

BAY-117082 Reduced NLRP3 Inflammasome Activation in CAL27 Xenograft Model
To confirm the effect of BAY-117082 on the NLRP3 inflammasome pathway, we decided to also investigate this pathway in the xenograft model by Western blot analysis. The results show that the control group was characterized by high expression of NLRP3, ASC, and caspase-1; however, treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce their expression ( Figure 8).

BAY-117082 Reduced NLRP3 Inflammasome Activation in CAL27 Xenograft Model
To confirm the effect of BAY-117082 on the NLRP3 inflammasome pathway, we decided to also investigate this pathway in the xenograft model by Western blot analysis. The results show that the control group was characterized by high expression of NLRP3, ASC, and caspase-1; however, treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce their expression (Figure 8).  Moreover, we decided to investigate, by immunohistochemistry staining, the expression of IL-1β, a pro-inflammatory cytokine related to cancer progression. Our results show that the control group was characterized by marked expression of IL-1β, while treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to reduce its expression in a dose-dependent manner (Figure 9), confirming the previously obtained in vitro data.
Moreover, we decided to investigate, by immunohistochemistry staining, the expression of IL-1β, a pro-inflammatory cytokine related to cancer progression. Our results show that the control group was characterized by marked expression of IL-1β, while treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to reduce its expression in a dosedependent manner (Figure 9), confirming the previously obtained in vitro data.

BAY-117082 Modulated NF-κB/IκB-α Pathway in CAL27 Xenograft Model
Accumulating evidence has suggested that the nuclear factor-κB (NF-κB) signaling pathway plays a critical role in oral carcinogenesis [24,25]. It has been demonstrated that NF-κB pathway activation promotes initiation and progression of oral cancer, increasing metastasis and cell invasion [25]. Therefore, we decided to evaluate the NF-κB/IκB-α pathway by Western blot analysis. Our data reveal that BAY-117082 at doses of 2.5 and 5 mg/kg was able to reduce NF-κB translocation into the nucleus and restore IκB-α cytosolic expression in a dose-dependent manner compared to the control group ( Figure 10).

BAY-117082 Modulated NF-κB/IκB-α Pathway in CAL27 Xenograft Model
Accumulating evidence has suggested that the nuclear factor-κB (NF-κB) signaling pathway plays a critical role in oral carcinogenesis [24,25]. It has been demonstrated that NF-κB pathway activation promotes initiation and progression of oral cancer, increasing metastasis and cell invasion [25]. Therefore, we decided to evaluate the NF-κB/IκB-α pathway by Western blot analysis. Our data reveal that BAY-117082 at doses of 2.5 and 5 mg/kg was able to reduce NF-κB translocation into the nucleus and restore IκB-α cytosolic expression in a dose-dependent manner compared to the control group ( Figure 10).

BAY-117082 Modulated CD4, CD8, and CD30 Expression in CAL27 Xenograft Model
Cancer development and its response to therapy are strongly influenced by innate and adaptive immunity, which can promote or attenuate tumorigenesis with opposite effects on the therapeutic outcome [26]. It has been demonstrated that aberrant immune system activation can influence the carcinogenesis process [26]. Therefore, in this study we decided to evaluate the effect of BAY-117082 on the immune system by evaluating CD4, CD8, and CD30 expression. Our data demonstrate that BAY-11082 treatment at doses of 2.5 and 5 mg/kg significantly reduced CD4, CD8, and C30 levels compared to the control group in a dose-dependent manner (Figure 11).

BAY-117082 Modulated Apoptosis in CAL27 Xenograft Model
Apoptosis plays a key role in cancer progression, therefore targeting apoptosis could be a relevant therapeutic approach in anti-cancer drug development [10]. Thus, we decided to also investigate in the xenograft model the effect of BAY-117082 on the apoptosis pathway, evaluating the apoptotic markers Bax, Bcl2, and Bcl-xL by Western blot analysis. Our results demonstrate that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to increase pro-apoptotic Bax protein expression and reduce anti-apoptotic Bcl2 and Bcl-xL protein expression in a dose-dependent manner compared to the control group (Figure 12), confirming the previously obtained in vitro data.

BAY-117082 Modulated Apoptosis in CAL27 Xenograft Model
Apoptosis plays a key role in cancer progression, therefore targeting apoptosis could be a relevant therapeutic approach in anti-cancer drug development [10]. Thus, we decided to also investigate in the xenograft model the effect of BAY-117082 on the apoptosis pathway, evaluating the apoptotic markers Bax, Bcl2, and Bcl-xL by Western blot analysis. Our results demonstrate that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to increase pro-apoptotic Bax protein expression and reduce anti-apoptotic Bcl2 and Bcl-xL protein expression in a dose-dependent manner compared to the control group (Figure 12), confirming the previously obtained in vitro data.

BAY-117082 Reduced Ki-67 Expression in CAL27 Xenograft Model
Ki-67 is a DNA-binding nuclear protein involved in cell proliferation [27], and is widely used as a prognostic and predictive indicator of cancer progression [28]. Clinically, Ki-67 has also been shown to correlate with metastasis and the clinical stage of tumors [27]. Therefore, in this study we decided to investigate Ki-67 expression as a marker of cancer cell proliferation by immunohistochemistry staining. Our data show that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce Ki-67 expression compared to the control group ( Figure 13A-C).

BAY-117082 Reduced Ki-67 Expression in CAL27 Xenograft Model
Ki-67 is a DNA-binding nuclear protein involved in cell proliferation [27], and is widely used as a prognostic and predictive indicator of cancer progression [28]. Clinically, Ki-67 has also been shown to correlate with metastasis and the clinical stage of tumors [27]. Therefore, in this study we decided to investigate Ki-67 expression as a marker of cancer cell proliferation by immunohistochemistry staining. Our data show that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce Ki-67 expression compared to the control group ( Figure 13A-C).

BAY-117082 Reduced Ki-67 Expression in CAL27 Xenograft Model
Ki-67 is a DNA-binding nuclear protein involved in cell proliferation [27], and is widely used as a prognostic and predictive indicator of cancer progression [28]. Clinically, Ki-67 has also been shown to correlate with metastasis and the clinical stage of tumors [27]. Therefore, in this study we decided to investigate Ki-67 expression as a marker of cancer cell proliferation by immunohistochemistry staining. Our data show that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce Ki-67 expression compared to the control group ( Figure 13A-C).

Discussion
Oral squamous cell carcinoma (OSCC), the most common oral cancer, arises from the mucosal lining of the oral cavity, with an incidence of 450,000 new cases per year [2]. Major risk factors for OSCC development include smoking and excessive alcohol consumption, but other factors such as human papillomavirus (HPV), nutritional deficiencies, and genomic alterations have also been associated [2,29]. Clinical treatment of oral cancer includes surgical resection, chemotherapy, and radiotherapy, however, additional therapies are needed [1]. Previous studies have demonstrated that aberrant innate immune system activation may promote oral carcinogenesis [21,30]. Inflammasomes are multiprotein complexes that are involved in the innate immune system and the inflammatory response [31]. Inflammasomes belong to a larger family of receptors known as pattern recognition receptors (PRRs); their function is to recognize pathogen-or danger-associated molecular patterns (PAMPs or DAMPs), causing the activation, maturation, and production of pro-inflammatory cytokines [32]. NLRP3 inflammasome is one of the most studied inflammasomes belonging to the NLR protein family [7].
Although the pathophysiology of oral cancer remains unclear, in vivo and in vitro studies have demonstrated that aberrant and excessive NLRP3 inflammasome activation significantly contributes to the initiation and progression of oral cancer [5,20]. Moreover, it has been proven that cell apoptosis also has a predominant role in oral cancer [10]. Specifically, evidence suggests that dysregulation in the apoptosis process may induce cancer progression, promoting cancer cell survival [9,33]. Recently, more attention has been given to the potential application of NLRP3 inhibitors for the treatment of cancer. BAY-117082, a sulfonic derivate, is a strong inhibitor of NLRP3 inflammasome, with neuroprotective and anti-inflammatory effects [6, 14,16]. An important study conducted by Chen et al. demonstrated that BAY-117082 exerts an anti-tumor effect through mitochondrial pathway modulation, suggesting its possible use as a promising treatment for cancer [17]. Therefore, based on the key roles of the NLRP3 inflammasome and apoptosis pathways in oral carcinogenesis, in this study we decided to evaluate the beneficial effect of BAY-117082 in in vitro and in vivo xenograft models of oral cancer.
First of all, we evaluated the cytotoxic effect of BAY-117082 at different concentrations in an in vitro model of OSCC using CAL27, HSC-2, and SCC-4 cell cultures. Clearly, our results demonstrate that BAY-117082 treatment only at higher concentrations significantly reduced CAL27, HSC-2, and SCC-4 cell viability in the same way.
Many papers have proven that NLRP3 inflammasome plays a key role in oral carcinogenesis [5,21]. NLRP3 inflammasome regulates innate immunity response through ASC and caspase-1 activation, consequently promoting the inflammatory response [6].
However, recent studies have demonstrated that excessive and aberrant NLRP3 inflammasome activation may contribute to oral cancer progression [4,20]. Thus, we decided to investigate the effect of BAY-117082 on the NLRP3 inflammasome pathway, showing that BAY-117082 at higher concentrations was able to significantly reduce NLRP3, ASC, and caspase-1 expression compared to the control group in CAL27 cell lysates.
Once activated in response to intracellular stimuli, NLRP3 inflammasome promotes the proteolytic processing of pro-IL-1β and pro-IL-18 into their bioactive forms IL-1β and IL-18, respectively [34]. Overexpression of pro-inflammatory cytokines such as IL-1β and IL-18 may promote cancer progression, increasing an excessive inflammatory response [22]. Our results demonstrate that the control group was characterized by marked expression of IL-1β and IL-18, whereas treatment with BAY-117082 at higher concentrations significantly reduced their expression compared to the control.
Among the mechanisms involved in cancer pathogenesis, apoptosis also has a fundamental role [33,35]. Programmed cell death occurs through two main signaling pathways, one defined as intrinsic and the other extrinsic [35]. The intrinsic pathway is regulated by the balance of two proteins, Bax and Bcl-2; Bax has a pro-apoptotic role, while Bcl-2 exerts anti-apoptotic activity. On the other hand, the extrinsic pathway is regulated by caspase cascade activation, which stimulates chromatin fragmentation [36,37]. Apoptosis appears to be a very interesting and complex phenomenon, thus further investigation into its mechanism is needed to discover and develop novel agents that could increase the sensitivity of cancer cells to programmed cell death [38]. Therefore, in this study we decided to investigate the effect of BAY-117082 on the apoptosis pathway, evaluating the pro-apoptotic markers Bax, Bad, and p53 and anti-apoptotic marker Bcl2. Our results show that the control group was characterized by low expression of Bax, Bad, and p53, whereas treatment with BAY-117082 at higher concentrations significantly increased their expression. Moreover, BAY-117082 treatment significantly reduced anti-apoptotic marker Bcl2 expression compared to the control group.
Despite the promising results obtained with an in vitro model on CAL27 cell culture, we decided to construct an in vivo xenograft model of oral cancer to confirm the beneficial effect of BAY-117082.
Our data demonstrate that BAY-117082 treatment significantly reduced subcutaneous tumor mass and tumor necrosis compared to the control group in a dose-dependent manner. Additionally, treatment with BAY-117082 significantly decreased tumor burden and tumor weight compared to the control group in a dose-dependent manner, without changing the animals' weight.
Moreover, considering the key role of NLRP3 inflammasome in oral carcinogenesis [20], we also investigated the effect of BAY-117082 in an in vivo xenograft model. Our results confirm that BAY-117082 was able to significantly reduce NLRP3 activation, also decreasing ASC and caspase-1 expression compared to the control group. Additionally, we investigated the expression of pro-inflammatory cytokine IL-1β, which is involved in the NLRP3 inflammasome pathway [39], showing that treatment with BAY-117082 was able to significantly reduce its expression in a dose-dependent manner.
It has been demonstrated that the NF-κB signaling pathway has a fundamental role in oral cancer pathogenesis [24]. NF-κB is a transcription factor that regulates different biological processes, including cell growth, proliferation, and apoptosis [25]. However, abnormal activation of NF-κB contributes to the development of various autoimmune, inflammatory, and malignant diseases such as cancer, promoting metastasis and invasiveness [25]. Thus, in this study we decided to evaluate the NF-κB/IκB-α signaling pathway, demonstrating that treatment with BAY-117082 significantly reduced NF-κB translocation into the nucleus and restored IκB-α cytosolic expression in a dose-dependent manner.
In addition, NF-κB plays a critical role in regulating the activation and differentiation of innate immune cells and inflammatory T cells [40]. In a malignant disease such as cancer, the response to chemotherapy is influenced by immune system activation, which can promote or attenuate tumorigenesis [26]. Recently, emphasis is increasingly being placed on the role of the immune system and its association with the occurrence and progression of cancer [41]. Despite the impressive successes in cancer immunotherapy, the response in patients is sometimes short-lived [41]. This is due to factors that hamper the immune response against cancer, such as the excessive and aberrant presence of CD4 T and CD8 T cells in the tumor microenvironment [41]. Therefore, based on these findings, we decided to investigate the effect of BAY-117082 on the immune system by evaluating CD4, CD8, and CD30 expression. In this context, our results demonstrate that BAY-117082 treatment reduced CD4, CD8, and CD30 levels compared to the control group in a dosedependent manner.
Furthermore, to confirm the data obtained in the in vitro model with regard to the apoptosis pathway, we decided to also investigate the effect of BAY-117082 on programmed cell death in the xenograft model, demonstrating that it was able to significantly increase pro-apoptotic Bax protein expression, while anti-apoptotic Bcl2 and Bcl-xL were significantly reduced following BAY-117082 treatment in a dose-dependent manner, in contrast to cell proliferation.
Another important hallmark of cancer is cell-cycle dysregulation [42]. During oral carcinogenesis, growth signaling become dysregulated and cancer cells can proliferate without exogenous stimulation [43]. Ki-67 is a nuclear protein closely associated with cell proliferation [28]. It is exclusively present within the nucleus during the interphase, and since it is present during all phases of the cell cycle (G1, S, G2, mitosis), it may represent a useful marker of cell growth [44]. In the last decade, Ki-67 has been widely used as a prognostic marker for cancer progression [28].
Therefore, in this study we decided to investigate Ki-67 expression, showing that the control group was characterized by elevated Ki-67 expression, whereas treatment with BAY-117082 was able to significantly reduce its appearance.
Thus, based on the results obtained, using BAY-117082 could be considered as a valid therapeutic strategy to reduce or counteract OSCC progression thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways. However, further investigations are needed to better understand the involvement of these pathways in oral carcinogenesis.
BALB/c nude male mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and housed in microisolator cages under pathogen-free conditions with 12 h light/12 h dark. Animals were fed with a standard diet and water ad libitum. This study was approved by the University of Messina Review Board, under project identification code 137/2017-PR, released on 9 February 2017. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (DM 116192) as well as EU regulations (OJ of EC L 358/1 18 December 1986).

Xenograft Tumor Model
The xenograft tumor model was established by subcutaneously inoculating 3 × 10 6 CAL27 cells per tumor in 0.2 mL of PBS and 0.1 mL Matrigel (BD Bioscience, Bedford, MA, USA) as previously described [47]. After tumor cell inoculation, animals were monitored daily for morbidity and mortality, and their body weight was monitored weekly to evaluate overall health. After 1 week of tumor induction, mice were divided randomly into 3 groups. When tumor size reached about 200-300 mm 3 , mice were treated with BAY-117082 at doses of 2.5 and 5 mg/kg every 3 days according to [17,48]. BAY-117082 was dissolved in PBS with 0.001% DMSO. The tumor size was monitored daily by a caliper and calculated as follows: V = W 2 × L/2, where W and L represent minor and major length. After 30 days, mice were sacrificed and tumors were excised and processed for analysis.

Experimental Groups
Mice were randomly divided into 3 groups: Santa Cruz Biotechnology, Dallas, TX, USA) in 1× PBS, 5% w/v dried nonfat milk and 0.1% Tween-20 (PMT) at 4 • C overnight. Membranes were incubated with peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) or peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. To establish that blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against β-actin protein (sc-8432, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA). Signals were revealed with an enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer's instructions (Thermo, Waltham, MO, USA, cat# 457). The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS+ software and standardized to β-actin levels as an internal control.

Materials
All compounds and other chemicals were obtained from Sigma-Aldrich (Milan, Italy). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Milan, Italy).

Statistical Analysis
Data were analyzed with GraphPad Prism 7.04 software using t-test analysis. All values are indicated as mean ± standard error of the mean (SEM) of N observations.

Conclusions
In conclusion, the results obtained offer new insights into the role of the NLRP3 inflammasome and apoptosis signaling pathways in oral carcinogenesis, showing that the use of BAY-117082, a strong NLRP3 inflammasome inhibitor, could represent a potential therapeutic treatment to counteract or reduce the progression of OSCC, which has been showing an increasing mortality rate.

Data Availability Statement:
The authors declare that all data and materials supporting the findings of this study are available within the article. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.