The Endocannabinoid, Anandamide, Acts as a Novel Inhibitor of LPS-Induced Inflammasome Activation in Human Gastric Cancer AGS Cell Line: Involvement of CB1 and TRPV1 Receptors

In ﬂ ammasome activation is a pivotal step for the maturation of IL-1 β , which is involved in the development and progression of gastric cancer (GC). Endocannabinoids, such as anandamide (AEA), are emerging as new anticancer therapeutic agents; however, their e ﬀ ects on in ﬂ ammasome components and underlying mechanisms have not been well elucidated. This study was designed to investigate the e ﬀ ects of AEA on the expression of in ﬂ ammasome components in lipopolysaccharide- (LPS-) stimulated AGS cells. Moreover, we explored the involvement of cannabinoid receptors (CRs), including CB1R and TRPV1R, in the observed e ﬀ ects of AEA. Our results showed that in ﬂ ammation was induced by LPS (10 μ g/ml) in AGS cells, and in ﬂ ammasome components (NLRP3, MLRC4, ASC, IL-18, and IL-1 β ) were overexpressed. Exposure to AEA (10 μ M, 24h) before or after in ﬂ ammation induction downregulated the expression of in ﬂ ammasome components and attenuated in ﬂ ammasome activation as demonstrated by cleavage of caspase 1 and matured IL-1 β secretion, although AEA pretreatment showed more reducing e ﬀ ects on the in ﬂ ammasome activation. In addition, blocking of CB1R and TRPV1R by application of AM-251 and AMG-9810 antagonists remarkably reversed the observed e ﬀ ects of AEA and revealed that NLRP3, NLRC4, and IL-1 β genes were mainly regulated via CB1R, while TRPV1R could only regulate the expression of IL-1 β and IL-18 genes. In conclusion, our results would indicate a novel anticancer e ﬀ ect of anandamide by attenuation of in ﬂ ammasome activation and consequently reducing IL-1 β production in human AGS cancer cell line via CB1R and TRPV1R.


Introduction
Gastric cancer (GC) is the fourth frequent malignancy and the second cause of cancer-related death worldwide [1,2]. Chronic inflammation has been accepted as one of the main causes for development and progression of GC [3]. Stomach infections, such as Helicobacter pylori, and chronic gastritis present strong etiological links between inflammation and gastric tumorigenesis [3]. Inflammasomes are intracellular protein complexes that drive inflammatory reactions in response to harmful stimuli such as pathogens. Inflammasomes are made up of NLR family pyrin domain-containing 1 (NLRP1), NLRP3, NLR family CARD domain-containing 4 (NLRC4), and absent in melanoma 2 (AIM 2) which consider as inflammasome sensor molecules, apoptosisassociated speck-like protein (ASC) in the role of an adaptor protein, and caspase 1 as an end activator of inflammasome pathway [4]. Upon these protein complexes formed, inflammasome switches caspase 1 to its active form and cleaves proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18, to their mature forms [4].
IL-1β not only is expressed highly in GC tissues but also correlates with clinical and pathological features of GC [5]. Stomach-specific overexpression of human IL-1β in transgenic mice leads to spontaneous gastric inflammation and cancer [5]. Increased levels of IL-1β are believed to contribute to the signs of inflammation such as the development of severe gastritis [6]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are mostly used to alleviate inflammation [7]. Despite their effectiveness, NSAID treatment may induce severe side effects such as gastrointestinal damage and respiratory abnormalities [8]. More recently, there has been increasing interest in finding more compatible compounds to alleviate gastric inflammation [9,10].
Endocannabinoids are a group of lipid neurotransmitters found in several tissues [11]. They are a subclass of cannabinoids, the active compounds of Cannabis sativa [11]. N-Arachidonoylethanolamide (anandamide, AEA) and 2arachidonylglicerol (2-AG) are two main endocannabinoids described to now. Since the 1970s, when it was found that cannabinoids inhibited Lewis lung adenocarcinoma growth, the anticancer effects of these compounds have received increasing attention [12,13]. Cannabinoids usually act via two distinct receptors including CB1 and CB2 receptors (CB1R and CB2R). CB1Rs are predominantly found in the peripheral and central nervous system, and CB2Rs are mostly expressed in the immune cells [14]. AEA can bind and activate both CB1 and CB2 receptors, but shows less intrinsic activity when binding CB2 receptor [15,16]. In recent years, a great deal of evidence has been shown that anandamide also activates the transient receptor potential type 1 receptor (TRPV1R) [17]. Recent investigations have shown that cannabinoid agonists induced cancer cell apoptosis [18] and enhanced anti-inflammatory cytokine production [19]. Nevertheless, the underlying molecular mechanisms in GC are still not fully understood.
Up to now, there is no data available about the involvement of endocannabinoids and inflammasome in GC. In light of these data, to identify the relationship among endocannabinoid system, inflammasome, and IL-1β secretion, this study was designed to characterize whether and how anandamide can influence inflammasome component transcription and IL-1β secretion in AGS cell line. Our results might provide more evidence for the therapeutic value of endocannabinoid-based therapies for the treatment of GC in the future.

Materials and Methods
2.1. Cell Line, Culture Conditions, and Preparation of Chemical Regents. The AGS human GC cell line was obtained from Pasteur Institute Cell Bank (Tehran, Iran) and cultured in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum (FBS) and 1% penicillinstreptomycin (Gibco, Carlsbad, CA, USA) in a CO 2 incubator at 37°C with 99% humidity and 5% CO 2 . The cells were starved 6 h with serum-free RPMI-1640 before treatment, and all treatments except LPS and ATP induction were performed in RPMI-1640 medium supplemented with 2% FBS. AEA (Sigma-Aldrich, MO, USA) was dissolved in absolute ethanol and made aliquots in dark microtubes and stored at -20°C. AM-251 and AMG-9810 (Tocris Bioscience, Bristol, UK) were dissolved in an absolute ethanol and stored in a refrigerator (2-8°C).
2.2. LPS-Induced Inflammation in AGS Cells. Inflammation was induced using LPS (Escherichia coli O111:B4; Sigma-Aldrich, MO, USA) as described previously [20]. Briefly, AGS cells were seeded in a 6-well plate at a density of 7 × 10 5 cells/well and cultured overnight; then, they were induced with LPS at the dose of 10 μg/ml for 6 h and treated with ATP (Honeywell Fluka™, Shanghai, China) at the final concentration of 5 mM for 30 min.
2.3. Cell Viability Assays. The viability of AGS cells was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. AGS cells were cultured in 96well culture plates (8 × 10 3 cells/well) in RPMI-1640 medium. The cells from the overnight culture were treated with LPS (10 μg/ml) for 6 h and various concentrations of AEA (2.5-40 μM) (Sigma-Aldrich, MO, USA) for 12 and 24 h. Ten microliters of MTT solution (5 mg/ml) (Sigma-Aldrich, MO, USA) was added to each well; after incubation at 37°C for 3 h, the medium was replaced with 100 μl of dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries, Ltd.). The absorbance of all wells was measured at 570 nm using a microplate reader (BioTek, USA). Data were displayed as the percentage of viable cells in treated groups compared to vehicle-treated cells and resulted from three independent experiments with triplicated wells.

Experimental
Design. AGS cells were seeded at a density of 7 × 10 5 cells/well in 6-well plates and divided into four groups including control, LPS, pretreatment (RT), and posttreatment (OT) groups. The blank control cells were cultured in RPMI-1640 medium and treated only with ATP (5 mM, 30 min). Cells in LPS control group were treated with LPS (10 μg/ml, 6 h) and ATP (5 mM, 30 min). In RT group, the cells that had been treated with 10 μM AEA for 24 h induced with LPS (10 μg/ml, 6 h) and ATP (5 mM, 30 min), and the cells in OT group were first treated with LPS (10 μg/ml, 6 h) and ATP (5 mM, 30 min) and then treated with AEA (10 μM, 24 h). At the end of experiments, cell-free supernatants and cell pellets were collected and stored at -70°C for subsequent analyses. Schematic diagram of our experimental protocol is shown in Figure 1. Mediators of Inflammation AEA on inflammasome components in RT group, inhibition of cannabinoid receptors was conducted only in this experimental group. Briefly, the cells were seeded in 6-well plates and pretreated with AM-251 (1 μM, 1 h) and AMG-9810 (1 μM, 30 min). Following AEA treatment (10 μM, 24 h) and LPS induction, cell-free supernatants and cell pellets were collected and stored at -70°C for subsequent analysis.
2.6. RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR (qRT-PCR). Total cellular RNA was extracted using Tripure Isolation Reagent (Roche, Basel, Switzerland) according to the manufacturer's procedure. All extractions were followed by DNase I (Thermo Fisher Scientific, Massachusetts, USA) treatment to remove contaminating genomic DNA. First-strand cDNA was synthesized from 2 μg RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc, Massachusetts, USA) with oligo (dT) and random hexamer primers according to manufacturer's protocol. Afterward, cDNA was amplified for qRT-PCR by the qPCR™ Green Master Kit for SYBR Green I® (Yektatajhiz, Tehran, Iran) using specific primers ( Table 1). The relative expression level of the target genes was compared to beta2-microglobulin (B2M) as a housekeeping gene. All reac-tions were performed in triplicate. Relative quantification was performed according to the comparative 2 -ΔΔCt method. Validation of assay to check that the primer for target genes and housekeeping gene had similar amplification efficiencies was performed as described previously [21].

Statistical Analysis.
All experiments reported in the present study were performed at least in triplicate. The data are reported as mean ± standard error of the mean ðSEMÞ. The statistical significance of the differences between groups was compared using the Student's t-test and one-way analysis of variance (ANOVA), and subsequent post hoc analysis was performed using Tukey's test. Differences with p < 0:05 were considered statistically significant.

Results
3.1. AEA Decreased Cell Viability Dose-Dependently. AGS cells were treated with LPS and/or AEA for 12 and 24 h, and the viability of cells was assayed using MTT assay. As exhibited in Figure 2, AEA showed a dose-dependent inhibitory effect on LPS-stimulated AGS cells (p < 0:05). The viability of AGS cells had no significant differences between 12 and 24 h exposure times, and therefore, the subsequent experiments were performed using 10 μM AEA for 24 h.

AEA Suppressed Inflammasome
Activation. The secretion of IL-1β and the activity of caspase 1 were determined in the medium or cell lysate of LPS-stimulated cells. The results revealed that the secretion of IL-1β and the activity of caspase 1 were increased after stimulation of AGS cells by 10 μg/ml LPS (p < 0:01; p < 0:05) ( Figure 4). Additionally, as shown in Figure 4, treatment of AGS cells with AEA significantly reduced IL-1β secretion in the RT group (p < 0:05) and caspase 1 activity (p < 0:01; p < 0:05) in both experimental groups. AEA in RT group had more reducing effects on IL-1β secretion (p < 0:05) and caspase 1 activity (p < 0:001) compared with OT group.

CB1 and TRPV1 Receptors Blockade
Reversed AEA-Mediated Inflammasome Inhibition. The effective concentrations of two CBR antagonists were selected based on IL-1β secretion in the culture media using ELISA. AM-251 reversed the AEA effect on IL-1β secretion dose-dependently, and hence, the highest concentration of AM-251 was chosen for the next experiments ( Figure 5(a)). Moreover, administration of 1 μM AMG-9810 led to a significant elevation in IL-1β secretion (Figure 5(a)). Accordingly, a concentration of 1 μM of AMG-9810 and AM-251 was selected for subsequent experiments.
Effect of CB1R and TRPV1R blockade using AM-251 and AMG-9810 on mRNA relative expression ( Figure 5(b)) and protein expression (Figure 5(c)) of inflammasome-related genes in AEA-treated AGS cells is shown in Figure 5. The  Mediators of Inflammation results showed that AM-251 reversed AEA action on NLRP3, NLRC4, and IL-1β (p < 0:05) in LPS-stimulated cells, which indicated that AEA regulated the expression of these genes via CB1 receptor. Although, ASC showed an upward trend after AM-251 treatment, the observed change was not significant. AMG-9810 administration inhibited AEA influence on IL-18 and IL-1β (p <0.05) expression in LPS-stimulated cells, while other tested genes demonstrated no significant changes in the presence of AMG-9810 ( Figure 5(b)). As illustrated in Figure 6, measurement of cleaved IL-1β (Figure 6(a)) and the amount of secreted IL-1β in the medium (Figure 6(b)) showed that AM-251 and AMG-9810 could significantly reverse the inhibitory effects of AEA (p < 0:01; p < 0:05). While the expression of procaspase 1 was not altered, the expression of the cleaved caspase 1 (Figure 6(c)) and activity of caspase 1 (Figure 6(d)) was boosted after the application of antagonists in LPSstimulated cells (p < 0:05; p < 0:01), indicated that AEA regulated these alterations via CB1R and TRPV1R.

Discussion
In the present study, we confirmed that the transcription levels of inflammasome component genes, including NLRP3, NLRC4, ASC, IL-1β, and IL-18, were increased in LPSstimulated AGS cells, and AEA significantly diminished the expression of examined genes. This inhibitory effect was observed when LPS-stimulated inflammation was induced before or after treatment of AGS cells with AEA. It was observed that this endogenous cannabinoid reduced LPSenhanced caspase 1 activity and IL-1β production and secretion in AGS cells. However, the application of AEA before LPS-mediated inflammation showed more profound effects on the downregulation of inflammasome-related genes and IL-1β secretion. Furthermore, it was found that the administration of selective CB1R antagonist (AM-251) attenuated AEA-induced downregulation of NLRP3 and NLRC4. Though AM-251 had no significant effect on the expression of IL-18 gene, AMG-9810 elevated its expression via blocking TRPV1R. IL-1β secretion was regulated via both CB1R and TRPV1R. Taken together, our data suggested that CB1R and TRPV1R may contribute to inhibitory effects of AEA on inflammasome activation and secretion of IL-1β in inflammatory AGS cells. expressed as means ± SEM of three independent experiments. * Indicates significantly fold changes compared to group, * p < 0:05, and * * p < 0:01; # compared to LPS group, # p < 0:05, and ## p < 0:01; $ compared to OT group and $ p < 0:05. All experiments were performed in triplicate, and the results were obtained from at least two independent experiments. Data are expressed as means ± SEM. * Indicates significantly changes compared to control group, * p < 0:05, and * * p < 0:1; # compared to LPS group, # p < 0:05 , and ## p < 0:01; $ compared to OT group, $ p < 0:05, and $$$ p < 0:001.

Mediators of Inflammation
Recent studies suggest that inflammasome disorders and abnormal activation of IL-1β are involved in the generation and development of different chronic inflammatory diseases, such as lung cancer [22], diabetic cardiomyopathy [23], and gastric carcinogenesis [24]. Gastric cancer is known as one of the most common and deadly malignancies globally. Over the past decade, many lines of evidence highlighted the role of inflammasome activation and IL-1β hypersecretion on the occurrence and development of GC. Inflammasomes induced such effects mainly through blocking gastric acid secretion, alterations in epigenetics, assisting angiogenesis, and releasing other inflammatory factors [5]. IL-1β not only is expressed highly in GC tissues but also its content is correlated with clinical and pathological features of GC [25,26]. Accordingly, pharmacological manipulation of inflammasome complex and subsequent IL-1β secretion may provide new therapeutic opportunities in further management of the occurrence, development, and metastasis of GC [27,28].
The endocannabinoid system, in particular AEA, has been suggested as a promising therapeutic agent in gastrointestinal cancer therapy [29,30], but their effect on inflammasome activity and subsequent IL-1β secretion in GC cells has not yet been fully uncovered.
Recently, some studies suggested profound antiinflammatory effects of cannabinoids on the modulation of inflammasome signaling, in particular via reduction of IL-1β level, NF-κB, and other inflammasome components [31]. Δ 9 -THC inhibited the secretion of IL-1β in LPSstimulated human osteosarcoma cell line MG-63 via CB2R [32]. CBD suppressed the inflammatory effects of LPSnigericin in THP-1 monocytes via inhibition of NLRP3 inflammasome activation [33]. In line with our observations, inhibitory effects of AEA pretreatment on CCL2 expression in human vascular smooth muscle cells [34], LPS-induced NO release in rat microglial [35], and production of TNF-α and IL-12 p40 in human keratinocytes [36] have been demonstrated. Moreover, the beneficial effects of AEA in various inflammatory disease models, such as LPS-induced pulmonary inflammation [37], liver damage [38], LPS-induced neuroinflammation [35], experimental periodontitis [39], intestinal inflammation [40,41], colonic inflammation [42], and LPS-stimulated changes in circulating cytokines in rat [43], have been reported. Our findings are consistent with previous studies in the literature which suggested endocannabinoids via cannabinoid receptors reduced inflammation and proinflammatory cytokines.
To the best of our knowledge, the present results strongly show the first experimental evidence regarding the inhibitory activity of AEA on LPS-induced inflammasome activation and IL-1β secretion in AGS gastric cancer cells.
There are limited studies regarding the effects of AEA in gastrointestinal inflammatory diseases. D'Argenio and colleagues reported that TNBS-induced colitis was inhibited by AEA treatment [44]. Inhibition of AEA degradation using All experiments were performed in triplicate, and the results were obtained from at least two independent experiments. Data are expressed as means ± SEM. * Indicates significantly changes compared to control group, * p < 0:05, and * * p < 0:01; # compared to LPS group, # p < 0:05, ## p < 0:01; $ compared to RT group and $ p < 0:05.

6
Mediators of Inflammation chemical agents against its degradative enzymes such as fatty acid amide hydrolase or using genetic knockout models improved inflammation [45], and ameliorated anandamide mediated antitumorigenic effect in some cancer models [46]. It was reported that pretreatment with WIN55,212,-2, a synthetic CB1 receptor agonist, inhibited the expansion of stress-induced gastric ulcers in rats [47]. The gastroprotective effect of AEA and reduction of IL-1β in serum have also been reported after exposure of gastric mucosa to AEA in gastric lesions induced by water immersion and restrain stress (WRS) [48]. Furthermore, AEA synergistically augmented paclitaxel-induced apoptosis in gastric cancer cell lines [18].

Conclusion
In conclusion, our findings showed that AEA potentially acted as an anti-inflammatory molecule in AGS cells by inhibition of expression or activation of inflammasome components and secretion of IL-1β via CB1R and TRPV1R. Collectively, our results provide more evidence for the therapeutic value of endocannabinoid-based therapeutics for the treatment of GC.

Data Availability
The data used to support the findings of this study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors declare that they have no conflicts of interest. All experiments were performed in triplicate, and the results were obtained from at least two independent experiments. Data are expressed as means ± SEM. * Indicates significantly changes compared to control group, * p < 0:05, and * * p < 0:01; # compared to LPS group and ## p < 0:01; $ compared to RT group, $ p < 0:05, and $$ p < 0:01.