Glyburide attenuates B(a)p and LPS‐induced inflammation‐related lung tumorigenesis in mice

Glyburide (Gly) could inhibit NLRP3 inflammasome, as well as could be treated with Type 2 diabetes as a common medication. Despite more and more studies show that Gly could influence cancer risk and tumor growth, it remains unclear about the effect of Gly in lung tumorigenesis. To evaluate whether Gly inhibited lung tumorigenesis and explore the possible mechanisms, a benzo(a)pyrene [B(a)p] plus lipopolysaccharide (LPS)‐induced non‐diabetes mice model was established with B(a)p for 4 weeks and once a week (1 mg/mouse), then instilled with LPS for 15 weeks and once every 3 weeks (2.5 μg/mouse) intratracheally. Subsequently, Gly was administered by gavage (10 μl/g body weight) 1 week before B(a)p were given to the mice until the animal model finished (when Gly was first given named Week 0). At the end of the experiment called Week 34, we analyzed the incidence, number and histopathology of lung tumors, and detected the expression of NLRP3, IL‐1β, and Cleaved‐IL‐1β protein. We found that vehicles and tricaprylin+Gly could not cause lung carcinogenesis in the whole process. While the incidence and mean tumor count of mice in B(a)P/LPS+Gly group were decreased compared with B(a)p/LPS group. Moreover, Gly could alleviate inflammatory changes and reduce pathological tumor nest numbers compared with mice administrated with B(a)p/LPS in histopathological examination. The B(a)p/LPS increased the expression of NLRP3, IL‐1β, and Cleaved‐IL‐1β protein significantly than Vehicle, whereas decreased in B(a)P/LPS+Gly (0.96 mg/kg) group compared with B(a)p/LPS group. Results suggested glyburide might inhibit NLRP3 inflammasome to attenuate inflammation‐related lung tumorigenesis caused by intratracheal instillation of B(a)p/LPS in non‐diabetes mice.


| INTRODUCTION
International Agency for Research on Cancer reported that the primary factor of cancer-related morbidity and death was lung cancer, 1 the estimated new cases of lung cancer ranked second and the estimated deaths of lung cancer ranked first in USA, 2020. 2 As an important and most investigated inflammatory regulator, NLRP3 inflammasome has been reported to have a key function in the occurrence of lung carcinoma, and recently studies show that inflammasomes may serve as preventing and treating cancer. 3,4 In our previous articles, we investigated the function of NLRP3 in inflammation-related lung carcinoma initiation, and found that the lack of NLRP3 inhibited B(a)p or B(a)p/LPS-related lung carcinoma initiation, 5 we also revealed that activated NLRP3 inflammasome could act on in lung cancer initiation in inflammation-related mice caused by B(a)p/LPS. 6 Glyburide, a NLRP3 inhibitor, plays an anti-inflammatory role to inhibit the NLRP3 inflammasome, decreases the production of proinflammatory cytokines, reduces the recruitment and migration of inflammatory cells, and production of nitricoxide. 7 In addition, glyburide is a second-generation oral antidiabetic drug known as sulphonylureas, which is one of insulin secretatogues widely used for non-insulin-dependent diabetes mellitus. Growing evidence demonstrated that glyburide played a critical role in antitumor effect through inhibiting KATP channels or ATP-binding cassette super-family. 8 In several clinical studies, there are contradictory results mentioning that sulfonylureas could increase or reduce cancer risks, some research indicated that glyburide was in relation to higher risks of cancer, [9][10][11] such as breast cancer, 12 while according to some other studies had adverse findings that glyburide could reduce cancer risks, 13 such as prostate cancer. 14,15 Furthermore, in some early studies about cancer cells, it demonstrated that glyburide might suppress the initiation of many cancers, such as prostate carcinoma, 16 bladder carcinoma, 17 liver carcinoma, 18,19 or colon carcinoma. 20 Moreover, few in vivo studies have discussed the function of glyburide in lung carcinoma initiation, especially in the non-diabetes mice model. One research observed the impact of chronic glyburide treatment on breast carcinoma growth in prediabetic obese rats. It showed that glyburide treatment alleviated tumor growth by 27% in rats, and demonstrated glyburide might inhibit tumor growth. 21 Another research about the impact of glyburide on mammary cancer induced by N-Nitroso-N-Methylureain diabetes and non-diabetes rats indicating the antitumor action in vivo of glyburide in nondiabetic rats. 22 However, it remains unclear that the role glyburide plays in the initiation of lung cancer.
Our aim was to explore whether glyburide inhibit the occurrence of lung cancer, so we established the inflammation-related lung cancer model in non-diabetes mice which were administrated with B(a)p/LPS as designed in our earlier study, and the glyburide treatment were administered from 1 week before intratracheal instillation with B(a)p.
This study could provide an important guiding significance for the medication with lung carcinoma.  , were administered B(a)p intratracheally for 4 weeks and once a week, and then administered LPS intratracheally for 15 weeks and once every 3 weeks. Glyburide treated during the whole procedure. At Week 34, the mice were anesthesia by penrobarbital sodium (1%), respectively. B(a)p, benzo(a)pyrene; Gly, glyburide; LPS, lipopolysaccharide measured before the modeling process, and were also weighted and measured about once a month in subsequent studies. At Week 34, the mice were anesthesia by penrobarbital sodium (1%) and then sacrificed to harvest lungs and observed the lung surface to count visible tumors. Left lungs of all groups were used for histopathological studies which were fixed by 4% paraformaldehyde, then the right lungs were preserved in À 80 C and used for studies that followed.

| Lung coefficient
At Week 34, the lung tissues were removed from sacrificed mice, washed with normal saline, absorbed moisture by filter paper, next weighted with electronic balance. One of the indicators of lung injury was expressed as the follow equation: Lung coefficient = lung weight/body weight.

| Lung pathological alterations
The left lungs were fixed overnight by 4% paraformaldehyde, then cut into sections (5 μm thickness) after embedded in paraffin. Eventually, the sections were observed by an electron microscope using hematoxylin and eosin (HE) staining. To evaluate the inflammation changes of lung tissues according to the literature published by Huang et al. 5 The pathological tumor nests were identified by two experienced pathologists in a blind manner and based on the previous papers published. 23

| Immunohistochemistry
The protein (NLRP3, IL-1β, IL-18) of lung tissues was determined by immunohistochemistry (IHC). First, the sections were blocked at room temperature for 30 min with goat serum, next, incubated at 4 C overnight with rabbit anti-mouse antibodies at a dilution of 1:100 including NLRP3, IL-1β, and IL-18 (above three antibodies were all from Servicebio, Wuhan, China), the following was incubated at 37 C for 50 min with biotinylated goat anti-rabbit at a dilution of 1:100. Quantification of positive IHC staining were carried out as brown staining and the AOD quantification (AOD = integrated optical density [IOD] SUM/area SUM) was under high-power vision fields reflected by Image-Pro Plus 6.0 software.

| Western blot
RIPA including 100 μg/ml PMSF was used to lyse Mice lung tissues, and BCA Protein Assay Kit (above three reagents were all from CWbio, Beijing, China) was applied to quantify protein concentration, loading buffer was used to mix protein samples and boiled for 5 min subsequently. SDS-PAGE (12 or 15%) was used to isolate proteins and then transferred onto polyvinylidene fluoride membranes. Milk (5%)-TBST was used to block membranes at room temperature for 2 h, the following was incubation with rabbit polyclonal anti-caspase-1 antibody, or mouse polyclonal anti-IL-1β antibody, or mouse polyclonal anti-IL-18 antibody or rabbit polyclonal anti-β-actin antibody (above four antibodies were all from Servicebio, Wuhan, China, 1:1000) at 4 C overnight, then secondary antibodies was applied to incubate membranes using goat anti-rabbit IgG antibody (1:5000) or goat anti-mouse IgG antibody (1:10000) (above two antibodies were both from Jackson ImmunoResearch, America) at room temperature for 1 h. An ECL Western blot detection system was applied to visualizing protein bands, and Image J software was applied to quantify the intensity.

| Statistical analysis
To compare the tumor incidence among different groups using Chisquare test, and the results of mean tumor count, lung coefficient and pathological tumor nests were presented as mean ± SEM, then both one-way analysis of variances and Student's t-test(two-tailed) were used to perform data analysis through SPSS21.0 (IBM, NC, USA). The p value (two-tailed) less than .05 was marked as statistical significantly.

| Effects of glyburide on fasting blood glucose and body weight alteration in mice
To observe the influence of glyburide on FBG and body weight alteration on mice which were administered 0.48 and 0.96 mg/kg glyburide by gavage three times a week, the mice treated with glyburide were fasted overnight, and then the FBG and body weight were measured the next morning about once a month.

| Glyburide treatment attenuates lung tumorigenesis induced by B(a)p plus LPS in mice
To determine if glyburide inhibits the occurrence of lung cancer, the incidence and mean tumor count in mice administrated with Gly or not were compared. Figure 3A

| Expression of Cleaved-IL-1β protein in different lung tissue samples
As shown in Figure 6, comparing with that administrated with Vehicle, cleaved-IL-1β protein level was increased administrated with B(a)p/PS in lung tissues (p < .05), but decreased significantly in B(a)P/LPS +Gly0.96 (mg/kg) group contrasted with B(a)p/LPS group (p < .05).

| DISCUSSION
There has been increasing evidence showing that glyburide is involved in the progression of cancer. However, glyburide has been studied mainly according to its pharmacological effects on the potassium channels but only a little part of its antitumor effects has been paid attention to in nondiabetic models of this drug systematically, also, the evidence of its effect in the occurrence of lung carcinoma remains unclear. Thus, we searched for the effect of glyburide in the occurrence of lung cancer which could contribute to a new therapeutic strategy for lung cancer using the non-diabetes mice model administrated with B(a)p/LPS.
Chronic glyburide treatment decreased the FBG significantly, and mice administrated with B(a)p/LPS obviously decreased weight contrasted with mice administrated with vehicle which may be attributed to the cancer cachexia. 25 Glyburide is treated with diabetes as a kind of medication, however, the FBG of the mice significantly decreased Previous studies show that glyburide could have antiproliferative effects on cancer cell growth in some cancer models, but its role was inconsistent with some clinical studies. Findings in vitro from preclinical studies demonstrated it could inhibit development of human prostate, 16 breast, 26 gastric, 27 bladder, 17 glioma, 28 hepatocellular, 19 and colon 20 cancer cell lines. Furthermore, for in vivo experiments, one article indicated that in breast tumor growth and invasion which was induced by Tientsin Albino 2 (TA2), the levels of metal matrix proteinase-9 (MMP-9) and proliferating cell nuclear antigen (PCNA) could be decreased by the glyburide in combination with cobalt chloride (CoCl 2 ) treatment, due to MMP-9 being involved in the tumor invasion and metastasis, as well as PCNA participation in the process of synthesis of DNA and metabolism of nucleic acid as an accessory protein, Zhe Rong et al. found this combined treatment inhibited TA2 spontaneous breast cancer. 29 One clinical study indicated the relationship between glyburide and higher cancer risk 11 and a retrospective study which were followed up about average of 5 years showed glyburide users had higher total cancer mortality than gliclazide users. 10 In our study, we found that glyburide could attenuate lung tumorigenesis, chronic glyburide treatment decreased tumor incidence and Moreover, immunohistochemical results demonstrated higher expression of NLRP3 and IL-1β protein in B(a)P/LPS group than those administrated with Vehicle, which were in accordance with our previous study. Comparing with B(a)P/LPS group, the expression of NLRP3 and IL-1β protein were both decreased after chronic glyburide treatment. IL-1β serves as a proinflammatory cytokine in downstream of NLRP3 inflammasome, and it also could promote the progression in lung tumor. 33 Glyburide has anti-inflammatory response mainly through depressing activation of NLRP3 inflammasome and decreasing IL-1β release. 34 Thus, we next examined cleaved-IL-1β protein levels in Gly (0.96 mg/kg)-treated group decreased significantly compared with B(a)P/LPS group. Taken together, glyburide might inhibit cleaved-IL-1β levels during lung carcinoma initiation induced by B(a)P/LPS, which may be attributed to attenuate inflammationrelated lung cancer initiation in mice. It has been reported that in an ex vivo model of human endotoxinemia, glyburide could reduce proinflammatory cytokines including LPS-induced releases of IL-1β and so on, 35 besides, upregulation of IL-1β could enhance the proliferation and migration of A549 lung adenocarcinoma cell due to the activation of NLRP3 inflammasome. 33,36 Therefore, it suggests that glyburide might inhibit NLRP3 inflammasome to attenuate the inflammation-related lung cancer in mice given B(a)p/LPS.

| CONCLUSION
In conclusion, our results demonstrated that chronic glyburide treatment attenuated inflammation-related lung tumorigenesis in nondiabetes mice which might by inhibiting NLRP3 inflammasome. However, we need to further explore the mechanism of glyburide alleviated lung tumorigenesis.

CONFLICT OF INTERESTS
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.
F I G U R E 6 NLRP3 inflammasome activation in lung tissues of mice exposed to B(a)p plus LPS. Expression of cleaved-IL-1β protein was determined by Western blot (A). The expression of IL-1β (B). Data were expressed as mean ± SEM. *: versus vehicle control, p < .05; # : versus B(a) P/LPS, p < .05. B(a)p, benzo(a)pyrene; LPS, lipopolysaccharide

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.