Glyburide Attenuates B(a)p and LPS-Induced In ammation-Related Lung Tumorigenesis in Mice

Mengyuan Li Zhengzhou University Hong Liu Zhengzhou University First A liated Hospital Hua Shao Zhengzhou university Peng Zhang Henan Cancer Hospital Min Gao Zhengzhou University Li Huang Zhengzhou University Pingping Shang Zhengzhou Tobacco Research Institute Qiao Zhang Zhengzhou University Wei Wang Zhengzhou University school Jing Wang Zhengzhou University First A liated Hospital Feifei Feng (  feifeifeng@zzu.edu.cn ) Zhengzhou University https://orcid.org/0000-0001-5534-5928


Introduction
International Agency for Research on Cancer reported that lung cancer is the primary factor of cancerrelated morbidity and death [1], the estimated new cases of lung cancer ranked second and the estimated deaths of lung cancer ranked rst in United States, 2020 [2]. A large of evidences have shown that occupational exposures are associated closely with lung cancer [3], and Polycyclic Aromatic Hydrocarbons serve as occupational exposures for people work in such occupational environments could increase the risk of lung cancer [4]. As an important and most investigated in ammatory regulator, NLRP3 in ammasome has been reported to play a critical role in tumorigenesis, and recently studies show that in ammasomes serve as a promising therapeutic target for the prevention and treatment of cancer [5,6].
In our previous study, we investigated the role of NLRP3 in mouse in ammation-related lung cancer initiation, and found that NLRP3 deletion inhibited lung cancer initiation induced by B(a)p or B(a)p plus LPS [7], we also found that NLRP3 in ammasome activation played an important role in B(a)p plus LPSinduced in ammation-related lung tumorigenesis in mice [8].
Glyburide, a NLRP3 inhibitor, plays an anti-in ammatory role to inhibit the NLRP3 in ammasome, decreases the production of proin ammatory cytokines, reduces the recruitment and migration of in ammatory cells, and production of nitricoxide [9]. In addition, glyburide is a second-generation oral antidiabetic drug known as sulphonylureas, which is one of insulin secretatogues widely used for noninsulin-dependent diabetes mellitus. Growing evidence demonstrated that glyburide played a critical role in antitumor effect, which basically by two kinds of mechanisms: a general inhibitor of the ABC protein super-family and an inhibitor of KATP channels [10]. There are con icting results which were reported in clinical studies regarding the potential effect of sulfonylureas increasing or decreasing cancer risks, some studies showed that glyburide was associated with higher cancer risks and mortality [11][12][13], such as breast cancer [14], while according to the other studies revealed opposite ndings that glyburide could reduce cancer risks [15], such as prostate cancer [16,17]. Furthermore, in some early studies about cancer cells, it demonstrated that glyburide could inhibit progression in many cancers such as bladder carcinoma [18], prostate cancer [19], colon cancer [20], and liver cancer [21,22].Moreover, few in vivo studies have discussed the involvement of glyburide in lung tumorigenesis, 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 tumor growth inhibition might be a more direct effect of glyburide [23]. 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 [24]. However, the involvement of glyburide in lung tumorigenesis remains unclear.
The objective of our study was to investigate whether glyburide inhibit the occurrence of lung cancer, so we established the non-diabetes mice model of in ammation-related lung cancer induced by B(a)p plus LPS which have been reported in our previous study [7], and the glyburide treatment were administered from one week before the mice were intratracheal instillation with B(a)p. This study could provide an important guiding signi cance for the medication with lung cancer.

Materials And Methods
Animals C57BL/6J mice (SPF degree, 6-8 week) were obtained from their heterozygous littermates in the college of Public Health of Zhengzhou University, Henan, China (temperature 22℃, lights on 08:00-20:00), and were kept in stainless steel cages with water and food ad libitum. All studies were approved by the life Science Institutional Review Board of Zhengzhou University and performed strictly in accordance with the Guideline of Zhengzhou University for Animal Experiments.
In ammation-related lung tumorigenesis mouse models and glyburide treatment The in ammation-related lung tumorigenesis models in the mouse were induced by intratracheal instillation with B(a)p and LPS. Mice were divided into 6 groups randomly: Group 1: Vehicle control group (n=16), Group 2: B(a)p plus LPS group (n=30), Group 3: tricaprylin+Gly(0.48mg/kg) group (n=19), Group 4: B(a)P/LPS+Gly(0.48mg/kg) (n=31), Group 5: tricaprylin+Gly(0.96mg/kg) group (n=16) and Group 6: B(a)P/LPS+Gly(0.96mg/kg) (n=34). As shown in Fig.1, mice in groups B(a)p plus LPS, B(a)P/LPS+Gly(0.48mg/kg) and B(a)P/LPS+Gly(0.96mg/kg) were instilled intratracheally with B(a)p (at a dose of 1mg/mouse in 50μl tricaprylin) once a week for 4 times. 3 weeks later, these mice were instilled intratracheally with LPS (at a dose of 2.5μg/mouse in 50μl physiological saline solution) once every three weeks for 5 times, whereas mice in group Vehicle control, tricaprylin+Gly(0.48mg/kg) and tricaprylin+Gly(0.96mg/kg) were given 50μl tricaprylin in a similar manner. All instillations were administered under iso urane (Sigma) anesthesia. To explore the potential of chronic Gly treatment as a preventive measure against tumorigenesis, mice in group tricaprylin+Gly(0.48mg/kg), B(a)P/LPS+Gly(0.48mg/kg), tricaprylin+Gly(0.96mg/kg), B(a)P/LPS+Gly(0.96mg/kg) were administered the Gly (0.48mg/kg or 0.96mg/kg, dissolved in 50μlsaline) treatment (gavage, three times a week) from one week before the mice were instilled intratracheally with B(a)p (the week of the rst time of Gly treatment named Week 0) until the animal model nished. The Gly-treated mice were weighed and their fast blood glucose (FBG) were 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 sacri ced to harvest lungs to count visible tumors on the surface of the lung tissues. The left lobes of the lungs of all groups were used for histopathological studies which were xed by 4% paraformaldehyde, then the right lobes of the lungs were preserved in -80℃ and used for subsequent studies.

Lung coe cient
At week 34, the lung tissues were removed from sacri ced mice, washed in physiological saline solution, sucked dry by lter paper, and then weighted with electronic balance. Lung coe cient could be one of the indicators of lung injury, which was expressed as the follow equation: Lung coe cient=lung weight/body weight.

Lung pathological alterations
The left lobes of the lungs were xed overnight by 4% paraformaldehyde, embedded in para n, then cut into sections having a thickness of 5μm. Eventually, the sections were observed by an electron microscope using haematoxylin and eosin(HE) staining. To evaluate the in ammation changes of lung tissues according to the literature published by Huang et al [25]. The pathological tumor nests were identi ed by two experienced pathologists in a blind manner and based on the previous papers published [26,27].
Positive IHC staining was re ected as brown staining and the AOD quanti cation was carried out using the Image-Pro Plus 6.0 software in the high-power vision elds (AOD = Integrated Optical Density [IOD] SUM/Area SUM).

Statistical analysis
Chi-square test was used to compare the tumor incidence among different groups. The results of mean tumor count, lung coe cient and pathological tumor nests were presented as mean±SEM, and the data analysis was performed by one-way ANOVAs and two-tailed Student's t-test using SPSS21.0 (IBM, NC, USA). The P value less than 0.05 for two-tailed was considered statistically signi cant.

Effects of glyburide on fasting blood glucose and body weight alteration in mice
To investigate the effects of glyburide on FBG and body weight alteration in C57BL/6 mice which were administered 0.48 mg/kg 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. Figure 2A and Fig. 2B indicated that chronic glyburide treatment signi cantly decreased FBG level in both dose at the end of the experiments (P < 0.05). Moreover, the FBG of mice treated with Gly (0.96 mg/kg) signi cantly decreased more earlier than the mice treated with Gly (0.48 mg/kg) compared with baseline of FBG level respectively. Figure 2C shows the body weight of all groups treated with Gly and B(a)P + LPS group decreased signi cantly compared with Vehicle group (P < 0.05). Also, the mice in B(a)P/LPS + Gly(0.96 mg/kg) group obviously decreased compared with B(a)P + LPS and B(a)P/LPS + Gly(0.48 mg/kg) group (P < 0.05).

Glyburide treatment attenuates lung tumorigenesis induced by B(a)p plus LPS in mice
In order to determine if glyburide inhibits lung tumorigenesis, we compared the incidence and mean tumor count of lung tumors in mice treated with B(a)p plus LPS or B(a)P/LPS + Gly in three sets of tumor bioassays. Figure 3A shows that mice exposed to B(a)p plus LPS could induce lung tumors, whereas there were still no visible lung tumors in Vehicle control group, Gly(0.48 mg/kg)-treated tricaprylin group or Gly(0.96 mg/kg)-treated tricaprylin group. Moreover, treatment with chronic glyburide could inhibit the occurrence of lung tumors induced by B(a)p + LPS in mice. Tumor incidence of mice in B(a)P/LPS + Gly(0.48 mg/kg) group (82.6%) and B(a)P/LPS + Gly(0.96 mg/kg) group (70.8%) were decreased compared with mice exposure to B(a)p plus LPS (89.4%), but there were no signi cant difference among these groups (Fig. 3B). In addition, the mice in B(a)P/LPS + Gly(0.48 mg/kg) group and B(a)P/LPS + Gly(0.96 mg/kg) group developed 3.22 ± 3.029, 2.13 ± 2.071 visible tumors/mouse on the surface of the lung respectively which decreased signi cantly compared with mice treated with B(a)p plus LPS (5.58 ± 4.538 tumors/mouse) (P < 0.05) (Fig. 3C).
Effect of glyburide on lung coe cient of mice induced by B(a)p plus LPS Lung coe cient can re ect lung injury in mice. As shown in the Fig. 3D, mice exposed to B(a)p plus LPS caused the rise of lung coe cient compared with that mice treated with Vehicles (P < 0.05). However, chronic glyburide treatment decreased this parameter in the mice of B(a)p plus LPS group that were treated with glyburide. Lung coe cient in mice exposed to B(a)P/LPS + Gly(0.96 mg/kg) was signi cantly decreased than mice exposed to B(a)p plus LPS (P < 0.05), but there was no signi cant difference between B(a)P/LPS + Gly(0.48 mg/kg) group and B(a)p plus LPS group. Besides, compared with the Vehicle control group, lung coe cient of mice induced by tricaprylin plus glyburide were both increased, whereas there was no signi cant difference among these groups.

Effects of glyburide on pathological alterations in the lungs of mice induced by B(a)p plus LPS
As shown in Fig. 4A, there was no pathological tumor nest in cross-section of the left lobes of lungs in Vehicle control group and Gly-treated tricaprylin group, whereas we could observe pathological tumor nest in both B(a)P/LPS + Gly(0.48 mg/kg) (0.38 ± 0.18) and B(a)P/LPS + Gly(0.96 mg/kg) (1.31 ± 0.35) group signi cantly decreased compared with B(a)p plus LPS group (3.56 ± 1.02) (P < 0.05) (Fig. 4B). Meanwhile, we found the signi cantly in ammatory changes in mice exposed to B(a)p plus LPS including in ltration of in ammatory cells, fractures of alveolar walls and injury of bronchial epithelium compared with Vehicles group (Fig. 4C). However, chronic glyburide treatment could reduce the in ammatory changes signi cantly in mice induced by B(a)p plus LPS.

The expression of NLRP3, IL-1β and IL-18 protein in lung tissues of mice in different groups
The expression of NLRP3, IL-1β and IL-18 protein in lung tissues was observed by using immunohistochemistry. Figure 5A shows that positive expression areas of NLRP3, IL-1β and IL-18 were in the cytoplasm. As shown in the Fig. 5B, the expression of NLRP3 and IL-1β protein in lung tissues of mice exposure to B(a)p plus LPS were higher than those in Vehicle control group (P < 0.05, respectively), however, the levels of NLRP3 and IL-1β protein in lung tissues of B(a)P/LPS + Gly0.96 (mg/kg)-treated mice were signi cant decreased compared with those in B(a)p plus LPS group (P < 0.05, respectively). Moreover, there was no signi cant difference on the level of IL-18 protein in lung tissues of B(a)P/LPS + Gly0.48/0.96 (mg/kg) compared with that in B(a)p plus LPS group (Fig. 5B). Above results indicated that the downregulation of the expression of NLRP3 and IL-1β protein could due to the chronic treatment in lung tissues of mice.
The expression of Cleaved-IL-1β protein in lung tissues of mice in different groups As shown in Fig. 6, the level of Cleaved-IL-1β protein were increased induced by B(a)p plus LPS in lung tissues of mice compared with that in Vehicle control group (P < 0.05), but the level of Cleaved-IL-1β protein in B(a)P/LPS + Gly0.96 (mg/kg) group decreased signi cantly than that in B(a)p plus LPS group (P < 0.05).

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 lung tumorigenesis remains unclear. Thus, we explored the role of glyburide in lung tumorigenesis which could contribute to a new therapeutic strategy for lung cancer using the non-diabetes mice model induced by B(a)p plus LPS.
Chronic glyburide treatment decreased the FBG signi cantly, and mice exposed to B(a)p plus LPS signi cantly decreased weight compared with mice in Vehicle control group which may be attributed to the cancer cachexia [28]. Glyburide is one of the most common medication used in the treatment of type 2 diabetes, however, the FBG of the mice signi cantly decreased during chronic glyburide treatment, this would be a side effect of glyburide treatment.
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 from preclinical studies demonstrated it could inhibit growth of human prostate [29], hepatocellular [22], breast [30], gastric [31], bladder [32], glioma [33] and colon [20] cancer cell lines in vitro. Furthermore, for in vivo experiments, one article indicated the impact of glyburide in combination with cobalt chloride (CoCl2) on the growth and invasiveness of Tientsin Albino 2 (TA2) breast cancer, and the expression of metal matrix proteinase-9 (MMP-9) and proliferating cell nuclear antigen (PCNA) could be decreased by the treatment, due to MMP-9 being involved in the cancer invasion and metastasis, while PCNA participation in nucleic acid metabolism and DNA synthesis as an accessory protein, Zhe Rong et al found this combined treatment inhibited TA2 spontaneous breast cancer growth and invasiveness [34]. Another study for non-diabetes rat models induced with N-Nitroso-N-Methylurea, in the presence of glyburide, breast cancer cell growth could also be inhibited [24]. One clinical study indicated an association between glyburide and higher cancer risk [13] and a retrospective study with a mean follow-up of 5 years, showed a higher all-cancer mortality in glyburide users compared to gliclazide users [12]. In our study, we found that glyburide could attenuate lung tumorigenesis, chronic glyburide treatment decreased tumor incidence and mean tumor count of visible tumors on the surface of lungs. In addition, the increase of lung coe cient is related with lung injury in mice, and B(a)p plus LPS exposure increased lung coe cient signi cantly. We found that chronic treatment glyburide signi cantly decreased lung coe cient compared with mice exposed to B(a)p plus LPS, and the reduced lung coe cient may be attributed to the number of tumor nodules decreased.
All these evidences demonstrated that glyburide could induce the inhibition of occurrence of lung tumors in nondiabetic mice which was in line with the above reports about the anti-tumor effect of glyburide.
Histopathological observation con rmed that the number of pathological tumor nest of the left lobes of lungs decreased signi cantly after glyburide treatment compared with the mice in B(a)p plus LPS group and lung in ammatory changes were relieved. As evidence indicated that glyburide could induce inhibition of occurrence of lung tumor through inhibiting the in ammation. It has been reported that all stages of tumorigenesis could be affected by in ammation [35], and the anti-in ammatory effects of glyburide have been indicated [36,37]. It is surprising that the number of pathological tumor nest in group of B(a)P/LPS + Gly(0.48 mg/kg) decreased compared with the mice in B(a)P/LPS + Gly(0.96 mg/kg) group. One reasonable explanation for this result is that we counted the number of pathological tumor nest only in the left lobes of lungs but not the whole lung tissues in mice.
Moreover, immunohistochemical results demonstrated that the expression of NLRP3 and IL-1β protein were higher in B(a)P + LPS group than those in Vehicle control group, which were in accordance with our previous study. Comparing with B(a)P + LPS group, the expression of NLRP3 and IL-1β protein both decreased after chronic glyburide treatment. IL-1β plays an important role in the downstream of NLRP3 in ammasome served as a proin ammatory cytokine, and it also could promote the progression in lung tumor [38]. Glyburide has anti-in ammatory response mainly by inhibiting the activation of NLRP3 in ammasome and decreasing IL-1β release [39]. Thus, we next examined the levels of cleaved-IL-1β protein in Gly(0.96 mg/kg)-treated group decreased signi cantly compared with B(a)P + LPS group. Taken together, glyburide could inhibit the expression of cleaved-IL-1β in lung tumorigenesis induced by B(a)P plus LPS, which may be attributed to attenuate in ammation-related lung tumorigenesis in mice. It has been reported that in an ex vivo model of human endotoxinemia, glyburide could reduce proin ammatory cytokines including LPS-induced releases of IL-1β and tumor necrosis factor α (TNF-α) [40], besides, the proliferation and migration of lung adenocarcinoma cell line A549 could be enhanced by the upregulation of IL-1β which were induced by the activation of NLRP3 in ammasome [38,41]. Therefore, it suggests that glyburide might inhibit NLRP3 in ammasome to attenuate the in ammationrelated lung cancer induced by B(a)p plus LPS.

Conclusion
In conclusion, our results demonstrated that chronic glyburide treatment attenuated in ammation-related lung tumorigenesis in non-diabetes mice which might by inhibiting NLRP3 in ammasome in this study. However, we need to further explore the mechanism of glyburide alleviated lung tumorigenesis.