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Lee, Lee, Jeon, Lee, Lee, Kim, and Lee: Regulation of Tumor Necrosis Factor-α-induced Airway Mucin Production and Gene Expression by Carbenoxolone, Prunetin, and Silibinin
Original Article
Tuberculosis and Respiratory Diseases

Tuberculosis and Respiratory Diseases 2010; 69(5): 348-353.

Published online: 30 November 2010

DOI: https://doi.org/10.4046/trd.2010.69.5.348

Regulation of Tumor Necrosis Factor-α-induced Airway Mucin Production and Gene Expression by Carbenoxolone, Prunetin, and Silibinin

Hyun Jae Lee, M.S.1, *, Su Yel Lee, B.S.1, *, Byeong Kyou Jeon, Ph.D.2, Jae Woo Lee, Ph.D.3, Mi Nam Lee, B.S.1, Ju-Ock Kim, M.D., Ph.D.4, Choong Jae Lee, Ph.D.1

1Department of Pharmacology, Chungnam National University School of Medicine, Daejeon, Korea.

2Department of Radiologic Technology, Daegu Health College, Daegu, Korea.

3LG Life Science, Seoul, Korea.

4Pulmonology Section, Department of Internal Medicine, Chungnam National University Hospital, Chungnam National University School of Medicine, Daejeon, Korea.

Address for correspondence: Choong Jae Lee, Ph.D. Department of Pharmacology, Chungnam National University College of Medicine, 6, Munhwa-dong, Jung-gu, Daejeon 301-747, Korea. Phone: 82-42-580-8255, Fax: 82-42-585-6627, LCJ123@cnu.ac.kr

Equal contributor:

*These two authors equally contributed to this work.

Received 24 August 2010       Accepted 14 September 2010

Copyright © 2010. The Korean Academy of Tuberculosis and Respiratory Diseases. All rights reserved.

Abstract

Background

In this study, we tried to investigate whether carbenoxolone, prunetin, and silibinin affect tumor necrosis factor (TNF)-α-induced MUC5AC mucin production and gene expression from human airway epithelial cells.

Methods

Confluent NCI-H292 cells were pretreated with each agent (carbenoxolone, prunetin, and silibinin) for 30 min and then stimulated with TNF-α for 24 hours. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively.

Results

Carbenoxolone, prunetin and silibinin inhibited the production of MUC5AC mucin protein induced by TNF-α; the 3 compounds also inhibited the expression of MUC5AC mucin gene induced by TNF-α.

Conclusion

This result suggests that carbenoxolone, prunetin and silibinin can inhibit mucin gene expression and production of mucin protein induced by TNF-α, by directly acting on airway epithelial cells.

Keywords: MUC5AC protein, human, Carbenoxolone, Prunetin, Silibinin

Introduction

Mucus in the airway epithelium plays a pivotal role in defensive mechanisms against airborne chemicals, particles and pathogenic microorganisms. The protective function of airway mucus is due mainly to the viscoelastic property of mucous glycoproteins or mucins. However, any abnormality in the quality or quantity of mucins not only cause altered airway physiology but may also impair host defenses often leading to serious airway pathology as exemplified in chronic bronchitis, cystic fibrosis, asthma, and bronchiectasis1. Therefore, we suggest it is valuable to find the possible activity of controlling (inhibiting) the excess mucin secretion (production) by the components from medicinal plants that have been used for the management of airway diseases. We have tried to investigate the possible activities of some natural products on mucin secretion from cultured airway epithelial cells. As a result of our trial, we previously reported that several natural compounds affected mucin secretion and/or production from airway epithelial cells2,3. According to folk medicine, Glycyrrhiza glabra L. (licorice) has been used for regulating diverse inflammatory diseases including pulmonary diseases4. Carbenoxolone, a steroid-like compound derived from Glycyrrhiza glabra L., was reported to have various biological effects including anti-inflammatory effect5-8. Prunetin, a flavonoid derived from Glycyrrhiza glabra L., showed antioxidant, inhibition of phosphodiesterase, inhibition of aldehyde dehydrogenase and alcohol dehydrogenase activities9-12. Also, according to a number of reports, flavonoids derived from milk thistle, Carduus marianus L., showed hepatoprotective, antioxidant, anti-inflammatory, anti-cancer and immunomodulatory effects13-19. However, to the best of our knowledge, there are no reports about the potential effects of carbenoxolone, prunetin and silibinin on tumor necrosis factor (TNF)-α-induced MUC5AC mucin production and gene expression from human airway epithelial cells. Therefore, in this study, we checked whether carbenoxolone, prunetin and silibinin affect airway mucin production and gene expression stimulated by TNF-α from NCI-H292 cells, a human pulmonary mucoepidermoid cell line.

Materials and Methods

1. Materials

All the chemicals and reagents used in this experiment including carbenoxolone (purity, 98.0%), prunetin (purity, 98.0%) and silibinin (purity, 97.0%) were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified.

2. Cell culture

NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 units/mL), streptomycin (100 µg/mL) and HEPES (25 mM) at 37℃ in a humidified, 5% CO2/95% air, water-jacketed incubator. For serum deprivation, confluent cells were washed twice with phosphate-buffered saline (PBS) and recultured in RPMI 1640 with 0.2% fetal bovine serum for 24 hours.

3. Treatment of cells with carbenoxolone, prunetin, and silibinin

After 24 hours of serum deprivation, cells were pretreated with carbenoxolone (1, 10, 100 µM), prunetin (1, 10, 100 µM), and silibinin (1, 10, 100 µM) for 30 minutes and treated with TNF-α (0.2 nM) for 24 hours in serum-free RPMI 1640. After 24 hours, cells were lysed with buffer solution containing 20 mM Tris, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and collected to measure the production of MUC5AC protein (in 24-well culture plate). The total RNA was extracted for measuring the expression of MUC5AC gene (in 6-well culture plate) by using reverse transcription-polymerase chain reaction (RT-PCR).

4. MUC5AC mucin analysis using ELISA

MUC5AC protein was measured by using enzyme-linked immunosorbent assay (ELISA). Cell lysates were prepared with PBS at 1 : 10 dilution, and 100 µL of each sample was incubated at 42℃ in a 96-well plate, until dry. Plates were washed three times with PBS and blocked with 2% BSA (fraction V) for 1 hour at room temperature. Plates were again washed three times with PBS and then incubated with 100 µL of 45M1, a mouse monoclonal MUC5AC antibody (NeoMarkers, Freemont, CA, USA) (1 : 200), which was diluted with PBS containing 0.05% Tween 20 and dispensed into each well. After 1 hour, the wells were washed three times with PBS, and 100 µL of horseradish peroxidase-goat anti-mouse IgG conjugate (1 : 3,000) was dispensed into each well. After 1 hour, plates were washed three times with PBS. Color reaction was developed with 3,3',5,5'-tetramethylbenzidine (TMB) peroxide solution and stopped with 1 N H2SO4. Absorbance was read at 450 nm.

5. Total RNA isolation and RT-PCR

Total RNA was isolated by using Easy-BLUE Extraction Kit (iNtRON Biotechnology Inc., Seongnam, Korea) and reverse transcribed by using AccuPower RT Premix (BIONEER Co., Daejeon, Korea) according to the manufacturer's instructions. 2 µg of total RNA was primed with 1 µg of oligo (dT) in a final volume of 50 µL (RT reaction). 2 µL of RT reaction product was PCR amplified in a 25 µL by using Thermorprime Plus DNA Polymerase (ABgene, Rochester, NY, USA). Primers for MUC5AC were (forward) 5'-TGA TCA TCC AGC AGG GCT-3' and (reverse) 5'-CCG AGC TCA GAG GAC ATA TGG G-3'. The size of expected fragment amplified by PCR was 458 bp. As quantitative controls, primers for Rig/S15 rRNA, which encodes a small ribosomal subunit protein, a housekeeping gene that was constitutively expressed, were used. Primers for Rig/S15 were (forward) 5'-TTC CGC AAG TTC ACC TAC C-3' and (reverse) 5'-CGG GCC GGC CAT GCT TTA CG-3'. The size of expected fragment amplified by PCR was 361 bp. The PCR mixture was denatured at 94℃ for 2 minutes followed by 40 cycles at 94℃ for 30 seconds, 60℃ for 30 seconds and 72℃ for 45 seconds. After PCR, 5 µL of PCR products were subjected to 1% agarose gel electrophoresis and visualized with ethidium bromide under a transilluminator.

6. Statistics

Means of individual group were converted to percent control and expressed as mean±SEM. The difference between groups was assessed using one-way ANOVA and Student's t-test for unpaired samples. p<0.05 was considered as significantly different.

Results

1. Effect of carbenoxolone on TNF-α-induced MUC5AC mucin production from NCI-H292 cells

As can be seen in Figure 1, carbenoxolone inhibited TNF-α-induced MUC5AC mucin production. The amounts of MUC5AC mucin in the cells of carbenoxolone-treated cultures were 100±10%, 1,250±98%, 1,235±108%, 1,124±76%, and 500±48% for control, TNF-α 0.2 nM only, carbenoxolone 10-6 M+TNF-α, carbenoxolone 10-5 M+TNF-α and carbenoxolone 10-4 M+TNF-α, respectively (Figure 1).

2. Effect of prunetin on TNF-α-induced MUC5AC mucin production from NCI-H292 cells

As can be seen in Figure 2, prunetin also inhibited TNF-α-induced MUC5AC mucin production, dose-dependently. The amounts of MUC5AC mucin in the cells of prunetin-treated cultures were 100±6%, 349±29%, 284±30%, 92±9%, and 69±5% for control, TNF-α 0.2 nM only, prunetin 10-6 M+TNF-α, prunetin 10-5 M+TNF-α and prunetin 10-4 M+TNF-α, respectively (Figure 2).

3. Effect of silibinin on TNF-α-induced MUC5AC mucin production from NCI-H292 cells

As can be seen in Figure 3, silibinin inhibited TNF-α-induced MUC5AC mucin production, dose-dependently. The amounts of MUC5AC mucin in the cells of silibinin-treated cultures were 100±7%, 317±29%, 257±30%, 198±13%, and 59±5% for control, TNF-α 0.2 nM only, silibinin 10-6 M+TNF-α, silibinin 10-5 M+TNF-α and silibinin 10-4 M+TNF-α, respectively (Figure 3).

4. Effects of carbenoxolone, prunetin and silibininon TNF-α-induced MUC5AC gene expression from NCI-H292 cells

As can be seen in Figure 4, carbenoxolone, prunetin and silibinin inhibited TNF-α-induced MUC5AC gene expression at the concentration of 10-4 M, respectively.

Discussion

MUC5AC was reported to be mainly expressed in goblet cells existing in the airway surface epithelium among the twenty genes coding human mucins (MUC)20. Also, TNF-α is a well-known stimulator of secretion and gene expression of airway mucin21-23. TNF-α level in sputum was reported to be increased, with further increases during exacerbation of diseases24,25. TNF-α converting enzyme (TACE) mediated MUC5AC mucin expression in cultured human airway epithelial cells22 and TNF-α induced MUC5AC gene expression in normal human airway epithelial cells23. It also induced mucin secretion from guinea pig tracheal epithelial cells21. On the basis of these reports, in this study, we tried to test the possible effects of carbenoxolone, prunetin and silibinin on TNF-α-induced MUC5AC mucin production and gene expression from NCI-H292 cells, a human pulmonary mucoepidermoid cell line, which are frequently used for the purpose of elucidating intracellular signaling pathways involved in airway mucin production and gene expression22,26. As can be seen in results, carbenoxolone, prunetin and silibinin inhibited the production of MUC5AC mucin protein induced by TNF-α. In the result of experiment shown in Figure 1, TNF-α induced MUC5AC mucin production more potently than mucin productions in the other two results of experiments shown in Figures 2 and 3. Actually, based on the preliminary experiments performed by our group, the efficacy of stimulation of mucin production by TNF-α varied depending on the experimental conditions, although the exact cause of this phenomenon is unclear at present. On the other hand, the three compounds inhibited the expression of MUC5AC mucin gene induced by TNF-α. This result suggests that carbenoxolone, prunetin and silibinin can regulate mucin gene expression and production of mucin protein, by directly acting on airway epithelial cells. The underlying mechanisms of action of these three compounds on MUC5AC production and gene expression are not clear at present, although we are trying to investigate whether these three compounds act as regulators of NF-kB signaling pathway activated by TNF-α in mucin-producing NCI-H292 cells. Taken together, the inhibitory actions of carbenoxolone, prunetin and silibinin on airway mucin production and gene expression might explain, at least in part, the traditional use of G. glabra L. as anti-inflammatory agent and mucoregulator for airway inflammatory diseases, in oriental medicine and the folk use of Carduus marianus L. as anti-inflammatory agent. We suggest it is valuable to find the natural products that have specific inhibitory effects on mucin production and/or gene expression - in view of both basic and clinical sciences - and the result from this study suggest a possibility of using carbenoxolone, prunetin and silibinin as new efficacious mucoregulators for respiratory diseases, although further studies are essential.

Figures and Tables

Figure 1
Effect of carbenoxolone on tumor necrosis factor (TNF)-α-induced MUC5AC mucin production from NCI-H292 cells. NCI-H292 cells were pretreated with various concentrations of carbenoxolone for 30 minutes and then stimulated with TNF-α (0.2 nM) for 24 hours. Cell lysates were collected for measurement of MUC5AC mucin production by enzyme-linked immunosorbent assay. Means of individual group were converted to percent control and expressed as mean±SEM. The difference between groups was assessed using one-way ANOVA and Student's t-test for unpaired samples. p<0.05 was considered as significantly different. Each bar represents a mean±SEM of 3~4 culture wells in comparison with that of control set at 100%. con: control; Car: carbenoxolone. *Significantly different from control (p<0.05), †Significantly different from TNF-α alone (p<0.05).
trd-69-348-g001
Figure 2
Effect of prunetin on tumor necrosis factor (TNF)-α-induced MUC5AC mucin production from NCI-H292 cells. NCI-H292 cells were pretreated with various concentrations of prunetin for 30 minutes and then stimulated with TNF-α (0.2 nM) for 24 hours. Cell lysates were collected for measurement of MUC5AC mucin production by enzyme-linked immunosorbent assay. Means of individual group were converted to percent control and expressed as mean±SEM. The difference between groups was assessed using one-way ANOVA and Student's t-test for unpaired samples. p<0.05 was considered as significantly different. Each bar represents a mean±SEM of 3~4 culture wells in comparison with that of control set at 100%. cont: control, Pru: prunetin. *Significantly different from control (p<0.05), †Significantly different from TNF-α alone (p<0.05).
trd-69-348-g002
Figure 3
Effect of silibinin on tumor necrosis factor (TNF)-α-induced MUC5AC mucin production from NCI-H292 cells. NCI-H292 cells were pretreated with various concentrations of silibinin for 30 minutes and then stimulated with TNF-α (0.2 nM) for 24 hours. Cell lysates were collected for measurement of MUC5AC mucin production by enzyme-linked immunosorbent assay. Means of individual group were converted to percent control and expressed as mean±SEM. The difference between groups was assessed using one-way ANOVA and Student's t-test for unpaired samples. p<0.05 was considered as significantly different. Each bar represents a mean±SEM of 3~4 culture wells in comparison with that of control set at 100%. cont: control; Sil: silibinin. *Significantly different from control (p<0.05), †Significantly different from TNF-α alone (p<0.05).
trd-69-348-g003
Figure 4
Effects of carbenoxolone, prunetin and silibinin on tumor necrosis factor (TNF)-α-induced MUC5AC mucin gene expression from NCI-H292 cells. NCI-H292 cells were pretreated with carbenoxolone, prunetin, and silibinin (100 µM), respectively, for 30 minutes and then stimulated with TNF-α (0.2 nM) for 24 hours. MUC5AC gene expression was measured by reverse transcription-polymerase chain reaction. cont: control; Car: carbenoxolone; Pru: prunetin; Sil: silibinin.
trd-69-348-g004

Acknowledgements

This study was partly supported by 2009 grant of Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

References

1. Voynow JA, Rubin BK. Mucins, mucus, and sputum. Chest. 2009. 135:505–512.
2. Lee CJ, Lee JH, Seok JH, Hur GM, Park YC, Seol IC, et al. Effects of baicalein, berberine, curcumin and hesperidin on mucin release from airway goblet cells. Planta Med. 2003. 69:523–526.
3. Heo HJ, Lee SY, Lee MN, Lee HJ, Seok JH, Lee CJ. Genistein and curcumin suppress epidermal growth factor-induced MUC5AC mucin production and gene expression from human airway epithelial cells. Phytother Res. 2009. 23:1458–1461.
4. Research Institute of Natural Products, Seoul National University. Treatise on Asian herbal medicines. 2003. Seoul: Haksulpyeonsugwan.
5. Tanaka Y, Kikuzaki H, Fukuda S, Nakatani N. Antibacterial compounds of licorice against upper airway respiratory tract pathogens. J Nutr Sci Vitaminol (Tokyo). 2001. 47:270–273.
6. Suzuki S, Koyama K, Darnel A, Ishibashi H, Kobayashi S, Kubo H, et al. Dexamethasone upregulates 11beta-hydroxysteroid dehydrogenase type 2 in BEAS-2B cells. Am J Respir Crit Care Med. 2003. 167:1244–1249.
7. Suzuki S, Matsuda Y, Sugawara T, Tabata T, Ishibashi H, Hoshikawa Y, et al. Effects of carbenoxolone on alveolar fluid clearance and lung inflammation in the rat. Crit Care Med. 2004. 32:1910–1915.
8. Asl MN, Hosseinzadeh H. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytother Res. 2008. 22:709–724.
9. Jung HA, Kim AR, Chung HY, Choi JS. In vitro antioxidant activity of some selected Prunus species in Korea. Arch Pharm Res. 2002. 25:865–872.
10. Ko WC, Shih CM, Lai YH, Chen JH, Huang HL. Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships. Biochem Pharmacol. 2004. 68:2087–2094.
11. Sheikh S, Weiner H. Allosteric inhibition of human liver aldehyde dehydrogenase by the isoflavone prunetin. Biochem Pharmacol. 1997. 53:471–478.
12. Keung WM. Biochemical studies of a new class of alcohol dehydrogenase inhibitors from Radix puerariae. Alcohol Clin Exp Res. 1993. 17:1254–1260.
13. Polyak SJ, Morishima C, Lohmann V, Pal S, Lee DY, Liu Y, et al. Identification of hepatoprotective flavonolignans from silymarin. Proc Natl Acad Sci U S A. 2010. 107:5995–5999.
14. Trappoliere M, Caligiuri A, Schmid M, Bertolani C, Failli P, Vizzutti F, et al. Silybin, a component of sylimarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells. J Hepatol. 2009. 50:1102–1111.
15. Singh RP, Raina K, Sharma G, Agarwal R. Silibinin inhibits established prostate tumor growth, progression, invasion, and metastasis and suppresses tumor angiogenesis and epithelial-mesenchymal transition in transgenic adenocarcinoma of the mouse prostate model mice. Clin Cancer Res. 2008. 14:7773–7780.
16. Li L, Gao Y, Zhang L, Zeng J, He D, Sun Y. Silibinin inhibits cell growth and induces apoptosis by caspase activation, down-regulating survivin and blocking EGFR-ERK activation in renal cell carcinoma. Cancer Lett. 2008. 272:61–69.
17. Ligeret H, Brault A, Vallerand D, Haddad Y, Haddad PS. Antioxidant and mitochondrial protective effects of silibinin in cold preservation-warm reperfusion liver injury. J Ethnopharmacol. 2008. 115:507–514.
18. Hogan FS, Krishnegowda NK, Mikhailova M, Kahlenberg MS. Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer. J Surg Res. 2007. 143:58–65.
19. Lee SO, Jeong YJ, Im HG, Kim CH, Chang YC, Lee IS. Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells. Biochem Biophys Res Commun. 2007. 354:165–171.
20. Rogers DF, Barnes PJ. Treatment of airway mucus hypersecretion. Ann Med. 2006. 38:116–125.
21. Fischer BM, Rochelle LG, Voynow JA, Akley NJ, Adler KB. Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro. Am J Respir Cell Mol Biol. 1999. 20:413–422.
22. Shao MX, Ueki IF, Nadel JA. Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells. Proc Natl Acad Sci U S A. 2003. 100:11618–11623.
23. Song KS, Lee WJ, Chung KC, Koo JS, Yang EJ, Choi JY, et al. Interleukin-1 beta and tumor necrosis factor-alpha induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells. J Biol Chem. 2003. 278:23243–23250.
24. Chung KF. Cytokines in chronic obstructive pulmonary disease. Eur Respir J Suppl. 2001. 34:50s–59s.
25. Cohn L, Whittaker L, Niu N, Homer RJ. Cytokine regulation of mucus production in a model of allergic asthma. Novartis Found Symp. 2002. 248:201–213.
26. Takeyama K, Dabbagh K, Lee HM, Agustí C, Lausier JA, Ueki IF, et al. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci U S A. 1999. 96:3081–3086.
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