Breast Cancer Research and Treatment

, Volume 125, Issue 1, pp 73–87 | Cite as

Nicotine-induced human breast cancer cell proliferation attenuated by garcinol through down-regulation of the nicotinic receptor and cyclin D3 proteins

  • Ching-Shyang Chen
  • Chia-Hwa Lee
  • Chang-Da Hsieh
  • Chi-Tang Ho
  • Min-Hsiung Pan
  • Ching-Shui Huang
  • Shih-Hsin Tu
  • Ying-Jan Wang
  • Li-Ching Chen
  • Yu-Jia Chang
  • Po-Li Wei
  • Yi-Yuan Yang
  • Chih-Hsiung Wu
  • Yuan-Soon Ho
Preclinical study


Previous studies have demonstrated that the persistent exposure of human bronchial epithelial cells to nicotine (Nic) through nicotinic acetylcholine receptors increases cyclin D1 promoter activity and protein expression. The main purpose of this study is to elucidate the carcinogenic role of cyclin D3, which is involved in breast tumorigenesis when induced by Nic. Real-time PCR analysis revealed that cyclin D3 is highly expressed at the mRNA level in surgically dissected breast tumor tissue, compared to the surrounding normal tissue (tumor/normal fold ratio = 17.93, n = 74). To test whether Nic/nicotinic acetylcholine receptor (nAChR) binding could affect cyclin D3 expression in human breast cancer cells, the transformed cell line MCF-10A-Nic (DOX) was generated from normal breast epithelial cells (MCF-10A) with inducible α9-nAChR gene expression, using the adenovirus tetracycline-regulated Tet-off system. Tet-regulated overexpression of α9-nAChR in MCF-10A-Nic (DOX) xenografted BALB/c-nu/nu mice resulted in a significant induction of cyclin D3. In contrast, cyclin D3 expression was down-regulated in α9-nAChR knock-down (siRNA) MDA-MB-231-xenografted tumors in NOD.CB17-PRKDC(SCID)/J(NOD-SCID) mice. Furthermore, we found that Nic-induced human breast cancer (MDA-MB-231) cell proliferation was inhibited by 1 μM of garcinol (Gar), isolated from the edible fruit Garcinia indica, through down-regulation of α9-nAChR and cyclin D3 expression. These results suggest that α9-nAChR-mediated cyclin D3 overexpression is important for nicotine-induced transformation of normal human breast epithelial cells. The homeostatic regulation of cyclin D3 has the potential to be a molecular target for antitumor chemotherapeutic or chemopreventive purposes in clinical breast cancer patients.


Smoking Cyclin D3 Garcinol Nicotinic acetylcholine receptors Breast cancer 



Activating protein-1


Chromatin immunoprecipitation analysis




Dulbecco’s modified Eagle’s medium






Fluorescence-activated cell sorter


Fetal calf serum


Glyceraldehyde-3-phosphate dehydrogenase














Nuclear factor kappa B




Nicotinic acetylcholine receptor




Phosphate-buffered saline


Reverse transcriptase polymerase chain reaction


Small interfering RNA



This study was supported by the National Science Council, Grant NSC 95-2320-B-038-016-MY3 to Dr. Ho and NSC 96-2314-B-038-002 to Dr. Wu, and by the Taipei Medical University Hospital (98TMU-TMUH-02-1) to Dr. Yang.

Supplementary material

10549_2010_821_MOESM1_ESM.doc (52 kb)
Supplementary material 1 (DOC 51 kb)


  1. 1.
    Lin Y, Kikuchi S, Tamakoshi K, Wakai K, Kondo T, Niwa Y, Yatsuya H, Nishio K, Suzuki S, Tokudome S, Yamamoto A, Toyoshima H, Mori M, Tamakoshi A (2008) Active smoking, passive smoking, and breast cancer risk: findings from the Japan Collaborative Cohort Study for Evaluation of Cancer Risk. J Epidemiol 18:77–83CrossRefPubMedGoogle Scholar
  2. 2.
    Slattery ML, Curtin K, Giuliano AR, Sweeney C, Baumgartner R, Edwards S, Wolff RK, Baumgartner KB, Byers T (2008) Active and passive smoking, IL6, ESR1, and breast cancer risk. Breast Cancer Res Treat 109:101–111CrossRefPubMedGoogle Scholar
  3. 3.
    Armitage AK, Dollery CT, George CF, Houseman TH, Lewis PJ, Turner DM (1975) Absorption and metabolism of nicotine from cigarettes. Br Med J 4:313–316CrossRefPubMedGoogle Scholar
  4. 4.
    Benowitz NL, Jacob P III (1984) Nicotine and carbon monoxide intake from high- and low-yield cigarettes. Clin Pharmacol Ther 36:265–270CrossRefPubMedGoogle Scholar
  5. 5.
    Lindell G, Farnebo LO, Chen D, Nexo E, Rask Madsen J, Bukhave K, Graffner H (1993) Acute effects of smoking during modified sham feeding in duodenal ulcer patients. An analysis of nicotine, acid secretion, gastrin, catecholamines, epidermal growth factor, prostaglandin E2, and bile acids. Scand J Gastroenterol 28:487–494CrossRefPubMedGoogle Scholar
  6. 6.
    Mei J, Hu H, McEntee M, Plummer H III, Song P, Wang HC (2003) Transformation of non-cancerous human breast epithelial cell line MCF10A by the tobacco-specific carcinogen NNK. Breast Cancer Res Treat 79:95–105CrossRefPubMedGoogle Scholar
  7. 7.
    Siriwardhana N, Choudhary S, Wang HC (2008) Precancerous model of human breast epithelial cells induced by NNK for prevention. Breast Cancer Res Treat 109:427–441CrossRefPubMedGoogle Scholar
  8. 8.
    Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, Cooke JP (2001) Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med 7:833–839CrossRefPubMedGoogle Scholar
  9. 9.
    Kellar KJ, Davila-Garcia MI, Xiao Y (1999) Pharmacology of neuronal nicotinic acetylcholine receptors: effects of acute and chronic nicotine. Nicotine Tob Res 1(Suppl 2):S117–S120 discussion S139–S140CrossRefPubMedGoogle Scholar
  10. 10.
    Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, Sixma TK (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411:269–276CrossRefPubMedGoogle Scholar
  11. 11.
    Schuller HM, Orloff M (1998) Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells. Biochem Pharmacol 55:1377–1384CrossRefPubMedGoogle Scholar
  12. 12.
    Ho YS, Chen CH, Wang YJ, Pestell RG, Albanese C, Chen RJ, Chang MC, Jeng JH, Lin SY, Liang YC, Tseng H, Lee WS, Lin JK, Chu JS, Chen LC, Lee CH, Tso WL, Lai YC, Wu CH (2005) Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFkappaB activation and cyclin D1 up-regulation. Toxicol Appl Pharmacol 205:133–148CrossRefPubMedGoogle Scholar
  13. 13.
    Wei PL, Chang YJ, Ho YS, Lee CH, Yang YY, An J, Lin SY (2009) Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor. Ann Surg 249:978–985CrossRefPubMedGoogle Scholar
  14. 14.
    Chen RJ, Ho YS, Guo HR, Wang YJ (2008) Rapid activation of Stat3 and ERK1/2 by nicotine modulates cell proliferation in human bladder cancer cells. Toxicol Sci 104:283–293CrossRefPubMedGoogle Scholar
  15. 15.
    Troncone G, Volante M, Iaccarino A, Zeppa P, Cozzolino I, Malapelle U, Palmieri EA, Conzo G, Papotti M, Palombini L (2009). Cyclin D1 and D3 overexpression predicts malignant behavior in thyroid fine-needle aspirates suspicious for Hurthle cell neoplasms. Cancer Cytopathol 117:522–529PubMedGoogle Scholar
  16. 16.
    Maier S, Daroqui MC, Scherer S, Roepcke S, Velcich A, Shenoy SM, Singer RH, Augenlicht LH (2009) Butyrate and vitamin D3 induce transcriptional attenuation at the cyclin D1 locus in colonic carcinoma cells. J Cell Physiol 218:638–642CrossRefPubMedGoogle Scholar
  17. 17.
    Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC, Sicinski P, Fauq A, Golde TE, Osborne BA (2009) Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 113:1689–1698CrossRefPubMedGoogle Scholar
  18. 18.
    Zhou Q, Stetler-Stevenson M, Steeg PS (1997) Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro. Oncogene 15:107–115CrossRefPubMedGoogle Scholar
  19. 19.
    Chu M, Guo J, Chen CY (2005) Long-term exposure to nicotine, via ras pathway, induces cyclin D1 to stimulate G1 cell cycle transition. J Biol Chem 280:6369–6379CrossRefPubMedGoogle Scholar
  20. 20.
    Yu X, Luo Y, Zhou Y, Zhang Q, Wang J, Wei N, Mi M, Zhu J, Wang B, Chang H, Tang Y (2008) BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4–p21WAF1/CIP1 pathway: analyses using a breast cancer cell line and tumoral xenograft model. Int J Oncol 33:555–563PubMedGoogle Scholar
  21. 21.
    Rudas M, Lehnert M, Huynh A, Jakesz R, Singer C, Lax S, Schippinger W, Dietze O, Greil R, Stiglbauer W, Kwasny W, Grill R, Stierer M, Gnant MF, Filipits M (2008) Cyclin D1 expression in breast cancer patients receiving adjuvant tamoxifen-based therapy. Clin Cancer Res 14:1767–1774CrossRefPubMedGoogle Scholar
  22. 22.
    Aaltonen K, Amini RM, Landberg G, Eerola H, Aittomaki K, Heikkila P, Nevanlinna H, Blomqvist C (2009) Cyclin D1 expression is associated with poor prognostic features in estrogen receptor positive breast cancer. Breast Cancer Res Treat 113:75–82CrossRefPubMedGoogle Scholar
  23. 23.
    Yamaguchi F, Ariga T, Yoshimura Y, Nakazawa H (2000) Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind. J Agric Food Chem 48:180–185CrossRefPubMedGoogle Scholar
  24. 24.
    Yamaguchi F, Saito M, Ariga T, Yoshimura Y, Nakazawa H (2000) Free radical scavenging activity and antiulcer activity of garcinol from Garcinia indica fruit rind. J Agric Food Chem 48:2320–2325CrossRefPubMedGoogle Scholar
  25. 25.
    Pan MH, Chang WL, Lin-Shiau SY, Ho CT, Lin JK (2001) Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells. J Agric Food Chem 49:1464–1474CrossRefPubMedGoogle Scholar
  26. 26.
    Tanaka T, Kohno H, Shimada R, Kagami S, Yamaguchi F, Kataoka S, Ariga T, Murakami A, Koshimizu K, Ohigashi H (2000) Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats. Carcinogenesis 21:1183–1189CrossRefPubMedGoogle Scholar
  27. 27.
    Yang CM, Lee IT, Lin CC, Yang YL, Luo SF, Kou YR, Hsiao LD (2009) Cigarette smoke extract induces COX-2 expression via a PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB pathway and p300 in tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 297:L892–L902CrossRefPubMedGoogle Scholar
  28. 28.
    Pan MH, Huang MC, Wang YJ, Lin JK, Lin CH (2003) Induction of apoptosis by hydroxydibenzoylmethane through coordinative modulation of cyclin D3, Bcl-X(L), and Bax, release of cytochrome c, and sequential activation of caspases in human colorectal carcinoma cells. J Agric Food Chem 51:3977–3984CrossRefPubMedGoogle Scholar
  29. 29.
    Chen LC, Liu YC, Liang YC, Ho YS and Lee WS (2009) Magnolol inhibits human glioblastoma cell proliferation through upregulation of p21/Cip1. J Agric Food Chem (in press)Google Scholar
  30. 30.
    Tu SH, Chang CC, Chen CS, Tam KW, Wang YJ, Lee CH, Lin HW, Cheng TC, Huang CS, Chu JS, Shih NY, Chen LC, Leu SJ, Ho YS, Wu CH (2009). Increased expression of enolase alpha in human breast cancer confers tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat (in press)Google Scholar
  31. 31.
    West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain SM, Harris C, Belinsky S, Dennis PA (2003) Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest 111:81–90PubMedGoogle Scholar
  32. 32.
    Narayan S, Jaiswal AS, Kang D, Srivastava P, Das GM, Gairola CG (2004) Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro. Oncogene 23:5880–5889CrossRefPubMedGoogle Scholar
  33. 33.
    Chen F, Kim E, Wang CC, Harrison LE (2005) Ciglitazone-induced p27 gene transcriptional activity is mediated through Sp1 and is negatively regulated by the MAPK signaling pathway. Cell Signal 17:1572–1577CrossRefPubMedGoogle Scholar
  34. 34.
    Wilkinson DS, Ogden SK, Stratton SA, Piechan JL, Nguyen TT, Smulian GA, Barton MC (2005) A direct intersection between p53 and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene. Mol Cell Biol 25:1200–1212CrossRefPubMedGoogle Scholar
  35. 35.
    Govind AP, Vezina P, Green WN (2009) Nicotine-induced upregulation of nicotinic receptors: underlying mechanisms and relevance to nicotine addiction. Biochem Pharmacol 78:756–765CrossRefPubMedGoogle Scholar
  36. 36.
    Nuutinen S, Ekokoski E, Lahdensuo E, Tuominen RK (2006) Nicotine-induced upregulation of human neuronal nicotinic alpha7-receptors is potentiated by modulation of cAMP and PKC in SH-EP1-halpha7 cells. Eur J Pharmacol 544:21–30CrossRefPubMedGoogle Scholar
  37. 37.
    Al-Wadei HA, Plummer HK 3rd, Schuller HM (2009) Nicotine stimulates pancreatic cancer xenografts by systemic increase in stress neurotransmitters and suppression of the inhibitory neurotransmitter gamma-aminobutyric acid. Carcinogenesis 30:506–511CrossRefPubMedGoogle Scholar
  38. 38.
    Ramazzotti G, Faenza I, Gaboardi GC, Piazzi M, Bavelloni A, Fiume R, Manzoli L, Martelli AM, Cocco L (2008) Catalytic activity of nuclear PLC-beta(1) is required for its signalling function during C2C12 differentiation. Cell Signal 20:2013–2021CrossRefPubMedGoogle Scholar
  39. 39.
    Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, Heinemann S (1994) Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 79:705–715CrossRefPubMedGoogle Scholar
  40. 40.
    Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA (2008) Receptor-mediated tobacco toxicity: acceleration of sequential expression of alpha5 and alpha7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke. FASEB J 22:1356–1368CrossRefPubMedGoogle Scholar
  41. 41.
    Noda M, Furutani Y, Takahashi H, Toyosato M, Tanabe T, Shimizu S, Kikyotani S, Kayano T, Hirose T, Inayama S et al (1983) Cloning and sequence analysis of calf cDNA and human genomic DNA encoding alpha-subunit precursor of muscle acetylcholine receptor. Nature 305:818–823CrossRefPubMedGoogle Scholar
  42. 42.
    Nef P, Oneyser C, Alliod C, Couturier S, Ballivet M (1988) Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors. EMBO J 7:595–601PubMedGoogle Scholar
  43. 43.
    Wada K, Ballivet M, Boulter J, Connolly J, Wada E, Deneris ES, Swanson LW, Heinemann S, Patrick J (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science 240:330–334CrossRefPubMedGoogle Scholar
  44. 44.
    Lustig LR (2006) Nicotinic acetylcholine receptor structure and function in the efferent auditory system. Anat Rec A Discov Mol Cell Evol Biol 288:424–434PubMedGoogle Scholar
  45. 45.
    Nguyen VT, Ndoye A, Grando SA (2000) Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by Pemphigus vulgaris autoimmunity. Am J Pathol 157:1377–1391PubMedGoogle Scholar
  46. 46.
    Vincler M, Wittenauer S, Parker R, Ellison M, Olivera BM, McIntosh JM (2006) Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc Natl Acad Sci USA 103:17880–17884CrossRefPubMedGoogle Scholar
  47. 47.
    Sgard F, Charpantier E, Bertrand S, Walker N, Caput D, Graham D, Bertrand D, Besnard F (2002) A novel human nicotinic receptor subunit, alpha10, that confers functionality to the alpha9-subunit. Mol Pharmacol 61:150–159CrossRefPubMedGoogle Scholar
  48. 48.
    Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J (2001) alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci USA 98:3501–3506CrossRefPubMedGoogle Scholar
  49. 49.
    Chernyavsky AI, Arredondo J, Vetter DE, Grando SA (2007) Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization. Exp Cell Res 313:3542–3555CrossRefPubMedGoogle Scholar
  50. 50.
    Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA (2007) Receptor-mediated tobacco toxicity: alterations of the NF-kappaB expression and activity downstream of alpha7 nicotinic receptor in oral keratinocytes. Life Sci 80:2191–2194CrossRefPubMedGoogle Scholar
  51. 51.
    Song P, Sekhon HS, Fu XW, Maier M, Jia Y, Duan J, Proskosil BJ, Gravett C, Lindstrom J, Mark GP, Saha S, Spindel ER (2008) Activated cholinergic signaling provides a target in squamous cell lung carcinoma. Cancer Res 68:4693–4700CrossRefPubMedGoogle Scholar
  52. 52.
    Sato T, Abe T, Nakamoto N, Tomaru Y, Koshikiya N, Nojima J, Kokabu S, Sakata Y, Kobayashi A, Yoda T (2008) Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line. Biochem Biophys Res Commun 377:126–130CrossRefPubMedGoogle Scholar
  53. 53.
    Duan R, Ginsburg E, Vonderhaar BK (2008) Estrogen stimulates transcription from the human prolactin distal promoter through AP1 and estrogen responsive elements in T47D human breast cancer cells. Mol Cell Endocrinol 281:9–18CrossRefPubMedGoogle Scholar
  54. 54.
    Cattaneo MG, Codignola A, Vicentini LM, Clementi F, Sher E (1993) Nicotine stimulates a serotonergic autocrine loop in human small-cell lung carcinoma. Cancer Res 53:5566–5568PubMedGoogle Scholar
  55. 55.
    Seger R, Krebs EG (1995) The MAPK signaling cascade. FASEB J 9:726–735PubMedGoogle Scholar
  56. 56.
    Guo J, Chu M, Abbeyquaye T, Chen CY (2005) Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation. J Biol Chem 280:30422–30431CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2010

Authors and Affiliations

  • Ching-Shyang Chen
    • 1
  • Chia-Hwa Lee
    • 2
  • Chang-Da Hsieh
    • 2
  • Chi-Tang Ho
    • 3
  • Min-Hsiung Pan
    • 4
  • Ching-Shui Huang
    • 1
    • 5
  • Shih-Hsin Tu
    • 1
    • 5
  • Ying-Jan Wang
    • 6
  • Li-Ching Chen
    • 2
  • Yu-Jia Chang
    • 1
    • 9
    • 10
  • Po-Li Wei
    • 1
    • 9
  • Yi-Yuan Yang
    • 1
    • 7
  • Chih-Hsiung Wu
    • 8
    • 9
  • Yuan-Soon Ho
    • 2
    • 7
    • 9
  1. 1.Department of Surgery and Center of Quality Management and Breast Health CenterTaipei Medical UniversityTaipeiTaiwan
  2. 2.Graduate Institute of Medical SciencesTaipei Medical UniversityTaipeiTaiwan
  3. 3.Department of Food ScienceRutgers UniversityNew BrunswickUSA
  4. 4.Department of Seafood ScienceNational Kaohsiung Marine UniversityKaohsiungTaiwan
  5. 5.Department of SurgeryCathay General HospitalTaipeiTaiwan
  6. 6.Department of Environmental and Occupational HealthNational Cheng Kung University Medical CollegeTainanTaiwan
  7. 7.Graduate Institute of Biomedical TechnologyTaipei Medical UniversityTaipei 110Taiwan
  8. 8.Department of Surgery, School of MedicineTaipei Medical University-Shuang Ho HospitalJhonghe CityTaiwan
  9. 9.Center of Excellence for Cancer ResearchTaipei Medical UniversityTaipeiTaiwan
  10. 10.Graduate Institute of Clinical MedicineTaipei Medical UniversityTaipeiTaiwan

Personalised recommendations