Skip to main content

Advertisement

SpringerLink
Log in

Reserpine Inhibit the JB6 P+ Cell Transformation Through Epigenetic Reactivation of Nrf2-Mediated Anti-oxidative Stress Pathway

  • Research Article
  • Theme: Natural Products Drug Discovery in Cancer Prevention
  • Published:
The AAPS Journal Aims and scope Submit manuscript

Abstract

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a crucial transcription factor that regulates the expression of defensive antioxidants and detoxification enzymes in cells. In a previous study, we showed that expression of the Nrf2 gene is regulated by an epigenetic modification. Rauvolfia verticillata, a traditional Chinese herbal medicine widely used in China, possesses anticancer and antioxidant effects. In this study, we investigated how Nrf2 is epigenetically regulated by reserpine, the main active component in R. verticillata, in mouse skin epidermal JB6 P+ cells. Reserpine induced ARE (antioxidant response element)-luciferase activity in HepG2-C8 cells. Accordingly, in JB6 P+ cells, it upregulated the mRNA and protein levels of Nrf2 and its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), while it only increased the protein level of UDP-glucuronosyltransferase 1A1 (UGT1A1). Furthermore, reserpine decreased the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced colony formation of JB6 cells in a dose-dependent manner. DNA sequencing and methylated DNA immunoprecipitation further demonstrated the demethylation effect of reserpine on the first 15 CpGs of the Nrf2 promoter in JB6 P+ cells. Reserpine also reduced the mRNA and protein expression of DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferases 3a), and DNMT3b (DNA methyltransferases 3b). Moreover, reserpine induced Nrf2 expression via an epigenetic pathway in skin epidermal JB6 P+ cells, enhancing the protective antioxidant activity and decreasing TPA-induced cell transformation. These results suggest that reserpine exhibits a cancer preventive effect by reactivating Nrf2 and inducing the expression of target genes involved in cellular protection, potentially providing new insight into the chemoprevention of skin cancer using reserpine.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Cakir BÖ, Adamson P, Cingi C. Epidemiology and economic burden of nonmelanoma skin cancer. Facial Plast Surg Clin N Am. 2012;20(4):419–22.

    Article  Google Scholar 

  2. Lauren E, Dubas AI. Nonmelanoma skin cancer. Facial Plast Surg Clin N Am. 2013;21(1):43–53.

    Article  Google Scholar 

  3. Leiter U, Garbe C. Epidemiology of melanoma and nonmelanoma skin cancer—the role of sunlight. Adv Exp Med Biol. 2008;624:89–103.

    Article  PubMed  Google Scholar 

  4. Diepgen TL, Mahler V. The epidemiology of skin cancer. Br J Dermatol. 2002;146:1–6.

    Article  PubMed  Google Scholar 

  5. Rebat M, Halder MD, Sharon B, Shah MD. Skin cancer in African Americans. Cancer. 1995;75:667–73.

    Article  Google Scholar 

  6. Gallagher RP, Lee TK, Bajdik CD, Borugian M. Ultraviolet radiation. Chronic Dis Can. 2010;29(1):51–68.

    PubMed  Google Scholar 

  7. Katiyar SK, Mukhtar H. Green tea polyphenol (−)-epigallocatechin-3-gallate treatment to mouse skin prevents UVB-induced infiltration of leukocytes, depletion of antigenpresenting cells, and oxidative stress. J Leukoc Biol. 2001;69(5):719–26.

    CAS  PubMed  Google Scholar 

  8. Frank RG, Henk JK, Leon HFM. UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer. J Photochem Photobiol Biol. 2001;63:19–27.

    Article  Google Scholar 

  9. Ichihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, et al. UV-induced skin damage. Toxicology. 2003;189:21–39.

    Article  CAS  PubMed  Google Scholar 

  10. Halliday GM, Byrne SN, Damian DL. Ultraviolet A radiation: its role in immunosuppression and carcinogenesis. Semin Cutan Med Surg. 2011;30(4):214–21.

    Article  CAS  PubMed  Google Scholar 

  11. Hussein MR. Ultraviolet radiation and skin cancer: molecular mechanisms. J Cutan Pathol. 2005;32(3):191–205.

    Article  PubMed  Google Scholar 

  12. Nandakumar V, Vaid M, Tollefsbol TO, Katiyar SK. Aberrant DNA hypermethylation patterns lead to transcriptional silencing of tumor suppressor genes in UVB-exposed skin and UVB-induced skin tumors of mice. Carcinogenesis. 2011;32(4):597–604.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rimbach G, Hohler D, Fischer A, Roy S, Virqili F, Pallauf J, et al. Methods to assess free radicals and oxidative stress in biological systems. Arch Tierernahr. 1999;52(3):203–22.

    Article  CAS  PubMed  Google Scholar 

  14. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med. 2010;49(11):1603–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nourazarian AR, Kangari P, Salmaninejad A. Roles of oxidative stress in the development and progression of breast cancer. Asian Pac J Can Prev. 2014;15(12):4745–51.

    Article  Google Scholar 

  16. Kobayashi A, Ohta T, Yamamoto M. Unique function of the Nrf2-Keap1 pathway in the inducible expression of antioxidant and detoxifying enzymes. Meth Enzym. 2004;378:273–86.

    Article  CAS  PubMed  Google Scholar 

  17. Kwak MK, Wakabayashi N, Kensler TW. Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers. Mutat Res. 2004;555:133–48.

    Article  CAS  PubMed  Google Scholar 

  18. Su ZY, Zhang C, Lee JH, Shu L, Wu TY, Khor TO, et al. Requirement and epigenetics re-programming of Nrf2 in suppression of tumor promoter TPA-induced mouse skin cell transformation by sulforaphane. Cancer Prev Res. 2014;7:319–29.

    Article  CAS  Google Scholar 

  19. Lee JH, Khor TO, Shu L, Su Z-Y, Fuentes F, Kong A-NT. Dietary phytochemicals and cancer prevention: Nrf2 signaling, epigenetics, and cell death mechanisms in blocking cancer initiation and progression. Pharmacol Ther. 2013;137(2):153–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Su ZY, Shu L, Khor TO, Lee JH, Fuentes F, Kong AN. A perspective on dietary phytochemicals and cancer chemoprevention: oxidative stress, nrf2, and epigenomics. Top Curr Chem. 2013;329:133–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang CY, Su ZY, Khor TO, Shu LM, Kong AN. Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation. Biochem Pharmacol. 2013;85(9):1398–404.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002;3(6):415–28.

    CAS  PubMed  Google Scholar 

  23. Shu L, Khor TO, Lee JH, Boyanapalli SS, Huang Y, Wu TY, et al. Epigenetic CpG demethylation of the promoter and reactivation of the expression of Neurog1 by curcumin in prostate LNCaP cells. AAPS J. 2011;13(4):606–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sathyanarayana UG, Moore AY, Li L, Padar A, Majmudar K, Stastny V, et al. Sun exposure related methylation in malignant and non-malignant skin lesions. Cancer Lett. 2007;245(1–2):112–20.

    Article  CAS  PubMed  Google Scholar 

  25. Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358:1148–59.

    Article  CAS  PubMed  Google Scholar 

  26. Devinoy E, Rijnkels M. Epigenetics in mammary gland biology and cancer. J Mammary Gland Biol Neoplasia. 2010;15:1–4.

    Article  PubMed  Google Scholar 

  27. Kim TY, Bang YJ, Robertson KD. Histone deacetylase inhibitors for cancer therapy. Epigenetics. 2006;1:14–23.

    Article  PubMed  Google Scholar 

  28. Walton TJ, Li G, Seth R, McArdle SE, Bishop MC, Rees RC. DNA demethylation and histone deacetylation inhibition co-operate to reexpress estrogen receptor beta and induce apoptosis in prostate cancer cell-lines. Prostate. 2008;68:210–22.

    Article  CAS  PubMed  Google Scholar 

  29. Hatzimichael E, Crook T. Cancer epigenetics: new therapies and new challenges. J Drug Deliv. 2013;2013:1–9.

    Article  Google Scholar 

  30. Katiyar SK, Singh T, Prasad R, Sun Q, Vaid M. Epigenetic alterations in ultraviolet radiation-induced skin carcinogenesis: interaction of bioactive dietary components on epigenetic targets. Photochem Photobiol. 2012;88(5):1066–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhu WW, Wang Q, Deng XC, Su D. Determination of reserpine in the R. vomitoria roots from different producing areas by HPLC. Chin J Spectrosc Lab. 2011;28:708–10.

    CAS  Google Scholar 

  32. Bemis DL, Capodice JL, Gorroochurn P, Katz AE, Buttyan R. Anti-prostate cancer activity of a β-carboline alkaloid enriched extract from Rauwolfia vomitoria. Intern J Oncol. 2006;29:1065–73.

    CAS  Google Scholar 

  33. Popov SV, Vinter VG, Patova OA, Markov PA, Nikitina IR, Ovodova RG, et al. Chemical characterization and anti-inflammatory effect of rauvolfian, a pectic polysaccharide of Rauvolfia callus. Biochem (Moscow). 2007;72:778–84.

    Article  CAS  Google Scholar 

  34. Al-Qirim TM, Zafir A, Banu N. Comparative anti-oxidant potential of Rauwolfia serpentine and withania somnifera on cardiac tissues. FASEB J. 2007;21:510–5.

    Google Scholar 

  35. Liu PH, Liu YY, Shi J. Antioxidant potential of water soluble alkaloids from rauvolfia study by DPPH in vitro assay. Lishizhen Med Mat Res. 2010;21(3):607–9.

    CAS  Google Scholar 

  36. Lin WJ, Chen TD, Liu CW, Chen JL, Chang FH. Synthesis of lactobionic acid-grafted-pegylated-chitosan with enhanced HepG2 cells transfection. Carbohydr Polym. 2011;83(2):898–904.

    Article  CAS  Google Scholar 

  37. Su ZY, Khor TO, Shu L, Lee JH, Saw CL, Wu TY, et al. Epigenetic reactivation of Nrf2 in murine prostate cancer TRAMP C1 cells by natural phytochemicals Z-ligustilide and Radix angelica sinensis via promoter CpG demethylation. Chem Res Toxicol. 2013;26:477–85.

    Article  CAS  PubMed  Google Scholar 

  38. Yu S, Khor TO, Cheung KL, Li W, Wu TY, Huang Y, et al. Nrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice. PloS ONE. 2010;5:e857.

    Google Scholar 

  39. Khor TO, Huang Y, Wu TY, Shu L, Lee J, Kong AN. Pharmacodynamics of curcumin as DNA hypomethylation agent in restoring the expression of Nrf2 via promoter CpGs demethylation. Biochem Pharmacol. 2011;82:1073–8.

    Article  CAS  PubMed  Google Scholar 

  40. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev. 1994;74:139–62.

    CAS  PubMed  Google Scholar 

  41. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer. 2007;121:2381–6.

    Article  CAS  PubMed  Google Scholar 

  42. Trouba KJ, Hamadeh HK, Amin RP, Germolec DR. Oxidative stress and its role in skin disease. Antioxid Redox Signal. 2002;4:665–73.

    Article  CAS  PubMed  Google Scholar 

  43. Ito N, Hirose M. Antioxidants—carcinogenic and chemopreventive properties. Adv Cancer Res. 1989;53:247–302.

    Article  CAS  PubMed  Google Scholar 

  44. Pillai CK, Pillai KS. Antioxidants in health. Indian J Phys Pharm. 2002;46:1–5.

    CAS  Google Scholar 

  45. Wattenberg LW. Chemoprevention of cancer. Cancer Res. 1985;45:1–8.

    Article  CAS  PubMed  Google Scholar 

  46. Talalay P, Dinkova KAT, Holtzclaw WD. Importance of phase 2 gene regulation in protection against electrophile and reactive oxygen toxicity and carcinogenesis. Adv Enzym Regul. 2003;43:121–34.

    Article  CAS  Google Scholar 

  47. Nakamura Y, Kozuka M, Naniwa K, Takabayashi S, Torikai K, Hayashi R, et al. Arachidonic acid cascade inhibitors modulate phorbol ester-induced oxidative stress in female ICR mouse skin: differential roles of 5-lipoxygenase and cyclooxygenase-2 in leukocyte infiltration and activation. Free Radic Biol Med. 2003;35:997–1007.

    Article  CAS  PubMed  Google Scholar 

  48. Dhar A, Young MR, Colburn NH. The role of AP-1, NF-kappaB and ROS/NOS in skin carcinogenesis: the JB6 model is predictive. Mol Cell Biochem. 2002;234–235:185–93.

    Article  PubMed  Google Scholar 

  49. Committee of Chinese Experts on Rational Drug Use. Guidelines for rational drug use in hypertension. Chin J Front Med Sci. 2015;7:22–65.

    Google Scholar 

  50. Sui GZ, Jing FB, Su ZG, Yu GY. Rational usage of reserpine. Chin J Med. 2011;46:81–4.

    Google Scholar 

  51. Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P, et al. Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci U S A. 2001;98:3410–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Khor TO, Huang MT, Prawan A, Liu Y, Hao X, Yu S, et al. Increased susceptibility of Nrf2 knockout mice to colitis-associated colorectal cancer. Cancer Prev Res (Phila). 2008;1:187–91.

    Article  CAS  Google Scholar 

  53. Kang KW, Lee SJ, Kim SG. Molecular mechanism of nrf2 activation by oxidative stress. Antioxid Redox Signal. 2005;7:1664–73.

    Article  CAS  PubMed  Google Scholar 

  54. Nguyen T, Nioi P, Pickett CB. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem. 2009;284:13291–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chen C, Kong AN. Dietary chemopreventive compounds and ARE/EpRE signaling. Free Radic Biol Med. 2004;36:1505–16.

    Article  CAS  PubMed  Google Scholar 

  56. Hu R, Saw CL, Yu R, Kong AN. Regulation of NF-E2-related factor 2 signaling for cancer chemoprevention: antioxidant coupled with antiinflammatory. Antioxid Redox Signal. 2010;13:1679–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kwak MK, Egner PA, Dolan PM, Ramos-Gomez M, Groopman JD, Itoh K, et al. Role of phase 2 enzyme induction in chemoprotection by dithiolethiones. Mutat Res. 2001;480–481:305–15.

    Article  PubMed  Google Scholar 

  58. Cullinan SB, Gordan JD, Jin J, Harper JW, Diehl JA. The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase. Mol Cell Biol. 2004;4:8477–86.

    Article  Google Scholar 

  59. Kobayashi M, Li L, Iwamoto N, Nakajima-Takagi Y, Kaneko H, Nakayama Y, et al. The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Mol Cell Biol. 2009;29:493–502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. McMahon M, Lamont DJ, Beattie KA, Hayes JD. Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals. Proc Natl Acad Sci U S A. 2010;107:18838–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Hu C, Eggler AL, Mesecar AD, van Breemen RB. Modification of keap1 cysteine residues by sulforaphane. Chem Res Toxicol. 2011;24:515–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Huang Y, Khor TO, Shu L, Saw CL, Wu TY, Suh N, et al. A gamma-tocopherol-rich mixture of tocopherols maintains Nrf2 expression in prostate tumors of TRAMP mice via epigenetic inhibition of CpG methylation. J Nutr. 2012;142:818–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Zhang C, Su ZY, Khor TO, Shu L, Kong AN. Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation. Biochem Pharmacol. 2013;85:1398–404.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Wu TY, Khor TO, Su ZY, Saw CL, Shu L, Cheung KL, et al. Epigenetic modifications of Nrf2 by 3,3′-diindolylmethane in vitro in TRAMP C1 cell line and in vivo TRAMP prostate tumors. AAPS J. 2013;15:864–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Paredes-Gonzalez X, Fuentes F, Su ZY, Kong AN. Apigenin reactivates Nrf2 anti-oxidative stress signaling in mouse skin epidermal JB6 P + cells through epigenetics modifications. AAPS J. 2014;16:727–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang L, Zhang C, Guo Y, Su ZY, Yang Y, Shu L, et al. Blocking of JB6 cell transformation by tanshinone IIA: epigenetic reactivation of Nrf2 antioxidative stress pathway. AAPS J. 2014;16:1214–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31:27–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Kozuka S. Inhibiting effect of reserpine and female sensitivity in hepatic tumor induction with 2,7-diacetamidofluorene in SMA-Ms strain mice. Cancer Res. 1970;30:1384–6.

    CAS  PubMed  Google Scholar 

  69. Lupulescu A. Reserpine and carcinogenesis: inhibition of carcinoma formation in mice. J Natl Cancer Inst. 1983;71:1077–83.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank all the members in Dr. A.-N.T. Kong’s laboratory for their helpful discussion and preparation of this manuscript.

Authors’ Contributions

Conception and design: B. Hong, Z.Y. Su, C.Y. Zhang, and A.-N.T. Kong.

Development of methodology: B. Hong and Z.Y. Su.

Acquisition of data (provided animals, provided facilities, etc.): B. Hong, Z.Y. Su, and W.J. Li.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and computational analysis): B. Hong, Yuqing Yang, and Yue Guo.

Writing, review, and revision of the manuscript: B. Hong, Z.Y. Su, W.J. Li, and A.-N.T. Kong.

Study supervision: A.-N.T. Kong.

Author information

Author notes

    Authors and Affiliations

    1. Department of Pharmacy, Qiqihar Medical University, 161006, Qiqihar, Heilongjiang, China

      Bo Hong & Wenjing Li

    2. Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey, 08854, USA

      Bo Hong, Zhengyuan Su, Chengyue Zhang, Yuqing Yang, Yue Guo & Ah-Ng Tony Kong

    3. Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan City, 32023, Taiwan, Republic of China

      Zhengyuan Su

    Authors
    1. Bo Hong
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Zhengyuan Su
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Chengyue Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Yuqing Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Yue Guo
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Wenjing Li
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Ah-Ng Tony Kong
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Ah-Ng Tony Kong.

    Ethics declarations

    Grant Support

    This work was supported by institutional funds of Rutgers to Ah-Ng Tony Kong and Grant 81403173 from the National Science Foundation of China to Bo Hong.

    Conflict of Interest

    The authors declare that they have no competing interests.

    Additional information

    Guest Editors: Ah-Ng Tony Kong and Chi Chen

    Bo Hong and Zhengyuan Su contributed equally to this work.

    ELECTRONIC SUPPLEMENTARY MATERIAL

    Below is the link to the electronic supplementary material.

    ESM 1

    (DOC 23,785 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Hong, B., Su, Z., Zhang, C. et al. Reserpine Inhibit the JB6 P+ Cell Transformation Through Epigenetic Reactivation of Nrf2-Mediated Anti-oxidative Stress Pathway. AAPS J 18, 659–669 (2016). https://doi.org/10.1208/s12248-016-9901-6

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1208/s12248-016-9901-6

    Keywords

    Search

    Navigation