Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 392, Issue 3, pp 381–391 | Cite as

2-Methoxyestradiol attenuates liver fibrosis in mice: implications for M2 macrophages

  • Thikryat NeamatallahEmail author
  • Ashraf B. Abdel-Naim
  • Basma G. Eid
  • Atif Hasan
Original Article


Liver fibrosis is a major health problem worldwide due to its serious complications including cirrhosis and liver cancer. 2-Methoxyestradiol (2-ME) is an end metabolite of estradiol with anti-proliferative, antioxidant, and anti-inflammatory activities. However, the protective role of 2-ME in liver fibrosis has not been fully investigated. The aim of this study was to determine the protective effect of 2-ME in carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Animals were injected intraperitoneally with CCl4 twice weekly for 6 weeks. 2-ME 50 mg/kg or 100 mg/kg was administrated intraperitoneally every day over the same period. Our data showed that 2-ME reduced the extent of liver toxicity and fibrosis due to CCl4 exposure. It restored the elevated serum liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels and ameliorated oxidative status. In addition, 2-ME significantly reduced collagen deposition and alpha-smooth muscle actin (α-SMA) protein expressions. Furthermore, 2-ME markedly lowered macrophage infiltration and macrophage alternative activation marker chitinase-like molecules (CHI3L3/YM1). The results of this study indicate an important protective activity of 2-ME in liver fibrosis and highlight the role of macrophage recruitment and alternative activation as a possible target.


2-Methoxyestradiol Liver fibrosis Carbon tetrachloride Macrophages Alternative activation 



The project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. G-252-249-38. The authors, therefore, acknowledge with thanks DSR for technical and financial support.

Author contribution

TN and AA conceived and designed research. AA and AH participated in the biochemical and immunohistochemical studies. BE contributed new reagents and antibodies. TN performed the real-time polymerase chain reaction. TN, BE, and AA analyzed the data. TN wrote the manuscript. All authors revised and approved the manuscript.

Funding information

The project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. (G-252-249-38).

Compliance with ethical standards

The study was carried out in accordance with ethical standards in all aspects.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

210_2018_1577_MOESM1_ESM.docx (171 kb)
ESM 1 (DOCX 170 kb)


  1. Abdel-Naim AB, Neamatallah T, Eid BG, Esmat A, Alamoudi AJ, Abd El-Aziz GS, Ashour OM (2018) 2-Methoxyestradiol Attenuates Testosterone-Induced Benign Prostate Hyperplasia in Rats through Inhibition of HIF-1α/TGF-β/Smad2 Axis. Oxidative Med Cell Longev 2018:4389484Google Scholar
  2. Aquino-Galvez A, Gonzalez-Avila G, Delgado-Tello J, Castillejos-Lopez M, Mendoza-Milla C, Zuniga J, Checa M, Maldonado-Martinez HA, Trinidad-Lopez A, Cisneros J, Torres-Espindola LM, Hernandez-Jimenez C, Sommer B, Cabello-Gutierrez C, Gutierrez-Gonzalez LH (2016) Effects of 2-methoxyestradiol on apoptosis and HIF-1alpha and HIF-2alpha expression in lung cancer cells under normoxia and hypoxia. Oncol Rep 35:577–583Google Scholar
  3. Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115:209–218Google Scholar
  4. Becker CM, Rohwer N, Funakoshi T, Cramer T, Bernhardt W, Birsner A, Folkman J, D’amato RJ (2008) 2-Methoxyestradiol inhibits hypoxia-inducible factor-1α and suppresses growth of lesions in a mouse model of endometriosis. Am J Pathol 172:534–544Google Scholar
  5. Beljaars L, Schippers M, Reker-Smit C, Martinez FO, Helming L, Poelstra K, Melgert BN (2014) Hepatic localization of macrophage phenotypes during fibrogenesis and resolution of fibrosis in mice and humans. Front Immunol 5:430Google Scholar
  6. Berg FD, Kuss E (1992) Serum concentration and urinary excretion of “classical” estrogens, catecholestrogens and 2-methoxyestrogens in normal human pregnancy. Arch Gynecol Obstet 251:17–27Google Scholar
  7. Bruce JY, Eickhoff J, Pili R, Logan T, Carducci M, Arnott J, Treston A, Wilding G, Liu G (2012) A phase II study of 2-methoxyestradiol nanocrystal colloidal dispersion alone and in combination with sunitinib malate in patients with metastatic renal cell carcinoma progressing on sunitinib malate. Investig New Drugs 30:794–802Google Scholar
  8. Campbell M, Yang YX, Reddy KR (2005) Progression of liver fibrosis in women infected with hepatitis C: long-term benefit of estrogen exposure. Hepatology 41:939 author reply 939-40Google Scholar
  9. Canbay A, Friedman S, Gores GJ (2004) Apoptosis: the nexus of liver injury and fibrosis. Hepatology 39:273–278Google Scholar
  10. Collazos J, Carton JA, Asensi V (2011) Gender differences in liver fibrosis and hepatitis C virus-related parameters in patients coinfected with human immunodeficiency virus. Curr HIV Res 9:339–345Google Scholar
  11. Dahut WL, Lakhani NJ, Gulley JL, Arlen PM, Kohn EC, Kotz H, McNally D, Parr A, Parr A, Nguyen D, Yang SX, Steinberg SM, Venitz J, Sparreboom A, Figg II W (2014) Phase I clinical trial of oral 2-methoxyestradiol, an antiangiogenic and apoptotic agent, in patients with solid tumors. Cancer Biology & Therapy 5(1):22–27.
  12. Deng J, Huang Q, Wang Y, Shen P, Guan F, Li J, Huang H, Shi C (2014) Hypoxia-inducible factor-1alpha regulates autophagy to activate hepatic stellate cells. Biochem Biophys Res Commun 454:328–334Google Scholar
  13. Deuffic-Burban S, Poynard T, Valleron AJ (2002) Quantification of fibrosis progression in patients with chronic hepatitis C using a Markov model. J Viral Hepat 9:114–122Google Scholar
  14. Dubey RK, Jackson EK (2009) Potential vascular actions of 2-methoxyestradiol. Trends Endocrinol Metab 20:374–379Google Scholar
  15. Dubey RK, Gillespie DG, Zacharia LC, Rosselli M, Korzekwa KR, Fingerle J, Jackson EK (2000) Methoxyestradiols mediate the antimitogenic effects of estradiol on vascular smooth muscle cells via estrogen receptor-independent mechanisms. Biochem Biophys Res Commun 278:27–33Google Scholar
  16. Friedman SL (2008) Mechanisms of hepatic fibrogenesis. Gastroenterology 134:1655–1669Google Scholar
  17. Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3:23–35Google Scholar
  18. Gordon S, Martinez FO (2010) Alternative activation of macrophages: mechanism and functions. Immunity 32:593–604Google Scholar
  19. Harrison MR, Hahn NM, Pili R, Oh WK, Hammers H, Sweeney C, Kim K, Perlman S, Arnott J, Sidor C, Wilding G, Liu G (2011) A phase II study of 2-methoxyestradiol (2ME2) NanoCrystal® dispersion (NCD) in patients with taxane-refractory, metastatic castrate-resistant prostate cancer (CRPC). Invest New Drugs 29:1465–1474Google Scholar
  20. Heymann F, Hammerich L, Storch D, Bartneck M, Huss S, Rüsseler V, Gassler N, Lira SA, Luedde T, Trautwein C, Tacke F (2012) Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice. Hepatology (Baltimore, Md.) 55:898–909Google Scholar
  21. Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, Van Rooijen N, Macdonald AS, Allen JE (2011) Local macrophage proliferation, rather than recruitment from the blood, is a signature of Th2 inflammation. Science (New York, NY) 332:1284–1288Google Scholar
  22. Jenkins SJ, Ruckerl D, Thomas GD, Hewitson JP, Duncan S, Brombacher F, Maizels RM, Hume DA, Allen JE (2013) IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. J Exp Med 210:2477–2491Google Scholar
  23. Kang S-H, Cho HT, Devi S, Zhang Z, Escuin D, Liang Z, Mao H, Brat DJ, Olson JJ, Simons JW, LaVallee TM, Giannakakou P, Van Meir EG, Shim H (2006) Antitumor effect of 2-Methoxyestradiol in a rat orthotopic brain tumor model. Cancer Res 66(24):11991–11997.
  24. Lakhani NJ, Sarkar MA, Venitz J, Figg WD (2003) 2-Methoxyestradiol, a promising anticancer agent. Pharmacotherapy 23:165–172Google Scholar
  25. Liedtke C, Luedde T, Sauerbruch T, Scholten D, Streetz K, Tacke F, Tolba R, Trautwein C, Trebicka J, Weiskirchen R (2013) Experimental liver fibrosis research: update on animal models, legal issues and translational aspects. Fibrogenesis Tissue Repair 6:19Google Scholar
  26. Lindquist JN, Marzluff WF, Stefanovic B (2000) Fibrogenesis. III. Posttranscriptional regulation of type I collagen. Am J Physiol Gastrointest Liver Physiol 279:G471–G476Google Scholar
  27. Liu QH, Li DG, Huang X, Zong CH, Xu QF, Lu HM (2004) Suppressive effects of 17beta-estradiol on hepatic fibrosis in CCl4-induced rat model. World J Gastroenterol 10:1315–1320Google Scholar
  28. López-Navarrete G, Ramos-Martínez E, Suárez-Álvarez K, Aguirre-García J, Ledezma-Soto Y, León-Cabrera S, Gudiño-Zayas M, Guzmán C, Gutiérrez-Reyes G, Hernández-Ruíz J, Camacho-Arroyo I, Robles-Díaz G, Kershenobich D, Terrazas LI, Escobedo G (2011) Th2-associated alternative Kupffer cell activation promotes liver fibrosis without inducing local inflammation. Int J Biol Sci 7:1273–1286.
  29. Mann DA, Smart DE (2002) Transcriptional regulation of hepatic stellate cell activation. Gut 50:891–896Google Scholar
  30. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25:677–686Google Scholar
  31. Miura K, Yang L, Van Rooijen N, Ohnishi H, Seki E (2012) Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2. Am J Physiol Gastrointest Liver Physiol 302:G1310–G1321Google Scholar
  32. Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958–969Google Scholar
  33. Mueck AO, Seeger H (2010) 2-Methoxyestradiol--biology and mechanism of action. Steroids 75:625–631Google Scholar
  34. Nair AB, Jacob S (2016) A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 7(2):27–31.
  35. Nair MG, Gallagher IJ, Taylor MD, Loke P, Coulson PS, Wilson RA, Maizels RM, Allen JE (2005) Chitinase and Fizz family members are a generalized feature of nematode infection with selective upregulation of Ym1 and Fizz1 by antigen-presenting cells. Infect Immun 73:385–394Google Scholar
  36. Naito M, Hasegawa G, Ebe Y, Yamamoto T (2004) Differentiation and function of Kupffer cells. Med Electron Microsc 37:16–28Google Scholar
  37. Parada-Bustamante A, Valencia C, Reuquen P, Diaz P, Rincion-Rodriguez R, Orihuela PA (2015) Role of 2-methoxyestradiol, an endogenous estrogen metabolite, in health and disease. Mini Rev Med Chem 15:427–438Google Scholar
  38. Pellicoro A, Ramachandran P, Iredale JP, Fallowfield JA (2014) Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nat Rev Immunol 14:181–194Google Scholar
  39. Popov Y, Sverdlov DY, Sharma AK, Bhaskar KR, Li S, Freitag TL, Lee J, Dieterich W, Melino G, Schuppan D (2011) Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice. Gastroenterology 140:1642–1652Google Scholar
  40. Poynard T, Ratziu V, Charlotte F, Goodman Z, Mchutchison J, Albrecht J (2001) Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis c. J Hepatol 34:730–739Google Scholar
  41. Poynard T, Mathurin P, Lai CL, Guyader D, Poupon R, Tainturier MH, Myers RP, Muntenau M, Ratziu V, Manns M, Vogel A, Capron F, Chedid A, Bedossa P, GROUP, P (2003) A comparison of fibrosis progression in chronic liver diseases. J Hepatol 38:257–265Google Scholar
  42. Pribluda VS, Gubish ER Jr, Lavallee TM, Treston A, Swartz GM, Green SJ (2000) 2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate. Cancer Metastasis Rev 19:173–179Google Scholar
  43. Puche JE, Saiman Y, Friedman SL (2013) Hepatic stellate cells and liver fibrosis. Compr Physiol 3:1473–1492Google Scholar
  44. Raes G, Noel W, Beschin A, Brys L, De Baetselier P, Hassanzadeh GH (2002) FIZZ1 and Ym as tools to discriminate between differentially activated macrophages. Dev Immunol 9:151–159Google Scholar
  45. Raes G, van den Bergh R, de Baetselier P, Ghassabeh GH (2005) Arginase-1 and Ym1 are markers for murine, but not human, alternatively activated myeloid cells. J Immunol 174:6561–6562Google Scholar
  46. Ramachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, Hartland SN, Snowdon VK, Cappon A, Gordon-Walker TT, Williams MJ, Dunbar DR, Manning JR, Van Rooijen N, Fallowfield JA, Forbes SJ, Iredale JP (2012) Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis. Proc Natl Acad Sci U S A 109:E3186–E3195Google Scholar
  47. Ricker JL, Chen Z, Yang XP, Pribluda VS, Swartz GM, van Waes C (2004) 2-Methoxyestradiol inhibits hypoxia-inducible factor 1α, tumor growth, and angiogenesis and augments paclitaxel efficacy in head and neck squamous cell carcinoma. Clin Cancer Res 10:8665–8673Google Scholar
  48. Rygiel KA, Robertson H, Marshall HL, Pekalski M, Zhao L, Booth TA, Jones DE, Burt AD, Kirby JA (2008) Epithelial-mesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease. Lab Investig 88:112–123Google Scholar
  49. Sato M, Suzuki S, Senoo H (2003) Hepatic stellate cells: unique characteristics in cell biology and phenotype. Cell Struct Funct 28:105–112Google Scholar
  50. Seki E, Brenner DA (2015) Recent advancement of molecular mechanisms of liver fibrosis. J Hepato-Biliary Pancreat Sci 22:512–518Google Scholar
  51. Tacke F, Zimmermann HW (2014) Macrophage heterogeneity in liver injury and fibrosis. J Hepatol 60:1090–1096Google Scholar
  52. Tevaarwerk AJ, Holen KD, Alberti DB, Sidor C, Arnott J, Quon C, Wilding G, Liu G (2009) Phase I trial of 2-methoxyestradiol NanoCrystal dispersion in advanced solid malignancies. Clin Cancer Res 15:1460–1465Google Scholar
  53. Tofovic SP, Zhang X, Jackson EK, Zhu H, Petrusevska G (2009) 2-methoxyestradiol attenuates bleomycin-induced pulmonary hypertension and fibrosis in estrogen-deficient rats. Vasc Pharmacol 51:190–197Google Scholar
  54. Vijayanathan V, Venkiteswaran S, Nair SK, Verma A, Thomas TJ, Zhu BT, Thomas T (2006) Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17β-estradiol in human breast cancer cells. Clin Cancer Res 12:2038–2048Google Scholar
  55. Wang L, Zheng Q, Yuan Y, Li Y, Gong X (2017) Effects of 17beta-estradiol and 2-methoxyestradiol on the oxidative stress-hypoxia inducible factor-1 pathway in hypoxic pulmonary hypertensive rats. Exp Ther Med 13:2537–2543Google Scholar
  56. Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214:199–210Google Scholar
  57. Wynn TA, Barron L (2010) Macrophages: master regulators of inflammation and fibrosis. Semin Liver Dis 30:245–257Google Scholar
  58. Wynn TA, Vannella KM (2016) Macrophages in tissue repair, regeneration, and Fibrosis. Immunity 44:450–462Google Scholar
  59. Xu JW, Gong J, Chang XM, Luo JY, Dong L, Hao ZM, Jia A, Xu GP (2002) Estrogen reduces CCL4-induced liver fibrosis in rats. World J Gastroenterol 8:883–887Google Scholar
  60. Xu JW, Gong J, Chang XM, Luo JY, Dong L, Jia A, Xu GP (2004) Effects of estradiol on liver estrogen receptor-alpha and its mRNA expression in hepatic fibrosis in rats. World J Gastroenterol 10:250–254Google Scholar
  61. Xu X, Roman JM, Issaq HJ, Keefer LK, Veenstra TD, Ziegler RG (2007) Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry. Anal Chem 79:7813–7821Google Scholar
  62. Xue J, Sharma V, Hsieh MH, Chawla A, Murali R, Pandol SJ, Habtezion A (2015) Alternatively activated macrophages promote pancreatic fibrosis in chronic pancreatitis. Nat Commun 6:7158Google Scholar
  63. Yan C, Shen Y, Sun Q, Yuan D, Tang H, Gao H (2017) 2-Methoxyestradiol protects against IgG immune complex-induced acute lung injury by blocking NF-kappaB and CCAAT/enhancer-binding protein beta activities. Mol Immunol 85:89–99Google Scholar
  64. Zhang CY, Yuan WG, He P, Lei JH, Wang CX (2016) Liver fibrosis and hepatic stellate cells: etiology, pathological hallmarks and therapeutic targets. World J Gastroenterol 22:10512–10522Google Scholar
  65. Zhao XY, Wang BE, Li XM, Wang TL (2008) Newly proposed fibrosis staging criterion for assessing carbon tetrachloride- and albumin complex-induced liver fibrosis in rodents. Pathol Int 58:580–588Google Scholar
  66. Zhu L, Song Y, Li M (2015) 2-Methoxyestradiol inhibits bleomycin-induced systemic sclerosis through suppression of fibroblast activation. J Dermatol Sci 77:63–70Google Scholar
  67. DOI: 10.7150/ijbs.7.1273Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Thikryat Neamatallah
    • 1
    Email author
  • Ashraf B. Abdel-Naim
    • 1
  • Basma G. Eid
    • 1
  • Atif Hasan
    • 2
  1. 1.Department of Pharmacology and Toxicology, Faculty of PharmacyKing Abdulaziz UniversityJeddahSaudi Arabia
  2. 2.Department of Anatomy and Embryology, Faculty of Veterinary MedicineKafrelsheikh UniversityKafrelsheikhEgypt

Personalised recommendations