Chemopreventive potential of esculetin in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis

  • Renganathan Selvasundaram
  • Shanmugam Manoharan
  • Rajamanickam Buddhan
  • Mani Neelakandan
  • Radhakrishnan Murali Naidu


7,12-Dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis is widely preferred to assess the tumor-inhibiting efficacy of the medicinal plants or their constituents. The present study explores the tumor-inhibiting potential of esculetin by utilizing the status of lipid peroxidation by products (thiobarbituric acid reactive substances), antioxidants (vitamin E, reduced glutathione, superoxide dismutase, catalase, and glutathione peroxidase), and phase I and phase II detoxification agents as biochemical end points and by using histopathological studies as well in DMBA-induced hamster buccal pouch carcinogenesis. Oral tumors developed in the buccal pouch were subjected to histopathological studies, and were confirmed as oral squamous cell carcinoma. Hamsters treated with DMBA alone showed an abnormal pattern of lipid peroxidation, antioxidants, and detoxification agents as compared to control hamsters. The status of the above-mentioned biochemical markers and histopathological abnormalities were found to be reversed in DMBA + esculetin-treated hamsters. The result of the present study thus indicates the tumor preventive potential of esculetin in DMBA-induced oral carcinogenesis.


Oral cancer Esculetin Lipid peroxidation Antioxidants 



Financial assistance from Indian Council of Medical Research, New Delhi is gratefully acknowledged. The authors gratefully acknowledge UGC-SAP and DST-FIST support in the form of providing necessary infrastructural facilities to carry out the work successfully.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


  1. 1.
    Sangle VA, Bijjaragi S, Shah N, Kangane S, Ghule HM, Rani SA (2016) Comparative study of frequency of micronuclei in normal, potentially malignant diseases and oral squamous cell carcinoma. J Nat Sci Biol Med 7(1):33–38CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Nagini S (2009) Of humans and hamsters: the hamster buccal pouch carcinogenesis model as a paradigm for oral oncogenesis and chemoprevention. Anticancer Agents Med Chem 9(8):843–852CrossRefPubMedGoogle Scholar
  3. 3.
    Manoharan S, Sindhu G, Nirmal MR, Vetrichelvi V, Balakrishnan S (2011) Protective effect of berberine on expression pattern of apoptotic, cell proliferative, inflammatory and angiogenic markers during 7,12 dimethylbenz(a)anthracene induced hamster buccal pouch carcinogenesis. Pak J Biol Sci 14(20):918–932CrossRefPubMedGoogle Scholar
  4. 4.
    Manimaran A, Buddhan R, Manoharan S (2017) Emodin downregulates cell proliferation markers during DMBA induced oral carcinogenesis in golden Syrian hamsters. Afr J Tradit Complement Altern Med 13(2):83–91 14(CrossRefGoogle Scholar
  5. 5.
    Manoharan S, Guillemin GJ, Abiramasundari RS, Essa MM, Akbar M, Akbar MD. (2016) The role of reactive oxygen species in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease: a mini review. Oxid Med Cell Longev 2016:1–15CrossRefGoogle Scholar
  6. 6.
    Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, Murata M, Kawanishi S (2013). DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013:1–9CrossRefGoogle Scholar
  7. 7.
    Lobo V, Patil A, Phatak A, Chandra N (2010) Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn Rev 4(8):118–126CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gu X, Manautou JE (2012) Molecular mechanisms underlying chemical liver injury. Expert Rev Mol Med. PubMedPubMedCentralGoogle Scholar
  9. 9.
    Rajasekaran D, Manoharan S, Prabhakar MM, Manimaran A (2015) Enicostemma littorale prevents tumor formation in 7,12 dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. Hum Exp Toxicol 34(9):911–921CrossRefPubMedGoogle Scholar
  10. 10.
    Venugopala KN, Rashmi V, Odhav B (2013) Review on natural coumarin lead compounds for their pharmacological activity. Biomed Res Int. PubMedPubMedCentralGoogle Scholar
  11. 11.
    Wang J, Lu ML, Dai HL, Zhang SP, Wang HX, Wei N (2015) Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway. Braz J Med Biol Res 48(3):245–253CrossRefPubMedGoogle Scholar
  12. 12.
    Arora R, Sawney S, Saini V, Steffi C, Tiwari M, Saluja D (2016) Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1. Mol Cancer 15:(64):1–15Google Scholar
  13. 13.
    Jeon YJ, Cho JH, Lee SY, Choi YH, Park H, Jung S, Shim JH, Chae JI (2016) Esculetin induces apoptosis through EGFR/PI3K/Akt signaling pathway and nucleophosmin relocalization. J Cell Biochem 117(5):1210–1221CrossRefPubMedGoogle Scholar
  14. 14.
    Cho JH, Shin JC, Cho JJ, Choi YH, Shim JH, Chae JI (2015) Esculetin (6,7-dihydroxycoumarin): a potential cancer chemopreventive agent through suppression of Sp1 in oral squamous cancer cells. Int J Oncol 46(1):265–271CrossRefPubMedGoogle Scholar
  15. 15.
    Wang C, Pei A, Chen J, Yu H, Sun ML, Liu CF, Xu X (2012) A natural coumarin derivative esculetin offers neuroprotection on cerebral ischemia/reperfusion injury in mice. J Neurochem 121(6):1007–1013CrossRefPubMedGoogle Scholar
  16. 16.
    Tien YC, Liao JC, Chiu CS, Huang TH, Huang CY, Chang WT, Peng WH (2011) Esculetin ameliorates carbon tetrachloride-mediated hepatic apoptosis in rats. Int J Mol Sci 12(6):4053–4067CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yum S, Jeong S, Lee S, Kim W, Nam J, Jung Y (2015) HIF-prolyl hydroxylase is a potential molecular target for esculetin-mediated anti-colitic effects. Fitoterapia 103:55–62CrossRefPubMedGoogle Scholar
  18. 18.
    Yagi K (1987) Lipid peroxides and human diseases. Chem Phys Lipids 45(2–4):337–351CrossRefPubMedGoogle Scholar
  19. 19.
    Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–355CrossRefPubMedGoogle Scholar
  20. 20.
    Kakkar P, Das B, Viswanathan PN (1984) A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 21(2):130–132PubMedGoogle Scholar
  21. 21.
    Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47(2):389–394CrossRefPubMedGoogle Scholar
  22. 22.
    Rotruck JT, Pope AL, Ganther HE (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179(4073):588–590CrossRefPubMedGoogle Scholar
  23. 23.
    Deasi ID (1984) Vitamin E analysis methods for animal tissues. Methods Enzymol 105(1):138–147CrossRefGoogle Scholar
  24. 24.
    Beutler E, Kelly BM (1963) The effect of sodium nitrite on red cell GSH. Experientia 19(2):96–97CrossRefPubMedGoogle Scholar
  25. 25.
    Omura T, Sato R (1964) The carbon monoxide binding pigment of liver microsomes. J Biol Chem 239(1):2379–2385PubMedGoogle Scholar
  26. 26.
    Habig WH, Pabst MJ, Jakoby WBC (1974) Glutathione-S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 249(22):7130–7139PubMedGoogle Scholar
  27. 27.
    Carlberg BM, Mannervik G (1985) Glutathione reductase. Methods Enzymol 113(1):484–490CrossRefPubMedGoogle Scholar
  28. 28.
    Ernster L (1967) DT-Diaphorase. In: Estabrook RW, Pullman ME (ed) Methods in enzymology, vol 10. Academic Press, New York, pp 309–317Google Scholar
  29. 29.
    Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27(3):502–522CrossRefPubMedGoogle Scholar
  30. 30.
    Waris G, Ahsan H (2006) Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 5(14):1–8Google Scholar
  31. 31.
    Niki E (2009) Lipid peroxidation: physiological levels and dual biological effects. Free Radic Biol Med 47(5):469–484CrossRefPubMedGoogle Scholar
  32. 32.
    Manoharan S, Kolanjiappan K, Suresh K, Panjamurthy K (2005) Lipid peroxidation & antioxidants status in patients with oral squamous cell carcinoma. Indian J Med Res 122(6):529–534PubMedGoogle Scholar
  33. 33.
    Karthikeyan S, Srinivasan R, Wani SA, Manoharan S (2013) Chemopreventive potential of chrysin in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis 3:46–53CrossRefGoogle Scholar
  34. 34.
    Manimaran A, Manoharan S (2018) Tumor preventive efficacy of Emodin in 7,12-dimethylbenz[a] anthracene-induced oral carcinogenesis: a histopathological and biochemical approach. Pathol Oncol Res 24(1):19–29CrossRefPubMedGoogle Scholar
  35. 35.
    Manoharan S, Balakrishnan S, Menon VP, Alias LM, Reena AR (2009) Chemopreventive efficacy of curcumin and piperine during 7,12 dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. Singap Med J 50(2):139–146Google Scholar
  36. 36.
    Manoharan S, Singh AK, Suresh K, Vasudevan K, Subhasini R, Baskaran N (2012) Anti-tumor initiating potential of andrographolide in 7,12-dimethylbenz[a] anthracene Induced hamster buccal pouch carcinogenesis. Asian Pac J Cancer Prev 13(11):5701–5708CrossRefPubMedGoogle Scholar
  37. 37.
    Silvan S, Manoharan S, Baskaran N, Anusuya C, Karthikeyan S, Prabhakar MM (2011) Chemopreventive potential of apigenin in 7,12-dimethylbenz(a)anthracene induced experimental oral carcinogenesis. Eur J Pharmacol 670(2–3):571–577CrossRefPubMedGoogle Scholar
  38. 38.
    Anand MA, Suresh K (2014) Biochemical profiling and chemopreventive activity of phloretin on 7,12-dimethylbenz (a) anthracene induced oral carcinogenesis in male golden Syrian hamsters. Toxicol Int 21(2):179–185CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kathiresan S, Govindhan A (2016) [6]-Shogaol, a novel chemopreventor in 7,12-dimethylbenz[a] anthracene-induced hamster buccal pouch carcinogenesis. Phytother Res 30(4):646–653CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Renganathan Selvasundaram
    • 1
  • Shanmugam Manoharan
    • 1
  • Rajamanickam Buddhan
    • 1
  • Mani Neelakandan
    • 1
  • Radhakrishnan Murali Naidu
    • 2
  1. 1.Department of Biochemistry and Biotechnology, Faculty of ScienceAnnamalai UniversityAnnamalainagarIndia
  2. 2.Department of Oral & Maxillofacial Pathology, Rajah Muthiah Dental College & HospitalAnnamalai UniversityAnnamalainagarIndia

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