, Volume 71, Issue 1, pp 461–473 | Cite as

Expression of P53, BAX, and BCL-2 in human malignant melanoma and squamous cell carcinoma cells after tea tree oil treatment in vitro

  • Mohammed A. Ramadan
  • Alaa E. ShawkeyEmail author
  • Mohamed A. Rabeh
  • Ashraf O. Abdellatif
Short Communication


Tea tree oil (TTO) is an essential oil obtained by steam distillation from the leaves of Melaleuca alternifolia (Myrtaceae). This oil has traditionally been used for the treatment of various skin infections. The present study aimed to investigate the cytotoxic effects of TTO against two representative types of human skin cancer, namely malignant melanoma (A-375) and squamous cell carcinoma (HEp-2).To outline the basic molecular mechanism involved in apoptosis induction in A-375 and HEp-2 cell lines, Annexin V/PI staining for apoptosis detection, cell cycle analysis were monitored using flow cytometry and mRNA expression levels of the apoptosis-regulatory genes P53, BAX, and BCL-2 were determined by real-time PCR and western blot after treatment with TTO. Results showed that TTO exhibited a strong cytotoxicity towards A-375 and HEp-2 cell lines, with IC50 values of 0.038% (v/v) and 0.024% (v/v) respectively. This cytotoxicity resulted from TTO induced apoptosis in both A-375 and HEp-2 cell lines as evidenced by morphological features of apoptosis and Annexin V/PI staining results in addition to the activation of caspase-3/7 and -9, upregulation of pro-apoptotic genes (P53 and BAX) and downregulation of the anti-apoptotic gene BCL-2. Additionally, cell cycle analysis showed that TTO caused cell cycle arrest mainly at G2/M phase. Taken together, the results of this study reveal that TTO is an effective apoptosis inducer in A-375 and HEp-2 cancer cell lines, indicating that it could be a promising chemopreventive candidate to be used in topical formulations against melanoma and squamous cell cancers; however, further in vivo studies may be warranted.


Tea tree oil Malignant melanoma Squamous cell carcinoma Cell cycle analysis Apoptosis Chemoprevention 


Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.


  1. Aburjai T, Natsheh FM (2003) Plants used in cosmetics. Phytother Res 17:987–1000CrossRefGoogle Scholar
  2. Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326CrossRefGoogle Scholar
  3. Altschul A, Heintze K, Obst-Gemueseverwert Z (1978) Composition of Australian tea tree oil (Melaleuca alternifolia). J Agric Food Chem 26:735Google Scholar
  4. Basu A, Haldar S (1998) The relationship between Bcl-2, Bax and p53: consequences for cell cycle progression and cell death. Mol Hum Reprod 4:1099–1109CrossRefGoogle Scholar
  5. Bursch W, Oberhammer F, Schulte-Hermann R (1992) Cell death by apoptosis and its protective role against disease. Trends Pharmacol Sci 13:245–251CrossRefGoogle Scholar
  6. Carson C, Hammer K, Riley T (2006) Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev 19:50–62CrossRefGoogle Scholar
  7. Cassady JM, Baird WM, Chang C-J (1990) Natural products as a source of potential cancer chemotherapeutic and chemopreventive agents. J Nat Prod 53:23–41CrossRefGoogle Scholar
  8. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16CrossRefGoogle Scholar
  9. Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A (2009) Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 30:1073–1081CrossRefGoogle Scholar
  10. Einspahr JG, Stratton SP, Bowden GT, Alberts DS (2002) Chemoprevention of human skin cancer. Crit Rev Oncol Hematol 41:269–285CrossRefGoogle Scholar
  11. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516CrossRefGoogle Scholar
  12. Fulda S, Debatin K (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811CrossRefGoogle Scholar
  13. Gloster HM, Brodland DG (1996) The epidemiology of skin cancer. Dermatol Surg 22:217–226Google Scholar
  14. Gogvadze V, Orrenius S, Zhivotovsky B (2006) Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta Bioenergy 1757:639–647CrossRefGoogle Scholar
  15. Gullett NP et al (2010) Cancer prevention with natural compounds. In: Seminars in oncology, vol 3. Elsevier, pp 258–281Google Scholar
  16. Hammer KA, Carson CF, Riley TV (1998) In-vitro activity of essential oils, in particular Melaleuca alternifolia (tea tree) oil and tea tree oil products, against Candida spp. J Antimicrob Chemother 42:591–595CrossRefGoogle Scholar
  17. Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N (2014) Apoptosis and molecular targeting therapy in cancer. Biomed Res Int 2014:150845Google Scholar
  18. Hawrot A, Alam M, Ratner D (2003) Squamous cell carcinoma. Curr Probl Dermatol 15:91–133CrossRefGoogle Scholar
  19. Hayes AJ, Leach DN, Markham JL, Markovic B (1997) In vitro cytotoxicity of Australian tea tree oil using human cell lines. J Essent Oil Res 9:575–582CrossRefGoogle Scholar
  20. Hussein MR (2008) Extracutaneous malignant melanomas. Cancer Invest 26:516–534CrossRefGoogle Scholar
  21. Hwang A, Muschel RJ (1998) Radiation and the G2 phase of the cell cycle. Radiat Res 150:S52–S59CrossRefGoogle Scholar
  22. Igney FH, Krammer PH (2002) Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2:277–288CrossRefGoogle Scholar
  23. Johnstone RW, Ruefli AA, Lowe SW (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell 108:153–164CrossRefGoogle Scholar
  24. Karikas G (2010) Anticancer and chemopreventing natural products: some biochemical and therapeutic aspects. J BUON 15:627–638Google Scholar
  25. Kirkin V, Joos S, Zörnig M (2004) The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta Mol Cell Res 1644:229–249CrossRefGoogle Scholar
  26. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132–1136CrossRefGoogle Scholar
  27. Korsmeyer SJ (1999) BCL-2 gene family and the regulation of programmed cell death. Cancer Res 59:1693s–1700sGoogle Scholar
  28. Kroemer G, Reed JC (2000) Mitochondrial control of cell death. Nat Med 6:513CrossRefGoogle Scholar
  29. Kuo H-M, Chang L-S, Lin Y-L, Lu H-F, Yang J-S, Lee J-H, Chung J-G (2007) Morin inhibits the growth of human leukemia HL-60 cells via cell cycle arrest and induction of apoptosis through mitochondria dependent pathway. Anticancer Res 27:395–405Google Scholar
  30. Liu X, Zu Y, Fu Y, Yao L, Gu C, Wang W, Efferth T (2009) Antimicrobial activity and cytotoxicity towards cancer cells of Melaleuca alternifolia (tea tree) oil. Eur Food Res Technol 229:247CrossRefGoogle Scholar
  31. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  32. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin Phenol reagent. J Biol Chem 193:265–275Google Scholar
  33. Martinou J-C, Youle RJ (2011) Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell 21:92–101CrossRefGoogle Scholar
  34. Matsumura Y, Ananthaswamy HN (2004) Toxic effects of ultraviolet radiation on the skin. Toxicol Appl Pharmacol 195:298–308CrossRefGoogle Scholar
  35. Mehta RG, Murillo G, Naithani R, Peng X (2010) Cancer chemoprevention by natural products: how far have we come? Pharm Res 27:950–961CrossRefGoogle Scholar
  36. Millimouno FM, Dong J, Yang L, Li J, Li X (2014) Targeting apoptosis pathways in cancer and perspectives with natural compounds from mother nature. Cancer Prev Res 7:1081–1107CrossRefGoogle Scholar
  37. Miyashita T, Harigai M, Hanada M, Reed JC (1994) Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res 54:3131–3135Google Scholar
  38. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  39. Norbury CJ, Zhivotovsky B (2004) DNA damage-induced apoptosis. Oncogene 23:2797–2808CrossRefGoogle Scholar
  40. Nylander K, Nilsson P, Mehle C, Roos G (1995) p53 mutations, protein expression and cell proliferation in squamous cell carcinomas of the head and neck. Br J Cancer 71:826CrossRefGoogle Scholar
  41. Ola MS, Nawaz M, Ahsan H (2011) Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol Cell Biochem 351:41–58CrossRefGoogle Scholar
  42. Oren M (1992) The involvement of oncogenes and tumor suppressor genes in the control of apoptosis. Cancer Metastasis Rev 11:141–148CrossRefGoogle Scholar
  43. Patil BS, Jayaprakasha G, Chidambara Murthy K, Vikram A (2009) Bioactive compounds: historical perspectives, opportunities, and challenges. J Agric Food Chem 57:8142–8160CrossRefGoogle Scholar
  44. Pezzuto JM (1997) Plant-derived anticancer agents. Biochem Pharmacol 53:121–133CrossRefGoogle Scholar
  45. Reed JC (1995) Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr Opin Oncol 7:541–546CrossRefGoogle Scholar
  46. Sporn MB, Suh N (2002) Chemoprevention: an essential approach to controlling cancer. Nat Rev Cancer 2:537–543CrossRefGoogle Scholar
  47. Takahashi R, Markovic SN, Scrable HJ (2014) Dominant effects of Δ40p53 on p53 function and melanoma cell fate. J Invest Dermatol 134:791–800CrossRefGoogle Scholar
  48. Thomas A, Giesler T, White E (2000) p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway. Oncogene 19:5259CrossRefGoogle Scholar
  49. Tong MM, Altman PM, Barnetson RS (1992) Tea tree oil in the treatment of tinea pedis. Australas J Dermatol 33:145–149CrossRefGoogle Scholar
  50. Vermes I, Haanen C, Reutelingsperger C (2000) Flow cytometry of apoptotic cell death. J Immunol Methods 243:167–190CrossRefGoogle Scholar
  51. Xia W, Spector S, Hardy L, Zhao S, Saluk A, Alemane L, Spector NL (2000) Tumor selective G2/M cell cycle arrest and apoptosis of epithelial and hematological malignancies by BBL22, a benzazepine. Proc Natl Acad Sci USA 97:7494–7499CrossRefGoogle Scholar
  52. Yip K, Reed J (2008) Bcl-2 family proteins and cancer. Oncogene 27:6398–6406CrossRefGoogle Scholar
  53. Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Mohammed A. Ramadan
    • 1
  • Alaa E. Shawkey
    • 1
    Email author
  • Mohamed A. Rabeh
    • 2
  • Ashraf O. Abdellatif
    • 1
    • 3
  1. 1.Department of Microbiology and Immunology, Faculty of PharmacyCairo UniversityGizaEgypt
  2. 2.Department of Pharmacognosy, Faculty of PharmacyCairo UniversityGizaEgypt
  3. 3.Department of Microbiology and Immunology, Faculty of PharmacyKarary UniversityKhartoumSudan

Personalised recommendations