Breast Cancer Research and Treatment

, Volume 101, Issue 1, pp 27–36 | Cite as

Novel triterpenoid 25-hydroxy-3-oxoolean-12-en-28-oic acid induces growth arrest and apoptosis in breast cancer cells

  • Thangaiyan Rabi
  • Liming Wang
  • Sipra Banerjee
Preclinical Study


25-Hydroxy-3-oxoolean-12-en-28-oic acid (Amooranin-AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A herbal preparation used for the treatment of cancer by the Ayurvedic system of medicine contains the stem bark of Amoora rohituka as one of the ingredients. In this paper, we show that AMR displays a strong inhibitory effect on survival of human breast carcinoma MDA-468, breast adenocarcinoma MCF-7 cells compared to breast epithelial MCF-10A control cells. A 50% decrease in cells (IC50) ranged from 1.8 to 14.6 µM and cell growth was suppressed by arresting cell cycle at G+ M phase. AMR effectively induces apoptosis and triggered a series of effects associated with apoptosis including cleavage of caspase-8, -9, -3, Bid and ER stress in MDA-468 cells and caspase- 8, -9, -6 and Bid in MCF-7 cells, release of cytochrome c from the mitochondria, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation with a concomitant upregulation of p53, Bax and down-regulation of Bcl-2 in MDA-468 cells, but Bax unchanged in MCF-7 cells. The use of caspase blocking peptides and acridine orange staining confirmed the involvement of primarily caspase-9 and -3 in MDA-468 cells with mutated p53 and primarily caspase-8, -9 and -6 in MCF-7 cells expressing wt p53. We also observed in MCF-7/p53siRNA cells AMR treatment caused reduced expression of Bcl-2 without affecting levels of Bax similar to MCF-7 cells treated with AMR and proteolytic activation of Bax in MDA-468 cells. These results suggest that AMR induces apoptosis in human breast carcinoma cells via caspase activation pathway and likely it is a p53-independent apoptosis.


Amooranin Breast cancer Apoptosis Caspase-3 p53 status 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the NCI/NIH, MD, USA through Oncology Research Faculty Development Program.


  1. 1.
    American Cancer Scociety. Cancer Statistics, 2005Google Scholar
  2. 2.
    Kim KB, Lotan R, Yue P, Sporn MB, Suh N, Gribble GW, Honda T, Wu GS, Hong W, Sun SY (2002) Identification of a novel synthetic triterpenoid, methyl-2-cyano-3, 12-dioxooleane-1,9-dien-28-oate, that potently induces caspase-mediated apoptosis in human lung cancer cells. Mol Cancer Ther 1:177–184PubMedGoogle Scholar
  3. 3.
    Rabi T (1986) Antitumor activity of amooranin from Amoora rohituka stem bark. Curr Sci 70:80–81Google Scholar
  4. 4.
    Prasad GC (1987) Studies on cancer in Ayurveda and its management. J Res Ayurveda Sidha 8:147–167Google Scholar
  5. 5.
    Rabi T, Ramachandran C, Fonseca HB, Alamo A, Melnick SJ, Escalon E (2003) Amooranin, a novel drug, induces apoptosis through caspase activity in human breast carcinoma cell lines. Breast Cancer Res Treat 80:321–330PubMedCrossRefGoogle Scholar
  6. 6.
    Ramachandran C, Rabi T, Fonseca HB, Melnick SJ, Escalaon EA (2003) Novel plant triterpenoid drug amooranin overcomes multidrug resistance in human leukemia and colon carcinoma cell lines. Int J Cancer 105:784–789PubMedCrossRefGoogle Scholar
  7. 7.
    Rabi T, Karunagaran D, Krishnan Nair M, Bhattathiri VN (2002) Cytotoxic activity of amooranin and its derivatives. Phytother Res 16:S84–86PubMedCrossRefGoogle Scholar
  8. 8.
    Li P, Nijhawan D, Budihardjo I, Srinivasulu SM, Ahmad M, Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489PubMedCrossRefGoogle Scholar
  9. 9.
    Srinivasulu SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES (1998) Autoactivation of procaspase-9 by Apaf-1 mediated oligomerization. Mol Cell 1:949–957CrossRefGoogle Scholar
  10. 10.
    Li H, Zhu X, Xu CJ, Yuan J (1998) Cleavage of Bid by caspase-8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501PubMedCrossRefGoogle Scholar
  11. 11.
    Luo X, Budihardjo I, Zou H, Slaughter C, Wang I (1998) Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mytochondria in response to activation of cell surface death receptors. Cell 94:481–490PubMedCrossRefGoogle Scholar
  12. 12.
    Tsujimoto Y, Shimizu (2000) Bcl-2 family: life-or-death switch. FEBS Lett 466:6–10PubMedCrossRefGoogle Scholar
  13. 13.
    Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME (1998) Two CD 95 (APO/Fas) signaling pathways. EMBO J 17:1675–1687PubMedCrossRefGoogle Scholar
  14. 14.
    Fribley A, Zeng Q, Wang CY (2004) Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress reactive oxygen species in head and neck squamous cell carcinoma cells. Mol Cell Biol 24:9695–9704PubMedCrossRefGoogle Scholar
  15. 15.
    Hansen MB, Nielsen SE, Berg K (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Meth 119:203–210CrossRefGoogle Scholar
  16. 16.
    Rudner AD, Murray AW (1996) The spindle assembly checkpoint. Curr Opin Cell Biol 8:773–780PubMedCrossRefGoogle Scholar
  17. 17.
    Cohen JJ, Duke RC (1984) Glucocorticoid activation of a calcium dependent endonuclease in thymocyte nucleic leads to cell death. J Immunol 132:38–42PubMedGoogle Scholar
  18. 18.
    Prasad KA, Church JG (1997) Characterization of DNA binding and transcriptional regulatory function of an endogenous mutant p53 in MDA-468 human breast cancer cells. Biochem Biophys Res Commun 232:14–19PubMedCrossRefGoogle Scholar
  19. 19.
    Robinson BW, Schewach DS (2001) Radiosensitization by Gemcitabine in p53 wild-type and mutant MCF-7 breast carcinoma cell lines. Clin Cancer Res 7:2581–2589PubMedGoogle Scholar
  20. 20.
    Dumaz N, Meek DW (1999) Serine 15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J 18:7002–10PubMedCrossRefGoogle Scholar
  21. 21.
    Tweddle DA, Malcolm AJ, Bown N, Pearson AD, Lunec J (2001) Evidence for the development of p53 mutations after cytotoxic therapy in a neurobalstoma cell line. Cancer Res 61:8–13PubMedGoogle Scholar
  22. 22.
    Janicke RU, Ng P, Sprengart MI, Porter AG (1998a) Caspase-3 is required for fodrin cleavage but dispensible for cleavage of other death substrates in apoptosis. J Biol Chem 273:15540–15545CrossRefGoogle Scholar
  23. 23.
    Rabi T, Gupta RC (1995) Antitumor and cytotoxic investigation of Amoora rohituka. Int J Pharmacogn 33:359–361CrossRefGoogle Scholar
  24. 24.
    Weinert TA, Hartwell LH (1990) Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationary in cell cycle arrest after DNA damage. Mol Cell Biol 10:6554–6564PubMedGoogle Scholar
  25. 25.
    O’Connor PM, Ferris DK, Pagano M, Draetta G, Pines J, Hunter T, Longo DL, Kohn KW (1993) G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2, and cyclin B1/cdc2-kinase complexes differently. J Biol Chem 268:8298–8303PubMedGoogle Scholar
  26. 26.
    Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362:847–849PubMedCrossRefGoogle Scholar
  27. 27.
    Mehmet H (2002) Apoptosis: caspase find a new place to hide. Nature 403:29–30CrossRefGoogle Scholar
  28. 28.
    Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776PubMedCrossRefGoogle Scholar
  29. 29.
    Weller M (1998) Predicting response to cancer chemotherapy: the role of p53. Cell Tissue Res 292:443–448CrossRefGoogle Scholar
  30. 30.
    Ferreira CG, Tolis C, Giaccone G (1999) p53 and chemosensitivity. Ann Oncol 10:1011–1021PubMedCrossRefGoogle Scholar
  31. 31.
    Tang D, Kidd VJ (1998) Cleavage of DFF-45/ICAD by multiple caspases is essential for its function during apoptosis. J Biol Chem 273:28549–28552PubMedCrossRefGoogle Scholar
  32. 32.
    Zapata JM, Krajewski S, Huang R, Takayama S, Wang H, Adamson E, Reed JC (1998) Expression of multiple apoptosis regulatory genes in human breast cancer cell lines and primary tumors. Breast Cancer Res Treat 47:129–140PubMedCrossRefGoogle Scholar
  33. 33.
    Zheng TS, Hunot S, Kuida K, Momoi T, Srinivasan A, Nicholson DW, Lazebnik Y, Flavell RA (2000) Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat Med 6:1241–1247PubMedCrossRefGoogle Scholar
  34. 34.
    Wolf BB, Schuler M, Echeverri F, Green DR (1999) Caspase-3 is the primary activator of apoptotic DNA fragmentation factor-45/Inhibitor of caspase-activated DNase inactivation. J Biol Chem 274:30651–30656PubMedCrossRefGoogle Scholar
  35. 35.
    Decker P, Isenberg D, Muller S (2000) Inhibition of caspase-3 mediated poly (ADP-ribose) polymerase (PARP) apoptotic cleavage by human PARP autoantibodies and effect on cells undergoing apoptosis. J Biol Chem 275:9043–9046PubMedCrossRefGoogle Scholar
  36. 36.
    Sellers WR, Fisher DE (1999) Apoptosis and cancer drug targeting. J Clin Invest 104:1655–1661PubMedCrossRefGoogle Scholar
  37. 37.
    Roth W, Reed JC (2002) Apoptosis and cancer. When BAX is TRAILing away. Nat Med 8:216–218PubMedCrossRefGoogle Scholar
  38. 38.
    Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–1308PubMedCrossRefGoogle Scholar
  39. 39.
    Adams JM, Cory S (1998) The bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326PubMedCrossRefGoogle Scholar
  40. 40.
    Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–615PubMedCrossRefGoogle Scholar
  41. 41.
    Zinszner H, Kuroda M, Wang X, Batchvarova N, Light-foot RT, Remotti H, Stevens JL, Ron D (1998) CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev 12:982–995PubMedGoogle Scholar
  42. 42.
    McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ (2001) Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl-2 and perturbing the cellular redox state. Mol Cell Biol 21:1249–1259PubMedCrossRefGoogle Scholar
  43. 43.
    Hitomi J, Katayama T, Eguchi Y (2004) Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death. J Cell Biol 165:347–356PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Department of Cancer BiologyLerner Research Institute, Cleveland Clinic FoundationClevelandUSA

Personalised recommendations