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Novel Drug Amooranin Induces Apoptosis Through Caspase Activity in Human Breast Carcinoma Cell Lines

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Abstract

Amooranin (AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A. rohituka stem bark is one of the components of a medicinal preparation used in the Indian Ayurvedic system of medicine for the treatment of human malignancies. We investigated the mechanism of cell death associated with AMR cytotoxicity in human mammary carcinoma MCF-7, multidrug resistant breast carcinoma MCF-7/TH and breast epithelial MCF-10A cell lines. AMR IC50 values ranged between 3.8–6.9 µg/ml among MCF-7, MCF-7/TH and MCF-10A cells. AMR induced oligonucleosome-sized DNA ladder formation characteristic of apoptosis when tumor cells were treated with 1–8µg/ml AMR for 48 h. In situ cell death detection assay indicated that AMR caused 37.3–72.1% apoptotic cells in MCF-7, 32–48.7% in MCF-7/TH and 0–37.1% in MCF-10A cells at 1–8µg/ml concentrations. The induction of apoptosis in AMR treated cells was accompanied by the elevation of total caspase and caspase-8 activities. Flow cytometric analysis showed that AMR induced caspase-8 activation in 40.8–71% MCF-7, 28.5–43.2% MCF-7/TH and 4–32.8% MCF-10A cells at 1–8 µg/ml concentrations. Our results suggest that AMR is a novel drug having potential for clinical development against human malignancies.

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References

  1. Rabi T: Antitumour activity of amooranin from Amoora rohituka stem bark. Curr Sci 70: 80–81, 1996

    Google Scholar 

  2. Prasad GC: Studies on cancer in Ayurveda and its management. J Res Ayurveda Sidha 8: 147–167, 1987

    Google Scholar 

  3. Rabi T, Karunagaran D, Krishnan Nair M, Bhattathiri VN: Cytotoxic activity of amooranin and its derivatives. Phytother Res 16: S84–S86, 2002

    Google Scholar 

  4. Skladanowski A, Konopa J: Adriamycin and daunomycin induce programmed cell death (apoptosis) in tumor cells. Biochem Pharmacol 46: 375–382, 1993

    Google Scholar 

  5. Ohmori T, Podack ER, Nishio K, Takahashi M, Miyahara Y, Takeda Y, Kubota N, Funayama Y, Ogasawara H, Ohira T, Ohta S, Saijo N: Apoptosis of lung cancer cells caused by some anti-cancer agents (MMC, CPT-11, ADM) is inhibited by bcl-2. Biochem Biophys Res Commun 192: 30–36, 1993

    Google Scholar 

  6. Ling Y-H, Priebe W, Perez-Soler R: Apoptosis induced by anthracycline antibiotics in P388 parent and multidrug resistant cells. Cancer Res 53: 1845–1852, 1993

    Google Scholar 

  7. Kolber MA, Broschat KO, Landa-Gonzalez B: Cytochalasin B induces cellular DNA fragmentation. FASEB J 4: 3021–3027, 1990

    Google Scholar 

  8. Klucer J, Al-Rubeai M: G2 cell cycle arrest and apoptosis are induced in Burkitt's lymphoma cells by the anticancer agent Oracin. FEBS Lett 400: 127–130, 1997

    Google Scholar 

  9. Green D: Apoptotic pathways: the roads to ruin. Cell 94: 695–698, 1998

    Google Scholar 

  10. Earnshaw WC, Martins LM, Kaufmann SH: Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383–424, 1999

    Google Scholar 

  11. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274: 20049–20052, 1999

    Google Scholar 

  12. Martin DS, Bertino JR, Koutcher JA: ATP depletion plus pyrimidine depletion can markedly enhance cancer therapy: fresh insight for a new approach. Cancer Res 60: 6776–6783, 2000

    Google Scholar 

  13. Budihardjo I, Oliver H, Lutter M, Luo X, Wang X: Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15: 269–290, 1999

    Google Scholar 

  14. Hansen MB, Nielsen SE, Berg K: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Meth 119: 203–210, 1989

    Google Scholar 

  15. Chen G, Ramachandran C, Krishan A: Thaliblastine, a plant alkaloid, circumvents multidrug resistance by direct binding to P-glycoprotein. Cancer Res 53: 2544–2547, 1993

    Google Scholar 

  16. Cohen JJ, Duke RC: Glucocorticoid activation of a calcium dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 132: 38–42, 1984

    Google Scholar 

  17. Ramachandran C, You W, Krishan A: Bcl-2 and mdr-1 gene expression during doxorubicin-induced apoptosis in murine leukemic P388 and P388/R84 cells. Anticancer Res 17: 3369–3376, 1997

    Google Scholar 

  18. Carcia-Calvo M, Peterson E, Leiting B, Ruel R, Nicholson D, Thornberry N: Inhibition of human caspases by peptidebased and macromolecular inhibitors. J Biol Chem 273: 32608–32613, 1998

    Google Scholar 

  19. Rabi T, Gupta RC: Antitumor and Cytotoxic investigation of Amoora rohituka. Int J Pharmacogn 33: 359–361, 1995

    Google Scholar 

  20. Rabi T, Ramachandran C, Melnick SJ, Fonseca HB, Escalon E, Jhabvala P: Amooranin overcomes cellular drug resistance and acts synergistically with doxorubicin in multidrug resistant human colon carcinoma and leukemic cell lines. In: Proceedings of AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications. October 29-November 2, 2001

  21. Cragg GM: Role of plants in the National Cancer Institute drug discovery and development program. In: Human Medicinal Agents from plants, ACS Symposium Series 534, Kinghorn AD, Balandrin MF (eds), American Chemical Society Books, Washington, DC, 1993, pp 149–169

    Google Scholar 

  22. Wall ME, Wani MC: Camptothecin and analogues: synthesis, biological in vitro and in vivo activities and clinical possibilities. In: Human Medicinal Agents from Plants, ACS Symposium Series 534, Kinghorn AD, Balandrin MF (eds) American Chemical Society Books, Washington, DC, 1993, pp 149–169

    Google Scholar 

  23. Konopleva Marina, Tsao, Twee, Ruvolo, Peter, Stiouf, Estrov, Zeev, Leysath CE, Zhao S, Harris WD, Chang S, Jackson CE, Munsell M, Suh N, Gribble G, Honda T, May S, Sporn MB, Andreef M: Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia. Blood 99: 326–335, 2002

    Google Scholar 

  24. Kim DK, Baek JH, Kang CM, Yoo MA, Sung JW, Kim DK, Chung HY, Kim ND, Choi YH, Lee SH, Kim KW: Apoptotic activity of ursolic acid may correlate with the inhibition of initiation of DNA replication. Int J Cancer 87: 629–636, 2000

    Google Scholar 

  25. Hoernlein RF, Orlikowsky TH, Zehrer C, Niethammer D, Sailer ER, Simmet TH, Dannecker GE: Acetyl-11-Keto-b-Boswellic acid induces apoptosis in HL-60 and CCRF-CEM cells and inhibits Topoisomerase 1. J Pharmacol Exp Ther 288: 613–619, 1999

    Google Scholar 

  26. Stadheim TA, Suh N, Ganju N, Sporn MB, Eastman A: The novel triterpenoid 2-cyano-3, 12-dioxoolean-1,9-dien-28-oic acid (CDDO) potentially enhances apoptosis induced by tumor necrosis factor in human leukemic cells. J Biol Chem 277: 16448–16455, 2002

    Google Scholar 

  27. Suh N, Wang Y, Honda T, Gribble GW, Dmitrovsky E, Hickey WF, Maue RA, Place AE, Porter DM, Spinella MJ, Williams CR, Wu G, Dannenberg AJ, Flanders KC, Letterio JJ, Mangelsdorf DJ, Nathan CF, Nguyen L, Porter WW, Ren RF, Roberts AB, Roche NS, Subbaramaiaqh K, Sporn MB: A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative and anti-inflammatory activity. Cancer Res 59: 336–341, 1999

    Google Scholar 

  28. Huang MT, Ho CT, Wang ZY, Ferraro T, Lou YR, Stauber K, Ma W, Georgiadis C, Laskin JD, Conney AH: Inhibition of skin tumorigenesis by rosemary and its constituents carnesol and ursolic acid. Cancer Res 54: 701–708, 1994

    Google Scholar 

  29. Nishino H, Nishino A, Takayasu J, Hasegawa T, Iwashima A, Hirabayashi K, Iwata S, Shibata S: Inhibition of the tumorpromoting action of 12-o-tetradecanoylphorbol-13-acetate by some oleanane-type triterpenoid compounds. Cancer Res 48: 5210–5215, 1988

    Google Scholar 

  30. Ito Y, Pandey P, Place A, Sporn MB, Gribble GW, Honda T, Kharbanda S, Kube D: The novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-oic acid induces apoptosis of human myeloid leukemic cells by a caspase-8 dependent mechanism. Cell Growth Differ 11: 261–267, 2000

    Google Scholar 

  31. Ito Y, Pandey P, Sporn MB, Datta R, Kharbanda S, Kube D: The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism. Mol Pharmacol 59: 1094–1099, 2001

    Google Scholar 

  32. Patel T, Gores GJ, Kaufman SH: The role of proteases during apoptosis. FASEB J 10: 587–597, 1997

    Google Scholar 

  33. Zhivotovsky B, Burgess DH, Vanags DM, Orrenius S: Involvement of cellular proteolytic machinery in apoptosis. Biochem Biophys Res Commun 230: 481–488, 1997

    Google Scholar 

  34. Cohen GM: Caspases: the executioners of apoptosis. Biochem J 326: 1–16, 1997

    Google Scholar 

  35. Nicholson DW, Thorberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME, Yamin TT, Yu VL, Miller DK: Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37–43, 1995

    Google Scholar 

  36. Rao L, White E: Bcl-2 and ICE family of apoptotic regulators: making a connection. Curr Opin Gene Dev 7: 52–58, 1997

    Google Scholar 

  37. Salvesen GS, Dixit VM: Caspases: intracellular signaling by proteolysis. Cell 91: 443–446, 1997

    Google Scholar 

  38. Thornberry NA, Lazebnik Y: Caspases: enemies within. Science (Washington, DC) 281: 1312–1316, 1998

    Google Scholar 

  39. Hu S, Snipas SJ, Vincenz C, Salvesen G, Dixit VM: Caspase-14 is a novel developmentally regulated protease. J Biol Chem 273: 29648–29653, 1998

    Google Scholar 

  40. Janicke RU, Sprengart ML, Wati MR, Porter AG: Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273: 9357–9360, 1998

    Google Scholar 

  41. Fernandes AT, Armstrong RC, Krebs J, Srinivasulu SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli KJ, Litwack G, Alnemri ES: In vitro activation of CPP32 and Mch3 by Mch4, a novel huma apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci USA 93: 7464–7469, 1996

    Google Scholar 

  42. Boldin MP, Goncharov TM, Goltsev YN, Wallach D: Involvement of MACH, a novel MORT1/FADD-Interacting protease, in Fas/Apo-1 and TNF receptor-induced cell death. Cell 85: 803–815, 1996

    Google Scholar 

  43. Bedner E, Smolewski P, Amstad P, Darzynkiewicz Z: Activation of caspases measured in situ by binding of fluorochromelabeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res 259: 308–313, 2000

    Google Scholar 

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Rabi, T., Ramachandran, C., Fonseca, H.B. et al. Novel Drug Amooranin Induces Apoptosis Through Caspase Activity in Human Breast Carcinoma Cell Lines. Breast Cancer Res Treat 80, 321–330 (2003). https://doi.org/10.1023/A:1024911925623

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