The Effects of Crinum asiaticum on the Apoptosis Induction and the Reversal of Multidrug Resistance in HL-60/MX2

  • Hyun, Jae-Hee ;
  • Kang, Jung-Il ;
  • Kim, Sang-Cheol ;
  • Kim, Elvira ;
  • Kang, Ji-Hoon ;
  • Kwon, Jung-Mi ;
  • Park, Doek-Bae ;
  • Lee, Young-Jae ;
  • Yoo, Eun-Sook ;
  • Kang, Hee-Kyoung
  • Published : 2008.03.31


The present study investigated the anti-proliferative and chemosensitizing effects of Crinum asiaticum var. japonicum against multi-drug resistant (MDR) cancer cells. The 80% methanol extract, chloroform ($CHCl_3$) fraction and butanol (BuOH) fraction of C. asiaticum inhibited the growth of mitoxantrone (MX) resistant HL-60 (HL-60/MX2) cells. When HL-60/MX2 cells were treated with the $CHCl_3$ and BuOH fractions, DNA ladder and sub-G1 hypodiploid cells were observed. Furthermore, the fractions reduced BcI-2 mRNA levels, whereas Bax mRNA levels were increased. These results suggest that the inhibitory effect of C. asiaticum on the growth of the HL-60/MX2 cells might arise from the induction of apoptosis. Treatment of HL-60/MX2 cells with the fractions markedly decreased the mRNA levels of the multi-drug resistance protein-1 and breast cancer resistance protein. The $CHCl_3$ fraction and hexane fraction increased MX accumulation in HL-60/MX2 cells. These results imply that the $CHCl_3$ fraction of C. asiaticum plays a pivotal role as a chemosensitizer. We suggest that components of C. asiaticum might have a therapeutic potential for the treatment of MDR leukemia.


HL-60/MX2;Crinum asiaticum;Apoptosis;Chemosensitizer;MRP-1;BCRP


  1. Allikmets, R., Gerrard, B., Hutchinson, A. and Dean, M. (1996). Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database. Hum. Mol. Genet., 5, 1649-1655
  2. Cakici, I., Ulug, H.Y., Inci, S., Tunctan, B., Abacioglu, N., Kanzik, I. and Sener, B. (1997). Antinociceptive effect of some amaryllidaceae plants in mice. J. Pharm. Pharmacol., 49, 828-830
  3. Choi, S.U., Lee, C.O., Kim, K.H., Choi, E.J., Park, S.H., Shin, H.S., Yoo, S.E., Jung, N.P. and Lee, B.H. (1998). Reversal of multidrug resistance by novel verapamil analogs in cancer cells. Anticancer Drugs, 9, 157-165
  4. Doyle, L.A. and Ross, D.D. (2003). Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene, 22, 7340-7358
  5. Garrigues, A., Loiseau, N., Delaforge, M., Ferte, J., Garrigos, M., Andre, F. and Orlowski, S. (2002). Characterization of two pharmacophores on the multidrug transpoter P-glycoprotein. Mol. Pharmacol., 62, 1288-1298
  6. Loor, F., Tiberghien, F., Wenandy, T., Didier, A. and Traber, R. (2002). Cyclosporins; structure activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC transporter. J. Med. Chem., 45, 4598-4612
  7. Min, B.S., Gao, J.J., Nakamura, N., Kim, Y.H. and Hattori, M. (2001). Cytotoxic alkaloids and a flavan from the bulbs of Crinum asiaticum var. japonicum. Chem. Pharm. Bull. (Tokyo), 49, 1217-1219
  8. Oltvai, Z.N., Millman, C.L. and Korsmeyer, S.J. (1993). Bc1-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74, 609-619
  9. Park, M.H. (2001). Chemical Constituents and Biological Activity of Crinum asiaticum var. japonicum, Chungnam National University, Taejon, pp. 79-80
  10. Pettit, G.R., Cragg, G.M., Singh, S.B., Duke, J.A. and Doubek, D.L. (1990). Antineoplastic agents, 162 Zephyranthes candida. J. Nat. Prod., 53, 176-178
  11. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning Laboratory Manual (2nd edition) Vol. III, Cold Spring Harbor Laboratory Press, New York, pp. 18-19
  12. Takagi, S. and Yamaki, M. (1977). Studies on the purgative drugs. III. On the constituents of the flowers of Prunus persica Batsch. Yakugaku Zasshi, 97, 1155-1157
  13. Yang, H.H., Ma, M.H., Vescio, R.A. and Berenson, J.R. (2003). Overcoming drug resistance in multiple myeloma: The emergence of therapeutic approaches to induce apoptosis. J. Clin. Oncol., 21, 4239-4247
  14. Allikmets, R., Schriml, L.M., Hutchinson, A., Romano-Spica V. and Dean M. (1998). A human placenta-specific ATP binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. Cancer Res., 58, 5337-5339
  15. Xie, J., Wang, Y., Freeman, M.E., Barlogie, B. and Yi, O. (2003). Beta 2-microglobulin as a negative regulator of the immune system: high concentrations of the protein inhibit in vitro generation of sunctional dendritic cells. Blood, 101, 4005-4012
  16. Abdel-Halim, O.B., Morikawa, T., Ando, S., Matsuda, H. and Yoshikawa, M. J. (2004). New crinine-type alkaloids with inhibitory effect on induction of inducible nitric oxide synthase from Crinum yemense. Nat. Prod., 67, 1119-1124
  17. Gottesman, M.M. and Pastan, I. (1993). Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem., 62, 385-427
  18. Comerford, K.M., Wallace, T.J., Karhausen, J., Louis, N.A., Montalto, M.C. and Colgan, S.P. (2002). Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res., 62, 3387-3394
  19. Fojo, T. and Bates, S. (2003). Strategies for reversing drug resistance. Oncogene, 22, 7512-7523
  20. Furusawa, E. and Furusawa, S. (1988). Effect of pretazettine and viva-natural, a dietary seaweed extract, on spontaneous AKR leukemia in comparison with standard drugs. Oncology, 45, 180-186
  21. Endicott, J.A. and Ling, V. (1989). The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu. Rev. Biochem., 58, 137-171
  22. Koch, S., Mayer, F., Honecker, F., Schittenhelm, M. and Bokemeyer, C. (2003). Efficacy of cytotoxic agents used in the treatment of testicular germ cell tumors under normoxic and hypoxic conditions in vitro. Br. J. Cancer., 89, 2133-2139
  23. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning Laboratory Manual (2nd edition) Vol. III, Cold Spring Harbor Laboratory Press, New York, pp. 18-19
  24. Kalra, R., Jones, A.M., Kirk, J., Adams, G.E. and Stratford, I.J. (1993). The effect of hypoxia on acquired drug resistance and response to epidermal growth factor in Chinese hamster lung fibroblasts and human breast cancer cells in vitro. Int. J. Cancer, 54, 650-655
  25. Sellers, W.R. and Fisher, D.E. (1999). Apoptosis in cancer drug targeting. J. Clin. Invest., 104, 1655-1661
  26. Heere-Ress, E., Thallinger, C., Lucas, T., Schlagbauer-Wadl, H., Wacheck, V., Monia, B.P., Wolff, K., Pehamberger, H. and Jansen, B. (2002). Bcl-X(L) is a chemoresistance factor in human melanoma cells that can be inhibited by antisense therapy. Int. J. Cancer, 99, 29-34
  27. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D. and Mitchell, J.B. (1987). Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res., 47, 943-946
  28. Likhitwitayawuid, K., Angerhofer, C.K., Chai, H., Pezzuto, J.M., Cordell, G.A. and Ruangrungsi, N. (1993). Cytotoxic and antimalarial alkaloids from the bulbs of Crinum amabile. J. Nat. Prod., 56, 1331-1338
  29. McNulty, J., Nair, J.J., Codina, C., Bastida, J., Pandey, S., Gerasimoff, J. and Griffin, C. (2007). Selective apoptosisinducing activity of crinum-type Amaryllidaceae alkaloids. Phytochemistry, 68, 1068-1074
  30. Pettersson, M., Jernberg-Wiklund, H., Larsson, L.G., Sundstrom, C., Givol, I., Tsujimoto, Y. and Nilsson, K. (1992). Expression of bcl-2 gene in human multiple myeloma cell lines and normal plasma cells. Blood, 79, 495-502
  31. Harker, W.G., Slade, D.L., Dalton, W.S., Meltzer, P.S. and Trent, J.M. (1989). Multidrug resistance in mitoxantroneselected HL-60 leukemia cells in the absence of P-glycoprotein overexpression. Cancer Res., 49, 4542-4549
  32. Larsen, A.K., Escargueil, A.E. and Skladanowski, A. (2000). Resistance mechanisms associated with altered intracellular distribution of anticancer agents. Pharmacol. Ther., 85, 217-229
  33. Boumendjel, A., Baubichon-Cortay, H., Trompier, D., Perrotton, T. and Di Pietro, A. (2005). Anticancer multidrug resistance mediated by MRP 1: Recent advances in the discovery of reversal agents. Med. Res. Rev., 25, 453-472
  34. Cole, S.P., Bhardwaj, G., Gerlach, J.H., Mackie, J.E., Grant, C.E., Almquist, K.C., Stewart, A.J., Kurz, E.U., Duncan, A.M. and Deely, R.G.. (1992). Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science, 258, 1650-1654
  35. List, A.F. (1996). The role of multidrug resistance and its pharmacological modulation in acute myeloid leukemia. Leukemia, 10, S36-38
  36. Miyashita, T. and Reed, J.C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell, 27, 293-299
  37. Gabrielsen, B., Monath, T.P., Huggins, J.W., Kefauver, D.F., Pettit, G.R., Groszek, G., Hollingshead, M., Kirsi, J.J., Shannon, W.M. and Schubert, E.M. (1992). Antiviral (RNA) activity of selected Amaryllidaceae isoquinoline constituents and synthesis of related substances. J. Nat. Prod., 55, 1569-1581
  38. Kim, Y.H., Park, E.J., Park, M.H., Badarch, U., Woldemichael, G.M. and Beutler, J.A. (2006). Crinamine from Crinum asiaticum var. japonicum Inhibits hypoxia inducible factor-1 activity but not activity of hypoxia inducible factor-2. Biol. Pharm. Bull., 29, 2140-2141
  39. Furusawa, E., Irie, H., Combs, D. and Wildman, W.C. (1980). Therapeutic activity of pretazettine on Rauscher leukemia: comparison with the related Amaryllidaceae alkaloids. Chemotherapy, 26, 36-45
  40. Zhou, S., Morris, J.J., Barnes, Y., Lan, L., Schuetz, J.D. and Sorrentino, B.P. (2002). Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc. Natl. Acad. Sci. USA 99, 12339-12344
  41. Cole, S.P. and Deeley, R.G.. (1993). Multidrug resistanceassociated protein: sequence correction. Science, 260, 879
  42. Hanahan, D. and Weinberg, R.A. (2000). The hallmarks of cancer. Cell, 100, 57-70
  43. Sherwood, S.W., Sheridan, J.P. and Schimke, R.T. (1994). Induction of apoptosis by the anti-tubulin drug colcemid: relationship of mitotic checkpoint control to the induction of apoptosis in HeLa S3 cells. Exp. Cell Res., 215, 373-379
  44. Xie, C., Kokubun, T., Houghton, P.J. and Simmonds, M.S. (2004). Antibacterial activity of the Chinese traditional medicine. Phytother. Res., 18, 497-500
  45. Kioka, N., Yamano, Y., Komano, T. and Ueda, K. (1992). Heat-shock responsive elements in the induction of the multidrug resistance gene (MDR1). FEBS Lett., 301, 37-40
  46. Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R. and Sikorska, M. (1993). Apoptosis death in epithelial cell: cleavage of DNA to 300 and/or 50 kb fragments prior to in the absence of internucleosomal fragmentation. EMBO J., 12, 3679-3684
  47. Riordan, J.R. and Ling, V. (1979). Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J. Biol. Chem., 254, 12701-12705
  48. Liang, B.C. (1996). Effects of hypoxia on drug resistance phenotype and genotype in human glioma cell lines. J. Neurooncol., 29, 149-155
  49. Monaghan, P., Robertson, D., Amos, T.A., Dyer, M.J., Mason, D.Y. and Greaves, M.F. (1992). Ultrastructural localization of BCL2 protein. J. Histochem. Cytochem., 40, 1819-1825
  50. Sakurai, K., Ishii, H., Kobayashi, S. and Iwao, T. (1976). Isolation of 4 beta-hydroxywithanolide E, a new withanolide from Physalis peruviana L. Chem. Pharm. Bull. (Tokyo), 24, 1403-1405