Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes.

Certain anti-cancer agents are known to induce apoptosis in human tumour cells. However, these agents are intrinsically cytotoxic against cells of normal tissue origin, including myelocytes and immunocytes. Here we show that a naturally occurring flavone of citrus origin, tangeretin (5,6,7,8,4'-pentamethoxyflavone), induces apoptosis in human promyelocytic leukaemia HL-60 cells, whereas the flavone showed no cytotoxicity against human peripheral blood mononuclear cells (PBMCs). The growth of HL-60 cells in vitro assessed by [3H]thymidine incorporation or tetrazolium crystal formation was strongly suppressed in the presence of tangeretin; the IC50 values range between 0.062 and 0.173 microM. Apoptosis of HL-60 cells, assessed by cell morphology and DNA fragmentation, was demonstrated in the presence of > 2.7 microM tangeretin. Flow cytometric analysis of tangeretin-treated HL-60 cells also demonstrated apoptotic cells with low DNA content and showed a decrease of G1 cells and a concomitant increase of S and/or G2/M cells. Apoptosis was evident after 24 h of incubation with tangeretin, and the tangeretin effect as assessed by DNA fragmentation or growth inhibition was significantly attenuated in the presence of Zn2+, which is known to inhibit Ca(2+)-dependent endonuclease activity. Ca2+ and Mg2+, in contrast, promoted the effect of tangeretin. Cycloheximide significantly decreased the tangeretin effect on HL-60 cell growth, suggesting that protein synthesis is required for flavonoid-induced apoptosis. Tangeretin showed no cytotoxicity against either HL-60 cells or mitogen-activated PBMCs even at high concentration (27 microM) as determined by a dye exclusion test. Moreover, the flavonoid was less effective on growth of human T-lymphocytic leukaemia MOLT-4 cells or on blastogenesis of PBMCs. These results suggest that tangeretin inhibits growth of HL-60 cells in vitro, partially through induction of apoptosis, without causing serious side-effects on immune cells.

[1]  E. Nelson.  The Occurrence of a Pentamethyl Flavonol in Tangerine Peel , 1934 .

[2]  E. Racker,et al.  The effect of flavonoids on aerobic glycolysis and growth of tumor cells. , 1975, Cancer research.

[3]  E. Racker,et al.  Inhibition of (Na+, K+)adenosine triphosphatase and its partial reactions by quercetin. , 1976, Biochemistry.

[4]  S. Collins,et al.  Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture , 1977, Nature.

[5]  R. F. Raffauf,et al.  Antineoplastic activity and cytotoxicity of flavones, isoflavones, and flavanones. , 1979, Journal of natural products.

[6]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[7]  L. C. Anderson,et al.  LIGNAN FORMATION IN MAN—MICROBIAL INVOLVEMENT AND POSSIBLE ROLES IN RELATION TO CANCER , 1981, The Lancet.

[8]  J. Dwyer,et al.  EXCRETION OF THE LIGNANS ENTEROLACTONE AND ENTERODIOL AND OF EQUOL IN OMNIVOROUS AND VEGETARIAN POSTMENOPAUSAL WOMEN AND IN WOMEN WITH BREAST CANCER , 1982, The Lancet.

[9]  J. Cohen,et al.  Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[10]  H. Adlercreutz.  Does fiber-rich food containing animal lignan precursors protect against both colon and breast cancer? An extension of the "fiber hypothesis". , 1984, Gastroenterology.

[11]  J. Cohen,et al.  Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. , 1984, Journal of immunology.

[12]  A. Wyllie.  The biology of cell death in tumours. , 1985, Anticancer research.

[13]  A. Falus,et al.  Induction and activation of tissue transglutaminase during programmed cell death , 1987, FEBS letters.

[14]  M. Shibuya,et al.  Genistein, a specific inhibitor of tyrosine-specific protein kinases. , 1987, The Journal of biological chemistry.

[15]  M. Imaizumi,et al.  Retinoic acid‐induced differentiation of the human promyelocytic leukemia cell line, HL‐60, and fresh human leukemia cells in primary culture: A model for differentiation inducing therapy of leukemia , 1987 .

[16]  M. Tenniswood,et al.  Characterization and cloning of androgen-repressed mRNAs from rat ventral prostate. , 1987, Biochemical and biophysical research communications.

[17]  F. Valeriote,et al.  Flavone acetic acid (NSC 347512)-induced DNA damage in Glasgow osteogenic sarcoma in vivo. , 1988, Cancer research.

[18]  M. A. Tanner,et al.  Inhibition of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin. , 1988, Cancer research.

[19]  K. Oka,et al.  Antiproliferative effects of synthetic and naturally occurring flavonoids on tumor cells of the human breast carcinoma cell line, ZR-75-1. , 1989, Research communications in chemical pathology and pharmacology.

[20]  K. Oka,et al.  Effects of synthetic and naturally occurring flavonoids on Na+,K+-ATPase: aspects of the structure-activity relationship and action mechanism. , 1989, Life sciences.

[21]  R. L. Baldwin,et al.  Second cytotoxic pathway of diphtheria toxin suggested by nuclease activity. , 1989, Science.

[22]  K. Oka,et al.  Effects of synthetic and naturally occurring flavonoids on mitogen-induced proliferation of human peripheral-blood lymphocytes. , 1989, Life sciences.

[23]  C. Taylor,et al.  Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. , 1989, British Journal of Cancer.

[24]  T. Rytömaa,et al.  UV light and ionizing radiations cause programmed death of rat chloroleukaemia cells by inducing retropositions of a mobile DNA element (L1Rn). , 1990, International journal of radiation biology.

[25]  W. Bursch,et al.  The biochemistry of cell death by apoptosis. , 1990, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[26]  A. Wyllie,et al.  Apoptosis. The role of the endonuclease. , 1990, The American journal of pathology.

[27]  A. Eastman,et al.  Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. , 1990, Biochemical pharmacology.

[28]  K. Oka,et al.  Antiproliferative activity of mammalian lignan derivatives against the human breast carcinoma cell line, ZR-75-1. , 1990, Cancer investigation.

[29]  J. Hickman,et al.  Drug-target interactions: only the first step in the commitment to a programmed cell death? , 1991, British Journal of Cancer.

[30]  Gerard I. Evan,et al.  Induction of apoptosis in fibroblasts by c-myc protein , 1992, Cell.

[31]  Z. Darżynkiewicz,et al.  Cytostatic and cytotoxic effects of fostriecin on human promyelocytic HL-60 and lymphocytic MOLT-4 leukemic cells. , 1992, Cancer research.

[32]  N. Shen,et al.  Apoptosis and DNA fragmentation precede TNF‐induced cytolysis in U937 cells , 1992, Journal of cellular biochemistry.

[33]  R. Stockley,et al.  The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils. , 1992, The Journal of biological chemistry.

[34]  S. Shack,et al.  Phenylacetate: a novel nontoxic inducer of tumor cell differentiation. , 1992, Cancer research.

[35]  Z. Darżynkiewicz,et al.  The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. , 1993, Cancer research.

[36]  K. Nicolaou,et al.  Cell-specific regulation of apoptosis by designed enediynes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[37]  H. Adlercreutz,et al.  Genistein, a dietary-derived inhibitor of in vitro angiogenesis. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[38]  K. Yanagihara,et al.  Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract. , 1993, Cancer research.

[39]  Z. Darżynkiewicz,et al.  Simultaneous analysis of DNA replication and apoptosis during treatment of HL-60 cells with camptothecin and hyperthermia and mitogen stimulation of human lymphocytes. , 1994, Cancer research.

[40]  M. Gotoh,et al.  Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells. , 1994, Life sciences.

[41]  S. Grant,et al.  Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. , 1994, Cancer research.

[42]  Genistein arrests cell cycle progression at G2-M , 1995 .