Pyranocoumarins Isolated from Peucedanum praeruptorum as Differentiation Inducers in Human Leukemic HL-60 Cells

Abstract

Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (±)-4′-O-acetyl-3′-O-angeloyl-cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 μg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27^kip1, but not p21^WAF1, was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia.

Abbreviations

APC:(±)-cis-3′-O-angeloyl-4′-O-acetyl-khellactone

MAPK:mitogen-activated protein kinase

ERK:extracellular signal-regulated kinase

MEK:MAPK/ERK kinase

NBT:nitroblue tetrazolium

RA:all-trans-retinoic acid

TPA:12-O-tetradecanoylphorbol 13-acetate

PE:R-phycoerythrin

FITC:fluorescein isothiocyanateide

HPLC:high-performance liquid chromatography

NMR:nuclear magnetic resonance

Key words

Pyranocoumarins - HL-60 - Differentiation - p27^kip1 - MEK/ERK pathways - Peucedanum praeruptorum - Apiaceae

References

• 1 Chen Z X, Huang B S, She Q L, Zeng G F. The chemical constituents of Bai-Hua-Qian-Hu, the root of Peucedanum praeruptorum Dunn (Umbelliferae) - four new coumarins.  Yao Xue Xue Bao (Acta Pharmaceutica Sinica). 1979;  14 486-96
• 2 Okuyama T, Shibata S. Studies on coumarins of a Chinese drug ”Qian-Hu”.  Planta Med. 1981;  42 89-96
• 3 Aida Y, Kasama T, Takeuchi N, Tobinaga S. The antagonistic effects of khellactones on platelet-activating factor, histamine, and leukotriene D₄ .  Chem Pharm Bull. 1995;  43 859-67
• 4 Nishino H, Okuyama T, Takata M, Shibata S, Tokuda H, Takayasu J, Hasegawa T, Nishino A, Ueyama H, Iwashima A. Studies on the anti-tumor-promoting activity of natural occurring substances. IV. Pd-II [(+)anomalin, (+)praeruptorin B], a seselin-type coumarin, inhibits the promotion of skin tumor formation by 12-O-tetradecanoylphorbol 13-acetate in 7,12-dimethylbenz[a]anthracene-initiated mice.  Carcinogenesis. 1990;  11 1557-61
• 5 Kawaii S, Tomono Y, Katase E,Ogawa K, Yano M. Effect of coumarins on HL60 cell differentiation.  Anticancer Res. 2000;  20 2505-12
• 6 Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay.  Planta Med. 2002;  68 445-8
• 7 Wang X, Studzinski G P. Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1,25-dihydroxyvitamin D₃-induced differentiation of HL60 cells.  J Cell Biochem . 2001;  80 471-82
• 8 Breitman T R, Selonick S E, Collins S J. Induction of differentiation of the human promyelocytic leukemia cell line (HL60) by retinoic acid.  Proc Natl Acad Sci USA. 1980;  77 2936-40
• 9 Marshall C J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.  Cell. 1995;  80 179-85
• 10 Nagata U, Rakahashi N, Davis R J, Todokoro K. Activation of p38 MAP kinase and JNK but not ERK is required for erythropoietin-induced erythroid differentiation.  Blood. 1998;  92 1859-69
• 11 Alessi D R, Cuenda A, Cohen P, Dudley D T, Saltiel A R. PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo .  J Biol Chem. 1995;  270 27 489-94
• 12 Menger H, Yuchen Y, Shurong C, Jinren C. Jia-xiang L, Lin Z, Longjun G, Zhenyi W. Use of all-trans-retinoic acid in the treatment of acute promyelocytic leukemia.  Blood. 1988;  72 567-72
• 13 Reiss M, Gamba-Vitalo C, Sartorelli A C. Induction of tumor cell differentiation as a therapeutic approach: Preclinical models for hematopoietic and solid neoplasms.  Cancer Treat Rep. 1986;  70 201-18
• 14 Hisatake J I, Kubota T, Hisatake Y, Uskokovic M, Tomoyasu S, Koeffler H P. 5,6-trans-16-ene-Vitamin D₃: A new class of potent inhibitors of proliferation of prostate, breast, an myeloid leukemic cells.  Cancer Res. 1999;  59 4023-9
• 15 Kharbanda S, Saleem A, Emoto Y. Stone R, Rapp U, Kufe D. Activation of Raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myloid leukemia cells.  J Biol Chem. 1994;  269 872-8
• 16 Yen A, Roberson M S, Varvayanis S, Lee A T. Retinoic acid induced mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase-dependent MAP kinase activation needed to elicit HL-60 cell differentiation and growth arrested.  Cancer Res. 1998;  58 3163-72
• 17 Yen A. Control of HL-60 myeloid differentiation. Evidence of uncoupled growth and differentiation control, S-phase specificity, and two-step regulation.  Exp Cell Res. 1985;  156 198-212