Pyranocoumarin (±)-4′-O-Acetyl-3′-O-angeloyl-cis-khellactone Induces Mitochondrial-Dependent Apoptosis in HL-60 Cells

Abstract

The pyranocoumarin (±)-4′-O-acetyl-3′-O-angeloyl-cis-khellactone (PC) isolated from Radix Peucedani (root of Peucedanum praeruptorum Dunn) showed a dose-dependent effect at 10 - 30 μg/mL on causing apoptotic DNA and nuclear fragmentations in HL-60 cells. After 24 h of PC treatment there were losses of mitochondrial membrane potential and cytochrome c. PC also increased total cellular and mitochondrial Bax protein, stimulated an increase in caspase-dependent Bcl-2 cleavage but showed no effect on Bcl-X_L. These observations strongly suggest activation of the mitochondria apoptotic pathway. The pan-specific caspase inhibitor, ZVAD-fmk, abolished the PC-induced apoptosis, whereas the caspase-8 inhibitor IETD-fmk showed no effect, implying the involvement of the caspase 9 pathway. PC caused a 2 to 12 hour transient increase in phospho-ERK, and a 72 h-long activation of JNK. Pre-treatment with the MEK inhibitor PD98059, which suppresses ERK activation, paradoxically promoted PC-induced mitochondrial cytochrome c release, procaspase-3 and -8 cleavage, and enhanced apoptosis. Our results show that PC triggers mitochondria-mediated apoptosis in HL-60 cells, and the involvement of ERK and JNK signal pathways in the process.

Abbreviations

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

MAPK:mitogen-activated protein kinase

ERK:extracellular signal-regulated kinase

MEK:MAPK/ERK kinase

ΔΨ_m:mitochondrial membrane potential

HPLC:high-performance liquid chromatography

NMR:nuclear magnetic resonance

Key words

4′-O-acetyl-3′-O-angeloyl-khellactone - Peucedanum praeruptorum - Umbelliferae - HL-60 - Apoptosis - mitogen-activated protein kinases - Bax, Bcl-2

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 Pharmaceut. Sinica) 1979;  14 486-96
• 2 Okuyama T, Shibata S. Studies on coumarins of a Chinese drug ”Qian-Hu”.  Planta Medica. 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 .  Bull Chem Pharmacol. 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 Wu J YC, Fong W F, Zhang J X, Leung C H, Kwong H L, Yang M S, Li D, Cheung H Y. Reversal of multidrug resistance by pyranocoumarin (±)-3′-angeloyl-4′-acetoxy-cis-khellactone isolated from Peucedanum praeruptorum Dunn.  Eur J Pharmacol. 2003;  473 9-17
• 6 Zhang J X, Fong W F, Wu J YC, Yang M S, Cheung H Y. Pyranocoumarin (±)-3′-angeloyl-4′-acetoxy-cis-khellactone isolated from Peucedanum praeruptorum Dunn as differentiation inducers in human leukemic HL-60 cells.  Planta Medica. 2003;  69 223-8
• 7 Cory S, Adams J M. The Bcl2 family: regulators of the cellular life-or-death switch.  Nat Rev Cancer. 2002;  2 647-56
• 8 Antonsoon B. Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways.  Mol Cell Biochem. 2004;  256 141-55
• 9 Cross T G, Scheel-Toellner D, Henriquez N V, Deacon E, Salmon M, Lord J M. Serine/threonine protein kinases and apoptosis.  Exp Cell Res. 2000;  256 34-41
• 10 Chang F, Steelman L S, Lee J T, Shelton J G, Navolanic P M, Blalock W L, Franklin R A, McCubrey J A. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention.  Leukemia. 2003;  17 1263-93
• 11 Cossarizza A, Baccarani Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocynanine iodide (JC-1).  Biochem Biophys Res Commun. 1993;  197 40-5
• 12 Wang Z, Van Tuyle G, Conrad D, Fisher P B, Dent P, Grant S. Dysregulation of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1/MDA6 increases the susceptibility of human leukemia cells (U937) to 1-beta-D-arabinofuranosylcytosine-medated mitochondrial dysfunction and apoptosis.  Cancer Res. 1999;  59 1259-67
• 13 Cheng E H, Kirshc D G, Clem R J, Ravi R, Kastan M B, Bedi A, Ueno K, Hardwick J M. Conversion of bcl-2 to a Bax-like death effector by caspase.  Science. 1997;  278 1966-8
• 14 Jurgensmeier J M, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed J C. Bax directly induces release of cytochrome c from isolated mitochondria.  Proc Natl Acad Sci USA. 1998;  95 4997-5002
• 15 Gross A, Yin X M, Wang K, Wei M C, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeryer S J. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.  J Biol Chem. 1999;  274 1156-63
• 16 Cheng E H, Kirsch D G, Clem R J, Ravi R, Kastan M B, Bedi A, Ueno K, Hardwick J M. Conversion of Bcl-2 to a Bax-like death effector by caspases.  Science. 1997;  278 1966-68
• 17 García-Fernández L F, Losada A, Alcaide V, Álvarez A M, Cuadrado A, González L, Nakayama K, Nakayama K I, Fernández-Sousa J M, Muñoz A, Sánchez-Puelles J M. Aplidin^TM induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase Cδ.  Oncogene. 2002;  21 7533-44
• 18 Yu C R, Wang Z L, Dent P, Grant S. MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of ΔΨm in HL-60 cells.  Biochem Biophy Res Commun. 2001;  288 1011-8
• 19 Lei K, Nimnual A, Zong W X, Kennedy N J, Flavell R A, Thompson C B, Bar-Sagi D, Davis R J. The Bax subfamily of Bcl2-related proteins in essential for apoptotic signal transduction by c-Jun NH₂-Terminal kinase.  Mol Cell Biol. 2002;  22 4929-42