Abstract
N-Butylidenephthalide (BP), isolated from the chloroform extract of Angelica sinensis, has been examined for its antitumor effects on glioblastoma multiforme brain tumors; however, little is known about its antitumor effects on hepatocellular carcinoma cells. Two hepatocellular carcinoma cell lines, HepG2 and J5, were treated with either N-butylidenephthalide or a vehicle, and cell viability and apoptosis were evaluated. Apoptosis-related mRNA and proteins expressed, including orphan receptor family Nurr1, NOR-1, and Nur77, were evaluated as well as the effect of N-butylidenephthalide in an in vivo xenograft model. N-Butylidenephthalide caused growth inhibition of both the cell lines at 25 μg/ml. Furthermore, N-butylidenephthalide-induced apoptosis seems to be related to Nur77 translocation from nucleus to cytosol, which leads to cytochrome c release and caspase-3-dependent apoptosis. N-Butylidenephthalide-related tumor apoptosis was associated with phosphatidylinositol 3-kinase/protein kinase B (AKT)/glycogen synthase kinase-3β rather than the mitogen-activated protein kinase or protein kinase C pathway. Blockade of AKT activation enhanced proliferation inhibition and the induction of phosphor-Bcl-2 and Nur77 proteins. Besides, the increasing apoptosis by BP via transfection wild-type cAMP-response element-binding protein (CREB) into tumor cell was suppressed by dominant phosphorylation site mutation of CREB. This finding suggested CREB pathway was also partly involved in tumor apoptosis caused by BP. Administration of N-butylidenephthalide showed similar antitumoral effects in both HepG2 and J5 xenograft tumors. N-Butylidenephthalide induced apoptosis in hepatocellular carcinoma cells, both in vitro and in vivo, suggesting a potential clinical use of this compound for improving the prognosis of hepatocellular carcinoma cells.
Footnotes
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This work was supported National Science Council of the Republic of China grants 96-2320-B-197-001-MY2 and 96-2320-B-303-001-MY3. S.-Z.L. and H.-J.H. contributed equally to this work.
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ABBREVIATIONS: HCC, hepatocellular carcinoma; AS, Angelica sinensis; Nur77, nerve growth factor-induced B; NGF, nerve growth factor; PB, N-butylidenephthalide; AKT, protein kinase B; TCR, T-cell receptor; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide; CD437, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid; LMB, leptomycin B; PD98059, 2-amino-3-methoxyflavone; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one; PKC, protein kinase C; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; Gö6983, 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide; PI3K, phosphatidylinositol-3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PARP, poly(ADP-ribose) polymerase; CREB, cAMP response element-binding protein; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; PI, propidium iodide; siRNA, small interfering RNA; AP-1, activator protein-1; CMV, cytomegalovirus; pCMV, phosphorylated cytomegalovirus; C133, CREB133; TV, tumor volume; GBM, glioblastoma multiforme; RTV, relative tumor volume.
- Received January 1, 2008.
- Accepted June 23, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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