cyclin-d1 and eupatilin

Artemisia asiatica Nakai has been used frequently in traditional Asian medicine for the treatment of inflammation and cancer. Eupatilin (5,7-dihydroxy-3',4', 6-trimethoxy-flavone) was shown to be a pharmacologically active ingredient of A. asiatica. In the present study, we found that expression of cyclin D1, a key protein that regulates G1/S progression, was decreased in MCF-10A-ras cells treated with eupatilin. Downregulation of cyclin D1 expression by eupatilin was accompanied by a reduced expression of c-Jun and the DNA binding activity of the transcription factor AP-l. The expression of p21waf1/Cip1 was also decreased by eupatilin treatment in both protein and the mRNA levels. We concluded that the inhibitory effect of eupatilin on p21waf1/Cip1 expression is likely to be associated with the downregulation of cyclin D1 expression and AP-1 activation, which play an important role in the cell cycle arrest of ras-transformed breast epithelial cells.

Topics: Cell Line, Transformed; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; DNA; Down-Regulation; Drugs, Chinese Herbal; Female; Flavonoids; G1 Phase; Genes, ras; Humans; Mammary Glands, Human; S Phase; Transcription Factor AP-1

Extracts of Artemisia asiatica Nakai (Asteraceae) possess anti-inflammatory and anti-oxidative activities. Eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone), one of the pharmacologically active ingredients derived from A. asiatica, was shown to induce apoptosis in human promyelocytic leukemia (HL-60) cells [Mutat Res 496 (2001) 191]. In the present study, we examined the cytostatic effects of eupatilin in H-ras-transformed human breast epithelial (MCF10A-ras) cells. Eupatilin inhibited the growth of MCF10A-ras cells in a concentration-dependent and time-related manner. To explore whether the anti-proliferative effects of eupatilin could be mediated through modulation of the cell cycle in MCF10A-ras, DNA contents were analyzed by the flow cytometry. Eupatilin inhibited the expression of cyclin D1, cyclin B1, Cdk2 and Cdc2 that are key regulators of the cell cycle. In addition, eupatilin treatment led to elevated expression of p53 and p27Kip1 that act as Cdk inhibitors. It has been known that the Ras-signaling pathway plays integral roles in the induction of cyclin D1. Eupatilin inhibited the activation of ERK1/2 as well as expression of Raf-1 and Ras in MCF10A-ras cells. Thus, the inhibitory effect of eupatilin on cyclin D1 expression appears to be mediated by targeting the Raf/MEK/ERK signaling cascades. Eupatilin did not change activation of Akt, an important component of cell-survival pathways. In conclusion, the anti-proliferative effect of eupatilin in MCF10A-ras cells is associated with its blockade of cell cycle progression which appears to be attributable in part to inhibition of ERK1/2 activation.

Topics: Artemisia; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Epithelial Cells; Flavones; Flavonoids; G1 Phase; Genes, ras; HL-60 Cells; Humans; Mammary Glands, Human; Mitogen-Activated Protein Kinases; Mitosis; Proto-Oncogene Proteins c-raf; S Phase; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation

Extracts of the whole herb of Artemisia asiatica Nakai (Asteraceae) have been used in traditional oriental medicine for the treatment of inflammation, cancer and other disorders. In the present work, we have evaluated the apoptosis-inducing capability of eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), a pharmacologically active ingredient of A. asiatica, in cultured human promyelocytic leukemia (HL-60) cells. Thus, eupatilin exhibited concentration-dependent inhibitory effects on viability and DNA synthesis capability of HL-60 cells. The anti-proliferative effect of eupatilin was attributable to its apoptosis-inducing activity as determined by characteristic nuclear condensation, in situ terminal end-labeling of fragmented DNA (TUNEL), release of mitochondrial cytochrome c into cytoplasm, proteolytic activation of caspases-9, -3, and -7, and cleavage of poly(ADP-ribose)polymerase. Eupatilin-induced HL-60 cell apoptosis does not appear to be mediated via alteration in Bcl-2/Bax-2. Taken together, the above findings suggest that eupatilin has chemopreventive and cytotoxic effects.

Topics: Antineoplastic Agents; Apoptosis; Artemisia; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Survival; Cyclin D1; Cytochrome c Group; DNA Replication; Dose-Response Relationship, Drug; Flavonoids; HL-60 Cells; Humans; In Situ Nick-End Labeling; Mitochondria; Plant Extracts; Plants, Medicinal; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2