| Summary: | 博士 === 慈濟大學 === 醫學研究所 === 94 === Lung cancer is the most common cancer and one of the leading causes of cancer death in the word. Any working for the improvements in prevention, early detection and therapeutic effects for lung cancer patients benefit to lung cancer achieve. Several studies showed that some herbal mixtures or pure compounds could inhibit cancer cells proliferation or transformation in vitro and in vivo. We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (AE-BS) 60 microg/ml has anti-proliferation activity and apoptosis effects on A549 human lung cancer cells. They can also cause tumor cell arrest in G2/M phase. To better understand its target protein in A549 cell, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry were applied. The modification of keratin 8 was identified. By immunoblot analysis, the expression of phosphorylated keratin 8 at Ser-73 was increased from 2.0 to 3.0-fold after AE-BS treatment 24 to 48 hr respectively as compared with untreated A549 control cells. Furthermore, the A549 cells were pretreated with 50 microM PD98059, a specific inhibitor of the upstreamregulator of ERK1/2, or with the p38 kinase inhibitor 20 microM SB203580 or JNK inhibitor 20 microM SP600125 for 30 min, followed by 24 h of incubation with BS-AE, PD98059 can inhibit K8-Ser-73 hyperphosphorylation and prevented cell apoptosis which was induced by AE-BS significantly. By immunoblot analysis, AE-BS also can induce ERK1/2 phosphorylation.
Bioactivity-directed fractionation and purification processes were used for further identified the active antiproliferative components of BS-AE. A novel lignan, isochaihulactone (4-benzo[1,3]dioxol-5-ylmethyl-3 (3,4,5-trimethoxyl-benzylidene)–dihydro-furan-2-one), was isolated from BS-AE and identified from spectral evidence (1H NMR, 13C NMR, IR, and MS) and by comparison with authentic synthetic standards. Isochaihulactone was cytotoxic (IC50 = 10-50 �慆icroM) in a variety of human tumor cell lines. In in vitro and in vivo microtubule assembly assays, it inhibited tubulin polymerization in a concentration-dependent manner. As determined by flow cytometry, isochaihulactone caused G2/M phase arrest and apoptosis in a time- and concentration-dependent manner. G2/M arrest was correlated with increased p21/WAF1 levels, downregulation of the checkpoint proteins cyclin B1/cdc2 and mobility shift of cdc25C. Moreover, isochaihulactone (30 and 50 mg/kg) inhibited the growth of non-small cell lung carcinoma A549 xenograft in nude mice.
The novel lignan isochaihulactone inhibits cell proliferation and is a very effective inducer of apoptosis in a variety of carcinoma cell lines. In order to realize the mechanism of isochaihulactone-induced growth arrest and apoptosis, we began to examine isochaihulactone-induced changes in gene expression by oligodeoxynucleotide-based microarray screening using human lung carcinoma A549 cells. This analysis identified several isochaihulactone-inducible genes, including the immediate-early genes Egr-1 and nonsteroidal anti-inflammatory drug-activated gene NAG-1. isochaihulacone was able to increase Egr-1 and NAG-1 mRNA and protein expression in a time- and concentration-dependent manner and the expression of EGR-1 was prior to NAG-1. The pure compound isochaihulactone induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2). The MEK1/2 inhibitor PD98059 reduced the induction of EGR-1 and NAG-1 mRNA and protein expression. Inhibition of isochaihulactone-induced NAG-1 expression by EGR-1 short interfering RNA (siRNA) blocked isochaihulactone-induced apoptosis in A549 cells, suggesting induction EGR-1 negatively affects A549 cell survival. RNA interference using double-stranded RNA specific for the NAG-1-1gene also inhibited isochaihulactone-induced apoptosis. The NAG-1 protein had no effect on cell proliferation and induced apoptosis. Our results suggest that the expression of NAG-1 was up-regulated by ischaihulactone in A549 cells through a ERK-dependent pathway involving the activation of EGR-1. Furthermore, our data showed that activation of EGR-1 and NAG-1 take part in the isochaihulactone-induced apoptosis. These findings indicate isochaihulactone is a promising new antimitotic anticancer compound with potential for clinical application in the future.
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