Acetylbritannilactone Inhibits The Proliferation In Human Colon HT-29 Cells

Acetylbritannilactone (ABL) is a new active compound isolated from Inula Britannica L, a traditional Chinese medicinal herb. It has been reported that ABL can inhibit the proliferation of vascular smooth muscle cells (VSMCs) and neointima formation after balloon injury in rats. ABL induces cell apotosis in breast and ovarian cancers. The present study was designed to elucidate the effect and the molecular mechanism of ABL on the proliferation and cell cycle in human colon cancer HT-29 cells.Methods and Results1 ABL inhibits HT-29 cell proliferationHT-29 cells were plated in a 96-well plate, and then treated with ABL for 24 h. The MTT assay was preformed to detect the cell proliferation. The reasults showed that ABL (0, 50, 100, 150, 200μmol/L) supressed the proliferation of HT-29 cells which reduced by 21 %, 38 %, 49 % and 61 %, respectively, compared with the control group. We counted the cells under the same conditions and found that the cell number decreased by 40 %, 52 %, 64 % and 76 %, compared with the control group respectively. Collectively, the results showed that ABL treatment resulted in a significant reduction of HT-29 cell proliferation in a dose-dependent manner.2 Effect of ABL on cell cycle in HT-29 cellsHT-29 cells treated with ABL were trypsinized and fixed with ice 70 % ethanol at 4℃overnight. Following two more washes with PBS, the cells were stained with 50μg/ml of propidium iodide (PI). Cell cycle distribution was then analyzed by flow cytometry. After ABL treatment (0, 50, 100, 150, 200μmol/L), the number of cells distributed in S phase decreased dramatically, while the cells in G0/G1 phase was increased by 3 %, 12 %, 19 % and 24 %, compared with control. The results suggested that the cell cycle of HT-29 cells was arrested in the G0/G1 phase by ABL in a does-dependent manner.3 ABL suppresses the expression of cyclinE and CDK4, and increases the expression of p21Western blot analysis showed that cyclinE and CDK4 protein levels in HT-29 cells were down-regulated after ABL treatment in a does-dependent manner. Howerer, CDK inhibitor p21 was up-regulated under the same conditions.4 The effect of ABL on KLF4 expression in HT-29 cellsKrüppel-like factor 4 (KLF4) is a transcription factor involved in regulating cell growth, differentiation, proliferation and apoptosis. Here, Western blot analysis showed that the expression of KLF4 was induced by ABL in a does-dependent manner. Cell immunofluorescence analysis indicated that KLF4 was mainly located in the cytoplasm, and the expression of KLF4 was significantly increased in HT-29 cells treated with ABL (200μmol/L) for 24 h.5 KLF4 overexpression inhibits HT-29 cell proliferationHT-29 cells were infected with recombinant adenovirus pAd-KLF4 for 48 h to confirm the relationship between KLF4 expression and proliferation inhibition induced by ABL. The MTT assay showed that the proliferation of HT-29 cells was inhibited by the overexpression of KLF4, which was the same effects as ABL.6 KLF4 inhibits the expression of cyclinE and CDK4, and increases the expression of p21In order to investigate the mechanism by which KLF4 mediates the inhibition of HT-29 cell proliferation induced by ABL, the expression of cell cycle-related proteins was detected in HT-29 cells after pAd-KLF4 infection. Western blot analysis showed that KLF4 overexpression resulted in decrease in cyclinE and CDK4 protein levels, and an increase in the expression of p21. These results suggested that ABL arrested the cell cycle and inhibited proliferation of HT-29 cells via activating KLF4.Conclusions: 1 ABL inhibits the proliferation of HT-29 cells in a does-dependent manner.2 ABL blocks the cell cycle in the G0/G1 phase through suppressing cyclinE and CDK2 expression and inducing p21 in HT-29 cells.3 ABL inhibits HT-29 cell proliferation via the activation of KLF4.