Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells

<div><p>Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β) induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.</p></div

Resibufogenin induces G1-phase arrest in HT-29 cells.

<p>(A) Cell cycle analysis of HT-29 cells treated with resibufogenin. Cells were treated with resibufogenin at the indicated concentrations for 24 h. The DNA content of propidium iodide-stained nuclei was analysed by flow cytometry. The percentages in G1 (black), S (dark gray) and G2/M (light gray) phases of the cell cycle were analysed using Modfit LT software. UT, untreated; DM, treated with DMSO. Points, means (n = 3); bars, SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01, compared with the DMSO-treated control. (B) Representative histogram of each treatment. (C) Cell cycle analysis of HT-29 cells treated with resibufogenin in synchronized cells by serum starvation. Cells were treated with resibufogenin at the indicated concentrations for 24 h after the 24 h serum starvation without FBS. The cell cycle analysis was similarly performed and shown as described in the figure legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129851#pone.0129851.g003" target="_blank">Fig 3A</a> above. (D) Representative histogram of each treatment. (E) BrdU incorporation analysis of HT-29 cells treated with resibufogenin. Cells were treated with resibufogenin at the indicated concentrations for 24 h. Subsequently, the cells were incubated with BrdU for 2 h. The incorporation of BrdU into DNA was measured using a cell proliferation enzyme-linked immunosorbent BrdU assay kit. The data obtained with DMSO was taken as 100%. UT, untreated; DM, treated with DMSO. Points, means (n = 3); bars, SD. **<i>P</i> < 0.01, compared with the DMSO-treated control.</p

Structural formula of resibufogenin.

<p>Structural formula of resibufogenin.</p

Resibufogenin inhibits the growth of human colon cancer HT-29 cells.

<p>(A) Growth inhibitory effect of resibufogenin by counting viable cell number. Cells were treated with resibufogenin at the indicated concentrations for 1, 2, or 3 days. Viable cell number was measured by a Guava EasyCyte plus flow cytometry. UT, untreated; DM, treated with DMSO. Points, means (n = 3); bars, SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01, compared with the DMSO-treated control. (B) The effect of resibufogenin on cell viability. Cells were treated and measured as shown in (A). Viability was calculated using the number of viable cells and dead cells. UT, untreated; DM, treated with DMSO. Points, means (n = 3) (C) The effect of resibufogenin on apoptosis by annexin V staining. Cells were treated with resibufogenin at the indicated concentrations for 24 h, and subjected to annexin V staining. The stained cells were measured by FACSCalibur. UT, untreated; DM, treated with DMSO. Celecoxib at 100 μM was used as a positive control (PC). Points, means (n = 3); bars, SD. *<i>P</i> < 0.05, **<i>P</i> < 0.01, compared with the DMSO-treated control.</p

Resibufogenin down-regulates cyclin D1 through proteasomal degradation.

<p>(A) The effect of MG132 on suppression of cyclin D1 expression by resibufogenin in HT-29 cells. Cells were treated by 5 μM resibufogenin with or without 20 μM MG132 for 24 h. (B) The effect of MG132 on suppression of cyclin D1 expression by resibufogenin in A549 cells. Cells were treated with 2.5 μM resibufogenin with or without 20 μM MG132 for 24 h. (C) The effect of SB216763 on suppression of cyclin D1 expression by resibufogenin in HT-29 cells. Cells were treated by 5 μM resibufogenin with or without 30 μM SB216763 for 3 h. (D) The effect of SB216763 on suppression of cyclin D1 expression by resibufogenin in A549 cells. Cells were treated with 2.5 μM resibufogenin with or without 30 μM SB216763 for 6 h. In all figures, GAPDH was used as a loading control for protein quantitation. UT, untreated; DM, treated with DMSO. The band intensity was measured and normalized by GAPDH, and the protein levels are shown at the bottom of each blot.</p

Resibufogenin converts RB protein to the unphosphorylated form with down-regulation of cyclin D1 expression.

<p>(A) The effect of resibufogenin on the expression of phosphorylated RB (p-RB (Ser⁷⁸⁰) and p-RB (Ser^807/811)), RB, cyclin D1 and cyclin E. Cells were treated with resibufogenin at the indicated concentrations for 24 h and analysed by Western blotting. (B) Time-course study on the phosphorylation status of RB protein and the expression of cyclin D1. Cells were treated with 5 μM resibufogenin (R) for 1, 2, 3, 6, 9 or 24 h and analysed by Western blotting. In both figures, RB or GAPDH was used as a loading control for protein quantitation. UT, untreated; DM, treated with DMSO. The band intensity was measured and normalized by RB or GAPDH, and the protein levels are shown at the bottom of each blot.</p