Metformin Causes G1-Phase Arrest via Down-Regulation of MiR-221 and Enhances TRAIL Sensitivity through DR5 Up-Regulation in Pancreatic Cancer Cells

<div><p>Although many chemotherapeutic strategies against cancer have been developed, pancreatic cancer is one of the most aggressive and intractable types of malignancies. Therefore, new strategies and anti-cancer agents are necessary to treat this disease. Metformin is a widely used drug for type-2 diabetes, and is also known as a promising candidate anti-cancer agent from recent studies <i>in vitro</i> and <i>in vivo</i>. However, the mechanisms of metformin’s anti-cancer effects have not been elucidated. We demonstrated that metformin suppressed the expression of miR-221, one of the most well-known oncogenic microRNAs, in human pancreatic cancer PANC-1 cells. Moreover, we showed that the down-regulation of miR-221 by metformin caused G1-phase arrest via the up-regulation of p27, one of the direct targets of miR-221. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also a promising agent for cancer treatment. While recent studies showed that treatment with only TRAIL was not effective against pancreatic cancer cells, the present data showed that metformin sensitized p53-mutated pancreatic cancer cells to TRAIL. Metformin induced the expressions of death receptor 5 (DR5), a receptor for TRAIL, and Bim with a pro-apoptotic function in the downstream of TRAIL-DR5 pathway. We suggest that the up-regulation of these proteins may contribute to sensitization of TRAIL-induced apoptosis. The combination therapy of metformin and TRAIL could therefore be effective in the treatment of pancreatic cancer.</p></div

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

The RAF inhibitor PLX4720 negated MEK inhibition by PD0325901 in a NRAS-mutated cell line.

<p>A, SK-MEL-2 cells were treated with PLX4720 in the presence or absence of CH5126766 or PD0325901 for 24 h. Cell extracts were analysed by immunoblotting for the indicated proteins. B, Clonogenic suppression by PD0325901 and/or PLX4720 in NRAS-mutated SK-MEL-2 cells. SK-MEL-2 cells were treated for 72 h with 100 nM of CH5126766, or 10 nM of PD0325901 in the presence or absence of 1 µM of PLX4720. After 14 days, colony numbers were counted. Data represent the means of triplicate with SD indicated. Significance was assessed by a Student's <i>t</i> test. *, P<0.05; **, P<0.01. CH; CH5126766, PD; PD0325901, PLX; PLX4720</p

Metformin up-regulates the expression of DR5 in p53 mutant PANC-1 cells.

<p>(A) PANC-1 cells were treated with the indicated concentrations of metformin for 48 hours. Western blotting for DR4, DR5 and Bim was performed. β-actin is a loading control. Arrow, nonspecific band. (B), (C) Cell surface expressions of DR4 and DR5 in PANC-1 cells treated with or without 40 mM metformin for 48 hours. Cells were stained with isotype control IgG and monoclonal antibodies against DR4 and DR5. Data were analyzed by flow cytometry. (B) Gray histogram, no treatment; white histogram, metformin treatment. (C) The Y-axis represents the geometric mean values of the cell populations in the histograms. Gray bar, no treatment; white bar, metformin treatment. (D) Quantitative real-time RT-PCR of DR5 mRNA in PANC-1 cells treated with the indicated concentrations of metformin for 48 hours. The internal control was β2MG. Values are fold change in the expression of DR5 mRNA /β2MG mRNA compared with untreated control. Data are the means ± SD of 3 determinations. **P<0.01.</p

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

Antitumor activity of CH5126766 in a xenograft model of SK-MEL-2 cells.

<p>No animals died or had more than 10% of their body weight gain or loss from baseline in these experiments. Vehicle or CH5126766 was administered orally every day for eleven days. Statistical analysis was conducted by using Dunnett test. *, P<0.05. ▾; Administration of vehicle or CH5126766</p

Metformin sensitizes TRAIL-resistant PANC-1 cells to TRAIL-induced apoptosis.

<p>(A) PANC-1 cells were treated with the indicated concentrations of TRAIL. After incubation for 72 hours, viable cells were evaluated using a Cell Counting Kit-8. (B), (C) PANC-1 cells were treated with the indicated concentrations of TRAIL (B) or metformin (C) for 48 hours. Sub-G1 populations were analyzed by flow cytometry. (D) Combined effects of 40 mM metformin and/or 10 ng/mL TRAIL for 48 hours. Sub-G1 populations were analyzed by flow cytometry. Data are the means ± SD of 3 determinations. *P<0.05, **P<0.01.</p

Structural formula of resibufogenin.

<p>Structural formula of resibufogenin.</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

Metformin induces G1-phase arrest in PANC-1 cells through down-regulation of miR-221.

<p>(A) PANC-1 cells were treated with the indicated concentrations of metformin for 48 hours. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. (B), (C) PANC-1 cells were treated with the indicated concentrations of metformin for 48 hours. (B) Western blotting for p27. β-actin is a loading control. (C) Real-time RT-PCR quantification of miR-221 expression. The internal control was RNU48. Values are fold change in the expression of miR-221/RNU48 compared with untreated control. (D), (E) PANC-1 cells were transfected with 5 nM miR-221 mimic or 5 nM negative control. After 24 hours, the cells were incubated with or without 40 mM metformin for 48 hours. (D) The percentage of cells in each phase of the cell cycle was determined by flow cytometry. (E) Western blotting for p27. β-actin is a loading control. Arrow, a nonspecific band. Data are the means ± SD of 3 determinations. **P<0.01.</p