Journal: PLoS ONE

Article Title: Vitamin E δ-Tocotrienol Induces p27 Kip1 -Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

doi: 10.1371/journal.pone.0052526

Figure Lengend Snippet: A , pancreatic cancer and HPDE6 C7 cells were treated with δ-tocotrienol at predetermined IC 50 for each cell line. Protein lysates from 0–48 hours were collected and analyzed by Western blot analysis for Ras oncogenic signaling targets, MEK and ERK, and p27 Kip1 . Results are representative of 3 independent experiments. δ-Tocotrienol selectively inhibits MAP kinase signaling and increases p27 Kip1 expression in transformed pancreatic cancer cells. B, MIAPaCa-2 cells were treated with δ-tocotrienol at predetermined IC 50 . Protein lysates from 0–24 hours were collected and analyzed by Western blot for AKT and a downstream target GSK-3β. C , MIAPaCa-2 cells were treated with vehicle or δ-tocotrienol in FBS for 12 hours, and pure nuclear and cytosolic fractions were isolated. Western blots show increased levels of p27 Kip1 in δ-tocotrienol treated cells with high concentrations in the nucleus. D, simultaneously, whole cells were stained with immunofluorescent p27 Kip1 antibody and analyzed by fluorescent microscopy for p27 Kip1 localization. δ-Tocotrienol localized p27 Kip1 to the nucleus similar to the starved state, whereas serum-treated cells showed equal levels of p27 Kip1 in the nucleus and cytoplasm. E, δ-tocotrienol (DT3) suppresses tumor cell growth of MIAPaCa-2 cells in the presence of added mevalonate, a metabolite of HMG-CoA reductase. MIAPaCa-2 cells (in 96-well plates) were treated for 24 hours with δ-tocotrienol or lovastatin in the absence or presence of increasing concentrations of mevalonate. MTT results demonstrate that mevalonate rescues the growth inhibitory effects of lovastatin, but not that of δ-tocotrienol. In E , lines 1 and 7 had no δ-tocotrienol (DT3) or lovastatin.

Article Snippet: Each well was transfected the following day with 2 μg of p27 Kip1 luciferase reporter plasmid (full-length p27 Kip1 -1609, different 5′ deletion mutants of mouse p27 Kip1 promoter luciferase reporter, or pGL-3 base cDNA empty vector; all plasmids provided by Dr. Pledger) along with siRNA E2F-1 or non-target siRNA (Santa Cruz).

Techniques: Western Blot, Expressing, Transformation Assay, Isolation, Staining, Microscopy

Journal: PLoS ONE

Article Title: Vitamin E δ-Tocotrienol Induces p27 Kip1 -Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

doi: 10.1371/journal.pone.0052526

Figure Lengend Snippet: A, p27 Kip1 siRNA attenuates δ-tocotrienol-mediated growth suppression in human MIAPaCa-2 pancreatic cancer cells. After transfection with p27 Kip1 siRNA or with noncoding siRNA for 24 hours, MIAPaCa-2 cells were incubated with fresh medium containing either δ-tocotrienol (IC 50 ) or vehicle for an additional 24 hours. Cells were taken from culture and divided into 2 aliquots. Immunoblots demonstrate inhibited p27 Kip1 expression with siRNA p27 Kip1 and rescue from inhibition of proliferation in siRNA p27 Kip1 -pretreated cells. B, MIAPaCa-2 cells expressing stable shRNA p27 Kip1 are protected from the growth inhibitory effects of δ-tocotrienol. Stable MIAPaCa-2 cells expressing shRNAp27 Kip1 or empty vector were treated with increasing concentrations of δ-tocotrienol or vehicle for 24 hours, with proliferation determined by MTT assay. MIAPaCa-2 cells expressing shRNAp27 Kip1 demonstrate resistance to growth inhibitory effects of δ-tocotrienol. C, p27 Kip1 knockout cells attenuate δ-tocotrienol-mediated growth inhibitory effects. Stable MEF p27 Kip1 (−/−) and MEF p27 Kip1 (+/+) were plated and incubated with either δ-tocotrienol at the indicated doses or with vehicle for 48 hours. Cell cultures were then collected in 2 aliquots and analyzed as reported previously for siRNA p27 Kip1 -treated cells. In the absence of p27 Kip1 expression, δ-tocotrienol exerts minimal growth inhibitory effects in mouse epithelial cells.

Article Snippet: Each well was transfected the following day with 2 μg of p27 Kip1 luciferase reporter plasmid (full-length p27 Kip1 -1609, different 5′ deletion mutants of mouse p27 Kip1 promoter luciferase reporter, or pGL-3 base cDNA empty vector; all plasmids provided by Dr. Pledger) along with siRNA E2F-1 or non-target siRNA (Santa Cruz).

Techniques: Transfection, Incubation, Western Blot, Expressing, Inhibition, shRNA, Plasmid Preparation, MTT Assay, Knock-Out

Journal: PLoS ONE

Article Title: Vitamin E δ-Tocotrienol Induces p27 Kip1 -Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

doi: 10.1371/journal.pone.0052526

Figure Lengend Snippet: A, MIAPaca-2 cells were treated with δ-tocotrienol at a predetermined IC 50 and then with cyclohexamide to block protein synthesis. p27 Kip1 turnover rate was examined by Western blot. B, densitometry plot for panel A using β-actin for density control, showing similar rates of degradation by δ-tocotrienol. C, RT-PCR confirms upregulation of p27 Kip1 at the mRNA level. MIAPaCa-2 cells were treated with δ-tocotrienol for 24 hours, and p27 Kip1 mRNA expression level was determined by RT-PCR at different amplification cycles. Representative result at cycle 40 is shown. D, activation of p27 Kip1 promoter by δ-tocotrienol. MIAPaCa-2 cells were transfected with 5′-deletion mutants of the mouse p27 Kip1 promoter luciferase reporter. After 24-hour transfection, cells were treated with δ-tocotrienol (IC 50 ) or vehicle for an additional 24 hours, and luciferase activity was determined. Deletion analysis of the mouse p27 Kip1 promoter suggests that the region between 326 and 615 contains sequences necessary for significant response to δ-tocotrienol in p27 Kip1 reporter assays. E, illustration of 5′ deletion mutants of the mouse p27 Kip1 promoter luciferase reporter. Sequence search of this region revealed several putative E2F-1 binding sites (TTTGGCTA, GCGCGGAG, GCGCCGAG) as demonstrated in the shaded area of the deletion mutant constructs. F , immunoblot showing suppressed E2F-1 expression using siRNA E2F-1, G , attenuated δ-tocotrienol mediated effects on the transfected full-length p27 Kip1 promoter activity using a luciferase reporter assay.

Article Snippet: Each well was transfected the following day with 2 μg of p27 Kip1 luciferase reporter plasmid (full-length p27 Kip1 -1609, different 5′ deletion mutants of mouse p27 Kip1 promoter luciferase reporter, or pGL-3 base cDNA empty vector; all plasmids provided by Dr. Pledger) along with siRNA E2F-1 or non-target siRNA (Santa Cruz).

Techniques: Blocking Assay, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Activation Assay, Transfection, Luciferase, Activity Assay, Sequencing, Binding Assay, Mutagenesis, Construct, Reporter Assay

Journal: PLoS ONE

Article Title: Vitamin E δ-Tocotrienol Induces p27 Kip1 -Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

doi: 10.1371/journal.pone.0052526

Figure Lengend Snippet: A, nu/nu mice were injected subcutaneously with a suspension of MIAPaCa-2 cells (1×10 6 ) combined with Matrigel in both flanks. Tumor volume was recorded every other day according to the formula V = ( l + w )/2 × ( l × w ) × 0.5236, where l is length and w is width. Tumors with equal growth rates and initial volumes between 250 and 300 mm 3 were then randomized to receive δ-tocotrienol (100 mg/kg) or vehicle (purified olive oil) daily by gavage. Data showing significant inhibition of tumor growth by δ-tocotrienol are representative of 5 tumors/treatment group repeated on 3 separate occasions ( P <0.003). B, tissue sections from tumors were fixed in paraformaldehyde and embedded in paraffin for immunohistochemical staining of Ki67, MAP kinase, and p27 Kip1 . Representative sections are shown. Areas of tumor necrosis (N) were consistently visualized in the δ-tocotrienol group in contrast to the vehicle group where only viable (V) tumor was present. H&E, hematoxylin and eosin. Numbers represent percentage of positive cells. H&E and immunohistochemistry, x200.

Article Snippet: Each well was transfected the following day with 2 μg of p27 Kip1 luciferase reporter plasmid (full-length p27 Kip1 -1609, different 5′ deletion mutants of mouse p27 Kip1 promoter luciferase reporter, or pGL-3 base cDNA empty vector; all plasmids provided by Dr. Pledger) along with siRNA E2F-1 or non-target siRNA (Santa Cruz).

Techniques: Injection, Suspension, Purification, Inhibition, Immunohistochemical staining, Staining, Immunohistochemistry