Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

• DOI: 10.1371/journal.pone.0052526
• Publication Date: 2013-02-05T22:42:30Z
• Authors: Pamela J. Hodul; Yanbin Dong; Kazim Husain; Jose M. Pimiento; Jiandong Chen; Anying Zhang; Rony Francois; Warren J. Pledger; Domenico Coppola; Said M. Sebti; Dung-Tsa Chen; Mokenge P. Malafa
• Keywords: 0301 basic medicine; Microscopy, Confocal; Reverse Transcriptase Polymerase Chain Reaction; Science; Q; Blotting, Western; R; Cell Cycle Checkpoints; Flow Cytometry; Immunohistochemistry; Cell Line; 3. Good health; Pancreatic Neoplasms; 03 medical and health sciences; Cell Line, Tumor; Medicine; Humans; Vitamin E; Cyclin-Dependent Kinase Inhibitor p27; E2F1 Transcription Factor; Research Article; Cell Proliferation

Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G(1) cell-cycle arrest and increased p27(Kip1) nuclear accumulation. This finding is significant considering that loss of nuclear p27(Kip1) expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27(Kip1) expression. To determine whether p27(Kip1) induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27(Kip1), in MIAPaCa-2 PDCA cells and demonstrated that p27(Kip1) silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27(Kip1) mRNA expression but not its protein degradation. p27(Kip1) gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27(Kip1) expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27(Kip1) induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27(Kip1) as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.