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

Pamela J Hodul,Warren J Pledger,Said M Sebti,Yanbin Dong,Jose M Pimiento,Domenico Coppola,Jiandong Chen,Kazim Husain,Anying Zhang,Dung-Tsa Chen,Rony Francois,Mokenge P Malafa,Wael El-Rifai

doi:10.1371/journal.pone.0052526

Abstract

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 G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 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 p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.

Highlights

Pancreatic cancer is one of the most lethal cancers in the United States, ranking fourth among the leading causes of cancer-related deaths [1]
The effects of d-tocotrienol ( 1A) on cell growth in MIAPaCa-2, BxPC-3, and SW1990 human pancreatic cancer cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay
The effects of dtocotrienol were evaluated in the non-transformed human pancreatic ductal epithelial cell line HPDE6 C7 to rule out possible cytotoxic effects of d-tocotrienol on non-neoplastic cells. dTocotrienol treatment inhibited anchorage-dependent cell proliferation in both a time- and concentration-dependent manner in human pancreatic cancer cells (Figure 1B); no significant growth inhibitory effects were noted in HPDE6 C7 cells. dTocotrienol treatment significantly inhibited colony formation in MIAPaCa-2 cells grown in soft agar from 2.5 mM (P = 0.02) (Figure 1C)

Summary

Introduction

Pancreatic cancer is one of the most lethal cancers in the United States, ranking fourth among the leading causes of cancer-related deaths [1]. The death rate for patients with pancreatic cancer has overall remained unchanged for decades. Studies have suggested that increased intake of dietary fruits, vegetables, and cereal grains may decrease pancreatic cancer risk [2,3,4]. Tocotrienols, found in cereal grains, comprise one of the most compelling groups of anti-tumor bioactive compounds [5]. Tocotrienols are a group of four (a-, b-, d-, c-) unsaturated, naturally occurring vitamin E compounds that inhibit the proliferation of a variety of human tumor cells, including breast, colon, lung, and hepatocellular [6,7,8], and exhibit chemopreventive properties [9,10].

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