Triolein from Coix Lacryma-jobi Induces Cell Cycle Arrest through P53/p21 Signaling Pathway

p53, a tumor suppressor protein, has important roles in DNA repair, cell cycle and apoptosis, is a one of the key events in cancer development. Coix lacryma-jobi seed has been used as a food and traditional medicine plant with anti-oxidant, anti-cancer and anti-diabetic effects. In currently research, we identified the most potent p53-increasing compound among 4 compounds (1 – 4) found in Coix lacryma-jobi and demonstrated its molecular mechanism in MCF-7 cells. Among the four isolated compounds (1 – 4), triolein most increased p53. Triolein treatment induced p53, p21, p27 and Bax in MCF-7 cells. Moreover, triolein caused S phase arrest through suppression of CDK1, phopho-Rb and E2F1 in dose-dependent manner. We also observed the decreasing of DNA synthesis by triolein. These data suggest that triolein may induced cell cycle restart involve DNA synthesis and apoptosis pathway in MCF-7 cells.


INTRODUCTION
Cancer is a complex disease with multiple genetic and epigenetic changes.Cancer cells proliferate without stopping and spread into surrounding tissues.Chemotherapy is the most common types of cancer treatment.p53 plays an important role in cancer treatment mainly by increasing cell cycle arrest, senescence, DNA damage and apoptosis and interconnecting with many cellular functions [1][2] .Targeting p53 provides many opportunities to fight against cancer.p53 regulates various responses to genotoxic stress by controlling the transcription of numerous target genes involved in cell cycle and DNA repair (p21, GADD45), apoptosis (Bax, Bcl2), and senescence (Sirt1) [3][4] .One promising cancer treatment strategy is the development natural products that induce p53 transcription activity and its target genes to induce growth arrest and apoptosis in p53 wild-type cancer cells.
Coix seed (Coix lachryma-jobi) is used in Vietnam and Chinese as a nutritious food and traditional medicine.Previous studies demonstrated that Coix seed extract exhibits anti-inflammatory, protects against oxidative stress, and anti-cancer activities [5][6] .Coix extract constituents inhibited NF-kB and protein kinase C signalling in cancer cells to suppress growth MDA-MB-231 xenograft in athymic nude mice, 7 inhibited COX-2 expression of human lung cancer cells 5 , activated PI3K/Akt to upregulate the Nrf2, HO-1 expression to protects against oxidative stress-induced injury 8 .
Triolein, one active component in Coix seed extract, exhibits anti-tumor activity.The anticancer activities of triolein are limited due to its use as a cell fuel.Triolein is degradated by lipoprotein and fatty acids released are used to provide energy via beta-oxidation 9 .However, mechanism investigations on impacts of triolein on cancer cells are necessary before drug development studies.Therefore, this study was performed to investigate impact of triolein on p53 and its target genes that involves in cell cycle arrest in breast cancer cells.We found that triolein from Coix seed extract induces p53 protein level in concentration-dependent manner in MCF-7 cells.Triolein up-regulated the expression of p53-dependent target genes that related to cell cycle arrest such as p21 and cyclin D1, reducing DNA content.This study provided scientific evidences for the use of Coix seed.

Western blot analysis
After protein determination using BCA protein assay kit (Pierce, Rockford, IL), aliquots of the lysates (10-30 ìg of protein) were boiled for 5 min and electrophoresed on 10% SDSpolyacrylamide gels.Proteins in the gels were transferred onto nitrocellulose membranes and then incubated with primary antibodies specified (p53, p21, Bax, p27, Cyclin D1, phospho-Rb, total-Rb, E2F1, â-actin).Bands were visualized using ECL (GE Healthcare Life Sciences) and images were developed using the LAS 3000.

BrdU assay
BrdU assay was performed as described 10 .

Plant materials and extraction
The seeds of Coix lachryma-jobi var.mayuen were collected in Yenbai province, Vietnam, in December 2014.The specimen of materials has been identified by one of authors Dr. Do Thi Ha (D.T.H).The specimen (HTV-211214) have been deposited with the Deparment of Phytochemistry, National Institute of Medicinal Materials, Viet Nam.

RESULTS AND DISCUSSION
p53 tumour suppressor protein has been demonstrated to induce apoptosis and cell cycle arrest in cancer cells [1] .To screen natural drug candidates against cancer, we first determined the effect of ethanol 96% extractions from Coix lachryma-jobi includes: adlay aril, adlay brain and adlay seed kernel (1 mg/ml) on p53 protein expression using western blot analysis.p53 expression up-regulated p21-cell cycle arrest inducer to induce cell cycle arrest and then repair the damaged DNA due to increase apoptosis in cancer cells [15] .Thus, we examine the effect of triolein on p21, p27 and Bax protein expression.As expect, p53, p21, p27 and Bax protein level were significant increased by triolein in dose-dependent (Fig. 2A).
Fig 1A indicated that adlay aril extraction most potential increased p53 in MCF-7 cells.From adlay aril extraction, 3 fractions include: n-hexan, ethyl acetate and water were prepared and further examine the effects of these fraction on p53 expression.As shown as in Fig 1B, n-hexan fraction most activated p53 protein expression in MCF-7 cells.From this fraction, we isolated 4 compounds: CLH1 (triolein), CLH3, CLH4 and CLH5.Using western blot analysis, we found triolein (100 ìM) compound most significant increase p53 protein expression level in MCF-7 cells (Fig 1C).