Hien T. T, Ha D. T, Truong D. M, Duc L. V, Dao T. T, Hai N. T. Triolein from Coix Lacryma-Jobi Induces Cell Cycle Arrest through P53/P21 Signaling Pathway. Biomed Pharmacol J 2016;9(2).

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Manuscript received on :April 10, 2016
Manuscript accepted on :June 25, 2016
Published online on: 12-08-2016

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Tran Thi Hien^1,5*, Do Thi Ha³, Do Minh Truong², Loi Vu Duc⁴, Trong Tuan Dao³ and Nguyen Thanh Hai⁴

¹Department of pharmacy, Thai Binh medical and pharmacy University, Vietnam.

²College of Sciences, Thai Nguyen University, Vietnam.

³National Institute of Medicinal Materials, 3B Quangtrung, Hanoi, Vietnam.

⁴School of Medicine and Pharmacy, Hanoi National University, 144 Xuan Thuy street, Cau Giay, Hanoi, Vietnam.

⁵Faculty of medicine, Lund University, Sweden.

*Corresponding Author E-mail: nguyenthanh.haihnu@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/967

Abstract

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.

Keywords

Triolein; Coix lacryma-jobi; MCF-7 cells; apoptosis; DNA synthesis

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 anti-cancer 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.

Experimental

Chemicals and Cell Culture

Most chemicals were purchased from Sigma (St. Louis, MO). p53, p21, Bax, p27, Cyclin D1, phospho-Rb, total-Rb, E2F1, anti-mouse and anti-rabbit were obtained from Cell Signaling. MCF-7 cells were obtained from the American type culture collection (Bethesda, MD) and incubated at 37 °C in a 5% CO₂/95% air atmosphere in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.

Western Blot Analysis

After protein determination using BCA protein assay kit (Pierce, Rockford, IL), aliquots of the lysates (10-30 μg of protein) were boiled for 5 min and electrophoresed on 10% SDS-polyacrylamide 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, β-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. ma-yuen 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.

Sample Preparation for Bioassays

Each sample (adlay aril (CL1), adlay brain (CL2) and adlay seed kernel (CL3), 50 g) was extracted with 96% EtOH (3 × 0.5 L) and then evaporated to dryness under vacuum at 40 ºC to obtain the 96 % CL1 (2.81 %; wt/wt), CL2 (11.64 %; wt/wt), CL3 (11.46 %; wt/wt). Adlay aril (the CL2 extract (5,32 g) was suspended in 100 mL H₂O, followed by sequential partitioning with hexane, ethyl acetate, and water, to give CL2H, 2.44 g), ethyl-acetate-soluble (CL2E, 0.1 g), water-soluble (CL2W, 2.78 g) extract. The extracts were stored at -20 ºC until tested.

Extraction and Isolation of Compounds from the Significant Bioactive Fraction

Adlay aril powder (5,85 kg) was extracted three times with 96% ethanol at boiling temperature (20l × 3 h × 3 times). The combined filtrates were evaporated to dryness in vacuo at 40 ^oC. The residue (681,1 g) was stirred in 1L MeOH and then suspended in 2 l H2O, followed by sequential partitioning with hexane (Hx), ethyl acetate (EtOAc), and water (W), to give hexane-soluble, ethyl-acetate-soluble, and water-soluble fractions. Hexan-soluble fraction (280 g) was then subjected to silica gel (230–400 mesh) column chromatography with successive elution by a Hex/EtOAc gradient (100:0, 70:1,…, 1:10,…) and MeOH solvent system. Subfractions with the same thin-layer chromatography (TLC) patterns were combined into one fraction; thus 9 fractions were obtained (AHH1A~AHH1I). Rechromatography of the first bioactive subfraction AHH1A (80 g) on silica gel columns using Hx-EtOAc gradient (100:0, 50:1, 30:1, 10:1) to yield compound 1 (3,00 g). Fraction T1CL1F (13,6 g) was subjected to silica gel (230–400 mesh) column chromatography, eluted with dichloromethan (DCM) to obtain five sub-fractions T1CL1F1~ T1CL1F5. The fraction T1CL1F5 (1,5 g) was purification by column chromatography with a gradient solvent system DCM-MeOH (50:1, 30:1, 20:1, 10:1) obtained compound 2 (88,8 mg). Compounds 3 (3,0 g) and 4 (190.8 mg)  were obtained by further purification of the fraction T1CL1F4 (13,6 g) using normal phase chromatography with a gradient solvent system DCM-MeOH (100:1~20:1) and reverse phase chromatography YMC using a solvent system aceton-H₂O (4:1).

CLH1

oil; APCI-MS: m/z: 886,8 [M+H]+ (C57H104O6, M = 885,45). 1H-NMR (500 MHz, CDCl3) δH: 5.34 (6H, m, H-8׳, H-9׳, H-8״, H-9״, H-8״׳, H-9״׳), 5.26 (1H, t, H-2), 4.29 (2H, dd, J = 4.0, 12.0 Hz, H-1), 4.15 (2H, dd,  J = 6.0, 12.0 Hz, H-3), 2.31 (6H, t, H-1׳, H-1״, H-1״׳), 2.0 (12H, H-7׳, H-10׳, H-7״, H-10״, H-7״׳, H-10״׳), 1.60 (6H, m, H-2׳, H-2״, H-2״׳), 1.30 (60H, CH2), 0.88 (9H, t, H-17׳, H-17״, H-17״׳). 13C-NMR (500 MHz, CDCl₃) δC: 173.3 (C-1׳, C-1״׳), 172.8 (C-1״), 130.0 (C-8׳, C-9׳, C-8״׳, C-9״׳), 129.7 (C-8״, C-9״), 68.9 (C-2), 62.1 (C-1, C-2), 14.1 (C-17׳, C-17״, C-17״׳), 22.7-34.2 (CH2). ¹H and ¹³C-NMR data were consistent with literature values of triolein. ^[11]

CLH3

A colorless powder; M.p. 290 – 294⁰C; ¹H-NMR (400 MHz, Pyridin-d₅) δ: 5.35 (1H, brd, H-6); 3.53 (1H, m, H-3); 1.01 (3H, s, H-19); 0.69 (3H, s, H-20); 0.92 (3H, d, J = 6,8 Hz, H-21); 0.83 (3H, d, J = 1.6 Hz, H-26); 0.81 (3H, brs, H-27); 0.85 (3H, brs, H-29); Glu: 5.07 (1H, d, J = 7.2 Hz, H-1′); 4.59 (1H, dd, J = 2.4; 11.7 Hz, H-6a’); 4.43 (1H, dd, J = 5.7; 11.7 Hz, H-6b’); 4.30 (2H, m, H-3′, 5′); 4.06 (1H, m, H-4′); 3.97 (1H, m, H-2′). ¹³C-NMR (100 MHz, pyridin-d₅) δ: 37.2 (C-1); 31.6 (C-2); 71.7 (C-3); 42.2 (C-4); 140.7 (C-5); 121.7 (C-6); 31.8 (C-7); 31.9 (C-8); 50.0 (C-9); 36.4 (C-10); 21.0 (C-11); 39.7 (C-12); 42.2 (C-13); 56.7 (C-14); 24.3 (C-15); 28.2 (C-16); 55.9 (C-17); 11.8 (C-18); 19.4 (C-19); 36.1 (C-20); 18.7 (C-21); 33.9 (C-22); 25.9 (C-23); 45.8 (C-24); 29.1 (C-25); 19.8 (C-26); 18.9 (C-27); 23.0 (C-28); 11.0 (C-29); Glc: 102.9 (C-1′); 79.0 (C-3′); 78.9 (C-5′); 75.7 (C-2′); 72.1 (C-4′); 63.3 (C-6′). ¹H and ¹³C-NMR data were consistent with literature values of daucosterol. ^[12]

CLH4

A viscous oil. ACPI pos m/z 842.3 [M+H]⁺ consistent with the chemical formula C₅₃H₉₂O₇; ESI neg 161.0 [M-C₁₈H₃₃O-C₂₉H₄₉O-H]^– consistent with a glucose unit C₆H₁₀O₅^– (162.05), m/z 413.3 [M-C₁₈H₃₃O-C₆H₁₀O₅]^– consistent with a sitosteryl unit (413.4); m/z 139.2 [M-C₄₃H₇₃O₇]⁺ consistent with an alkene unit (the =C-C- bond fragmentation at position 8 and 9 broken) (C₁₀H₁₉) of an oleyl unit_. ¹H-NMR (500 MHz, CDCl₃) δ_H: 5.36 (2H, m, -CH=CH-), 5.34 (1H, m, H-6), 4.44 (1H, dd, J = 5.5, 12.0 Hz, H-6’a), 4.38 (1H, d, J = 7.0 Hz, H-1′), 4.25 (1H, dd, J = 2.0, 12.0 Hz, H-6’b), 3.56 (1H, m, H-5′), 3.54 (1H, dd, 8.0, 8.5 Hz, H-5′), 3.46 (1H, m, H-6’a), 3.44 (1H, m, H-4′), 3.38 (1H, dd, J = 9.0, 11.5 Hz, H-2′), 3.55 (2H, dd, J = 6.0, 11.5 Hz, H-H3b), 2.35 (2H, m, H-2″), 1.28 (30H, m, (CH₂)₁₅, 1.09 (3H, s, H-19), 0.98 (3H, d, J = 6.0 Hz, Me-18”), 0.95 (3H, d, J = 6.8 Hz, Me-21), 0.89 (3H, t, J = 7.0 Hz, Me-29), 0.88 (3H, d, J = 7.0 Hz, Me-26), 0.68 (3H, s, Me-18). ¹³C-NMR (500 MHz, CDCl₃) δ_C: 174.6 (C-1’׳), olefinic carbons: 140.3 (C-5), 130.2 (C-9’׳), 130.0 (C-10”), 121.1 (C-6), 73.5 (C-3), 56.8 (C-14), 56.1 (C-17), 50.2 (C-9), 45.8 (C-23), 42.4 (C-13), 39.8 (C-12), 37,3 (C-1), 36,7 (C-10), 36,1 (C-20), 34,2 (C-22), 31,9 (C-8), 31,8 (C-7), 31,5 (C-2), 29,1 (C-25), 28,3 (C-16), 25,2 (C-23), 24,3 (C-15), 23,1 (C-28), 21,0 (C-11), 19,4 (C-26), 19,3 (C-19), 19.0 (C-27), 18,8 (C-21), 11,8 (C-18), 11,0 (C-29); Glc: 100.2 (C-1′), 79.6 (C-5′), 76.0 (C-3′), 73.9 (C-2′), 70.2 (C-4′), 63.3 (C-6′), 174.6 (C-1″), 33.9 (C-2″), 31.8 (C-8″), 31.7 (C-10″), 29.2-29.7 (-CH₂-, C-4″, 5″, 6″, 7″, 12″, 13″, 14″, 15″, 16″), 25.7 (C-3″), 22.7 (C-17″), 14.1 (C-18″). ¹H and ¹³C-NMR data were consistent with literature values of sitosterol-3-O-6-oleoyl-b-D-glucopyranoside. ^[13]

CLH5

Colorless gum; APCI-MS pos: m/z 355.2 [M+H]⁺ and ESI pos: m/z 394.1 [M+Na]⁺ consistent with molecular formular C₂₁H₃₈O₄. ¹H-NMR (500 MHz, CDCl₃) δ_H: 5.34 (2H, m, H-9׳, H-12׳), 4.08-4.11 (2H, dd, J = 6.0, 11.0 Hz, H-1a, H-1b), 3.87 (1H, m, H-2), 3.63 (1H, dd, 4.5, 11.5 Hz, H-3a),  3.55 (2H, dd,  J = 6.0, 11.5 Hz, H-H3b), 2.77 (2H, dd, J =  6.5, 7.0 Hz, H-11′), 2.34 (2H, t, J = 7.5 Hz, H-2׳), 2.00 (4H, H-8׳, H-13׳), 1.62 (2H, m, H-3׳), 1.31 (20H, 10CH₂), 0.89 (3H, t, H-18׳). ¹³C-NMR (500 MHz, CDCl₃) δ_C: 174.6 (C-1׳), olefinic carbons: 130.4 (C-9׳), 130.2 (C-13′), 129.9 (C-10׳), 128.2 (C-12′), 70.1 (C-2), 65.3 (C-1), 63.4 (C-3), 34.2 (C-11′), 32.0 (C-8′, 14′), 31.7 (C-2′), 29.2-29.8 (7 CH₂), 27.3 (C-4′), 25.0 (C-3′), 14.1 (C-18׳). ¹H and ¹³C-NMR data were consistent with literature values of 2,3-dihydroxypropyl octadecadienoate (1-monolinolein). ^[14]

P-values were calculated by Student’s t-test or one-way analysis of variance followed by Bonferroni post-hoc testing using GraphPad Prism 5 (GraphPad Software Inc.). P < 0.05 was considered statistically significant. Data are expressed as mean ± S.E.M. *, p<0.05; **, p<0.01; ***, p<0.001.

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. 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 μM) compound most significant increase p53 protein expression level in MCF-7 cells (Fig 1C).

Figure 1: Trioelin activates p53 in MCF-7 cells (A) Effect of p53 by 96% ethanol extraction from Coix lacryma-jobi (adlay aril, adlay brain and adlay seed kernel) at 1mg/ml. Cells were incubated with different extractions for 24h. Cells were then harvested and analysis by western blot analysis. (B) Effect of three fraction: n-hexan, ethyl acetate and water from adlay aril extraction on p53 protein expression. (C) Effect of four compounds isolated from n-hexan fraction on p53 protein expression. (n=3)   Click here to View figure

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).

Figure 2: (A) Effect of trioelin on p53, p27, Bax and p21 protein expression in dose-dependent manner in MCF-7 cells. Cells were treated with various concentrations of triolein and then cells were harvested to subject to western blot analysis (n=3). (B) Effect of triolein in dose-dependent on Cyclin D1, phospho-Rb, Rb and E2F1 protein expression (n=3). (C) Cell viability after treatment of triolein in MCF-7 cells by MTT assay (n=4). (D) Effect of triolein on DNA synthesis by BrdU assay (n=4). *, p<0.05; **, p<0.01; ***, p<0.001 relative to control.   Click here to View figure

Figure 3: Molecular mechanism of triolein in cell cycle in MCF-7 cells.   Click here to View figure

Cyclin D1 is an important regulator in G1/S phase of cell cycle, cell proliferation and apoptosis. E2F1 was reported to induce apoptosis result from the inhibition of Rb. By using western blot analysis, Fig. 2B presented Cyclin D, phospho-Rb and E2F1 were significant suppressed by triolein in dose-dependent manner (10 μM-300 μM). Moreover, we observed the cell cytotoxicity by triolein at 300 μM. p53/p21 plays the important key event in the control of cell cycle, apoptosis and cellular stability (ref). During DNA damage process, wild-typep53 is expressed to increase cell cycle arrest at several checkpoints to repair the damaged DNA. Our study demonstrated that triolein inhibited DNA synthesis in dose-dependent mamner. These data suggest triolein isolated from Coix lacryma-jobi induces cell cycle arrest through p53/p21 signaling pathway

Declaration of Interest

These authors have declared that there is no conflict of interest. This research is funded by Hanoi National University under Tay Bac Program Grant number KHCN-TB.05C/13-18

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