Î³-Tocotrienol-Inhibited Cell Proliferation of Human Gastric Cancer by

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r-Tocotrienol inhibits cell proliferation of human gastric cancer by regulating nuclear factor-kB activity Wen-Guang Sun, Rui-Peng Song, Yong Wang, Sheng Ge, Ya-Hui Zhang, Hai-Xia Wang, Jiaren Liu, and Lian-Xin Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05832 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Journal of Agricultural and Food Chemistry

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-Tocotrienol inhibits cell proliferation of human gastric cancer by regulating nuclear factor-B activity Wen-Guang Sun†, #, Rui-Peng Song‡, #, Yong Wang&, #, Ya-Hui Zhang†, Hai-Xia Wang†, Sheng Ge†, Jia-Ren Liu@,$, Lian-Xin Liu‡,$,* †

International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai JiaoTong University, 910 Hengshan Rd, Shanghai 200030, People's Republic of China, E-mail: [email protected] @

Department of Clinical Laboratory, the Forth Affiliated Hospital of Harbin Medical University, 37 YiYuan Street, NanGang District, Harbin 150001, People's Republic of China, E-mail: [email protected] ‡

Department of General Surgery, the First Affiliated Hospital of University of Science and Technology, 17 LuJiang Road, LuYang District, HeFei 230031, People's Republic of China &

Harbin Center for Disease Control and Prevention, 30 WeiXing Road, DaoWai District, Harbin 150056, People’s Republic of China

#

Co-first authors

$

Co-corresponding authors

* Corresponding author Professor Lian-Xin Liu at the Department of General Surgery, the First Affiliated Hospital of University of Science and Technology, 17 LuJiang Road, LuYang District, HeFei 230031, People's Republic of China E-mail: [email protected] Subtitle: -Tocotrienol inhibits NF-B activity

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Abstract

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-Tocotrienol (-T3) exhibits the activity of anti-cancer via regulating cell signaling

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pathways. Nuclear factor-B (NF-B), one of crucial pro-inflammatory factors,

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involved in the regulation of cell proliferation, apoptosis, invasion and migration of

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tumor. In the present study, NF-B activity inhibited by -T3 was investigated in

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gastric cancer cells. Cell proliferation, NF-B activity, active protein phosphatase

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type 2A (PP2A), and ataxia-telangiectasia mutated (ATM) protein were explored

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using MTT, methylene blue, ELISA, malachite green, luciferase and Western blotting

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assays. The effects of -T3 on tumor growth, the expression of NF-B and PP2A

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proteins were also further examined by implanting human gastric cancer cells in a

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BALB/c nude mouse model. The results showed that -T3 significantly inhibited the

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cell proliferation and attenuated the NF-B activity in vitro and in vivo. -T3

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dramatically increased PP2A activity and protein expression, which suppressed ATM

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phosphorylation and its translocation to the cytoplasm in gastric cancer cells. Thus,

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our findings may provide mechanistic insight into effects of -T3 on the regulation of

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NF-B activity by a PP2A-dependent mechanism and suggest that PP2A may serve as

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a molecular target for a potential chemopreventive agent.

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Key words: -Tocotrienol, NF-B activity, ATM, PP2A


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Introduction

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Gastric cancer is a common cancer in China. It has the second highest incidence and

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the third highest mortality among cancer patients in China1. It has been estimated that

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new gastric cancer cases and cancer deaths were 312 and 221 per million Chinese

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people in 2011, respectively1. According to the World Cancer Research Foundation

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2007 report2, an estimated 35% of the cancer incidence worldwide could be

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attributable to lifestyle factors such as food and physical activity. Increasing evidence

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has demonstrated that nutrients and non-nutrients from foods such as vitamins and

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phytochemicals have a protective effect against cancer3-5. As a subgroup of vitamin E,

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tocotrienols include 4 different forms, i.e., α-, β-, γ- and δ-forms. Tocotrienols are

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commonly derived from many dietary sources including rice bran, palm oil and wheat

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germ. In particular, -tocotrienol (-T3) is one of the most abundant forms of

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tocotrienols in these foods6. The available data have shown that tocotrienols and a

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tocotrienol-rich fraction (TRF) had the activity of anti-cancer in vitro and in vivo7-9.

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-T3 has anti-proliferative and apoptotic attribution associated with the inhibition of

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transcription factors of the nuclear factor B (NF-B) activity in MDA-MB-231 and

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MCF-7 cells10. In our previous studies, -T3 has shown potent anti-proliferative

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ability and apoptotic induction in the human gastric cancer SGC-7901 cells through a

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mitochondria-dependent pathway4. It also inhibits the proliferation of human colon

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carcinoma HT-29 and SW620 cells and induces paraptosis-like cell death through

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Wnt-dependent pathways9, 11, 12. In addition, -T3 inhibits the invasion and migration

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of SGC-7901 cells by down-regulating the expression of MMP-2 and MMP-913. -T3



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also inhibits angiogenesis in human umbilical vein endothelial cells that was induced

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by the conditioned medium from cancer cells7. These results suggested that -T3 has

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potential anti-cancerous and anti-metastatic activity in cancer cells.

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NF-B, one of crucial pro-inflammatory factors, plays a pivotal role in the important

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physiological function including immune response, inflammation, cell proliferation

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and survival, cell invasion and development14. NF-B activity is found in human

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chronic diseases including cancers15. NF-B is constitutively activated in human

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gastric cancer tissues and is associated with tumor progression16. NF-B activity also

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suppresses cell apoptosis and causes potential resistance in cancer cells induced by

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chemotherapeutic agents17, 18. Many potential food-derived chemotherapeutic agents

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could inhibit NF-B activity in cancer cells19, 20. Thus, inhibition of NF-B activity

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has become one of the major targets for the development of chemotherapeutic

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agents21. In a study, -T3 inhibited the proliferation of human cancer cells via

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inactivation of NF-B22. However, its exact mechanism is still fully understood. In

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previous studies, ataxia telangiectasia mutated (ATM), one of nuclear factors, can

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induce activation of NF-B, in which is response to DNA damage23. ATM plays an

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important role in NF-B activity during cell stress23. Once ATM is activated, it

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directly phosphorylates NF-B-essential modulator to activate NF-B activity23. This

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procession is also negatively regulated by protein phosphatase 2A (PP2A)23,

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However, whether γ-T3 inhibited NF-B activity by ATM mediating pathways, it is

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still unclear. Thus, the objectives of this study were to determine how -T3 inhibited

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the proliferation of cancer cells via inhibiting the NF-B signaling cascade in gastric


24.

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cancer cells.

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Materials and Methods

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Reagents and treatment

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-Tocotrienol (-T3) (97%) was purchased from Hygeia Industries Inc (Wilmette, IL).

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3-[4, 5-Dimethylthiazol-2-yl]x-2,5-diphenyl tetrazolium bromide (MTT), 4′,6-

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diamidino-2-phenylindole (DAPI), HEPES, okadaic acid (OA), methylene blue, (-)

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isoproterenol hydrochloride (ISO) and tumor necrosis factor-alpha (TNF-α) were

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purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Antibodies to

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phosphorylated-ATM(S1981), ATM, phosphorylated-p65 (S536 and S311), p65,

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PP2A-A, B, and C, β-actin, GAPDH and anti-rabbit or anti-mouse secondary

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antibodies conjugated with horseradish peroxidase were bought from Cell Signaling

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Technology (Danver, MA). The phosphate detection kit (DY996) was bought from

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R&D Systems (Minneapolis, MN). NF-B p65 siRNA was bought from Cell

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Signaling Technology. All reagents were of analytical grade unless otherwise noted.

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The treatment time and doses of-T3 were chosen according to our previous studies8,

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9, 11, 13, 25.

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and stored at −20 °C. For the cell toxicity tests25, the cells were treated with 15, 30, 45

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or 60 μmol/L of -T3 for 24 hours or 30 μmol/L of -T3 for 2, 4, 6, 12 or 24 hours.

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Equal volumes of the ethanol vehicle (0.1%, v/v) were included in the experimental

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and control groups. Each experiment was performed in triplicate.

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Animal Care and Treatment

Briefly, -T3 was dissolved in absolute ethanol to make a 1.0 mol/L solution



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The protocols for all experimental procedures and animal care were approved by the

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Committee for Ethics of Harbin Medical University (Harbin, China). Seven-week-old

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BALB/c nu/nu female mice were purchased from the Shanghai SLAC Experimental

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Animal Company (Shanghai, China). This model was used according our previous

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studies with a little modification12, 26. Briefly, the mice were housed in a room with a

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12 h light/12 h dark cycle and adapted the surroundings and the sterile basic

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supplementary diet for 1 week. Mice were randomly assigned to two groups (n =

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5/group). -T3 was dissolved in corn oil. The experimental or control groups were

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given -T3 at a dose of 25 mg/kg body weight (b.w.) or an equal volume of corn oil,

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respectively, by intraperitoneal (ip) daily for one week. The mice in the experimental

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or control groups were injected with human gastric cancer SGC-7901 cells (3.0 × 106)

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into the right-side subcutaneous gluteal region to generate the human gastric cancer

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xenograft. Mice were then continuously treated with -T3 or control oil daily for 25

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days. The mice were checked daily to monitor their breathing and movements as

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reflections of their health status. The body weight of mice was measured every 3 days.

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On the 7th d following the xenograft implantation, the volumes of the tumors were

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measured every 3 days using a caliper to measure the length and width of tumor and

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calculate the tumor volume27. The mice were euthanized if they became moribund

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during the experiment and were necropsied if they died prior to the terminal sacrifice

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time. At the termination of the experiments, the tumor tissues were removed from the

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mice under anesthesia (50 mg/kg pentobarbital, ip). Part of tumor tissues were fixed

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in 10% paraformaldehyde and embedded in paraffin for immunohistochemical


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analysis26, 28. The remaining tissues were snap frozen in liquid nitrogen and stored at

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−80 °C for Western blotting analysis.

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Cell culture

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Human gastric cancer SGC-7901 and MGC-803 cell lines was purchased from the

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Cancer Institute of Chinese Academy of Medical Science (Beijing, China). SGC-7901

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cells and MGC-803 cells were cultured at 37 °C in a 5% CO2 incubator in 100-mm

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culture dishes with RPMI 1640 media (Gibco) containing 2 mmol/L of L-glutamine,

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10 mM of HEPES and 2 g/L of sodium bicarbonate (Gibco) and supplemented with

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10% fetal bovine serum (FBS) and antibiotics (2AA) (Gibco). On reaching

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approximately 70% confluence, the cells were trypsinized using a mixture of 0.25%

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trypsin and EDTA. Cells in the logarithmic phase of growth were used in all

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experiments.

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Cell viability

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The cell viability of SGC-7901 and MGC-803 cells treated with various

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concentrations of -T3 was determined using MTT and methylene blue (MB) assays4,

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29, 30.

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the culture medium was changed to fresh complete culture medium containing 0, 15,

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30, 45 or 60 μmol/L of -T3. The control group was treated with 100% ethanol as a

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vehicle control (0.1% v/v). For MTT assay, after incubation for 2, 4, 6, 12 or 24 hours,

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20 μL of MTT (2.0 mg/mL in phosphate buffered saline (PBS)) was added to each

The cells were seeded in 96-well microtiter plates. After incubation overnight,



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well, and the cells were incubated for 4 additional hours at 37 °C. After incubation for

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4 hours, the medium was carefully discarded and then 200 μL of dimethyl sulfoxide

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(DMSO) was added to each well. After 15 minutes of shaking at room temperature,

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the absorbance values were measured at a wavelength of 570 nm in a Microplate

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Reader (ELX800, BIO-TEK Instruments INC). For MB assay, after incubation for 2, 4,

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6, 12 or 24 hours, the medium in each well was discarded, 50 L of MB solution was

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added each well, continued to culture cells for another 1 hour at 37 °C and followed

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by our previous study29,

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proliferation where the vehicle-treated cells were taken as 100% viable.

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Phosphate determination in medium

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Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase,

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which is involved in the dephosphorylation of cellular protein in the regulation of cell

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signal transduction31. To determine the released phosphate (Pi) reflecting to PP2A

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activity, the total Pi was measured in the medium. Pi release to medium was

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quantitated by Malachite Green assay. Briefly, the cells were seeded in 60 mm-dishes

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overnight. The cells were washed several times using phosphate-free RPMI 1640

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medium and then serum-free phosphate-free medium containing 30 µmol/L of -T3

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for 2, 4, 6, 12 and 24 hours or various concentrations of -T3 for 24 hours. The culture

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supernatant in each treatment was collected to determine the concentrations of Pi. The

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quantity of Pi in the media was measured by a phosphate detection kit following the

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manufacturer’s instruction. The results were expressed as nmoles of Pi.

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The cell viability was assessed as percentage for cell


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Protein phosphatase 2A (PP2A) activity assay

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PP2A activity was measured using a Malachite Green phosphatase kit as described a

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previous study23. Briefly, after being treated with -T3, the cells were harvested and

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centrifuged at 1000 rpm for 5 minutes at 4 °C. The PP2A activity in the cell lysates

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was measured using an active PP2A kit (DYC3309, R&D Systems) according to the

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manufacturer’s instruction. PP2A activity was expressed as phosphate (nmoles)/mg

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total protein. The positive controls included okadaic acid (OA, 100 nmol/L) and (-)

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isoproterenol hydrochloride (ISO, 1.0 µmol/L).

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Cell transfection and NF-κB reporter assays

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SGC-7901 cells were transfected and carried out using FuGENE HD (Roche Applied

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Science, Indianapolis, IN) according to the manufacturer’s instruction23. After

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transfected with pNF-B-Luc (pNRE; Clontech) for 72 hours, the cells were treated

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with 20 ng/mL of tumor necrosis factor  (TNF-α) at 37 °C in 5% CO2 for 30 minutes

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with or without -T3. The NF-B activity was measured in the cell lysates using the

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Dual-Glo Luciferase assay system (Promega Biosciences, Inc. San Luis Obispo, CA)

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and assessed with a Luminometer (Turner Biosystems). The NF-B activity was

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expressed as firefly (LUC)/mg total protein.

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Immunofluorescence

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NF-B is primarily activated via the phosphorylated ATM (p-ATM) translocation

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from nuclear to cytoplasm23. To measure the translocation of p65 (p-p65, NF-B) and



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phosphorylated-ATM (p-ATM, S1981), SGC-7901 cells were seeded in 4-well glass

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chamber slides (Nalgene Nunc International) and grown to 70% confluence. The cells

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were exposed to -T3 for 12 hours and then treated with TNF-α, an antagonist of

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PP2A-okadaic acid (OA) or agonist of PP2A-(-) isoproterenol hydrochloride (ISO).

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The cells were fixed in 1% paraformaldehyde overnight at 4 °C and permeabilized

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with pre-chilled PBS containing 0.2% Triton X-100 and 1% bovine serum albumin

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(BSA). The cells were incubated with rabbit anti-p-p65 (1:100; Rockland

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Immunochemicals), or anti-p-ATM (S1981) in 1% normal goat serum in PBS

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overnight followed by FITC-anti-rabbit or FITC-anti-mouse (1:100, Santa Cruz

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Biotechnology) for 90 minutes. Cell images were captured using a fluorescence

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microscope.

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Immunoprecipitation

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After the treatments, the total protein was extracted from SGC-7901 cells using a cell

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lysis buffer (Cell Signaling Technology) containing a protease inhibitor mixture

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(Roche Applied Science), phenylmethylsulfonyl fluoride (1 mol/L) and sodium

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orthovanadate (1 mol/L). The concentrations of total protein were measured using

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the BCA assay (Thermo Scientific, San Diego, CA). After preclearing with 60 μL of

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immobilized protein A/G beads (Thermo Scientific, Rockford, IL) for 2 hours, rabbit

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anti-ATM antibody (Cell Signaling Technology) and normal rabbit serum

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(Sigma-Aldrich) were added to the lysate and gently shaken overnight. Sixty

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microliters of immobilized protein A/G beads were then added to each tube, incubated


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for 4 hours at 4 °C, and centrifuged at 6000 rpm for 5 minutes. The complex-bound

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gel was then collected and washed 4 times with cell lysis buffer. Finally, 70 μL of

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SDS loading buffer was added to each tube, boiled at 95 °C for 5 minutes, centrifuged

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at 14,000 rpm for 30 seconds, loaded onto a 10% gel, and subjected to the Western

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blotting steps described below.

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Western blotting

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The total protein was isolated from SGC-7901 cells or tumor tissues using cell lysis

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buffer (Cell Signaling Technology) containing protease inhibitors and phosphatase

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inhibitor cocktails (Sigma-Aldrich Chemical Co.). The methods for total protein

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determination and gel electrophoresis were performed as described in our previous

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study29,

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rabbit anti-p-p65(S536 or S311), rabbit anti-p65, rabbit anti-PP2A-A, B, and C,

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mouse anti-p-ATM(S1981), rabbit anti-ATM, mouse anti-GAPDH or rabbit

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anti-β-actin overnight at 4 °C with slow shaking. Subsequently, the blots were

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incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary

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antibodies at room temperature for 2 hours. The protein bands in membranes were

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visualized using enhanced chemiluminescence (Thermo Scientific). The intensity of

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target protein bands was measured using a FluorChem Imaging System (Alpha

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Innotech).

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Statistical analysis

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The membranes were incubated with the primary antibodies including



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The data are expressed as the means ± standard deviation (S.D.). The data analyses

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were performed using SPSS for Windows Version 22.0 (SPSS Inc., Chicago, IL). The

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differences were analyzed using Student’s t-test for the cell viability, tumor weight,

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and tumor volume, and One-Way ANOVA tests with the Bonferroni post hoc

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multiple comparisons were used to assess the levels of Pi release, PP2A and NF-B

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activity as well as protein expression in the various groups. Statistical significance

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was set at P < 0.05, and all P values were adjusted for multiple comparisons.

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Results

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-T3 inhibited the growth of SGC-7901 cells

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As shown in Figure 1, -tocotrienol (-T3) significantly inhibited the proliferation of

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SGC-7901 cells and MGC-803 cells (Figures 1) in a dose-dependent and

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time-dependent manner (p

Î³-Tocotrienol-Inhibited Cell Proliferation of Human Gastric Cancer by

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