Inhibition of gastric cancer cell growth, invasion and metastasis by tocotrienolic amide

Purpose: To investigate the effect of tocotrienolic amide on gastric cancer (GC) cell growth and metastasis, and the underlying mechanism of action. Methods: Gastric cancer (GC) cell lines MKN28 and NCI-N87 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin solution at 37  ̊C in a humidified atmosphere of 5 % CO2 and 95 % air. Cell invasion and migration were determined using Transwell and wound healing assays, respectively. Realtime quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used for the determination of changes in the levels of expression of Snail, E-cadherin, vimentin, and microRNA-1955p (miR-195-5p). In vivo tumor growth inhibition was determined 45 days after establishment of GC xenografts in nude mice. Results: Treatment of MKN28 and NCI-N87 cells with tocotrienolic amide significantly and dosedependently reduced their invasiveness and migratory capacity (p < 0.05). It also significantly and dosedependently downregulated the mRNA and protein expressions of Snail and vimentin, but significantly upregulated E-cadherin expression (p < 0.05). The mRNA expression of miR-195-5p was significantly and dose-dependently upregulated in MKN28 and NCI-N87 cells treated with tocotrienolic amide, but was downregulated after transfection with miR-195-5p inhibitor (p < 0.05). Transfection of MKN28 and NCI-N87 cells with miR-195-5p inhibitor significantly and dose-dependently upregulated mRNA and protein expressions of Snail (p < 0.05). Moreover, treatment of GC mice with TCTA led to significant and dose-dependent reduction in tumor weight and volume (p < 0.05). Conclusion: These results suggest that tocotrienolic amide inhibits the growth and metastasis of GC cells by directly targeting Snail and vimentin genes, and thus can potentially be developed for the management of gastric cancer.


INTRODUCTION
Gastric cancer (GC), a malignant tumor that develops in the inner lining of the stomach, is the fifth most common cancer worldwide. It is one of the leading causes of cancer-related death globally [1]. The incidence of GC is higher in developing countries, and accounts for approximately 70 % of total diagnosed cases [2]. Less than 5 % of GC occur in people under 40 years of age, and most of these patients are in the age range of 30 to 39 years, while the others are aged 20 to 29 years. China alone accounts for 25 % of all GC-related deaths. This disease has a quiet onset and progresses slowly, thereby making its early detection difficult since early symptoms are usually not obvious. Gastric cancer (GC) is characterized by poor prognosis, with 5-year survival less than 40 % [3]. The pathogenesis of GC is complex, but it is thought to involve an interplay of factors such as diet, environment, genetics, lifestyle and H. pylori infection [2]. Resistance to traditional medicines, high metastasis of gastric tumor cells and recurrence are mainly responsible for poor prognosis of GC, despite availability of improved techniques [4]. Therefore, an understanding of the molecular pathways involved and discovery of efficient therapeutic agents are of great importance for GC treatment.
MicroRNAs (miRNAs) block gene translation by interaction with 3'untranslated portions of mRNAs [5]. Epithelial to mesenchymal transition (EMT) as well as many other processes such as cell migration and proliferation are influenced by miRNAs [6]. Several studies on the molecular mechanisms involved in different kinds of cancers have revealed that the expression of miR-195-5p is dysregulated [7]. During oncogenesis, a process in which normal cells transform into carcinoma cells in situ, EMT plays a crucial role [8]. Moreover, carcinoma cells invade and migrate in situ into the blood and lymph vessels, leading to metastasis to remote organs [8]. The EMT process and cell metastasis are promoted by Snail, a zinc-finger protein transcription factor, through a shift in equilibrium between E-cadherin and N-cadherin [9]. A previous study reported the clinical importance of Snail in GC [9]. The present study investigated the inhibitory effect of tocotrienolic amide on the migration and invasion of gastric cancer cells.

EXPERIMENTAL Materials
Bicinchoninic acid (BCA) protein assay kit was a product of Biyuntian Biotechnology Institute. Rabbit anti-human Snail, vimentin, E-cadherin, β-actin and horseradish peroxidase-labeled goat anti-rabbit IgG were bought from Abcam Inc. (UK). Trizol reagent was obtained from Thermo Fisher Scientific, Inc. Polymerase chain reaction (PCR) primers were purchased from Qiagen (USA), while SYBR Premix Ex Tap was obtained from Toyobo Co. Ltd. (Japan). Enhanced chemiluminescence system was a product of Life Sciences, UK.

Cell lines and culture conditions
Gastric cancer cell lines (MKN28 and NCI-N87) were obtained from Biochemistry and Cell Biology Institute, Shanghai, China. The cells were cultured in DMEM supplemented with 10 % FBS and 1 % penicillin/streptomycin solution at 37 °C for 24 h in a humidified atmosphere of 5 % CO2 and 95 % air until they attained 80 % confluency. The medium was replaced with fresh one every two days. After 1 week of incubation, the adherent confluent cells were trypsinized with 0.25 % trypsin-EDTA (2 mL), cultured again, and passaged for later use. Cells in logarithmic growth phase were selected and used in this study.

In vitro cell migration assay (scratch test)
Gastric cancer cells (MKN28 and NCI-N87) in logarithmic growth phase were seeded in 24-well plates at a density of 1 × 10 6 cells/mL and cultured in DMEM for 24 h. Tocotrienolic amide (2 -10 mΜ) was added to the cells and incubated for 24 h. After achieving 80 % confluency, scratches were made on cell monolayers. The cells were further cultured for 24 h after washing thrice with serum-free medium, and then observed and photographed.

In vitro cell invasion assay
The degrees of invasiveness of MKN28 and NCI-N87 cells were determined using Transwell invasion assay. The cells were placed at a density of 1 × 10 6 cells/mL in 24-well plates containing serum-free DMEM in Transwell chamber. The DMEM (800 μL) mixed with 10 % FBS was added the basolateral chamber, whereas 200 μL of the cell suspension was added to the upper chamber. Incubation with tocotrienolic amide at 2, 5 and 10 μM for 24 h was followed by removal of the medium. The migrated cells were fixed for 35 min with 5 % glutaraldehyde, followed by staining with crystal violet for 25 min, photographing and counted under an inverted microscope. Cell invasion was determined by counting the invaded cells in five randomly chosen fields under the microscope.

Western blotting
The MKN28 and NCI-N87 cells were treated with varied concentrations of tocotrienolic amide (2 -10 mΜ) for 24 h. Changes in expression levels of Snail, E-cadherin and vimentin were determined using Western blotting. The cells were washed with phosphate-buffered saline (PBS) and lysed with ice-cold radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitor (1 mM phenylmethanesulfonyl fluoride). The resultant lysate was centrifuged at 12, 000 rpm for 25 min at 4 °C, and the protein concentration of the supernatant was determined using BCA protein assay kit.
A portion of total cell protein (40 μg) from each sample was separated on 12 % sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and transferred to a fixed polyvinylidene fluoride membrane at 110 V and 90 ° C for 90 min. Subsequently, the membrane was incubated with non-fat milk powder (5 %) in Trisbuffered saline containing 0.2 % Tween-20 (TBS-T), with gentle shaking at 37 o C to block nonspecific binding of the blot. Thereafter, the blots were incubated overnight at 4 ∘ C with primary antibodies for Snail, E-cadherin, vimentin and βactin, each at a dilution of 1 to 1000.
Then, the membrane was washed thrice with TBS-T, and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 90 min at room temperature. The blot was developed using an Xray film. Grayscale analysis of the bands was performed using ImageJ Launcher software. The respective protein expression levels were normalized to that of standard β-actin which served as standard.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Trizol RNA extraction reagent was used to extract total RNA from the cells, while cDNA synthesis kit was used to perform cDNA synthesis reaction according to the instructions of the manufacturer. Light Cycler 1536 RT-PCR detection system was used for the measurement of the expression levels of Snail, E-cadherin, vimentin, and miR-195-5p. Variation in the cDNA content was normalized using glyceraldehyde 3phosphate dehydrogenase (GAPDH). The qRT-PCR reaction conditions were: pre-denaturation at 94 o C for 3 min, PCR reaction at 93 o C for 26 sec, 58 o C for 26 sec, and 71 ˚C for 26 sec, and a total of 39 cycles. The PCR reaction mixture (20 µL) consisted of 6.4 μL of dH2O, 1.6 μL of gene-specific primer (10 μM), 2 μL of synthesized cDNA, and 10 μL of SYBR Premix Ex Taq™ II. The Ct value of U6 was taken as the internal parameter, and 2 -ΔΔCt method was used for calculating the relative expression levels of the proteins. The forward and reverse sequences of miR-195-5P primer were 5'-CTGGAGCAGC ACAGCCAATA-3' and 5'AGCT TCCCTGGCTC TAGCA-3', respectively.

Determination of effect of miR-195-5p inhibitor on tocotrienolic amide-induced expression of snail
Gastric cancer (GC) cells were cultured in serum-free medium with equal volume of miR-195-5p inhibitor or control, each at concentration of 10 μmol/L. Incubation was carried out at room temperature for 6 h. Lipofectamine 2000 was dissolved in serum-free medium and incubated at room temperature for 10 min to form a mixture. The mixture was then added to cells in each group, and cultured at 37 o C in a humidified atmosphere of 5 % CO2 and 95 % air for 48 h. Normal cell culture without miR-195-5p inhibitor or control served as normal control group. The transfection efficiency was determined with qRT-PCR.

Mice
BALB/c male mice (n = 40) aged 6 weeks (mean weight = 30 ± 3 g) were obtained from Haisco Pharmaceutical Group, Sichuan, China. The mice were housed in plastic cages in a laboratory with 12-h light/12-h dark cycle, average temperature of 25 ˚C and 50 % humidity. They were allowed free access to standard feed and clean drinking water. The study protocol was approved by the Institutional Animal Ethics Committee of the People's Hospital of Suzhou New District (no. 2018-182). The study procedures were carried out according to the guidelines of National Institute of Health (NIH) [8].

Establishment of GC xenografts in nude mice
Gastric cancer (GC) cells (NCI-B87) were trypsinized, washed thrice with RPMI medium, and re-suspended in the same medium. The mice were anaesthetized with ether and placed in supine position prior to inoculation with NCI-B87 cells. A small incision of about 6 -8 mm in diameter was made in the anorectal region of each mouse through the anorectal wall at the anterior end. A 27-gauge (20 mm) needle was used for subcutaneous injection of NCI-B87 cells (2 x 10 5 cells/mL) suspended in RPMI, into the posterior wall of the mice. The mice were then randomly assigned to 4 groups (10 mice/group): sham, GC, 2 mg tocotrienolic amide/kg body weight (bwt) and 5 mg tocotrienolic amide/kg bwt groups. Mice in sham and GC groups received equivalent volumes of physiological saline in place of tocotrienolic amide. The mice were sacrificed via cervical dislocation on the 45 th day of tumor inoculation, under pentobarbital sodium anaesthesia.

Determination of tumor growth in nude mice
The tumors formed in nude mice were excised and weighed to determine the effect of tocotrienolic amide on tumor growth in the treatment groups.

Statistical analysis
Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS version 21.0. Groups were compared using Tukey's multiple comparison test. Statistical significance was assumed at p < 0.05.

Effect of tocotrienolic amide on metastasis of GC cells
Treatment of MKN28 and NCI-N87 cells with tocotrienolic amide significantly and dosedependently reduced their invasiveness and migratory capacity (p < 0.05; Figure 1).

Effect of tocotrienolic amide on mRNA and protein expressions of Snail, E-cadherin and vimentin in GC cells
The results of Western blotting and qRT-PCR showed that tocotrienolic amide treatment significantly and dose-dependently downregulated the mRNA and protein expressions of Snail and vimentin, but it significantly upregulated the mRNA and protein expressions of E-cadherin (p < 0.05). The level of expression of E-cadherin was significantly higher in MKN28 cells than in NCI-N87 cells. These results are shown in Figure 2.

Effect of miR-195-5p inhibitor on tocotrienolic amide-induced suppression of expression of snail
As shown in Figure 4, transfection of MKN28 and NCI-N87 cells with miR-195-5p inhibitor significantly and dose-dependently upregulated mRNA and protein expressions of Snail (p < 0.05).

DISCUSSION
Gastric cancer (GC) is the fifth most common cancer worldwide [1]. Helicobacter pylori infection is the most common cause of GC, and accounts for 60 % of diagnosed cases. Other common causes include consumption of pickled vegetables, smoking and genetics. Early symptoms of the disease include heartburn, upper abdominal pain, nausea, loss of appetite, difficulty swallowing, and blood in the stool (hematochezia) [2]. Gastric cancer is treated with surgery, chemotherapy, radiation therapy, and targeted therapy.
However, the outcomes are often poor, with 5-year survival less than 10 % globally [2]. The high metastatic nature of the GC contributes to poor prognosis of patients [10]. The pathogenesis of GC involves several signaling pathways and abnormal gene expression patterns which are often accompanied by inactivation of tumor suppressor genes and activation of oncogenes [2]. This study investigated the effect of tocotrienolic amide on GC cell growth and metastasis, and the mechanism involved. Epithelial -mesenchymal transition (EMT) contributes to tumor progression, and a switch from E-cadherin to N-cadherin expression is indicative of EMT. Upregulation of snail protein expression and subsequent downregulation of E-cadherin occur during EMT [11]. Another prominent gene that contributes significantly to EMT is vimentin [12]. The results of previous studies revealed that the upregulation of protein expressions of snail and E-cadherin inhibited EMT in lung cancer cells (A549) [13]. In this study, tocotrienolic amide significantly and dosedependently inhibited the invasion and migration of MKN28 and NCI-N87 cells. It also significantly and dose-dependently downregulated the mRNA and protein expressions of Snail and vimentin. These results suggest that tocotrienolic amide may inhibit GC cell metastasis via the regulation of EMT.
The role of miR-195-5p in tumor suppression has been reported. Abnormal expression level of this protein leads to chemo-resistance and thus, poor prognosis [14]. It has been reported that in GC patients, miR-195-5p protein level was markedly reduced, while its upregulation resulted in cell apoptosis [15]. MicroRNA-195-5p (miR-195-5p) has been demonstrated to inhibit GC cell proliferation and metastasis by directly targeting fibroblast growth factor (FGF) in nude mice [15]. Overexpression of miR-195-5p suppressed GC cell proliferation and metastasis via inhibition of several signaling pathways [16]. The results of this study indicate that tocotrienolic amide significantly and dosedependently upregulated miR-195-5p expressions in MKN28 and NCI-N87 cells.
However, transfection of the cells with miR-195-5p inhibitor markedly reversed the effect of tocotrienolic amide. Protein and mRNA expressions of Snail were enhanced after transfection of MKN28 and NCI-N87 cells with miR-195-5p inhibitor. It is likely that Snail may be directly involved in EMT. These results are in agreement with those of previous studies [11,12]. Epithelial-mesenchymal transition (EMT) is partially enhanced by Snail via direct interaction with E-cadherin [17]. Downregulation of E-cadherin expression is considered to be responsible for poor clinical outcomes in cancer patients [17]. High expression level of Snail is indicative of GC progression [18]. In this study, treatment of GC mice with tocotrienolic amide led to significant and dose-dependent reductions in tumor weight and volume.