The effects of vitamin E succinate on the expression of c-jun gene and protein in human gastric cancer SGC-7901 cells

Abstract

AIM: To investigate the effects of vitamin E succinate (VES) on the expression of c-jun gene and protein in human gastric cancer SGC-7901 cells.

METHODS: After SGC-7901 cells were treated with VES at different doses (5, 10, 20 mg•L^-1) at different time, reverse transcription-PCR technique was used to detect the level of c-jun mRNA; Western Blot was applied to measure the expression of c-jun protein.

RESULTS: After the cells were treated with VES at 20 mg•L^-1 for 3 h, the expression rapidly reached its maximum that was 3.5 times of UT control (P < 0.01). The level of c-jun mRNA was also increased following treatment of VES for 6 h. However, the expression after treatment of VES at 5 mg•L^-1 for 24 h was 1.6 times compared with UT control (P < 0.01). Western blot analysis showed that the level of c-jun protein was obviously elevated in VES-treated SGC-7901 cells at 20 mg•L^-1 for 3 h. The expression of c-jun protein was gradually increased after treatment of VES at 20 mg•L^-1 for 3, 6, 12 and 24 h, respectively, with an evident time-effect relationship.

CONCLUSION: The levels of c-jun mRNA and protein in VES-treated SGC-7901 cells were increased in a dose- and time-dependent manner; the expression of c-jun was prolonged by VES, indicating that c-jun is involved in VES-induced apoptosis in SGC-7901 cells.

INTRODUCTION

RRR-α-tocopheryl succinate (vitamin E succinate, VES), a derivative of natural vitamin E, has been shown to be a potent growth inhibitor of various cancer cell types in vitro and in vivo[1-7]. Growth inhibition by VES is attributed to cell cycle blockage[4,8-10], induced cellular differentiation[11,12], increased expression of biologically active transforming growth factor-βs (TGF-βs) and their type II cell surface receptors[1,13,14] and the induction of apoptosis[15-18]. VES is noteworthy not only for its antiproliferative effects on tumor cells, but also for its non-toxic effect on normal cell types.

Gastric cancer is one of the most common tumors in China[19-28]. Up to date, the exact mechanisms of tumorigenesis is still unclear, but our previous studies showed that VES can block cell cycle, arrest DNA synthesis and induce apoptosis in human gastric cancer SGC-7901 cells, therefore inhibiting the cell growth[29-32]. In addition, our in vivo research in our laboratory demonstrated that VES inhibited benzo (a) pyrene (B (a) P)-induced forestomach carcinogenesis in female mice[33]. The exact mechanisms of apoptosis are not clearly known, but we found that VES can secrete and activate biologically active TGF-β and then TGF-β increases the kinase activity of c-jun N-terminal kinase (JNK) followed by phosphorylation of c-jun, and finally activated c-jun triggers apoptosis in human gastric cancer SGC-7901 cells[34]. In this study, the expression of c-jun mRNA was detected using reverse-transcription polymerase chain reaction (RT-PCR) technique and the level of c-jun protein was measured using western blot in order to further investigate the mechanisms of VES-triggered apoptosis.

MATERIALS AND METHODS

Materials

VES was purchased from Sigma Co. Ltd. RPMI 1640 media and TRIzol total RNA isolation kit were obtained from Gibco BRL, TITANIUM^TM one-step RT-PCR kit from Clontech. Inc. c-jun (H79) rabbit polyclonal antibody was from Santa Cruz Biotechnologies.

Methods

Cell culture Human gastric cancer cell lines SGC-7901 were maintained in RPMI 1640 medium supplemented with 100 mL•L^-1 fetal calf serum (FCS), 100 kU•L^-1 penicillin, 100 mg•L^-1 streptomycin and 2 mmol•L^-1 L-glutamine under 50 mL•L^-1 CO₂ in a humidified incubator at 37 °C. SGC-7901 cells were incubated for different time periods in the presence of VES at 5, 10 and 20 mg•L^-1 (VES was dissolved in absolute ethanol and diluted in RPMI 1640 complete condition media correspondingly to a final concentration of VES and 1 mL•L^-1 ethanol), succinic acid, vitamin E and ethanol equivalents as vehicle (VEH) control and condition media only as untreated (UT) control.

RT-PCR After SGC-7901 cells were treated with VES for 3, 6 and 24 h, respectively, total cellular RNA was isolated by using TRIzol Reagent according to the manufacturer’s instructions. The concentration and purity of total RNA were determined by DU^R 640 nucleic acid and protein analyzer (Beckman, USA). One-step RT-PCR was carried out following the manufacturer's instructions. RT-PCR mixture was heated 1 h at 50 °C for reverse transcription and 5 min at 95 °C for pre-denaturation, then into 34 PCR cycles of 30 s at 94 °C for denaturation, 30 s at 60 °C for annealing, 30 s at 72 °C for extension in PTC-100 programmable thermal controller (MJ Research, USA). The corresponding fragment of c-jun gene was amplified with specific primers synthesized[35]. β-actin gene was designed as an internal standard with purpose to remove false negative outcome (Table 1).

Table 1.

Nucleotide sequence and size of the expected PCR products for oligonucleotide primers used for RT-PCR

Genes	Sequence	Size (bp)
c-jun	Upstream: 5'-GGAAACGACCTTCTATGACGAGCCC-3'	315
	Downstream: 5'-GAACCCCTCCTGCTCATCTGTCAGG-3'
β-actin	Upstream: 5'-GTGGGCCGCTCTAGGCACCAA-3'	540
	Downstream: 5'-CTCTTTGATGTCACGCACGATTTC-3'

The amplified products were seperated in 20 g•L^-1 agorose gel stained with ethidium bromide. After electrophoresis, the gel was observed and photographed under ultraviolet reflector. The density and area of each band were analyzed using ChemiImager^TM 4000 Digital System (Alpha Innotech Corporation, USA).

Western blot SGC-7901 cells treated with VES were harvested, washed in PBS and lyzed in lysis buffer containing 150 mmol•L^-1 NaCl, 1 mL•L^-1 NP-40, 5 mg•L^-1 sodium deoxycholate, 1 g•L^-1 SDS, 50 mmol•L^-1 Tris (pH7.4), 1 mmol•L^-1 DTT, 0.5 mmol•L^-1 Na₃VO₄, 10 mmol•L^-1 phenylmethylsulfonyl fluoride (PMSF), 10 mg•L^-1 trypsin, 10 mg•L^-1 aprotinin and 5 mg•L^-1 leupeptin. Following the centrifugation of 12000 × g for 30 min at 4 °C, the amount of protein in the supernatant was determined using Biorad DC protein assay. Equal amount of protein was separated on 10% SDS-PAGE and transferred to nitrocellulose filter (Gibco BRL, USA) overnight. Blocked with 50 g•L^-1 defatty milk, the filter was incubated with c-jun (H79) rabbit polyclonal antibody and horseradish peroxidase-conjugated IgG, finally developed with DAB.

Statistical analysis

The data were expressed as ¯x ± s. Statistical analysis was performed using student’s t-test. P < 0.05 was considered significant.

RESULTS

Effect of VES on the expression of c-jun mRNA in SGC-7901 cells

1 μg of total cellular RNA from groups of control, succinate, vitamin E, VES at 5, 10 and 20 mg•L^-1 was added to amplify c-jun and β-actin genes by RT-PCR. Baseline expression of c-jun mRNA was observed in SGC-7901 cells (Figure 1). After the cells were treated with VES at 20 mg•L^-1 for 3 h, the expression rapidly reached its maximum that was 3.5 times of UT control (P < 0.01). The level of c-jun mRNA was also increased following treatment of VES for 6 h. However, the expression after treatment of VES at 5 mg•L^-1 for 24 h was 1.6-fold increase compared with UT control (P < 0.01), while there was no significant difference between 10 and 20 mg•L^-1 VES groups and UT control group (Table 2).

Figure 1.

Effect of VES on the expression of c-jun mRNA in SGC-7901 cells for different time points by RT-PCR. A: treatment of VES for 3 h; B: treatment of VES for 6 h; C: treatment of VES for 24 h.1: UT control; 2: succinate; 3: vitamin E; 4: VES at 5 mg•L^-1; 5: VES at 10 mg•L^-1; 6: VES at 20 mg•L^-1; M: molecular weight marker.

Table 2.

The relative expression of c-jun mRNA in SGC-7901 cells (n = 6, ¯x ± s)

Groups	Ratio of c-jun/β-actin
	3 h	6 h	24 h
UT control	0.469 ± 0.092	0.432 ± 0.095	0.368 ± 0.104
succinate	0.426 ± 0.082	0.408 ± 0.078	0.361 ± 0.083
vitamin E	0.514 ± 0.101	0.430 ± 0.081	0.367 ± 0.075
5 mg•L-1 VES	0.550 ± 0.115	0.621 ± 0.086b	0.584 ± 0.097b
10 mg•L-1 VES	0.471 ± 0.086	0.584 ± 0.101a	0.421 ± 0.077
20 mg•L-1 VES	1.663 ± 0.109b	0.905 ± 0.099b	0.411 ± 0.094

^aP < 0.05,

^bP < 0.01, vs UT control.

Effect of VES on the expression of c-Jun protein in SGC-7901 cells

Western blot analysis showed that the level of c-Jun protein was obviously elevated in VES-treated SGC-7901 cells at 20 mg•L^-1 for 3 h in a significant dose-dependent manner (Figure 2A, 2B). Meanwhile, compared with the cells in UT control group, the VES-treated cells at 20 mg•L^-1 exhibited 1.8-, 2.0-, 2.3- and 2.8-fold increases in the expression of c-jun protein for 3, 6, 12 and 24 h, respectively, with an evident time-effect relationship (Figure 3A, 3B).

Figure 2.

The expression of c-jun protein in SGC-7901 cells following treatment of VES for 3 h. Lane1: Molecular weight marker; Lane2: UT control; Lane3: succinate; Lane4: vitamin E; Lane5: VES at 5 mg•L^-1; Lane6: VES at 10 mg•L^-1; Lane7:VES at 20 mg•L^-1.

Figure 3.

The expression of c-jun protein in SGC-7901 cells following treatment of VES at 20 mg•L^-1 for different time poits. Lane1: Molecular weight marker; Lane2: 3 h; Lane3: 6 h; Lane4: 12 h; Lane5: 24 h.

DISCUSSION

The oncogene, c-jun, belongs to an immediate early gene and can be rapidly and transiently induced in response to multiple extracellular stimuli[36-38]. The product of c-jun gene is a nuclear transcription factor, an important composition of activation protein 1 (AP-1) dimmers, involved in signal transduction and regulation of many kinds of genes[39-41].

Transcription of c-jun mRNA rises after exposure of cells to a number of treatment including ultraviolate, irradition, heat shock, H₂O₂, TNF-α and other apoptosis-associated factors[42-46]. In addition to this transcriptional mode of regulation, c-jun activity can also be modulated directly at the protein level. In certain cell types, induction of c-jun is observed during apoptosis. There is some evidence that prolonged expression of c-jun in selected vulnerable cells suggests neuronal cell death[47].

Apoptosis is an innate program of cell suicide that is required for removal of unnecessary or damaged cells from bodily structures. Apoptosis is complex and regulated by a variety of factors[48-58]. Previous studies showed that the induction of apoptosis in tumor cells is one of the important mechanisms of VES-induced cell growth inhibition[59-61]. In the present study, the expression of c-jun mRNA and protein was measured in human gastric cancer SGC-7901 cells treated with VES at different doses for different time points. We found that the expression of c-jun mRNA was evidently promoted after 3 h of VES treatment at 20 mg•L^-1 and reduced to the normal level after 24 h of treatment; whereas in the case of VES treatment at 5 mg•L^-1, that was also increased after 3 h and remained a high level after 24 h. The data above showed that the c-jun activation was enhanced and prolonged by VES, therefore indicating that c-jun is involved in VES-triggered apoptosis in SGC-7901 cells. The results from western blot ananlysis showed that the level of c-jun protein was elevated following SGC-7901 cells were treated with VES at different doses for 3 h and with VES at 20 mg•L^-1 for different time in a dose- and time-dependent manner.

The diversity of signals and signaling pathways that are directed toward c-jun is also reflected in the biological responses, in which the transcription factors have been implicated. It is reported that the mainly biological functions of c-jun are blockage of cell cycle and induction of apoptosis[62-64]. The study presented here demonstrated that VES can obviously increase the expression of c-jun mRNA and protein in human gastric cancer SGC-7901 cells, implicating that c-jun is involved in VES-induced apoptosis.