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.

Key Words: $[Keywords]

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.