Introduction
Gastric cancer is the second leading cause of cancer-related deaths worldwide. In general, high degree of mortality and short median survival time in gastric cancer is due to the fact that by the time diagnosis is arrived at it is too late, and that, in part, due to the advanced stage at which patients seek medical attention, which can be ascribed to its asymptomatic nature in early stages (1–3). Previously, it was reported that selenium-binding protein 1 (SBP1) may serve as an independent prognostic marker of human gastric cancer. It is known that SBP1 levels may correlate with differentiation, TNM stage, and lymph node metastasis in several types of cancer (3,4). For instance, decreased expression of SBP1 had been reported in prostate, stomach, colon, esophagus, uterus, ovary and lung cancers. Due to its wide expression in a variety of cancers, SBP1 could be an attractive target for cancer therapy (5–10). Low levels of expression of SBP1 indicates poor prognosis in gastric, lung adenocarcinoma and colorectal cancers (10,11). In contrast, high level of SBP1 expression is found in normal colonic epithelia (10) while its decreased expression is a late event in colorectal cancer (12) that may indicate the rapid progression of colorectal carcinoma (12). Furthermore, SBP1 is overexpressed in the chemosensitive tissues (13) suggesting that its expression could be considered as an important predictor of response of cancer to chemotherapeutic drugs (6). It has been suggested that SBP1 could form an important independent risk factor to predict overall survival and disease recurrence (14).
SBP1 is an α-β protein present throughout the cell and localizes at several places in the cell depending on the cell types (15,16). SBP1 is predominantly present in the cytosol and participates in the late stages of intra-Golgi protein transport and participates in the intracellular transport of selenium (17). SBP1 binds to selenium via cysteine (Cys57) (16) and is thus, involved in ubiquitination/deubiquitination pathways. Selenium by virtue of its incorporation into selenoproteins shows a wide range of pleiotropic effects, including its ability to prevent cancer and its progression (6,10,18). Diminished expression of SBP1 in human cancers is due to methylation of the SBP1 promoter and by alternative splicing of SBP1 mRNA (6). However, the exact relationship between selenium levels and SBP1 expression in cancer progression is yet to be clarified. Previous studies suggested that selenium may exert its growth inhibitory action by modifying the function of pre-existing proteins. SBP1 is a target of the hypoxia-inducible factor-1 α (HIF1α) through which selenium may modify SBP1 expression (19), enhances glutathione peroxidase 1 (GPX1) activity without altering its expression, and directly interact with von Hippel-Lindau protein (pVHL) that plays a role in the proteasomal degradation pathway in a selenium-dependent manner (6,15,19). However, the exact mechanisms of SBP1 regulation and its anticancer effects is not clear and needs to be investigated.
In the present study, the differences in protein expression among normal gastric mucosa, early gastric carcinoma and the corresponding gastric carcinoma tissues were investigated in a cohort of patients by two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). These evaluations have showed that the gradual SBP1 loss was associated with an increased malignant grade. Then we evaluated the role of SELENBP1 in gastric cancer by studying the expression of SBP1 and its function employing gene knockout and overexpression techniques especially its role in the proliferation, migration, senescence and chemoresistance of gastric cancer cells to cisplatin.
Materials and methods
Cell culture
SGC7901 and BGC823 cancer cells, used in the present study, were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic antimyocytic (Gibco) and maintained at 37°C in a humidified atmosphere containing 5% CO2. Exponentially growing cells were removed from the culture flasks using trypsin/EDTA, centrifuged at 800 rpm for 5 min, resuspended, and counted for use in subsequent experiments. Stock cultures of each cell line were routinely sub-cultured at least once a week and the media were changed every 2–3 days.
Construction of SELENBP1 stable cell line
The pEGFP-SELENBP1, SELENBP1 small interfering RNA (siRNA) and pEGFP vector (control) were purchased from Genepharma Co. Ltd (Shanghai, China) and were stably transfected into SGC7901 and BGC823 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Stably transfected cells were selected with G418. Selected clones were maintained in growth medium containing 400 μg/ml geneticin.
Cell proliferation assay
Approximately 3,000 cells in 100 μl of medium were plated in 96-well plates and allowed to attach for 24 h. Subsequently, cells were treated with the cell proliferation reagent MTS (Promega, Madison, WI, USA) to spectrophotometrically evaluate cell proliferation, viability and chemosensitivity in accordance with the manufacturer’s directions. Cultures were collected at different time-points (24, 48, 72, 96, 120 h). Relative proliferation rates were calculated as a percentage of the initial T0 reading within each cell line.
Flat plate colony formation assay
The cells transfected steadily with pEGFP-siRNA/pEGFP-SELENBP1 or empty vector were harvested and then 500 cells were plated in RPMI-1640/10% FBS on 6-well plates per well, and were cultured for 2 weeks in the incubator at 37°C. The clones were stained by 1 ml 0.1% crystal violet for 20 min. The number of the clones in 10 randomly chosen fields was assessed by a microscope.
Cell migration assay
Cells were trypsinized and resuspended in RPMI-1640 medium supplemented with 0.5% FBS. The cell suspension was adjusted to a concentration of 1×106 cells/ml, and 200 μl of cells were pipetted into the upper chamber of a Transwell plate (Costar, Cambriged, MA, USA). The cells at 37°C in 5% CO2 were allowed to attach for 1 h and the lower chamber was filled with 600 μl of RPMI-1640 medium containing 20% FBS. After 24 h of incubation at 37°C, the filter side of the upper chamber was scraped with a cotton tip to eliminate cells that had not migrated, the filter was removed and stained with 600 μl 0.1% crystal violet for 20 min. The cell number in 10 randomly chosen fields was determined using a light microscope.
RNA preparation, complementary DNA synthesis and quantitative real-time PCR
Total RNA was isolated from SGC7901 and BGC823 cell lysates using an RNeasy mini kit according to the manufacturer’s instructions. Total RNA was then treated with DNase I in the presence of anti-RNase to remove DNA contamination before complementary DNA synthesis. The complementary DNA was synthesized with random primers and avian myeloblastosis virus reverse transcriptase (Promega). Real-time PCR (power SYBR-Green, ABI, Warrington, UK) analysis was performed using an ABI Prism 7500 sequence detector according to the manufacturer’s protocol. Primer sequences were as follows: for SELENBP1, 5′-ATCACCGACATCCTGCTCT-3′ (forward), 5′-GACTTTAGTTCCTCGTCCTCC-3′ (reverse); and for β-actin, 5′-ATCATGTTTGAGACCTTCAA-3′ (forward), 5′-CATCTCTTGCTCGAAGTCCA-3′ (reverse). Fold changes in the genes of interest were calculated after normalisation with the endogenous control β-actin and using the comparative threshold cycle (Ct) method.
Protein extraction and immunoblot analysis
Cells were scraped from culture plates, and the final protein concentration of the cell lysates was determined using the bicinchoninic acid (BCA) method with bovine serum albumin as the standard. Equivalent cell extracts (20–40 μg of protein) were boiled in 5X sodium dodecyl sulfate (SDS) at 95°C for 5 min, cooled on ice, and then total protein extracts were separated by 10–12% SDS-PAGE (20–50 per lane), and electro-transferred to polyvinylidene fluoride membranes. Anti-SELENBP1 (1:1,000, Abcam, Cambridge, UK) and anti-β-actin (1:1,000, Abcam) antibodies were diluted in TBST (TBST/tween; 5% BSA powder) and incubated with the membranes at 4°C overnight. The appropriate secondary antibody was applied (1:1,000, anti-rabbit) at room temperature for 1 h. Immunoreactive proteins were visualized by enhanced chemiluminescence.
Cell cycle analysis by flow cytometry
SGC7901 and BGC823 cells (2.5×105/well) were seeded onto 6-well cell culture plates and incubated with 20 μm of CDDP for 24 h. The cells were harvested and washed by centrifugation. For apoptosis determination, cells were fixed by 70% ethanol in −20°C overnight and then re-suspended in PBS containing 40 μg/ml PI in 37°C for 30 min and then added 100 μg/ml RNase A in 4°C dark room for 30 min. The cell apoptosis was determined by flow cytometry (FACSCalibur™, Becton Dickinson, Franklin Lakes, NJ, USA).
Senescence-associated β-galactosidase (SA-b-Gal) activity
Cells for this assay were seeded in the dishes, washed twice with PBS and fixed in PBS that contained 2% formaldehyde/0.2% glutaraldehyde for 7 min at room temperature. SA-b-Gal staining was performed in fresh senescence-associated X-Gal staining solution at 37°C (no CO2). Incubation typically lasted for 12 h. Cells were rinsed in PBS and stored in PBS with 70% glycerol. Cells were then examined under a microscope for blue-green staining of the cytoplasm that was indicative of senescence. Digital imagines were taken at ×20 optical magnification, and stained cells were counted and expressed as a percentage of total cell number in three independent fields per well per treatment group to obtain an average value for β-galactosidase staining activity.
Xenograft assay
Cells were collected by trypsinization and washed twice before injection. Cells (2×106 cells in 150 μl PBS) were injected subcutaneously into nude mice. All injected mice formed tumours. Tumour volumes were measured twice every week from week 2 to week 6 and calculated using the following formula: 0.5236 × L1 × (L2)2, where L1 is the long axis and L2 is the short axis of the tumour. Seven weeks after the inoculation of the cancer cells, tumors were isolated and tumor volume and weight were determined. Tissues were fixed in 10% buffered formaldehyde solution and SBP1 expression level was determined by western blot analysis and immunohistochemistry.
Experimental statistical analysis
Graphpad Prism5 and SPSS were used for statistical analysis. Statistical comparison between two groups was performed using the Student’s t-test. For comparison of more than three groups, we used one-way analysis of variance, followed by Tukey’s multiple comparison. P-values <0.05 were considered statistically significant.
Results
Construction of SELENBP1 and siRNA stable cell line and its confirmation
As reported previously, we observed that a subset of tumor samples had higher expression of SBP1. To examine the biological significance of SBP1 in gastric cancer, BGC823 and SGC7901 cells were stably transfected with either pEGFP-SELENBP1/pEGFP-siRNA or corresponding empty vector plasmid, and the levels of SBP1 expression were determined by immunoblot analysis and real-time PCR. Clone SELENBP1 expressed high levels, whereas siRNA expressed low levels of SBP1 compared with empty vector and untransfected BGC823 and SGC7901 cells (Fig. 1). When BGC823 and SGC7901 cells were transfected with pEGFP-SELENBP1, the expression of SBP1 was enhanced by 4-fold in SGC7901 cells and 5-fold in BGC823 cells, while when transfected with pEGFP-siRNA, the expression of SBP1 was suppressed by at least 70% in both SGC7901 and BGC823 cells.
Roles of SBP1 on cell growth in vivo and in vitro
To determine the role of SBP1 on the proliferation of cells, we conducted MTS assays after the transfection of pEGFP-SELENBP1/ pEGFP-siRNA and the empty vector. Cultures were collected at different time points for analysis of cell proliferation. SBP1 downregulation significantly enhanced the proliferation of SGC7901 and BGC823 cells, while SBP1 upregulation remarkably reduced the proliferation of both cell lines (Fig. 3). The cell proliferation curve showed that the proliferation of pEGFP-siRNA transfected cells decreased at 24 h in both cell lines. Cell growth was much reduced at 48, 72, 96 and 120 h, compared with the control groups (P<0.01) (Fig. 3A and B). The stable transfectants expressing SELENBP1 had incomplete inhibition but moderate proliferation retardation (P<0.01) (Fig. 3C and D).
To confirm the effect of pEGFP-siRNA and pEGFP-SELENBP1 in vivo, we subcutaneously inoculated nude mice with BGC823 and SGC7901 which were either stably transfected with pEGFP-siRNA/pEGFP-SELENBP1 or empty vector that formed the control. We observed significant rate of enhancement of growth of cancer cells that were stably transfected with pEGFP-siRNA and decreased growth of cancer cells transfected with pEGFP-SELENBP1 (Fig. 4). Consistent with in vitro data, mice injected with SBP1 overexpressing SGC7901 and BGC823 cells showed significantly smaller tumor volume than mice injected with control. Examination of tumour tissues of xenografts showed significantly reduced expression of SBP1 in SiRNA group while SELENBP1 group showed enhanced expression compared with the control group (Fig. 5).
To examine the effect of downregulation and overexpression of SBP1 on the colony formation of cells, stably transfected SGC7901 and BGC823 cells were inoculated into 6-well plates (500 cells). Number of colonies formed by SGC7901 and BGC823 cells transfected with pEGFP-siRNA were enhanced by 147% (P<0.01) and 170% (P<0.001), respectively, compared to coresponding cells transfected with empty vector (Fig. 2A and C). The colonies formed by the pEGFP-SELENBP1 transfected SGC7901 and BGC823 cells were suppressed to 64% (P<0.01) and 60% (P<0.05), respectively, compared to control (Fig. 2B and D).
In addition, we also studied the role of SBP1 in cancer cell senescence. It was noted that a significant difference exists between cells transfected with empty vector and pEGFP-siRNA/pEGFP-SELENBP1 in gastric cancer cell line SGC7901. SBP1 significantly enhanced the senescence of SGC7901 cells as assayed by β-galactosidase staining. Downregulation of SBP1 decreased senescence of SGC7901 gastric carcinoma cells (Fig. 6).
Effects of SELENBP1 on cell migration
We next examined the role of SBP1 in cancer cell migration. We observed a significant difference between cells transfected with empty vector and pEGFP-siRNA/pEGFP-SELENBP1. SBP1 downregulation by pEGFP-siRNA (SiRNA) enhanced SGC7901 and BGC823 cell migration to 159% (P<0.01) and 185% (P<0.05), respectively (Fig. 7A and C), while SGC7901 and BGC823 stably transfected with pEGFP-SELENBP1 (SELENBP1) cells showed reduced migration by almost half the normal [57% (P<0.001) and 45% (P<0.001), respectively] (Fig. 7B and D).
Roles of SBP1 on chemosensitivity to CDDP
To determine if the pEGFP-siRNA and pEGFP-SELENBP1 transfected SGC823 and BGC7901 cells could change the cytotoxicity of CDDP, stably transfected SGC7901 and BGC823 cells with pEGFP-siRNA and pEGFP-SELENBP1 were exposed to different concentrations of cisplatin and cell viability was analyzed at the end of 24 h of treatment by MTS assay. It was observed that pEGFP-siRNA transfected gastric cancer cells treated with 20 and 40 μmol/l cisplatin showed significantly enhanced viability compared with control, while the viability pEGFP-SELENBP1 transfected cells was reduced (Fig. 8).
Apoptosis was measured by flow cytometry, but no significant difference in the index of apoptosis was detected between pEGFP siRNA/SELENBP1 transfected SGC823 and BGC7901 cells and control (Fig. 9A and B). However, when pEGFP-siRNA and pEGFP-SELENBP1 transfected SGC7901 and BGC823 cells were treated with 20 μmol/l cisplatin for 24 h, downregulation of SBP1 decreased the apoptosis of SGC7901 and BGC823 cells in comparison with the empty vector-transfected control. On the other hand, overexpression of SBP1 enhanced cisplain-mediated apoptosis of both cell lines (Fig. 9C and D).
Discussion
Gastric cancer is common in Asian countries. Five year survival rate of the advanced gastric cancer is only 30–40%, whereas when gastric cancer is detected early, it is about 90%. The mechanism of the development of gastric cancer is still not clear. Previous studies suggested that SBP1 has the potential to be a novel biomarker in predicting prognosis of gastric cancer. SBP1 level can be correlated with differentiation, TNM stage, and lymph node metastasis of gastric cancer. The 3-year survival rate of patients with high expression of SBP1 was significantly higher than that of those with low expression. Suppression of SBP1 may be a crucial event that may play a significant role in the progression of gastric cancer (3,20) that implies that it has the potential as a reliable diagnostic marker of gastric cancer (21). In the present study, we attempted a systematic study to understand the biological significance of selenium-binding protein 1 in gastric cancer in vitro and in vivo.
The observation that decreased expression of SBP1 occurs in human cancers led to the suggestion that it (SBP1) may have tumor suppressive action. In the present study, we explored that the probable tumor suppressor action of SBP1 by silencing and overexpressing endogenous SBP1 expression in SGC7901 and BGC823 cells and analyzed phenotypic changes of stably transfected cells. We could achieve almost complete downregulation and significant upregulation of SBP1 expression using the transfection of SGC7901 and BGC823 cells by pEGFP-siRNA/pEGFP-SELENBP1 plasmids.
Uncontrolled cell proliferation and colony formation are important hallmarks of cancer. Previously, it was reported that overexpression of SBP1 in HCT116 cells suppressed cell proliferation and inhibited tumor growth in vivo(6). In esophageal adenocarcinoma model, it was noted that overexpression of SBP1 potentiated the antiproliferative response of selenium supplementation, particularly those cells that showed higher SBP1 expression (9).
Inhibition of SBP1 effectively increased cell motility, promoted cell proliferation in hepatocellular carcinoma (14). In the present study, we observed for the first time that SBP1 regulates proliferation of gastric cancer cells SGC7901 and BGC823. SBP1 downregulation in SGC7901 and BGC823 cells led to a significant inhibition of proliferation and colony formation, while SBP1 upregulation promoted proliferation and colony formation of both cell lines. SBP1 downregulation and upregulation seems to be a determining factor that alters SGC7901 and BGC823 cell growth in vitro that, in turn, is supported by the consistent results obtained in vivo. Mice injected with SBP1 overexpressing SGC7901 and BGC823 cells had a significantly smaller tumor volume than the mice injected with control cells. Besides, SBP1 expression was significantly increased in tumor tissues injected with pEGFPSELENBP1 transfected cells and decreased in tumor tissues injected with pEGFP-siRNA transfected cells. These results are in tune with the suggestion that SBP1 has the potential to suppress gastric tumor formation. We propose that one mechanism by which SBP1 regulates cell proliferation is by virtue of its ability to covalently bind to selenium. Population-based studies suggested higher serum selenium concentration is inversely related to the incidence of a variety of epithelial malignancies (22). But the exact relationship between selenium levels and SBP1 expression in cancer progression is not known.
Overexpression of SBP1 accelerated senescence in mice (9,23). Selenium supplements were suggested to work in conjunction with SBP1 to activate senescence in human esophageal adenocarcinoma and thus, retard their growth. To determine if SBP1 overexpression activated cellular senescence in human gastric cancer cells, we studied stably transfected SGC7901 and BGC823 cells for the expression of senescence-associated β-galactosidase which revealed that that enhanced expression of SBP1 in SGC7901 cells, but not BGC823, increased senescence as determined by β-galactosidase staining. It was also noted that diminished expression of SBP1 decreased senescence of SGC7901 gastric carcinoma cells. However, we noted that only few BGC cells showed staining for β-galactosidase. We suspected that this negative results could have resulted from either BGC823 cells not being sensitive to β-galactosidase staining or β-galactosidase expression was not high enough to be detected in BGC823 cell lines. Nonetheless, SBP1-transfected cells showed increased senescence. These data suggest that SBP1 activates the senescence pathway in some gastric cancer cells.
Downregulation of SBP1 increased the migration of SGC7901 and BGC823 gastric cancer cells, while pEGFP-SELENBP1 transfected SGC7901 and BGC823 cells showed decreased migration. These results emphasize the important role of SBP1 in its ability to suppress tumor growth and metastasis. SBP1 is known to suppress colorectal cancer cell migration (6). Decreased expression of SBP1 promoted tumor invasiveness by increasing GPX1 activity and diminishing HIF-1α expression in hepatocellular carcinoma (14). Regulation of cancer cell migration by SBP1 may explain greater lymph node metastasis and poor prognosis observed in patients with gastric cancers with low SBP1 expression.
The significance of SBP1 in response of cancer cells to anticancer drugs observed in the present study is rather interesting. SGC7901 and BGC823 cells showed decreased vitality in response to cis-platinum treatment. Overexpression of SELENBP1 enhanced cisplatin-mediated apoptosis. We suggest that enhanced SBP1 expression in gastric cancer may increase the chemosensitivity of gastric cancer cells via apoptotic signaling pathways. This is supported by the observation that SBP1 overexpression in colorectal cancer cells sensitized them to H2O2-induced growth inhibition (9). Decreased SBP1 expression effectively inhibited apoptosis under oxidative stress and greatly enhanced glutathione peroxidase 1 (GPX1) activity without altering GPX1 expression and downregulated hypoxia-inducible factor-1α (HIF-1α) expression. SBP1 and GPX1 formed nuclear bodies and are co-localized under oxidative stress (14). Similar results have been reported in esophageal adenocarcinoma where it was noted that diminished SBP1 expression blunted the cellular response to selenium supplementation (6). Based on these results, we propose that SBP1 could serve as a reliable target for gastric carcinoma therapy. It has been suggested that enhanced oxidative stress in cancer cells would lead to cellular apoptosis due to the inhibition of GPX1 activity by the upregulated SBP1 (14). We suspect that similar mechanism may be occurring in gastric cancer cells.
It is evident from the results of previous studies and the present investigation that diminished SBP1 expression may indicate poor prognosis in the gastric cancer. SBP1 overexpression in gastric cancer may increase the chemosensitivity of gastric cancer cells via apoptotic signaling pathways. However, further studies are needed to explore other possible mechanisms of the anticancer action of SBP1.