Multidrug resistance (MDR) is one of the major reasons for the failure of chemotherapy-based gastric carcinoma (GC) treatments, hence, biologically based therapies are urgently needed. Gastrin (GAS), a key gastrointestinal (GI) hormone, was found to be involved in tumor formation, progression, and metastasis. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining analysis revealed a high level of expression of GAS in drug-insensitive GC tissues (P<0.01) and similar results were revealed in GC cell lines SGC7901 and its multidrug-resistant variants SGC7901/VCR and SGC7901/ADR. We constructed a eukaryotic expression vector pCDNA3.1(+)/GAS for GAS overexpression and recombinant lentiviral vectors for specific siRNA (siGAS). Transfection of pCDNA3.1(+)/GAS increased (P<0.05) while transfection of siGAS (P<0.05) and co-treated with paclitaxel (TAX) and vincristine (VCR) combination (TAX-VCR) decreased (P<0.01) the cell viability of SGC7901, SGC7901/VCR and SGC7901/ADR. Apoptosis rates of SGC7901/VCR and SGC7901/ADR were reduced by pCDNA3.1(+)/GAS and increased by siGAS (P<0.05). The apoptosis rates of SGC7901/VCR, SGC7901/ADR and SGC7901 were all upregulated (P<0.01) when cells were co-treated with a combination of siGAS and TAX-VCR. Additionally, siGAS significantly downregulated the expression of Bcl-2 and multidrug-resistant associate protein (MRP1) and P-glycoprotein (Pgp) (P<0.05) in SGC7901/VCR and SGC7901/ADR cells. Moreover, GAS overexpression in SGC7901 cells significantly inhibited p27Kip1 expression but increased phosphorylation levels of p27Kip1 on Thr (187) and Ser (
Gastric carcinoma (GC) is one of the most common malignant tumors. Currently, combined surgical operation and chemotherapy play an important role in the comprehensive treatment of GC (
It has been reported that there are several mechanisms whereby GCs become resistant to chemotherapeutic agents. One mechanism is that P-glycoprotein (P-gp, 170 kDa) and its coding gene multidrug resistance protein 1 (MDR1) play a role in drug-excreted pump function (
A large number of studies have demonstrated that p27Kip1 plays an important role in the modulation of MDR through its degradation. In drug-resistant human myeloma cell lines, it was found that facilitation of S-phase kinase-associated protein 2 (Skp2) expression promoted cell cycle progression and p27Kip1 degradation (
GAS, an important gastrointestinal (GI) hormone, stimulates parietal cells to secret gastric acid (HCl) and participate in epithelial proliferation of the GI tract (
Although GAS has been shown to regulate the growth of GC cells, the latest studies of GAS in our laboratory have found that GAS accelerated cell cycle progression and proliferation of GC and is associated with promoting the migration and invasion through the β-catenin/T-cell factor-4 (TCF-4) pathway (
Surgical specimens of cancer tissues were collected from 17 patients with advanced GC. All patients (ten males and seven females with an average age of 58.3 years, ranging from 48 to 71 years) received neoadjuvant chemotherapy based on docetaxel (DTX) and cisplatin (CDDP) prior to surgical removal of the tumors (
The human gastric adenocarcinoma cell line SGC7901 (obtained from Xijing Hospital, the Fourth Military Medical University, Xi'an, Shaanxi, China) and its multidrug-resistant variants SGC7901/VCR and SGC7901/ADR (established and maintained in our laboratory) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL, Grand Island, NY, USA) in a humidified atmosphere containing 5% CO2 at 37°C. To maintain the MDR phenotype, VCR (final concentration 1
Total RNA from GC tissues or cells was extracted using TRIzol (Invitrogen), and cDNA was synthesized using an M-MLV Reverse Transcriptase kit (Invitrogen). Then, the detection of specific products was performed using SYBR green (Applied Biosystems, Foster City, CA, USA) technology. Amplification involved a denaturation step (95°C for 5 min, 1 cycle), and amplification and quantification were repeated for 40 cycles (95°C for 5 sec and 60°C for 1 min, respectively). The data of the relative mRNA expression levels were calculated using the 2−∆∆Ct method and are presented as the fold change of transcripts for genes. The sample mean value was calculated and expressed as the cycle threshold (Ct). mRNA expression was calculated as the difference (∆Ct) between the Ct value of the target gene and the Ct value of the inner control. 2−∆∆Ct means the fold change in the target mRNA expression. The mRNA expression expressed as the mean ± SD. Primer sequences were as follows: GAS, forward, 5′-GAGCTACCCTGGCTGGAGCAGCAG-3′; reverse, 5′-CTCATCCTCAGCACTGCGGCGGCC-3′. Pgp, forward, 5′-GCAAGAGGAGCAGCTTAT-GAAG-3′; reverse, 5′-ACTCCCTACCTTCAAGTTGAGG-3′. β-actin, forward, 5′-AGGTCATCACCATTGGCAAT-3′; reverse, 5′-ACTCGTCATACTCCTGCTTG-3′.
Total proteins from tissue and cell samples were extracted using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% NP-40 and 0.1% SDS). For nuclear protein and cytoplasmic protein extraction, protein was isolated using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime, Haimen, China) according to the manufacturer's instructions. Briefly, cells were harvested and washed with PBS, and then centrifuged at 8,000 g for 10 min. The cell sediments were collected and resuspended in 20 ml of PBS and mixed with 200 ml of buffer A containing 1 mM phenylmethylsulfonyl fluoride (PMSF). After vortexing, cells were placed in an ice bath for 10 min. Thereafter, 10 ml of buffer B was added and the cells were again vortexed and placed in an ice bath for 1 min followed by centrifugation at 12,000 g for 5 min at 4°C. The supernatants containing cytoplasmic protein were collected and used for further study. The remaining sediments were collected and resuspended in 50 ml of a nuclear protein extraction agent and subjected to an ice bath for 30 min with vortexing at intervals of 2 min. After centrifugation (12,000 g for 10 min at 4°C), the supernatants containing nuclear protein were collected and stored at −70°C for further analysis.
The protein quantification was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Proteins (20
We transfected SGC7901 cells with pCDNA3.1(+)/GAS or pCDNA3.1(+)/GAS -NC. At 48 h after the transfection, the cells were fixed in 3% paraformaldehyde in PBS at room temperature for 8 min, then permeabilized with 0.2% Triton X-100 for 15 min at room temperature. After washing in PBS, the cells were incubated with Skp2 (1:400, Abcam) or KPC (1:600, Abcam) primary antibody at 4°C overnight. After washing, the cells were incubated with FITC-labeled secondary antibody (Pierce, Rockford, IL, USA) at room temperature for 2 h avoiding light. The nuclear was stained with DAPI for 5 min and washed with PBS at three times. Adding anti-quenching reagent, and mounting was performed avoiding light. The cells were examined under a Nikon fluorescence microscope (Image Systems, Columbia, MD, USA).
GC tissues were fixed in neutral buffered formalin for 2 h at room temperature and for 18 h at 4°C and then tissues were embedded in paraffin and sliced. The thickness of the tissue serial sections was 3.5
The construction of the GAS over-expression vector [pCDNA3.1(+)/GAS], and recombinant lentiviral vectors for siRNA specific for GAS (siGAS) and Skp2 (siSkp2) was performed by Sangon Biotechnology Co. Ltd. (Shanghai, China). All vectors were transfected into cells according to the manufacturer's instructions and following previous studies (
To determine the drug sensitivity of the cells, SGC7901/VCR, SGC7901/ADR and SGC7901 cells were plated in a 96-well plate at a density of 5×103 cells/well in RPMI-1640 containing 10% FBS and were transfected respectively with the above-mentioned vectors for 48 h. For the SGC7901/VCR and SGC7901/ADR groups, 20
Each group of cells was collected and detected using an Annexin V Fluorescein Isothiocyanate kit (AV-FITC, BD Pharmingen, San Diego, CA, USA) on a BD FACSCalibur™ system (BD, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. In brief, the groups of cells were consistent with the MTT assays and cells from the different conditions were suspended in 100 ml of binding buffer, at a density of 1×106 cells per ml. Then, cells were incubated with AV-FITC and propidium iodide (PI) for 15 min, and analyzed using Beckman CXP software (Beckman Coulter, Brea, CA, USA) on a FC-500 flow cytometer (Beckman Coulter).
Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Data were analyzed using Student's t-test between two groups, and categorical data were examined using the χ2 test. Data are presented as the mean ± SD. P<0.05 was considered statistically significant.
Recently, GAS was demonstrated to participate in the growth and metastasis of GC cells (
Additionally, GAS-positive cells were stained brown. Obviously, GAS-positive cells in the N/P-R group are much more numerous than in the M-R group (
We constructed the GAS overexpression vector pCDNA3.1(+)/GAS and its silencing vector siGAS, and the effects of transfection were tested. After 48 h of transfection, we found that GAS was significantly upregulated in pCDNA3.1(+)/GAS vector-transfected SGC7901/VCR, SGC7901/ADR and SGC7901 cells (
To further investigate whether GAS contributes to MDR in GC, it was force expressed or knocked down in GC cell lines. MTT results showed that the transfection of pCDNA3.1(+)/GAS markedly increased the cell viability of the drug-resistant GC cell lines including SGC7901/VCR and SGC7901/ADR compared with pCDNA3.1(+)/GAS-NC-transfected cells (P<0.05) (
Previous studies have revealed that GAS play the role of anti-apoptosis in a variety of cancer cell types (
Previous studies have shown that GAS can inhibit the expression of p27Kip1 and p27Kip1 can participate in drug resistance in a variety of cancer cells (
Studies have shown that depletion of Skp2 could inhibit the degradation of p27Kip1 (
These results indicated that inhibition of p27Kip1 degradation could attenuate the effects of GAS overexpression. All of these results suggested that GAS induces the MDR of SGC7901 via the degradation of p27Kip1.
In this study, we demonstrated a stimulatory role of GAS in MDR in human GC cells. Firstly, we provided evidence that GAS is upregulated in the SGC7901/VCR and SGC7901/ADR cell lines and in non- or poorly-responding gastric tumor patients. Secondly, further analysis demonstrated that the overexpression of GAS partly abolished the drug sensitivity of SGC7901 cells while the silencing of GAS increased the drug sensitivity of SGC7901, SGC7901/VCR and SGC7901/ADR cells. A mechanistic assay confirmed that the degradation of p27Kip1 was the downstream mechanism of GAS in regulating MDR in GC cells. These findings suggest a potentially important role for GAS in regulating MDR.
MDR is a great impediment to the success of chemotherapy for GC. It is believed that GAS plays an important role in the process of cell migration and might be involved in the regulation of cancer development and drug resistance in digestive tract and other cancers (
SGC7901/VCR and SGC7901/VCR are the MDR variants of SGC7901, and we found remarkable upregulation of GAS along with MRP1, Pgp and Bcl-2 overexpression, while the downregulation of p27Kip1 was also demonstrated in these two cell lines compared with SGC7901. Interestingly, we overexpressed GAS using pCDNA3.1(+)/GAS in SGC7901, and found the overexpression of GAS upregulated MRP1, Pgp and Bcl-2, but accelerated the degradation of p27Kip1 with a coincident upregulation of the phosphorylation levels of p27Kip1 on Thr (187) and Ser (
A large number of studies have demonstrated that the degradation of p27Kip1 plays an important role in the modulation of MDR. First, in drug-resistant human myeloma cell lines, it was found that facilitation of Skp2 expression promoted cell cycle progression and suppressed p27Kip1 expression (
In the present study, we found a significant accumulation of Skp2 in the nucleus and accumulation of KPC in the cytoplasm of GC cells. Our results are consistent with previous studies. It has been shown that the accumulation of p27Kip1 was increased in Skp2 defect-mice. Skp2 can interact with p27Kip1 ubiquitinating it (
In conclusion, our findings indicated that GAS promotes the introduction of MDR in GC cells by p27Kip1 degradation through a mechanism promoting phosphorylation levels of p27Kip1 on Thr (187) and Ser (
The authors would like to thank the members of the Second Affiliated Hospital of Xi'an Jiaotong University and Xi'an Central Hospital for providing technical support and helpful discussions concerning the present study.
The expression of gastrin in tissues and the gancer cell line SGC7901. M-R, moderate responders; N/P-R, non-responders or poor responders. (A) The mRNA levels of gastrin in non-drug-resistant tissues and drug-resistant tissues in 17 patients with gastric cancer. (B) The expression of gastrin was detected by immunohistochemistry in 17 patients with gastric cancer (×400); arrows indicate the positive marks. (C) The mRNA levels of gastrin in SGC7901 and drug-resistant gastric cancer cell lines SGC7901/VCR and SGC7901/ADR. (D) The effects of gastrin overexpression plasmid [pCDNA3.1(+) GAS] on mRNA level were tested in three cell lines. (E) The effects of pCDNA3.1(+)GAS on protein level of gastrin were tested in SGC7901, SGC7901/VCR and SGC7901/ADR cell lines. (F) The effects of gastrin silence plasmid (siGAS) on mRNA level of gastrin were tested in SGC7901, SGC7901/VCR and SGC7901/ADR cell lines. (G) The effects of siGAS on protein level of gastrin were tested in SGC7901, SGC7901/VCR and SGC7901/ADR cell lines.
Effects of pCDNA3.1(+)GAS or siGAS on the gastric cancer cell viability and apoptosis rates in SGC7901, SGC7901/VCR and SGC7901/ADR cell lines. (A) Cell viability was detected by MTT assay. (B) Cell viability was detected by MTT assay. (C) Cell apoptosis rates were detected by flow cytometry. (D) Cell apoptosis rates were detected by flow cytometry.
Effects of pCDNA3.1(+)GAS or siGAS on the multidrug resistance marker proteins in SGC7901/VCR and SGC7901/ADR cells and SGC7901 treated with TAX-VCR. (A and F) The expression of Bcl-2, MRP1 and Pgp were detected in the SGC7901 cells by western blotting. [(B–D) and (G–I)] Relative protein expression levels were quantified using ImageJ 1.43 software and normalized to β-actin. (E and J) Relative mRNA expression levels of Pgp and normalized to β-actin.
Gastrin promotes the degradation of p27Kip1 during the drug resistance of gastric cancer cells. (A-C) The protein level changes of p27Kip1 and the phosphorylation of p27Kip1 at Thr (187) and Ser (
Effects of pCDNA3.1(+)GAS and Skp2 silence (siSkp2) combination on cell viability and apoptosis of gastric cancer cell lines and the level changes of MRP1 and Pgp. (A) The protein level changes of p27Kip1 and the phosphorylation of p27Kip1 at Thr (187) and Ser (
Clinicopathological variables and the expression of GAS in GC patients.
Clinicopathological variable | Cases | GAS expression | t-value | P-value |
---|---|---|---|---|
Age (mean ± SD), 58.3±7.1 years (range) (48–71) | 0.828 | 0.421 | ||
<59 | 9 | 1.488±0.495 | ||
≥59 | 8 | 1.281±0.536 | ||
Sex | 1.050 | 0.310 | ||
Female | 7 | 1.56±0.51 | ||
Male | 10 | 1.69±0.26 | ||
Chemotherapy regimen | 1.428 | 0.174 | ||
5-Fluorouracil | 11 | 1.517±0.474 | ||
Irinotecan | 6 | 1.159±0.531 | ||
Docetaxel (DTX) | 17 | |||
Cisplatin (CDDP) | 17 | |||
Response to chemotherapy | 3.933 | 0.001 | ||
No or poor | 12 | 1.62±0.46 | ||
Moderate | 5 | 0.68±0.72 | ||
Metastasis | 0.475 | 0.642 | ||
Yes | 14 | 1.58±0.23 | ||
No | 3 | 1.20±0.70 |