This article is mentioned in:

Introduction

Pancreatic cancer is an aggressive gastrointestinal tumor with a poor prognosis. Tissue hypoxia, due to uneven distribution of blood vessels, is a potent micro-environmental stress during tumor evolution and is a common feature of the majority of solid tumors (1). Several studies have demonstrated that hypoxia is associated with prevention of apoptosis, epithelial mesenchymal transition and angiogenesis of tumor cells, which promotes tumor proliferation and metastasis (2). Hypoxia may also reduce the efficacy of radiotherapy, chemotherapy and other therapeutic approaches (3).

The KAI1 gene was originally isolated in prostate cancer cells (4). Reduced KAI1 mRNA expression levels were reported to correlate with the formation of metastases in pancreatic and colorectal cancer (5,6). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase/Akt constitutes an important signaling pathway regulating multiple biological processes, including cell proliferation, apoptosis, metabolism and cell growth. Abrogated PTEN activity, through mutations, deletions or promoter methylation silencing, occurs at high frequency in numerous primary and metastatic human cancer types (7,8).

The aim of the present study was to imitate the hypoxic environment in the ASPC-1 pancreatic cancer cell line, and investigate the effects of tumor suppressor gene PTEN and tumor metastasis suppressor gene KAI1 double-transfection on the proliferation, metastasis and radiosensitivity of ASPC-1 cells under hypoxic conditions. This may provide a theoretical foundation for controlling pancreatic cancer cell proliferation and metastasis via combined gene therapy.

Materials and methods

Materials

The following materials were used in the present study: ASPC-1 cell line (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China), Dulbecco’s modified Eagle’s medium (Gibco-BRL, Carlsbad, CA, USA), fetal calf serum (Gibco-BRL), Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), plasmid extraction kit (Qiagen, Hilden, Germany), PTEN antibody (Abcam, Cambridge, MA, USA), KAI1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), MTT (Biyuntian, Shanghai, China), Giemsa (Biyuntian), Transwell chamber system (Corning Inc, Acton, MA, USA), Annexin V Apoptosis Detection kit (Chemicon, Temecula, CA, USA). The pEAK8 plasmid carrying the PTEN gene and adenovirus carrying the KAI1 gene were prepared according to procedures described previously (9,10).

Western blot analysis of PTEN and KAI1 protein overexpression in ASPC-1 cells under hypoxic conditions

The hypoxic environment was imitated by continuous mechanical ventilation with 1% O2, 5% CO2 and 94% N2 in a completely closed square box. ASPC-1 cells in logarithmic growth phase were cultured under hypoxic conditions for one week. Subsequent to cell proliferation and division, the cells were seeded into 6-well plates and transfected with pEAK8 plasmids carrying the PTEN gene and adenoviruses carrying the KAI1 gene using Lipofectamine 2000. Following screening and further cultivation of the recombinant cells, 2×106 cells were collected by trypsin and rinsed with phosphate-buffered saline (PBS) twice. The cell total protein was denatured at 95°C and quantified using the Bradford assay. A total of 50 μg denatured protein was separated on a 12% polyacrylamide gel by electrophoresis and transferred to nitrocellulose membranes. The primary antibodies (PTEN and KAI1 antibody) and secondary antibodies [goat anti-mouse IgG-horseradish peroxidase (HRP) and goat anti-rabbit IgG-HRP antibodies] were successively incubated. The blotted membranes were treated using the SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology Inc., Rockford, IL, USA) and signals were detected using a Las-4000 mini CCD camera (GE Healthcare, Buckinghamshire, UK). Enhanced chemiluminescence was used to develop the immunoblots. GAPDH served as an internal control to normalize PTEN and KAI1 expression levels.

MTT assay of ASPC-1 cell proliferation following double transfection with PTEN and KAI1 genes under hypoxic conditions

The ASPC-1 cells double-transfected by PTEN and KAI1 genes and the control cells were cultured under hypoxic conditions for 1 week and then cultured in 96-well plates (2×103 cell/well). MTT (5 mg/ml) was added to the wells (10 μl/well) on day two following transfection and the plate was incubated at 37°C for 4 h. The supernatants in the wells were removed and replaced with dimethyl sulfoxide (100 μl/well) for 5 min. The optical density (OD) value of each well was measured using a microculture plate reader (eLX-800; BioTek Instruments Inc., Winooski, VT, USA) at 490 nm. MTT detection was performed on five consecutive days and five parallel wells were designed at each time point. The cell proliferation curve was drawn using time as the X-axis and the OD value at A490 nm as the Y-axis.

Tumor colony-forming assay of ASPC-1 cells following double transfection with PTEN and KAI1 genes under hypoxic conditions

The ASPC-1 cells double-transfected by PTEN and KAI1 genes and the control cells were cultured under hypoxic conditions for 1 week and then transferred to 6-well plates (200 cells/well) with three parallel wells in each group. The cells were further cultured for another 14 days and changed with fresh medium every 3–4 days. The cells were rinsed twice at the end of the experiment and fixed in paraformaldehyde, followed by Giemsa staining for 10 min. Subsequent to washing with deionized water three times, the tumor colonies were counted in each well and images of the colonies were captured using a microscope (IX51; Olympus, Tokyo, Japan).

Transwell assay of ASPC-1 cells following double transfection with PTEN and KAI1 genes under hypoxic conditions

A transwell assay was performed according to the manufacturer’s instructions. Subsequently, 30% fetal calf serum medium was added to the lower chamber. Serum-free suspensions of ASPC-1 double-transfected cells and control cells were prepared and 2×104 cells were seeded into the upper chamber and incubated for 8 h in the incubator. The small chamber was turned upside-down and placed on absorbent paper to air-dry the medium. Non-invasive cells in the upper chamber were gently removed with a cotton swab and stained with Giemsa for 30 min, then, subsequent to rinsing several times, images of the cells were captured through microscopes (IX51; Olympus). The cells were then dissolved in 10% acetic acid to measure the OD value at 570 nm using a microplate reader (BioTek Instruments Inc.).

Annexin V flow cytometric assay of the apoptotic rate of X-ray-treated ASPC-1 cells

The ASPC-1 double-transfected cells and the control cells were cultured under hypoxic conditions and seeded into 6-well plates with three parallel wells for each group. When the cells reached 90% confluency, they were irradiated once at 8 Gy radiation dose (dose rate 2 Gy/min) by using an X-ray irradiator (MBR-1520R; Hitachi, Tokyo, Japan). The cells were cultured for a further 24 h and trypsinized. Following centrifugation and washing the cell precipitation with PBS, the cells were resuspended in 0.5 ml 1X binding buffer, 5 ml Annexin V-APC (BD Biosiences, Franklin Lakes, NJ, USA) at 1×106 cells/ml. The cells were incubated for 15 min at room temperature and then analyzed by flow cytometry (Cytomics™ FC500; Beckman Coulter, Miami, FL, USA).

Statistical analysis

All data are expressed as the mean ± standard deviation. Data were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The differences between groups were assessed with Student’s paired t-test and P<0.05 was considered to indicate a statistically significant difference.

Results

PTEN and KAI1 protein expression efficiency

Subsequent to transfection of the ASPC-1 cells with the PTEN and KAI1 genes, the expression levels of PTEN and KAI1 protein were significantly increased (Fig. 1). However, no significant differences were detected in PTEN and KAI1 protein expression levels between the control cells transfected with empty vector and the ASPC-1 blank control cells.

Figure 1 (A) PTEN and (B) KAI1 protein expression efficiency in ASPC-1 pancreatic cancer cells, analyzed by western blotting. Blank, blank control; CTRL, negative control transfected with empty vector; OE, cells double-transfected with PTEN and KAI1 genes.

ASPC-1 cell growth curve following double-transfection with PTEN and KAI1 under hypoxic conditions

The ASPC-1 cells were separately co-transfected with PTEN and KAI1 overexpression vectors or empty vectors, and an MTT assay was performed to detect the OD value in each group 1–5 days after transfection. Statistical analysis found that the proliferation rate of ASPC-1 cells co-transfected with PTEN and KAI1 over-expression vectors was significantly reduced, compared with that of the control group (Fig. 2). Notably, the OD values at 2–5 days after transfection were significantly lower than those in the control cells transfected with empty vectors (P<0.05).

Figure 2 Proliferation of ASPC-1 pancreatic cancer cells under hypoxic conditions. CTRL, negative control cells transfected with empty vector; OE, cells double-transfected with PTEN and KAI1 genes; *P<0.05.

Tumor colony-forming assay of ASPC-1 cells following double transfection with PTEN and KAI1 genes under hypoxic conditions

The results of the tumor colony-forming assay are shown in Fig. 3A. The tumor colony-forming ability was significantly inhibited in ASPC-1 cells transfected with PTEN and KAI1 genes compared with the ASPC-1 cells transfected with the empty vector. Giemsa staining and counting further confirmed that the number of tumor cells forming tumor colonies with >50 cells per colony was significantly reduced (P<0.05; Fig. 3B). The number of cells in single clones of ASPC-1 cells transfected with PTEN and KAI1 genes was significantly reduced compared with that of the control group (P<0.05).

Figure 3 Colony-forming ability of ASPC-1 pancreatic cancer cells under hypoxic conditions. (A) Colony formation was observed under a microscope 2 weeks after incubation. (B) The number of colonies was calculated. Representative results from three independent experiments. CTRL, cells transfected with empty vector (negative control); OE, cells double-transfected with PTEN and KAI1 genes; *P<0.05, compared with control.

Transwell assay of ASPC-1 cells following double transfection with PTEN and KAI1 genes under hypoxic conditions

In the present study, the tumor cell migration ability of the cells was evaluated using a Transwell assay. Giemsa staining of the migrated metastatic cells in the Transwell chamber revealed that the migratory ability of ASPC-1 cells transfected with PTEN and KAI1 genes was significantly reduced compared with that of the ASPC-1 cells transfected with empty vector (Fig. 4A). The cell migratory rate was calculated by the ratio between the OD value of migrated cells at the bottom of the chamber and the OD value of cells when seeded. This cell migratory rate was significantly reduced following double-transfection of ASPC-1 cells with the PTEN and KAI1 genes (P<0.05; Fig. 4B).

Figure 4 Migration of ASPC-1 pancreatic cancer cells under hypoxic conditions. (A) Migration of ASPC-1 pancreatic cancer cells was observed under a microscope. Cells were stained with Giemsa as described in Materials and methods. (B) The invasion rate was determined by using a microplate reader as described in Materials and methods. Representative results of three independent experiments are shown. CTRL, cells transfected with empty vector (negative control); OE, cells double-transfected with PTEN and KAI1 genes; *P<0.05, compared with control.

Annexin V flow cytometric assay of the apoptotic rate of X-ray-treated ASPC-1 cells

Using Annexin V staining of the cell membrane as an apoptotic index, the apoptotic rate of ASPC-1 cells double-transfected with PTEN and KAI1 genes or ASPC-1 cells transfected with empty vector was analyzed by flow cytometry (Fig. 5A). The results revealed that the apoptotic rate of ASPC-1 cells transfected with PTEN and KAI1 genes was significantly increased, when compared with that of the control cells (P<0.05; Fig. 5B). This demonstrated that PTEN and KAI1 genes promoted ASPC-1 cell apoptosis during the X-ray treatment process.

Figure 5 Apoptotic rate of X-ray treated ASPC-1 pancreatic cancer cells under hypoxic conditions. (A) Apoptosis of ASPC-1 cells, which were stably transfected with empty vector or co-transfected with PTEN and KAI1 genes. (B) Percentage of apoptotic ASPC-1 cells. CTRL, cells transfected with empty vector (negative control); OE, cells double-transfected by PTEN and KAI1 genes; *P<0.05, compared with control.

Discussion

With improvements in lifestyle and an aging population, the morbidity of pancreatic cancer has notably increased worldwide. In 2008, an estimated 37,680 cases of pancreatic cancer were diagnosed in the USA with 34,290 fatalities (11). The clinical manifestation of pancreatic cancer commonly presents as non-specific symptoms, thus the majority of patients are diagnosed with advanced or locally advanced pancreatic cancer, which is unresectable. With the development of radiotherapy, three-dimensional conformal radiotherapy and intensity-modulated radiation therapy have been recently recommended as the main therapeutic approaches for pancreatic cancer. However, the blood supply is commonly uneven during the tumor growth process and thus hypoxia may occur, which reduces the efficacy of radiotherapy, and promotes tumor proliferation and metastasis (12,13). Accounting for these factors, the effect of combination gene therapy on the proliferation and metastasis of pancreatic cancer cells was investigated in the present study.

PTEN is a conservative tumor suppressor gene identified following the identification of p53, which is closely associated with tumor progression. PTEN is located on chromosome 10q23.3, contains nine exons and eight introns, and has been shown to exert a pivotal regulatory role in cell cycle arrest, cell proliferation and possibly cell migration suppression (14–16). Previous studies have demonstrated that the PTEN gene arrested ASPC-1 cell growth at the G2/M =phase, promoted hypoxia-induced cell apoptosis and X-ray-induced G2/M phase cell arrest, and inhibited ASPC-1 cell proliferation under normoxic and hypoxic conditions (17,18).

KAI1/CD82, a tumor suppressor gene, was first isolated from a prostate carcinoma cell line in 1995 (4). KAI1 is a member of the transmembrane-4 superfamily and encodes a 29.6-kDa transmembrane glycoprotein, which is important in regulating cell motility and differentiation, and inhibiting tumor metastasis (19). Previous studies have demonstrated that the KAI1 gene is closely associated with pancreatic cancer metastasis; pancreatic cancer cell growth and migratory ability were significantly restrained following transfection with the KAI1 gene (20,21). In vivo studies also revealed significantly reduced lesion metastasis number and size in liver and lung mouse tumors following injection with a KAI1-expressing plasmid, compared with that in the control group (20–22).

Tumor proliferation and metastasis involve an interaction network among multiple genes. Previous studies regarding the regulatory effect of PTEN and KAI1 transfection on the proliferation and metastasis of pancreatic cancer focused only on single-gene efficacies (9,22). Thus, the effect of combination gene therapy in pancreatic cancer progression and development has not been examined. In addition, the hypoxic conditions that affect the prognosis and treatment sensitivity of pancreatic cancer are rarely investigated. In the present study, ASPC-1 cells double-transfected with PTEN and KAI1 genes under hypoxic conditions were selected to use in the experiments. Hypoxic conditions partially emulate the natural growth environment of tumor cells. This combination gene therapy may provide a theoretical foundation its use in clinical applications.

In conclusion, the results of the present study demonstrated that double transfection with PTEN and KAI1 genes significantly inhibited ASPC-1 cell proliferation and colony formation, reduced invasion and metastasis, promoted X-ray induced tumor cell apoptosis and improved radiosensitivity. However, further in vivo studies are required to confirm these results.

Acknowledgements

This study was supported by the Liaoning Province Natural Science Foundation of China (grant no. 201102238) and the Scientific Research Fund of Liaoning Province Education Department (grant no. L2010627).

References