Abstract
OBJECTIVE Recent studies have shown that hepsin, a type of transmembrane serine protease, is highly upregulated in prostate cancer, but, little is known about its role in progression and invasion of this cancer. We constructed a hepsin-expressing plasmid and transfected it into PC-3 cells to investigate the effect of the hepsin gene on the biological behavior of the PC-3 cells.
METHODS Plasmid pHepsin-IRES2 was transfected into prostate cancer PC-3 cells using Fugene6, and the cells with stable hepsin expression were screened and selected with Zeocin (600 mg/L). The hepsin mRNA level was measured by real-time PCR and the growth curve of the PC-3-transfected cells assessed using MTT and BrdU assays. A Boyden chamber was used to examine the difference in invasion and metastases between transfected and non-transfected cells.
RESULTS The hepsin mRNA level in pHepsin-IRES2 transfected -PC-3 cells was significantly higher than that found in the control PC -3 cells. While the growth curve of the hepsin gene transfected PC -3 cells showed that there was no significant effect on proliferation, the invasive ability of the pHepsin-IRES2 transfected PC-3 cells, as compared with control cells, was significantly increased (P<0.05).
CONCLUSION The results suggest that even though hepsin has no effect on the proliferation of prostate cancer PC-3 cells, it does promote cellular invasion and metastasis.Therefore hepsin may have a role in the development of prostate cancer.
keywords
Prostate cancer is the most common malignancy of men in the United States. In 2003, it was estimated that 220,900 new cases of prostate cancer were diagnosed, among which 28,900 men died.[1] The molecular mechanism leading to the development and progression of prostate cancer is not understood. Recent studies have shown increased hepsin expression in prostate cancers, and it has been identified as one of the most highly up-regulated genes,[2-4] but little is known about its role in prostate cancer development. In order to understand the functional consequences of hepsin expression in prostate cells, we constructed a hepisn gene-expressing plasmid which was transfected into PC-3 cells to investigate its biological effects in PC-3 cells.
MATERIALS AND METHODS
Ceil lines and vector
PC-3 and LNCap cells were obtained from the Cell Institute of the Chinese Medical Academy of Science, Shanghai, China. For routine maintenance, cells were cultured in 75 cm2 flasks at 37°C in humidified air containing 5% C02. The standard medium consisted of a 1:1 mixture of RPMI 1640 and Ham’s F-12, supplemented with 10% heat-inactivated fetal calf serum (FCS). A pIRES2-EGFP vector was obtained from BD Biosciences.
Chief reagents
3-(4, 5-dimethylthiazol-2yl)- 2, 5-diphenyl tetrazolium (MTT), RPMI 1640, and dimethylsulfoxide (DMSO) were obtained from Promega. The Fugene6 Transfection Reagent and BrdU kit were purchased from Roche. Diagnostics Boyden Chamber (pore size 8 μm; diameter 6.5 mm) were obtained from Coming Corstar. Zeocin was acquired from the Sigma Co.
Plasmid constructs
The full-length human hepsin sequence was amplified from cDNA which was obtained from LNCap cells. The EcoRI and Sail restriction sites were used to clone hepsin into pIRES2-EGFP. The 1.5-kb polymerase chain reaction (PCR) product was then ligated in the pIRES2-EGFP vector and the recombinant named pHepsin-IRES2. The plasmid expressing pHepsin-IRES2 was constructed and identified by sequencing.
Stable transfection assay
The PC-3 cells were seeded in 6-well plates (1.5×105 cells/well). When the cells reached 90% confluence, the 6-well plates were divided into 2 groups to transfect the pHepsin-IRES2 with empty vector used to obtain control cells. The Fugeneó reagent kit was used to transfect the cells. Cells were selected in the presence of Zeocin (600 mg/L) and cloned by limiting dilution to obtain hepsin stable transfectants. After 5 weeks, Zeocin-riesistent colonies were selected. The presence of stable hepsin expression was tested by real-time PCR assay. Total RNA was extracted with Trizol (Gibco-BRL) and used as the template for first-strand cDNA synthesis by random priming with reverse transcriptase. We mixed the cDNA with Sybr Green PCR Master Mix and analyzed the mixture by real-time PCR using the MJ Opticon II(MJ Research USA). Real-time PCR amplification was performed. Primers used for amplification were as follows : hespin 5'-CGG GAC CCC AAC AGC GAG GAG AAC-3' (sense); 5'-TCG GGG TAG CCA GCA CAG AAC ATC-3'(antisense); and GAPDH: 5'-ATG GGG AAG GTG AAG GTC GGA GTC (sense)-3’; 5'-GAC GCC TGC TTC ACC ACC TTC TTG-3' (antisene).
Measurement of cellular proliferation (MTT and BrdU assay)
Cells were plated in 24-well plates (1×104 cells/well) for measurement on days 1, 2, 3, 4, 5, and 6. Every day, 80 μ1 of the MTT solution was added to the culture medium and the PC-3 cells were subsequently incubated for 3 h. MTT-containing medium was then removed, and the formazan crystals formed within the cells were solubilized by the addition of 800 pi of DMSO with gentle agitation. The optical density of the solution was read at 540 nm. There were 6 replicates for each group and each experiment was repeated 4 times. In addition to the colorimetric assay, cell proliferation was assessed using the colorimetric 5-bromo-2'-deoxyuridine (BrdU) DNA incorporation assay. Approximately 2×103 PC-3 cells were seeded into 96-well microtiterplates. Then the cells were cultured in DMEM/Ham’s F12 (1:1) hybrid medium (GIBCO-BRL) containing 10% fetal bovine serum (FBS; GIBCO-BRL). Cellular proliferation by BrdU incorporation was also measured on days 1, 2, 3, 4, 5 and 6. Six-well cells were pulse-labeled with BrdU, fixed with FixDenat solution, and incubated (30 min). Fix-Denat was removed by tapping the microplates, followed by the addition of Anti-BrdU-POD and incubation for 90 min. The microplates were washed 3 times (15 min), then substrate added and the cells incubated for 20 min. Absorbance was measured using an ELISA plate reader. Data are expressed as percentages of control values (mean±SD).
Matrigel invasion assay
Inset chambers of a trans well system were coated with a dilution of 50 pg of Matrigel in 50 pi DMEM/F12, and the NIH3T3 cells cultured under serum-free conditions. The serum-free DMEM fluid was obtained with addition of chemotatic factor fluid (600 pl/well) in the lower compartment of the transwell system. After incubation for 14 h, the cells were fixed for 5 min using 4% formaldehyde followed by washing in PBS. Then the cells were stained with hematoxylin for 3 min and washed with tap water. Cells from the upper side of the inset were carefully removed using a cotton swab. After extensive washing, the membranes were dissected with a scalpel and placed on a glass slide with the upper side down. Coverslips were mounted and the number of cells penetrating the Matrigel was quantified by counting 5 visual fields at 200x and calculating the mean.
Statistical analysis
Statistical assessment was performed with SPSS 10.0 for Windows. The BrdU and MTT assays were evaluated using One-Way ANOVA. The invasive capacity of PC-3 cells in the Martrigel invasion assay was analyzed using a Student t-test. P value of <0.05 was considered significant.
RESULTS
The result of real-time PCR showed that the eukaryot-ic pHepsin-EGFP expression vector had successfully transfected the hepsin gene into the PC-3 cells. Quantitative real-time-PCR showed that hepsin expression was higher in the PC-3 cells transfected with pHepsin-IRES2, while the control PC-3 transfected empty vector did not express hepsin Fig.1.
PCR results of hepsin expression in PC3 stable cells transfected with pHepsin-EGFP. M Marker DL2000; 1,PC3 transfected with an empty vector plRES2; 2,PC3 transfected pHepsin-IRES2 cells.
The MTT colorimetric assay showed that proliferation of the PC-3 line was not enhanced by hepsin transfection, suggesting that hepsin was not sufficient to stimulate cell proliferation by itself Fig.2
The proliferation curve of the transfectied PC-3 cell line with hepsin and the control PC-3 cell line by the MTT assay.
Studies of proliferation by the ELISA and BrdU (colorimetric) assays showed that there was no significant difference between the control PC-3 and PC-3 cell with high hepsin expression Fig.3
The proliferation of hepsin transfected PC-3 cells and control PC-3 cells by the BrdU assay.
To determine whether hepsin affects the invasion of PC-3, we performed a Matrigel invasion assay. The membranes covered with PC-3 cells that had penetrated the Matrigel were 9.1 ±1.2 relative to 3.5 ±0.83 in controls (Figs. 4,5) This indicates that hepsin significantly up-regulates the invasive capacity of the PC-3 cells (F<0.01; Student’s t-test).
Invasion cells of stable hepsin transfected cells.
Invasion of control PC3 cells transfected with an empty vector.
DISCUSSION
Hepsin is a type of cellsurface-expressed chymotrypsin-like serine protease and a member of the family of type II transmembrane serine proteases.[5] The hepsin gene encodes a 417 amino acid single chain protein with a molecular mass of 51 kDa.[6] Several recent DNA microarrays and gene expression studies have revealed that hepsin is overexpressed in prostate cancers.[7] In situ hybridization demonstrated that hepsin is specifically overexpressed in prostate carcinoma cells compared to the adjacent stroma.[8] This indicates that hepsin can be a clinical biomarker, but its biologic role in prostate progression is elusive.
We constructed transfected PC-3 cells using a pHepsin-IRES2 vector and produced stable PC-3 cells expressing hepsin. Proliferation monitored by means of MTT colorimetric assay and BrdU demonstrated that hepsin had no significant effect on proliferation of the PC-3 cells. The Matrigel invasion assay was used to examine the PC-3 cellular invasion ability.The results demonstrated that hepsin significantly promoted invasion by the PC-3 cells
Degradation of the extracellular matrix (ECM) and basement membrane proteins is a critical event during local cellular invasion. On one hand, as hepsin is a serine protease, it is likely that it acts through proteolytic digestion of specific substrate proteins. On the other hand, hepsin may activate other proteases and thereby activate a proteolytic cascade resulting in degradation of the ECM. Results of our study showed that hepsin has no effect on PC-3 cellular proliferation, but it promotes its invasion ability. One recent study demonstrated that hepsin is a potent activator of pro-HGF, which suggests a functional link between hepsin and the HGF/Met pathway, therefore contributing to tumor progression.[9] It is clear that the HGF/Met pathway plays an important role in the progression of prostate and ovarian cancers,[10] but the precise role of hepsin in prostate cancer progression is still unknown. Studies emphasizing on the down-stream signaling pathway from hepsin and specific inhibitors of hepsin will aid in confirming its function.
- Received May 24, 2006.
- Accepted July 4, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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