Sublethal Irradiation Promotes Migration and Invasiveness of Prostate Cancer PC-3 Cells:Implications for Radiotherapy of Human Prostate Cancer

Cell culture and reagents

The PC-3 prostate cancer cell line used in this study was provided by the Academy of Military Medical Sciences. The cells were maintained in RPMI-1640(Sigma Chemical Co.) supplemented with 10% fetal bovine serum at 37°C with humidified 95% air and 5% CO2. The number of cells was counted with a particle distribution counter.

Irradiation

The cells were irradiated with does of 1,3 or 5 Gy at room temperature using a ⁶⁰Co source(γ-rays, FC-7000A, ShangDong Xinhua Ltd. China).

Cell proliferation assay

Cell proliferation was evaluated by the MTT assay^[5]. Briefly, cells were seeded in 96-well plates at a density of 3×10⁴/well. After overnight incubation, the cells were irradiated and cultured for 7 days. DMSO(150 μl) was added to each well 4 hr after the addition of MTT (20 μl), and the culture media examined by a multiwell plate-reader at 490 nm. The cell proliferation rate was calculated based on the OD at 490 nm of each well at the time point indicated at the day of irradiation.

Matrigel invasion assay

Invassion of the PC-3 cells was measured by the invasion of cells through Matrigel-coated transwell inserts ^[6,7]. Briefly, transwell inserts with 8 μm pores were coated with Matrigel(40 μg/well). Cell suspensions(500 μl;1×10⁵/ml)were added to the upper chambers while 750 μl of the same medium was placed in the lower wells. Thereafter, the cells were irradiated and incubated for 24 h. Cells that had invaded into the lower surface of the Matrigel-coated membrane were fixed with 70% ethanol, stained with H&E, and counted in five random fields under a light microscope.

Flow cytometry

For the MMP2 and MMP9 analysis, the PC-3 cells were irradiated, washed with PBS, incubated with trypsin for 30 s at room temperature and harvested. The cells(5×10⁵) were incubated with 200 μl(1:50) of either MMP2 or MMP9 mouse monoclonal antibodies(Zhongshan Biotechnology Co Ltd. China) for 120 min at 4°C with protection from light. Then 100 μl(1:100) of mouse IgG isotype antibody with FITC(Zhongshan Biotechnology Co Ltd. China) was added followed by incubation for 45 min at 4°C in the dark. The cells were then washed twice with PBS and analyzed on a FITC calibur flow cytometer using Cell Quest acquisition and analysis software(Stata 7.0 American).

RT-PCR

Total RNA was purified with ISOGEN(Nippon Gene, Tokyo, Japan) RNA extraction reagent. Complementary DNA(cDNA) was made by reverse transcription(RT) of 1 μg total RNA for each assay by using a Ready-To-GoTMT-Primed First-Strand Kit(Amershan Pharmmacia Biotech, Inc., Piscataway, NJ). Each cDNA sample was amplified with 10 μl of PLATINUM polymerase chain reaction(PCR) Supermix(Life Technologies, Inc., Rockville, MD). The PCR reactions for glyceraldehydes-3-phosphate dehydrogenase(GAPDH; 94°C for 15s; 56°C for 30s; 72°C for 30s), MMP2 and MMP9 (94°C for 2min; 92°C for 30s; 62°C for 30s; 72°C for 1min) were carried out by using the following forward and reverse primers to detect MMP2, MMP9 and GAPDH(housekeeping gene, positive control). The primer pairs were as follows:

• MMP2

• 5ˋ-AGA GTT GGC AGT GCA ATA CC-3ˋ

• 5ˋ-TTG CCA TAC TTC ACA CGG AC-3ˋ

• MMP9

• 5ˋ-CGG AGC ACG GAG ACG GCT AT-3ˋ

• 5ˋ-TGA AGG GGA AGA CGC ACA GC-3ˋ

• GAPDH

• 5ˋ-ACC ACA GTC CAT GCC ATC AC-3ˋ

• 5ˋ-TCA CCA CCC TGT TGC TGT TA-3ˋ

Each of the amplified products for MMP2, MMP9 and GAPDH were of the expected sizes (520, 537 and 543 bp, respectively) as determined by 1.5% agarose gel electrophoresis.

Western blotting

The proteins (80 μg/lane) from the soluble fraction of the PC-3 cells were fractionated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was incubated with 1:500 dilutions of polyclonal antibody for human MMP2 and MMP9 (Zhongshan Biotechnology Co Ltd. China) and then probed with antigoat IgG conjugated with horseradish peroxidase (Zhongshan Biotechnology Co Ltd. China). Immunoblots were detected by enhanced chemiluminescence (Amersham International, Buckinghamshire, United Kingdom).

Statistical analysis

Statistical analyses were performed by using ANOVA and unpaired Student’s t test. All of the statistics were performed on a two-sided test. P<0.05 was considered as significant. Each experiment was repeated three times.