The GPI-PLD Overexpression And Release Of GPI-anchored PSCA To Prostate Cancer Cell And Its Biological Effects

Objective1. To preliminaryly research the effect on overexpression of GPI-PLD gene in prostate cancer cell line PC-3.2. To investigate changes in cell biological functions after GPI-PLD release of GPI-anchored the prostate stem cell antigen (PSCA).3. To explore the action of PSCA in prostate cancer onset, to use of GPI-PLD gene therapy for prostate cancer and other neoplastic diseases as well as laying a solid foundation.Methods1. The GPI-PLD enzyme activity was mearsured after collected peripheral blood serum from26cases of normal human and18cases of patients with prostate cancer.2. The eukaryon expression vector pcDNA3.1(+)/GPI-PLD produced by our laboratory was transfected into PC-3cells by Sofast cationic polymer transfection, PC-3transfected with pcDNA3.1(+) and PC-3untransfected were used as control. The positive cell clones for stable expression of GPI-PLD were got after screening with800μg/mL G418.3. Expression of GPI-PLD mRNA in PC-3cells was detected by RT-PCR. With placental alkaline phosphatase (PLAP) which produced by our laboratory as the substrate, the GPI-PLD activity was estimated. The PSCA released from cells membrane before and after transfection with pcDNA3.1(+)/GPI-PLD were detected by ELISA. GPI-anchored PSCA on cell membrane before and after transfection with pcDNA3.1(+)/GPI-PLD were analyzed by flow cytometer. The cell cycle and apoptosis were detected by flow cytometer. The cell growth curve was measured by cell counting method.Results1. The GPI-PLD activity with the percentage to conversion of substrate PLAP in peripheral blood serum from prostate cancer patients and normal control group were29.79%±4.13%and38.27%±5.73%, respectively. The GPI-PLD activity from prostate cancer patients than from normal control group was significantly decreased, the difference was statistically significant (P<0.01).2. RT-PCR results and GPI-PLD enzyme activity showed that the PC-3cell line with steady overexpression of GPI-PLD gene was established.①Compared with pcDNA3.1(+)/PC-3cells and PC-3cells, the expression of GPI-PLD mRNA was significantly up-regulated in pcDNA3.1(+)/GPI-PLD/PC-3cells (P<0.01) with IA valued0.976±0.041while the other two groups were0.549±0.032and0.522±0.047.②Compared with pcDNA3.1(+)/PC-3cells and PC-3cells, GPI-PLD enzyme activity was signally increased in pcDNA3.1(+)/GPI-PLD/PC-3cells, the GPI-PLD activity (the percentage of PLAP transformation) in pcDNA3.1(+)/GPI-PLD/PC-3cells was24.47%±6.89%while the other two groups were11.63%±6.04%and10.10%±5.38%, separately (P<0.01).3. ELIS A assay for the detection of cell culture medium supernatant concentrations of PSCA released from GPI-anchored PSCA on cell membrane revealed that the concentration of PSCA in culture medium supernatant from pcDNA3.1(+)/GPI-PLD/PC-3cells was significantly increased and the value was146.96+3.32pg/mL, while that from pcDNA3.1(+) group and the untransfected group of this one value respectively90.66+3.19pg/mL and89.24+1.77pg/mL, differences are statistically significant (P<0.01).4. Flow cytometry detection of cell surface GPI anchoring protein PSCA levels showed, compared with pcDNA3.1(+)/PC-3cells and PC-3cells, the average fluorescence intensity was significantly reduced in pcDNA3.1(+)/GPI-PLD/PC-3cells, statistical significant differences (P<0.01). The values of average fluorescence intensity of pcDNA3.1(+)/GPI-PLD/PC-3cells was59.79±0.92, while the other two of the control group were85.63±1.30and85.97±1.23, separately.5. Cell cycle detected by flow cytometry showed that, compared with pcDNA3.1(+)/PC-3cells and PC-3cells, pcDNA3.1(+)/GPI-PLD/PC-3cells significantly increased in G0-G1phase, markedly decreased in S phase, and weakly decreased in M phase, the difference was statistically significant (P<0.05). Cell count and cell growth curve showed that the growing speed of pcDNA3.1(+)/GPI-the PLD/PC-3cell groups was significantly slowed down compared with pcDNA3.1(+)/PC-3cell groups and PC-3cell groups At the same time, the results showed that apoptosis of pcDNA3.1(+)/GPI-PLD/PC-3cell group relative to the other two groups, the apoptotic rate was significantly increased, the difference was statistically significant (P<0.01).Conclusion1. The GPI-PLD activity in peripheral blood serum from prostate cancer patients compared with normal people was significantly reduced, suggesting that GPI-PLD gene expression level in prostate cancer patients is down-regulated.2. Successfully constructed the PC-3cell line with stable overexpression of GPI-PLD gene.3. The GPI-PLD stable overexpression in PC-3cell line is able to significantly increase the GPI-PLD enzyme activity and induce the release of GPI-anchored PSCA on the cell membrane, therefore increase the level of lose-anching PSCA in the culture supernatants and reduce the GPI-anchored PSCA level on the membrane.4. The overexpression of GPI-PLD gene in prostate cancer PC-3cell lines is able to inhibit its cell cycle, increase its apoptosis rate and delay cell growth.