Combination of TMPRSS2 promoter regulation of CD-5-FC gene system and phytochemical drugs effect prostate cancer 22RV1 in vitro

Abstract

Objective To explore the effects of TMPRSS2 promoted suicide gene CD-5-FC combined with phytochemicals on the proliferation and apoptosis of prostate cancer (PCa) specific cell-line 22RV1. Methods From March 2016 to October 2016'TMPRSS2-VISA-CD/UPRT' vector was used to deliver pro-drug 5-FC(5-Fluorocytosine) in to PCa specific cell-line 22RV1. Transfection effect was verified by Western-blotting. 5-FC, 5-FU(5-Fluorouracil) and four phytochemicals epigallocatechin gallate(ECGC), Genistein, Daidzein, and Equol were selected in this study. MTS assay was performed to select dosages, which can kill 10%-40% of 22RV1 cells, from their pre-set drug concentrations, respectively. According to their select concentrations, either 5-FC plasmid or 5-FU in combination with one of four phytochemicals were added in 22RV1 cells in orders, and then cultured them together. Besides, these four phytochemicals were added in 22RV1 cells individually regarding their selecting concentrations and cultured at the same time. Cell viability was detected by MTS assay on 24hrs, 48hrs and 72hrs. Comparing the cell killing rates between the combination groups and each single drug groups on cell-line 22RV1. Results 'TMPRSS2-VISA-CD/UPRT' plasmid with 5-FC was transfected on PCa specific cell-line 22RV1 successfully. The most obvious transfection occurred on 48hrs. The selected concentration of the pro-drug 5-FC were 83.32 μmol/L and 833.20 μmol/L, 5-FU were 76.87 mol/L and 768.70 μmol/L. The concentrations of four phytochemical agents were 2.00 μmol/L, 10.00 μmol/L and 20.00 μmol/L. In term of the cell killing numbers, results showed that the combination treatment of 83.32 μmol/L 5-FC/76.87μmol/L 5-FU and 10.00 μmol/L/20.00 μmol/L four phytochemicals compared with 10.00 μmol/L/20.00 μmol/L single treatment of four phytochemicals, respectively, there was no significant difference (P>0.05), while the combination treatment of 833.20 μmol/L 5-FC/768.70 μmol/L 5-FU and 2.00 μmol/L/10.00 μmol/L four phytochemicals compared with 2.00 μmol/L/10.00 μmol/L single treatment of four phytochemicals, respectively, there was a significant difference (P 0.05). Thus the suicide gene transfection was successful, and its function was the same as the effect of cytotoxic drugs. Conclusions This study has proved that this plasmid with 5-FC suicide gene system can be successfully and efficiently transfected into PCa cells 22RV1. As the pro-drug of 5-FU, 5-FC got similar treatment effect with 5-FU, and inhibited cell proliferation. There was no synergistic reaction of enhancing cell apoptosis after combined phytochemical drugs EGCG, Genistein, Daidzen, Equol with suicide gene therapy on 22RV1 cell-line. Key words: Prostate cancer; Suicide gene; Phytochemicals