Liu Jian, Zhang Ji-min, Gao Qing, Wang Qi-wen, Ye Dian-jun, Liu Ying
Department of Gastrointestinal Surgery, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China.
Zhonghua Wai Ke Za Zhi. 2013 Jul;51(7):636-40.
To study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells.
TP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay.
The colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01).
The stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.
研究转染胸苷磷酸化酶（TP）基因的人结肠癌细胞系SW480和LOVO中5'-脱氧氟尿苷单磷酸（5'-DFUR）转化为氟尿嘧啶（5-FU）的能力变化。并探讨5'-DFUR对SW480和LOVO细胞的抗癌活性。
用慢病毒载体pLenti6.3_MCS_IRES2-EGFP将TP cDNA转染到人结肠癌细胞系SW480和LOVO中。通过流式细胞仪分析转染效率，用RT-PCR检测TP的mRNA表达，用蛋白质免疫印迹法检测TP蛋白表达，并用高效液相色谱法（HPLC）检测两种细胞及其培养基中5'-DFUR转化为5-FU的量。通过MTT法评估5'-DFUR对这两种野生型和TP转染细胞的结肠癌细胞系的50%抑制浓度（IC50）。
对转染人TP cDNA的结肠癌细胞系SW480和LOVO进行了5代监测，转染效率约为95%。与野生型细胞SW480和LOVO相比，SW480-TP和LOVO-TP的mRNA表达RQ值分别为（695±171）倍（t=-7.00，P=0.002）和（282±87）倍（t=-5.61，P=0.030）。蛋白质免疫印迹显示，SW480-TP和LOVO-TP中的TP蛋白表达也高于其亲本细胞。与亲本细胞相比，培养基中培养的SW480-TP和LOVO-TP中5'-DFUR转化为5-FU的量分别增加（t=19.406-66.921，P<0.01），而在野生型和TP转染细胞中均未检测到5-FU。转染TP cDNA后，5'-DFUR对SW480-TP和LOVO-TP的IC50分别为（587±17）μmol/L和（1088±89）μmol/L，显著低于其亲本细胞（t=-32.59和-8.52，P<0.01）。
用慢病毒载体可构建稳定转染且TP表达较高的SW480和LOVO细胞。将TP cDNA转染到SW480和LOVO中，可提高TP mRNA和TP蛋白的表达，增加培养基中5'-DFUR转化为5-FU的量，并增强对这两种细胞的抗癌作用。
