| Objective Thymidylate synthase (TS) is the only limiting enzyme in the de novo synthesis of thymine nucleotide, one of the precursors for DNA synthesis. Decrease in TS catalytic activity directly leads to inhibition of DNA synthesis and cell growth, which makes the TS an ideal target for development of anticancer drugs. Nolatrexed is a newly-developed non-classic folate analog, showing potent antiproliferative effect on many kinds of tumors in phase II clinical trials. Although a few studies of the antiproliferative mechanisms of Nolatrexed have demonstrated that the drug takes effect mainly by inhibiting TS, the dynamic changes at TS mRNA and protein levels are yet unclear. In this study, whether there is difference in the cellular uptake of the drug between different kinds of cell lines was determined and the dynamic changes in the levels of TS mRNA and protein expression in cells treated with Nolatrexed was also addressed in an attempt to explore the diversity of antiproliferative effect of the drug.Methods Rat C6 cell line, mouse SRS-82 cell line, human LoVo cell line, and human normal Lo2 cell line were used in this study. The sensitivity of the 4 cell lines to Nolatrexed was determined by MTT assay. At the level of cell membrane, the intracellular concentrations of Nolatrexed in the 3 tumor cell lines treated with the drug in different concentrations for indicated time intervals were determined by HPLC to investigate if there is difference in cellular uptake between tumor cell lines. At the TS mRNA level, all cell lines were exposed to nolatrexed in different concentrations for indicated time intervals and then total RNA was obtained for RT-PCR. TS and B -actinmRNA were amplified and the dynamic changes in TS mRNA were reflected by TS/B - actin ratio. At the protein level, TS expression in LoVo and Lo2 cells treated with nolatrexed in different concentrations for indicated time intervals was evaluated by flow cytometry, confocal microscopy and Western blot'.Results (1) The MTT assay revealed that the ICso values for Nolatrexed to inhibit growth of C6, SRS-82, LoVo and Lo2 cells were 0.37+0.09 u mol l-1, 2.50 +0.42 u mol l-1, 5.10 + 0.78 u mol l-1 and 54.90 + 4.70 u mol l-1, respectively. And ICpo values of C6, SRS-82, LoVo, Lo2 cells were 0.60+0.11 u mol l-1, 4.90 +0.42 u mol l-1, 10.46+1.51 u mol l-1 and 79.10+ 5.80 u mol 1-1, respectively. Compared with the normal cell line Lo2, the three tumor cell lines were mori sensitive to Nolatrexed and C6 was by far the most. IC50 values of'SRS-82 and LoVo were 6.8 and 13.8 fold'higher than that of C6 (PO.05). i(2) The cellular uptake mode of Nolatrexed in the three tumor cell lines was similar in quality as shown by HPLC. The intracellular drug concentration reached steady state within 5 minutes. After treatment of the cells with Nolatrexed in-concentrations ranged from 0 to 40 uM for 10 minutes, the intra- and extra-cellular drug concentrations had a linear relationship in all the three tumor cell lines. The intracellular Nolatrexed concentration in C6 cells was significantly higher than that in the other two cell lines (PO.05), namely, 4.96 and 4.52 fold higher than that of LoVo and SRS-82 cells, respectively. Between the LoVo and SRS-82 cells, their intra-cellular steady concentrations had no statistical significance. (3) The RT-PCR results demonstrated that in the time course of the 4 cell lines, except for normal Lo2 in which the TSmRNA maintained almost unchanged, TS/P -actin ratio in drug-treated cells as compared with control did has dynamicchange. Interestingly, there were different changing patterns in the three tumor cell lines, the ratio in C6 was elevated at first, and then reached a plateau, the ratio in SRS-82 was initially increased, and the went down to form a peak, whereas the ratio in LoVo were up all along within the time period. In the dosing group, except for LoVo in which the ratio had no obvious change, the changing patterns of the ratio in the other three cell lines were similar, that is, treatment with Nolatrexed in concen...
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