多种人结肠癌5-氟尿嘧啶耐药细胞株的建立及耐药机制初步研究

目的:通过建立多种5-氟尿嘧啶(5-FU)耐药的人结肠癌细胞株,探讨耐药的结肠癌细胞的生物学特性与耐药机制。方法:选用人结肠癌HT-29、LoVo和SW480细胞,通过高浓度5-FU反复接触结合药物浓度递增法建立耐药株HT-29/5-FU、LoVo/5-FU和SW480/5-FU。不同浓度5-FU作用所建立的耐药细胞株及其亲本细胞后,分别用MTT法、流式细胞术、qRT-PCR、Westernblot检测细胞对5-FU的敏感性、周期分布、耐药相关分子P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)、ATP结合盒超家族G成员2(ABCG2)]及第十号染色体缺失的磷酸酶和张力蛋白同源物(PTEN)与蛋白激酶B(Akt)的mRNA和蛋白的表达,并用Akt活性检测试剂盒检测细胞Akt活性。结果:与各自的亲本细胞比较,构建的HT-29/5-FU、LoVo/5-FU和SW480/5-FU对5-FU的IC50均明显升高(均P0.05),耐药指数分别为7.213、5.849和15.940。随着5-FU处理浓度的升高,亲本细胞和耐药细胞G0/G1期细胞数量均明显增加(均P0.05),但同一浓度5-FU处理下,各耐药株G0/G1期细胞数量均明显少于其对应亲本株(均P0.05)。与各自的亲本细胞比较,对应耐药株的P-gp、MRP1、ABCG2、Akt的mRNA和蛋白表达水平均明显升高,而PTEN表达均明显降低(均P0.05),且Akt活性均明显提高(均P0.05)。结论:成功建立了结肠癌5-FU耐药细胞株,其耐药机制可能与PTEN下调所致的PI3K/Akt降低PI3K/Akt通路活化有关。

Establishment of several human colon cancer cell lines resistant to 5-fluorouracil and preliminary analysis of the mechanism for drug resistance

Objective: Through establishing several types of 5-fluorouracil (5-FU) resistant human colon cancer cell lines, to investigate the biological characteristics of the 5-FU resistant colon cancer cells and the mechanism for drug resistance. Methods: Using human colon cancer HT-29, LoVo and SW480 cells, the 5-FU resistant colon cancer cell lines HT-29/5-FU, LoVo/5-FU and SW480/5-FU were established by repeated exposure to excessive and increasing concentrations of 5-FU. In the established drug-resistant cell lines and their parent cells after treatment with different concentrations of 5-FU, the sensitivities to 5-FU, the cell cycle distributions, the mRNA and protein expressions of drug resistance-related molecules P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette superfamily G member 2 (ABCG2)] and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and protein kinase B (Akt) were determined by MTT assay, flow cytometry, qRT-PCR and Western blot respectively, and the Akt activities were measured by Akt kinase assay kit. Results: Compared with their corresponding parent cell line, the IC50 values to 5-FU in HT-29/5-FU, LoVo/5-FU and SW480/5-FU were all significantly increased (all P<0.05), with the drug resistance indexes of 7.213, 5.849 and 15.940, respectively. The numbers of cells in G0/G1 phase in both drug-resistant cell lines and their parent cell lines were increased with the increase of the treatment concentration of 5-FU (all P<0.05), but the number of cells in G0/G1 phase in each drug-resistant cell line was significantly less than that in their respective corresponding parent cell line under the same concentration of 5-FU (all P<0.05). Compared with their corresponding parent cell line, the mRNA and protein expressions of P-gp, MRP1, ABCG2 and Akt were significantly increased and the mRNA and protein expressions of PTEN were significantly decreased in all the drug-resistant cell lines (all P<0.05), and the Akt activities were significantly enhanced (P<0.05). Conclusion: The 5-FU resistant colon cancer cell lines are successfully established. The mechanism for drug-resistance may probably be associated with the activation of PI3K/Akt pathway resulted from PTEN down-regulation.