Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Cancer Lett. 2011 Oct 28;309(2):228-35. doi: 10.1016/j.canlet.2011.06.006. Epub 2011 Jun 24.
Pemetrexed (PEM), a multitargeted antifolate with manageable toxicity, is active against non-squamous non-small cell lung cancer; however, most patients eventually acquire resistance to PEM. To elucidate the resistant mechanism, we established PEM-resistant lung adenocarcinoma cell lines. Two parental cell lines, PC-9 and A549, were treated with step-wise increasing concentrations of PEM. Growth inhibition was determined by the 3-[4,5-dimethyl-thizol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction. The four PC-9 sublines were more resistant than the PC-9 cell line to PEM (2.2-, 2.9-, 8.4-, and 14.3-fold, respectively). The four A549 sublines also showed more resistance to PEM (7.8-, 9.6-, 42.3-, and 42.4-fold, respectively) than the parent cell line. All resistant sublines showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine, 5-fluorouracil, or the active metabolite of irinotecan, SN-38. All PEM-resistant sublines expressed more TS than the parental cells, by polymerase chain reaction and Western blotting. DHFR was significantly increased in the four PEM-resistant A549 sublines. GARFT did not correlate with resistance to PEM. In summary, PEM-resistant cells remained sensitive to docetaxel, vinorelbine, 5-fluorouracil, and irinotecan. TS expression appeared to be associated with resistance to PEM.
培美曲塞（PEM）是一种多靶点抗叶酸药物，毒性可控制，对非鳞状非小细胞肺癌有效；然而，大多数患者最终会对 PEM 产生耐药性。为了阐明耐药机制，我们建立了 PEM 耐药的肺腺癌细胞系。将两个亲本细胞系 PC-9 和 A549 用逐步增加浓度的 PEM 处理。通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐测定法测定生长抑制率。通过定量实时逆转录聚合酶链反应分析编码胸苷酸合成酶（TS）、二氢叶酸还原酶（DHFR）和甘氨酰胺核糖核苷酸 formyltransferase（GARFT）的基因的表达。四个 PC-9 亚系对 PEM 的耐药性强于 PC-9 细胞系（分别为 2.2、2.9、8.4 和 14.3 倍）。四个 A549 亚系对 PEM 的耐药性也强于亲本细胞系（分别为 7.8、9.6、42.3 和 42.4 倍）。所有耐药亚系对顺铂均表现出交叉耐药性，但对多西他赛、长春瑞滨、5-氟尿嘧啶或伊立替康的活性代谢物 SN-38 均无耐药性。所有 PEM 耐药亚系的聚合酶链反应和 Western blot 均显示 TS 表达高于亲本细胞。DHFR 在四个 PEM 耐药的 A549 亚系中显著增加。GARFT 与 PEM 耐药性无关。总之，PEM 耐药细胞对多西他赛、长春瑞滨、5-氟尿嘧啶和伊立替康仍然敏感。TS 表达似乎与 PEM 耐药性相关。
