叶酰聚谷氨酸合成酶基因在甲氨蝶呤对映体获得性耐药A549细胞株中的表达差异

目的 研究不同甲氨蝶呤(MTX)对映体耐药与叶酰聚谷氨酸合成酶(FPGS)基因水平表达的关系.方法 用大剂量冲击递增结合低剂量持续诱导法诱导获得两组含不同构型15～55μmol/L浓度的MTX对映体[L-(+)-MTX和D-(-)-MTX]耐药的细胞系,细胞为人源非小细胞性肺癌A549细胞,用四甲基偶氮唑盐(MTT)法检测各细胞系的耐药指数;用实时荧光定量聚合酶链反应(RFQ-PCR)方法检测这两组各细胞系中胞质型FPGS(cFPGS)和线粒体型FPGS(mFPGS)基因的相对含量.结果 D-(-)-MTX耐药细胞组耐药指数高于L-(+)-MTX耐药细胞组(32.7±9.3比11.5±2.9,P＜0.05),L-(+)-MTX/A549细胞系耐药指数均在5～15之间,为中度耐药,而D-(-)-MTX/A549细胞系耐药指数均＞15,为高度耐药.在D-(-)-MTX和L-(+)-MTX两组耐药细胞系中,mFPGS表达水平仪在MTX为15 μmol/L时差异无统计学意义,在MTX其他各浓度点两组间差异均有统计学意义(25 μmol/L:2.3±0.9比1.3±0.7,35 μnol/L:2.6±0.3比1.1±0.9,45 μmol/L:1.4±0.8比1.0±1.0,55 μmol/L;1.0±0.2比0.2±0.1均P＜0.05);cFPGS表达水平在MTX为15μmol/L时两组间差异也同样无统计学意义,在25～55 μmol/L浓度区间内D-(-)-MTX/A549细胞系的cFPGS表达与耐药指数呈现高度负相关(r=-0.95,P＜0.05).结论 在A549细胞中MTX对映体初次剂量15 μmol/L冲击法诱导获得的对映体耐药与再次接受更大剂量(≥25 μmol/L)MTX诱导获得耐药的机制不同,D-(-)-MTX/A549耐药细胞系表现为更高的耐药性,提示临床使用MTX时应考虑该药物存在手性对映体问题.

Abstract：

Objective To investigate the relationship between the resistance of methotrexate (MTX) enantiomer and the gene expression levels of folylpolyglutamate synthetase (FPGS).Methods The cell lines of MTX enantiomer resistance from 15 -55 μmol/L were obtained when the A549 cell lines were exposed intermittently and progressively to an incremental dose of each MTX enantiomer.The resistant index of MTX resistance cell lines were detected by MTT.The gene expressions of FPGS in cytoplasm and mitochondria were detected by real-time quantitative polymerase chain reaction (PCR).Results The resistance indice of D-( - )-MTX resistant cell lines were higher than those of L-( + )-MTX resistant cells (32.7±9.3 vs 11.5 ±2.9,P ＜0.05).The resistant indice of L-( + )-MTX /A549 were from 5 to 15,which mean the middle resistance.The resistant indice of D-( - )-MTX/A549 were more than 15,which mean the severe resistance.The expression of mFPGS had difference between resistant cell lines of L-( + )and D-( - )-MTX except at 15 μmol/L MTX (at 25 μmol/L,1.3 ±0.7 vs.2.3 ±0.9;at 35 μmol/L,1.1 ±0.9 vs.2.6±0.3;at 45 μmol/L,1.0±1.0 vs.1.4±0.8;at 55 μmol/L,0.2±0.1 vs.1.0±0.2;all P＜0.05).The expressions of cFPGS had no difference between resistant cell lines of L-( + ) and D-( - )-MTX at 15 μmol/L MTX,while at 25 -55 μmol/L,the cFPGS levels and resistance indice of D-( - )-MTX/A549 resistant cell lines showed a highly negative correlation ( r = - 0.95,P＜0.05 ).Conclusion There may be a different mechanism between the first time treatment with 15 μmol/L dosage and the continual treatment with more than 25 μmol/L dosage in A549 cell lines.There had higher resistant index in D-( - )-MTX/A549 cell line than in L-( + )-MTX/A549 cell line.The results indicated that the difference in chirality should be considered in clinical treatment with MTX.

Differential gene expression of folylpolyglutamate synthetase in cytoplasm and mitochondria in acquired methotrexate enantiomers resistant to lung cancer A549 cell lines

Objective To investigate the relationship between the resistance of methotrexate (MTX) enantiomer and the gene expression levels of folylpolyglutamate synthetase (FPGS).Methods The cell lines of MTX enantiomer resistance from 15 -55 μmol/L were obtained when the A549 cell lines were exposed intermittently and progressively to an incremental dose of each MTX enantiomer.The resistant index of MTX resistance cell lines were detected by MTT.The gene expressions of FPGS in cytoplasm and mitochondria were detected by real-time quantitative polymerase chain reaction (PCR).Results The resistance indice of D-( - )-MTX resistant cell lines were higher than those of L-( + )-MTX resistant cells (32.7±9.3 vs 11.5 ±2.9,P ＜0.05).The resistant indice of L-( + )-MTX /A549 were from 5 to 15,which mean the middle resistance.The resistant indice of D-( - )-MTX/A549 were more than 15,which mean the severe resistance.The expression of mFPGS had difference between resistant cell lines of L-( + )and D-( - )-MTX except at 15 μmol/L MTX (at 25 μmol/L,1.3 ±0.7 vs.2.3 ±0.9;at 35 μmol/L,1.1 ±0.9 vs.2.6±0.3;at 45 μmol/L,1.0±1.0 vs.1.4±0.8;at 55 μmol/L,0.2±0.1 vs.1.0±0.2;all P＜0.05).The expressions of cFPGS had no difference between resistant cell lines of L-( + ) and D-( - )-MTX at 15 μmol/L MTX,while at 25 -55 μmol/L,the cFPGS levels and resistance indice of D-( - )-MTX/A549 resistant cell lines showed a highly negative correlation ( r = - 0.95,P＜0.05 ).Conclusion There may be a different mechanism between the first time treatment with 15 μmol/L dosage and the continual treatment with more than 25 μmol/L dosage in A549 cell lines.There had higher resistant index in D-( - )-MTX/A549 cell line than in L-( + )-MTX/A549 cell line.The results indicated that the difference in chirality should be considered in clinical treatment with MTX.