甲氨蝶呤对映体诱导肺癌细胞耐药后引起血管内皮细胞分化差异的研究

Chiral selectivity in differentiation of lung cancer A549 cells to vascular endothelial cells after drug resistance induced by D- or L-methotrexate enantiomers

目的研究甲氨蝶呤（MTX）对映体大剂量冲击作用肺癌A549细胞后，MTX对映体耐药细胞血管分化是否具有手性选择性。方法用大剂量冲击法诱导获得A549对映体耐药肺癌细胞，流式细胞双色分析细胞CD44／CD31以及P-170表达；皮下接种裸鼠，成瘤后取瘤体切片，进行CD34组化染色，计数新生微血管密度（MVD）。以A549亲本细胞（未获得耐药）作为对照组。结果分别建立耐药浓度为15μmol／L的L—MTX和D—MTX的A549耐药细胞株；它们与各自对映体的耐药指数分别为12．4±1．2和20．14-2．3；A549亲本细胞、L—MTX耐药细胞和D．MTX耐药细胞中：CD31’阳性率分别为55．5％±9．7％、32．9％±8．O％和91．9％±3．2％，差异有统计学意义（F=511．92，P=0．000）；P-170阳性率分别为85．5％±4．6％、71．5％±8．2％和87．0％±8．9％，L—MTX耐药细胞P-170的阳性率低于D．MTX耐药细胞（t=6．13，P=0．002）；3种细胞植入裸鼠成瘤后切片免疫标记CD34统计新生MVD，A549亲本细胞为3．6±1．2，L-MTX耐药细胞为7．24-1．7，D-MTX耐药细胞为55．9±11．9，D．MTX耐药细胞显著高于L—MTX耐药细胞（t=13．37，P=0．000）。结论MTX对映体诱导肺癌A549耐药细胞向血管内皮细胞分化能力不同，D—MTX耐药细胞经血管转移高于L．MTX耐药细胞，这提示MTX制剂中D型组分不能简单当成污染物而忽视，应尽量选用单-对映体的L-MTX药物制剂，减少D—MTX带来的副作用。

Objective To study the chiral selectivity in vascular endothelial differentiation in drug resistant lung cancer cells induced by high-dose L- or D-methotrexate （MTX） enantiomer. Methods Human lung cancer cells of the line A549 were co-cultured with high-dose （ 15μmol/L） L- or D- MTX enantiomer so as to develop cancer cells resistant to MTX. MTT method was used to detect the drug resistant index. Flow cytometry was used to detect the expression of CD44, a transmembrane glycoprotein reflecting the migration ability of cells, CD31, a marker of vascular endothelium, and P-170 protein. Fifteen BALB/c nude mice were inoculated with the parent A549 cells, L-MTX-resistant A549 cells induced by L-MTX enantiomer, and D-MTX-resistant A549 cells induced by D-MTX enantiomer. Four weeks later the mice were killed to take out the tumor masses. Immunohistochemistry with CD34 staining was used to detect the microvascular density （MVD）. Results The drug resistant index of the D-MTX induced drug resistant A549 cells was 20.1 ± 2.3, significantly higher than that of the L-MTX-induced cells （ 12.4±1.2, P = 0.000）. The CD44 positive rate of the D-MTX induced A549 cells was 97.0% ± 0.9%, not significantly different from that of the L-MTX-induced A549 cells （96.7% ±1. 4%, P = 0. 544）. The CD31 positive rate of the D-MTX induced A549 cells was 91.9% ± 3.2%, significantly higher than that of the L-MTX-induced A549 cells （32.9% ±8.0%, P =0.000）. The P-170 protein positive rate of the parent cells was 85.5% ± 4.6%, and the P-170 protein positive rate of the D-MTX-induced A549 cells was 87.0% ± 8.9%, significantly higher than that of the L-MTX-induced cells （71.5% ± 8.2%, P =0.002）. The MVD of the D-MTX-induced cells was 55.9± 11.9, significantly higher than that of the L-MTX-induced cells （7. 2 ±1.7, P = 0. 000）. MVD was significantly positively correlated with the CD31 level （ r = 0. 462, P = 0.007）, and not correlated with P-170 protein and CD34 levels. Conclusion The MTX enantiomers have different chiral selectivity on human lung cancer cell, D-MTX resistant cells shows a potential of differentiation from cancer ceils to vascular endothelial cells. D-MTX is not be regarded just as a pollutant in the drug MTX, MTX with single enantiomer （L-MTX） should be selected clinically so as to decrease the side effects of D-MTX.