[Differential invasion and metastasis capacities of methotrexate enantiomer-resistant A549 cell lines].

OBJECTIVE: To study the differential in vitro motor and invasion capacities of methotrexate (MTX) enantiomer-resistant tumor cells. METHODS: The incremental concentrations and successive low-dose induction were employed to acquire the cell series resistant to 15 µmol/L MTX enantiomer, namely L-(+)-MTX/A549 and D-(-)-MTX/A549. Their drug-resistant indices were determined by MTT assay and their migration capacities by wound healing assay. Double soft-agar clone formation was used to detect the colony efficiency and size. And Transwell was employed to detect the in vitro movement and invasion capacity of three cell types. RESULTS: The resistance indices of L-(+)-MTX/A549 and D-(-)-MTX/A549 were (6.1 ± 1.0) and (20.3 ± 1.8) respectively. At 72 hours after wound healing assay, the number of L-(+)-MTX/A549 entering scratch zone was fewer than that of D-(-)-MTX/A549; The numbers of colony formation in D-(-)-MTX/A549, L-(+)-MTX/A549 and parental cells were (50 ± 7), (44 ± 6), (52 ± 7) and the rates of colony formation (1.68% ± 0.23%), (1.49% ± 0.18%), (1.73% ± 0.23%) respectively. And there was no significant significance among three groups (P > 0.05). But the size of D-(-)-MTX/A549 was larger than that of L-(+)-MTX/A549. Transwell detected infiltration and invasion through artificial basement membrane Matrigel. The numbers of D-(-)-MTX/A549, L-(+)-MTX/A549 and parent cells were (267 ± 30), (106 ± 16) and (134 ± 16) respectively. The data were significant between D-(-)-MTX/A549 cells and L-(+)-MTX/A549 or parent cells (P < 0.05) but not significant between L-(+)-MTX/A549 and parent cells (P > 0.05). CONCLUSION: The D-(-)-MTX-induced NSCLC A549 cells have greater motor and invasion capacities than those of L-(+)-MTX-induced ones. It suggests that MTX enantiomer has different capacities of tumor invasion and metastasis after acquiring resistance.

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

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 (15 micromol/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 cells 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.

High-performance capillary electrophoretic method for the quantification of global DNA methylation: application to methotrexate-resistant cells.

Global DNA hypomethylation in tumor tissue is a common characteristic in a variety of malignancies such as breast, colon, oral, lung, and blood cancers. A rapid and sensitive method has been developed for the determination of global DNA methylation in cells. Five substances--2'-deoxycytidine (dC), 5-methyl 2'-deoxycytidine (mdC), 2'-deoxyadenosine (dA), 2'-deoxythymidine (dT), and 2'-deoxyguanosine (dG)--were completely separated by high-performance capillary electrophoresis in 10 min. Intraday coefficient of variation was less than 1%, and interday coefficient of variation was less than 2%. The minimal detection limit was 1 microM. Acquired drug resistance to methotrexate (MTX) is one of the most serious problems in cancer chemotherapy. Under optimal conditions, we analyzed global DNA methylation levels in A549 and A549/MTX cells, and only 10(5) cells are needed to obtain reliable results. The percentage of 5-methyl-2'-deoxycytidine (5-mC) was 4.80+/-0.52% in A549 cells, and this decreased to 4.20+/-0.44% in A549/MTX cells. It was considered as statistically significant. This demonstrated that the mechanisms of acquired drug resistance to MTX might be concerned with DNA methylation.