Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

Abstract

Objective: The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX) in human choriocarcinoma cells regarding DNA damage response.

Methods: Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot.

Results: Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63) but not in MTX-resistant cancer cells (A2780 and Hela) after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR) repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53.

Conclusion: The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma.

Keywords: DNA double-strand break; RAD51; chemotherapy hypersensitivity; choriocarcinoma; p53.

Figure 1

The sensitivity to MTX of cancer cell lines was evaluated by MTT assays. Notes: (A) The viability of JAR, JEG-3 and MG63 cells after 48 h treatment of MTX with different concentrations. (B) The viability of A2780 and Hela cells after 48 h treatment of MTX with different concentrations. Abbreviations: MTX, methotrexate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

MTX-induced DNA damage in cancer cell lines. Notes: (A) The representative images of comet assays in JAR, JEG-3, MG63, A2780 and Hela cell lines. (B) The statistical histogram of tail length and tail moment in JAR, JEG-3, MG63, A2780 and Hela cell lines. (C) Western blot detected the γH2AX protein level in cancer cell lines after treatment with MTX. **P<0.01. Abbreviations: MTX, methotrexate; PBS, phosphate-buffered saline; IC₅₀, half maximal inhibitory concentration.

Figure 3

Results of qRT-PCR showed the mRNA levels of HR-related genes (RAD51, RAD52, BRCA1 and BRCA2) in cancer cells treated with MTX. Notes: The mRNA levels of HR-related genes in JAR (A), JEG-3 (B), MG63 (C), A2780 (D) and Hela (E). *P<0.05 and **P<0.01. Abbreviations: qRT-PCR, quantitative reverse transcription polymerase chain reaction; HR, homologous recombination; MTX, methotrexate.

Figure 4

Western blot assays showed the RAD51 and p53 protein levels in cancer cells treated with MTX. Abbreviations: MTX, methotrexate; IC₅₀, half maximal inhibitory concentration.