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The functional status of DNA repair pathways determines the sensitization effect to cisplatin in non-small cell lung cancer cells

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Abstract

Purpose

Cisplatin can cause a variety of DNA crosslink lesions including intra-strand and inter-strand crosslinks (ICLs), which are associated with the sensitivity of cancer cells to cisplatin. Here, we aimed to assess the contribution of the Fanconi anemia (FA), homologous recombination (HR) and nucleotide excision repair (NER) pathways to cisplatin resistance in non-small cell lung cancer (NSCLC)-derived cells.

Methods

The expression of FA, HR and NER pathway-associated genes was assessed by RT-qPCR and Western blotting. siRNAs were used to knock down the expression of these genes. CCK-8 and flow cytometry assays were used to assess the viability and apoptotic rate of NSCLC-derived cells, respectively. Immunofluorescence and alkaline comet assays were used to assess the repair of ICLs.

Results

We found that acquired cisplatin-resistant NSCLC-derived A549/DR cells exhibited markedly enhanced FA and HR repair pathway capacities compared to its parental A549 cells and another independent NSCLC-derived cell line, Calu-1, which possesses a moderate innate resistance to cisplatin. siRNA-mediated silencing of the FA-associated genes FANCL and RAD18 and the HR-associated genes BRCA1 and BRCA2 significantly potentiated the sensitivity of A549/DR cells to cisplatin compared to A549 and Calu-1 cells, suggesting that the acquired cisplatin resistance in A549/DR cells may be attributed to enhanced FA and HR pathway capacities responsible for ICL repair. Although we found that expression knockdown of the NER-associated genes XPA and ERCC1 sensitized the three NSCLC-derived cell lines to cisplatin, the sensitization effect was more significant in Calu-1 cells than in A549 and A549/DR cells, implying that the innate cisplatin resistance in Calu-1 cells may result from an increased NER activity.

Conclusions

Our results indicate that the functional status of DNA repair pathways determine the sensitivity of NSCLC cells to cisplatin. Direct targeting of the pathway that is involved in cisplatin resistance may be an effective strategy to surmount cisplatin resistance in NSCLC.

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Authors and Affiliations

  1. Department of Pulmonary Medicine, Affiliated Hospital of Jiangsu University, 438 North Jiefang Street, Zhenjiang, 212001, China

    Ping Chen, Jian Li, Yu-Jiao Chen & Jin-Yu Su

  2. Institute of Medical Science, Jiangsu University, Zhenjiang, 212013, China

    Yong-Chang Chen, Hai Qian, Min Wu & Ting Lan

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  1. Ping Chen
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  2. Jian Li
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Correspondence to Jian Li.

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Supplementary Fig. 1

The validation of siRNAs used in this study. Representative Western blotting showing FANCL, RAD18, BRCA1, BRCA2, XPA and ERCC1 expression in A549/DR cells. Cells were transfected with the indicated siRNAs separately for 48 h. Whole cell lysates were prepared and subjected to Western blotting for detecting the protein expressions of these factors. (GIF 81 kb)

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Supplementary Fig. 2

The expression of cleaved caspase-3 and cleaved PARP were elevated in A549/DR cells after depletion of the FA and HR factors. (a) A549/DR cells were treated with 10 μM cisplatin for 4 h following transfection with non-targeting control siRNA (NC), siRNAs against FANCL, RAD18, BRCA1, BRCA2, XPA and ERCC1. Then cells were harvested and subjected to Western blotting with specific antibodies as indicated. (b) The relative expression levels of cleaved caspase-3 and cleaved PARP protein were quantified. (GIF 298 kb)

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Chen, P., Li, J., Chen, YC. et al. The functional status of DNA repair pathways determines the sensitization effect to cisplatin in non-small cell lung cancer cells. Cell Oncol. 39, 511–522 (2016). https://doi.org/10.1007/s13402-016-0291-7

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