Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆

Abstract

Bulky cisplatin lesions are repaired primarily by nucleotide excision repair (NER), in which the structure specific endonuclease XPF–ERCC1 is a critical component. It is now known that the XPF–ERCC1 complex has repair functions beyond NER and plays a role in homologous recombination (HR). It has been suggested that expression of ERCC1 correlates with cisplatin drug resistance in non-small cell lung cancer (NSCLC). In our study, using NSCLC, ovarian, and breast cancer cells, we show that the XPF–ERCC1 complex is a valid target to increase cisplatin cytotoxicity and efficacy. We targeted XPF–ERCC1 complex by RNA interference and assessed the repair capacity of cisplatin intrastrand and interstrand crosslinks by ELISA and alkaline comet assay, respectively. We also assessed the repair of cisplatin-ICL-induced double-strand breaks (DSBs) by monitoring γ-H2AX focus formation. Interestingly, XPF protein levels were significantly reduced following ERCC1 downregulation, but the converse was not observed. The transcript levels were unaffected suggesting that XPF protein stability is likely affected. The repair of both types of cisplatin-DNA lesions was decreased with downregulation of XPF, ERCC1 or both XPF–ERCC1. The ICL-induced DSBs persist in the absence of XPF–ERCC1. The suppression of the XPF–ERCC1 complex significantly decreases the cellular viability which correlates well with the decrease in DNA repair capacity. A double knockdown of XPF–ERCC1 displays the greatest level of cellular cytotoxicity when compared with XPF or ERCC1 alone. The difference in cytotoxicity observed is likely due to the level of total protein complex remaining. These data demonstrate that XPF–ERCC1 is a valid target to enhance cisplatin efficacy in cancer cells by affecting cisplatin-DNA repair pathways.

Novel Role of Base Excision Repair in Mediating Cisplatin Cytotoxicity

“…Transfection with siRNAs-Transfections were carried out as described previously (20). Briefly, MDA-MB-231 cells were seeded in 6-well plates, and two transfections were done at 24-h intervals to target XPF and ERCC1 based on the manufacturer's instructions.…”

“…Cisplatin Interstrand Cross-link Measurement-An alkaline comet assay was used to analyze the repair of cisplatin ICLs as described (20,21). Cell suspensions (ϳ10,000 cells) were embedded on a microscopic slide, lysed, and incubated in icecold alkaline solution for 20 min.…”

“…The reaction was stopped with 1 M sulfuric acid, and absorbance was measured at 450 nm (Spectramax M5 plate reader, Molecular Devices). The percentages of intrastrand adducts were calculated using A 450 nm where the 0-h time point was used as 100% intrastrand adducts in each cell line as described previously (20).…”

“…These data indicate that the increased cisplatin resistance that we observe during defective BER is not due to increased intrastrand adduct DNA repair. It should be noted that cells deficient in NER are defective in the repair of the intrastrand adducts (20,21).…”

“…The comet assay has been used to evaluate DNA interstrand cross-link induction and repair in vivo at the single cell level (20). The repair kinetics of cisplatin ICLs was evaluated over a period of 0, 24, 48, and 72 h after treatment and was expressed as the percentage of cross-links that remained at that particular time point (Fig.…”