Downregulation of XPF-ERCC1 enhances cisplatin efficacy in cancer cells.
Downregulation of XPF-ERCC1 enhances cisplatin efficacy in cancer cells.
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DOI：
10.1016/j.dnarep.2010.03.010
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发表时间：
2010-07-01
期刊：
影响因子：
3.8
通讯作者：
Patrick SM
中科院分区：
文献类型：
--
作者：
Arora S;Kothandapani A;Tillison K;Kalman-Maltese V;Patrick SM
大体积顺铂损伤主要通过核苷酸切除修复（NER）修复，其中结构特异性内切核酸酶XPF-ERCC 1是关键组分。目前已知XPF-ERCC 1复合物具有NER以外的修复功能，并在同源重组（HR）中发挥作用。ERCC 1的表达与非小细胞肺癌（NSCLC）顺铂耐药相关。在我们的研究中，使用NSCLC，卵巢癌和乳腺癌细胞，我们表明XPF-ERCC 1复合物是增加顺铂细胞毒性和疗效的有效靶点。我们通过RNA干扰技术靶向XPF-ERCC 1复合物，并通过ELISA和碱性彗星试验分别评估顺铂链内和链间交联的修复能力。我们还通过监测γ-H2 AX焦点形成来评估顺铂-ICL诱导的双链断裂（DSB）的修复。有趣的是，XPF蛋白水平在ERCC 1下调后显著降低，但没有观察到相反的情况。转录水平未受影响，表明XPF蛋白稳定性可能受到影响。两种类型的顺铂-DNA损伤的修复随着XPF、ERCC 1或XPF-ERCC 1两者的下调而减少。ICL诱导的DSB在不存在XPF-ERCC 1的情况下持续存在。XPF-ERCC 1复合物的抑制显著降低了细胞活力，这与DNA修复能力的降低密切相关。当与单独的XPF或ERCC 1相比时，XPF-ERCC 1的双敲低显示出最大水平的细胞毒性。观察到的细胞毒性差异可能是由于剩余的总蛋白复合物水平所致。这些数据表明，XPF-ERCC 1是通过影响顺铂-DNA修复途径来增强顺铂在癌细胞中的功效的有效靶标。
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.
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