Identification Of Natural Compound Derivative For Inhibition Of XLF And Overcoming Chemoresistance In Colorectal Cancer Cells

Abstract

Purpose

A previous study has identified that XRCC4-like factor (XLF) is a potential target to overcome resistance to 5-fluorouracil (5-Fu) and oxaliplatin (OXA) in colorectal cancer (CRC). The purpose of this study is to develop potent XLF inhibitors to chemoresistance in CRC.

Methods

Virtual screening was adopted to identify novel XLF-binding compounds by initially testing 6800 molecules in Chemical Entities of Biological Interest library. Hit compounds were further validated by Western blot assay. Cell sensitivity to 5-Fu and OXA was measured using sulforhodamine B assay. The effect of XLF inhibitor on DNA repair efficiency was evaluated by comet assay, fluorescent-based nonhomologous end joining (NHEJ) and homologous recombination (HR) reporter assays. DNA-binding activity of NHEJ key factors was examined by chromatin fractionation assay.

Results

We identified G3, a novel and potent XLF inhibitor (IC₅₀ 0.47±0.02 µM). G3 induced XLF protein degradation in CRC cells. Significantly, G3 improved cell sensitivity to 5-Fu and OXA in chemoresistant CRC cell lines. Mechanistically, G3 depleted XLF expression, severely compromised NHEJ efficiency by up to 65% and inhibited NHEJ key factor assembly on DNA. G3 also inhibited HR efficiency in a time-dependent manner.

Conclusion

These results suggest that G3 overcomes 5-Fu and OXA resistance in CRC cells by inhibiting XLF expression. Thus, XLF is a promising target and its inhibitor G3 is a potential candidate for treatment of chemoresistant CRC patients.

Keywords:

• virtual screening
• XLF inhibitor
• chemoresistance
• colorectal cancer

Abbreviations

CRC, colorectal cancer; 5-Fu, 5-florouracil; OXA, oxaliplatin; NHEJ, nonhomologous end joining; HR, homologous recombination; DSB, DNA double-strand break; Ku, Ku70/80 heterodimer; WT, wild type; DNA-PKcs, DNA-PK catalytic subunit; MRN, CtIP/MRE11-RAD50-NBS1; RPA, replication protein A; ssDNA, single-strand DNA; XRCC4, X-ray cross-complementing 4; XLF, XRCC4-like factor.

Disclosure

This work was supported by funding from Jilin Province Science and Technology Institute (20160101034JC and 20170204050SF to Zhuo Liu and Dongxin Wang, respectively). The authors report no conflicts of interest in this work.