Multiple Strategies Reverse ABCG2 Mediated Multidrug Resistance In Colorectal Cancer

Objective:Colorectal cancer is a common malignancy with the third highest incidence and second highest mortality rate among all cancers in the world.Chemotherapy resistance in colorectal cancer is an essential factor leading to the high mortality rate.The ATP-binding cassette(ABC)superfamily G member 2(ABCG2),a transmembrane transporter,widely located in various kinds of cells and organs,extruding phospholipids,lipophilic drugs,cholesterol,and other small molecules to extracellular in an ATP dependent manner that protects hosts against xenobiotic molecules.ABCG2 has ectopically expressed in a range of cancer cells,resulting in multidrug resistance to many chemotherapeutic agents through decreasing their intracellular content.Until now,ABCG2 inhibitors still not be used in clinics,so the development of novel inhibitors or modulators of ABCG2 with high effectivity and bioavailability is helpful for overcoming ABCG2-mediated multidrug resistance.Methods:Using CRISPR-Cas9 technology to establish an ABCG2 stable knock-out human colorectal cancer cell line,providing a valuable tool for screening select ABCG2 inhibitors.Potential ABCG2 inhibitors were predicted based on structure-activity relationship.Then the cytotoxic and reversing effect of putative inhibitors were detected by MTT assay.Drug accumulation assay was used to study potential inhibitors whether inhibit the pumping activity of ABCG2,resulting in the augment of intracellular content of substrate drugs.Next,the effect of inhibitors on ABCG2 expression level was detected by Western blot assay.Finally,a molecular docking assay in silico was conducted to simulate the binding site and intermolecular interactions between inhibitors and ABCG2.Result:1.ABCG2 stable knock-out monoclonal colorectal cancer cell line,S1-M1-80 sg ABCG2,was acquired by CRISPR-Cas9 genome-editing technology;2.Cytotoxicity assay showed that KU55933 and AZ32(≤1 μM)did not inhibit the growth of all cell lines used in this study;3.KU55933 and AZ32 profoundly reversed the resistance of Mitoxantrone and Doxorubicin to ABCG2-overexpressing cell lines in a concentration dependent manner.KU5593 is even more potent than the well-known selective ABCG2 inhibitor FTC,while the effect of AZ32 is a little weaker than FTC.However,the effect of the non-substrate drug Cisplatin was not changed by these inhibitors;4.KU55933 and AZ32 increased the intracellular level of ABCG2 substrate drugs,Mitoxantrone, Doxorubicin,and Rhodamine 123 by inhibiting the effluxing activity of ABCG2;5.KU55933 and AZ32 functional as a static inhibitor of ABCG2,did not change the protein level of ABCG2;6.Molecular docking assay in silico showed that KU55933 and AZ32 could bind within the putative multidrug-binding pocket of ABCG2,and the conformations are stabilized by hydrophobic interactions.Conclusion:In this study,we found that ATM kinase inhibitors KU55933 and AZ32 had a moonlighting role in inhibiting the effluxing activity of ABCG2 without altering the protein expression level of ABCG2.KU55933 and AZ32 could re-sensitive ABCG2-overexpressing MDR colon cancer cell lines to ABCG2 substrate drugs,such as Mitoxantrone and Doxorubicin,by retaining them in the cell.Therefore,KU55933 and AZ32 synergistic with other antineoplastic drugs may as an attractive modality to overcome multidrug resistance in colorectal cancer.