CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

Document Type : Original Article

Authors

1 Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

2 Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

3 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Department of Biochemistry and Genetic, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

Abstract

Objective(s): Multidrug resistance (MDR) is a major obstacle in the successful chemotherapy of ovarian cancer. Inhibition of P-glycoprotein (P-gp), a member of ATP-binding cassette (ABC) transporters, is a well-known strategy to overcome MDR in cancer. The aim of this study was to investigate the efficiency and ability of CRISPR/Cas9 genome editing technology to knockdown ABCB1 gene expression in adriamycin resistant (A2780/ADR) ovarian cancer cell line and evaluate the sensitivity changes to doxorubicin.
Materials and Methods: Three single-guide RNAs (sgRNAs) targeting the fourth and fifth exons of human ABCB1 gene were designed in this study. Expression level of ABCB1 was detected using quantitative real time PCR (qRT-PCR) after co-transfection of all three sgRNAs into A2780/ADR cell line and subsequent antibiotic selection. Drug sensitivity to doxorubicin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Results: The results showed that CRISPR/Cas9 system could significantly reduce the expression of P-gp. The dramatic decline in ABCB1 gene expression was associated with increased sensitivity of cells transfected with sgRNAs to doxorubicin.
Conclusion: Based on the results of this study, it is concluded that the CRISPR-based systems, used in the present study, effectively down-regulated the target gene and acted as an ideal and cost-effective tool for gene editing of A2780/ADR cell line resulting in restoration of nonmalignant phenotype.

Keywords

Main Subjects

References

1. Sankaranarayanan R, Ferlay J. Worldwide burden of gynaecological cancer: The size of the problem. Best Pract Res Cl Ob 2006; 20: 207–225.
2. Beaufort CM, Helmijr JC, Piskorz AM, Hoogstraat M, Ruigrok-Ritstier K, Besselink N, et al. Ovarian cancer cell line panel (OCCP): clinical importance of in vitro morphological subtypes. PLoS One 2014; 9: e103988.
3. Kobayashi A, Yokoyama Y, Osawa Y, Miura R, Mizunuma H. Gene therapy for ovarian cancer using carbonyl reductase 1 DNA with a polyamidoamine dendrimer in mouse models. Cancer Gene Ther 2016; 23: 24–28.
4. Cho KR, Shih leM. Ovarian Cancer. Annu Rev Pathol 2009; 4: 287–313.
5. Reinartz S, Finkernage F, Adhikary T, Rohnalter V, Schumann T, Schober Y, et al. A transcriptome-based global map of signaling pathways in the ovarian cancer microenvironment associated with clinical outcome. Genome Biol 2016; 17: 108.
6. Banerjee S, Kaye SB. New strategies in the treatment of ovarian cancer: current clinical perspectives and future potential. Clin Cancer Res 2013; 19: 961–968.
7. Markman M. Current standards of care for chemotherapy of optimally cytoreduced advanced epithelial ovarian cancer. Gynecol Oncol 2013; 131: 241–245.
8. Baguley BC. Multiple drug resistance mechanisms in cancer. Mol Biotechnol 2010; 46: 308–316.
9. Wu CP, Calcagno AM, Ambudkar SV. Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: evaluation of current strategies. Curr Mol Pharmacol 2008; 1: 93–105.
10. Binkhathlan Z, Lavasanifar A. P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: current status and future perspectives. Curr Cancer Drug Targets 2013; 13: 326–346.
11. Follit CA, Brewer FK, Wise JG, Vogel PD. In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture. Pharmacol Res Perspect 2015; 3: e00170.
12. Gottesman MM, Ling V. The molecular basis of multidrug resistance in cancer: The early years of P-glycoprotein research. FEBS Lett 2006; 580: 998–1009.
13. Sui H, Fan ZZ, Li Q. Signal transduction pathways and transcriptional mechanisms of ABCB1/Pgp-mediated multiple drug resistance in human cancer cells. J Int Med Res 2012; 40: 426–435.
14. Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013; 31: 397–405.
15. Torres-Ruiz R, Rodriguez-Perales S. CRISPR-Cas9: A revolutionary tool for cancer modelling. Int J Mol Sci 2015; 16: 22151–22168.
16. Chang H, Yi B, Ma R, Zhang X, Zhao H, Xi Y. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Sci Rep 2016; 6: 22312.
17. Zhang H, Wang J, Cai K, Jiang L, Zhou D, Yang C, et al. Downregulation of gene MDR1 by shRNA to reverse multidrug-resistance of ovarian cancer A2780 cells. J Cancer Res Ther 2012; 8: 226–231.
18. Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 2010; 649: 247-256.
19. Li YL, Ye F, Hu Y, Lu WG, Xie X. Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction. Anal Biochem 2009; 394: 110–116.
20. Jacob F, Guertler R, Naim S, Nixdorf S, Fedier A, Hacker NF, et al. Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS One 2013; 8: e59180.
21. Xie Z, Cao L, Zhang J. miR-21 modulates paclitaxel sensitivity and hypoxia-inducible factor-1-alpha expression in human ovarian cancer cells. Oncol Lett 2013; 6: 795–800.
22. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome editing using CRISPR/Cas system. Science 2013; 339: 819-823.
23. Savic N, Schwank G. Advances in therapeutic CRISPR/Cas9 genome editing. Transl Res 2016; 168: 15–21.
24. Bikard D, Marraffini LA. Control of gene expression by CRISPR-Cas systems. F1000 Prime Rep 2013; 5: 47.
25. Liu T, Li Zh, Zhang Q, De Amorim Bernstein K, Lozano-Calderon S, Choy E, et al. Targeting ABCB1 (MDR1) in multi-drug resistant osteosarcoma cells using the CRISPR-Cas9 system to reverse drug resistance. Oncotarget 2016; 7: 83502–83513.
26. Yang Y, Qiu JG, Li Y, Di JM, Zhang WJ, Jiang QW, et al. Targeting ABCB1-mediated tumor multidrug resistance by CRISPR / Cas9-based genome editing. Am J Transl Res 2016; 8: 3986–3994.
27. Wunder JS, Bull SB, Aneliunas V, Lee PD, Davis AM, Beauchamp CP, et al. MDR1 gene expression and outcome in osteosarcoma: a prospective, multicenter study. J Clin Oncol. 2000; 18: 2685–2694.
28. Baldini N, Scotlandi K, Barbanti-Bròdano G, Manara MC, Maurici D, Bacci G, et al. Expression of P-glycoprotein in high-grade osteosarcomas in relation to clinical outcome. N Engl J Med 1995; 333: 1380–1385.
29. Jekerle V, Klinkhammer W, Scollard DA, Breitbach K, Reilly RM, Piquette-Miller M, et al. In vitro and in vivo evaluation of WK-X-34, a novel inhibitor of P-glycoprotein and BCRP using radio imaging techniques. Int J Cancer 2006; 119: 414–422.
30. Simoff I, Karlgren M, Backlund M, Lindstrom AC, Gaugaz FZ, Matsson P, et al. Complete knockout of endogenous Mdr1 (Abcb1) in MDCK cells by CRISPR-Cas9. J Pharm Sci 2016; 105: 1017–1021.
31. Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 2015; 16: 299–311.
32. Ha JS, Byun J, Ahn DR. Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption. Sci Rep 2016; 6: 22847.
33. Sims JT, Ganguly SS, Bennett H, Friend JW, Tepe J, Plattner R. Imatinib reverses doxorubicin resistance by affecting activation of STAT3-dependent NF-kB and HSP27/p38/AKT pathways and by inhibiting ABCB1. PLoS One 2013; 8: e55509.
34. Callaghan R, Luk F, Bebawy M. Inhibition of the multidrug resistance P-glycoprotein: time for a change of strategy?. Drug Metab Dispos 2014; 42: 623–631.
35. Kaymaz BT, Kosova B. Advances in therapeutic approaches using RNA interference as a gene silencing tool. Adv Tech Biol Med 2013; 1: 108.
36. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152: 1173–1183.
37. Porteus MH. Towards a new era in medicine: therapeutic genome editing. Genome Biol 2015; 16: 286.
Iranian Journal of Basic Medical Sciences
Volume 21, Issue 2
February 2018
Pages 181-187

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