Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2016 Mar 10:6:22847.
doi: 10.1038/srep22847.

"V体育ios版" Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption

Jong Seong Ha  1 , Juyoung Byun  2 , Dae-Ro Ahn  1   2
Affiliations

Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption

Jong Seong Ha et al. Sci Rep. .

Abstract

In this study, Cas9 system was employed to down-regulate mdr1 gene for overcoming multidrug resistance of cancer cells. Disruption of the MDR1 gene was achieved by delivery of the Cas9-sgRNA plasmid or the Cas9-sgRNA ribonucleoprotein complex using a conventional gene transfection agent and protein transduction domain (PTD). Doxorubicin showed considerable cytotoxicity to the drug-resistant breast cancer cells pre-treated with the RNA-guided endonuclease (RGEN) systems, whereas virtually non-toxic to the untreated cells. The potency of drug was enhanced in the cells treated with the protein-RNA complex as well as in those treated with plasmids, suggesting that mutation of the mdr1 gene by intracellular delivery of Cas9-sgRNA complex using proper protein delivery platforms could recover the drug susceptibility. Therefore, Cas9-mediated disruption of the drug resistance-related gene can be considered as a promising way to overcome multidrug resistance in cancer cells VSports手机版. .

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Schematic presentation of Cas9-sgRNA delivery system for down regulation of MDR1.
Cas9-sgRNA system was introduced into the multidrug resistant cells (MCF-7/ADR) via transfection of the cells with the plasmid for expression of Cas9-sgRNA and intracellular delivery of the Cas9-sgRNA ribonucleoprotein complex using either a PTD peptide or Lipofectamine.
Figure 2
Figure 2. Disruption of mdr1 using Cas9-sgRNA plasmid.
(A) The pSpCas9(BB)-2A-Puro plasmid containing Cas9 and sgRNA scaffolds. The target sequence for mdr1 was inserted into the plasmid using BbsI sites. (B) The T7E1 cleavage assay to detect disruption in the target gene. (C) Western blot showing MDR1 protein levels in MCF-7/ADR cells transfected with the mock vector and the Cas9-sgRNA plasmid from three independent experiments. The protein level of the drug-sensitive MCF cells was also compared. (D) Densitometric analysis of the Western blot was performed for quantitative evaluation of the protein level after normalization by the expression level of β–actin. The data represent the mean ± s.d. (n = 3) ***P ≤ 0.001 vs. the mock vector-treated MCF-7/ADR cell.
Figure 3
Figure 3. Disruption of mdr1 by intracellular delivery of the Cas9-sgRNA ribonucleocomplex.
(A) The T7E1 cleavage assay to detect the gene disruption and (B) Western blot to measure MDR1 protein levels in MCF-7/ADR cells treated with the Cas9-sgRNA ribonucleocomplex using LF3K. (C) Densitometric analysis of the Western blot was performed for quantitative evaluation of the protein level after normalization by the expression level of β–actin. The data represent the mean ± s.d. (n = 3). **P ≤ 0.01 vs. PBS-treated MCF-7/ADR cell. (D) The T7E1 cleavage assay to detect the gene disruption and (E) Western blot to measure MDR1 protein levels in MCF-7/ADR cells treated with the Cas9-sgRNA ribonucleocomplex using TAT. (F) Densitometric analysis of the Western blot was performed for quantitative evaluation of the protein level after normalization by the expression level of β–actin. The data represent the mean ± s.d. (n = 3). ns P > 0.05 vs. PBS-treated MCF-7/ADR cell.
Figure 4
Figure 4. Intracellular accumulation of DOX in the drug-resistant cells after mdr1 gene disruption by Cas9-sgRNA.
(A) Fluorescence microscopic images showing DOX accumulation in MCF-7 (drug-sensitive) and MCF-7/ADR (drug-resistant) cells. (B) Fluorescence microscopic images showing DOX accumulation in MCF-7/ADR cells treated with Cas9-sgRNA (scale bar: 20 μm). (C) Flow cytometric analysis of intracellular DOX accumulation. The data represent the mean ± s.d. (n = 12). ****P ≤ 0.0001 vs. untreated MCF-7/ADR cells.
Figure 5
Figure 5. Enhanced potency of DOX in the drug-resistant cells after mdr1 gene disruption by Cas9-sgRNA.
Cell viability of MCF-7/ADR cells at various concentrations of DOX was examined after treatment with Cas9-sgRNA using the plasmid (blue bars), LF3K (green bars) and TAT (yellow bars), and compared with that of untreated MCF-7 (white bars) and MCF-7/ADR cells (red bars). The data represent the mean ± s.d. (n = 7). ns P > 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 vs. untreated MCF-7 cells.
See this image and copyright information in PMC

References

    1. Longley D. B. & Johnston P. G. Molecular mechanisms of drug resistance. J Pathol 205, 275–92 (2005). - PubMed
    1. Borowski E., Bontemps-Gracz M. M. & Piwkowska A. Strategies for overcoming ABC-transporters-mediated multidrug resistance (MDR) of tumor cells. Acta Biochim Pol 52, 609–27 (2005). - PubMed
    1. Szakacs G., Paterson J. K., Ludwig J. A., Booth-Genthe C. & Gottesman M. M. Targeting multidrug resistance in cancer. Nat Rev Drug Discov 5, 219–34 (2006). - PubMed
    1. Patil R. R., Guhagarkar S. A. & Devarajan P. V. Engineered nanocarriers of doxorubicin: a current update. Crit Rev Ther Drug Carrier Syst 25, 1–61 (2008). - VSports最新版本 - PubMed
    1. Wong H. L. et al. A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system. J Pharmacol Exp Ther 317, 1372–81 (2006). - PubMed

Publication types

MeSH terms

Substances