Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .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.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Aug 7;15(8):e0228002.
doi: 10.1371/journal.pone.0228002. eCollection 2020.

CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer

Hiroya Matsuoka  1 , Koji Ando  1 , Emma J Swayze  2 , Elizabeth C Unan  2 , Joseph Mathew  2 , Quingjiang Hu  1 , Yasuo Tsuda  1 , Yuichiro Nakashima  1 , Hiroshi Saeki  3 , Eiji Oki  1 , Ajit K Bharti  2 , Masaki Mori  1
Affiliations

CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer

Hiroya Matsuoka et al. PLoS One. .

Abstract

Irinotecan specifically targets topoisomerase I (topoI), and is used to treat various solid tumors, but only 13-32% of patients respond to the therapy. Now, it is understood that the rapid rate of topoI degradation in response to irinotecan causes irinotecan resistance. We have published that the deregulated DNA-PKcs kinase cascade ensures rapid degradation of topoI and is at the core of the drug resistance mechanism of topoI inhibitors, including irinotecan. We also identified CTD small phosphatase 1 (CTDSP1) (a nuclear phosphatase) as a primary upstream regulator of DNA-PKcs in response to topoI inhibitors. Previous reports showed that rabeprazole, a proton pump inhibitor (PPI) inhibits CTDSP1 activity. The purpose of this study was to confirm the effects of rabeprazole on CTDSP1 activity and its impact on irinotecan-based therapy in colon cancer. Using differentially expressing CTDSP1 cells, we demonstrated that CTDSP1 contributes to the irinotecan sensitivity by preventing topoI degradation. Retrospective analysis of patients receiving irinotecan with or without rabeprazole has shown the effects of CTDSP1 on irinotecan response. These results indicate that CTDSP1 promotes sensitivity to irinotecan and rabeprazole prevents this effect, resulting in drug resistance. To ensure the best chance at effective treatment, rabeprazole may not be a suitable PPI for cancer patients treated with irinotecan.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. CTDSP1 regulates irinotecan sensitivity in colon cancer cell lines.
A, Cell lysates from four colon cancer lines, HCT15, DLD1, LoVo and HT29, were immunoblotted with anti-CTDSP1 and anti-β-actin. B, HCT116 and HT29 cells were treated with 2.5 μM SN-38 and harvested after 90 and 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin. C, HCT116 and HT29 cells were treated with various concentrations of SN-38 and clonogenic assays were performed to determine the relative number of colonies.
Fig 2
Fig 2. Silencing of CTDSP1 enhances topoI degradation and irinotecan resistance.
A, Cells transfected with CTDSP1 or control siRNA were lyzed and the cells’ lysates were immunoblotted with anti-CTDSP1 and anti-β-actin antibodies. B, HCT116-siRNA CTDSP1 or control siRNA, treated with 2.5 μM SN-38 were harvested after 90 and 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin antibodies. Cells’ lysates were immunoblotted with anti-topoI and anti-β-actin antibodies. C, EGFP was integrated with topoI in HCT116 cells using CRISPR/Cas9 system and CTDSP1 was knocked down in this cell line by siRNA. Cells were treated with 2.5uM SN-38 for 60 min and the topoI-EGFP signal was imaged by Leica SP5 confocal microscope. D, HCT116-siRNA-CTDSP1 or control siRNA were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by detecting the luminescence. E, HCT116-siRNA-CTDSP1 or control siRNA cells were plated in a 6-well plate and treated with various concentrations of SN-38 for 24 h. Then, 50 cells per well were plated in a 6-well plate. After 14 days, when colonies were apparent (right panel), colonies were counted and the relative number of colonies was determined (left panel).
Fig 3
Fig 3. CTDSP1 restores irinotecan sensitivity in HCT116 cells.
A, CTDSP1-overexpressing and control cells were lyzed and the cell lysates were immunoblotted with anti-CTDSP1 and anti-β-actin antibodies. B, CTDSP1-overexpressing and control cells were treated with 2.5 μM SN-38 and harvested after 90 or 180 min. Cells lysates were immunoblotted with anti-topo I and anti-β-actin antibodies. C, CTDSP1-overexpressing and control cells were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by luminescence detection.
Fig 4
Fig 4. CTDSP1 activates DNA-PKcs and enhances DNA-PKcs dependent topoI degradation in response to irinotecan.
A, HCT116 and HT29 cell lysates were immunoblotted with anti-DNAPKcs-pS2056 and anti-GAPDH antibodies. B, CTDSP1 was silenced by CTDSP1 siRNA in HCT116 cells, and control as well as CTDSP1-silenced cell lysates were subjected to immunoblot with anti-CTDSP1 and anti-β-actin. C, Control and CTDSP1 knockdown cells were treated with SN38 and analyzed by immunofluorescence staining with anti-DNA-PKcs–pS2056). The cells were analyzed by Leica SP5 confocal microscope.
Fig 5
Fig 5. Rabeprazole promotes topo I degradation and irinotecan resistance.
A, HCT116 cells were plated in a 6-well plate and treated with various concentrations of rabeprazole (0, 10, 20 μM) for 72 h, and then with 2.5 μM SN-38 and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-topoI and anti-β-actin. B, Genomically edited HCT116 cells with TopoI-EGFP fusion proteins were treated with 5 and 10 μM of Rabeprazole for 48 hours and topoI-GFP protein level was analyzed by confocal microscope. C. HCT116 cells were plated in a 6-well plate, treated with 40 μM rabeprazole or DMSO for 72 h, and then with 2.5 μM SN-38, and harvested after 90 or 180 min. Cell lysates were immunoblotted with anti-pDNA-PKcs and anti-β-actin. D, HCT 116 cells were treated with rabeprazole, control and treated cells were analyzed by immunofluorescence analysis with anti-phospho-DNA-PKcs-pS2056 and confocal microscopy. E. HCT116 cells were plated in a 6-well plate and treated with rabeprazole or DMSO for 72 h. Then, 50 cells were plated in each well of a 6-well plate and treated with various concentrations of SN-38 for 24 hours. Cell colonies were counted after 14 days. F. HCT116 cells were plated in a 6-well plate and treated with 40 μM rabeprazole or DMSO for 72 h. Then, cells were plated in a 96-well plate and treated with various concentrations of SN-38 for 72 h. Cell viability was determined by luminescence detection.
Fig 6
Fig 6. Flow chart depicting the process of patient selection.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hsiang YH, Liu LF. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer research 1988; 48: 1722–1726. - PubMed
    1. Liu LF, Desai SD, Li TK, Mao Y, Sun M, Sim SP. Mechanism of action of camptothecin. Annals of the New York Academy of Sciences 2000; 922: 1–10. 10.1111/j.1749-6632.2000.tb07020.x - DOI - PubMed
    1. Ando K, Shah AK, Sachdev V, Kleinstiver BP, Taylor-Parker J, Welch MM, et al. Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway. Oncotarget 2017; 8: 43733–43751. 10.18632/oncotarget.16376 - DOI - PMC - PubMed
    1. Maliepaard M, van Gastelen MA, de Jong LA, Pluim D, van Waardenburg RC, Ruevekamp-Helmers MC, et al. Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. Cancer research 1999; 59: 4559–4563. - PubMed
    1. Brangi M, Litman T, Ciotti M, Nishiyama K, Kohlhagen G, Takimoto C, et al. Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance half-transporter (MXR), and potential for glucuronidation in MXR-expressing cells. Cancer research 1999; 59: 5938–5946. - PubMed

MeSH terms

Grants and funding

The authors received no specific funding for this work.
-