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.
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.
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.
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.
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.
Flow chart depicting the process of patient selection.
Table 1.
The overall response in the rabeprazole and non-rabeprazole group.