Govindarajah, Narendranath, Bowden, david, Sutton, paul, Parsons, jason and Vimalachandran, dale
(2022)
The role of acid ceramidase in the radiotherapy response of an in vitro model of rectal cancer.
Doctor of Medicine thesis, University of Liverpool.
|
Text
201229551_October2022.pdf - Author Accepted Manuscript Download (4MB) | Preview |
Abstract
The role of acid ceramidase in the radiotherapy response of an in vitro model of rectal cancer N Govindarajah, P Sutton, D Bowden, JL Parsons, D Vimalachandran. Background: Chemo radiotherapy (CRT) is often employed to treat locally advanced rectal cancer with highly variable response, emphasizing the necessity for predictive response biomarkers. Our initial proteomic and immune-histochemical work demonstrated that acid ceramidase (AC) expression correlated with poorer CRT responses in rectal cancer. We described that higher AC expression correlates with radio resistance in colorectal cancer cells and improved radio sensitivity through siRNA inhibition of AC. The mechanisms behind AC expression, radio resistance and apoptosis remain unknown in colorectal cancer. AC is known to affect apoptosis and the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme that is also cleaved into specific fragments during apoptosis. Aims: To elucidate a potential mechanism linking AC expression with radio resistance in colorectal cancer cells. Methods: Differential AC protein expression of four colorectal cell lines was confirmed by Western blotting. Radio sensitivity of these cell lines was examined using standard clonogenic assays by counting individual colony survival post-exposure to increasing doses of ionizing radiation. siRNA knockdown of AC was performed with further clonogenic assays to establish the impact of AC inhibition on radio sensitivity. HT29 and HCT cells were then treated with non-targeting control siRNA and AC siRNA, irradiated at increased doses of radiation then harvested at specific time points (2,6,24h). Western blotting was then performed to detect and measure for specific PARP-1 cleavage fragments as specific apoptotic markers. Results: Clonogenic assays confirmed that cell lines with greater cellular AC protein expression (LIM1215/MDST8) demonstrated higher colony survival compared to those with lower AC expression (HT29/HCT 116) post irradiation. siRNA AC knockdown improved radio sensitivity by reducing colony formation efficiency (CFE) in three cell lines: HT29(0.52 CFE control vs 0.13 CFE knockdown at 1Gyp=0.00004); HCT116(0.24 CFEcontrolvs0.09 CFE knockdown at 1Gyp=0.026); LIM1215 (0.88 CFE control vs 0.43 CFE knockdown at 0.25Gyp=0.001).Western blotting confirmed that HT29,HCT116 and LIM1215cells treated with AC siRNA displayed significantly higher levels of the 24kD PARP-1 cleavage fragments compared to control therefore indicating increased apoptosis. Conclusion: Higher AC expression correlates with radio resistance in several colorectal cell lines and radio sensitivity was successfully improved through biological (siRNA) inhibition of AC. Initial mechanistic work has confirmed that siRNA inhibition of AC causes increased apoptosis.
| Item Type: | Thesis (Doctor of Medicine) |
|---|---|
| Divisions: | Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 10 Nov 2022 14:32 |
| Last Modified: | 18 Jan 2023 20:37 |
| DOI: | 10.17638/03165268 |
| Supervisors: |
|
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3165268 |
Dimensions
Dimensions
