title: COX-2 selective inhibitors as an adjunct to radiotherapy
Creators: name: King, Liam Denson
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Radiotherapy
subject: COX-2 inhibitors
subject: Radiosensitisation
subject: Cancer cells
subject: PSA velocity

description: Background Radiotherapy is a common treatment modality for many solid state cancers including prostate cancer (PCa). Unfortunately, up to 50% of patients that undergo radiotherapy for localised PCa will develop biochemical failure. Acute and delayed toxicities and tumour resistance are two key factors limiting the effectiveness of many cancer treatments, including radiotherapy. Toxicity rates in PCa patients that accompany radiotherapy are high with 80% of men experiencing some degree of urinary frequency, 40% bowel frequency and chronic impotence is usual. Furthermore, tumour radioresistance is driven by a number factors with tumour hypoxia playing a key role. In addition to hypoxia driving tumourigenesis the lack of oxygen reduces the ability of ionizing radiation to produce reactive oxygen species (ROS) necessary to produce DNA damage that results in tumour death. Hypoxia also increases the expression of a number of genes including cyclooxygenase (COX) -2. COX-2 is an inducible enzyme that is upregulated in inflammation and carcinomas, making it a suitable target for new cancer treatment options. COX-2 expression has been shown to be upregulated in a number of cancers including PCa and found to be involved in proliferation, invasion, apoptosis, host immune response and angiogenesis. Past research has also demonstrated that the major product of COX-2, prostaglandins (PG), provide radioprotection to cancer cells. As well as the ability of COX-2 to directly impact radiosensitivity, it has the ability to influence other cellular products that impact survival and apoptosis. One product that has been linked to COX-2 is the tumour suppressor p53. The p53 protein protects against genomic instability and the development of cancer by inducing cell cycle arrest and apoptosis. COX-2 expression has been shown to be induced by p53 and in turn results in inhibition of p53 mediated apoptosis. To our knowledge no studies have investigated the use of COX-2 inhibitors, at clinically relevant doses, as an adjunct to radiotherapy. In addition, while the direct mechanisms of COX-2 mediated radiosensitivity have been well investigated, research into the indirect mechanisms and also the effect of COX-2 inhibitors as radiosensitising agents in hypoxia is lacking. This study aims to demonstrate the ability of COX-2 inhibitors to improve treatment outcomes post-radiotherapy for PCa in human patients and also to investigate the mechanisms behind COX-2 inhibitor mediated radiosensitivity in normoxia and hypoxia. Method The ability of COX-2 to produce radiosensitisation in cancer cells was investigated in two ways in this research. Firstly, a retrospective human study investigated if the use of the COX-2 inhibitors meloxicam and celecoxib during radiotherapy improves treatment outcomes in PCa patients. Secondly, an in vitro model was developed that investigated the mechanisms behind COX-2 mediated radiosensitivity in human cancer cell lines. The retrospective human study examined the patient database at Genesis Cancer Care to identify patients that received radiotherapy for primary treatment of localised PCa. Screening of the database then identified patients that had used meloxicam or celecoxib during radiotherapy treatment. Three treatment outcomes were measured; the percentage of patients that demonstrated biochemical relapse at 2 and 5 years post-treatment, the time to biochemical relapse and the prostate specific antigen (PSA) velocity of each group post-treatment. The in vitro model used HeLa (cervical), PC3 (Prostate), MCF7 (breast) and MeWo (melanoma) cells to investigate, mechanistically, how structurally unrelated COX-2 inhibitors impact radiosensitivity in normoxia and hypoxia. This model utilised resazurin reduction to measure proliferation, siCOX-2 RNA to produce COX-2 knockdown cells and enzyme-linked immunosorbent assays (ELISAs) to measure p53 phosphorylation and prostaglandin E2 (PGE2) production. Results PSA velocity was found to be 0.197ng/mL/year (0.939) for the meloxicam group and 0.828 ng/mL/year (3.15) for the celecoxib group. The two treatment groups were found to have significantly lower (p&lt;0.05) PSA velocities than the control group, 1.12 ng/mL/year (3.05). In addition, at the two year time point meloxicam was found to have no patients to have relapsed and the celecoxib and control groups had percentage relapse rates of 6.7% (n=4) and 8.6% (n=5). The percentage relapse at five years post-treatment was 18.9% (n=10), 18.3% (n=11) and 21.0% (n=31%) for the meloxicam, celecoxib and control groups respectively. Mean time to biochemical relapse was found to be 54.15 months (16.08) in the meloxicam group and 46.20 months (31.70) in the celecoxib group, in contrast the control group demonstrated a mean time to biochemical relapse of 35.53 months (20.21). The in vitro model aimed to provide an insight to the mechanisms behind the results seen in the retrospective analysis. NS398 (10μM), a highly specific COX-2 inhibitor, selectively sensitised hypoxic HeLa (p&lt;0.01) and MCF7 (p&lt;0.05) cells to ioninsing radiation, however did not affect sensitisation in PC3 or MeWo cells. Celecoxib (20μM) and meloxicam (20μM) failed to produce radiosensitisation in any cell line in normoxia or hypoxia. Based on these findings further investigations were carried out in HeLa cells using NS398. Investigations using COX-2 siRNA demonstrated that knockdown of the COX-2 enzyme did not produce radiosensitisation but resulted in the loss of radiosensitising ability of NS398. Furthermore, PGE2 release was significantly increased in response to hypoxia (p&lt;0.05) and irradiation (p&lt;0.001) and treatment with NS398 significantly (p&lt;0.001) attenuated PGE2. Treatment with misoprostol, a prostaglandin analogue, significantly (p&lt;0.01) increased cell survival in normoxic HeLa cells in response to ionising radiation, but had no effect on hypoxic HeLa cells. The interaction between COX-2 and p53 was then explored. It was discovered that COX-2 knockdown HeLa cells had significantly (p&lt;0.001) higher levels of p53 protein than wild-type HeLa cells. Furthermore, hypoxia was able to significantly (p&lt;0.001) attenuate p53 phosphorylation at both 30 minutes and 4 hours post-irradiation and treatment of hypoxic HeLa cells with NS398 (10μM) resulted in a significant (p&lt;0.01) increase in phosphorylated p53. The p53 inhibitor pifithrin-α did not effect the radiosensitivity of wild-type HeLa cells in either hypoxia or normoxia, however it significantly (p&lt;0.01) increased cell survival in response to ionizing radiation in HeLa cells transfected with COX-2 siRNA. Discussion The findings from the retrospective analysis demonstrated that the use of the COX-2 inhibitors celecoxib and meloxicam may improve treatment outcomes in patients that receive radiation therapy for PCa. Importantly we were able to demonstrate that patients using these agents displayed a significantly lower PSA velocity than those who were not. Importantly, the PSA velocity was found to fall below the threshold of 0.35ng/mL, with PSA velocities that sit above this threshold having been found to have an approximate 5.3 to 10 fold increased risk of PCa. This retrospective analysis also found reduced biochemical relapse rates and reduced time to biochemical relapse in the celecoxib and meloxicam groups in comparison to the control. The in vitro model was able to demonstrate the ability of COX-2 inhibitors to increase radiosensitivity, however this effect was not demonstrated by all COX-2 inhibitors or all cell lines. This finding suggested that this effect may be drug and cell line specific. Furthermore, COX-2 was found to be necessary for NS398 induced radiosensitivity but knockdown of COX-2 alone did not affect radiosensitivity. Based on these findings it was suggested that COX-2 inhibitor mediated radiosensitivity occurs through both direct and indirect COX-2 mechanisms. We also demonstrated the relationship between COX-2 and p53, with COX-2 knockdown cells demonstrating increased p53 expression. Furthermore, the inhibition of COX-2 by NS398 resulted in increased p53 phosphorylation. Results from both the retrospective and in vitro studies provide evidence that further study into the role of COX-2 inhibitors as an adjunct treatment option in radiotherapy is warranted. Further studies, including large prospective human studies, are needed to confirm these findings. These large studies should collect tumour biopsies to allow for histological investigations. The research provides further evidence to the potential for COX-2 inhibitors to be used as an adjunct to radiotherapy in cancer.