Activation of Guanylate Cyclase-C With Heat-Stable Enterotoxin Fails to Potentiate Celecoxib-Induced Reduction in Colorectal Cancer Cell Growth in Vitro
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CitationSagartz, Samuel John. 2016. Activation of Guanylate Cyclase-C With Heat-Stable Enterotoxin Fails to Potentiate Celecoxib-Induced Reduction in Colorectal Cancer Cell Growth in Vitro. Master's thesis, Harvard Extension School.
AbstractThis study evaluated the use of heat-stable enterotoxin (ST), a guanylate cyclase receptor type C (GC-C) agonist, in combination with celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, for the prevention of colorectal cancer (CRC) growth in vitro. GC-C is a membrane-bound enzyme found in the lumen of the intestines and is responsible for the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). Activation of GC-C elevates intracellular cGMP which controls fluid-ion homeostasis and enterocyte differentiation along the crypt-villus axis. GC-C function can regulate colonic cell proliferation and induce cell cycle arrest. COX-2 is a cytosolic enzyme that catalyzes the formation of prostaglandins from arachidonic acid and inhibition of this enzyme is known to inhibit proliferation of human cancer cells. Previous research demonstrated that inhibition of the primary enzyme responsible for degradation of cGMP potentiated the cytostatic effects of celecoxib. This thesis tested if GC-C activation can enhance the antiproliferative effects of celecoxib.
Cell proliferation was assessed using a thymidine incorporation protocol, in which cells are cultured with tritiated thymidine after treatment and the amount of measured incorporated radioactivity correlates with cell division. Celecoxib was found to inhibit proliferation of T84 cells, which are derived from a human colorectal adenocarcinoma. This was consistent with previous studies that demonstrated an anti-proliferative effect of celecoxib in other human cancer cell lines. However, the addition of ST to celecoxib was unable to potentiate the anti-proliferative effects of celecoxib. ST also had no effect on proliferation when used as a monotherapy.
Changes in COX-2 activity, as well as the presence of COX-2 enzyme, were measured to assess whether the effect of celecoxib in T84 cells was dependent on its inhibitory activity on COX-2. No changes in cyclooxygenase activity were observed and COX-2 was not detected in T84 cells. In addition, celecoxib and the combination of celecoxib and ST were unable to produce a detectable amount of Caspase-3, a marker of cell death, suggesting that these drugs do not have a cytotoxic effect in T84 cells.
This research showed that ST does not have an effect on proliferation of T84 cells, which conflicts with previous research conducted by Pitari et al. (2001). It is possible that an effect of ST on adenocarcinomas in the gastrointestinal tract may vary based on the location of the targeted cells. Therefore, while ST did not reduce proliferation on cells derived from the colon, it may have an effect on proliferation of epithelial cells that are located in the small intestine upstream from the colon. Celecoxib was confirmed to have an anti-proliferative effect, and this was shown to be independent of the drugs enzymatic target, implying that there are additional mechanisms through which celecoxib exerts its activity. Additional research is needed to clarify these mechanisms.
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