Alexandra Anker, Tobias Welponer, Sami Judeeba, MD, John Geibel, DSc, MD, MSc. Yale Medical School, Department of Surgery
Introduction:
Many studies prove the effect of aspirin on reducing the risk of colorectal cancer, polyps and adenoma in terms of incidence and recurrence especially among FAP patients. In fact, there is some evidence that aspirin prevents hereditary non-polyposis colorectal cancer. Experts are still arguing about the optimal dose and duration of aspirin to exert its anti-tumorigenic activity. Some studies however could demonstrate a benefit with any type of NSAID at any dose.
In our study we try to improve our understanding of the mechanism by which aspirin decreases the risk of colorectal cancer as it is still unclear. We analyzed the impact of low-dose Aspirin (7.9µM) on crypt cells of rat distal colon with particular focus on Na+/H+ exchanger (NHE) activity and pH changes.
Materials and methods:
Male Sprague-Dawley rats weighing 325-425 g were dissected according to approved protocols of the Animal and Use Committee at Yale University. Isolated crypt cells from rat distal colon were transferred to a coverslip that was pretreated with the fibrin sealant Cell-Tak. The coverslip was attached to a perfusion chamber and incubated at room temperature for 18 min with HEPES-buffered Ringer solution containing 10µM of BCECF-AM (2',7'-bis-(Carboxyethyl)-5(6')-carboxyfluorescein Acetoxymethyl Ester, Santa Cruz Biotech) a fluorescent indicator for the measurement of intracellular pH. Afterwards the chamber was placed on the temperature-controlled (37°C) stage of an inverted microscope (Olympus IX71). A minimum of two midportion regions of each crypt were selected and monitored using a digital monitoring system during the experiment. BCECF was excited at 490nm ± 10nm and 440nm ± 10nm and the resultant fluorescent emission signal was measured at 535nm ± 10nm every 15s using a digital camera and specific software (Metafluor). The cells were perfused continuously with each of the following solutions for at least 5 minutes: standard HEPES full-sodium buffer solution – 30mM NH4Cl solution (initial alkalization of the cell) – sodium-free HEPES solution (acidification of the cell) – reperfusion with standard HEPES full-sodium solution. NHE activity was determined by monitoring pH recovery (d pHi/s) during reperfusion. To calculate pHi values from image data (intensity ratio 490/440) cells were calibrated using the high-K+/Nigericin calibration technique. It could be demonstrated that crypts exposed to Aspirin show a significantly higher rate of recovery compared to a control without Aspirin.
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