Abstract
Sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colorectal cancer. However, the optimal drug, period of efficacy and mechanism(s) of action are unknown. Experiments were undertaken to determine which of several NSAIDs would modulate colon crypt cell proliferation or apoptosis when given during the initiation phase of 1,2-dimethylhydrazine (DMH)-induced rat colon cancer. Colon crypts located both away from and over an aggregate of lymphoid nodules (ALN) were examined. Rats were injected with aspirin, indomethacin, nabumetone, sodium salicylate, 16,16-dimethyl prostaglandin E2 or saline for 3 days and DMH or DMH vehicle on day 4 of each week for 8 weeks, then killed 3 days after the last DMH injection. At the time of killing, DMH had significantly increased crypt cell proliferation but not apoptosis. There was significantly more cell proliferation and apoptosis in crypts over the ALN than away from the ALN. Aspirin and salicylate increased proliferation and apoptosis in crypts over the ALN. Finally, the distributional peaks of cell proliferation and apoptosis were shifted significantly closer together after DMH. Thus, DMH increases proliferation and alters the distribution of proliferating and apoptotic cells in colon crypts early in carcinogenesis. Aspirin may suppress tumour incidence via salicylate by enhancing apoptosis in carcinogen-initiated cells.
Highlights
MethodsMale Sprague-Dawley rats were purchased at 2 months of age from Harlan Sprague-Dawley (Indianapolis, IN, USA) 4 weeks before the experiment
The following materials were used in these studies: DMH, acetylsalicylic acid (ASA, aspirin), sodium salicylate, indomethacin, and 16,16-dimethyl-prostaglandin E, were purchased from Sigma (St Louis, MO, USA); nabumetone was a generous gift from SmithKline Beecham, King of Prussia, PA, USA; an antiproliferating cell nuclear antigen (PCNA) monoclonal antibody (PC10 clone) was purchased from Signet (Dedham, MA, USA); and the Apoptag kit was used in the terminal deoxynucleotidal transferase (TdT) UDP nucleotide end-labelling (TUNEL) assay for fragmented DNA ends
Two-way analysis of variance (ANOVA) evaluating the influence of location of crypts either over or away from the aggregate of lymphoid nodules (ALN) and the influence of non-steroidal anti-inflammatory drugs (NSAIDs) treatment on cell proliferation in DMH-treated rats revealed that both variables independently and significantly increased the number of proliferating cells per crypt, with a significant interaction between the two variables
Summary
Male Sprague-Dawley rats were purchased at 2 months of age from Harlan Sprague-Dawley (Indianapolis, IN, USA) 4 weeks before the experiment. Rats had free access to Teklad ML485 Rat Diet (Teklad/HSD, Madison, WI, USA) and water before and throughout these studies. The following materials were used in these studies: DMH, acetylsalicylic acid (ASA, aspirin), sodium salicylate, indomethacin, and 16,16-dimethyl-prostaglandin E, (dmPGE2, a stable PGE2 analogue) were purchased from Sigma (St Louis, MO, USA); nabumetone was a generous gift from SmithKline Beecham, King of Prussia, PA, USA; an antiproliferating cell nuclear antigen (PCNA) monoclonal antibody (PC10 clone) was purchased from Signet (Dedham, MA, USA); and the Apoptag kit (purchased from Oncor, Gaithersburg, MD, USA) was used in the TdT UDP nucleotide end-labelling (TUNEL) assay for fragmented DNA ends. DMH vehicle contained 0.9% sodium chloride and 0.18% EDTA, pH 6.5, and all other solutions were prepared and diluted with normal saline (0.9% codium chloride solution, pH 6.5).
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