Vol. 4, 239-244, April/May 1995 Cancer Epidemiology, Biomarkers & Prevention 239
Measurement of Prostaglandin E2 in Rectal Mucosa in Human Subjects: A Method Study1
;;I i;:I-R. Finley, Cheryl 1. Bogert, David S. Alberts,2 adults with incidence increasing to as high as 50% in Janine Einspahr, David 1. Earnest, Gina Blackwell, persons over 70 years of age (1 ). Multiple studies have and Ken Girodias shown that certain NSAIDs3 inhibit cyclooxygenase and the
Section of Clinical Pathology, Department of Pathology (P. R. Fl. Arizona synthesis of prostaglandmns and can reduce the formation of Cancer Center IC. L. B., D. S. A., J. E., G. B., K. G.l, Section of both colon polyps and cancers in experimental animals Hematology and Oncology, Department of Medicine ID. S. Al, given known carcinogens (2, 3). Two recent case-control and Section of Gastroenterology, Department of Medicine ED. L. E., G. B.l, drug surveillance studies and one large cohort study found University of Arizona, College of Medicine, Tucson, Arizona 85724 that patients with regular aspirin use had a reduced mci- dence or decreased death rate from colorectal cancer (4- Abstract 6). Most recently, the results of a randomized clinical trial showed that the NSAID Sulindac promotes regression and It has been demonstrated and confirmed that certain inhibits recurrence of adenomatous colon polyps in patients nonsteroidal anti-inflammatory drugs which inhibit with familial adenomatous polyposis (7). cyclooxygenase and the synthesis of prostaglandins and Further evidence that there is a relationship between other eicosanoids, can reduce the formation of both the concentration of PGE2 in tissue and the development of colon polyps and cancers in experimental animals given colonectal cancer was reported by Earnest et a!. (8). Tissue known carcinogens. Additionally, the results of several concentration of PGE2 was significantly increased in cob- epidemiologic studies have suggested that nonsteroidal rectal adenomas compared to normal flat mucosa and was anti-inflammatory drugs may reduce the risk of colon even greater (P< 0.05) in malignant colon polyps and gross polyp occurrence and/or colon cancer mortality. We cancer. Nanisawa et a!. (9) showed a high level of PGE2 in have carried out a study to evaluate the methodology of local venous blood-draining colon carcinomas and in the the measurement of prostaglandin E2 (PGE2) in human peripheral blood in patients with liver or lung metastases. colonic mucosa because its concentration may serve as a Tissue analysis showed a significantly larger amount of valuable intermediate marker of the pharmacological PGE2 production in carcinomatous versus normal cobonic activity in Phase II studies of nonsteroidal anti- mucosal tissue. These investigators suggested that increased inflammatory drugs as colon cancer preventive agents. local blood PGE2 could enhance metastasis formation, and We studied all aspects of the actual measurement of that increased peripheral blood PGE2 might be useful in the PGE2 including the extradion efficiency of the PGE2 detection of such metastases in colorectab cancer. from the mucosa, the precision of the assay and The purpose of this study was to establish strict criteria calculation of the PGE2 content in terms of milligrams of for the measurement of PGE2 in normal appearing rectal protein in the sample, the inhibition of PGE2 by mucosal biopsies and to control all ofthe variables that may indomethacin over time, the reproducibility of the affect the accuracy and precision of the assay. Such accu- measurement within one homogenate, the rate of PGE2 racy and precision are critical to the validity of prospective production over time, the effect of adding indomethacin Phase II cancer prevention trials that use tissue bevels of versus snap freezing on PGE2 production, the stability of PGE2 as an intermediate marker of NSAID pharmacological PGE2 fl tissues over time stored in liquid nitrogen, and effect. the variability of the measurement of PGE2 in separate biopsies from one individual. Our studies indicated that the most reliable method for accurate and consistent Materials and Methods measurements of PGE2 was to add the mucosal tissue Colon Biopsy Sample Collection. Samples were collected instantly after biopsy to an indomethacin buffer that by one of several assisting gastroenterobogists working with effectively inhibited the in vitro formation of PGE2. us. Patients were prepped using two 1 50-mb tap water enemas 30 min-2 h before the procedure. After the sig- moidoscope was inserted into the rectum, biopsies were Introduction taken perpendicular to the mucosal surface from the upper Cancer of the colon is the second most prevalent malig- half of the rectum (1 2-1 8 cm from the anus). Because the nancy in the United States. There is strong evidence that amount of endogenous PGE2 can be determined by the adenomatous colon polyps are precursor lesions of colon depth of the biopsy (1 0), routine-sized forceps were used to cancer. Such polyps are found in approximately 10% of ensure a continuity of biopsy depth into the mucosa. Samples were placed in either empty cryovials or cryoviabs
Received 6/28/94; revised 12/21/94; accepted 12/21/94. This work was supported in part by NIH Grants CA-41 1 08 and CA-23074. 2 To whom requests for reprints should be addressed, at Arizona Cancer 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; Center, 1 501 N. Campbell Avenue, Tucson, AZ 85724. PGE2, prostaglandmn E2; RIA, radioimmunoassay.
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containing aqueous indomethacin and snap frozen in liquid [3H]prostaglandin tracers (1 2). Appropriate dilutions were nitrogen with approximately the same time period between made to the samples to fit the values into the curve range. biopsy removal and freezing for each biopsy. Aqueous Samples were counted on the Micromedic 1 0/6000 Plus indomethacin is Indocin IV. (Merck, Sharp, & Dohme, Apex Series gamma counter (Micromedic Systems Division, West Point, PA) dissolved in double distilled water to a final ICN Biomedicals, Inc., Costa Mesa, CA). The standard concentration of 5 pg/mI. All tissue samples remained curve was reduced using four-parameter logit curve fitting. stored in liquid nitrogen until the time ofexpenimentation or Protein Assay. Protein aliquots were analyzed using the extraction. A single biopsy had an approximate wet weight bicinchoninic acid assay (Pierce, Rockfond, IL). When com- of 1 7.9 mg (range, 1 2.0-22.0 mg). Only biopsies stored in pared to other protein determination methods, the bicin- aqueous indomethacin were weighed. choninic acid assay provides improved sensitivity, easily Tissue Homogenization. Tissue was homogenized in either modified assay parameters, less difficulty with interfering ice-cold (0-4#{176}C) extraction buffer or ice-cold indometha- substances, and increased stability in the working reagent cm buffer depending on the experiment to be performed. (1 3). Due to the small amount of sample taken for protein Extraction buffer is 0.05 M Tnizma base (Sigma Chemical determination, the microtiter plate protocol was used. Be- Co., St. Louis, MO) titrated to pH 7.4 with concentrated sides saving on reagents and providing a greater number of hydrochloric acid (FisherChem, Fair Lawn, NJ). Indometha- protein values per sample (6 wells/sample), this protocol cm buffer is Indocin IV. dissolved in extraction buffer to a allows for a sensitivity that is approximately 10-fold greaten final concentration of 5, 1 0, or 20 pg/mb depending on the than the manufacturer’s standard test tube assay procedure procedure to be followed. Tissue was homogenized by (1 4). The standard protein concentration ranges used were hand in a siliconized (reagent grade Sigmacote; Sigma) 200-1200 and 10-200 pg/mI. Appropriate dilutions were Duall glass-glass tissue grinder (Kontes Glass, Vineland, CT) made to the samples as needed to fit the values within an for approximately 30 s or until there were no visible pieces assay curve. The plates were incubated at 37#{176}Cfor 30 mm of tissue. When multiple rectal mucosal biopsies were ho- and read immediately on the Biomek 1000 (Beckman In- mogenized together, they immediately were diluted after struments, Inc., Fullerton, CA) at an absorbance wavelength homogenization in additional indomethacin buffer to attain of 540 nm. a consistent ratio of 1-2 biopsies/mI buffer. Extradion Efficiency [1251]. 125I-PGE2 (0.1 ml) from the Sample Extraction. The extraction procedure used was the RIA kit was diluted with extraction buffer to a final volume procedure outlined by Powell (1 1 ). The homogenate was of 2.0 ml. Two 0.1 -ml aliquots were placed in two test tubes placed in a 50.0-mb polypropylene tube. Two milliliters and capped (total counts). Two additional 0.1-mb aliquots 1 00% ethanol (Quantum Chemical Corp., Cincinnati, OH) were diluted with extraction buffer to a final volume of 1 .0 was added and the sample was vortexed and allowed to ml and extracted. Both samples were ebuted with 10.0 ml stand on ice for 5 mm. A 100-mg barge capacity reservoir methyl fonmate into three separate tubes. Each tube was C18 column (Alltech Associates, Inc., Deerfield, IL) was evaporated to dryness under nitrogen and all tubes were washed with i00% ethanol (20 ml) followed by double counted. The three values for the three tubes from each deionized water (20 ml) to remove excess ethanol. After sample were added. The two values from the total counts adding 1 0.3 ml ice-cold double deionized water to the were averaged. The two values from the extracted samples tissue homogenate to give a final concentration of 1 5% were averaged. The extraction efficiency was calculated. ethanol, the sample was vortexed vigorously and centni- fuged at 4#{176}Cat3000 rpm for 10 mm. The supemnatant was Total extracted counts Extraction efficiency = x 100 removed, the pellet discarded, and the pH of the supemna- Total counts tant adjusted to 3.0 with 0.25 M hydrochloric acid. The sample was then applied to the prepared column and ebuted The extraction efficiency for this experiment was 92%. under gentle vacuum with a Vac-Elut (Vanian Sample Prep- Extradion Efficiency [3H]. 1 5 p1 [3HIPGE2 (DuPont New aration Products, Harbor City, CA). The column was rinsed England Nuclear) was diluted with extraction buffer to a with 1 5% ethanol (20 ml) followed by 20.0 ml reagent final volume of 4.0 ml. One 1 .0-mI aliquot was placed in a grade petroleum ether (EM Science, Gibbstown, NJ). The scintillation vial and 10.0-mb scintillation cocktail was PGE7 was gravity eluted with 1 0.0 ml reagent grade methyl added. Two additional 1 .0-mI abiquots were extracted. The formate (Sigma) into a siliconized glass or polypropylene two samples were eluted into two separate scintillation vials test tube. The entire elution process occurred at a rate of and 1 0.0-mI scintillation cocktail were added to each. Each approximately 0.5 mI/mm. The methyl formate was divided of the three samples was counted for 1 mm on a Beckman into four 2.5-mI aliquots and evaporated to dryness under a LS 3801 liquid scintillation counter (Beckman Instruments, gentle stream of nitrogen using a Mini-Vap Six-port con- Inc.). The values for the two eluted samples were averaged centrator/evaporator (Chemical Research Supplies, Addi- and the extraction efficiency was calculated. Extraction son, IL). Samples were stored at -80#{176}Cuntil the time ofthe efficiency was calculated in the same manner as the 1251 experiment when they were reconstituted in assay buffer efficiency experiment. The extraction efficiency for this from the RIA kit. All tubes and glassware used during the experiment was 88%. extraction were polypropylene or siliconized glass. PGE2 Controls. Five milliliters each of 25, 100, and 200 Radioimmunoassay. The 112511 PGE2 RIA kit (DuPont New pg/mb PGE2 standard from the RIA kit were extracted. England Nuclear, Boston, MA) was used to determine the After the elution step, the methyl formate eluent was PGE2 content in the extracted samples and was performed placed in 0.5-mI aliquots in labeled microfuge tubes as per the manufacturer’s instructions. An iodinated PGE2 using a positive displacement pipettor. The samples were RIA was used due to a 25-30-fold increased sensitivity over evaporated to dryness under nitrogen and stoned at gas liquid chromatography coupled to mass spectrometry -20#{176}C. Each time an experiment was run, one sample and a 5-10-fold increase in sensitivity as compared to from each concentration was removed from the freezer,
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- 0- 5p ?mL Indomethacln 10 jg/ml indomethacin in the ability to inhibit prostag- 3000 -O-lOpglmL Indomethecln landin production. C . 2500 Rate of PGE2 Produdion over Time. Five rectal mucosal 0 2000 biopsies from one patient were homogenized in 1 .0 ml ice-cold (0-4#{176}C) extraction buffer and diluted with addi- E 1500 tional extraction buffer to a final volume of 6.0 ml. At time 1o00 0, 5, 15, 30, 45, 60, 90, 120, and 180 mm, 0.5 ml of homogenate was diluted in a microfuge tube in 0.5 ml 500 indomethacin buffer (20 pg/mb). The homogenate remained 0 5 10 15 20 25 30 35 40 45 50 55 60 at room temperature during the 180-mm incubation. Each TIme (MInutes) sample was vortexed, snap frozen in liquid nitrogen, and stoned at -80#{176}C.At a later date, the samples were thawed Fig. 1. PGE2 production rate in rectal mucosal tissue with addition of 5 or and two SO-p1 aliquots were removed from each for protein 10 pg/mI indomethacin. Five or 10 pg/mI indomethacin was added after 0, determination. The remaining 0.9 ml was transferred to a 5, 1 5, 30, or 60 mm of incubation. SO-mi polypropylene tube and extracted. (The undiluted homogenate remained at room temperature during the en- tine procedure.) This experiment was repeated using tissue from the same patient. The tissue was homogenized at room reconstituted in 0.25 ml RIA assay buffer, and assayed temperature but was incubated at 37#{176}Cforthe remainder of with the extracted samples. the procedure. The control values as well as all extracted samples Fig. 2 indicates the production of PGE2 over time in a were corrected using an average 90% extraction efficiency mucosal homogenate incubated either at room temperature as determined by the 1251 and 3H extraction efficiency or at 37#{176}Cwith prostaglandin production inhibited by in- expeni ments. domethacin added at specific time points. As shown, there pg PGE2/mg protein is no difference in PGE2 production between the two incu- Corrected sample value = 0.9 bation temperatures. Prostaglandmn production does not ap- pear to be stimulated by an increase of temperature above Calculation of PGE2/mg protein room temperature. Figs. 1 and 2 both indicate an apparent = pg PGE2/ml sample mg protein/mb sample plateau of approximately 30-60 mm after tissue removal for maximum PGE2 production. Both figures show the rapid but consistent linear rise in PGE2 content over the first Results 20-30 mm of incubation. PGE2 content appears to remain Reproducibility, Sensitivity, and Linearity. The corrected stable for at least 2.5 h after the plateau is reached. 25 pg/mI control value was 25.7 ± 2.0 (1 SD). The Effed of Adding Indomethacin versus Snap Freezing on corrected 1 00 pg/mI control value was 93.7 ± 7.82 (1 PGE2 Produdion. Five rectal mucosal tissue biopsies from SD). The corrected 200 pg/mI control value was 199 ± the same patient were homogenized in 1 .0-mI ice-cold 23.1 (1 SD). These values were obtained over a period of (0-4#{176}C) extraction buffer, and the final volume was brought 1 8 months with 23 separate runs. The lowest detectable up to 5.0 ml with additional extraction buffer. The homo- limit was demonstrated to be 2.5 pg/mI. The analytical genate remained at room temperature during the incubation range of the RIA is 2.5-250 pg/mI. Several suitable dilu- tions of the homogenate are made in each run to insure period. At time 0, 5, 60, and 180 mm, two 0.5-mI aliquots that results fall within the measurable analytical range. were removed. The first was diluted in a 1 .5-mI microfuge Serial dilutions of homogenates from many patient sam- tube in 0.5 ml aqueous indomethacin and snap frozen in pIes show linearity on the assay within the confines of the liquid nitrogen. The second was diluted in a 1 .5-mI mi- analytical range. crofuge tube in 0.5 ml extraction buffer and snap frozen in liquid nitrogen. All samples were thawed enough to enable PGE2 Inhibition by Indomethacin over Time. Fifteen rectal mucosal biopsies from one patient were homogenized in dropping the frozen pellet directly into 2.0 ml of 100% ethanol. The remainder of the extraction procedure was 3.5-mI ice-cold (0-4#{176}C) extraction buffer for 1 .0 mm, and the homogenate was vortexed. A 2.0-mb homogenate ali- performed. Of the remaining homogenate (0.2 ml) was quot was immediately placed in 4.0 ml extraction buffer diluted with 0.2 ml extraction buffer and eight SO-pb ali- quots made for protein determination. This experiment was (sample 1 ). This aliquot remained on ice for the duration of the incubation. At 0, 5, 15, 30, and 60 mm, two 0.5-mb repeated three times using tissue from separate patients. There was no significant difference in the inhibition of abiquots were removed from sample 1 . One aliquot was placed in 0.5 ml 10 pg/mI indomethacin buffer and the PGE2 production whether indomethacin was added before other placed in 0.5 ml 20 pg/mI indomethacin buffer. After snap freezing the tissue in liquid nitrogen. The values for the final time point was reached, five SO-p1 abiquots were the three experiments showed a wide range for 2,189.4- removed from the remaining sample 1 homogenate for 1 3,231 .7 pg PGE2/mg protein. When PGE2 content was protein determination. The protein abiquots were diluted converted to percentage of control for each of the three with 50 p1 double deionized water to give the same tissue experiments (control is the PGE7 content at the 1 80-mm concentrations as the time point samples. All samples were time point for the snap frozen sample in that experiment), extracted the same day. the three experiments showed similar production rates. The Fig. 1 indicates the effects of different concentrations use of indomethacin allows the investigator some freedom of indomethacin on PGE2 production at a series of time during the manipulation of the rectal mucosal tissue. Stor- points. There is no significant difference between 5 and ing the tissue(s) in indomethacin is preferable for procedural
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Control .. . D- 3 C 100 -0-Room Temperature - Continuing ..- -S 16000 Inhibition . 14000